Preface

Bacteriophages are viruses that target and infect bacterial cells. As such, they have long attracted interest as potential therapeutic agents to control microbial pathogenesis. Since their discovery they have been proven to be promising antimicrobials, but caution has been mixed into that hope, because they are also capable of invoking pathogenicity in commensal and environmental species of bacteria. Study and cumulative knowledge of the ingenious factors that these viruses use to survive in the wild and to prevail over their host cells, has contributed greatly to many diverse facets of molecular biology and to the rise of synthetic biology.

There is once again great promise that bacteriophages will play a prominent role in healthcare, not least fuelled by the rise in microbial antibiotic resistance globally. However, this hope also presents challenges. To understand the challenges, it is as well to also regard the problems bacteriophages could pose and how these can be solved. To realise their applied promise, bacteriophages will require production at unprecedented industrial scales. Exploring efficiency of mass production, as well as how to ensure purity from other bacteriophages that could contaminate medical-grade stocks, will be essential. In this volume, the chapters contributed respectively, by authors Zahn and Halter, and Ward et al., bring these matters into focus.

In a wider healthcare context, the structural properties of bacteriophages become important factors for consideration. The chapter in this volume contributed by White and Orlova gives a detailed overview of what we know so far about the structure and mechanics of some of the better-known phage particle types. Engineering the surfaces of these viruses is a mature technology, but its deployment in personalised medical imaging is now technologically feasible and demonstrated as effective. The contributed chapter by Khondee and Piyawattanametha illuminates how surgery to remove metastatic tumours is given greater precision via engineered phage particle avidity. Exploiting the unusual properties of bacteriophage-encoded enzymes can allow new approaches in gene therapies and cell and tissue engineering. In fact, applications enabled by such unusual enzymatic activities could revolutionise data storage via biological macromolecules. The chapters contributed to this volume by Pinheiro, and the chapter by Knott, Milsom, and Rothwell, showcase unusual enzymatic processes which bacteriophages utilise for their adaptation and survival.

Taking all these themes together, it is possible to envision bacteriophages as potentially versatile nanoscale entities, with scope to be repurposed for a variety of interventions involving bacterial cells, or in entirely unrelated aims, encompassing aspects of healthcare and electronics. The macromolecular tools utilised by replicating bacteriophages can further enrich such endeavours.

This book presents facets of current knowledge relevant to realising those broader future aims.

> **Renos Savva** Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck, University of London, United Kingdom

Chapter 1

Renos Savva

1. Resurgence

scientific discovery [7].

2. Repurposed

1

Synthetic Future

phages resembled prototypic lunar landing vehicles.

Introductory Chapter: Nature'

Ancient Nanomachines and Their

Bacteriophages are generally considered to be the most prevalent biological entities on planet Earth [1], with astronomical estimates of their myriad abundance. These fascinatingly diverse viruses, which infect bacterial cells, were discovered in the second decade of the twentieth century. Under the light microscope, bacterial cells were seen to be apparently eaten away, hence the scientific Greek naming of these viruses meaning literally "bacteria eaters". The middle decades of that century gave us a first glimpse of these viral particles, via their imaging using the technique of negative stain electron microscopy. The morphology of the first studied particles was symbolic of the so-called Space Age in which these discoveries took place: The archetypal Phi X 174 evoked the first artificial satellites, while the T-even

Such latter-day futuristic symbolism may seem amusingly outdated by the time of

In a commercial sense, phages are regarded as cheap to manufacture, because they will naturally multiply within their target bacteria. However, process control at

writing. However, the undoubtedly ancient bacteriophages are now riding a new wave of technological advancement: synthetic biology. Interestingly, this returns us to the potential revolution in healthcare that surrounded the initial discovery of bacteriophages. The simple fact is that phages, which is a common abbreviation used when referring to these viruses, are capable of selective killing of bacterial cells of a given species or strain. In other words, phages are exquisitely specific microbial control agents. In the current antimicrobial resistance era [2], exemplified by the ESKAPE organisms, which is to name only the vanguard of untreatable pathogenic microbes, phages offer a potential panacea for the treatment of pathogenic bacterial infection. Indeed, deployment of phages to treat bacterial infections in animals and humans was the first exploitative use of these virus entities. Their promise as therapeutic agents was, nevertheless, soon eclipsed by the rise of antibiotics. However, partly by economic necessity, the refinement of such phage-mediated treatments persisted in the former USSR [3]. Today, the fruit of that legacy provides effective alternative treatment in an era of antibiotic-resistant microorganisms [4]. Including the long-standing cases of treatment achieved within the pioneering hospitals in the former Soviet territory of Georgia [5], there have also been recent high-profile cases [6], as well as a rise in commercial offerings based upon sound

s

#### Chapter 1

## Introductory Chapter: Nature' s Ancient Nanomachines and Their Synthetic Future

Renos Savva

#### 1. Resurgence

Bacteriophages are generally considered to be the most prevalent biological entities on planet Earth [1], with astronomical estimates of their myriad abundance. These fascinatingly diverse viruses, which infect bacterial cells, were discovered in the second decade of the twentieth century. Under the light microscope, bacterial cells were seen to be apparently eaten away, hence the scientific Greek naming of these viruses meaning literally "bacteria eaters". The middle decades of that century gave us a first glimpse of these viral particles, via their imaging using the technique of negative stain electron microscopy. The morphology of the first studied particles was symbolic of the so-called Space Age in which these discoveries took place: The archetypal Phi X 174 evoked the first artificial satellites, while the T-even phages resembled prototypic lunar landing vehicles.

Such latter-day futuristic symbolism may seem amusingly outdated by the time of writing. However, the undoubtedly ancient bacteriophages are now riding a new wave of technological advancement: synthetic biology. Interestingly, this returns us to the potential revolution in healthcare that surrounded the initial discovery of bacteriophages. The simple fact is that phages, which is a common abbreviation used when referring to these viruses, are capable of selective killing of bacterial cells of a given species or strain. In other words, phages are exquisitely specific microbial control agents. In the current antimicrobial resistance era [2], exemplified by the ESKAPE organisms, which is to name only the vanguard of untreatable pathogenic microbes, phages offer a potential panacea for the treatment of pathogenic bacterial infection.

Indeed, deployment of phages to treat bacterial infections in animals and humans was the first exploitative use of these virus entities. Their promise as therapeutic agents was, nevertheless, soon eclipsed by the rise of antibiotics. However, partly by economic necessity, the refinement of such phage-mediated treatments persisted in the former USSR [3]. Today, the fruit of that legacy provides effective alternative treatment in an era of antibiotic-resistant microorganisms [4]. Including the long-standing cases of treatment achieved within the pioneering hospitals in the former Soviet territory of Georgia [5], there have also been recent high-profile cases [6], as well as a rise in commercial offerings based upon sound scientific discovery [7].

#### 2. Repurposed

In a commercial sense, phages are regarded as cheap to manufacture, because they will naturally multiply within their target bacteria. However, process control at industrial scales presents challenges: most notably in efficiency and reproducibility but also via complications in terms of sterility and purity [7, 8]. Knowing how to keep prevalent diverse phage types out of a production facility will doubtless be as important as manufacturing commercially relevant types efficiently. When considering efficiency of phage production, the ultimate cost depends upon the measures that have to be taken, from plant and tooling to culture volume and starter culture characteristics. Investigations of multiplicity of infection, and economics of scale, plus processing of waste effluent are all factors that will impact costs and affordability [9]. Upscale and purity of phage will have an impact on any type of technology or application that is currently envisaged for phage, from antimicrobial therapy to phage particles as nanomaterials.

applied biotechnology. The numbers of completely sequenced diverse phage genomes expand at a prolific rate, and the mysteries of their encoded protein

Introductory Chapter: Nature's Ancient Nanomachines and Their Synthetic Future

Phage biology then, played a central role in the elucidation of some of the major genetic insights of the twentieth century. Other ground-breaking studies in that century concerned phage genome replication, the structures of phage particles and their assembly, and mechanisms of bacterial cell immunity and its viral subversion. Modern techniques in genomics, proteomics, and structural biology are adding novel insights even now [13, 15]. This combination of precision studies illuminates the plasticity of macromolecular and cellular biology in this perennial cauldron of

In terms of replicative strategies, phage activity in a cell involves an interesting sideway taken on biological processes. The enzymatic agents encoded by phages have applications in laboratory research technologies, applied molecular evolution techniques, and also novel ways to make DNA for therapeutic and technological use: free of the heretofore necessity for bacterial cell passage to propagate this DNA and thus of bacterial propagation sequences, including antibiotic resistance genes. The insights from phages therefore bring us to the brink of revolutionising applications as diverse as human gene therapy, cell and regenerative medicine, and DNA as data

Finally, looking beyond what we have discovered and tested, beyond this volume and into that potential future of synthetically modified bacteriophages with

Bacteriophages can be envisaged as natural microbial control agents, as well as machines for targeted synthetic genetic programming. The encoded proteins, as well as the structures of phages, offer a multitude of possibilities

as outlined in this introductory chapter and detailed in the volume.

manifest may yet take a long time and a lot of care to unravel.

evolutionary cat and mouse.

DOI: http://dx.doi.org/10.5772/intechopen.90384

storage (Figure 1).

4. Rebooted

Figure 1.

3

We can consider that phage genetic and structural insights are opening doors in nanotechnology and synthetic biology applications, which have a translation potential back into healthcare. That medical relevance is not limited to antimicrobial applications but also encompasses cancer and gene therapy. Indeed, phage display technologies are revolutionising the high-resolution visualisation of metastatic tumours in surgical settings by enabling unambiguous contrast.

Regarding the structure of phage particles, as nanoscale parts assembling into a mechanised vehicle for DNA packaging and delivery, their production as potential therapeutic agents might well be due a synthetic makeover. The concept of a phage cocktail is now very well tested: namely, several phage types recognising the same bacterial species but attacking that cell type in different ways. Phage cocktails are known to make it less likely that the targeted cells can survive via adaptation, as is known to be the case via treatment with a single phage type. Lately, the reprogramming of a phage DNA payload to ensure target cell death rather than phage latent persistence in a population of cells has also been found to be effective [6]. Indeed, such engineering approaches have been used to alter the DNA payload injected by the phage particle in order to make phages more effective at cell killing, rather than infection per se [6, 10].

Nevertheless, from an efficiency and medical regulatory perspective, it could be advantageous to have one or just a few medically approved phage chassis. These might be envisaged as phages known for their low immunogenicity profiles in patients. Perhaps these may be patient-specific, in which the therapeutic DNA payload is installed in a phage chassis matched to the current patient's immune tolerance. Then one can envisage a programmable targeting built in via a synthetic biology approach of re-engineering components of these standardised phages to recognise any desired bacterial target. Demonstrations of swapping out the targeting structures (i.e., tails and adsorption features) have been equally impressive in principle [11, 12]. Thus, furthering a detailed knowledge of phage structure beyond well-studied phage types [13] is incentivised.

#### 3. Reimagined

One of the most fascinating things about phages is that they are hotbeds of molecular adaptation. An eclectic and presently largely arcane phage-encoded protein panoply supports the survival and success of the diverse bacteriophage types. The exploitation of deciphered elements of this phage protein repertoire was central to development of the recombinant biotechnology revolution. Circuitously, these very molecular tools are bringing about the technological revolution that promises to lend a starring role of phage in a biotechnologically repurposed guise. In fact, looking into that mesmerising pool of proteins of unknown function [14], it is easy to believe that the phages' encoded box of tricks isn't yet done revolutionising

#### Introductory Chapter: Nature's Ancient Nanomachines and Their Synthetic Future DOI: http://dx.doi.org/10.5772/intechopen.90384

applied biotechnology. The numbers of completely sequenced diverse phage genomes expand at a prolific rate, and the mysteries of their encoded protein manifest may yet take a long time and a lot of care to unravel.

Phage biology then, played a central role in the elucidation of some of the major genetic insights of the twentieth century. Other ground-breaking studies in that century concerned phage genome replication, the structures of phage particles and their assembly, and mechanisms of bacterial cell immunity and its viral subversion. Modern techniques in genomics, proteomics, and structural biology are adding novel insights even now [13, 15]. This combination of precision studies illuminates the plasticity of macromolecular and cellular biology in this perennial cauldron of evolutionary cat and mouse.

In terms of replicative strategies, phage activity in a cell involves an interesting sideway taken on biological processes. The enzymatic agents encoded by phages have applications in laboratory research technologies, applied molecular evolution techniques, and also novel ways to make DNA for therapeutic and technological use: free of the heretofore necessity for bacterial cell passage to propagate this DNA and thus of bacterial propagation sequences, including antibiotic resistance genes. The insights from phages therefore bring us to the brink of revolutionising applications as diverse as human gene therapy, cell and regenerative medicine, and DNA as data storage (Figure 1).

#### Figure 1.

industrial scales presents challenges: most notably in efficiency and reproducibility but also via complications in terms of sterility and purity [7, 8]. Knowing how to keep prevalent diverse phage types out of a production facility will doubtless be as important as manufacturing commercially relevant types efficiently. When considering efficiency of phage production, the ultimate cost depends upon the measures that have to be taken, from plant and tooling to culture volume and starter culture characteristics. Investigations of multiplicity of infection, and economics of scale, plus processing of waste effluent are all factors that will impact costs and affordability [9]. Upscale and purity of phage will have an impact on any type of technology or application that is currently envisaged for phage, from antimicrobial therapy

We can consider that phage genetic and structural insights are opening doors in

Regarding the structure of phage particles, as nanoscale parts assembling into a mechanised vehicle for DNA packaging and delivery, their production as potential therapeutic agents might well be due a synthetic makeover. The concept of a phage cocktail is now very well tested: namely, several phage types recognising the same bacterial species but attacking that cell type in different ways. Phage cocktails are known to make it less likely that the targeted cells can survive via adaptation, as is

Nevertheless, from an efficiency and medical regulatory perspective, it could be advantageous to have one or just a few medically approved phage chassis. These might be envisaged as phages known for their low immunogenicity profiles in patients. Perhaps these may be patient-specific, in which the therapeutic DNA payload is installed in a phage chassis matched to the current patient's immune tolerance. Then one can envisage a programmable targeting built in via a synthetic biology approach of re-engineering components of these standardised phages to recognise any desired bacterial target. Demonstrations of swapping out the targeting structures (i.e., tails and adsorption features) have been equally impressive in principle [11, 12]. Thus, furthering a detailed knowledge of phage structure

One of the most fascinating things about phages is that they are hotbeds of molecular adaptation. An eclectic and presently largely arcane phage-encoded protein panoply supports the survival and success of the diverse bacteriophage types. The exploitation of deciphered elements of this phage protein repertoire was central to development of the recombinant biotechnology revolution. Circuitously, these very molecular tools are bringing about the technological revolution that promises to lend a starring role of phage in a biotechnologically repurposed guise. In fact, looking into that mesmerising pool of proteins of unknown function [14], it is easy to believe that the phages' encoded box of tricks isn't yet done revolutionising

nanotechnology and synthetic biology applications, which have a translation potential back into healthcare. That medical relevance is not limited to antimicrobial applications but also encompasses cancer and gene therapy. Indeed, phage display technologies are revolutionising the high-resolution visualisation of meta-

static tumours in surgical settings by enabling unambiguous contrast.

known to be the case via treatment with a single phage type. Lately, the reprogramming of a phage DNA payload to ensure target cell death rather than phage latent persistence in a population of cells has also been found to be effective [6]. Indeed, such engineering approaches have been used to alter the DNA payload injected by the phage particle in order to make phages more effective at cell killing,

to phage particles as nanomaterials.

Bacteriophages - Perspectives and Future

rather than infection per se [6, 10].

3. Reimagined

2

beyond well-studied phage types [13] is incentivised.

Bacteriophages can be envisaged as natural microbial control agents, as well as machines for targeted synthetic genetic programming. The encoded proteins, as well as the structures of phages, offer a multitude of possibilities as outlined in this introductory chapter and detailed in the volume.

#### 4. Rebooted

Finally, looking beyond what we have discovered and tested, beyond this volume and into that potential future of synthetically modified bacteriophages with

diverse uses, what might we find? Perhaps we might find nanomachines inspired in their design by the multifarious forms and mechanisms of both known and newly studied types of these viruses, lightning-fast genotyping of bacterial infections at point of care, and efficiently timely synthetic tooling of medically approved phages to rapidly quell those infections, even on the scale of a few hours. We might also find synthetic phages reprogramming the microbiome, both by selective population control and by targeted and firewalled genetic modification in situ, and even the possibility of tooling wild species of bacteria for bioremediation purposes via reversible and firewalled genetic modification. There could be phage-encoded elements brought together in new and as yet unimagined combinations to effect all manner of building and alteration performed at the macromolecular scale. The future is always imagined yet unseen: with phages in mind that refers both to its dazzling scale of possibility and in its infinitesimal scale of operation. The bacteriophages will be just as much a major part of that synthetic future, as they have ever seemingly been in nature to this day.

References

jry024

[1] Clokie MRJ, Millard AD, Letarov AV,

DOI: http://dx.doi.org/10.5772/intechopen.90384

Introductory Chapter: Nature's Ancient Nanomachines and Their Synthetic Future

[9] Krysiak-Baltyn K, Martin GJO, Gras SL. Computational modelling of large scale phage production using a two-stage batch process. Pharmaceuticals (Basel). 2018;11(2):31. DOI:

[10] Kilcher S, Studer P, Muessner C, Klumpp J, Loessner MJ. Cross-genus rebooting of custom-made, synthetic bacteriophage genomes in L-form bacteria. Proceedings of the National Academy of Sciences of the United States of America. 2018;115(3):567-572.

DOI: 10.1073/pnas.1714658115

10.1016/j.cels.2015.08.013

molcel.2017.04.025

[12] Yosef I, Goren MG, Globus R, Molshanski-Mor S, Qimron U. Extending the host range of bacteriophage particles for DNA transduction. Molecular Cell. 2017; 66(5):721-728. DOI: 10.1016/j.

[13] Xu J, Wang D, Gui M, Xiang Y. Structural assembly of the tailed bacteriophage φ29. Nature

Communications. 2019;10:2366. DOI:

[14] Lima-Mendez G, Toussaint A, Leplae R. Analysis of the phage sequence space: The benefit of

structured information. Virology. 2007;

[15] Hrebík D, Štveráková D, Škubník K, Füzik T, Pantůček R, Plevka P. Structure and genome ejection mechanism of Staphylococcus aureus phage P68. Science Advances.2019;5:eaaw7414. DOI: 10.1126/sciadv.aaw7414

10.1038/s41467-019-10272-3

365:241-249. DOI: 10.1016/j.

virol.2007.03.047

[11] Ando H, Lemire S, Pires DP, Lu TK. Engineering modular viral scaffolds for targeted bacterial population editing. Cell Systems. 2015;1(3):187-196. DOI:

10.3390/ph11020031

Bacteriophage. 2011;1(1):31-45. DOI:

[3] Myelnikov D. An alternative cure:

Heaphy S. Phages in nature.

[2] Pendleton JN, Gorman SP, Gilmore BF. Clinical relevance of the ESKAPE pathogens. Expert Review of Anti-Infective Therapy. 2013;11(3): 297-308. DOI: 10.1586/eri.13.12

The adoption and survival of bacteriophage therapy in the USSR, 1922–1955. Journal of the History of Medicine and Allied Sciences. 2018; 73(4):385-411. DOI: 10.1093/jhmas/

[4] Morozova VV, Vlassov VV, Tikunova NV. Applications of bacteriophages in the treatment of localized infections in humans.

DOI: 10.3389/fmicb.2018.01696

[5] Parfitt T. Georgia: An unlikely stronghold for bacteriophage therapy. Lancet. 2005;365(9478):2166-2167. DOI:

10.1016/S0140-6736(05)66759-1

10.1038/s41591-019-0437-z

0133-z

05.014

5

[7] Schmidt C. Phage therapy's latest makeover. Nature Biotechnology. 2019; 37:581-586. DOI: 10.1038/s41587-019-

[8] Malika DJ, Sokolova IJ, Vinner GK, et al. Formulation, stabilisation and encapsulation of bacteriophage for phage therapy. Advances in Colloid and Interface Science. 2017;249: 100-133. DOI: 10.1016/j.cis.2017.

[6] Dedrick RM, Guerrero-Bustamante CA, Garlena RA, et al. Engineered bacteriophages for treatment of a patient with a disseminated drugresistant Mycobacterium abscessus. Nature Medicine. 2019;25:730-733. DOI:

Frontiers in Microbiology. 2018;9:1696.

10.4161/bact.1.1.14942

#### Conflict of interest

Author Sophie E. Knott is a co-author of the included chapter The unusual linear plasmid generating systems of prokaryotes and has recently started a collaborative research partly funded by Touchlight Genetics Ltd., towards a PhD in the editor's laboratory: This fact presents no conflicts of interest to the presently published work.

### Author details

Renos Savva

Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck, University of London, London, UK

\*Address all correspondence to: r.savva@mail.cryst.bbk.ac.uk

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introductory Chapter: Nature's Ancient Nanomachines and Their Synthetic Future DOI: http://dx.doi.org/10.5772/intechopen.90384

#### References

diverse uses, what might we find? Perhaps we might find nanomachines inspired in their design by the multifarious forms and mechanisms of both known and newly studied types of these viruses, lightning-fast genotyping of bacterial infections at point of care, and efficiently timely synthetic tooling of medically approved phages to rapidly quell those infections, even on the scale of a few hours. We might also find synthetic phages reprogramming the microbiome, both by selective population control and by targeted and firewalled genetic modification in situ, and even the possibility of tooling wild species of bacteria for bioremediation purposes via reversible and firewalled genetic modification. There could be phage-encoded elements brought together in new and as yet unimagined combinations to effect all manner of building and alteration performed at the macromolecular scale. The future is always imagined yet unseen: with phages in mind that refers both to its dazzling scale of possibility and in its infinitesimal scale of operation. The bacteriophages will be just as much a major part of that synthetic future, as they have ever

Author Sophie E. Knott is a co-author of the included chapter The unusual

collaborative research partly funded by Touchlight Genetics Ltd., towards a PhD in the editor's laboratory: This fact presents no conflicts of interest to the presently

Institute of Structural and Molecular Biology, Department of Biological Sciences,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

linear plasmid generating systems of prokaryotes and has recently started a

seemingly been in nature to this day.

Bacteriophages - Perspectives and Future

Conflict of interest

published work.

Author details

Birkbeck, University of London, London, UK

provided the original work is properly cited.

\*Address all correspondence to: r.savva@mail.cryst.bbk.ac.uk

Renos Savva

4

[1] Clokie MRJ, Millard AD, Letarov AV, Heaphy S. Phages in nature. Bacteriophage. 2011;1(1):31-45. DOI: 10.4161/bact.1.1.14942

[2] Pendleton JN, Gorman SP, Gilmore BF. Clinical relevance of the ESKAPE pathogens. Expert Review of Anti-Infective Therapy. 2013;11(3): 297-308. DOI: 10.1586/eri.13.12

[3] Myelnikov D. An alternative cure: The adoption and survival of bacteriophage therapy in the USSR, 1922–1955. Journal of the History of Medicine and Allied Sciences. 2018; 73(4):385-411. DOI: 10.1093/jhmas/ jry024

[4] Morozova VV, Vlassov VV, Tikunova NV. Applications of bacteriophages in the treatment of localized infections in humans. Frontiers in Microbiology. 2018;9:1696. DOI: 10.3389/fmicb.2018.01696

[5] Parfitt T. Georgia: An unlikely stronghold for bacteriophage therapy. Lancet. 2005;365(9478):2166-2167. DOI: 10.1016/S0140-6736(05)66759-1

[6] Dedrick RM, Guerrero-Bustamante CA, Garlena RA, et al. Engineered bacteriophages for treatment of a patient with a disseminated drugresistant Mycobacterium abscessus. Nature Medicine. 2019;25:730-733. DOI: 10.1038/s41591-019-0437-z

[7] Schmidt C. Phage therapy's latest makeover. Nature Biotechnology. 2019; 37:581-586. DOI: 10.1038/s41587-019- 0133-z

[8] Malika DJ, Sokolova IJ, Vinner GK, et al. Formulation, stabilisation and encapsulation of bacteriophage for phage therapy. Advances in Colloid and Interface Science. 2017;249: 100-133. DOI: 10.1016/j.cis.2017. 05.014

[9] Krysiak-Baltyn K, Martin GJO, Gras SL. Computational modelling of large scale phage production using a two-stage batch process. Pharmaceuticals (Basel). 2018;11(2):31. DOI: 10.3390/ph11020031

[10] Kilcher S, Studer P, Muessner C, Klumpp J, Loessner MJ. Cross-genus rebooting of custom-made, synthetic bacteriophage genomes in L-form bacteria. Proceedings of the National Academy of Sciences of the United States of America. 2018;115(3):567-572. DOI: 10.1073/pnas.1714658115

[11] Ando H, Lemire S, Pires DP, Lu TK. Engineering modular viral scaffolds for targeted bacterial population editing. Cell Systems. 2015;1(3):187-196. DOI: 10.1016/j.cels.2015.08.013

[12] Yosef I, Goren MG, Globus R, Molshanski-Mor S, Qimron U. Extending the host range of bacteriophage particles for DNA transduction. Molecular Cell. 2017; 66(5):721-728. DOI: 10.1016/j. molcel.2017.04.025

[13] Xu J, Wang D, Gui M, Xiang Y. Structural assembly of the tailed bacteriophage φ29. Nature Communications. 2019;10:2366. DOI: 10.1038/s41467-019-10272-3

[14] Lima-Mendez G, Toussaint A, Leplae R. Analysis of the phage sequence space: The benefit of structured information. Virology. 2007; 365:241-249. DOI: 10.1016/j. virol.2007.03.047

[15] Hrebík D, Štveráková D, Škubník K, Füzik T, Pantůček R, Plevka P. Structure and genome ejection mechanism of Staphylococcus aureus phage P68. Science Advances.2019;5:eaaw7414. DOI: 10.1126/sciadv.aaw7414

**7**

**Chapter 2**

**Abstract**

Bacteriophages: Their Structural

Viruses are infectious particles that exist in a huge variety of forms and infect practically all living systems: animals, plants, insects and bacteria. Viruses that infect and use bacterial resources are classified as bacteriophages (or phages) and represent the most abundant life form on Earth. A phage can be described as a specific type of nano-machine that is able to recognise its environment, find a host cell, start infection, self-assemble and safeguard its genome until the next cycle of replication is initiated. Remarkable results have been obtained by combining cryo-EM, X-ray analysis and bioinformatics in structural studies of these nano-machines. In this review we will describe results of structural studies of phages that uncover their organisation in different conformations, thus facilitating our understanding of the functional mechanisms in supramolecular assemblies and helping us understand the usage of phages in medical treatments. Currently, antibiotic resistance is an enormous challenge that we face. The tailed phages could be used in place of antibiotics due to their high specificity to host cells, but more knowledge of their organisation and function is required.

**Keywords:** viruses, bacteriophage, structural organisation, infectivity, function,

All living systems have many diseases that are often caused by small organisms such as bacteria or infectious particles consisting of proteins, nucleic acids and sometimes lipids. These particles are called viruses, use the resources of living cells for their own propagation and can be transmitted from one organism to another. Each type of particle infects its own host cells, and they can survive outside living organisms in very harsh conditions. some of them continue to replicate with cells despite the host's defence mechanisms and remain dormant (latent) in their host cell, e.g. herpesviruses which reactivate at a later date to produce further attacks of

Bacteriophages (or phages) are viruses that infect and use bacterial resources for their own reproduction. They are characterised by a high specificity to bacteria at infection and are very common in all environments. Their number is directly related to the number of bacteria present. It is estimated that there are more than 1030 tailed phages in the biosphere [2]. Phages are common in soil and readily isolated from faeces and sewage, as well as being very abundant in freshwater and oceans with an estimate of more than 10 million virus-like particles in 1 mL of seawater [3, 4]. Why study the structure-function relationship of phages? Currently, there are substantial problems with diseases caused by bacteria, especially in hospitals.

Organisation and Function

*Helen E. White and Elena V. Orlova*

structural methods, electron microscopy

the disease if the host's defence system weakens [1].

**1. Introduction**

#### **Chapter 2**

## Bacteriophages: Their Structural Organisation and Function

*Helen E. White and Elena V. Orlova*

#### **Abstract**

Viruses are infectious particles that exist in a huge variety of forms and infect practically all living systems: animals, plants, insects and bacteria. Viruses that infect and use bacterial resources are classified as bacteriophages (or phages) and represent the most abundant life form on Earth. A phage can be described as a specific type of nano-machine that is able to recognise its environment, find a host cell, start infection, self-assemble and safeguard its genome until the next cycle of replication is initiated. Remarkable results have been obtained by combining cryo-EM, X-ray analysis and bioinformatics in structural studies of these nano-machines. In this review we will describe results of structural studies of phages that uncover their organisation in different conformations, thus facilitating our understanding of the functional mechanisms in supramolecular assemblies and helping us understand the usage of phages in medical treatments. Currently, antibiotic resistance is an enormous challenge that we face. The tailed phages could be used in place of antibiotics due to their high specificity to host cells, but more knowledge of their organisation and function is required.

**Keywords:** viruses, bacteriophage, structural organisation, infectivity, function, structural methods, electron microscopy

#### **1. Introduction**

All living systems have many diseases that are often caused by small organisms such as bacteria or infectious particles consisting of proteins, nucleic acids and sometimes lipids. These particles are called viruses, use the resources of living cells for their own propagation and can be transmitted from one organism to another. Each type of particle infects its own host cells, and they can survive outside living organisms in very harsh conditions. some of them continue to replicate with cells despite the host's defence mechanisms and remain dormant (latent) in their host cell, e.g. herpesviruses which reactivate at a later date to produce further attacks of the disease if the host's defence system weakens [1].

Bacteriophages (or phages) are viruses that infect and use bacterial resources for their own reproduction. They are characterised by a high specificity to bacteria at infection and are very common in all environments. Their number is directly related to the number of bacteria present. It is estimated that there are more than 1030 tailed phages in the biosphere [2]. Phages are common in soil and readily isolated from faeces and sewage, as well as being very abundant in freshwater and oceans with an estimate of more than 10 million virus-like particles in 1 mL of seawater [3, 4].

Why study the structure-function relationship of phages? Currently, there are substantial problems with diseases caused by bacteria, especially in hospitals. Many pathogenic bacteria exist such as *Mycobacterium tuberculosis*, *Enterococcus faecalis*, *Staphylococcus aureus*, *Acinetobacter baumannii*, *Pseudomonas aeruginosa* and methicillin-resistant *S. aureus* (MRSA) and have become modified in hospitals due to the overuse of antibiotics. Bacteria have become resistant to some of the most potent drugs used in modern medicine, and this causes treatment problems [5–7]. It appears that the pathogenic bacteria adapt quicker to antibiotics than the new ones that can be produced. The number of new antibiotics being introduced has decreased since their first introduction [8].

A powerful method to circumvent this resistance is the use of phages in the treatment of bacterial infections [9]. Most current studies of phage therapy have focussed on acute infections in animals [10]. In order to regulate the mechanisms of phage infection, we need to know not only the phage structure but also the phage-cell surface interaction mechanism and the process of switching the cell replication machinery for phage propagation. One important factor that has to be considered is how phages are reproduced. Phages have two ways of propagation: lytic and lysogenic [11]. In the first case, phages cause the compete lysis of a cell, where it breaks open and subsequently dies after phage replication. In the second type of replication, a phage integrates its genome into the host bacterium's genome or forms a circular replicon in the bacterial cytoplasm. The bacterium then continues to live and reproduce normally, but the phage genome is transmitted to progeny cells at each subsequent cell division. Changes in cell conditions such as radiation or certain chemicals can release the phage genome, causing proliferation of new phages via the lytic cycle. Therefore, for medical treatments we need to use only lytic phages, so they will exist in an organism, while the pathogenic bacteria are around but only infect those bacteria that have the appropriate receptors in the outer membrane. This is an important factor that can be used to affect specific bacteria without harming those ones that are essential for the health of humans and animals [10]. In this review we will focus on tailed phages as they are abundant and well studied and could be beneficial to medicine [12]. We will describe the general organisation and structural features of their components revealed by current structural methods.

#### **2. Phages and their classification**

Virus classification is based on characteristics such as morphology, type of nucleic acid, replication mode, host organism and type of disease. The International Committee on Taxonomy of Viruses (ICTV) has produced an ordered system for classifying viruses (https://talk.ictvonline.org/taxonomy/). Phages are found in a variety of morphologies: filamentous phages, phages with a lipid-containing envelope and phages with lipids in the particle shell (**Figure 1A**). They have a genome, either DNA or RNA, which can be single or double stranded, and contain information on the proteins that constitute the particles, additional proteins that are responsible for switching cell molecular metabolism in favour of viruses and, therefore, the information on the self-assembly process. The genome can be one or multipartite and is located inside the phage capsid. Nearly 5500 bacterial viruses have been characterised by electron microscopy (EM) [15]. The shape of viruses is closely related to their genome, and a large genome indicates a large capsid and therefore a more complex organisation. The most studied group of phages is the tailed phages (order *Caudovirales*) which are classified by the type of tail; *Siphoviridae* have a long non-contractile tail, *Podoviridae* have a short non-contractile tail and *Myoviridae* have a complex contractile tail (**Figure 1B**).

**9**

**3.1 X-ray crystallography**

**Figure 1.**

*Bacteriophages: Their Structural Organisation and Function*

**3. Methods used for structural studies of viruses**

The first ideas on how viruses infect cells were based on results obtained by microbiology and bacteriology during the last century. Understanding the function of viruses and how this can be regulated and modified requires knowledge of their structural organisation. However, investigation of structure-function relationships needs a combination of different techniques. Microbiology has identified viruses as infectious agents, while bacteriology and light microscopy enabled us to identify specificity between viruses and host cell interactions and to recognise a level of survival of bacteria in the presence of different phages. In order to understand interactions at the molecular level, one needs to know the structural features of the viruses and their components at an atomic level. Different structural techniques are often utilised for smaller components, and the results fitted into larger EM structures.

*(A) Representation of prokaryote bacteriophage morphotypes [13]. (B) Members of the Caudovirales family [14].*

X-ray crystallography was the first method used to study proteins at the atomic

level, which is essential to reveal protein-ligand interactions that can boost or suppress protein activity. It is based on the principles of beam scattering within a crystal. By using specific software packages, a 3D electron density map of the protein that forms the crystal can be calculated [16]. However, to produce protein crystals, we need solutions of a protein at high concentration. The proteins have to be stable, and often mutations are made to remove their flexible parts, but this may produce different conformations to those that are required for their natural activity. X-ray analysis is an efficient tool for analysis of protein complexes from a few kDa to hundreds of kDa in size. In order to study the structure of a large protein or a complex of several proteins, the process of crystallisation becomes a more challenging step. The development of cryoprotection in X-ray crystallography, where the crystals are flash frozen, has improved the quality of the data and often resulted in higher resolution. Nowadays, many structures of large protein complexes (up to

*DOI: http://dx.doi.org/10.5772/intechopen.85484*

*Bacteriophages: Their Structural Organisation and Function DOI: http://dx.doi.org/10.5772/intechopen.85484*

**Figure 1.**

*Bacteriophages - Perspectives and Future*

decreased since their first introduction [8].

rent structural methods.

contractile tail (**Figure 1B**).

**2. Phages and their classification**

Many pathogenic bacteria exist such as *Mycobacterium tuberculosis*, *Enterococcus faecalis*, *Staphylococcus aureus*, *Acinetobacter baumannii*, *Pseudomonas aeruginosa* and methicillin-resistant *S. aureus* (MRSA) and have become modified in hospitals due to the overuse of antibiotics. Bacteria have become resistant to some of the most potent drugs used in modern medicine, and this causes treatment problems [5–7]. It appears that the pathogenic bacteria adapt quicker to antibiotics than the new ones that can be produced. The number of new antibiotics being introduced has

A powerful method to circumvent this resistance is the use of phages in the treatment of bacterial infections [9]. Most current studies of phage therapy have focussed on acute infections in animals [10]. In order to regulate the mechanisms of phage infection, we need to know not only the phage structure but also the phage-cell surface interaction mechanism and the process of switching the cell replication machinery for phage propagation. One important factor that has to be considered is how phages are reproduced. Phages have two ways of propagation: lytic and lysogenic [11]. In the first case, phages cause the compete lysis of a cell, where it breaks open and subsequently dies after phage replication. In the second type of replication, a phage integrates its genome into the host bacterium's genome or forms a circular replicon in the bacterial cytoplasm. The bacterium then continues to live and reproduce normally, but the phage genome is transmitted to progeny cells at each subsequent cell division. Changes in cell conditions such as radiation or certain chemicals can release the phage genome, causing proliferation of new phages via the lytic cycle. Therefore, for medical treatments we need to use only lytic phages, so they will exist in an organism, while the pathogenic bacteria are around but only infect those bacteria that have the appropriate receptors in the outer membrane. This is an important factor that can be used to affect specific bacteria without harming those ones that are essential for the health of humans and animals [10]. In this review we will focus on tailed phages as they are abundant and well studied and could be beneficial to medicine [12]. We will describe the general organisation and structural features of their components revealed by cur-

Virus classification is based on characteristics such as morphology, type of nucleic acid, replication mode, host organism and type of disease. The International Committee on Taxonomy of Viruses (ICTV) has produced an ordered system for classifying viruses (https://talk.ictvonline.org/taxonomy/). Phages are found in a variety of morphologies: filamentous phages, phages with a lipid-containing envelope and phages with lipids in the particle shell (**Figure 1A**).

They have a genome, either DNA or RNA, which can be single or double

stranded, and contain information on the proteins that constitute the particles, additional proteins that are responsible for switching cell molecular metabolism in favour of viruses and, therefore, the information on the self-assembly process. The genome can be one or multipartite and is located inside the phage capsid. Nearly 5500 bacterial viruses have been characterised by electron microscopy (EM) [15]. The shape of viruses is closely related to their genome, and a large genome indicates a large capsid and therefore a more complex organisation. The most studied group of phages is the tailed phages (order *Caudovirales*) which are classified by the type of tail; *Siphoviridae* have a long non-contractile tail, *Podoviridae* have a short non-contractile tail and *Myoviridae* have a complex

**8**

*(A) Representation of prokaryote bacteriophage morphotypes [13]. (B) Members of the Caudovirales family [14].*

#### **3. Methods used for structural studies of viruses**

The first ideas on how viruses infect cells were based on results obtained by microbiology and bacteriology during the last century. Understanding the function of viruses and how this can be regulated and modified requires knowledge of their structural organisation. However, investigation of structure-function relationships needs a combination of different techniques. Microbiology has identified viruses as infectious agents, while bacteriology and light microscopy enabled us to identify specificity between viruses and host cell interactions and to recognise a level of survival of bacteria in the presence of different phages. In order to understand interactions at the molecular level, one needs to know the structural features of the viruses and their components at an atomic level. Different structural techniques are often utilised for smaller components, and the results fitted into larger EM structures.

#### **3.1 X-ray crystallography**

X-ray crystallography was the first method used to study proteins at the atomic level, which is essential to reveal protein-ligand interactions that can boost or suppress protein activity. It is based on the principles of beam scattering within a crystal. By using specific software packages, a 3D electron density map of the protein that forms the crystal can be calculated [16]. However, to produce protein crystals, we need solutions of a protein at high concentration. The proteins have to be stable, and often mutations are made to remove their flexible parts, but this may produce different conformations to those that are required for their natural activity.

X-ray analysis is an efficient tool for analysis of protein complexes from a few kDa to hundreds of kDa in size. In order to study the structure of a large protein or a complex of several proteins, the process of crystallisation becomes a more challenging step. The development of cryoprotection in X-ray crystallography, where the crystals are flash frozen, has improved the quality of the data and often resulted in higher resolution. Nowadays, many structures of large protein complexes (up to

2–3 MDa) have been determined by X-ray analysis, but these projects have required decades to obtain high-quality crystals [17].

Viruses are much bigger particles and often have flexible components. The large size of the complexes results in significantly bigger unit cells, which results in technical challenges in obtaining fine structural details. Viruses with a rigid icosahedral lattice of the capsid have been studied successfully by X-ray crystallography at nearatomic resolution. The first viral structure was that of the *Blue tongue virus* (700 Å diameter) determined at a resolution of 3.5 Å which was the largest virus structure determined at that time [18]. The capsid of the *Siphoviridae* phage HK97 (without a portal protein) was determined at a resolution of 3.5 Å [19]. Later studies have shown that the fold of the HK97 phage capsid protein, which forms the envelope to protect viral genome from the harsh outer environment, represents a conservative fold found nowadays in nearly all dsDNA viruses so far studied.

#### **3.2 Nuclear magnetic resonance**

Nuclear magnetic resonance (NMR) is an important technique that resolves structures of small proteins that are not suitable for crystallisation due to their flexibility. This method is based on exploiting the electrical charges and spins of the nuclei in a molecule. If an external magnetic field is applied, energy is transferred to the nuclei changing their state from the level of base energy to a higher energy. This energy is emitted when the spin returns back to its base level at a frequency corresponding to radio frequencies1 *.* The signal that matches this transfer is measured and processed in order to yield a NMR spectrum [17, 20]. This technique is typically used for proteins of less than 200 amino acids and an upper weight limit of about 50 kDa, so it is unsuitable for the structural determination of complete viruses. However, it can be used to analyse flexibility of bigger complexes [21]. The NMR structures can be docked into low-resolution cryo-EM structures.

#### **3.3 Electron microscopy**

Light microscopy has been used for several centuries to study objects that are hardly visible to the naked eye. In conventional microscopy, resolution is mostly restricted according to the theoretical context of the Rayleigh criterion [22]. This limit is defined by the diffraction properties of light in lenses and has restricted our view to objects bigger than 250 nm. New developments in technology and advances in optical quality, electronics and software have delivered new options and extended the field of applications for electron microscopes allowing visualisation of single molecules. Electron microscopes use a beam of electrons (wavelength of less than 0.1 nm) instead of visible light *(*wavelength 400–700 nm)*.* Due to their charge, the electrons can be focused using an electromagnetic field, which is why the optical system of the electron microscope (EM) is similar to the general optical system of light [23]*.* The short wavelength of the electron beam allows details of small objects less than 0.1 nm in size to be seen. However, biological samples are not stable in the vacuum necessary to create an image using electrons that would otherwise become absorbed by air, and, moreover, biological samples are sensitive to the electron irradiation. These factors reduce the level of achievable resolution.

At the very beginning of EM evolvement, a method called negative staining was used for visualisation of biological complexes. In this case a drop of biocomplex solution is placed on a support grid and embedded in a heavy atom salt, usually uranyl acetate [24]. Since the specific density of the negative stain is much higher than

**11**

*Bacteriophages: Their Structural Organisation and Function*

the density of the biological molecules in the microscope, we can see the cast of the molecule merged into the surrounding stain. Where the stain did not penetrate into the molecule, one can see light spots in the image as the stain has blocked electrons. Sample preparation is fast and produces very high contrast. However, this technique does not allow fine details to be seen, and the particle becomes distorted due to the drying procedure required. The stain has a relatively large grain (up to 1.5 nm) that

Nearly four decades ago, a cryo-technique for sample preparation was introduced that allows biocomplexes to be kept at nearly native conditions. A thin layer of sample on a grid is flash frozen at liquid nitrogen temperatures, thus trapping molecules in a native, hydrated state within a thin layer of amorphous ice [25]. This technique is used to study the structural organisation of biocomplexes by cryo-electron microscopy (cryo-EM) or electron tomography (cryo-ET). Until two decades ago, all data in EM was collected on films that had to be developed and digitised, which was timeconsuming. The advent of charge-coupled devices (CCDs) allowed direct digital acquisition of images and the collection of large numbers of particles giving rise to structures of higher resolution. Later, direct electron detectors were introduced into EM and are now used in all high-end electron microscopes [26]. Together with new approaches in microtechnology and the automation of data collection, the results from image analysis have improved tremendously. Cryo-EM is now approaching the near-atomic resolution that had only been achieved by X-ray crystallography. New maps obtained by cryo-EM provide information on the main polypeptide chains and often reveal the positions of side chains. The current highest resolution of structures currently deposited in the EMDB is 1.5 Å [27], with many others at a resolution between 3.5 and 4 Å. At this resolution atomic models can be built and refined using

In cryo-ET the samples are also flash frozen, but data is collected by tilting the grid with the sample between −60 and 60° around the horizontal axis (perpendicular to the optical axis of the microscope) with an increment typically of 2°. The 2D images taken at each angle are combined to calculate a 3D map of the object. The limitation in the range of the tilt results in a cone of missing data [28]. The resolution in structures obtained by cryo-ET is lower than that in single-particle analysis. However, this approach allows visualisation of important organelles within cells. If there are multiple small structures such as ribosomes or viruses, then each structure can be extracted and averaged. This is called subtomogram averaging and will give

Phages may have different shapes and sizes (**Figure 1A**). The most studied group

The functional phage is a result of a multistep process that starts with all the necessary proteins produced by the host cell after infection: capsid, portal, tail, scaffolding, terminase, etc. (**Figure 2**). The capsids of the dsDNA phages often have fivefold or icosahedral symmetries [30], which are broken at one of the fivefold axes by the head-to-tail interface (HTI). The main component of the HTI is a dodecameric portal protein (PP) within the capsid. The PP represents the DNA-packaging motor,

is that of tailed phages with a dsDNA genome, and it also represents the largest group (**Figure 1B**). The tailed phages have three major components: a capsid where the genome is packed, a tail that serves as a pipe during infection to secure transfer of genome into host cell and a special adhesive system (adsorption apparatus) at the very end of the tail that will recognise the host cell and penetrate its wall. Cell

*DOI: http://dx.doi.org/10.5772/intechopen.85484*

obscures details of the molecules under study.

the crystallographic methods.

higher-resolution structures [29].

**4. Overall structural organisation of phages**

resources are used for the phage reproduction.

<sup>1</sup> chem.ch.huji.ac.il/nmr/whatisnmr/whatisnmr.html

#### *Bacteriophages: Their Structural Organisation and Function DOI: http://dx.doi.org/10.5772/intechopen.85484*

*Bacteriophages - Perspectives and Future*

**3.2 Nuclear magnetic resonance**

responding to radio frequencies1

**3.3 Electron microscopy**

<sup>1</sup> chem.ch.huji.ac.il/nmr/whatisnmr/whatisnmr.html

decades to obtain high-quality crystals [17].

fold found nowadays in nearly all dsDNA viruses so far studied.

structures can be docked into low-resolution cryo-EM structures.

2–3 MDa) have been determined by X-ray analysis, but these projects have required

Nuclear magnetic resonance (NMR) is an important technique that resolves structures of small proteins that are not suitable for crystallisation due to their flexibility. This method is based on exploiting the electrical charges and spins of the nuclei in a molecule. If an external magnetic field is applied, energy is transferred to the nuclei changing their state from the level of base energy to a higher energy. This energy is emitted when the spin returns back to its base level at a frequency cor-

and processed in order to yield a NMR spectrum [17, 20]. This technique is typically used for proteins of less than 200 amino acids and an upper weight limit of about 50 kDa, so it is unsuitable for the structural determination of complete viruses. However, it can be used to analyse flexibility of bigger complexes [21]. The NMR

Light microscopy has been used for several centuries to study objects that are hardly visible to the naked eye. In conventional microscopy, resolution is mostly restricted according to the theoretical context of the Rayleigh criterion [22]. This limit is defined by the diffraction properties of light in lenses and has restricted our view to objects bigger than 250 nm. New developments in technology and advances in optical quality, electronics and software have delivered new options and extended the field of applications for electron microscopes allowing visualisation of single molecules. Electron microscopes use a beam of electrons (wavelength of less than 0.1 nm) instead of visible light *(*wavelength 400–700 nm)*.* Due to their charge, the electrons can be focused using an electromagnetic field, which is why the optical system of the electron microscope (EM) is similar to the general optical system of light [23]*.* The short wavelength of the electron beam allows details of small objects less than 0.1 nm in size to be seen. However, biological samples are not stable in the vacuum necessary to create an image using electrons that would otherwise become absorbed by air, and, moreover, biological samples are sensitive to the electron irradiation. These factors reduce the level of achievable resolution. At the very beginning of EM evolvement, a method called negative staining was used for visualisation of biological complexes. In this case a drop of biocomplex solution is placed on a support grid and embedded in a heavy atom salt, usually uranyl acetate [24]. Since the specific density of the negative stain is much higher than

*.* The signal that matches this transfer is measured

Viruses are much bigger particles and often have flexible components. The large size of the complexes results in significantly bigger unit cells, which results in technical challenges in obtaining fine structural details. Viruses with a rigid icosahedral lattice of the capsid have been studied successfully by X-ray crystallography at nearatomic resolution. The first viral structure was that of the *Blue tongue virus* (700 Å diameter) determined at a resolution of 3.5 Å which was the largest virus structure determined at that time [18]. The capsid of the *Siphoviridae* phage HK97 (without a portal protein) was determined at a resolution of 3.5 Å [19]. Later studies have shown that the fold of the HK97 phage capsid protein, which forms the envelope to protect viral genome from the harsh outer environment, represents a conservative

**10**

the density of the biological molecules in the microscope, we can see the cast of the molecule merged into the surrounding stain. Where the stain did not penetrate into the molecule, one can see light spots in the image as the stain has blocked electrons. Sample preparation is fast and produces very high contrast. However, this technique does not allow fine details to be seen, and the particle becomes distorted due to the drying procedure required. The stain has a relatively large grain (up to 1.5 nm) that obscures details of the molecules under study.

Nearly four decades ago, a cryo-technique for sample preparation was introduced that allows biocomplexes to be kept at nearly native conditions. A thin layer of sample on a grid is flash frozen at liquid nitrogen temperatures, thus trapping molecules in a native, hydrated state within a thin layer of amorphous ice [25]. This technique is used to study the structural organisation of biocomplexes by cryo-electron microscopy (cryo-EM) or electron tomography (cryo-ET). Until two decades ago, all data in EM was collected on films that had to be developed and digitised, which was timeconsuming. The advent of charge-coupled devices (CCDs) allowed direct digital acquisition of images and the collection of large numbers of particles giving rise to structures of higher resolution. Later, direct electron detectors were introduced into EM and are now used in all high-end electron microscopes [26]. Together with new approaches in microtechnology and the automation of data collection, the results from image analysis have improved tremendously. Cryo-EM is now approaching the near-atomic resolution that had only been achieved by X-ray crystallography. New maps obtained by cryo-EM provide information on the main polypeptide chains and often reveal the positions of side chains. The current highest resolution of structures currently deposited in the EMDB is 1.5 Å [27], with many others at a resolution between 3.5 and 4 Å. At this resolution atomic models can be built and refined using the crystallographic methods.

In cryo-ET the samples are also flash frozen, but data is collected by tilting the grid with the sample between −60 and 60° around the horizontal axis (perpendicular to the optical axis of the microscope) with an increment typically of 2°. The 2D images taken at each angle are combined to calculate a 3D map of the object. The limitation in the range of the tilt results in a cone of missing data [28]. The resolution in structures obtained by cryo-ET is lower than that in single-particle analysis. However, this approach allows visualisation of important organelles within cells. If there are multiple small structures such as ribosomes or viruses, then each structure can be extracted and averaged. This is called subtomogram averaging and will give higher-resolution structures [29].

#### **4. Overall structural organisation of phages**

Phages may have different shapes and sizes (**Figure 1A**). The most studied group is that of tailed phages with a dsDNA genome, and it also represents the largest group (**Figure 1B**). The tailed phages have three major components: a capsid where the genome is packed, a tail that serves as a pipe during infection to secure transfer of genome into host cell and a special adhesive system (adsorption apparatus) at the very end of the tail that will recognise the host cell and penetrate its wall. Cell resources are used for the phage reproduction.

The functional phage is a result of a multistep process that starts with all the necessary proteins produced by the host cell after infection: capsid, portal, tail, scaffolding, terminase, etc. (**Figure 2**). The capsids of the dsDNA phages often have fivefold or icosahedral symmetries [30], which are broken at one of the fivefold axes by the head-to-tail interface (HTI). The main component of the HTI is a dodecameric portal protein (PP) within the capsid. The PP represents the DNA-packaging motor,

#### **Figure 2.**

*Self-assembly pathway of phages. Multiple copies of the capsid/scaffold complex bind the portal protein to form the procapsid; then, the scaffold proteins are ejected, and DNA is packaged into the procapsid, which expands to the size of the mature capsid. The head completion proteins (the stopper and the adaptor) are bound to the portal complex preventing DNA leakage. Next, decoration proteins bind to the capsid, and the tail, assembled separately or after DNA packaging, is attached; thus, the final infectious phage is produced. The preassembled tail attaches in Myoviridae and Siphoviridae, while in Podoviridae the tail assembles at the stopper.*

which is the crucial part of these nano-machines. The HTI also includes oligomeric rings of head completion proteins that play dual roles: (1) making an additional interface to molecules of ATP which provide energy for DNA packaging and (2) then connecting the portal protein and the tail. Some HTIs also serve as valves that close the exit channel preventing leakage of genome from the capsid but opening as soon as the phage is attached to the host cell. However, symmetries other than dodecameric have been found for nearly all PPs in vitro if the PPs are assembled under naive conditions, without any other phage protein components [31–35]. Typically, the main phage proteins have conservative folds despite low sequence similarity, although they may have different additional domains [36, 37].

The phage tail is the structural component of the phage that is essential during infection. Its adsorption apparatus located on the distal end of the tail recognises a receptor, or the envelope chemistry, of the host cell and ensures genome delivery to the cell cytoplasm. In *Myoviridae* and *Siphoviridae*, the tail is composed of a series of stacked rings with the host recognition device being located at the end of the tail. In *Podoviridae* the adsorption apparatus is bound immediately to the HTI. The adsorption apparatus is surrounded in many phages by fibrils that ensure a tight connection to the host cell (**Figure 2**).

#### **4.1 Procapsids**

The capsid of a phage has a precursor formation, named the procapsid, during the assembly process (**Figure 2**). Scaffolding proteins (SPs) drive the assembly process by chaperoning major capsid protein (MCP) subunits to build an icosahedral procapsid that is later filled with dsDNA. The SPs are bound to the portal

**13**

contacts.

**Table 1.**

**4.2 Capsids**

subunits are named as hexons.

*Bacteriophages: Their Structural Organisation and Function*

**Capsid protein**

**No. of residues**

282 (AC)

299 (AC)

SPP1 *Sipho* gp13 324 35 8.8 (C) EM [53] TP901-1 *Sipho* ORF36 272 29 15 EM [54] TW1 *Sipho* gp57\* 352 39 7 EM [55] φ29 *Podo* gp8 448 50 8 EM [56]

ε15 *Podo* gp7 335 37 4.5 EM [50]

HSV-1 *virus* VP5 1374 149 4.2 (C) EM [63]

λ *Sipho* gpE 341 38 6.8 (C),13.3

T7 *Podo* gp10 345 37 4.6 (PC)

P22 *Podo* gp5 430 47 3.8 (PC)

gp24\* gp23 gp23\* **M. Mass (kDa)**

42 3.44 (C)

12 (PC)

(PC)

3.6 (C)

4.0 (C) 3.3 (C)

2.9 (monomer) 3.3 (EM)

51 9 (C) EM [52]

**Resolution (Å) Structure** 

**analysis**

X-ray [42] EM [51]

EM [47]

EM [57]

EM [58–60]

X-ray [61] EM [62]

complex during formation of a procapsid with scaffolding inside. The sequence of conformational changes from a procapsid to the phage capsid where genome has been packed is named as the maturation process and goes through a series of intermediates [19, 38–40]. Some phages like HK97 and T5 do not have a separate SP; instead, the capsid protein is fused with a scaffolding domain at the N-terminus. As soon as the procapsid is assembled, the scaffolding domain is cleaved off and then like the separate SP will be removed from the capsid to make room for the genome [38, 39]. Structures of procapsids and mature virions have been determined for a number of phages (**Table 1**). The spherical capsid shell expands during maturation and becomes thinner due to alterations in the inter- and intra-subunit

Most tailed phages have capsids of an icosahedral shape formed by multiple copies of one or more proteins. Icosahedral capsids are characterised by 12× fivefold, 20× threefold and 30× twofold axes, which give rise to 60 copies of the major independent parts [41]. A triangulation number (T number) describes the number of copies of the same protein within the independent part of the icosahedral lattice. The overall number of proteins in the virus corresponds to the T number multiplied by 60; for example, a T = 3 virus has 180 subunits [41]. Oligomers of the proteins that are located on the fivefold axes are referred to as pentons, while those complexes that are located on the faces of the icosahedron and form oligomers from six

*DOI: http://dx.doi.org/10.5772/intechopen.85484*

HK97 *Sipho* gp5 385

Т5 *Sipho* pb8 458

**Phage Type of** 

**phage**

T4 *Myo* gp24

*AC—after cleavage; C—capsid; PC—procapsid*

*Phage procapsids and capsids.*


*Bacteriophages: Their Structural Organisation and Function DOI: http://dx.doi.org/10.5772/intechopen.85484*

#### **Table 1.**

*Bacteriophages - Perspectives and Future*

which is the crucial part of these nano-machines. The HTI also includes oligomeric rings of head completion proteins that play dual roles: (1) making an additional interface to molecules of ATP which provide energy for DNA packaging and (2) then connecting the portal protein and the tail. Some HTIs also serve as valves that close the exit channel preventing leakage of genome from the capsid but opening as soon as the phage is attached to the host cell. However, symmetries other than dodecameric have been found for nearly all PPs in vitro if the PPs are assembled under naive conditions, without any other phage protein components [31–35]. Typically, the main phage proteins have conservative folds despite low sequence similarity,

*Self-assembly pathway of phages. Multiple copies of the capsid/scaffold complex bind the portal protein to form the procapsid; then, the scaffold proteins are ejected, and DNA is packaged into the procapsid, which expands to the size of the mature capsid. The head completion proteins (the stopper and the adaptor) are bound to the portal complex preventing DNA leakage. Next, decoration proteins bind to the capsid, and the tail, assembled separately or after DNA packaging, is attached; thus, the final infectious phage is produced. The preassembled tail attaches in Myoviridae and Siphoviridae, while in Podoviridae the tail assembles at* 

The phage tail is the structural component of the phage that is essential during infection. Its adsorption apparatus located on the distal end of the tail recognises a receptor, or the envelope chemistry, of the host cell and ensures genome delivery to the cell cytoplasm. In *Myoviridae* and *Siphoviridae*, the tail is composed of a series of stacked rings with the host recognition device being located at the end of the tail. In *Podoviridae* the adsorption apparatus is bound immediately to the HTI. The adsorption apparatus is surrounded in many phages by fibrils that ensure a tight

The capsid of a phage has a precursor formation, named the procapsid, during the assembly process (**Figure 2**). Scaffolding proteins (SPs) drive the assembly process by chaperoning major capsid protein (MCP) subunits to build an icosahedral procapsid that is later filled with dsDNA. The SPs are bound to the portal

although they may have different additional domains [36, 37].

connection to the host cell (**Figure 2**).

**12**

**4.1 Procapsids**

**Figure 2.**

*the stopper.*

*Phage procapsids and capsids.*

complex during formation of a procapsid with scaffolding inside. The sequence of conformational changes from a procapsid to the phage capsid where genome has been packed is named as the maturation process and goes through a series of intermediates [19, 38–40]. Some phages like HK97 and T5 do not have a separate SP; instead, the capsid protein is fused with a scaffolding domain at the N-terminus. As soon as the procapsid is assembled, the scaffolding domain is cleaved off and then like the separate SP will be removed from the capsid to make room for the genome [38, 39]. Structures of procapsids and mature virions have been determined for a number of phages (**Table 1**). The spherical capsid shell expands during maturation and becomes thinner due to alterations in the inter- and intra-subunit contacts.

#### **4.2 Capsids**

Most tailed phages have capsids of an icosahedral shape formed by multiple copies of one or more proteins. Icosahedral capsids are characterised by 12× fivefold, 20× threefold and 30× twofold axes, which give rise to 60 copies of the major independent parts [41]. A triangulation number (T number) describes the number of copies of the same protein within the independent part of the icosahedral lattice. The overall number of proteins in the virus corresponds to the T number multiplied by 60; for example, a T = 3 virus has 180 subunits [41]. Oligomers of the proteins that are located on the fivefold axes are referred to as pentons, while those complexes that are located on the faces of the icosahedron and form oligomers from six subunits are named as hexons.

*HK97 phage.* The first structure of a phage capsid was solved for the *Siphoviridae* **HK97** phage (a mutant without the PP and tail, diameter ~650 Å, T = 7) at 3.6 Å resolution [19] by X-ray crystallography. This structure revealed a new type of protein fold, which has been found in many other phage capsid proteins (CP) despite low sequence identity (**Table 1**). Later, the structure was improved to 3.45 Å resolution [42]. This fold is also found in distantly related icosahedral tailed viruses that infect halophilic archaea [43] and human pathogens such as *Herpesviridae* [44–46]. The characteristic features of the HK97-fold are an N-arm, a peripheral P-domain with a long helix and a β-sheet, an axial A-domain and a long E-loop that fills the region between adjacent threefold axes (**Figure 3A**). The HK97 capsid is held together by molecular chain mail [19]. The two antiparallel β-strands in the E-loop are terminated by a loop, containing Lys169. This residue forms an isopeptide bond with the P-domain residue Asn356 of an adjacent subunit. The third residue involved is the catalytic residue Glu363 from a third subunit (**Figure 3A**). Conformational changes between HK97 procapsids and capsids were assessed by flexible fitting of atomic models [40]. The capsid subunits are skewed around a pseudo-twofold axis in the procapsid but form more symmetric pentons and hexons in the mature virion.

*λ phage*. The mature capsid of the *Siphoviridae* phage **λ** (diameter ~600 Å, T = 7) is stabilised with the help of a decoration protein gpD which is attached to the threefold vertices. A procapsid structure was determined with a resolution between 13.3 and 14.5 Å and a mature capsid at 6.8 Å [47]. The capsid protein gpE of the mature capsid shows clearly the HK97-fold so the HK97 model was used for rigid body fitting into the capsid [47]. However, the A-domain gave a poor fit so it was treated as a separate rigid body and was found to be rotated 15° clockwise compared with HK97. There was unassigned density, which could fit the extra 59 residues in

#### **Figure 3.**

*Structural organisation of the major capsid proteins. (A) Siphophage HK97 (1OHG). The catalytic residues are shown in brown and circled. (B) Podophage ε15 (3 J40). (C) Podophage P22 (5UU5). (D) MyophageT4 gp23\* (5VF3). The N-arm is dark blue, the P-domain is red, the A-domain is light blue and the E-loop is yellow. Extra inserted domains seen in P22 and T4 are magenta. The yellow linker in T4 is topologically equivalent to the E-loop seen in the other phages.*

**15**

*Bacteriophages: Their Structural Organisation and Function*

the λ phage. The crystal structure of gpD (1.1 Å) [48] was fitted into the cryo-EM capsid map and, when combined with the HK97 model, showed how gpD is held in

*ε15 phage.* The structure of gp7, the MCP of the *Salmonella* phage **ε15**, was determined by cryo-EM (4.5 Å) [49] and showed a fold similar to that in HK97 (**Figure 3B**). Later, the same group obtained a better resolution structure (~3.5 Å) [50] and, using their previous 4.5 Å structure for gp7, computationally analysed possible different gp7 conformations within EM density obtained from averaging the individual subunits in one asymmetric unit. They found that two models could be fitted in the structure: one that was consistent with the previous interpretation [49], and the other had a strand swap of the β-strands in the P-domain before the two helices in the A-domain. The strand order was 4-3-1-5-2, but in the swapped model, it was now 5-4-2-3-1. The model with the strand swap provided a better visual fit in the

*P22 phage*. Structures of a procapsid and a heat-expanded capsid of the *Podoviridae P22* phage (diameter ~620 Å, T = 7) were determined by cryo-EM at 9.1 and 8.2 Å, respectively [58]. This heat-expanded capsid was composed only of hexons with wide openings where the pentons would be. The MCP gp5 revealed the HK97-fold, but there was an extra domain (residues 223–349) with an immunoglobulin-like telokin domain [64]. Later, the procapsid and capsid structures were determined by cryo-EM at 3.8 and 4.0 Å resolution, respectively [59]. It was also found that gp5 has the HK97-fold with extra density above the E-loop corresponding to the telokin domain (**Figure 3C**). A model built from a cryo-EM map of P22 (3.3 Å) [60] identified the interactions that stabilise the capsid. In the mature virion, the N-arm forms an antiparallel β-strand pair between neighbouring subunits around the threefold axis. A second set of interactions involve hydrogen bonds and salt bridges between adjacent subunits involving the A-domain, the

*φ29 phage*. The podovirus **φ29** is a relatively small phage (prolate icosahedra, height ~540 Å, diameter ~450 Å) that requires pRNA along with the ATPase gp16 to provide enough energy for the DNA translocation. The procapsid consists of the MCP gp8, the SP gp7, the head fibre protein gp8.5, the connector gp10 and a pRNA. After DNA packaging, the pRNA and ATPase come off the procapsid and are replaced by the gp11 and gp13 underneath the connector, gp29 at the end of the tail and the appendages (gp12\*) which are attached to gp11 and gp13 [65]. During maturation an 18 kDa fragment of gp12 is cleaved off to give gp12\* found in the appendages whose role is to adsorb the virion on the host cell. A fibreless, isometric φ29 variant capsid cryo-EM structure (7.9 Å) showed that gp8 has a structure with the HK97-fold but with extra density [66]. Domain profile-searching algorithms [67] showed that residues 348–429 at the C-terminus were found to be 32% identical to a BIG2 domain consensus sequence (group 2 bacterial immunoglobulin-like domains). An asymmetric reconstruction of fibreless full (7.8 Å) and empty (9.3 Å) capsids by cryo-EM revealed the interactions between the capsid shell and DNA [56]. All these interactions were in similar or identical locations in most of the gp8 subunits. The most prominent contact was at the end of a long tubular piece of density, which after fitting a HK97 homology model could be assigned to the

*T4 phage*. The protein elements of the *Myoviridae* phage **T4** (prolate icosahedra, height ~2000 Å, diameter ~900 Å) and its overall organisation have been extensively studied by X-ray and EM [62, 68–70]. The procapsid of T4 contains two proteins gp23 and gp24 which have 22% sequence identity. During maturation the gp21 protease cleaves off 65 N-terminal residues from the capsid protein gp23 and 10 N-terminal residues from the vertex protein gp24 to produce gp23\* and gp24\*,

*DOI: http://dx.doi.org/10.5772/intechopen.85484*

place at the capsid threefold axis.

refined map [50].

E-loop and the I-domain [60].

N-terminal end of the HK97 long helix.

*Bacteriophages - Perspectives and Future*

and hexons in the mature virion.

*HK97 phage.* The first structure of a phage capsid was solved for the *Siphoviridae* **HK97** phage (a mutant without the PP and tail, diameter ~650 Å, T = 7) at 3.6 Å resolution [19] by X-ray crystallography. This structure revealed a new type of protein fold, which has been found in many other phage capsid proteins (CP) despite low sequence identity (**Table 1**). Later, the structure was improved to 3.45 Å resolution [42]. This fold is also found in distantly related icosahedral tailed viruses that infect halophilic archaea [43] and human pathogens such as *Herpesviridae* [44–46]. The characteristic features of the HK97-fold are an N-arm, a peripheral P-domain with a long helix and a β-sheet, an axial A-domain and a long E-loop that fills the region between adjacent threefold axes (**Figure 3A**). The HK97 capsid is held together by molecular chain mail [19]. The two antiparallel β-strands in the E-loop are terminated by a loop, containing Lys169. This residue forms an isopeptide bond with the P-domain residue Asn356 of an adjacent subunit. The third residue involved is the catalytic residue Glu363 from a third subunit (**Figure 3A**). Conformational changes between HK97 procapsids and capsids were assessed by flexible fitting of atomic models [40]. The capsid subunits are skewed around a pseudo-twofold axis in the procapsid but form more symmetric pentons

*λ phage*. The mature capsid of the *Siphoviridae* phage **λ** (diameter ~600 Å, T = 7)

*Structural organisation of the major capsid proteins. (A) Siphophage HK97 (1OHG). The catalytic residues are shown in brown and circled. (B) Podophage ε15 (3 J40). (C) Podophage P22 (5UU5). (D) MyophageT4 gp23\* (5VF3). The N-arm is dark blue, the P-domain is red, the A-domain is light blue and the E-loop is yellow. Extra inserted domains seen in P22 and T4 are magenta. The yellow linker in T4 is topologically equivalent to* 

is stabilised with the help of a decoration protein gpD which is attached to the threefold vertices. A procapsid structure was determined with a resolution between 13.3 and 14.5 Å and a mature capsid at 6.8 Å [47]. The capsid protein gpE of the mature capsid shows clearly the HK97-fold so the HK97 model was used for rigid body fitting into the capsid [47]. However, the A-domain gave a poor fit so it was treated as a separate rigid body and was found to be rotated 15° clockwise compared with HK97. There was unassigned density, which could fit the extra 59 residues in

**14**

**Figure 3.**

*the E-loop seen in the other phages.*

the λ phage. The crystal structure of gpD (1.1 Å) [48] was fitted into the cryo-EM capsid map and, when combined with the HK97 model, showed how gpD is held in place at the capsid threefold axis.

*ε15 phage.* The structure of gp7, the MCP of the *Salmonella* phage **ε15**, was determined by cryo-EM (4.5 Å) [49] and showed a fold similar to that in HK97 (**Figure 3B**). Later, the same group obtained a better resolution structure (~3.5 Å) [50] and, using their previous 4.5 Å structure for gp7, computationally analysed possible different gp7 conformations within EM density obtained from averaging the individual subunits in one asymmetric unit. They found that two models could be fitted in the structure: one that was consistent with the previous interpretation [49], and the other had a strand swap of the β-strands in the P-domain before the two helices in the A-domain. The strand order was 4-3-1-5-2, but in the swapped model, it was now 5-4-2-3-1. The model with the strand swap provided a better visual fit in the refined map [50].

*P22 phage*. Structures of a procapsid and a heat-expanded capsid of the *Podoviridae P22* phage (diameter ~620 Å, T = 7) were determined by cryo-EM at 9.1 and 8.2 Å, respectively [58]. This heat-expanded capsid was composed only of hexons with wide openings where the pentons would be. The MCP gp5 revealed the HK97-fold, but there was an extra domain (residues 223–349) with an immunoglobulin-like telokin domain [64]. Later, the procapsid and capsid structures were determined by cryo-EM at 3.8 and 4.0 Å resolution, respectively [59]. It was also found that gp5 has the HK97-fold with extra density above the E-loop corresponding to the telokin domain (**Figure 3C**). A model built from a cryo-EM map of P22 (3.3 Å) [60] identified the interactions that stabilise the capsid. In the mature virion, the N-arm forms an antiparallel β-strand pair between neighbouring subunits around the threefold axis. A second set of interactions involve hydrogen bonds and salt bridges between adjacent subunits involving the A-domain, the E-loop and the I-domain [60].

*φ29 phage*. The podovirus **φ29** is a relatively small phage (prolate icosahedra, height ~540 Å, diameter ~450 Å) that requires pRNA along with the ATPase gp16 to provide enough energy for the DNA translocation. The procapsid consists of the MCP gp8, the SP gp7, the head fibre protein gp8.5, the connector gp10 and a pRNA. After DNA packaging, the pRNA and ATPase come off the procapsid and are replaced by the gp11 and gp13 underneath the connector, gp29 at the end of the tail and the appendages (gp12\*) which are attached to gp11 and gp13 [65]. During maturation an 18 kDa fragment of gp12 is cleaved off to give gp12\* found in the appendages whose role is to adsorb the virion on the host cell. A fibreless, isometric φ29 variant capsid cryo-EM structure (7.9 Å) showed that gp8 has a structure with the HK97-fold but with extra density [66]. Domain profile-searching algorithms [67] showed that residues 348–429 at the C-terminus were found to be 32% identical to a BIG2 domain consensus sequence (group 2 bacterial immunoglobulin-like domains). An asymmetric reconstruction of fibreless full (7.8 Å) and empty (9.3 Å) capsids by cryo-EM revealed the interactions between the capsid shell and DNA [56]. All these interactions were in similar or identical locations in most of the gp8 subunits. The most prominent contact was at the end of a long tubular piece of density, which after fitting a HK97 homology model could be assigned to the N-terminal end of the HK97 long helix.

*T4 phage*. The protein elements of the *Myoviridae* phage **T4** (prolate icosahedra, height ~2000 Å, diameter ~900 Å) and its overall organisation have been extensively studied by X-ray and EM [62, 68–70]. The procapsid of T4 contains two proteins gp23 and gp24 which have 22% sequence identity. During maturation the gp21 protease cleaves off 65 N-terminal residues from the capsid protein gp23 and 10 N-terminal residues from the vertex protein gp24 to produce gp23\* and gp24\*,

respectively. In the mature capsid, gp23\* forms 120 hexons, and 11 capsid vertices are formed by gp24\* proteins, while the 12th is occupied by a dodecamer of gp20 PP. T4 has two decoration proteins Soc and Hoc [62]. A crystal structure of gp24 (2.9 Å) [61] showed a domain with the HK97-fold and a 60 residue insertion I-domain located on the outer capsid surface (**Figure 3D**). The 3.3 Å cryo-EM reconstruction of the isometric capsid of T4 [62] allowed the structure of gp23\* and gp24\* to be determined. The I-domain linker, missing from the crystal structure of gp24, could be seen and interacts with a neighbouring gp24\* molecule to stabilise the capsid. The structure of gp23\* is similar to gp24\* but with an extra compact region formed by residues 66–93, termed the "N-fist" prior to the N-arm residues 94–110 [62].

Crystal structures were obtained for the Hoc protein from the T4-like phage RB49 with the capsid-binding C-terminal domain 4 missing [71] and Soc protein from the T4-like phage RB49 [72]. The Soc molecules, which are required for capsid stability, interact with three gp23\* subunits [62] although not all binding sites were fully occupied possibly due to differences in the gp23\* I-domain linkers. The immunogenic outer capsid Hoc protein was found in two different sites within the asymmetric unit: at the centre of the hexon near the icosahedral threefold axis and in the hexon close to the fivefold axis [62]. The density of Hoc near the threefold axis was less interpretable than that near the fivefold axis.

*HSV-1 virus*. Although it is not a phage, the human herpesvirus **HSV-1** capsid (1250 Å, T = 16) is a close relative and undergoes a pathway of self-assembly similar to that of dsDNA phages [73]. The virion is characterised by the following features: envelope, tegument, capsid and the viral genome. There are three types of HSV capsids: A-capsids have neither DNA nor the SP, B-capsids have the SP but no DNA and C-capsids contain DNA but no SP. The MCP VP5 forms pentons and hexons and VP26 binds to the VP5 hexons. A triplex of VP19C and VP23 found between capsomers [45]. The upper domain of residues 451–1054 was crystallised and the structure was determined at 2.9 Å. The structure of the whole virion of HSV-1 was determined at ~7 Å resolution [45]. The model of HK97 capsid protein was fitted to the lower domain of VP5 where the E-loop and N-arm were visible, the spline helix was longer and the central channel was wider. Unlike HK97, the E-loop does not form the covalent cross-links or reach an adjacent capsomer. Instead, it interacts with adjacent subunits, lower and middle domains of same VP5 subunit and a triplex molecule. A structure of HSV-1 with its capsid-associated tegument complex (CATC) has been obtained at 4.2 Å [63]. VP5 had the HK97-fold with six additional domains. The two β-barrels in HSV Tri1 (VP19c) and Tri2 (VP23) resemble the homotrimers found in proteins like gpD of phage λ.

#### **4.3 Connectors**

In phages and herpesviruses, one of the fivefold vertices of the capsid is replaced by a *head-to-tail interface (*HTI*)* [30], which is a multi-protein complex (connector). In all phages the HTI provides a platform for docking of preassembled tails in *Sipho*or *Myoviridae* or initiates the assembly of a short tail in *Podoviridae* [30]. The HTI comprises a portal complex (PP) and head completion proteins (**Figure 2**) that serve as a valve for closing the channel and keeping the phage genome inside the capsid at high pressure and only opens to allow genome release from the capsid (under natural conditions) as soon as the phage becomes tightly attached to a host cell.

All currently known PPs are homo-dodecamers when extracted from the viral capsids, as that symmetry is imposed during self-assembly in vivo. However, naive assemblies in vitro of the PP complexes have some variations in their rotational symmetry with 13-mers being observed for SPP1, T7 and HK97 [31, 33, 74]. HSV has been shown to have 11-fold, 12-fold, 13-fold and sometimes even 14-fold symmetry [34].

**17**

**Table 2.**

*Phage portal proteins.*

*Bacteriophages: Their Structural Organisation and Function*

While monomers of the different PPs vary in size, all of them share a common fold shown by EM and X-ray structures that were obtained for the φ29, SPP1 and P22 portals [75–78] and by cryo-EM for T7 and T4 (**Table 2**) [69, 79]. All known PP monomers are characterised by four domains: clip, stem, wing and crown (**Figure 4**) [77]. The clip domain is exposed to the capsid exterior and involved in binding to the terminase for DNA packaging [75, 80, 81] and later to a head completion protein during the HTI assembly [82]. The first high-resolution structure of a phage PP was obtained for the φ29 phage (**Figure 4A,** [75]). The clip domain is linked to the wing region through a stem that comprises typically two α-helices and the outer loops (**Figure 4B, C**). X-ray structures of PP from φ29 and SPP1 phages revealed major helical components that form the central channel through which DNA enters and exits the capsid. The structures of other PPs obtained later have confirmed that this is a conserved element characteristic for all known PPs. The wing domain radiates outwards from the central axis and has an α-helix, which is the longest one and serves as a spine of the wing. It has an α/β sub-fold at its periphery [77]. The crown domain consists of α-helices and is relatively small in SPP1 and surprisingly long

*SPP1 phage*. The **SPP1 phage** connector has been studied for nearly two decades; the SPP1 PP structure was determined by X-ray crystallography, and all other portal complexes are compared with it (**Figures 4B, C** and **5A, B**), but this structure was a 13-mer [31]. The HTI of SPP1 extracted from the capsid was a stable complex composed of the PP gp6, the adaptor protein (AP) gp15 and the stopper (SP) gp16, all organised as three stacked cyclical homo-oligomers [82–84] (**Figure 5A, B**). The cryo-EM structure of the SPP1 HTI before and after DNA release was obtained by cryo-EM at ~7 Å resolution, where the HTI is bound to the tail [82] with gp16 acting as a docking platform for the SPP1 preassembled tail [85, 86]. Binding of the tail induces changes in the position of the gp16 residues Ile9 to Thr33 that close the

*HK97 phage.* There is no structure of the PP gp3 of the **HK97 phage**, but structures of gp6 and gp7 that correspond to the AP gp15 and SP gp16 of SPP1, respectively, were determined by X-ray analysis [74]. The 2.1 Å crystal structure of the gp6 AP revealed that it forms a 13-mer during crystallisation. A model for a

P22 gp1 725 83 10.5 (EM)

**Phage PP No. of residues M. Mass (kDa) Resolution (Å) Structure analysis** HK97 gp3 424 47 none T5 pb7 403 45 none λ gpB 533 59 none SPP1 gp6 503 57 3.4 (X-ray), ~7 (EM) X-ray [77], EM [82] TP901-1 ORF32 452 52 20 EM [54] TW1 gp24 459 51 21 EM [55] φ29 gp10 309 36 2.1 X-ray [76] T7 gp8 536 59 8, 12 EM [87]

ε15 gp4 556 61 20 EM [89] T4 gp20 524 61 3.6 EM [69] HSV-1 pUL6 676 74.2 8 EM [90]

3.25 (X-ray)

EM [88] X-ray [78]

*DOI: http://dx.doi.org/10.5772/intechopen.85484*

(213 aa) in phage P22 (**Figure 4B, D**, **Table 2**).

central channel of the connector.

#### *Bacteriophages: Their Structural Organisation and Function DOI: http://dx.doi.org/10.5772/intechopen.85484*

*Bacteriophages - Perspectives and Future*

respectively. In the mature capsid, gp23\* forms 120 hexons, and 11 capsid vertices are formed by gp24\* proteins, while the 12th is occupied by a dodecamer of gp20 PP. T4 has two decoration proteins Soc and Hoc [62]. A crystal structure of gp24 (2.9 Å) [61] showed a domain with the HK97-fold and a 60 residue insertion I-domain located on the outer capsid surface (**Figure 3D**). The 3.3 Å cryo-EM reconstruction of the isometric capsid of T4 [62] allowed the structure of gp23\* and gp24\* to be determined. The I-domain linker, missing from the crystal structure of gp24, could be seen and interacts with a neighbouring gp24\* molecule to stabilise the capsid. The structure of gp23\* is similar to gp24\* but with an extra compact region formed by residues 66–93, termed the "N-fist" prior to the N-arm residues 94–110 [62].

Crystal structures were obtained for the Hoc protein from the T4-like phage RB49 with the capsid-binding C-terminal domain 4 missing [71] and Soc protein from the T4-like phage RB49 [72]. The Soc molecules, which are required for capsid stability, interact with three gp23\* subunits [62] although not all binding sites were fully occupied possibly due to differences in the gp23\* I-domain linkers. The immunogenic outer capsid Hoc protein was found in two different sites within the asymmetric unit: at the centre of the hexon near the icosahedral threefold axis and in the hexon close to the fivefold axis [62]. The density of Hoc near the threefold

*HSV-1 virus*. Although it is not a phage, the human herpesvirus **HSV-1** capsid (1250 Å, T = 16) is a close relative and undergoes a pathway of self-assembly similar to that of dsDNA phages [73]. The virion is characterised by the following features: envelope, tegument, capsid and the viral genome. There are three types of HSV capsids: A-capsids have neither DNA nor the SP, B-capsids have the SP but no DNA and C-capsids contain DNA but no SP. The MCP VP5 forms pentons and hexons and VP26 binds to the VP5 hexons. A triplex of VP19C and VP23 found between capsomers [45]. The upper domain of residues 451–1054 was crystallised and the structure was determined at 2.9 Å. The structure of the whole virion of HSV-1 was determined at ~7 Å resolution [45]. The model of HK97 capsid protein was fitted to the lower domain of VP5 where the E-loop and N-arm were visible, the spline helix was longer and the central channel was wider. Unlike HK97, the E-loop does not form the covalent cross-links or reach an adjacent capsomer. Instead, it interacts with adjacent subunits, lower and middle domains of same VP5 subunit and a triplex molecule. A structure of HSV-1 with its capsid-associated tegument complex (CATC) has been obtained at 4.2 Å [63]. VP5 had the HK97-fold with six additional domains. The two β-barrels in HSV Tri1 (VP19c) and Tri2 (VP23) resemble the

In phages and herpesviruses, one of the fivefold vertices of the capsid is replaced by a *head-to-tail interface (*HTI*)* [30], which is a multi-protein complex (connector). In all phages the HTI provides a platform for docking of preassembled tails in *Sipho*or *Myoviridae* or initiates the assembly of a short tail in *Podoviridae* [30]. The HTI comprises a portal complex (PP) and head completion proteins (**Figure 2**) that serve as a valve for closing the channel and keeping the phage genome inside the capsid at high pressure and only opens to allow genome release from the capsid (under natural

All currently known PPs are homo-dodecamers when extracted from the viral capsids, as that symmetry is imposed during self-assembly in vivo. However, naive assemblies in vitro of the PP complexes have some variations in their rotational symmetry with 13-mers being observed for SPP1, T7 and HK97 [31, 33, 74]. HSV has been shown to have 11-fold, 12-fold, 13-fold and sometimes even 14-fold symmetry [34].

conditions) as soon as the phage becomes tightly attached to a host cell.

axis was less interpretable than that near the fivefold axis.

homotrimers found in proteins like gpD of phage λ.

**16**

**4.3 Connectors**

While monomers of the different PPs vary in size, all of them share a common fold shown by EM and X-ray structures that were obtained for the φ29, SPP1 and P22 portals [75–78] and by cryo-EM for T7 and T4 (**Table 2**) [69, 79]. All known PP monomers are characterised by four domains: clip, stem, wing and crown (**Figure 4**) [77]. The clip domain is exposed to the capsid exterior and involved in binding to the terminase for DNA packaging [75, 80, 81] and later to a head completion protein during the HTI assembly [82]. The first high-resolution structure of a phage PP was obtained for the φ29 phage (**Figure 4A,** [75]). The clip domain is linked to the wing region through a stem that comprises typically two α-helices and the outer loops (**Figure 4B, C**). X-ray structures of PP from φ29 and SPP1 phages revealed major helical components that form the central channel through which DNA enters and exits the capsid. The structures of other PPs obtained later have confirmed that this is a conserved element characteristic for all known PPs. The wing domain radiates outwards from the central axis and has an α-helix, which is the longest one and serves as a spine of the wing. It has an α/β sub-fold at its periphery [77]. The crown domain consists of α-helices and is relatively small in SPP1 and surprisingly long (213 aa) in phage P22 (**Figure 4B, D**, **Table 2**).

*SPP1 phage*. The **SPP1 phage** connector has been studied for nearly two decades; the SPP1 PP structure was determined by X-ray crystallography, and all other portal complexes are compared with it (**Figures 4B, C** and **5A, B**), but this structure was a 13-mer [31]. The HTI of SPP1 extracted from the capsid was a stable complex composed of the PP gp6, the adaptor protein (AP) gp15 and the stopper (SP) gp16, all organised as three stacked cyclical homo-oligomers [82–84] (**Figure 5A, B**). The cryo-EM structure of the SPP1 HTI before and after DNA release was obtained by cryo-EM at ~7 Å resolution, where the HTI is bound to the tail [82] with gp16 acting as a docking platform for the SPP1 preassembled tail [85, 86]. Binding of the tail induces changes in the position of the gp16 residues Ile9 to Thr33 that close the central channel of the connector.

*HK97 phage.* There is no structure of the PP gp3 of the **HK97 phage**, but structures of gp6 and gp7 that correspond to the AP gp15 and SP gp16 of SPP1, respectively, were determined by X-ray analysis [74]. The 2.1 Å crystal structure of the gp6 AP revealed that it forms a 13-mer during crystallisation. A model for a


**Table 2.** *Phage portal proteins.*

**Figure 4.**

*Structures of portal proteins. One chain of the PP is highlighted in red. (A) gp10 of φ29 (1FOU). (B) gp6 of SPP1 (2JES). (C) A gp6 SPP1 monomer with the crown, stem, wing and clip domains indicated. (D) gp1 of P22 (3LJ5). (E) gp8 of T7 (3J4A). (F) gp24 of T4 (3JA7).*

dodecameric ring of gp6 was constructed from a monomer taken from the 13-mer and fitted into a cryo-EM map of SPP1 [83]. This fitted well in size and shape, but the helices of HK97 gp6 did not fit well into the densities of the SPP1 connector EM map which suggested that the assembly into a 13-mer in the absence of other phage components may produce a different conformation [74].

*P22 phage*. The HTI of the **P22 phage** consists of two proteins, the PP gp1 and the AP gp4. The first structural organisation of the P22 HTI complex was obtained by cryo-EM at a resolution of 9.4 Å [91]. A crystal structure (3.25 Å) was obtained for the PP by using low-resolution EM data for phasing (**Figure 5C, D** [78]). The complete polypeptide chain was traced apart from a loop between residues 464 and 492, and loop modelling was used to build this area from a 9.2 Å cryo-EM map [88]. The overall height is ~300 Å with the gp4 AP forming a dodecameric ring below the PP. The PP of P22 has the same fold in its central channel as SPP1 [90] and φ29 [88, 89] (**Figures 4D, 5D**). The crystal structure of the full-length PP revealed that the C-terminal domain forms a ~200 Å long, α-helical barrel. At the same time, an asymmetric reconstruction of the entire P22 virion has been determined by cryo-EM at 7.8 Å resolution [92]. The 150 Å coiled-coil barrel structure extends from the PP to near the centre of the capsid. Fitting the crystal structure of the coregp4 complex into the 7.8 Å virion density map revealed a stretch of about 21 gp4 C-terminal residues that lie wedged between the capsid and portal [92]. Overlap between gp4 and the MCP gp5 indicates that gp4 must undergo significant conformational change during phage assembly when the tail is added. A comparison of the portal position in the procapsid and the virion shows that the portal increases its contact with the capsid shell during maturation. It was proposed that a portion of the scaffold remains in place during dsDNA packaging to allow access of the gp4

**19**

*Bacteriophages: Their Structural Organisation and Function*

C-terminus to the bottom of the portal [92]. When gp4 binds, the SP is displaced allowing the final conformational change implied by the position gp4 C-terminal

*Structures of the HTI. (A) The cryo-EM map of the SPP1 HTI coloured according to protein with the gp6 PP (blue), adaptor gp15 (brown) and stopper (purple) (EMD-1021 [83]). (B) Cutaway view of the SPP1 HTI with models gp6 (2JES), gp15 (2KBZ) and gp16 (2KCA) fitted into EMD-1021 (from [84]). (C) P22 connector complex determined by X-ray crystallography without barrel domain, PP pb1 (blue) and AP gp4 (red).* 

*T7 phage*. The HTI of **T7** was determined at 8 Å resolution [79], and the dodecameric T7 PP was found to be structurally similar to the PPs from other phages (**Figure 4E**). A ~12 Å resolution structure of a recombinant HTI (gp8-gp11-gp12) complex of T7 was determined by cryo-EM [87], and pseudo-atomic models were obtained using gp1 and gp4 of P22 for fitting and analysis of the T7 gp8 (PP) and gp11 (AP), respectively [78]. The T7 gp8 model was superimposed on gp10 from φ29 [75, 76], gp1 from P22 [78] and gp6 from SPP1 [77] which confirmed the presence of two stem helices, but the fold of the clip domain varies between the different PPs [87]. Previous structures of the head completion proteins have shown them to contain four helices, and when the T7 gp11 model was superimposed on gp4 from P22 and gp6 from HK97 [74, 78], the position of

*T4 phage*. The structure of the **T4** HTI region was determined by cryo-EM for the fully assembled capsid (~17 Å) [93]. The neck region, which connects the tail to the dodecameric PP (gp20), comprises adaptor proteins gp3, gp15, gp13, gp14 and gp *wac* (fibritin). It was assumed that the T4 HTI would have a similar PP organisation to other phages; therefore, the crystal structure of the φ29 PP was tentatively docked into the cryo-EM map of the T4 HTI region [75, 93]. Recently, the structure of the gp20 PP from T4 was determined by cryo-EM (3.6 Å) (**Figure 4F**, [69]). Interestingly, a gp20-N74 construct with the 73 N-terminal residues truncated contained 95% dodecamers and 5% 13-mers in solution [69]. The dodecameric PP was ~120 Å in height and varied in diameter from 90 Å (top) to 170 Å (middle) and to 80 Å close to the capsid, while the central channel goes from 90 to 28 Å near the middle of the channel. The connection to the capsid was by the PP wing domains,

polypeptide that is wedged between the capsid and the HTI.

these four helices was conserved, but the C-termini were flexible.

and the interactions are partially hydrophobic and partially polar [69].

*φ29 phage*. The asymmetric 3D reconstructions of *φ29* with and without DNA obtained by cryo-EM at 7.8 and 9.3 Å, respectively, showed that the PP gp10 dodecamer has elongated densities lining the central channel and tilted at ~30° to the central axis of the virion [56]. These cylindrical densities correspond to the α-helices seen in the crystal structure of the φ29 PP [76]. The density of the clip domain in the cryo-EM map lies further away from the PP axis than in the crystal structure possibly due to a different conformation of the PP within the fully assembled phage. Two cylindrical columns of high density were observed within the virions: one in the upper part and another at the bottom of the HTI. These densities were assigned to DNA based on the diameter, intensity and their location. The φ29 DNA is visible as ringlike densities below the PP, and then DNA stretches into the tail to about

*DOI: http://dx.doi.org/10.5772/intechopen.85484*

**Figure 5.**

*(D) Central slice of C [91].*

*Bacteriophages: Their Structural Organisation and Function DOI: http://dx.doi.org/10.5772/intechopen.85484*

#### **Figure 5.**

*Bacteriophages - Perspectives and Future*

dodecameric ring of gp6 was constructed from a monomer taken from the 13-mer and fitted into a cryo-EM map of SPP1 [83]. This fitted well in size and shape, but the helices of HK97 gp6 did not fit well into the densities of the SPP1 connector EM map which suggested that the assembly into a 13-mer in the absence of other phage

*Structures of portal proteins. One chain of the PP is highlighted in red. (A) gp10 of φ29 (1FOU). (B) gp6 of SPP1 (2JES). (C) A gp6 SPP1 monomer with the crown, stem, wing and clip domains indicated. (D) gp1 of P22 (3LJ5).* 

*P22 phage*. The HTI of the **P22 phage** consists of two proteins, the PP gp1 and the AP gp4. The first structural organisation of the P22 HTI complex was obtained by cryo-EM at a resolution of 9.4 Å [91]. A crystal structure (3.25 Å) was obtained for the PP by using low-resolution EM data for phasing (**Figure 5C, D** [78]). The complete polypeptide chain was traced apart from a loop between residues 464 and 492, and loop modelling was used to build this area from a 9.2 Å cryo-EM map [88]. The overall height is ~300 Å with the gp4 AP forming a dodecameric ring below the PP. The PP of P22 has the same fold in its central channel as SPP1 [90] and φ29 [88, 89] (**Figures 4D, 5D**). The crystal structure of the full-length PP revealed that the C-terminal domain forms a ~200 Å long, α-helical barrel. At the same time, an asymmetric reconstruction of the entire P22 virion has been determined by cryo-EM at 7.8 Å resolution [92]. The 150 Å coiled-coil barrel structure extends from the PP to near the centre of the capsid. Fitting the crystal structure of the coregp4 complex into the 7.8 Å virion density map revealed a stretch of about 21 gp4 C-terminal residues that lie wedged between the capsid and portal [92]. Overlap between gp4 and the MCP gp5 indicates that gp4 must undergo significant conformational change during phage assembly when the tail is added. A comparison of the portal position in the procapsid and the virion shows that the portal increases its contact with the capsid shell during maturation. It was proposed that a portion of the scaffold remains in place during dsDNA packaging to allow access of the gp4

components may produce a different conformation [74].

**18**

**Figure 4.**

*(E) gp8 of T7 (3J4A). (F) gp24 of T4 (3JA7).*

*Structures of the HTI. (A) The cryo-EM map of the SPP1 HTI coloured according to protein with the gp6 PP (blue), adaptor gp15 (brown) and stopper (purple) (EMD-1021 [83]). (B) Cutaway view of the SPP1 HTI with models gp6 (2JES), gp15 (2KBZ) and gp16 (2KCA) fitted into EMD-1021 (from [84]). (C) P22 connector complex determined by X-ray crystallography without barrel domain, PP pb1 (blue) and AP gp4 (red). (D) Central slice of C [91].*

C-terminus to the bottom of the portal [92]. When gp4 binds, the SP is displaced allowing the final conformational change implied by the position gp4 C-terminal polypeptide that is wedged between the capsid and the HTI.

*T7 phage*. The HTI of **T7** was determined at 8 Å resolution [79], and the dodecameric T7 PP was found to be structurally similar to the PPs from other phages (**Figure 4E**). A ~12 Å resolution structure of a recombinant HTI (gp8-gp11-gp12) complex of T7 was determined by cryo-EM [87], and pseudo-atomic models were obtained using gp1 and gp4 of P22 for fitting and analysis of the T7 gp8 (PP) and gp11 (AP), respectively [78]. The T7 gp8 model was superimposed on gp10 from φ29 [75, 76], gp1 from P22 [78] and gp6 from SPP1 [77] which confirmed the presence of two stem helices, but the fold of the clip domain varies between the different PPs [87]. Previous structures of the head completion proteins have shown them to contain four helices, and when the T7 gp11 model was superimposed on gp4 from P22 and gp6 from HK97 [74, 78], the position of these four helices was conserved, but the C-termini were flexible.

*T4 phage*. The structure of the **T4** HTI region was determined by cryo-EM for the fully assembled capsid (~17 Å) [93]. The neck region, which connects the tail to the dodecameric PP (gp20), comprises adaptor proteins gp3, gp15, gp13, gp14 and gp *wac* (fibritin). It was assumed that the T4 HTI would have a similar PP organisation to other phages; therefore, the crystal structure of the φ29 PP was tentatively docked into the cryo-EM map of the T4 HTI region [75, 93]. Recently, the structure of the gp20 PP from T4 was determined by cryo-EM (3.6 Å) (**Figure 4F**, [69]). Interestingly, a gp20-N74 construct with the 73 N-terminal residues truncated contained 95% dodecamers and 5% 13-mers in solution [69]. The dodecameric PP was ~120 Å in height and varied in diameter from 90 Å (top) to 170 Å (middle) and to 80 Å close to the capsid, while the central channel goes from 90 to 28 Å near the middle of the channel. The connection to the capsid was by the PP wing domains, and the interactions are partially hydrophobic and partially polar [69].

*φ29 phage*. The asymmetric 3D reconstructions of *φ29* with and without DNA obtained by cryo-EM at 7.8 and 9.3 Å, respectively, showed that the PP gp10 dodecamer has elongated densities lining the central channel and tilted at ~30° to the central axis of the virion [56]. These cylindrical densities correspond to the α-helices seen in the crystal structure of the φ29 PP [76]. The density of the clip domain in the cryo-EM map lies further away from the PP axis than in the crystal structure possibly due to a different conformation of the PP within the fully assembled phage. Two cylindrical columns of high density were observed within the virions: one in the upper part and another at the bottom of the HTI. These densities were assigned to DNA based on the diameter, intensity and their location. The φ29 DNA is visible as ringlike densities below the PP, and then DNA stretches into the tail to about

~100 Å [56]. The DNA appears to contact with the PP crown domain and then with the density corresponding to the tunnel loops located between the PP crown and stem domains. These tunnel loops in the narrowest part of the connector channel were found in the SPP1 phage PP and are believed that they play a role in DNA translocation [77, 94]. The φ29 DNA appears to be clamped at the top of the tail tube [56].

*HSV-1 virus*. Herpes simplex virus (**HSV-1**) has its DNA packaged into the capsid through a portal channel of the PP complex (pUL6) [95]. The structure of a dodecameric HSV-1 PP has been determined by cryo-EM at 16 Å resolution from purified portals [34]. The structure showed a close resemblance to the SPP1 PP [83]. The PP is about the same size as the pentons that occupy the other fivefold vertices, which explains the difficulty in localising the portal density in images of HSV capsids unlike in phages, which have a tail. The PP, pUL6, was well defined in the A-capsid structure located at one fivefold vertex on the outer surface of the capsid as shown by cryo-ET [96]. There is also a strong density within the portal channel and inside of the capsid, which is interpreted as the end of DNA as seen in φ29 and SPP1 [56, 82].

#### **4.4 Tails**

The tail organisation in phages depends on their type: *Siphoviridae* have long flexible tails, and *Podoviridae* have very short tails that mostly consist of the adhesive


**21**

**Figure 6.**

*Bacteriophages: Their Structural Organisation and Function*

device, while *Myoviridae* have rigid long contractile tails that consist of a number of different proteins forming the inner rigid tube and the outer contractile sheath. *Siphoviridae* and *Myoviridae* have an independent pathway for assembly of their tails and are attached to the capsid after it has been packed with genome. However, in *Podoviridae* the tails are assembled on the capsids after DNA packaging as the last step of self-assembly (**Figure 2**, **Table 3**). The long tails of *Siphoviridae* are composed of tail proteins (TPs) that form circular oligomeric rings with three- or sixfold rotational symmetry. The rings are assembled around a tape measure protein (TMP) that defines the length of the tail and are stacked on the top of each other with helical symmetry. A tail terminator protein (TrP) caps the tail when it reaches the length defined by the TMP; the TrP serves as an interface with the capsid. When the phage interacts with the host receptor, the HTI opens and the TMP is pushed out by DNA as a result of the inner pressure of the capsid. Most long tails have a smooth outer surface, but

some have appendages that protrude outwards from the tail surface.

*T5 phage*. The **T5** phage has a long tail (1600 Å) with a diameter of 90 Å. A crystal structure has been obtained for the TP pb6 of T5 (2.2 Å) [97]. The protein consists of two domains: an N-terminal domain with two subdomains both of which comprise a β-sandwich flanked by an α-helix and a long loop **(Figure 6A**) and a C-terminal domain with an immunoglobulin-like fold [98]. The overall tail structure has threefold symmetry (**Figure 6B**) [99, 100]. Cryo-EM was used to

*Bacteriophage tails. (A) The crystal structure of a monomer of T5 pb6 (5NGJ). The extra immunoglobulin domain is coloured yellow. (B) A slice of the combined EM map of T5 (EMD-3692) showing the fold symmetry of the tail. (C) The crystal structure of the N-terminal domain of the P22 TP gpV (2K4Q ). (D) Cryo-EM map of SPP1 tails (gp17.1). (E) Cryo-EM map of SPP1 tails (gp17.1\*). The protrusions are the size of an immunoglobulin domain. (F) The T4 cryo-EM map (EMD-8767) with fitted coordinates (5W5F). Alternate subunits in the central ring are coloured red and blue. The red circle in B and rectangles in D, E and F indicate* 

*the inner tail tube, γ—rotation between adjacent tail rings.*

*DOI: http://dx.doi.org/10.5772/intechopen.85484*

**Table 3.** *Phage tail structures.*

#### *Bacteriophages: Their Structural Organisation and Function DOI: http://dx.doi.org/10.5772/intechopen.85484*

*Bacteriophages - Perspectives and Future*

**Phage Tail proteins No. of** 

component IPR010064, gp10

gpH (TMP) gpU (terminator)

> gp17\* (TP) gp18 (TMP)

> gp14 (TMP)

gp12 (tailspike)

gp12 (TP) gp17 (fibres)

gp9 (tailspike)

gp15 (terminator)

HK97 putative tail-

λ gpV (TP)

SPP1 gp17 (TP)

TW1 gp12 (TP)

φ29 gp9 (knob)

T7 gp11 (TP)

P22 gp10 (hub)

T4 gp19 (TP)

**4.4 Tails**

~100 Å [56]. The DNA appears to contact with the PP crown domain and then with the density corresponding to the tunnel loops located between the PP crown and stem domains. These tunnel loops in the narrowest part of the connector channel were found in the SPP1 phage PP and are believed that they play a role in DNA translocation [77, 94]. The φ29 DNA appears to be clamped at the top of the tail tube [56]. *HSV-1 virus*. Herpes simplex virus (**HSV-1**) has its DNA packaged into the capsid through a portal channel of the PP complex (pUL6) [95]. The structure of a dodecameric HSV-1 PP has been determined by cryo-EM at 16 Å resolution from purified portals [34]. The structure showed a close resemblance to the SPP1 PP [83]. The PP is about the same size as the pentons that occupy the other fivefold vertices, which explains the difficulty in localising the portal density in images of HSV capsids unlike in phages, which have a tail. The PP, pUL6, was well defined in the A-capsid structure located at one fivefold vertex on the outer surface of the capsid as shown by cryo-ET [96]. There is also a strong density within the portal channel and inside of the capsid, which is interpreted as the end of DNA as seen in φ29 and SPP1 [56, 82].

The tail organisation in phages depends on their type: *Siphoviridae* have long flexible tails, and *Podoviridae* have very short tails that mostly consist of the adhesive

> **M. Mass kDa**

> > 26 92 15

15 28 111

> 18 72

68 92

22 89 62

52 72

18 32 **Resolution (Å)**

6

n/a 2.7

n/a 14

2.04 1.8–2.05 7.8

12.0 2.0 (X-ray)

> 9.4 2.0

4.11 3.4 15.0 3.2

not defined not known n/a None

**Structure analysis**

X-ray [97] EM [97]

NMR [98, 102] X-ray [103]

NMR (gp17) [104] EM [85]

X-ray [105, 106] EM [56]

EM (gp11,12,17) [87] X-ray (gp17) [107]

EM, tomography [88, 91], X-ray [108, 109]

EM [111, 112] X-ray [113]

23 EM [55]

**residues**

246 853 131

134 264 1032

> NF 675

599 854

196 794 553

472 667

163 272

ε15 gp20 (tailspike) 1070 116 20n/r EM [89, 110]

HSV does not have the tail n/a n/a n/a n/a

Т5 pb6 464 50 2.2

**20**

**Table 3.**

*Phage tail structures.*

device, while *Myoviridae* have rigid long contractile tails that consist of a number of different proteins forming the inner rigid tube and the outer contractile sheath. *Siphoviridae* and *Myoviridae* have an independent pathway for assembly of their tails and are attached to the capsid after it has been packed with genome. However, in *Podoviridae* the tails are assembled on the capsids after DNA packaging as the last step of self-assembly (**Figure 2**, **Table 3**). The long tails of *Siphoviridae* are composed of tail proteins (TPs) that form circular oligomeric rings with three- or sixfold rotational symmetry. The rings are assembled around a tape measure protein (TMP) that defines the length of the tail and are stacked on the top of each other with helical symmetry. A tail terminator protein (TrP) caps the tail when it reaches the length defined by the TMP; the TrP serves as an interface with the capsid. When the phage interacts with the host receptor, the HTI opens and the TMP is pushed out by DNA as a result of the inner pressure of the capsid. Most long tails have a smooth outer surface, but some have appendages that protrude outwards from the tail surface.

*T5 phage*. The **T5** phage has a long tail (1600 Å) with a diameter of 90 Å. A crystal structure has been obtained for the TP pb6 of T5 (2.2 Å) [97]. The protein consists of two domains: an N-terminal domain with two subdomains both of which comprise a β-sandwich flanked by an α-helix and a long loop **(Figure 6A**) and a C-terminal domain with an immunoglobulin-like fold [98]. The overall tail structure has threefold symmetry (**Figure 6B**) [99, 100]. Cryo-EM was used to

#### **Figure 6.**

*Bacteriophage tails. (A) The crystal structure of a monomer of T5 pb6 (5NGJ). The extra immunoglobulin domain is coloured yellow. (B) A slice of the combined EM map of T5 (EMD-3692) showing the fold symmetry of the tail. (C) The crystal structure of the N-terminal domain of the P22 TP gpV (2K4Q ). (D) Cryo-EM map of SPP1 tails (gp17.1). (E) Cryo-EM map of SPP1 tails (gp17.1\*). The protrusions are the size of an immunoglobulin domain. (F) The T4 cryo-EM map (EMD-8767) with fitted coordinates (5W5F). Alternate subunits in the central ring are coloured red and blue. The red circle in B and rectangles in D, E and F indicate the inner tail tube, γ—rotation between adjacent tail rings.*

determine the tail structure of T5 at ~6 Å resolution before and after DNA ejection [97] and the atomic model of pb6 used to interpret the results. No differences were found between the two structures, apart from the absence of the tape measure protein pb2 after DNA ejection [97].

*λ phage.* In the phage **λ**, the tail has sixfold symmetry and is composed of gpV (TP) and gpH (TMP) [101]. The N-terminal domain of gpV (gpVN) is required for assembly of the functional phage, and the structure was determined by solution NMR [102]. There are seven β-strands, arranged into two antiparallel sheets which fold into a twisted β-sandwich (**Figure 6C**). The single α-helix is located at the side of the sandwich. The C-terminus of gpV (gpVC) was shown by solution NMR to have an Ig-like fold [98].

*SPP1 phage*. The structures of the tails from the *Siphoviridae* **SPP1** before and after DNA ejection were determined using negative stain EM at ~14.5 Å resolution [85]. This tail is ~1600 Å long and consists of three proteins gp17 and gp17\* that form the tail tube and the TMP gp18. Even at low resolution, the structures revealed movements in the positions of inner domains gp17 and gp17\* after DNA ejection [85]. The ratio of proteins gp17 and gp17\* within the tail complex suggests that the tail has to have threefold symmetry. Reconstructions of mutants comprising either gp17 or gp17\* were obtained and demonstrated sixfold symmetry. A comparison of structures indicated that protein gp17\* has an immunoglobulin domain located on the outer surface of the tail (**Figure 6D, E**) (Orlova, Personal communication).

*T4 phage*. *Myoviridae* have contractile tails with a sheath that surrounds a central tail tube. On infection, the tail sheath contracts allowing the tail tube to penetrate the outer membrane of the host cell. The structure of the **T4** phage (a representative of *Myoviridae*) tail is well studied. It consists of a rigid tube, composed of multiple copies of gp19, surrounded by contractile sheath from gp18 subunits [93]. A structure of the central tube at 3.4 Å has been obtained by cryo-EM and showed sixfold symmetry [112] with an axial rise for the helical unit of ~40 Å. This resolution revealed the strands of the β-sheets indicating that four strands from one subunit become part of a continuous helical β-sheet lining the inner channel of the tail (**Figure 6F**). The structure of the T4 contracted tail was obtained at 17 Å resolution, and the tail sheath protein gp18 was found to be arranged into 23 hexameric rings [93]. A crystal structure of a gp18 mutant missing the C-terminal domain [114] was used as the basis for identifying domains in gp18. A homology model based on other *Myoviridae* tail sheath structures [115] was used to localise the C-terminal domain [113].

#### **4.5 Adsorption apparatus**

Most *Siphoviridae* phages have an oligomeric ring formed by distal tail proteins (DTPs), which is attached to the last ring of the tail tube [116, 117]. The DTP ring usually serves as an apparatus to recognise and connect to receptor-binding proteins; sometimes, this interaction is assisted by tail fibres found in T4, T5 and other phages. The DTP of SPP1 does not have the fibres [118]. Many phages that infect Gram-negative bacteria have lysozyme-like proteins on the ends of their tails, which enter the periplasm to digest the peptidoglycan barrier [119].

*P22 phage*. *Podoviridae* phage tails have a central needle with trimeric appendages (or spikes) around it, and the part of the tail attached to the capsid could be considered as the baseplate. The short tail of the podovirus **P22** has been studied by cryo-EM of the entire virion [120, 121]. The structure of the P22 tail was obtained at 9.4 Å and aided understanding of its organisation [91]. The 2.8 MDa multisubunit complex is formed by the dodecameric PP gp1, 6 trimeric tailspikes of gp9, 12 copies of the tail accessory protein gp4, a hexon of gp10 and a trimer of the tail needle gp26 (**Figure 7A**). The proteins gp4 and gp10 form a HTI on which the tail

**23**

*one in gp27 is coloured in magenta, red and cyan.*

**Figure 7.**

*Bacteriophages: Their Structural Organisation and Function*

crystal structures and models of gp1, gp4 and gp10 [92].

assembles and are attached to the PP, while the N-terminal head-binding domain of the outer tailspikes (gp9) attaches to the interface of the gp4 and gp10 subunits. The second larger receptor-binding domain of gp9 contacts and destroys the cell surface lipopolysaccharide [121, 122]. Crystal structures have been obtained for both gp26 [123] and gp9 [108, 109], and these were fitted into the cryo-EM map to find how the different proteins hold the complex together. The tail needle gp26 is folded as triple-stranded coiled coil. The gp9 head-binding domain has a β-helical domain, and the receptor-binding domain is a triangular β-prism (**Figure 7B**) [91]. The asymmetric cryo-EM structure of P22 (7.8 Å) allowed a detailed analysis of the interactions between gp9 and the gp4–gp10 interface by fitting the gp9 and gp26

*SPP1 phage*. The *Siphoviridae* **SPP1** phage has the long flexible tail. A negative stain reconstruction of the adsorption apparatus revealed a hinge connection between the tip and the tail tube, allowing bending angles as high as 50° [85]. The gp24 protein keeps the tail closed before DNA ejection by forming a cap and is located between the tail and the tip. The first protein in the tip after the cap is gp21,

*Adsorption apparatus. (A) The EM map (EMD-5051) of P22 coloured according to the constituent proteins. The PP (gp1), the gp4 12-mer, the gp10 6-mer, the gp9 tailspikes and the gp26 cell-puncturing needle are in purple, green, red, blue and brown, respectively. (B) The crystal structures for the N-terminal head-binding domain (1LKT) and the C-terminal receptor-binding domain (1TYU) of P22 gp9 docked into the cryo-EM density (EMDB-5051). (C) The receptor-binding carboxy-terminal domain of T5 tail fibre pb1 (5AQ5). The five different domain regions are labelled. The N- and C-termini are indicated. (D) The EM map (EMD-8868) of the TW1 tail showing the TP gp12 (in blue), gp15 (in green), gp16, gp18, gp27 (together in light brown) and the gp19 tailspikes (in purple). (E) The φ29 tailspike protein gp12 (3SUC). The four domains D1\*, D2, D3 and D4 are labelled. (F) The crystal structure (1 K28) of the cell-puncturing device of T4 (gp27 ± gp5\* ± gp5C)3. Each monomer within in the gp5 trimer is coloured in blue, green and orange, and each* 

*DOI: http://dx.doi.org/10.5772/intechopen.85484*

#### *Bacteriophages: Their Structural Organisation and Function DOI: http://dx.doi.org/10.5772/intechopen.85484*

*Bacteriophages - Perspectives and Future*

protein pb2 after DNA ejection [97].

have an Ig-like fold [98].

**4.5 Adsorption apparatus**

determine the tail structure of T5 at ~6 Å resolution before and after DNA ejection [97] and the atomic model of pb6 used to interpret the results. No differences were found between the two structures, apart from the absence of the tape measure

*λ phage.* In the phage **λ**, the tail has sixfold symmetry and is composed of gpV (TP) and gpH (TMP) [101]. The N-terminal domain of gpV (gpVN) is required for assembly of the functional phage, and the structure was determined by solution NMR [102]. There are seven β-strands, arranged into two antiparallel sheets which fold into a twisted β-sandwich (**Figure 6C**). The single α-helix is located at the side of the sandwich. The C-terminus of gpV (gpVC) was shown by solution NMR to

*SPP1 phage*. The structures of the tails from the *Siphoviridae* **SPP1** before and after DNA ejection were determined using negative stain EM at ~14.5 Å resolution [85]. This tail is ~1600 Å long and consists of three proteins gp17 and gp17\* that form the tail tube and the TMP gp18. Even at low resolution, the structures revealed movements in the positions of inner domains gp17 and gp17\* after DNA ejection [85]. The ratio of proteins gp17 and gp17\* within the tail complex suggests that the tail has to have threefold symmetry. Reconstructions of mutants comprising either gp17 or gp17\* were obtained and demonstrated sixfold symmetry. A comparison of structures indicated that protein gp17\* has an immunoglobulin domain located on the outer surface of the tail (**Figure 6D, E**) (Orlova, Personal communication). *T4 phage*. *Myoviridae* have contractile tails with a sheath that surrounds a central tail tube. On infection, the tail sheath contracts allowing the tail tube to penetrate the outer membrane of the host cell. The structure of the **T4** phage (a representative of *Myoviridae*) tail is well studied. It consists of a rigid tube, composed of multiple copies of gp19, surrounded by contractile sheath from gp18 subunits [93]. A structure of the central tube at 3.4 Å has been obtained by cryo-EM and showed sixfold symmetry [112] with an axial rise for the helical unit of ~40 Å. This resolution revealed the strands of the β-sheets indicating that four strands from one subunit become part of a continuous helical β-sheet lining the inner channel of the tail (**Figure 6F**). The structure of the T4 contracted tail was obtained at 17 Å resolution, and the tail sheath protein gp18 was found to be arranged into 23 hexameric rings [93]. A crystal structure of a gp18 mutant missing the C-terminal domain [114] was used as the basis for identifying domains in gp18. A homology model based on other *Myoviridae* tail sheath

structures [115] was used to localise the C-terminal domain [113].

enter the periplasm to digest the peptidoglycan barrier [119].

Most *Siphoviridae* phages have an oligomeric ring formed by distal tail proteins (DTPs), which is attached to the last ring of the tail tube [116, 117]. The DTP ring usually serves as an apparatus to recognise and connect to receptor-binding proteins; sometimes, this interaction is assisted by tail fibres found in T4, T5 and other phages. The DTP of SPP1 does not have the fibres [118]. Many phages that infect Gram-negative bacteria have lysozyme-like proteins on the ends of their tails, which

*P22 phage*. *Podoviridae* phage tails have a central needle with trimeric appendages (or spikes) around it, and the part of the tail attached to the capsid could be considered as the baseplate. The short tail of the podovirus **P22** has been studied by cryo-EM of the entire virion [120, 121]. The structure of the P22 tail was obtained at 9.4 Å and aided understanding of its organisation [91]. The 2.8 MDa multisubunit complex is formed by the dodecameric PP gp1, 6 trimeric tailspikes of gp9, 12 copies of the tail accessory protein gp4, a hexon of gp10 and a trimer of the tail needle gp26 (**Figure 7A**). The proteins gp4 and gp10 form a HTI on which the tail

**22**

assembles and are attached to the PP, while the N-terminal head-binding domain of the outer tailspikes (gp9) attaches to the interface of the gp4 and gp10 subunits. The second larger receptor-binding domain of gp9 contacts and destroys the cell surface lipopolysaccharide [121, 122]. Crystal structures have been obtained for both gp26 [123] and gp9 [108, 109], and these were fitted into the cryo-EM map to find how the different proteins hold the complex together. The tail needle gp26 is folded as triple-stranded coiled coil. The gp9 head-binding domain has a β-helical domain, and the receptor-binding domain is a triangular β-prism (**Figure 7B**) [91]. The asymmetric cryo-EM structure of P22 (7.8 Å) allowed a detailed analysis of the interactions between gp9 and the gp4–gp10 interface by fitting the gp9 and gp26 crystal structures and models of gp1, gp4 and gp10 [92].

*SPP1 phage*. The *Siphoviridae* **SPP1** phage has the long flexible tail. A negative stain reconstruction of the adsorption apparatus revealed a hinge connection between the tip and the tail tube, allowing bending angles as high as 50° [85]. The gp24 protein keeps the tail closed before DNA ejection by forming a cap and is located between the tail and the tip. The first protein in the tip after the cap is gp21,

#### **Figure 7.**

*Adsorption apparatus. (A) The EM map (EMD-5051) of P22 coloured according to the constituent proteins. The PP (gp1), the gp4 12-mer, the gp10 6-mer, the gp9 tailspikes and the gp26 cell-puncturing needle are in purple, green, red, blue and brown, respectively. (B) The crystal structures for the N-terminal head-binding domain (1LKT) and the C-terminal receptor-binding domain (1TYU) of P22 gp9 docked into the cryo-EM density (EMDB-5051). (C) The receptor-binding carboxy-terminal domain of T5 tail fibre pb1 (5AQ5). The five different domain regions are labelled. The N- and C-termini are indicated. (D) The EM map (EMD-8868) of the TW1 tail showing the TP gp12 (in blue), gp15 (in green), gp16, gp18, gp27 (together in light brown) and the gp19 tailspikes (in purple). (E) The φ29 tailspike protein gp12 (3SUC). The four domains D1\*, D2, D3 and D4 are labelled. (F) The crystal structure (1 K28) of the cell-puncturing device of T4 (gp27 ± gp5\* ± gp5C)3. Each monomer within in the gp5 trimer is coloured in blue, green and orange, and each one in gp27 is coloured in magenta, red and cyan.*

and a structural similarity has suggested that SPP1 gp21 has a fold like the P22 tailspike [85]. When the P22 tailspike is fitted to the SPP1 density, the main domain of the P22 tailspike [124] occupies ~70% of the broad flattened area. The most remote protein in the tip is gp19.1, and the predicted secondary structure was structurally similar to the head-binding domain of the P22 tailspike [124]; and fitting three copies of the P22 trimer accounted for all the density in that region. During infection the tip is lost so DNA can pass into the cell and the cap remains in an open state.

*T5 phage*. The crystal structure of the **T5** DTP pb9 has shown that it has two domains. The A-domain has a barrel-like fold with structural similarity to the N-domains of other phage DTPs [118, 125, 126]. In spite of low sequence identity, these proteins form a hexameric ring that occupies the central core of the baseplate. The peripheral B-domain has an oligosaccharide-/oligonucleotide-binding (OB) fold [127]. The attachment of phage T5 to the host cell is assisted by three side tail fibres attached to the distal end of the tail [107, 127, 128], and they are homo-trimers of the pb1 (1396 aa). The trimeric structure of the receptor-binding carboxyterminal domain 970–1263(aa) was determined at 2.3 Å using X-ray crystallography [107] and could be divided into five different regions (**Figure 7C**) based on the structure of the P22 spike [124]. The N-terminal region (989-1009 aa) is shaped by the β-strands of the three monomers that wrap around each other to form a threefold beta-helix [124, 129]. The first "interdigitated" region (**ir1**) is followed by a triangular domain (1010–1129 aa) where three concave β sheets form a β-prism (**td1**). The second interdigitated region **ir2** (1130–1160 aa) also forms a short triple beta-helix. A second triangular domain **td2** (1161–1238 aa) is a β-prism like **td1**. At the distal end of the fibre, the third interdigitated region (aa1239–1263), **ir3**, forms a tapered triple-helical structure making the end of the structure pointed (**Figure 7C**). There is some similarity in the structure with the P22 tailspike [124] as both have a β-helical domain, an **ir** region, a triangular beta-prism domain and a second **ir** domain (called caudal fin). The triangular β-prism of P22 is the most similar to **td2** of pb1 and has the same topology.

*TW1 phage*. *TW1* has an unusual tail organisation for a siphophage, as a cryo-EM reconstruction of the tail (23.5 Å) [55] revealed six spikes on the distal end from the head. They are attached to the central tail tube, similar to the spikes seen in podophages P22 and Sf6 [120, 130] (**Figure 7D**). The TW1 gp19 tailspike (TS) protein is homologous to the TS protein of the podophage HK620 [131] so the crystal structure of the HK620 TS protein was fitted into the TW1 appendages. The TW1 gp19 TSs are thought to be attached to the phage via the DTP gp15 protein. However, the size of TW1 gp15 and the EM density suggest that this protein does not have a peripheral OB-fold domain as seen in the DTP of phage T5 [127]. Below gp15 are gp16 and gp18, which form the central tip of the phage tail (**Figure 7D**) and are similar to phage λ proteins gpL and gpJ, respectively [132]. At the tip of the tail is gp27 (**Figure 7D**) which is homologous to peptidoglycan-degrading enzymes. Many phages that infect Gram-negative bacteria have lysozyme-like proteins in their tails which enter the periplasm to digest the peptidoglycan barrier [119].

*φ29 phage*. The tail of *φ29* has 12 appendages, which are similar to the tailspikes of phage P22 and are attached to the bulge of densities close to the capsid [133]. Each appendage is a trimer of gp12\* (the cleavage product of gp12 which during maturation loses an 18 kDa C-terminal fragment). Although there is no sequence similarity with the P22 tailspike, the P22 tailspike domain structure gave a good fit into the peripheral component of the φ29 appendages [65, 124]. A construct of gp12 residues 89–854 was cleaved in vivo to give an N-terminal fragment (up to Ser691) and a C-terminal fragment (from Asp692) and crystal structures obtained for each [105]. They are both trimers, and the N-terminal part attaches to the virion and has three domains: D1\* is a coiled coil, D2 is mostly a β-helix and D3

**25**

*Bacteriophages: Their Structural Organisation and Function*

is also a β-helix (**Figure 7E**). The C-terminal domain D4 acts as a chaperone for trimer assembly and is cleaved by autocatalysis. The φ29 structure attached to the lipid bilayer has been obtained by cryo-ET (34 Å) [134]. The structure is comparable to cryo-EM structures of mature φ29 [56, 133, 135]. Tomographic reconstructions demonstrated the different stages of infection [134]. In the adsorption stage, the phage is tilted to the cell wall, and both the appendages and the tail seem to contact the cell surface. The tail tip protein helps the phage penetrate the cell wall. When it contacts the cytoplasmic membrane, a pore is created which allows the

*T4 phage*. The structure of the baseplate in *Myoviridae* is complex as illustrated by the **T4** phage. The sixfold symmetric baseplate is 270 Å long and about 520 Å in diameter at the base and is connected to the distal end of the tail [136, 137]. It is composed at least by 16 different proteins [137]. A star-shaped baseplate is formed by sequential binding of four different proteins to form a wedge shape [137]. Six wedges are arranged around the independently assembled hub. Finally, other proteins are added to form the complete baseplate. Once gp48 and gp54 have bound to the top of the central hub, polymerisation of the tail tube is initiated, and after gp25 has attached to them, then polymerisation of the sheath is initiated [138]. Crystal structures of these constituent proteins were fitted into EM structure, and this showed the location of the proteins [137]. Six long fibres and six short fibres are attached to the baseplate. The long fibres reversibly interact with the cell surface receptors [139]. After recognition, the baseplate comes closer to the cell surface allowing the six short tail fibres to bind irreversibly to the cell outer membrane. This process is accompanied by a large conformational change in the baseplate from a "high-energy" to a "low-energy" structure [93, 140]. This induces contraction of the tail sheath and allows the inner tail tube to pierce the outer host cell membrane and penetrate the inner membrane so that the genome is transferred directly to the

The structure of the T4 baseplate was assembled in vitro from gp10, gp7, gp8, gp6 and gp53, and the crystal structure was determined (4.2 Å) [141]. This indicated interesting differences compared to the structures when they are separately crystallised. However, about two-thirds of the structure was missing, but a cryo-EM structure of the same construct (3.8 Å) provided the positions of these missing parts [142]. The structures of T4 baseplate in its pre- and post-host attachment states were determined at 4.11 and 6.77 Å, respectively, by cryo-EM [111]. By combining high-resolution structures of the individual baseplate proteins, the authors were able to build a pseudoatomic model for the baseplate proteins. The crystal structure at 2.9 Å of the gp5–gp27 cell-puncturing device was fitted into the EM structure (**Figure 7F**) [143]. Positions of gp27, gp5C (the C-terminal β-helix domain of gp5) and gp5\* (the N-terminal OB-fold domain and the lysozyme middle domain) were identified. A monomeric protein gp5.4 caps the tip of the gp5 β-helix to sharpen the central spike [144]. During infection this spike punctures the cell membrane, and the lysozyme domain of gp5 digests the

*ε15 phage.* A 20 Å cryo-EM map of **ε15** showed six gp20 tailspikes extending out from one of the fivefold capsid vertices. Each tailspike is composed of two domains [89] and has slightly different orientations with respect to the capsid. Cryo-ET has been used to show the interaction of ε15 phage with the cell and to visualise the process of how ε15 infects its host *Salmonella anatum* [110]*.* Initially, the tailspikes attach to the host cell followed by the tail hub attaching to a putative cell receptor. A bowl-shaped density was observed beneath the tail hub at the beginning of infection. It was proposed that phage indents the host outer membrane looking for a secondary receptor or for puncturing the membrane. A tunnel is established

*DOI: http://dx.doi.org/10.5772/intechopen.85484*

genome to be injected into the cell.

host's cytoplasm.

peptidoglycan in the *E. coli* periplasm.

through the cell wall which allows the DNA to enter the cell.

*Bacteriophages - Perspectives and Future*

of pb1 and has the same topology.

and a structural similarity has suggested that SPP1 gp21 has a fold like the P22 tailspike [85]. When the P22 tailspike is fitted to the SPP1 density, the main domain of the P22 tailspike [124] occupies ~70% of the broad flattened area. The most remote protein in the tip is gp19.1, and the predicted secondary structure was structurally similar to the head-binding domain of the P22 tailspike [124]; and fitting three copies of the P22 trimer accounted for all the density in that region. During infection the tip is lost so DNA can pass into the cell and the cap remains in an open state. *T5 phage*. The crystal structure of the **T5** DTP pb9 has shown that it has two domains. The A-domain has a barrel-like fold with structural similarity to the N-domains of other phage DTPs [118, 125, 126]. In spite of low sequence identity, these proteins form a hexameric ring that occupies the central core of the baseplate. The peripheral B-domain has an oligosaccharide-/oligonucleotide-binding (OB) fold [127]. The attachment of phage T5 to the host cell is assisted by three side tail fibres attached to the distal end of the tail [107, 127, 128], and they are homo-trimers

of the pb1 (1396 aa). The trimeric structure of the receptor-binding carboxyterminal domain 970–1263(aa) was determined at 2.3 Å using X-ray crystallography [107] and could be divided into five different regions (**Figure 7C**) based on the structure of the P22 spike [124]. The N-terminal region (989-1009 aa) is shaped by the β-strands of the three monomers that wrap around each other to form a threefold beta-helix [124, 129]. The first "interdigitated" region (**ir1**) is followed by a triangular domain (1010–1129 aa) where three concave β sheets form a β-prism (**td1**). The second interdigitated region **ir2** (1130–1160 aa) also forms a short triple beta-helix. A second triangular domain **td2** (1161–1238 aa) is a β-prism like **td1**. At the distal end of the fibre, the third interdigitated region (aa1239–1263), **ir3**, forms a tapered triple-helical structure making the end of the structure pointed (**Figure 7C**). There is some similarity in the structure with the P22 tailspike [124] as both have a β-helical domain, an **ir** region, a triangular beta-prism domain and a second **ir** domain (called caudal fin). The triangular β-prism of P22 is the most similar to **td2**

*TW1 phage*. *TW1* has an unusual tail organisation for a siphophage, as a cryo-EM reconstruction of the tail (23.5 Å) [55] revealed six spikes on the distal end from the head. They are attached to the central tail tube, similar to the spikes seen in podophages P22 and Sf6 [120, 130] (**Figure 7D**). The TW1 gp19 tailspike (TS) protein is homologous to the TS protein of the podophage HK620 [131] so the crystal structure of the HK620 TS protein was fitted into the TW1 appendages. The TW1 gp19 TSs are thought to be attached to the phage via the DTP gp15 protein. However, the size of TW1 gp15 and the EM density suggest that this protein does not have a peripheral OB-fold domain as seen in the DTP of phage T5 [127]. Below gp15 are gp16 and gp18, which form the central tip of the phage tail (**Figure 7D**) and are similar to phage λ proteins gpL and gpJ, respectively [132]. At the tip of the tail is gp27 (**Figure 7D**) which is homologous to peptidoglycan-degrading enzymes. Many phages that infect Gram-negative bacteria have lysozyme-like proteins in their tails

*φ29 phage*. The tail of *φ29* has 12 appendages, which are similar to the tailspikes of phage P22 and are attached to the bulge of densities close to the capsid [133]. Each appendage is a trimer of gp12\* (the cleavage product of gp12 which during maturation loses an 18 kDa C-terminal fragment). Although there is no sequence similarity with the P22 tailspike, the P22 tailspike domain structure gave a good fit into the peripheral component of the φ29 appendages [65, 124]. A construct of gp12 residues 89–854 was cleaved in vivo to give an N-terminal fragment (up to Ser691) and a C-terminal fragment (from Asp692) and crystal structures obtained for each [105]. They are both trimers, and the N-terminal part attaches to the virion and has three domains: D1\* is a coiled coil, D2 is mostly a β-helix and D3

which enter the periplasm to digest the peptidoglycan barrier [119].

**24**

is also a β-helix (**Figure 7E**). The C-terminal domain D4 acts as a chaperone for trimer assembly and is cleaved by autocatalysis. The φ29 structure attached to the lipid bilayer has been obtained by cryo-ET (34 Å) [134]. The structure is comparable to cryo-EM structures of mature φ29 [56, 133, 135]. Tomographic reconstructions demonstrated the different stages of infection [134]. In the adsorption stage, the phage is tilted to the cell wall, and both the appendages and the tail seem to contact the cell surface. The tail tip protein helps the phage penetrate the cell wall. When it contacts the cytoplasmic membrane, a pore is created which allows the genome to be injected into the cell.

*T4 phage*. The structure of the baseplate in *Myoviridae* is complex as illustrated by the **T4** phage. The sixfold symmetric baseplate is 270 Å long and about 520 Å in diameter at the base and is connected to the distal end of the tail [136, 137]. It is composed at least by 16 different proteins [137]. A star-shaped baseplate is formed by sequential binding of four different proteins to form a wedge shape [137]. Six wedges are arranged around the independently assembled hub. Finally, other proteins are added to form the complete baseplate. Once gp48 and gp54 have bound to the top of the central hub, polymerisation of the tail tube is initiated, and after gp25 has attached to them, then polymerisation of the sheath is initiated [138]. Crystal structures of these constituent proteins were fitted into EM structure, and this showed the location of the proteins [137]. Six long fibres and six short fibres are attached to the baseplate. The long fibres reversibly interact with the cell surface receptors [139]. After recognition, the baseplate comes closer to the cell surface allowing the six short tail fibres to bind irreversibly to the cell outer membrane. This process is accompanied by a large conformational change in the baseplate from a "high-energy" to a "low-energy" structure [93, 140]. This induces contraction of the tail sheath and allows the inner tail tube to pierce the outer host cell membrane and penetrate the inner membrane so that the genome is transferred directly to the host's cytoplasm.

The structure of the T4 baseplate was assembled in vitro from gp10, gp7, gp8, gp6 and gp53, and the crystal structure was determined (4.2 Å) [141]. This indicated interesting differences compared to the structures when they are separately crystallised. However, about two-thirds of the structure was missing, but a cryo-EM structure of the same construct (3.8 Å) provided the positions of these missing parts [142]. The structures of T4 baseplate in its pre- and post-host attachment states were determined at 4.11 and 6.77 Å, respectively, by cryo-EM [111]. By combining high-resolution structures of the individual baseplate proteins, the authors were able to build a pseudoatomic model for the baseplate proteins. The crystal structure at 2.9 Å of the gp5–gp27 cell-puncturing device was fitted into the EM structure (**Figure 7F**) [143]. Positions of gp27, gp5C (the C-terminal β-helix domain of gp5) and gp5\* (the N-terminal OB-fold domain and the lysozyme middle domain) were identified. A monomeric protein gp5.4 caps the tip of the gp5 β-helix to sharpen the central spike [144]. During infection this spike punctures the cell membrane, and the lysozyme domain of gp5 digests the peptidoglycan in the *E. coli* periplasm.

*ε15 phage.* A 20 Å cryo-EM map of **ε15** showed six gp20 tailspikes extending out from one of the fivefold capsid vertices. Each tailspike is composed of two domains [89] and has slightly different orientations with respect to the capsid. Cryo-ET has been used to show the interaction of ε15 phage with the cell and to visualise the process of how ε15 infects its host *Salmonella anatum* [110]*.* Initially, the tailspikes attach to the host cell followed by the tail hub attaching to a putative cell receptor. A bowl-shaped density was observed beneath the tail hub at the beginning of infection. It was proposed that phage indents the host outer membrane looking for a secondary receptor or for puncturing the membrane. A tunnel is established through the cell wall which allows the DNA to enter the cell.

#### **5. Conclusions**

Structural studies of the currently known tailed phages have shown a common organisation, which implies that they have a single ancestor and diversity has arisen through evolution [37]. All phages have a similar pathway of self-assembly: a procapsid formed with the help of a SP (or sometimes a scaffolding domain); conformational changes induced by release of the SP create a space for the DNA, and assisted by DNA terminases, the genome is packaged into the procapsid. This step is typically named as the maturation of the capsid. The tail is then attached or assembled on the capsid to form the infectious virion. The MCPs are characterised by the HK97 capsid protein fold. However, phages have a very low sequence similarity, which leads to differences in how the capsid stability is arranged to withstand the high inner pressure of the genome. In some phages like HK97 and SPP1, the interactions between capsid proteins are strong and hold the capsid intact. In many phages the process of capsid maturation is linked to attachment of additional proteins that are named as auxiliary or decoration proteins. They are often essential to enhance the capsid stability. The HK97 capsid is held together by chain mail covalent links between the MCPs; in SPP1 and T5, the decoration proteins enhance stability of the capsid, but in λ, T4 and ε15 phages, these proteins are essential for keeping DNA inside the capsid [19, 52, 53].

The HTIs play an important role in all tailed phages as they provide a channel for DNA to enter and exit the capsid and at the same time provide a covalent connection to either the preassembled tails or tails assembled on the capsid. They all contain a dodecameric PP positioned within the capsid at one of the fivefold vertices and that acts as a gatekeeper holding the DNA within the capsid even in very harsh environments. Like the capsid proteins, the PPs have a common fold with the conserved elements being involved in interactions with DNA [145]. They have mostly α-helical domains in their central part and β-layers in the wing domains that interact with the capsid to fix the PP position. Head completion proteins below the PP also have similar folds to each other.

A much higher level of divergence is reflected in phage tail structures. The most common feature in all long-tailed phages is a central tube with a large number (30–40) of three- or sixfold circular rings of the major TPs. There is structural similarity between these major TPs: they have a similar fold of a β-sandwich flanked by alpha-helices and loops that provide links between adjacent rings. The helical tails have a typical rise of about 40 Å and rotation of around 20° between adjacent rings. Some tails also have appendages, which appear to have an immunoglobulinlike fold. Very little is known about the organisation of tail sheaths that have some similarities with type VI secretion systems, but sometimes they have extra appendages like immunoglobulin domains to help phages recognise their host cells. There is also some structural similarity of the TP with the tail terminator proteins and proteins in the T4 sheath.

Even higher diversity is found in the adsorption apparatus which are responsible for the recognition of the host cells and signalling the opening of the gate for the genome release. The tip of phage SPP1 recognises its receptor; induces the tail to be attached to the outer membrane of the host cell after disconnection of the tip. At the same time this interaction generates a signal that open the PP gate keeper. The T4 phage has a significantly more complex system of a baseplate which undergoes several steps of complex conformational changes.

Interestingly, the receptor-binding proteins also a have similar organisation: they are all trimers, usually intertwined with β-helical regions, and use their N-terminal domain to bind to the phage. Spikes and fibres are also found in many phages. However, the number of spikes or fibres varies significantly between phages. Podophages have

**27**

*Bacteriophages: Their Structural Organisation and Function*

trimeric tailspikes to recognise the specific host cell for infection. Like other phage components, they vary from six fibres in phage T7 to 12 in phage φ29, but they all have a β-helical fold. The fibres can have different roles within a phage, for instance, T4 has six long fibres that serve as host recognition and six short fibres which then

Antibiotics (especially of the broad-spectrum type) are very effective at killing infectious bacteria; however, they kill typically multiple bacterial species indiscriminately, thus destroying beneficial bacteria of the host microbiome as well. Since phages are specific to one species of bacteria, they are unlikely to perturb microbiome bacterial species. Current problems with antibiotic resistance require new approaches, and here phages can be used [12]. For medicinal purposes it is necessary to design a phage that will recognise the specific bacteria we want to eliminate [146]. Phages can be modified for high specificity in the recognition of pathogens. The high level of phage specificity is based on recognition of receptor characteristic for a given type of bacteria which is where the differences in the adsorption systems of different phages play a crucial role. The important task in studying phages is to find those that are able to kill only antibiotic-resistant bacteria. Here, the lytic phages are of most interest, since rather than stopping bacteria from producing a certain type of protein that will slow down the bacterium proliferation, like in the case of antibiotics, these phages destroy the bacteria's cell wall and cell membrane completely. In addition, many bacteria develop biofilm—a thick layer of viscous materials that protect them from antibiotics. Some phages are equipped with tools that can digest this biofilm [147]. There are some problems with phages, since they are easy to use for topical applications, but often specific medications have to be administered internally. For phages to be used for delivery of drugs, they need to be more precise in their action. Consequently, we need to modify them so that the infectivity will be efficient by replacing the genome with DNA encoding specific enzymes and the adsorption apparatus made more effective. To develop these medical approaches, we need to know the phage organisation and the interactions between protein components at the atomic level. To achieve this hybrid, methods should be used including structural biology, biochemistry and microbiology [21].

The authors are grateful to Dr. D. Houldershaw and Mr. Y. Goudetsidis for their computer support. The authors apologise for the incomplete coverage of known phage structures and have drawn on a limited subset, owing to space constraints.

*DOI: http://dx.doi.org/10.5772/intechopen.85484*

extend and bind to the cell.

**Acknowledgements**

**Conflict of interest**

The authors declare no conflict of interest.

#### *Bacteriophages: Their Structural Organisation and Function DOI: http://dx.doi.org/10.5772/intechopen.85484*

*Bacteriophages - Perspectives and Future*

keeping DNA inside the capsid [19, 52, 53].

the PP also have similar folds to each other.

several steps of complex conformational changes.

proteins in the T4 sheath.

Structural studies of the currently known tailed phages have shown a common organisation, which implies that they have a single ancestor and diversity has arisen through evolution [37]. All phages have a similar pathway of self-assembly: a procapsid formed with the help of a SP (or sometimes a scaffolding domain); conformational changes induced by release of the SP create a space for the DNA, and assisted by DNA terminases, the genome is packaged into the procapsid. This step is typically named as the maturation of the capsid. The tail is then attached or assembled on the capsid to form the infectious virion. The MCPs are characterised by the HK97 capsid protein fold. However, phages have a very low sequence similarity, which leads to differences in how the capsid stability is arranged to withstand the high inner pressure of the genome. In some phages like HK97 and SPP1, the interactions between capsid proteins are strong and hold the capsid intact. In many phages the process of capsid maturation is linked to attachment of additional proteins that are named as auxiliary or decoration proteins. They are often essential to enhance the capsid stability. The HK97 capsid is held together by chain mail covalent links between the MCPs; in SPP1 and T5, the decoration proteins enhance stability of the capsid, but in λ, T4 and ε15 phages, these proteins are essential for

The HTIs play an important role in all tailed phages as they provide a channel for DNA to enter and exit the capsid and at the same time provide a covalent connection to either the preassembled tails or tails assembled on the capsid. They all contain a dodecameric PP positioned within the capsid at one of the fivefold vertices and that acts as a gatekeeper holding the DNA within the capsid even in very harsh environments. Like the capsid proteins, the PPs have a common fold with the conserved elements being involved in interactions with DNA [145]. They have mostly α-helical domains in their central part and β-layers in the wing domains that interact with the capsid to fix the PP position. Head completion proteins below

A much higher level of divergence is reflected in phage tail structures. The most

Even higher diversity is found in the adsorption apparatus which are responsible for the recognition of the host cells and signalling the opening of the gate for the genome release. The tip of phage SPP1 recognises its receptor; induces the tail to be attached to the outer membrane of the host cell after disconnection of the tip. At the same time this interaction generates a signal that open the PP gate keeper. The T4 phage has a significantly more complex system of a baseplate which undergoes

Interestingly, the receptor-binding proteins also a have similar organisation: they are all trimers, usually intertwined with β-helical regions, and use their N-terminal domain

the number of spikes or fibres varies significantly between phages. Podophages have

to bind to the phage. Spikes and fibres are also found in many phages. However,

common feature in all long-tailed phages is a central tube with a large number (30–40) of three- or sixfold circular rings of the major TPs. There is structural similarity between these major TPs: they have a similar fold of a β-sandwich flanked by alpha-helices and loops that provide links between adjacent rings. The helical tails have a typical rise of about 40 Å and rotation of around 20° between adjacent rings. Some tails also have appendages, which appear to have an immunoglobulinlike fold. Very little is known about the organisation of tail sheaths that have some similarities with type VI secretion systems, but sometimes they have extra appendages like immunoglobulin domains to help phages recognise their host cells. There is also some structural similarity of the TP with the tail terminator proteins and

**5. Conclusions**

**26**

trimeric tailspikes to recognise the specific host cell for infection. Like other phage components, they vary from six fibres in phage T7 to 12 in phage φ29, but they all have a β-helical fold. The fibres can have different roles within a phage, for instance, T4 has six long fibres that serve as host recognition and six short fibres which then extend and bind to the cell.

Antibiotics (especially of the broad-spectrum type) are very effective at killing infectious bacteria; however, they kill typically multiple bacterial species indiscriminately, thus destroying beneficial bacteria of the host microbiome as well. Since phages are specific to one species of bacteria, they are unlikely to perturb microbiome bacterial species. Current problems with antibiotic resistance require new approaches, and here phages can be used [12]. For medicinal purposes it is necessary to design a phage that will recognise the specific bacteria we want to eliminate [146]. Phages can be modified for high specificity in the recognition of pathogens. The high level of phage specificity is based on recognition of receptor characteristic for a given type of bacteria which is where the differences in the adsorption systems of different phages play a crucial role. The important task in studying phages is to find those that are able to kill only antibiotic-resistant bacteria. Here, the lytic phages are of most interest, since rather than stopping bacteria from producing a certain type of protein that will slow down the bacterium proliferation, like in the case of antibiotics, these phages destroy the bacteria's cell wall and cell membrane completely. In addition, many bacteria develop biofilm—a thick layer of viscous materials that protect them from antibiotics. Some phages are equipped with tools that can digest this biofilm [147]. There are some problems with phages, since they are easy to use for topical applications, but often specific medications have to be administered internally. For phages to be used for delivery of drugs, they need to be more precise in their action. Consequently, we need to modify them so that the infectivity will be efficient by replacing the genome with DNA encoding specific enzymes and the adsorption apparatus made more effective. To develop these medical approaches, we need to know the phage organisation and the interactions between protein components at the atomic level. To achieve this hybrid, methods should be used including structural biology, biochemistry and microbiology [21].

#### **Acknowledgements**

The authors are grateful to Dr. D. Houldershaw and Mr. Y. Goudetsidis for their computer support. The authors apologise for the incomplete coverage of known phage structures and have drawn on a limited subset, owing to space constraints.

### **Conflict of interest**

The authors declare no conflict of interest.

*Bacteriophages - Perspectives and Future*

#### **Author details**

Helen E. White and Elena V. Orlova\* Institute of Structural and Molecular Biology, Birkbeck College, London, United Kingdom

\*Address all correspondence to: e.orlova@mail.cryst.bbk.ac.uk

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**29**

*Bacteriophages: Their Structural Organisation and Function*

phage therapy. Advances in Colloid and Interface Science. 2017;**249**:100-133

prokaryotic viruses. FEMS Microbiology

[12] Criscuolo E, Spadini S, Lamanna J, Ferro M, Burioni R. Bacteriophages

[14] Orlova EV. Bacteriophages and Their Structural Organisation. In: Kurtböke İ, editor. Bacteriophages. IntechOpen; 2012. DOI: 10.5772/1065. ISBN:

[15] Ackermann HW. Classification of bacteriophages. In: Calendar R, editor. The Bacteriophages. New York, USA: Oxford University Press; 2006. pp. 8-16.

[16] Drenth J. Principles of Protein X-Ray Crystallography. New York:

Orlova EV. The ribosome and its role in protein folding: Looking through a magnifying glass. Acta Crystallographica. Section D, Structural Biology. 2017;**73**(Pt 6):509-521. DOI:

10.1107/S2059798317007446

[17] Javed A, Christodoulou J, Cabrita LD,

[18] Grimes JM, Burroughs JN, Gouet P, Diprose JM, Malby R, Ziéntara S, et al. The atomic structure of the bluetongue virus core. Nature. 1998;**395**:470-478

[11] Weinbauer MG. Ecology of

Reviews. 2004;**28**:127-181

and their immunological applications against infectious threats. Journal of Immunology Research. 2017;**2017**:3780697. Published online: Apr 6, 2017. DOI:

10.1155/2017/3780697

2014;**22**:6334-6344

978-953-51-0272-4

ISBN: 0-19-514850-9

Springer-Verlag; 2007

[13] Pietilä MK, Demina TA, Atanasova NS, Oksanen HM, Bamford DH. Archaeal viruses and bacteriophages: Comparisons and contrasts. Trends in Microbiology .

*DOI: http://dx.doi.org/10.5772/intechopen.85484*

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[4] Breitbart M. Marine viruses: Truth or dare. Annual Review of Marine Science.

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**Author details**

United Kingdom

provided the original work is properly cited.

Helen E. White and Elena V. Orlova\*

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Institute of Structural and Molecular Biology, Birkbeck College, London,

\*Address all correspondence to: e.orlova@mail.cryst.bbk.ac.uk

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*Bacteriophages - Perspectives and Future*

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str.2018.04.004

bs.mie.2016.04.014

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biomolecular complexes by combination

Current Opinion in Structural Biology. 2017;**43**:104-113. DOI: 10.1016/j.

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crystallography, and NMR. Journal of Structural and Functional Genomics.

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[118] Veesler D, Robin G, Lichière J, Auzat I, Tavares P, Bron P, et al. Crystal structure of bacteriophage SPP1 distal tail protein (gp19.1): A baseplate hub paradigm in Gram-positive infecting phages. The Journal of Biological Chemistry. 2010;**285**:36666-36673

[119] Xiang Y, Morais MC, Cohen DN, Bowman VD, Anderson DL, Rossmann MG. Crystal and cryoEM structural studies of a cell wall degrading enzyme in the bacteriophage phi29 tail. Proceedings of the National Academy of Sciences of the United States of America. 2008;**105**(28):9552-9557. DOI: 10.1073/pnas.0803787105

[120] Tang L, Marion WR, Cingolani G, Prevelige PE, Johnson JE. Threedimensional structure of the bacteriophage P22 tail machine. The EMBO Journal. 2005;**24**:2087-2095

[121] Chang J, Weigele P, King J, Chiu W, Jiang W. Cryo-EM asymmetric reconstruction of bacteriophage P22 reveals organization of its DNA packaging and infecting machinery. Structure. 2006;**14**(6):1073-1082

**37**

*Bacteriophages: Their Structural Organisation and Function*

[129] Mitraki A, Papanikolopoulou K, van Raaij MJ. Natural triple β-stranded fibrous folds. Advances in Protein Chemistry. 2006;**73**:97-124

[130] Parent KN, Gilcrease EB, Casjens SR, Baker TS. Structural evolution of the P22-like phages: comparison of Sf6 and P22 procapsid and virion architectures.

[131] Barbirz S, Muller JJ, Uetrecht C, Clark AJ, Heinemann U, Seckler R. Crystal structure of Escherichia coli phage HK620 tailspike: Podoviral tailspike endoglycosidase modules are evolutionarily related. Molecular Microbiology. 2008;**69**:303-316

[132] Rajagopala SV, Casjens S, Uetz P. The protein interaction map of bacteriophage lambda. BMC Microbiology. 2011;**11**:213

[133] Morais MC, Tao Y, Olson NH, Grimes S, Jardine PJ, Anderson DL, et al. Cryoelectron-microscopy image reconstruction of symmetry mismatches

in bacteriophage φ29. Journal of Structural Biology. 2001;**135**:38-46

[134] Farley MM, Tu J, Kearns DB, Molineux IJ, Liu J. Ultrastructural analysis of bacteriophage Φ29 during infection of Bacillus subtilis. Journal of Structural Biology. 2017;**197**(2):163-171.

DOI: 10.1016/j.jsb.2016.07.019

[135] Tao Y, Olson NH, Xu W,

[136] Leiman PG, Kanamaru S,

Kostyuchenko VA, Aksyuk AA, Kanamaru S, et al. Morphogenesis

Anderson DL, Rossmann MG, Baker TS. Assembly of a tailed bacterial virus and its genome release studied in three dimensions. Cell. 1998;**95**:431-437

Mesyanzhinov VV, Arisaka F, Rossmann MG. Structure and morphogenesis of bacteriophage T4. Cellular and Molecular Life Sciences. 2003;**60**(11):2356-2370

[137] Leiman PG, Arisaka F, van Raaij MJ,

Virology. 2012;**427**:177-188

*DOI: http://dx.doi.org/10.5772/intechopen.85484*

[122] Tang L, Gilcrease EB, Casjens SR, Johnson JE. Highly discriminatory binding of capsid-cementing proteins

[123] Olia AS, Casjens S, Cingolani G.

envelope-penetrating needle. Nature Structural & Molecular Biology.

[124] Steinbacher S, Seckler R, Miller S,

Steipe B, Huber R, Reinemer P. Crystal structure of P22 tailspike protein: Interdigitated subunits in a thermostable trimer. Science.

[125] Sciara G, Bebeacua C, Bron P, Tremblay D, Ortiz-Lombardia M, Lichière J, et al. Structure of lactococcal phage p2 baseplate and its mechanism of activation. Proceedings of the National Academy of Sciences of the United States of America.

[126] Veesler D, Spinelli S, Mahony J, Lichière J, Blangy S, Bricogne G, et al. Structure of the phage TP901-1

alternative host adhesion mechanism. Proceedings of the National Academy of Sciences of the United States of America. 2012;**109**:8954-8958. DOI: 10.1073/pnas.1200966109

[127] Flayhan A, Vellieux FM, Lurz R, Maury O, Contreras-Martel C, Girard E, et al. Crystal structure of pb9, the distal tail protein of bacteriophage T5: A conserved structural motif among all siphophages. Journal of Virology.

[128] Zivanovic Y, Confalonieri F, Ponchon L, Lurz R, Chami M, Flayhan A,

et al. Insights into bacteriophage T5 structure from analysis of its morphogenesis genes and protein components. Journal of Virology.

1.8MDabaseplate suggests an

in bacteriophage L. Structure.

Structure of phage P22 cell

2007;**14**(12):1221-1226

1994;**265**:383-386

2010;**107**:6852-6857

2014;**88**:820-828

2014;**88**:1162-1174

2006;**14**(5):837-845

*Bacteriophages: Their Structural Organisation and Function DOI: http://dx.doi.org/10.5772/intechopen.85484*

[122] Tang L, Gilcrease EB, Casjens SR, Johnson JE. Highly discriminatory binding of capsid-cementing proteins in bacteriophage L. Structure. 2006;**14**(5):837-845

*Bacteriophages - Perspectives and Future*

[108] Steinbacher S, Baxa U, Miller S, Weintraub A, Seckler R, Huber R. Crystal structure of phage P22 tailspike protein complexed with Salmonella sp. O-antigen receptors. Proceedings of the National Academy of Sciences of the United States of America. 1996;**93**(20):10584-10588

[115] Aksyuk AA, Kurochkina LP, Fokine A, Forouhar F, Mesyanzhinov

conservation of the Myoviridae phage tail sheath protein fold. Structure.

common evolutionary origin for tailed bacteriophage functional modules and bacterial machineries. Microbiology and Molecular Biology Reviews.

[117] Davidson AR, Cardarelli L, Pell LG,

Long noncontractile tail machines of bacteriophages. Advances in Experimental Medicine and Biology. 2012;**726**:115-142. DOI: 10.1007/978-1-4614-0980-9\_6

[118] Veesler D, Robin G, Lichière J, Auzat I, Tavares P, Bron P, et al. Crystal structure of bacteriophage SPP1 distal tail protein (gp19.1): A baseplate hub paradigm in Gram-positive infecting phages. The Journal of Biological Chemistry. 2010;**285**:36666-36673

[119] Xiang Y, Morais MC, Cohen DN, Bowman VD, Anderson DL, Rossmann MG. Crystal and cryoEM structural studies of a cell wall degrading enzyme in the bacteriophage phi29 tail. Proceedings of the National Academy of Sciences of the United States of America.

2008;**105**(28):9552-9557. DOI: 10.1073/pnas.0803787105

[120] Tang L, Marion WR, Cingolani G, Prevelige PE, Johnson JE. Threedimensional structure of the

bacteriophage P22 tail machine. The EMBO Journal. 2005;**24**:2087-2095

Jiang W. Cryo-EM asymmetric reconstruction of bacteriophage P22 reveals organization of its DNA packaging and infecting machinery. Structure. 2006;**14**(6):1073-1082

[121] Chang J, Weigele P, King J, Chiu W,

VV, Tong L, et al. Structural

[116] Veesler D, Cambillau C. A

Radford DR, Maxwell KL.

2011;**19**:1885-1894

2011;**75**:423-433

[109] Steinbacher S, Miller S, Baxa U, Budisa N, Weintraub A, Seckler R, et al. Phage P22 tailspike protein: crystal structure of the head-binding domain at 2.3 Å, fully refined structure of the endorhamnosidase at 1.56 Å resolution, and the molecular basis of O-antigen recognition and cleavage. Journal of Molecular Biology.

[110] Chang JT, Schmid MF, Haase-Pettingell C, Weigele PR, King JA, Chiu W. Visualizing the structural changes of bacteriophage Epsilon15 and its Salmonella host during

infection. Journal of Molecular Biology. 2010;**402**(4):731-740. DOI: 10.1016/j.

[111] Taylor NM, Prokhorov NS, Guerrero-Ferreira RC, Shneider MM, Browning C, Goldie KN, et al. Structure of the T4 baseplate and its function in triggering sheath contraction. Nature.

[112] Zheng W, Wang F, Taylor NMI, Guerrero-Ferreira RC, Leiman PG, Egelman EE. Refined Cryo-EM structure of the T4 tail tube: Exploring the lowest dose limit. Structure. 2017;**25**:1436-1441

[113] Fokine A, Zhang Z, Kanamaru S, Bowman VD, Aksyuk AA, Arisaka F, et al. The molecular architecture of the bacteriophage T4 neck. Journal of Molecular Biology. 2013;**425**:1731-1744

[114] Aksyuk AA, Leiman PG, Shneider MM, Mesyanzhinov VV, Rossmann MG. The structure of gene product 6 of bacteriophage T4, the hinge-pin of the baseplate. Structure. 2009;**17**:800-808

1997;**267**:865-880

jmb.2010.07.058

2016;**533**:346-352

**36**

[123] Olia AS, Casjens S, Cingolani G. Structure of phage P22 cell envelope-penetrating needle. Nature Structural & Molecular Biology. 2007;**14**(12):1221-1226

[124] Steinbacher S, Seckler R, Miller S, Steipe B, Huber R, Reinemer P. Crystal structure of P22 tailspike protein: Interdigitated subunits in a thermostable trimer. Science. 1994;**265**:383-386

[125] Sciara G, Bebeacua C, Bron P, Tremblay D, Ortiz-Lombardia M, Lichière J, et al. Structure of lactococcal phage p2 baseplate and its mechanism of activation. Proceedings of the National Academy of Sciences of the United States of America. 2010;**107**:6852-6857

[126] Veesler D, Spinelli S, Mahony J, Lichière J, Blangy S, Bricogne G, et al. Structure of the phage TP901-1 1.8MDabaseplate suggests an alternative host adhesion mechanism. Proceedings of the National Academy of Sciences of the United States of America. 2012;**109**:8954-8958. DOI: 10.1073/pnas.1200966109

[127] Flayhan A, Vellieux FM, Lurz R, Maury O, Contreras-Martel C, Girard E, et al. Crystal structure of pb9, the distal tail protein of bacteriophage T5: A conserved structural motif among all siphophages. Journal of Virology. 2014;**88**:820-828

[128] Zivanovic Y, Confalonieri F, Ponchon L, Lurz R, Chami M, Flayhan A, et al. Insights into bacteriophage T5 structure from analysis of its morphogenesis genes and protein components. Journal of Virology. 2014;**88**:1162-1174

[129] Mitraki A, Papanikolopoulou K, van Raaij MJ. Natural triple β-stranded fibrous folds. Advances in Protein Chemistry. 2006;**73**:97-124

[130] Parent KN, Gilcrease EB, Casjens SR, Baker TS. Structural evolution of the P22-like phages: comparison of Sf6 and P22 procapsid and virion architectures. Virology. 2012;**427**:177-188

[131] Barbirz S, Muller JJ, Uetrecht C, Clark AJ, Heinemann U, Seckler R. Crystal structure of Escherichia coli phage HK620 tailspike: Podoviral tailspike endoglycosidase modules are evolutionarily related. Molecular Microbiology. 2008;**69**:303-316

[132] Rajagopala SV, Casjens S, Uetz P. The protein interaction map of bacteriophage lambda. BMC Microbiology. 2011;**11**:213

[133] Morais MC, Tao Y, Olson NH, Grimes S, Jardine PJ, Anderson DL, et al. Cryoelectron-microscopy image reconstruction of symmetry mismatches in bacteriophage φ29. Journal of Structural Biology. 2001;**135**:38-46

[134] Farley MM, Tu J, Kearns DB, Molineux IJ, Liu J. Ultrastructural analysis of bacteriophage Φ29 during infection of Bacillus subtilis. Journal of Structural Biology. 2017;**197**(2):163-171. DOI: 10.1016/j.jsb.2016.07.019

[135] Tao Y, Olson NH, Xu W, Anderson DL, Rossmann MG, Baker TS. Assembly of a tailed bacterial virus and its genome release studied in three dimensions. Cell. 1998;**95**:431-437

[136] Leiman PG, Kanamaru S, Mesyanzhinov VV, Arisaka F, Rossmann MG. Structure and morphogenesis of bacteriophage T4. Cellular and Molecular Life Sciences. 2003;**60**(11):2356-2370

[137] Leiman PG, Arisaka F, van Raaij MJ, Kostyuchenko VA, Aksyuk AA, Kanamaru S, et al. Morphogenesis

of the T4 tail and tail fibers. Virology Journal. 2010;**7**:355. DOI: 10.1186/1743-422X-7-355

[138] Arisaka F, Yap ML, Kanamaru S, Rossmann MG. Molecular assembly and structure of the bacteriophage T4 tail. Biophysical Reviews. 2016;**8**(4):385-396. DOI: 10.1007/s12551-016-0230-x

[139] Crawford JT, Goldberg EB. The function of tail fibers in triggering baseplate expansion of bacteriophage T4. Journal of Molecular Biology. 1980;**139**(4):679-690

[140] Kostyuchenko VA, Leiman PG, Chipman PR, Kanamaru S, van Raaij MJ, Arisaka F, et al. Three-dimensional structure of bacteriophage T4 baseplate. Nature Structural Biology. 2003;**10**(9):688-693

[141] Yap ML, Rossmann MG. Structure and function of bacteriophage T4. Future Microbiology. 2014;**9**(12): 1319-1327. DOI: 10.2217/fmb.14.91

[142] Yap ML, Klose T, Arisaka F, Speirc JA, Veeslerc D, Fokine A, et al. The role of bacteriophage T4 baseplate in regulating assembly and infection. Proceedings of the National Academy of Sciences of the United States of America. 2016;**113**(10):2654-2659

[143] Kanamaru S, Leiman PG, Kostyuchenko VA, Chipman PR, Mesyanzhinov VV, Arisaka F, et al. Structure of the cell-puncturing device of bacteriophage T4. Nature. 2002;**415**:553-557. DOI: 10.1038/415553a

[144] Shneider MM, Buth SA, Ho BT, Basler M, Mekalanos JJ, Leiman PG. PAAR-repeat proteins sharpen and diversify the type VI secretion system spike. Nature. 2013;**500**:350-353. DOI: 10.1038/nature1245

[145] Tavares P, Zinn-Justin S, Orlova EV. Genome gating in tailed bacteriophage capsids. Advances in Experimental Medicine and Biology. 2012;**726**:585-600. DOI: 10.1007/978- 1-4614-0980-9\_25. [Review. PMID: 22297531]

[146] Ando H, Lemire S, Pires DP, Lu TK. Engineering modular viral scaffolds for targeted bacterial population editing. Cell Systems. 2015;**1**(3):187-196

[147] Abedon ST. Ecology of anti-biofilm agents II: Bacteriophage exploitation and biocontrol of biofilm bacteria. Pharmaceuticals (Basel). 2015;**8**(3): 559-589. DOI: 10.3390/ph8030559

**39**

**Chapter 3**

Race

**Abstract**

*Vitor B. Pinheiro*

solely by researcher ingenuity.

**1. Introduction**

readily available.

tion and release [3].

Biotechnology Tools Derived from

The long association and intense competition between bacteria and their viruses

The relationship between bacteriophages and bacteria is often explained in terms of an arms race: each 'developing' measures and countermeasures for attacking and defending itself from the other [1, 2]. The imagery of an arms race is a powerful metaphor to summarise the relationship between possibility and availability that have constrained the emergence, evolution and diversification of life on Earth. Life is limited by what is possible. For instance, life can only exist because chemical information storage is possible. On the other hand, life as we know it has evolved around DNA and RNA because they are informational molecules that could function in the environment of the early Earth and whose building blocks were

The relationship between bacteria and bacteriophages is similarly constrained. The emergence (or availability) of bacterial cells capable of establishing a rich internal environment (compared to the outside of the cell) creates the possibility for other organisms to evolve predatory or parasitic survival strategies, including bacteriophages. Once phages emerge, they alter the dynamics of the ecological niche and create an advantage to bacterial hosts that can reduce the success of phage infection—whether by hindering phage access to the cell cytoplasm, by interfering with phage survival or replication in the cell or by interfering with phage matura-

Bacterial defences arise from any function already available in the host (e.g. uracil-DNA glycosylase involved in DNA repair) or that can be co-opted from available genetic resources in the cell or in the environment

have created a fertile ground for evolution to develop numerous tools for DNA modification, assembly and degradation. Many of these tools underpin the past 50 years of molecular biology, and others show great potential in shaping the next 50 years of the field. Here, I present some of the tools that have come out of the bacteria-bacteriophage arms race and discuss some of the concepts that may shape their future use. Molecular biology remains a fast-growing area increasingly limited

**Keywords:** molecular biology tools, orthogonality, DNA modifications

the Bacteriophage/Bacteria Arms

#### **Chapter 3**

*Bacteriophages - Perspectives and Future*

[138] Arisaka F, Yap ML, Kanamaru S, Rossmann MG. Molecular assembly and structure of the bacteriophage T4 tail. Biophysical Reviews. 2016;**8**(4):385-396. bacteriophage capsids. Advances in Experimental Medicine and Biology. 2012;**726**:585-600. DOI: 10.1007/978- 1-4614-0980-9\_25. [Review. PMID:

[146] Ando H, Lemire S, Pires DP, Lu TK. Engineering modular viral scaffolds for targeted bacterial population editing. Cell Systems. 2015;**1**(3):187-196

[147] Abedon ST. Ecology of anti-biofilm agents II: Bacteriophage exploitation and biocontrol of biofilm bacteria. Pharmaceuticals (Basel). 2015;**8**(3): 559-589. DOI: 10.3390/ph8030559

22297531]

DOI: 10.1007/s12551-016-0230-x

[139] Crawford JT, Goldberg EB. The function of tail fibers in triggering baseplate expansion of bacteriophage T4. Journal of Molecular Biology.

[140] Kostyuchenko VA, Leiman PG, Chipman PR, Kanamaru S, van Raaij MJ, Arisaka F, et al. Three-dimensional structure of bacteriophage T4 baseplate. Nature Structural Biology.

[141] Yap ML, Rossmann MG. Structure and function of bacteriophage T4. Future Microbiology. 2014;**9**(12): 1319-1327. DOI: 10.2217/fmb.14.91

[142] Yap ML, Klose T, Arisaka F, Speirc JA, Veeslerc D, Fokine A, et al. The role of bacteriophage T4 baseplate in regulating assembly and infection. Proceedings of the National Academy of Sciences of the United States of America. 2016;**113**(10):2654-2659

[143] Kanamaru S, Leiman PG, Kostyuchenko VA, Chipman PR, Mesyanzhinov VV, Arisaka F, et al. Structure of the cell-puncturing device of bacteriophage T4. Nature. 2002;**415**:553-557. DOI:

[144] Shneider MM, Buth SA, Ho BT, Basler M, Mekalanos JJ, Leiman PG. PAAR-repeat proteins sharpen and diversify the type VI secretion system spike. Nature. 2013;**500**:350-353. DOI:

10.1038/415553a

10.1038/nature1245

[145] Tavares P, Zinn-Justin S, Orlova EV. Genome gating in tailed

of the T4 tail and tail fibers. Virology Journal. 2010;**7**:355. DOI:

10.1186/1743-422X-7-355

1980;**139**(4):679-690

2003;**10**(9):688-693

**38**

## Biotechnology Tools Derived from the Bacteriophage/Bacteria Arms Race

*Vitor B. Pinheiro*

#### **Abstract**

The long association and intense competition between bacteria and their viruses have created a fertile ground for evolution to develop numerous tools for DNA modification, assembly and degradation. Many of these tools underpin the past 50 years of molecular biology, and others show great potential in shaping the next 50 years of the field. Here, I present some of the tools that have come out of the bacteria-bacteriophage arms race and discuss some of the concepts that may shape their future use. Molecular biology remains a fast-growing area increasingly limited solely by researcher ingenuity.

**Keywords:** molecular biology tools, orthogonality, DNA modifications

#### **1. Introduction**

The relationship between bacteriophages and bacteria is often explained in terms of an arms race: each 'developing' measures and countermeasures for attacking and defending itself from the other [1, 2]. The imagery of an arms race is a powerful metaphor to summarise the relationship between possibility and availability that have constrained the emergence, evolution and diversification of life on Earth.

Life is limited by what is possible. For instance, life can only exist because chemical information storage is possible. On the other hand, life as we know it has evolved around DNA and RNA because they are informational molecules that could function in the environment of the early Earth and whose building blocks were readily available.

The relationship between bacteria and bacteriophages is similarly constrained. The emergence (or availability) of bacterial cells capable of establishing a rich internal environment (compared to the outside of the cell) creates the possibility for other organisms to evolve predatory or parasitic survival strategies, including bacteriophages. Once phages emerge, they alter the dynamics of the ecological niche and create an advantage to bacterial hosts that can reduce the success of phage infection—whether by hindering phage access to the cell cytoplasm, by interfering with phage survival or replication in the cell or by interfering with phage maturation and release [3].

Bacterial defences arise from any function already available in the host (e.g. uracil-DNA glycosylase involved in DNA repair) or that can be co-opted from available genetic resources in the cell or in the environment (e.g. restriction-modification systems). Defences can also emerge from loss of function (e.g. mutations to the maltose porin LamB in *E. coli*, which make it resistant to bacteriophage λ infections [4]).

Given the prevalence of bacteria and phages in the environment, and given the evolutionary scale time of their arms race, the variety, complexity and efficiency of these attack and defence strategies are huge and can range from silent integration into the host genome (i.e. lysogeny) to enacting a hostile molecular takeover of the bacterial host cell. Despite our current efforts to map the genetic diversity available on Earth, it remains likely that new strategies are still to be identified and characterised.

Nonetheless, many of these defence and attack strategies have also been harnessed for biotechnology applications, significantly beyond the simple use of bacteriophages (or bacteriophage proteins) as bacterial control agents [5–7]. Restriction-modification (RM) systems found in bacteria were among the very first tools isolated from the bacteria-phage arms race [8, 9]. They *de facto* represent the start of modern molecular biology, and they have remained key tools for over 50 years (**Figure 1**).

**41**

*Biotechnology Tools Derived from the Bacteriophage/Bacteria Arms Race*

**2. Common molecular biology tools and orthogonality**

hybrid promoters, with their specific host and phage dependencies.

Because of the wide range of bacteriophage infection strategies available, it becomes difficult to introduce simple classification without recreating the complexity of approaches taken by phages. For instance, bacteriophage promoters can rely exclusively on host proteins (e.g. T4 early promoters), on a mixture of host and phage proteins (e.g. T4 middle promoters or PL promoter from λ phage) or exclusively on phage-derived factors (e.g. T7 RNA polymerase promoters). This provides a continuum that can be further dissected by analysing the mechanism of the

That continuum maps how independent a phage system is from the host while still active within the host, i.e. it is a measure of the orthogonality of the system. Having evolved to survive in a changing environment, bacteria have complex layers of gene expression regulation with multiple feedback systems which are not necessarily easy to control independently, despite our advances in understanding bacterial metabolism [29, 30]. In that context, phage systems that have reduced dependencies on the host machinery (i.e. increased orthogonality) provide isolated systems that can be simpler to regulate and are, at least in part, shielded from variations in the cellular machinery—an approach that has dominated biotechnology

Many of the common phage-derived biotechnology tools have been developed from such systems, none more so than T7 RNA polymerase [31]. Isolated from T7 bacteriophage, this monomeric RNA polymerase can recognise a specific promoter sequence. The core T7 promoter sequence (TAATACGACTCACTATAG) is sufficient to trigger transcription in cells harbouring a T7 RNA polymerase gene. This is the strategy set up behind pET vectors, which contain a T7 promoter and rely on an *E. coli* host carrying a T7 RNA polymerase under an inducible promoter (e.g. *lacUV*)—usually the result of the introduction of a DE3 phage [32, 33].

Nevertheless, the context of the T7 promoter can have a significant impact on the expression level of the downstream genes, and at high polymerase concentrations, it is possible to drive transcription from suboptimal promoters—highlighting

Given its monomeric structure and orthogonal role in cellular transcription, T7 RNA polymerase became not only a useful tool in biotechnology but also an important model system for the study of transcription (reviewed in [34]). Because of its orthogonality, T7 RNA polymerase (and its promoter) can be harnessed for the regulation of transcription in a wide range of hosts beyond *E. coli*, including Gram-positive bacterial hosts [35], yeast [36, 37] and human cells [38, 39]. Its role in the regulation of transcription has also been expanded through the creation of more complex systems using split T7 RNA polymerase proteins. Surprisingly for a mesophilic highly dynamic enzyme, T7 RNA polymerase can be expressed in two [40, 41] or more [42] fragments that *in vivo* are able to reassemble

While orthogonality can be a desirable feature for *in vivo* applications, it is wholly unnecessary for in vitro applications, where the key constraint lies on identifying reaction conditions in which expressed and purified proteins are sufficiently active to carry out the desired function. That is the case with T4 DNA ligase and T4 polynucleotide kinase, which were originally isolated from the *E. coli* T4 phage and

T4 DNA ligase has a central role in the replication and repair of the phage genome during its infection of *E. coli* [43]. This also entails coping with DNA modifications such as the full substitution of cytosine for 5-hydroxymethylcytosine and glucosyl-5-hydroxymethylcytosine that naturally occurs *in vivo* [44, 45]—this

that the orthogonality of the system is limited.

and function as viable RNA polymerases.

remain important tools in molecular biology.

*DOI: http://dx.doi.org/10.5772/intechopen.90367*

until recently.

#### **Figure 1.**

*Potential biotechnology applications derived from bacteriophages. Almost every aspect of the bacteriophage life cycle can be exploited for the development of biotechnology tools. Aside from their natural bactericidal role, phages can be used for the delivery of engineered circuits [10, 11] and in directed evolution through phage display [12] and PACE [13]. Individual phage systems have been also successfully developed as tools.*

More recently, another tool derived from bacterial defence has been harnessed, with potentially equally transformative impact on how we interact with biology: clustered regularly interspersed palindromic repeats (CRISPR) [26–28]. CRISPR forms part of an adaptive immunity system in prokaryotes, but it is being harnessed to deliver a wide range of research and therapeutic tools.

Although RM systems and CRISPR are deservedly acknowledged as having a significant impact on molecular biology and biotechnology, many other tools have been or are being developed based on components isolated from the bacteria-phage arms race. This chapter focuses on some of those tools—their mechanisms, current and potential applications.

*Bacteriophages - Perspectives and Future*

50 years (**Figure 1**).

resistant to bacteriophage λ infections [4]).

(e.g. restriction-modification systems). Defences can also emerge from loss of function (e.g. mutations to the maltose porin LamB in *E. coli*, which make it

Given the prevalence of bacteria and phages in the environment, and given the evolutionary scale time of their arms race, the variety, complexity and efficiency of these attack and defence strategies are huge and can range from silent integration into the host genome (i.e. lysogeny) to enacting a hostile molecular takeover of the bacterial host cell. Despite our current efforts to map the genetic diversity available on Earth, it remains likely that new strategies are still to be identified and characterised. Nonetheless, many of these defence and attack strategies have also been harnessed for biotechnology applications, significantly beyond the simple use of bacteriophages (or bacteriophage proteins) as bacterial control agents [5–7]. Restriction-modification (RM) systems found in bacteria were among the very first tools isolated from the bacteria-phage arms race [8, 9]. They *de facto* represent the start of modern molecular biology, and they have remained key tools for over

More recently, another tool derived from bacterial defence has been harnessed, with potentially equally transformative impact on how we interact with biology: clustered regularly interspersed palindromic repeats (CRISPR) [26–28]. CRISPR forms part of an adaptive immunity system in prokaryotes, but it is being harnessed

*Potential biotechnology applications derived from bacteriophages. Almost every aspect of the bacteriophage life cycle can be exploited for the development of biotechnology tools. Aside from their natural bactericidal role, phages can be used for the delivery of engineered circuits [10, 11] and in directed evolution through phage display [12] and PACE [13]. Individual phage systems have been also successfully developed as tools.*

Although RM systems and CRISPR are deservedly acknowledged as having a significant impact on molecular biology and biotechnology, many other tools have been or are being developed based on components isolated from the bacteria-phage arms race. This chapter focuses on some of those tools—their mechanisms, current

to deliver a wide range of research and therapeutic tools.

**40**

**Figure 1.**

and potential applications.

#### **2. Common molecular biology tools and orthogonality**

Because of the wide range of bacteriophage infection strategies available, it becomes difficult to introduce simple classification without recreating the complexity of approaches taken by phages. For instance, bacteriophage promoters can rely exclusively on host proteins (e.g. T4 early promoters), on a mixture of host and phage proteins (e.g. T4 middle promoters or PL promoter from λ phage) or exclusively on phage-derived factors (e.g. T7 RNA polymerase promoters). This provides a continuum that can be further dissected by analysing the mechanism of the hybrid promoters, with their specific host and phage dependencies.

That continuum maps how independent a phage system is from the host while still active within the host, i.e. it is a measure of the orthogonality of the system. Having evolved to survive in a changing environment, bacteria have complex layers of gene expression regulation with multiple feedback systems which are not necessarily easy to control independently, despite our advances in understanding bacterial metabolism [29, 30]. In that context, phage systems that have reduced dependencies on the host machinery (i.e. increased orthogonality) provide isolated systems that can be simpler to regulate and are, at least in part, shielded from variations in the cellular machinery—an approach that has dominated biotechnology until recently.

Many of the common phage-derived biotechnology tools have been developed from such systems, none more so than T7 RNA polymerase [31]. Isolated from T7 bacteriophage, this monomeric RNA polymerase can recognise a specific promoter sequence. The core T7 promoter sequence (TAATACGACTCACTATAG) is sufficient to trigger transcription in cells harbouring a T7 RNA polymerase gene. This is the strategy set up behind pET vectors, which contain a T7 promoter and rely on an *E. coli* host carrying a T7 RNA polymerase under an inducible promoter (e.g. *lacUV*)—usually the result of the introduction of a DE3 phage [32, 33].

Nevertheless, the context of the T7 promoter can have a significant impact on the expression level of the downstream genes, and at high polymerase concentrations, it is possible to drive transcription from suboptimal promoters—highlighting that the orthogonality of the system is limited.

Given its monomeric structure and orthogonal role in cellular transcription, T7 RNA polymerase became not only a useful tool in biotechnology but also an important model system for the study of transcription (reviewed in [34]). Because of its orthogonality, T7 RNA polymerase (and its promoter) can be harnessed for the regulation of transcription in a wide range of hosts beyond *E. coli*, including Gram-positive bacterial hosts [35], yeast [36, 37] and human cells [38, 39].

Its role in the regulation of transcription has also been expanded through the creation of more complex systems using split T7 RNA polymerase proteins. Surprisingly for a mesophilic highly dynamic enzyme, T7 RNA polymerase can be expressed in two [40, 41] or more [42] fragments that *in vivo* are able to reassemble and function as viable RNA polymerases.

While orthogonality can be a desirable feature for *in vivo* applications, it is wholly unnecessary for in vitro applications, where the key constraint lies on identifying reaction conditions in which expressed and purified proteins are sufficiently active to carry out the desired function. That is the case with T4 DNA ligase and T4 polynucleotide kinase, which were originally isolated from the *E. coli* T4 phage and remain important tools in molecular biology.

T4 DNA ligase has a central role in the replication and repair of the phage genome during its infection of *E. coli* [43]. This also entails coping with DNA modifications such as the full substitution of cytosine for 5-hydroxymethylcytosine and glucosyl-5-hydroxymethylcytosine that naturally occurs *in vivo* [44, 45]—this

is a phage defence mechanism discussed below. Although its structure has only recently been determined [46], the mechanism of action of this enzyme has long been characterised [47]. Even in the absence of other phage genes, it is active *in vivo* [48], but its main application in molecular biology remains its in vitro activity that, coupled with restriction endonucleases, has underpinned modern molecular biology—allowing a molecular cut and paste approach to DNA assembly.

Ligase in vitro activity, particularly its ability to accept modified ligands, has been extensively explored for the assembly of heavily modified DNA sequences for aptamer selection [49, 50] and to explore a wider range of nucleic acid modifications, such as sugar-modified nucleic acids [51].

#### **3. Second-generation tools and applications**

The combination of different enzymatic functions has created novel applications, such as the large-scale DNA assembly in Gibson assembly through the combination of exonuclease, polymerase and ligase activities [52]. However, a whole range of novel applications are possible by harnessing additional bacteriophage proteins that have not yet been extensively explored.

Recombinases and integrases, enzymes that catalyse the sequence-specific insertion of a phage genome into the host chromosome [53, 54], were identified early in bacteriophage research (e.g. the λ integrase) but were not immediately harnessed for applications. These came substantially later once recombinases were shown to facilitate DNA assembly, whether by increasing the efficiency of subcloning such as in Gateway cloning [55], or enabling multipart assemblies needed for metabolic engineering [56].

In general, recombinases bind DNA specifically, as dimers, on recognition sites that are relatively short (usually between 30 and 50 bases) and partially palindromic, termed *attP* and *attB* (originally to distinguish phage and bacterial origins). These two sites have different sequences which contribute to making the recombination process unidirectional. The recombinases facilitate the break and religation of double-stranded DNA within the *att* sites resulting in chimeric sites that are then labelled *attR* and *attL* (from right and left sides, respectively). Insertion of a phage genome into the host is efficient and stable, but it can also be reversed with the contribution of a single host factor (reviewed in [57]).

Because of the high efficiency of integration, recombinases have been also developed as systems to facilitate homologous recombination in higher eukaryotes, such as mammalian cells [58], *Drosophila* organisms [59] and plants (reviewed in [60]). In the latter, recombinases were of particular interest because of their potential to remove transformation and selection markers from engineered crops, leaving behind only the genes responsible for the engineered trait. This idea of using recombinases to directly alter an organism's genome has been vastly expanded in the Yeast 2.0 project, through the design implementation of multiple *loxP* (the equivalent to *att* sites for Cre recombinase) in the synthetic yeast genome that can be activated, leading to large-scale genome rearrangement—termed synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) [61].

By enabling controllable chromosome rearrangements between the designed *loxP* sites, a synthetic yeast can delete, duplicate and reorder many of its genes, allowing *in vivo* selection for desirable traits such as increased alkali tolerance [62]. Alternatively, the system can be coupled to heterologously expressed genes allowing the rapid optimisation of pathways [63].

Like SCRaMbLE, protein-directed evolution relies on cycles of mutagenesis (to introduce diversity into a population) and selection (to reduce diversity towards

**43**

*Biotechnology Tools Derived from the Bacteriophage/Bacteria Arms Race*

functional proteins) [64] which in some platforms can be achieved continuously *in vivo*—e.g. in PACE [13] or in some continuous culture approaches [65]. In both examples, mutation can be controlled by stressors or the induction of error-prone replication but are not necessarily limited to the area of interest (e.g. a single gene). Greater control of targeting is possible and has been reported through the use of an error-prone PolI [66]—which is necessary for the replication of some bacterial plasmids and can be used to drive diversification *in vivo* in the vicinity of plasmid replication initiation—and protein fusions that target an error-prone polymerase to a particular region of the genome [e.g. EvolvR and MutaT7 (reviewed in [67])]. However, one such system, termed diversity-generating retroelements (DGRs), has naturally emerged in bacteriophages and was first implicated in the tropism switching of *Bordetella* phages [68, 69] but that has since been identified in a wide range of bacterial and archaeal genomes [70, 71]. The system relies on an error-prone reverse transcriptase (RT) and on two DNA repeats—one operating as a template (template repeat) and the other as the target (variable repeat). RNA synthesised from the template is used to guide the error-prone synthesis of a DNA by the RT, which also coordinates its insertion at the target site. The errorprofile of DGR RTs results in adenines being replaced with other bases, creating a directionality to the evolution that is always anchored by the template repeat [72]. Nevertheless, changes in DNA sequences involved in the targeting of the retroelement, termed initiation of mutagenic homing (IMH), can lead to both template and variable repeats being allowed to evolve at different rates—removing some of the directionality in evolution and freeing the system to explore the sequence space

Despite its potential, it remains to be seen whether such a system can be harnessed for protein engineering. If the DGR systems can themselves be engineered, their targeting and error rate may be amenable to modulation opening possibilities

Chemical modification of the phage's own genome is a widespread strategy that emerged multiple times in evolution to circumvent (or at least slow down) bacterial defence mechanisms that target the invading DNA for degradation: restriction endonucleases, exonucleases and CRISPR-Cas systems [44, 74]. Those modifications have been reported not only on the nucleobases, akin to eukaryotic epigenetic

Reported nucleobase modifications suggest that the overwhelming majority of any such modifications is targeted to the C5 position in pyrimidines. They range from small modifications, such as methylation, to bulky modifications, such as putrescine and even carbohydrate moieties. While such modifications have long been known, new sequencing platforms capable of reading the DNA sequence without an amplification step, such as nanopore sequencing, hold great promise in enabling a more systematic mapping and characterisation of DNA modifications in

DNA modifications, particularly modifications that bring chemical functionality not available in natural bases, such as glycosylation in *Bacillus subtilis* SP-15 phage [76], can be harnessed for function as has been achieved through the chemical modification of DNA bases and systematic evolution of ligands by exponential enrichment (SELEX) [50, 77]. Despite characterisation of the biosynthetic pathway for multiple-phage DNA modification systems, none have been implemented

to compete with the most recent Cas9-derived gene editors [73].

*DOI: http://dx.doi.org/10.5772/intechopen.90367*

more thoroughly [68].

**4. Xenobiotic nucleic acids**

phage genomes (**Figure 2**) [75].

in vitro for applications.

markers, but also on the nucleic acid backbone.

#### *Biotechnology Tools Derived from the Bacteriophage/Bacteria Arms Race DOI: http://dx.doi.org/10.5772/intechopen.90367*

*Bacteriophages - Perspectives and Future*

tions, such as sugar-modified nucleic acids [51].

**3. Second-generation tools and applications**

that have not yet been extensively explored.

the rapid optimisation of pathways [63].

engineering [56].

is a phage defence mechanism discussed below. Although its structure has only recently been determined [46], the mechanism of action of this enzyme has long been characterised [47]. Even in the absence of other phage genes, it is active *in vivo* [48], but its main application in molecular biology remains its in vitro activity that, coupled with restriction endonucleases, has underpinned modern molecular

Ligase in vitro activity, particularly its ability to accept modified ligands, has been extensively explored for the assembly of heavily modified DNA sequences for aptamer selection [49, 50] and to explore a wider range of nucleic acid modifica-

The combination of different enzymatic functions has created novel applications, such as the large-scale DNA assembly in Gibson assembly through the combination of exonuclease, polymerase and ligase activities [52]. However, a whole range of novel applications are possible by harnessing additional bacteriophage proteins

Recombinases and integrases, enzymes that catalyse the sequence-specific insertion of a phage genome into the host chromosome [53, 54], were identified early in bacteriophage research (e.g. the λ integrase) but were not immediately harnessed for applications. These came substantially later once recombinases were shown to facilitate DNA assembly, whether by increasing the efficiency of subcloning such as in Gateway cloning [55], or enabling multipart assemblies needed for metabolic

In general, recombinases bind DNA specifically, as dimers, on recognition sites that are relatively short (usually between 30 and 50 bases) and partially palindromic, termed *attP* and *attB* (originally to distinguish phage and bacterial origins). These two sites have different sequences which contribute to making the recombination process unidirectional. The recombinases facilitate the break and religation of double-stranded DNA within the *att* sites resulting in chimeric sites that are then labelled *attR* and *attL* (from right and left sides, respectively). Insertion of a phage genome into the host is efficient and stable, but it can also be

reversed with the contribution of a single host factor (reviewed in [57]).

Because of the high efficiency of integration, recombinases have been also developed as systems to facilitate homologous recombination in higher eukaryotes, such as mammalian cells [58], *Drosophila* organisms [59] and plants (reviewed in [60]). In the latter, recombinases were of particular interest because of their potential to remove transformation and selection markers from engineered crops, leaving behind only the genes responsible for the engineered trait. This idea of using recombinases to directly alter an organism's genome has been vastly expanded in the Yeast 2.0 project, through the design implementation of multiple *loxP* (the equivalent to *att* sites for Cre recombinase) in the synthetic yeast genome that can be activated, leading to large-scale genome rearrangement—termed synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) [61].

By enabling controllable chromosome rearrangements between the designed *loxP* sites, a synthetic yeast can delete, duplicate and reorder many of its genes, allowing *in vivo* selection for desirable traits such as increased alkali tolerance [62]. Alternatively, the system can be coupled to heterologously expressed genes allowing

Like SCRaMbLE, protein-directed evolution relies on cycles of mutagenesis (to introduce diversity into a population) and selection (to reduce diversity towards

biology—allowing a molecular cut and paste approach to DNA assembly.

**42**

functional proteins) [64] which in some platforms can be achieved continuously *in vivo*—e.g. in PACE [13] or in some continuous culture approaches [65]. In both examples, mutation can be controlled by stressors or the induction of error-prone replication but are not necessarily limited to the area of interest (e.g. a single gene). Greater control of targeting is possible and has been reported through the use of an error-prone PolI [66]—which is necessary for the replication of some bacterial plasmids and can be used to drive diversification *in vivo* in the vicinity of plasmid replication initiation—and protein fusions that target an error-prone polymerase to a particular region of the genome [e.g. EvolvR and MutaT7 (reviewed in [67])].

However, one such system, termed diversity-generating retroelements (DGRs), has naturally emerged in bacteriophages and was first implicated in the tropism switching of *Bordetella* phages [68, 69] but that has since been identified in a wide range of bacterial and archaeal genomes [70, 71]. The system relies on an error-prone reverse transcriptase (RT) and on two DNA repeats—one operating as a template (template repeat) and the other as the target (variable repeat). RNA synthesised from the template is used to guide the error-prone synthesis of a DNA by the RT, which also coordinates its insertion at the target site. The errorprofile of DGR RTs results in adenines being replaced with other bases, creating a directionality to the evolution that is always anchored by the template repeat [72]. Nevertheless, changes in DNA sequences involved in the targeting of the retroelement, termed initiation of mutagenic homing (IMH), can lead to both template and variable repeats being allowed to evolve at different rates—removing some of the directionality in evolution and freeing the system to explore the sequence space more thoroughly [68].

Despite its potential, it remains to be seen whether such a system can be harnessed for protein engineering. If the DGR systems can themselves be engineered, their targeting and error rate may be amenable to modulation opening possibilities to compete with the most recent Cas9-derived gene editors [73].

#### **4. Xenobiotic nucleic acids**

Chemical modification of the phage's own genome is a widespread strategy that emerged multiple times in evolution to circumvent (or at least slow down) bacterial defence mechanisms that target the invading DNA for degradation: restriction endonucleases, exonucleases and CRISPR-Cas systems [44, 74]. Those modifications have been reported not only on the nucleobases, akin to eukaryotic epigenetic markers, but also on the nucleic acid backbone.

Reported nucleobase modifications suggest that the overwhelming majority of any such modifications is targeted to the C5 position in pyrimidines. They range from small modifications, such as methylation, to bulky modifications, such as putrescine and even carbohydrate moieties. While such modifications have long been known, new sequencing platforms capable of reading the DNA sequence without an amplification step, such as nanopore sequencing, hold great promise in enabling a more systematic mapping and characterisation of DNA modifications in phage genomes (**Figure 2**) [75].

DNA modifications, particularly modifications that bring chemical functionality not available in natural bases, such as glycosylation in *Bacillus subtilis* SP-15 phage [76], can be harnessed for function as has been achieved through the chemical modification of DNA bases and systematic evolution of ligands by exponential enrichment (SELEX) [50, 77]. Despite characterisation of the biosynthetic pathway for multiple-phage DNA modification systems, none have been implemented in vitro for applications.

#### **Figure 2.**

*Examples of XNAs in phages. Nucleobases are composed of three different chemical moieties, and change to any one of them has the potential to alter duplex conformation and how easily they are recognised by natural nucleic acid processing enzymes [14–25]. The base, sugar and backbone discussed in the chapter are shown here.*

One potential bottleneck lies on how the phage and the host handle those chemically modified DNAs. Upon infection, the mature phage DNA needs to be modified if that is an evolutionary strategy being exploited to slow down or avoid *in vivo* degradation. On the other hand, those chemical modifications can affect DNA structure and biophysical properties which may also be detrimental to bacteriophage replication—since this would require a DNA polymerase capable of processing such heavily modified genomes.

It is known that at least in some cases, this chemical cloaking is removed upon infection, such as in SP-15 [78], before unmodified DNA is replicated *in vivo*. But, given viral polymerases more permissive substrate specificity, it is possible that some systems can be replicated directly by highly adapted phage polymerases—either to DNA followed by reinstalling the chemical modifications or directly from modified DNA to modified DNA. In the case of SP-15, the bulkier modifications are rapidly removed prior to replication of DNA [78]. T4 seems to follow a similar pattern where glycosylation is removed, and DNA is replicated containing only the simpler 5-hydroxymethylation modification. This is further supported by the biochemical evidence that glycosylation is 'installed' on the replicated T4 DNA [44, 74]. Nevertheless, early T4 transcription occurs rapidly, and it is carried out by the host RNA polymerase, suggesting that natural RNA polymerases can still use the hypermodified bases in that template.

Notably, although phage polymerases replicate phage DNA *in vivo* harbouring simple chemical modifications, such as (in *Synechocystis* S-2 L phage) 2-aminoadenine [79] and uracil (in *Bacillus* phages AR9, PBS1 and PBS2, *Yersinia* phage PhiR1-37 and *Staphylococcus* phage S6), no viral polymerase has been described that is capable of selectively incorporating the modified bases. That is, although some bacteriophages make use of modified nucleobases and have evolved systems that lead to 100% incorporation of the modified bases in their genomes, their DNA polymerases have not specialised towards being able to only incorporate the modified nucleobases—they remain able to recognise unmodified triphosphates.

**45**

**Author details**

Vitor B. Pinheiro1,2

London, UK

1 Rega Institute for Medical Research, KU Leuven, Leuven, Belgium

\*Address all correspondence to: vitor.pinheiro@kuleuven.be

provided the original work is properly cited.

2 Department of Structural and Molecular Biology, University College London,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Biotechnology Tools Derived from the Bacteriophage/Bacteria Arms Race*

Still, the increased substrate flexibility of phage DNA polymerases may at least in part justify why a *Bacillus subtilis* Phi29 DNA polymerase required a single mutation for the synthesis of anhydrohexitol nucleic acids (HNA) [80] while an archaeal

Bacteriophages remain a rich source of novel functionalities that can be harnessed to advance molecular biology (and synthetic biology). The examples here provided represent only a small fraction of the potential applications available, which also include medical applications from phage proteins [82, 83] and engi-

In addition, bacteriophages have had a close relationship with directed evolution, either as a vehicle such as in phage display [85, 86] or by providing (in addition to the examples above) proteins to accelerate strain engineering, such as in multi-

Finally, bacteriophages may also become an important tool in harnessing new non-model organisms in synthetic biology, as pre-optimised DNA delivery nano-

*DOI: http://dx.doi.org/10.5772/intechopen.90367*

**5. Conclusion**

neered phages [10, 11, 84].

machines for custom circuits.

enzyme required in excess of seven mutations [81].

plex automated genome engineering (MAGE) [87].

Still, the increased substrate flexibility of phage DNA polymerases may at least in part justify why a *Bacillus subtilis* Phi29 DNA polymerase required a single mutation for the synthesis of anhydrohexitol nucleic acids (HNA) [80] while an archaeal enzyme required in excess of seven mutations [81].

### **5. Conclusion**

*Bacteriophages - Perspectives and Future*

ing such heavily modified genomes.

**Figure 2.**

One potential bottleneck lies on how the phage and the host handle those chemically modified DNAs. Upon infection, the mature phage DNA needs to be modified if that is an evolutionary strategy being exploited to slow down or avoid *in vivo* degradation. On the other hand, those chemical modifications can affect DNA structure and biophysical properties which may also be detrimental to bacteriophage replication—since this would require a DNA polymerase capable of process-

*Examples of XNAs in phages. Nucleobases are composed of three different chemical moieties, and change to any one of them has the potential to alter duplex conformation and how easily they are recognised by natural nucleic acid processing enzymes [14–25]. The base, sugar and backbone discussed in the chapter are shown here.*

It is known that at least in some cases, this chemical cloaking is removed upon infection, such as in SP-15 [78], before unmodified DNA is replicated *in vivo*. But, given viral polymerases more permissive substrate specificity, it is possible that some systems can be replicated directly by highly adapted phage polymerases—either to DNA followed by reinstalling the chemical modifications or directly from modified DNA to modified DNA. In the case of SP-15, the bulkier modifications are rapidly removed prior to replication of DNA [78]. T4 seems to follow a similar pattern where glycosylation is removed, and DNA is replicated containing only the simpler 5-hydroxymethylation modification. This is further supported by the biochemical evidence that glycosylation is 'installed' on the replicated T4 DNA [44, 74]. Nevertheless, early T4 transcription occurs rapidly, and it is carried out by the host RNA polymerase, suggesting that natural RNA polymerases can still use the hypermodified bases in that template.

Notably, although phage polymerases replicate phage DNA *in vivo* harbouring simple chemical modifications, such as (in *Synechocystis* S-2 L phage) 2-aminoadenine [79] and uracil (in *Bacillus* phages AR9, PBS1 and PBS2, *Yersinia* phage PhiR1-37 and *Staphylococcus* phage S6), no viral polymerase has been described that is capable of selectively incorporating the modified bases. That is, although some bacteriophages make use of modified nucleobases and have evolved systems that lead to 100% incorporation of the modified bases in their genomes, their DNA polymerases have not specialised towards being able to only incorporate the modified nucleobases—they remain able to recognise unmodified triphosphates.

**44**

Bacteriophages remain a rich source of novel functionalities that can be harnessed to advance molecular biology (and synthetic biology). The examples here provided represent only a small fraction of the potential applications available, which also include medical applications from phage proteins [82, 83] and engineered phages [10, 11, 84].

In addition, bacteriophages have had a close relationship with directed evolution, either as a vehicle such as in phage display [85, 86] or by providing (in addition to the examples above) proteins to accelerate strain engineering, such as in multiplex automated genome engineering (MAGE) [87].

Finally, bacteriophages may also become an important tool in harnessing new non-model organisms in synthetic biology, as pre-optimised DNA delivery nanomachines for custom circuits.

### **Author details**

Vitor B. Pinheiro1,2

1 Rega Institute for Medical Research, KU Leuven, Leuven, Belgium

2 Department of Structural and Molecular Biology, University College London, London, UK

\*Address all correspondence to: vitor.pinheiro@kuleuven.be

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Sivaamnuaiphorn S, Xu M, Doulatov S, et al. Diversity-generating retroelement homing regenerates target sequences for repeated rounds of codon rewriting and protein diversification. Molecular Cell.

2015;**6**(1):6585

2008;**31**(6):813-823

s41586-019-1711-4

[64] Tizei PAG, Csibra E, Torres L, Pinheiro VB. Selection platforms for directed evolution in synthetic biology. Biochemical Society Transactions. 2016;**44**(4):1165-1175. DOI: 10.1042/

[65] Marlière P, Patrouix J, Döring V, Herdewijn P, Tricot S, Cruveiller S, et al. Chemical evolution of a bacterium's genome. Angewandte Chemie,

BST20160076

International Edition. 2011;**50**(31):7109-7114

[66] Camps M, Naukkarinen J, Johnson BP, Loeb LA. Targeted gene evolution in *Escherichia coli* using a highly error-prone DNA polymerase I. Proceedings of the National Academy of Sciences of the United States of America. 2003;**100**(17):9727-9732

[67] Yang J, Kim B, Kim GY,

Biofuels. 2019;**12**(1):113

Jung GY, Seo SW. Synthetic biology for evolutionary engineering: From perturbation of genotype to acquisition of desired phenotype. Biotechnology for

[68] Doulatov S, Hodes A, Dal L, Mandhana N, Liu M, Deora R, et al. Tropism switching in *Bordetella* bacteriophage defines a family of diversity-generating retroelements. Nature. 2004;**431**(7007):476-481

[69] Liu M, Deora R, Doulatov SR, Gingery M, Eiserling FA, Preston A, et al. Reverse transcriptase-mediated tropism switching in *Bordetella* bacteriophage. Science. 2002;**295**(5562):2091-2094

[70] Paul BG, Burstein D, Castelle CJ, Handa S, Arambula D, Czornyj E, et al. Retroelement-guided protein diversification abounds in vast lineages

of bacteria and archaea. Nature Microbiology. 2017;**2**(6):17045

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[79] Kirnos MD, Khudyakov IY, Alexandrushkina NI, Vanyushin BF. 2-Aminoadenine is an adenine substituting for a base in S-2L cyanophage DNA. Nature. 1977;**270**(5635):369-370

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[81] Pinheiro VB, Taylor AI, Cozens C, Abramov M, Renders M, Zhang S, et al. Synthetic genetic polymers capable of heredity and evolution. Science. 2012;**336**(6079):341-344. Available from: http://www.sciencemag.org/ content/336/6079/341%5Cnhttp://www. ncbi.nlm.nih.gov/pubmed/22517858

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[83] São-José C. Engineering of phagederived lytic enzymes: Improving their potential as antimicrobials. Antibiotics. 2018;**7**(2):29

[84] Pires DP, Cleto S, Sillankorva S, Azeredo J, Lu TK. Genetically engineered phages: A review of advances over the last decade. Microbiology and Molecular Biology Reviews. 2016;**80**(3):523-543

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**53**

**Chapter 4**

**Abstract**

Touchlight Genetics

**1. Introduction**

Prokaryotes

The Unusual Linear Plasmid

*Sophie E. Knott, Sarah A. Milsom and Paul J. Rothwell*

and *Borellia* will be discussed in terms of mechanism of action.

**Keywords:** protelomerase, telomere resolvase, linear plasmid, doggybone DNA,

The study of DNA, its structure and how it is replicated has been intensifying since the 1900s. Recent advances in DNA sequencing, bioinformatics and highresolution imaging has increased our understanding of the variations that exist between different DNA replication systems. In general, the genetic material of bacterial cells is in the form of circular DNA molecules. Infecting bacteriophage may integrate their DNA into the host genome, or maintain it independently as a viral episome; usually this plasmid is also circular. These structures have no free ends and are therefore not susceptible to exonuclease degradation and do not suffer from the end replication problem, whereby genetic material at the tip of a chromosome is lost during each round of replication. However, some prokaryotic cells have been identified as harboring closed linear DNA chromosomes. The ends of these structures

Linear DNA is vulnerable to exonuclease degradation and suffers from genetic loss due to the end replication problem. Eukaryotes overcome these problems by locating repetitive telomere sequences at the end of each chromosome. In humans and other vertebrates this noncoding terminal sequence is repeated between hundreds and thousands of times, ensuring important genetic information is protected. In most prokaryotes, the end-replication problem is solved by utilizing circular DNA molecules as chromosomes. However, some phage and bacteria do store genetic information in linear constructs, and the ends of these structures form either invertrons or hairpin telomeres. Hairpin telomere formation is catalyzed by a protelomerase, a unique protein that modifies DNA by a two-step transesterification reaction, proceeding via a covalent protein bound intermediate. The specifics of this mechanism are largely unknown and conflicting data suggests variations occur between different systems. These proteins, and the DNA constructs they produce, have valuable applications in the biotechnology industry. They are also an essential component of some human pathogens, an increased understanding of how they operate is therefore of fundamental importance. Although this review will focus on phage encoded protelomerase, protelomerases found from *Agrobacterium*

Generating Systems of

#### **Chapter 4**

## The Unusual Linear Plasmid Generating Systems of Prokaryotes

*Sophie E. Knott, Sarah A. Milsom and Paul J. Rothwell*

#### **Abstract**

Linear DNA is vulnerable to exonuclease degradation and suffers from genetic loss due to the end replication problem. Eukaryotes overcome these problems by locating repetitive telomere sequences at the end of each chromosome. In humans and other vertebrates this noncoding terminal sequence is repeated between hundreds and thousands of times, ensuring important genetic information is protected. In most prokaryotes, the end-replication problem is solved by utilizing circular DNA molecules as chromosomes. However, some phage and bacteria do store genetic information in linear constructs, and the ends of these structures form either invertrons or hairpin telomeres. Hairpin telomere formation is catalyzed by a protelomerase, a unique protein that modifies DNA by a two-step transesterification reaction, proceeding via a covalent protein bound intermediate. The specifics of this mechanism are largely unknown and conflicting data suggests variations occur between different systems. These proteins, and the DNA constructs they produce, have valuable applications in the biotechnology industry. They are also an essential component of some human pathogens, an increased understanding of how they operate is therefore of fundamental importance. Although this review will focus on phage encoded protelomerase, protelomerases found from *Agrobacterium* and *Borellia* will be discussed in terms of mechanism of action.

**Keywords:** protelomerase, telomere resolvase, linear plasmid, doggybone DNA, Touchlight Genetics

#### **1. Introduction**

The study of DNA, its structure and how it is replicated has been intensifying since the 1900s. Recent advances in DNA sequencing, bioinformatics and highresolution imaging has increased our understanding of the variations that exist between different DNA replication systems. In general, the genetic material of bacterial cells is in the form of circular DNA molecules. Infecting bacteriophage may integrate their DNA into the host genome, or maintain it independently as a viral episome; usually this plasmid is also circular. These structures have no free ends and are therefore not susceptible to exonuclease degradation and do not suffer from the end replication problem, whereby genetic material at the tip of a chromosome is lost during each round of replication. However, some prokaryotic cells have been identified as harboring closed linear DNA chromosomes. The ends of these structures

are protected by either invertron or hairpin telomeres. Invertron telomeres consist of inverted terminal repeats and covalently attached capping proteins, essential for priming DNA replication. These structures are distinct from hairpin telomeres, which have covalently closed hairpin ends.

The first linear genome of prokaryotes was obtained in 1964, when V. Ravin isolated the *Escherichia coli* phage N15 [1]. The genetic material of this phage is unusual, because it is not maintained as an independent circular entity nor is it integrated into the host genomic material. Instead, upon entry into the host, the N15 genome circularizes and is then processed into linear structures by an atypical cutting and re-joining enzyme, called a protelomerase, or telomere resolvase. Since this discovery, protelomerases have also been characterised in *Klebsiella oxytoca* phage φKO2, *Yersinia enterocolitica* phage PY54, *Vibrio parahaemolyticus* phage VP882 [2] and *Halomonas aquamarina* phage ΦHAP-1.

In addition to phage, these unusual enzymes have been isolated from certain bacteria. The best studied being ResT from *Borrelia burgdorferi* [3], the causative agent of Lyme disease. Linear chromosomes are now described as a hallmark of *Borrelia* [4] and protelomerases have been purified from *B. hermsii*, *B. parkeri*, *B. recurrentis*, *B. turicatae*, and *B. anserine* [5]. More recently, they have also been discovered in cyanobacteria [6] and the plant pathogen *Agrobacterium tumefaciens C58*, which contains a circular and linear chromosome as well as circular plasmids [7]. These proteins are clearly more widespread than initially believed and it is likely future research into other prokaryotes will identify additional members of the family.

Although currently under debate, it has been suggested protelomerases are tyrosine-recombinase-like enzymes. It remains to be determined whether the bacterial protelomerases have their origin in phage as both, to some extent, share a common substrate recognition and a DNA cleavage/rejoining mechanism. In addition, some protelomerases have been identified as having roles in vivo unrelated to telomere resolution such as single strand annealing [8] and ATP-dependent helicase activities [9]. Further research into this important class of enzymes should help elucidate the significance of single strand annealing and DNA unwinding activities during closed linear chromosome replication.

Not only are protelomerases essential for the organisms in which they reside, but their unique functionality makes them valuable to the biotechnology industry. DNA constructs produced using protelomerase are being marketed by Lucigen as improved cloning vectors for highly repetitive sequences [10, 11]. Linear structures are not susceptible to supercoiling, thus making them more stable and less susceptible to genetic loss during replication [10]. Protelomerases can also be expressed in engineered *E. coli* cells to produce linear eukaryotic vectors that contain no bacterial sequences [12]. In addition, protelomerases are a central component to Touchlight Genetics' DNA amplification platform that produces large quantities of high-quality DNA using a cell-free process, for therapeutic and industrial applications [13, 14]. Unlike plasmid DNA, the incumbent technology for therapeutic applications, Touchlight's doggybone DNA (dbDNA) platform contains no extraneous bacterial DNA sequences. The resulting minimal vector has an improved safety profile from a regulatory perspective due to elimination of antibiotic resistance genes. The small amounts of plasmid DNA required for this in vitro manufacturing process makes the dbDNA process well suited to scale production of "difficult" structured or repetitive DNA sequences or constructs that cause cell toxicity. Linear, minimal vectors may be valuable as nonviral gene therapy vectors and as DNA vaccines, both modalities gaining increasing focus and investment in the biotechnology market.

**55**

in different systems.

*The Unusual Linear Plasmid Generating Systems of Prokaryotes*

There is high interest in the study of this protein family due to their utility and potential value for biotechnology. To date, research has largely focused on characterizing protelomerase recognition sequences, solving 3-dimensional structures and exploring the effects of protein mutations on activity. An improved understanding of how protelomerases function will enhance their value for applications in synthetic biology, and may provide the opportunity to invent new and novel

Despite the diversity of organisms in which protelomerases reside, important features have been identified that unify and define this class of protein. The protelomerase target site, denoted as *telRL*, is a palindromic sequence of double stranded DNA. The substrate differs between protelomerases and to date only ResT, the bacterial protelomerase from *Borrelia*, has been shown to have specificity for more than one target sequence [15]. All protelomerases are thought to function as a dimer and it is widely believed that none require the addition of cofactors such as ATP or divalent cations. However, it has been shown that concentrations of EDTA >10 mm inhibit the N15 protelomerase, TelN, and the sequence of this protein predicts a

Current models propose that protelomerases bind nonspecifically to DNA and scan the sequence until finding the target site, or coming into contact with another monomer, at which point the protein immobilizes [17]. Immobilization occurs upon dimerization, whether this forms at the substrate target sequence or not. However, only when at the correct site will the reaction of telomere formation be catalyzed. This phenomenon can be observed in vitro, where a high concentration of TelK (over 400 nm) results in the condensation of DNA and inhibition of telomere formation [17]. Protelomerase concentration in vivo therefore, must be carefully controlled. This notion has been explored in phage N15, where negative control is

Protelomerases catalyzes a two-step transesterification reaction, and all are thought to initiate DNA cleavage using an active site tyrosine residue. This residue performs nucleophilic attack on the phosphodiester bond to form a 3′ covalently attached protein-DNA intermediate and a free 5′-OH. The protein bound intermediate is vital for avoiding deleterious double strand breaks and prevents the premature abortion of reactions [19]. The DNA cleavage reaction happens in a staggered formation 3-bp either side of the symmetrical target site center. This leaves a 6-nucleotide overhang that loops back and is ligated to form the covalently closed hairpin end. The DNA cleaving and re-joining reactions are isoenergetic and, in principle, each step in the reaction is reversible [19]. As DNA hairpins are unable to form complete base pairings [20], they are less stable than the starting material. In this case directionality is determined by the loop processing step. This part of the reaction is poorly understood and data available indicates conflicting mechanisms

**Figure 1a** is a model for telomere resolution by the protelomerase TelK from phage φKO2. An interlocked protein dimer forms at the *telRL* site and induces a sharp, roughly 73°, bend in the DNA, which displaces its helical structure and buckles the base pairs between the scissile phosphates [21]. This is described as "spring loading"; the energy stored in the distorted DNA drives the reaction forward, enabling spontaneous hairpin formation and protein dimer separation [19]. The mechanisms proposed for the bacterial protelomerases, TelA and ResT

*DOI: http://dx.doi.org/10.5772/intechopen.86882*

**2. Mechanism of telomere resolution**

binding motif for divalent cations [16].

used to regulate the levels of protein [18].

applications.

*Bacteriophages - Perspectives and Future*

which have covalently closed hairpin ends.

and *Halomonas aquamarina* phage ΦHAP-1.

during closed linear chromosome replication.

and investment in the biotechnology market.

members of the family.

are protected by either invertron or hairpin telomeres. Invertron telomeres consist of inverted terminal repeats and covalently attached capping proteins, essential for priming DNA replication. These structures are distinct from hairpin telomeres,

The first linear genome of prokaryotes was obtained in 1964, when V. Ravin isolated the *Escherichia coli* phage N15 [1]. The genetic material of this phage is unusual, because it is not maintained as an independent circular entity nor is it integrated into the host genomic material. Instead, upon entry into the host, the N15 genome circularizes and is then processed into linear structures by an atypical cutting and re-joining enzyme, called a protelomerase, or telomere resolvase. Since this discovery, protelomerases have also been characterised in *Klebsiella oxytoca* phage φKO2, *Yersinia enterocolitica* phage PY54, *Vibrio parahaemolyticus* phage VP882 [2]

In addition to phage, these unusual enzymes have been isolated from certain bacteria. The best studied being ResT from *Borrelia burgdorferi* [3], the causative agent of Lyme disease. Linear chromosomes are now described as a hallmark of *Borrelia* [4] and protelomerases have been purified from *B. hermsii*, *B. parkeri*, *B. recurrentis*, *B. turicatae*, and *B. anserine* [5]. More recently, they have also been discovered in cyanobacteria [6] and the plant pathogen *Agrobacterium tumefaciens C58*, which contains a circular and linear chromosome as well as circular plasmids [7]. These proteins are clearly more widespread than initially believed and it is likely future research into other prokaryotes will identify additional

Although currently under debate, it has been suggested protelomerases are tyrosine-recombinase-like enzymes. It remains to be determined whether the bacterial protelomerases have their origin in phage as both, to some extent, share a common substrate recognition and a DNA cleavage/rejoining mechanism. In addition, some protelomerases have been identified as having roles in vivo unrelated to telomere resolution such as single strand annealing [8] and ATP-dependent helicase activities [9]. Further research into this important class of enzymes should help elucidate the significance of single strand annealing and DNA unwinding activities

Not only are protelomerases essential for the organisms in which they reside, but their unique functionality makes them valuable to the biotechnology industry. DNA constructs produced using protelomerase are being marketed by Lucigen as improved cloning vectors for highly repetitive sequences [10, 11]. Linear structures are not susceptible to supercoiling, thus making them more stable and less susceptible to genetic loss during replication [10]. Protelomerases can also be expressed in engineered *E. coli* cells to produce linear eukaryotic vectors that contain no bacterial sequences [12]. In addition, protelomerases are a central component to Touchlight Genetics' DNA amplification platform that produces large quantities of high-quality DNA using a cell-free process, for therapeutic and industrial applications [13, 14]. Unlike plasmid DNA, the incumbent technology for therapeutic applications, Touchlight's doggybone DNA (dbDNA) platform contains no extraneous bacterial DNA sequences. The resulting minimal vector has an improved safety profile from a regulatory perspective due to elimination of antibiotic resistance genes. The small amounts of plasmid DNA required for this in vitro manufacturing process makes the dbDNA process well suited to scale production of "difficult" structured or repetitive DNA sequences or constructs that cause cell toxicity. Linear, minimal vectors may be valuable as nonviral gene therapy vectors and as DNA vaccines, both modalities gaining increasing focus

**54**

There is high interest in the study of this protein family due to their utility and potential value for biotechnology. To date, research has largely focused on characterizing protelomerase recognition sequences, solving 3-dimensional structures and exploring the effects of protein mutations on activity. An improved understanding of how protelomerases function will enhance their value for applications in synthetic biology, and may provide the opportunity to invent new and novel applications.

#### **2. Mechanism of telomere resolution**

Despite the diversity of organisms in which protelomerases reside, important features have been identified that unify and define this class of protein. The protelomerase target site, denoted as *telRL*, is a palindromic sequence of double stranded DNA. The substrate differs between protelomerases and to date only ResT, the bacterial protelomerase from *Borrelia*, has been shown to have specificity for more than one target sequence [15]. All protelomerases are thought to function as a dimer and it is widely believed that none require the addition of cofactors such as ATP or divalent cations. However, it has been shown that concentrations of EDTA >10 mm inhibit the N15 protelomerase, TelN, and the sequence of this protein predicts a binding motif for divalent cations [16].

Current models propose that protelomerases bind nonspecifically to DNA and scan the sequence until finding the target site, or coming into contact with another monomer, at which point the protein immobilizes [17]. Immobilization occurs upon dimerization, whether this forms at the substrate target sequence or not. However, only when at the correct site will the reaction of telomere formation be catalyzed. This phenomenon can be observed in vitro, where a high concentration of TelK (over 400 nm) results in the condensation of DNA and inhibition of telomere formation [17]. Protelomerase concentration in vivo therefore, must be carefully controlled. This notion has been explored in phage N15, where negative control is used to regulate the levels of protein [18].

Protelomerases catalyzes a two-step transesterification reaction, and all are thought to initiate DNA cleavage using an active site tyrosine residue. This residue performs nucleophilic attack on the phosphodiester bond to form a 3′ covalently attached protein-DNA intermediate and a free 5′-OH. The protein bound intermediate is vital for avoiding deleterious double strand breaks and prevents the premature abortion of reactions [19]. The DNA cleavage reaction happens in a staggered formation 3-bp either side of the symmetrical target site center. This leaves a 6-nucleotide overhang that loops back and is ligated to form the covalently closed hairpin end. The DNA cleaving and re-joining reactions are isoenergetic and, in principle, each step in the reaction is reversible [19]. As DNA hairpins are unable to form complete base pairings [20], they are less stable than the starting material. In this case directionality is determined by the loop processing step. This part of the reaction is poorly understood and data available indicates conflicting mechanisms in different systems.

**Figure 1a** is a model for telomere resolution by the protelomerase TelK from phage φKO2. An interlocked protein dimer forms at the *telRL* site and induces a sharp, roughly 73°, bend in the DNA, which displaces its helical structure and buckles the base pairs between the scissile phosphates [21]. This is described as "spring loading"; the energy stored in the distorted DNA drives the reaction forward, enabling spontaneous hairpin formation and protein dimer separation [19]. The mechanisms proposed for the bacterial protelomerases, TelA and ResT

#### **Figure 1.**

*Models of telomere resolution by TelK, TelA and ResT. (a) TelK monomers composed of N-terminal muzzle and C-terminal stirrup domains dimerise at the target site. This induces bending of the DNA, the spontaneous release of stored energy drives hairpin formation and dimer dissolution. (b) TelA cleaves the DNA and transient electrostatic interactions stabilize the transition state. Hairpin formation occurs within the protein dimer (c) ResT catalyzes telomere resolution with the aid of its hairpin-binding module. The final step of this reaction is product release, which is not observed for TelK or TelA [19].*

(from *Agrobacterium tumefaciens C58* and *Borrelia*, respectively) are fundamentally different to that of TelK. In TelA and ResT reactions, strand refolding is enzyme-mediated, as opposed to spontaneous. A key element of the TelA mechanism is the refolding intermediate that exists before hairpin formation. This conformation is stabilized by multiple protein-DNA and DNA-DNA interactions, which drive the reaction forward by virtue of changes in binding energies. TelA binds even more strongly to the final hairpin product, thus favoring its formation. The mechanisms for TelK and TelA have been deduced from structures solved by X-ray crystallography [21, 22]. There is no structure of ResT and the mechanism proposed in **Figure 1c** is a result of research involving structure prediction, substrate modifications and protein mutations. In ResT catalyzed telomere resolution, the protein binds and distorts the DNA by underwinding at the dimer interface [19]. This is consistent with the observation that ResT has a hairpin-binding module, that presumably stabilizes the conformation of prehairpin DNA [23]. Hydrolysis of base pairs between the scissile phosphates promotes strand ejection following DNA cleavage. The exact mechanism of strand refolding is yet to be determined, but it is suggested to occur before dissolution of the dimer [24]. This concept of a "spring-loaded" pre-cleavage intermediate is analogous to that of TelK.

#### **3. Substrate sequences**

Identifying the natural target site of protelomerases is not straightforward. A logical strategy is to determine the nucleotide sequence of the resultant telomere and deduce from this the starting material. However, sequencing telomeres is notoriously difficult as the hairpin ends are incapable of ligating to the vector during sequencing library construction [7]. An adapted method has been used,

**57**

**Figure 2.**

*The Unusual Linear Plasmid Generating Systems of Prokaryotes*

sequences, which may be confirmed by in vitro studies [25].

whereby a nuclease opens the closed ends to make them compliant for ligation [25]. This does not always give absolute results, but can provide predictions for the target

In general, protelomerases are highly specific and only process one target sequence. The exception to this is ResT, which is far less stringent and can resolve nine different telomere sequences found in the *B. burgdorferi* group of bacteria [15]. A conserved feature among all protelomerases is the palindromic nature of their substrate, with one protein molecule binding either side of the axis of symmetry to form a dimer. Interestingly, the TATAAT sequence of telomeres from N15 and φKO2 is also found in *Borrelia*. The significance of this is unconfirmed, although it has been suggested the nucleotides are important for protelomerase recognition [15]. For ResT, substitution of this sequence abolishes telomere resolution and mutating it to TTTAAT reduces the initial rate significantly [15]. Mutating the 6th and 7th nucleotide of this sequence within the TelN recognition site also produces a substrate the protein cannot process [26]. Despite functioning in different systems, TelN and TelK process highly similar target sequences, both of which are shown in **Figure 2**. These sites differ in length, but are identical in the center, and both protelomerases are capable of resolving each other's natural substrate [27]. Given the high sequence similarity (86.9%) between TelN and TelK, this observation is not

Comparison of the TelN and TelK *telRL* sequences, to the 42 base pair (bp) recognition site of the PY54 protelomerase indicates limited homology and this DNA cannot be processed by any of the other protelomerases [27]. However, Huang and colleagues found altering positions 15 and 16 of the PY54 target in the top strand, plus residues 28 and 27 of the bottom strand results in a substrate that is processed, although with limited efficiency, by TelK. They went on to suggest that TelN and TelK not only recognize these specific nucleotides, but also a cruciform DNA structure that is formed [27]. Although crystal structures of TelK have since discredited the suggestion that a cruciform structure is formed [21], this work is important in that it identifies the key nucleotides that are essential for telomere

*Protelomerase recognition sequences. (a) The* tos *site for TelN. Gray boxes indicate three regions of repeated sequences flanking the telRL site, which contains the central 22-bp telO site highlighted in cyan. Figure adapted from [16]. (b) The cognate sequence of TelN (top) and TelK (bottom). Both protelomerases are capable of processing each other's substrate. Two single point variations are shown in bold. In addition, the TelN natural* 

*DOI: http://dx.doi.org/10.5772/intechopen.86882*

hugely surprising.

resolution by these enzymes.

*substrate has six residues on each end.*

#### *The Unusual Linear Plasmid Generating Systems of Prokaryotes DOI: http://dx.doi.org/10.5772/intechopen.86882*

*Bacteriophages - Perspectives and Future*

(from *Agrobacterium tumefaciens C58* and *Borrelia*, respectively) are fundamentally different to that of TelK. In TelA and ResT reactions, strand refolding is enzyme-mediated, as opposed to spontaneous. A key element of the TelA mechanism is the refolding intermediate that exists before hairpin formation. This conformation is stabilized by multiple protein-DNA and DNA-DNA interactions, which drive the reaction forward by virtue of changes in binding energies. TelA binds even more strongly to the final hairpin product, thus favoring its formation. The mechanisms for TelK and TelA have been deduced from structures solved by X-ray crystallography [21, 22]. There is no structure of ResT and the mechanism proposed in **Figure 1c** is a result of research involving structure prediction, substrate modifications and protein mutations. In ResT catalyzed telomere resolution, the protein binds and distorts the DNA by underwinding at the dimer interface [19]. This is consistent with the observation that ResT has a hairpin-binding module, that presumably stabilizes the conformation of prehairpin DNA [23]. Hydrolysis of base pairs between the scissile phosphates promotes strand ejection following DNA cleavage. The exact mechanism of strand refolding is yet to be determined, but it is suggested to occur before dissolution of the dimer [24]. This concept of a "spring-loaded" pre-cleavage intermediate is

*reaction is product release, which is not observed for TelK or TelA [19].*

*Models of telomere resolution by TelK, TelA and ResT. (a) TelK monomers composed of N-terminal muzzle and C-terminal stirrup domains dimerise at the target site. This induces bending of the DNA, the spontaneous release of stored energy drives hairpin formation and dimer dissolution. (b) TelA cleaves the DNA and transient electrostatic interactions stabilize the transition state. Hairpin formation occurs within the protein dimer (c) ResT catalyzes telomere resolution with the aid of its hairpin-binding module. The final step of this* 

Identifying the natural target site of protelomerases is not straightforward. A logical strategy is to determine the nucleotide sequence of the resultant telomere and deduce from this the starting material. However, sequencing telomeres is notoriously difficult as the hairpin ends are incapable of ligating to the vector during sequencing library construction [7]. An adapted method has been used,

**56**

**Figure 1.**

analogous to that of TelK.

**3. Substrate sequences**

whereby a nuclease opens the closed ends to make them compliant for ligation [25]. This does not always give absolute results, but can provide predictions for the target sequences, which may be confirmed by in vitro studies [25].

In general, protelomerases are highly specific and only process one target sequence. The exception to this is ResT, which is far less stringent and can resolve nine different telomere sequences found in the *B. burgdorferi* group of bacteria [15]. A conserved feature among all protelomerases is the palindromic nature of their substrate, with one protein molecule binding either side of the axis of symmetry to form a dimer. Interestingly, the TATAAT sequence of telomeres from N15 and φKO2 is also found in *Borrelia*. The significance of this is unconfirmed, although it has been suggested the nucleotides are important for protelomerase recognition [15]. For ResT, substitution of this sequence abolishes telomere resolution and mutating it to TTTAAT reduces the initial rate significantly [15]. Mutating the 6th and 7th nucleotide of this sequence within the TelN recognition site also produces a substrate the protein cannot process [26]. Despite functioning in different systems, TelN and TelK process highly similar target sequences, both of which are shown in **Figure 2**. These sites differ in length, but are identical in the center, and both protelomerases are capable of resolving each other's natural substrate [27]. Given the high sequence similarity (86.9%) between TelN and TelK, this observation is not hugely surprising.

Comparison of the TelN and TelK *telRL* sequences, to the 42 base pair (bp) recognition site of the PY54 protelomerase indicates limited homology and this DNA cannot be processed by any of the other protelomerases [27]. However, Huang and colleagues found altering positions 15 and 16 of the PY54 target in the top strand, plus residues 28 and 27 of the bottom strand results in a substrate that is processed, although with limited efficiency, by TelK. They went on to suggest that TelN and TelK not only recognize these specific nucleotides, but also a cruciform DNA structure that is formed [27]. Although crystal structures of TelK have since discredited the suggestion that a cruciform structure is formed [21], this work is important in that it identifies the key nucleotides that are essential for telomere resolution by these enzymes.

#### **Figure 2.**

*Protelomerase recognition sequences. (a) The* tos *site for TelN. Gray boxes indicate three regions of repeated sequences flanking the telRL site, which contains the central 22-bp telO site highlighted in cyan. Figure adapted from [16]. (b) The cognate sequence of TelN (top) and TelK (bottom). Both protelomerases are capable of processing each other's substrate. Two single point variations are shown in bold. In addition, the TelN natural substrate has six residues on each end.*

#### **3.1 Minimal substrate**

In vitro studies have also involved truncating target sites in order to identify the minimum sequence required for protelomerase binding and telomere resolution. To date, the minimal site identified that can be resolved, is a 26-bp substrate of TelA [7]. This was found by systematically deleting residues from both sides of the target sequence, until no product was produced. Similar studies have been performed on the TelN substrate. **Figure 2** shows the complete telomere occupancy (*tos*) site, which consists of a 56-bp palindromic sequence flanked by a series of inverted repeats. Initially, it was believed that *telO* is insufficient for processing by TelN, and the reaction requires the whole *telRL* site [16]. However, it has since been found that at greater TelN concentrations, roughly 50-fold higher than those required for *telRL*, the *telO* substrate is processed [26]. This indicates *telO* contains all the necessary elements for telomere resolution, but the protein requires additional sequence for binding and recognition. The binding affinity of TelN is greater still when the whole *tos* site is included in the substrate [16]. Experiments performed using ResT have explored whether the protelomerase is able to mediate cleavage on half a target site. When this half site was in a plasmid, the assay failed to produce reaction products, therefore suggesting dimer formation is essential for activity and the whole palindromic sequence is required [28].

#### **4. Linear genomes**

In order to further appreciate how protelomerases function, it is necessary to understand their role in relation to the whole phage or bacterial cell life-cycle. Bacteriophage N15 has been extensively characterised and **Figure 3** illustrates the different structures its genetic material forms upon infection of *E. coli*. The phage

#### **Figure 3.**

*Forms of bacteriophage N15 DNA. Having infected an* E. coli *cell, the virion DNA circularizes, via complementary* cos *sites. Lytic or lysogenic replication can be initiated from the circular intermediate. Shown is the pathway for lysogeny, whereby the telRL is processed by protelomerases to form linear prophage DNA with covalently closed hairpin ends. Figure adapted from [1].*

**59**

**Figure 4.**

*into left- and right-hand arms. Adapted from [34].*

*The Unusual Linear Plasmid Generating Systems of Prokaryotes*

DNA is a 46.4 kb chromosome that has two cohesive end sites (*cos*) consisting of 12-nucleotide overhangs at each 5′-end. These sites are complementary and can be ligated to form circular DNA. This circular intermediate then acts as the starting material for either lytic (not shown in **Figure 3**) or lysogenic development. During lysogenic development the *telRL* site is recognized and processed by protelomerases, this reaction forms a linear DNA structure with covalently closed ends. A similar genome arrangement has been identified for φKO2 [27], VP58.5 [29], VP882 [2] and PY54 [30]. These prophages all have cohesive (*cos*) ends that presumably enable the formation of similar structures as those described for N15. Interestingly, no *cos* site has been identified in the ΦHAP1 genome [31], therefore indicating a different mechanism of DNA packing. The discovery of terminase genes [31], suggests that headful packing may occur, whereby concatermeric DNA is packed into the phage

Various models have been proposed to describe the replication and processing of linear DNA with hairpin telomeres [33]. Uncertainties arise about the specific mode of replication and whether it occurs uni- or bi-directionally. Other important factors that need to be determined are, where in the plasmid replication is initiated from and what the replication intermediates are. Bacteriophage N15 can be used as the model system to explore these questions, the general organization of its genome is shown in **Figure 4**. Genes have been largely identified by homology inferred from sequence similarity to other bacteriophages; mainly lambda, HK97 and HK002 [1]. The division between the left- and right-hand side of the N15 genome is marked by *telRL*. The left arm encodes structural proteins required for N15 head and tail assembly. The right-arm contains more unusual genes and only 10 of the 35 have identified homologs in other lambdoid phage [1]. These are therefore much harder to characterize, and it is yet to be determined how they all function during N15

*RepA* is the only gene essential for replication of prophage N15 DNA [18]. It encodes a large, multifunctional protein that has both primase and helicase activities [35]. Sequence alignments have highlighted regions of RepA with similarities to both plasmid and viral DNA replication proteins [1]. Most notably, the phage P4 alpha protein [1] also has combined primase and helicase activities [36]. Phage P4 replication occurs by a theta-mechanism [37]. The similarities between alpha protein and RepA, combined with studies measuring amplification rates of DNA markers [18], strongly suggests that typical bidirectional theta-replication also occurs in N15 prophage. The origin of replication (*ori*) resides within the *repA* gene

The gene *telN* encodes a 71 kDa protein that has partial homology to integrases and an amino acid sequence characteristic of those that bind DNA as homo- or hetero- dimers [38, 39]. This was correctly identified as the protelomerase encoding

*The chromosome of bacteriophage N15. 46.4 kb double stranded DNA with 12-bp single stranded cohesive termini (cosL and cosR). Arrows indicate the direction of transcription and the telRL site divides the sequence* 

[18], which is located closer to the left hairpin end of the plasmid.

*DOI: http://dx.doi.org/10.5772/intechopen.86882*

capsid until it is full [32].

replication.

**5. Bacteriophage N15 replication**

*The Unusual Linear Plasmid Generating Systems of Prokaryotes DOI: http://dx.doi.org/10.5772/intechopen.86882*

*Bacteriophages - Perspectives and Future*

the whole palindromic sequence is required [28].

In vitro studies have also involved truncating target sites in order to identify the minimum sequence required for protelomerase binding and telomere resolution. To date, the minimal site identified that can be resolved, is a 26-bp substrate of TelA [7]. This was found by systematically deleting residues from both sides of the target sequence, until no product was produced. Similar studies have been performed on the TelN substrate. **Figure 2** shows the complete telomere occupancy (*tos*) site, which consists of a 56-bp palindromic sequence flanked by a series of inverted repeats. Initially, it was believed that *telO* is insufficient for processing by TelN, and the reaction requires the whole *telRL* site [16]. However, it has since been found that at greater TelN concentrations, roughly 50-fold higher than those required for *telRL*, the *telO* substrate is processed [26]. This indicates *telO* contains all the necessary elements for telomere resolution, but the protein requires additional sequence for binding and recognition. The binding affinity of TelN is greater still when the whole *tos* site is included in the substrate [16]. Experiments performed using ResT have explored whether the protelomerase is able to mediate cleavage on half a target site. When this half site was in a plasmid, the assay failed to produce reaction products, therefore suggesting dimer formation is essential for activity and

In order to further appreciate how protelomerases function, it is necessary to understand their role in relation to the whole phage or bacterial cell life-cycle. Bacteriophage N15 has been extensively characterised and **Figure 3** illustrates the different structures its genetic material forms upon infection of *E. coli*. The phage

*Forms of bacteriophage N15 DNA. Having infected an* E. coli *cell, the virion DNA circularizes, via* 

*covalently closed hairpin ends. Figure adapted from [1].*

*complementary* cos *sites. Lytic or lysogenic replication can be initiated from the circular intermediate. Shown is the pathway for lysogeny, whereby the telRL is processed by protelomerases to form linear prophage DNA with* 

**3.1 Minimal substrate**

**4. Linear genomes**

**58**

**Figure 3.**

DNA is a 46.4 kb chromosome that has two cohesive end sites (*cos*) consisting of 12-nucleotide overhangs at each 5′-end. These sites are complementary and can be ligated to form circular DNA. This circular intermediate then acts as the starting material for either lytic (not shown in **Figure 3**) or lysogenic development. During lysogenic development the *telRL* site is recognized and processed by protelomerases, this reaction forms a linear DNA structure with covalently closed ends. A similar genome arrangement has been identified for φKO2 [27], VP58.5 [29], VP882 [2] and PY54 [30]. These prophages all have cohesive (*cos*) ends that presumably enable the formation of similar structures as those described for N15. Interestingly, no *cos* site has been identified in the ΦHAP1 genome [31], therefore indicating a different mechanism of DNA packing. The discovery of terminase genes [31], suggests that headful packing may occur, whereby concatermeric DNA is packed into the phage capsid until it is full [32].

#### **5. Bacteriophage N15 replication**

Various models have been proposed to describe the replication and processing of linear DNA with hairpin telomeres [33]. Uncertainties arise about the specific mode of replication and whether it occurs uni- or bi-directionally. Other important factors that need to be determined are, where in the plasmid replication is initiated from and what the replication intermediates are. Bacteriophage N15 can be used as the model system to explore these questions, the general organization of its genome is shown in **Figure 4**. Genes have been largely identified by homology inferred from sequence similarity to other bacteriophages; mainly lambda, HK97 and HK002 [1]. The division between the left- and right-hand side of the N15 genome is marked by *telRL*. The left arm encodes structural proteins required for N15 head and tail assembly. The right-arm contains more unusual genes and only 10 of the 35 have identified homologs in other lambdoid phage [1]. These are therefore much harder to characterize, and it is yet to be determined how they all function during N15 replication.

*RepA* is the only gene essential for replication of prophage N15 DNA [18]. It encodes a large, multifunctional protein that has both primase and helicase activities [35]. Sequence alignments have highlighted regions of RepA with similarities to both plasmid and viral DNA replication proteins [1]. Most notably, the phage P4 alpha protein [1] also has combined primase and helicase activities [36]. Phage P4 replication occurs by a theta-mechanism [37]. The similarities between alpha protein and RepA, combined with studies measuring amplification rates of DNA markers [18], strongly suggests that typical bidirectional theta-replication also occurs in N15 prophage. The origin of replication (*ori*) resides within the *repA* gene [18], which is located closer to the left hairpin end of the plasmid.

The gene *telN* encodes a 71 kDa protein that has partial homology to integrases and an amino acid sequence characteristic of those that bind DNA as homo- or hetero- dimers [38, 39]. This was correctly identified as the protelomerase encoding

#### **Figure 4.**

*The chromosome of bacteriophage N15. 46.4 kb double stranded DNA with 12-bp single stranded cohesive termini (cosL and cosR). Arrows indicate the direction of transcription and the telRL site divides the sequence into left- and right-hand arms. Adapted from [34].*

gene and in 2000, Deneke and colleagues purified its protein product [16]. TelN is capable of processing the 56-bp *telRL* site in both linear and circular supercoiled DNA [16]. To decipher the mechanism of N15 genomic replication, mutants deficient in this protein have been created [40]. In protelomerase-deficient cells, unprocessed replicative intermediates accumulate, the structures of which have been characterised as circular head-to-head dimer molecules [40].

**Figure 5** describes how these linear N15 constructs may be replicated and processed; it is consistent with the data cited above and proposes structures that have been validated by electron microscopy [18]. In pathway A, following replication of the *telL* site, TelN processes the DNA to create a Y-shaped structure. After duplication of *telR*, the right telomere is also modified to form the final linear product. Alternatively, in pathway B the whole DNA molecule has been replicated, producing a head-to-head circular dimer that is then resolved. Interestingly, this mechanism of replication is distinct from that described for eukaryotic replicons, even those with similar telomeric ends, therefore suggesting an independent evolution [18].

#### **5.1 Lytic replication**

A model of how N15 lytic replication could occur is proposed in pathway C of **Figure 2**. The DNA is duplicated and resolved into two circular monomers, as opposed to linear structures. These circular molecules are the starting material for subsequent cycles of amplification. This style of lytic replication is similar to that of phage lambda. This bacteriophage also circularizes its DNA upon entering the host cell, it has *cos* sites analogous to those of N15 [1]. Further similarities between N15 and lambda include: genome length, burst size, latent period, lysogenization frequency and phage particle and plaque morphology [38]. Their structural and packing proteins are also highly analogous, making it likely that N15 DNA packing follows a pathway similar to that of lambda [1]. It has even

#### **Figure 5.**

*Model for lytic and lysogenic replication of N15 linear prophage. Bi-directional theta-replication begins at the internal* ori *site. A: Duplicated* telL *sites are processed before complete replication of genome. B: The template is completely duplicated prior to processing by TelN. C: Lytic replication, whereby circular monomers are produced that then undergo subsequent rounds of replication [18].*

**61**

*The Unusual Linear Plasmid Generating Systems of Prokaryotes*

**5.2 N15 as a model for the replication of other linear plasmids**

model comparable to that proposed for N15.

To what extent can the model of bacteriophage N15 be extended to describe the replication of other replicons with hairpin ends? Genomic sequence analysis of phage encoded protelomerases reveal little overall sequence similarity [43]. However, the organization of functional domains is analogous and they appear to have conserved regulatory regions [43]. This would suggest a shared mechanism of plasmid replication and lysogeny control [43]. Virions of φKO2, VP58.5, VP882 and PY54 have cohesive ends [27, 29, 2], which facilitate circularization and enable the formation of similar structures as those described for N15. The absence of *cos* sites in the ΦHAP1 genome has already been discussed and suggests a different mechanism of DNA packing [31]. Importantly, these phages all have homologs of the N15 protelomerase and replication protein RepA. The genes encoding these proteins are found between the lysogeny control region and structural gene cluster, as is the case is N15 [43]. Although yet to be confirmed by in vitro studies, given these similarities it is sensible to suggest that replication of these linear phage plasmids follows a

Further comparisons can be made between the suggested phage model and that of bacterial linear chromosome replication. *B. burgdorferi* has a linear chromosome [44], and it is replicated in a bi-directional manner to produce circular, head-tohead intermediates [45], which are then processed by telomere resolution [46]. Here replication is also initiated at an internal *ori* site and the protelomerase, ResT,

been demonstrated that the N15 specific terminase can package lambda DNA

The key difference between N15 and lambda bacteriophage is that lambda integrates its DNA into the host genome, whereas N15 does not. Although protelomerases share some sequence homology with lambda integrases, and both appear to have comparable roles in helping establish prophage DNA, these proteins are not functional analogues. During lytic replication the lambda integrase is dispensable, in comparison the protelomerase of N15 is essential. This phenomenon has been proven by experiments showing N15 deficient in protelomerases are incapable of infecting *E. coli* cells [35], although why this is the case remains unclear. As the establishment of lytic growth requires the conversion of linear plasmid molecules to circular ones, it could be presumed a protelomerase mediated "telomere fusion" reaction occurs. However, TelK, which is highly analogous to TelN, is incapable of catalyzing this in vitro [21], it is therefore highly unlikely wild-type TelN is functioning in this way. The possibility of an unknown factor modifying the protelomerase and/or its target site to prevent the usual processing reaction cannot be ruled out. In lambda, Xis, assisted by the host factor Fis, is necessary to induce excision during induction of a lysogeny [42]. Potentially, an analogue could be encoded by one of the N15 late genes [35], although experimental evidence to support this theory is yet to be provided. However, it has been demonstrated that mutating histidine 415 of TelN to an alanine results in accumulation of circular head-to-tail monomers [35]. This histidine is important for catalytic activity and is believed to coordinate the scissile phosphate [16]. Interestingly, its mutation does not have the same effect as mutating the catalytic tyrosine 424, which acts as the nucleophile in telomere resolution. When this residue is changed to an alanine, accumulation of circular dimers does occur, but in this case, they are "head-to-head" as opposed to "headto-tail" [35]. The significance of this observation is currently unknown. Given that TelN cannot be recycled [27], it has also been proposed that the protein's depletion will result in fewer linear molecules being produced and a natural accumulation of head-to-head dimers, which can then be processed to circular structures [35].

*DOI: http://dx.doi.org/10.5772/intechopen.86882*

with reasonable efficiency [41].

#### *The Unusual Linear Plasmid Generating Systems of Prokaryotes DOI: http://dx.doi.org/10.5772/intechopen.86882*

*Bacteriophages - Perspectives and Future*

**5.1 Lytic replication**

gene and in 2000, Deneke and colleagues purified its protein product [16]. TelN is capable of processing the 56-bp *telRL* site in both linear and circular supercoiled DNA [16]. To decipher the mechanism of N15 genomic replication, mutants deficient in this protein have been created [40]. In protelomerase-deficient cells, unprocessed replicative intermediates accumulate, the structures of which have

**Figure 5** describes how these linear N15 constructs may be replicated and processed; it is consistent with the data cited above and proposes structures that have been validated by electron microscopy [18]. In pathway A, following replication of the *telL* site, TelN processes the DNA to create a Y-shaped structure. After duplication of *telR*, the right telomere is also modified to form the final linear product. Alternatively, in pathway B the whole DNA molecule has been replicated, producing a head-to-head circular dimer that is then resolved. Interestingly, this mechanism of replication is distinct from that described for eukaryotic replicons, even those with

similar telomeric ends, therefore suggesting an independent evolution [18].

A model of how N15 lytic replication could occur is proposed in pathway C of **Figure 2**. The DNA is duplicated and resolved into two circular monomers, as opposed to linear structures. These circular molecules are the starting material for subsequent cycles of amplification. This style of lytic replication is similar to that of phage lambda. This bacteriophage also circularizes its DNA upon entering the host cell, it has *cos* sites analogous to those of N15 [1]. Further similarities between N15 and lambda include: genome length, burst size, latent period, lysogenization frequency and phage particle and plaque morphology [38]. Their structural and packing proteins are also highly analogous, making it likely that N15 DNA packing follows a pathway similar to that of lambda [1]. It has even

*Model for lytic and lysogenic replication of N15 linear prophage. Bi-directional theta-replication begins at the internal* ori *site. A: Duplicated* telL *sites are processed before complete replication of genome. B: The template is completely duplicated prior to processing by TelN. C: Lytic replication, whereby circular monomers are* 

*produced that then undergo subsequent rounds of replication [18].*

been characterised as circular head-to-head dimer molecules [40].

**60**

**Figure 5.**

been demonstrated that the N15 specific terminase can package lambda DNA with reasonable efficiency [41].

The key difference between N15 and lambda bacteriophage is that lambda integrates its DNA into the host genome, whereas N15 does not. Although protelomerases share some sequence homology with lambda integrases, and both appear to have comparable roles in helping establish prophage DNA, these proteins are not functional analogues. During lytic replication the lambda integrase is dispensable, in comparison the protelomerase of N15 is essential. This phenomenon has been proven by experiments showing N15 deficient in protelomerases are incapable of infecting *E. coli* cells [35], although why this is the case remains unclear. As the establishment of lytic growth requires the conversion of linear plasmid molecules to circular ones, it could be presumed a protelomerase mediated "telomere fusion" reaction occurs. However, TelK, which is highly analogous to TelN, is incapable of catalyzing this in vitro [21], it is therefore highly unlikely wild-type TelN is functioning in this way. The possibility of an unknown factor modifying the protelomerase and/or its target site to prevent the usual processing reaction cannot be ruled out. In lambda, Xis, assisted by the host factor Fis, is necessary to induce excision during induction of a lysogeny [42]. Potentially, an analogue could be encoded by one of the N15 late genes [35], although experimental evidence to support this theory is yet to be provided. However, it has been demonstrated that mutating histidine 415 of TelN to an alanine results in accumulation of circular head-to-tail monomers [35]. This histidine is important for catalytic activity and is believed to coordinate the scissile phosphate [16]. Interestingly, its mutation does not have the same effect as mutating the catalytic tyrosine 424, which acts as the nucleophile in telomere resolution. When this residue is changed to an alanine, accumulation of circular dimers does occur, but in this case, they are "head-to-head" as opposed to "headto-tail" [35]. The significance of this observation is currently unknown. Given that TelN cannot be recycled [27], it has also been proposed that the protein's depletion will result in fewer linear molecules being produced and a natural accumulation of head-to-head dimers, which can then be processed to circular structures [35].

#### **5.2 N15 as a model for the replication of other linear plasmids**

To what extent can the model of bacteriophage N15 be extended to describe the replication of other replicons with hairpin ends? Genomic sequence analysis of phage encoded protelomerases reveal little overall sequence similarity [43]. However, the organization of functional domains is analogous and they appear to have conserved regulatory regions [43]. This would suggest a shared mechanism of plasmid replication and lysogeny control [43]. Virions of φKO2, VP58.5, VP882 and PY54 have cohesive ends [27, 29, 2], which facilitate circularization and enable the formation of similar structures as those described for N15. The absence of *cos* sites in the ΦHAP1 genome has already been discussed and suggests a different mechanism of DNA packing [31]. Importantly, these phages all have homologs of the N15 protelomerase and replication protein RepA. The genes encoding these proteins are found between the lysogeny control region and structural gene cluster, as is the case is N15 [43]. Although yet to be confirmed by in vitro studies, given these similarities it is sensible to suggest that replication of these linear phage plasmids follows a model comparable to that proposed for N15.

Further comparisons can be made between the suggested phage model and that of bacterial linear chromosome replication. *B. burgdorferi* has a linear chromosome [44], and it is replicated in a bi-directional manner to produce circular, head-tohead intermediates [45], which are then processed by telomere resolution [46]. Here replication is also initiated at an internal *ori* site and the protelomerase, ResT,

is known to be essential [47]. These findings indicate a shared fundamental mechanism of genomic replication between N15 and *Borrelia*. Nonetheless, discrepancies between the different systems have been highlighted. For one, in these bacterial cells the protelomerase is encoded, not on the same DNA construct it processes, but on a different circular plasmid, cp26 [3]. In addition, the possibility of *Borrelia* accessory factors influencing telomere resolution was suggested, following the observation that differential processing occurs in vitro compared to in vivo [15]. Potentially this could be a result of in vitro conditions not completely reconstructing those occurring in vivo. However, if correct it would indicate important discrepancies between how bacterial and phage protelomerases are regulated.

#### **6. Structural data**

#### **6.1 X-ray crystallography**

The X-ray structures described for both TelK and TelA have greatly enhanced our understanding of the protelomerase mechanism [21, 22]. The structure of TelK (from phage φKO2), is shown in **Figure 6**; crystallized in a dimer conformation

#### **Figure 6.**

*Crystal structure of TelK dimer complexed with DNA. (a) Two monomers of TelK dimers at the recognition site and are held together by multiple transient protein-protein and protein-DNA interactions. The structure of each monomer is largely alpha helical, with mixed beta strands at the catalytic domain. (b) The same complex viewed from the N-terminus. The helical linker fits in the major groove of the DNA, it contacts the DNA on the opposite side to the rest of the protein. The structures presented were generated with using ChimeraX (Goddard et al., 2018) PDB ID: 2v6e [21].*

**63**

**Figure 7.**

*important for site recognition. Adapted from [21].*

*The Unusual Linear Plasmid Generating Systems of Prokaryotes*

complexed with the minimal cognate DNA sequence of 44-bp [PDB: 2V6E]. TelK has been divided into three core domains, all of which make contact with the DNA. These include, the muzzle at the N-terminus, the catalytic domain in the center, and the stirrup domain at the C-terminus. A long alpha-helical linker is also

One of the most striking observations of this structure is the level distortion: DNA is bent at roughly 73° parallel to the axis of symmetry [21]. This provides a valuable insight into the mechanism of telomere resolution by TelK and would appear to refute previous theories that the DNA is forced into a cruciform conformation [27]. Core substrate binding occurs at the N-terminus and the muzzle makes

*TelK substrate recognition. One half of the TelK recognition sequence, nucleotides forming hydrogen bonds to protein are highlighted in cyan and those forming van der Waals interactions, in pink. Multiple amino acids interact with the DNA backbone and are not shown, those forming bonds with the bases are illustrated in red. Adenine at position 42 is circled, this residue forms hydrogen bonds with serine 68 of TelK and is therefore* 

highlighted, and this connects the core catalytic and N-terminal domains.

*DOI: http://dx.doi.org/10.5772/intechopen.86882*

*The Unusual Linear Plasmid Generating Systems of Prokaryotes DOI: http://dx.doi.org/10.5772/intechopen.86882*

*Bacteriophages - Perspectives and Future*

**6. Structural data**

**6.1 X-ray crystallography**

is known to be essential [47]. These findings indicate a shared fundamental mechanism of genomic replication between N15 and *Borrelia*. Nonetheless, discrepancies between the different systems have been highlighted. For one, in these bacterial cells the protelomerase is encoded, not on the same DNA construct it processes, but on a different circular plasmid, cp26 [3]. In addition, the possibility of *Borrelia* accessory factors influencing telomere resolution was suggested, following the observation that differential processing occurs in vitro compared to in vivo [15]. Potentially this could be a result of in vitro conditions not completely reconstructing those occurring in vivo. However, if correct it would indicate important discrepancies between how bacterial and phage protelomerases are regulated.

The X-ray structures described for both TelK and TelA have greatly enhanced our understanding of the protelomerase mechanism [21, 22]. The structure of TelK (from phage φKO2), is shown in **Figure 6**; crystallized in a dimer conformation

**62**

**Figure 6.**

*et al., 2018) PDB ID: 2v6e [21].*

*Crystal structure of TelK dimer complexed with DNA. (a) Two monomers of TelK dimers at the recognition site and are held together by multiple transient protein-protein and protein-DNA interactions. The structure of each monomer is largely alpha helical, with mixed beta strands at the catalytic domain. (b) The same complex viewed from the N-terminus. The helical linker fits in the major groove of the DNA, it contacts the DNA on the opposite side to the rest of the protein. The structures presented were generated with using ChimeraX (Goddard* 

complexed with the minimal cognate DNA sequence of 44-bp [PDB: 2V6E]. TelK has been divided into three core domains, all of which make contact with the DNA. These include, the muzzle at the N-terminus, the catalytic domain in the center, and the stirrup domain at the C-terminus. A long alpha-helical linker is also highlighted, and this connects the core catalytic and N-terminal domains.

One of the most striking observations of this structure is the level distortion: DNA is bent at roughly 73° parallel to the axis of symmetry [21]. This provides a valuable insight into the mechanism of telomere resolution by TelK and would appear to refute previous theories that the DNA is forced into a cruciform conformation [27]. Core substrate binding occurs at the N-terminus and the muzzle makes

#### **Figure 7.**

*TelK substrate recognition. One half of the TelK recognition sequence, nucleotides forming hydrogen bonds to protein are highlighted in cyan and those forming van der Waals interactions, in pink. Multiple amino acids interact with the DNA backbone and are not shown, those forming bonds with the bases are illustrated in red. Adenine at position 42 is circled, this residue forms hydrogen bonds with serine 68 of TelK and is therefore important for site recognition. Adapted from [21].*

#### *Bacteriophages - Perspectives and Future*

extensive contacts with the opposite subunit, strengthening the protein's structure and pushing the DNA into this strained conformation. The catalytic site is formed at the dimer interface, it binds to the opposite side of the substrate relative to the N-terminus and the helical linker that connects these two domains is fixed in the major groove. Extensive electrostatic interactions mediate the interaction.

Closer examination of the interactions between the DNA and protein reveal seven nucleotides that form hydrogen bonds with nearby residues (shown in cyan in **Figure 7**) and are presumed key for substrate recognition. This model is supported by the previously cited studies, whereby the natural PY54 substrate was effectively mutated into a sequence that could be processed by TelK. Adenine at position 42 is circled; this is one of the bases identified as forming hydrogen bonds with TelK and is one of the points that required mutating in order to form a cognate sequence. High salt has been shown to inhibit telomere resolution by protelomerases [16], this is possibly reflected by the extensive hydrophilic protein-DNA interactions that would be disrupted by an excess of ions.

#### **Figure 8.**

*Dimers of TelK and TelA complexed with DNA. The DNA is forced into the same disjointed structure down its helical axis [22]. The structures presented were generated with UCSF Chimera (Pettersen, at al., 2004) using PDB 2v6e (TelK) [21] and 4e0p (TelA) [22].*

**65**

**Figure 9.**

*The Unusual Linear Plasmid Generating Systems of Prokaryotes*

The stirrup domain of TelK has a winged helix-turn-helix motif [21], it makes few contacts with the rest of the protein but extends the DNA binding interface. In stabilizing the strained substrate conformation, this part of the protein aids hairpin formation; however, it is nonessential for the cleavage reaction [21]. Following strand cleavage, the stored energy is released, and this drives dimer dissolution which is proceeded by spontaneous hairpin formation. The stirrup is not conserved among protelomerases and this provides further evidence to support the theory that

The structure of the bacterial protelomerase, TelA has also been described via X-ray crystallography. TelA is considerably smaller than TelK, it lacks the stirrup and only consists of the catalytic and N-terminal domains. This design is similar to that of tyrosine recombinases, which are also typically composed of two domains [48]. The N-terminal 100 residues are poorly resolved in comparison to the rest of the protein, this area of low electron density suggests flexibility of the polypeptide chain. Comparing dimer substrate complexes of TelA to those of TelK (**Figure 8**) reveals a similar DNA conformation at the dimer interface. Extensive hydrogen bonds and van der Waals interactions are also involved in dictating the substrate specificity of TelA and the DNA exhibits the same disjunction down its

The catalytic domain of TelK is a mixed alpha beta structure. **Figure 9** shows the core catalytic residues of TelK R275, K300, K380, R383 and H416. These act together to maintain catalytic activity and coordinate nucleophilic attack of the tyrosine. Side chains of the basic amino acids at positions one, three and four provide a hydrogen bonding network that coordinates the scissile phosphate and stabilizes the transition state [22]. In type IB topoisomerases, the pentad is usually composed of RKKRH/N [49], with basic residues 1 and 3 having the same stabilization effect. In these proteins, the second lysine residue has been shown to donate a proton to the

*Conformation of protelomerase active site residues. The residues of TelK, R275, K300, K380, R383 and H416 maintain the active site conformation and coordinate nucleophilic attack of the tyrosine. The structures* 

*presented were generated with ChimeraX (Goddard, T et al., 2018) using PDB 2v6e [21].*

the mechanism of telomere resolution varies between different systems.

*DOI: http://dx.doi.org/10.5772/intechopen.86882*

helical axis [22].

**6.2 Catalytic domain**

*The Unusual Linear Plasmid Generating Systems of Prokaryotes DOI: http://dx.doi.org/10.5772/intechopen.86882*

The stirrup domain of TelK has a winged helix-turn-helix motif [21], it makes few contacts with the rest of the protein but extends the DNA binding interface. In stabilizing the strained substrate conformation, this part of the protein aids hairpin formation; however, it is nonessential for the cleavage reaction [21]. Following strand cleavage, the stored energy is released, and this drives dimer dissolution which is proceeded by spontaneous hairpin formation. The stirrup is not conserved among protelomerases and this provides further evidence to support the theory that the mechanism of telomere resolution varies between different systems.

The structure of the bacterial protelomerase, TelA has also been described via X-ray crystallography. TelA is considerably smaller than TelK, it lacks the stirrup and only consists of the catalytic and N-terminal domains. This design is similar to that of tyrosine recombinases, which are also typically composed of two domains [48]. The N-terminal 100 residues are poorly resolved in comparison to the rest of the protein, this area of low electron density suggests flexibility of the polypeptide chain. Comparing dimer substrate complexes of TelA to those of TelK (**Figure 8**) reveals a similar DNA conformation at the dimer interface. Extensive hydrogen bonds and van der Waals interactions are also involved in dictating the substrate specificity of TelA and the DNA exhibits the same disjunction down its helical axis [22].

#### **6.2 Catalytic domain**

*Bacteriophages - Perspectives and Future*

would be disrupted by an excess of ions.

extensive contacts with the opposite subunit, strengthening the protein's structure and pushing the DNA into this strained conformation. The catalytic site is formed at the dimer interface, it binds to the opposite side of the substrate relative to the N-terminus and the helical linker that connects these two domains is fixed in the major groove. Extensive electrostatic interactions mediate the interaction.

Closer examination of the interactions between the DNA and protein reveal seven nucleotides that form hydrogen bonds with nearby residues (shown in cyan in **Figure 7**) and are presumed key for substrate recognition. This model is supported by the previously cited studies, whereby the natural PY54 substrate was effectively mutated into a sequence that could be processed by TelK. Adenine at position 42 is circled; this is one of the bases identified as forming hydrogen bonds with TelK and is one of the points that required mutating in order to form a cognate sequence. High salt has been shown to inhibit telomere resolution by protelomerases [16], this is possibly reflected by the extensive hydrophilic protein-DNA interactions that

**64**

**Figure 8.**

*PDB 2v6e (TelK) [21] and 4e0p (TelA) [22].*

*Dimers of TelK and TelA complexed with DNA. The DNA is forced into the same disjointed structure down its helical axis [22]. The structures presented were generated with UCSF Chimera (Pettersen, at al., 2004) using* 

The catalytic domain of TelK is a mixed alpha beta structure. **Figure 9** shows the core catalytic residues of TelK R275, K300, K380, R383 and H416. These act together to maintain catalytic activity and coordinate nucleophilic attack of the tyrosine. Side chains of the basic amino acids at positions one, three and four provide a hydrogen bonding network that coordinates the scissile phosphate and stabilizes the transition state [22]. In type IB topoisomerases, the pentad is usually composed of RKKRH/N [49], with basic residues 1 and 3 having the same stabilization effect. In these proteins, the second lysine residue has been shown to donate a proton to the

#### **Figure 9.**

*Conformation of protelomerase active site residues. The residues of TelK, R275, K300, K380, R383 and H416 maintain the active site conformation and coordinate nucleophilic attack of the tyrosine. The structures presented were generated with ChimeraX (Goddard, T et al., 2018) using PDB 2v6e [21].*

5′-OH leaving group, which aids its removal during cleavage [50]. It is feasible that the lysine in the protelomerase's active site, with its side chain positioned between the DNA O5' and nonbridging oxygen, also functions in this way and protonates the leaving group [21].

These crystal structures are invaluable when trying to decipher the mechanism of protelomerase-catalyzed telomere resolution. TelN and TelK have highly homologous sequences and can process the same target site [27], it is therefore likely the structures of these enzymes are analogous and information about TelN can be derived from examining the TelK structure. However, is important to note that the crystallized structure of TelK is not full length and lacks 100 residues from the C-terminus. This does not appear to significantly affect protein activity in vitro [21], but the significance in vivo is unconfirmed.

#### **7. Evolutionary history**

It is widely believed Tyrosine recombinases and type IB topoisomerases have arisen from a common ancestor [50]. These structurally and mechanically analogous proteins catalyze important DNA rearrangements, they use an active site tyrosine residue and form covalently bound protein-DNA intermediates. Similarities between this mechanism, and that of protelomerases, has led to the suggestion that these protein classes have a shared evolutionary relationship [51].

Key to the function of tyrosine recombinases is the conserved catalytic motif: "RKHRH" [52, 53]. Crystal structures and sequence alignments reveal that protelomerases share this catalytic pentad [27], with exception of the middle residue, which varies between systems. Deviation at this central point is also observed in tyrosine recombinases, where the central histidine may be replaced by an arginine, asparagine, lysine or tyrosine. In protelomerases; TelN and TelK both have a methionine at this position, PY54 contains a lysine, VHML has a histidine and the bacterial protelomerases of *B. burgdorferi* and *A. tumefaciens*, have a tyrosine. It has been found substituting the tyrosine to a histidine or lysine in TelA is completely tolerable and results in no loss of activity [7]. Whether these variations are of significance is yet to be determined.

Structural comparisons outside of the catalytic domain reveal low overall sequence homology at both the N- and C- terminus. ResT is smaller than the phage proteins, partial proteolysis separates the 449 amino acid protein into two domains [54], and sequence analysis predicts its architecture is more comparable to that typical of tyrosine recombinases. In addition, this protein has the unusual and surprising ability to synapse Holliday Junctions [51]. The reaction appears to be favored in conditions that are counterproductive to telomere resolution, such as negative supercoiling, or unsymmetrical *telR* sites [51]. This observation provides compelling evidence for the argument that ResT may have evolved from a recombinase [51]. Although the significance of Holliday Junction formation by ResT is still under investigation, it has been suggested this could be an obsolete ancestral property of the protein [4]. Additional evidence highlighting a relationship between tyrosine recombinases and protelomerases comes from the Flp recombinase and lambda integrase. It has been found that, under specific conditions, these enzymes have the ability to form hairpin products [55, 56]. Thus, indicating the relative ease with which recombinases may be converted to telomere resolving enzymes.

In ResT, additional catalytic residues outside of the core pentad are required for telomere resolution [57]. This indicates digression from a typical tyrosine recombinase type mechanism. Furthermore, mutation of ResT histidine 324, the fifth residue of the catalytic motif does not result in total loss of activity [57]. This would

**67**

*The Unusual Linear Plasmid Generating Systems of Prokaryotes*

mark an obvious disparity between these two classes of enzymes, if it was not for the observation that this residue is also not essential in Flp recombinases [58]. Here it appears the final amino acid has a structural rather than catalytic role [58], which

protelomerases and tyrosine recombinases. Aside from ResT, which it has been suggested can act on more than one target site, protelomerases are specific and have stringent substrate sequence requirements. The target site of one recombinase often includes many other related points, such as phage or bacterial attachment sites. Furthermore, the reaction is intramolecular and can require auxiliary proteins [59]. This implies a mechanism more involved and complex than that of telomere resolution. The reaction catalyzed by type IB topoisomerases is considerably simpler, as these proteins act as monomers [60] and do not display the same sequence specific-

In conclusion, there are key differences between the catalytic mechanism of protelomerases and tyrosine recombinases/type IB topoisomerases. However, there are also significant similarities and whether these proteins have evolved from a common ancestor is difficult to determine. It is expected that the conversion of a tyrosine recombinase to an enzyme capable of telomere resolution would be accompanied by the linearization of plasmid DNA [4]. The data suggesting this could be achieved with relative ease adds support to the argument these proteins are related. Under certain conditions, recombinases can have topoisomerase activity, and topoisomerases can affect DNA strand exchanges [27]. This raises an interesting question as to whether protelomerases, under the correct conditions, may also

The unique properties of protelomerases, and the DNA structures they produce, makes them a valuable class of protein that have important applications in synthetic biology and biotechnology. Linear DNA does not exhibit the supercoiling associated with plasmids, as the ends are free to rotate. This makes them stable vectors invaluable for cloning difficult sequences. DNA that is rich in adenine and thymidine, or contains lots of short tandem repeats is typically hard, if not impossible, to clone into circular vectors [10]. A commercially available cloning vector, pJAZZ from Lucigen, is based on the linear N15 phage genome. pJAZZ is sold as part of a cloning system (BigEasy Kit), enabling the insertion of otherwise unclonable sequences into the vector for the creation of viable linear plasmids that can be transformed into cells. pJAZZ vectors encode RepA and TelN, essential for bidirectional replication and telomere resolution. Transcriptional terminators flank the cloning site, thus minimizing interference and preventing transcription into and out of this region. These modifications extend the cloning possibilities and allow for the inser-

The pJAZZ system has been further modified to specifically enhance its efficiency for the production of in vitro transcribed (IVT) mRNA. IVT mRNA is a powerful therapeutic tool, enabling the transient expression of heterologous proteins [61, 62]. However, in order to optimize translation efficiency and mRNA stability, the poly(A) tail length of the mRNA needs to be defined and optimized [63]. In particular, mRNA with poly(A) tails >300 nucleotides and purines at the 3′ end have been demonstrated as highly effective and appear to exhibit improved translation properties compared to those with shorter poly(A) tracts [64]. Due to its linear structure, a pJAZZ derived plasmid, called p(Extended Variable Length)

Analysis of the DNA substrates reveal further fundamental differences between

*DOI: http://dx.doi.org/10.5772/intechopen.86882*

be able to exhibit topoisomerase activity [27].

**8. Applications in biotechnology**

tion of large cDNAs or operons [10].

could be mirrored in ResT.

ity as protelomerases.

*Bacteriophages - Perspectives and Future*

[21], but the significance in vivo is unconfirmed.

leaving group [21].

**7. Evolutionary history**

significance is yet to be determined.

5′-OH leaving group, which aids its removal during cleavage [50]. It is feasible that the lysine in the protelomerase's active site, with its side chain positioned between the DNA O5' and nonbridging oxygen, also functions in this way and protonates the

These crystal structures are invaluable when trying to decipher the mechanism of protelomerase-catalyzed telomere resolution. TelN and TelK have highly homologous sequences and can process the same target site [27], it is therefore likely the structures of these enzymes are analogous and information about TelN can be derived from examining the TelK structure. However, is important to note that the crystallized structure of TelK is not full length and lacks 100 residues from the C-terminus. This does not appear to significantly affect protein activity in vitro

It is widely believed Tyrosine recombinases and type IB topoisomerases have arisen from a common ancestor [50]. These structurally and mechanically analogous proteins catalyze important DNA rearrangements, they use an active site tyrosine residue and form covalently bound protein-DNA intermediates. Similarities between this mechanism, and that of protelomerases, has led to the suggestion that

Key to the function of tyrosine recombinases is the conserved catalytic motif: "RKHRH" [52, 53]. Crystal structures and sequence alignments reveal that protelomerases share this catalytic pentad [27], with exception of the middle residue, which varies between systems. Deviation at this central point is also observed in tyrosine recombinases, where the central histidine may be replaced by an arginine, asparagine, lysine or tyrosine. In protelomerases; TelN and TelK both have a methionine at this position, PY54 contains a lysine, VHML has a histidine and the bacterial protelomerases of *B. burgdorferi* and *A. tumefaciens*, have a tyrosine. It has been found substituting the tyrosine to a histidine or lysine in TelA is completely tolerable and results in no loss of activity [7]. Whether these variations are of

Structural comparisons outside of the catalytic domain reveal low overall sequence homology at both the N- and C- terminus. ResT is smaller than the phage proteins, partial proteolysis separates the 449 amino acid protein into two domains [54], and sequence analysis predicts its architecture is more comparable to that typical of tyrosine recombinases. In addition, this protein has the unusual and surprising ability to synapse Holliday Junctions [51]. The reaction appears to be favored in conditions that are counterproductive to telomere resolution, such as negative supercoiling, or unsymmetrical *telR* sites [51]. This observation provides compelling evidence for the argument that ResT may have evolved from a recombinase [51]. Although the significance of Holliday Junction formation by ResT is still under investigation, it has been suggested this could be an obsolete ancestral property of the protein [4]. Additional evidence highlighting a relationship between tyrosine recombinases and protelomerases comes from the Flp recombinase and lambda integrase. It has been found that, under specific conditions, these enzymes have the ability to form hairpin products [55, 56]. Thus, indicating the relative ease

with which recombinases may be converted to telomere resolving enzymes.

In ResT, additional catalytic residues outside of the core pentad are required for telomere resolution [57]. This indicates digression from a typical tyrosine recombinase type mechanism. Furthermore, mutation of ResT histidine 324, the fifth residue of the catalytic motif does not result in total loss of activity [57]. This would

these protein classes have a shared evolutionary relationship [51].

**66**

mark an obvious disparity between these two classes of enzymes, if it was not for the observation that this residue is also not essential in Flp recombinases [58]. Here it appears the final amino acid has a structural rather than catalytic role [58], which could be mirrored in ResT.

Analysis of the DNA substrates reveal further fundamental differences between protelomerases and tyrosine recombinases. Aside from ResT, which it has been suggested can act on more than one target site, protelomerases are specific and have stringent substrate sequence requirements. The target site of one recombinase often includes many other related points, such as phage or bacterial attachment sites. Furthermore, the reaction is intramolecular and can require auxiliary proteins [59]. This implies a mechanism more involved and complex than that of telomere resolution. The reaction catalyzed by type IB topoisomerases is considerably simpler, as these proteins act as monomers [60] and do not display the same sequence specificity as protelomerases.

In conclusion, there are key differences between the catalytic mechanism of protelomerases and tyrosine recombinases/type IB topoisomerases. However, there are also significant similarities and whether these proteins have evolved from a common ancestor is difficult to determine. It is expected that the conversion of a tyrosine recombinase to an enzyme capable of telomere resolution would be accompanied by the linearization of plasmid DNA [4]. The data suggesting this could be achieved with relative ease adds support to the argument these proteins are related. Under certain conditions, recombinases can have topoisomerase activity, and topoisomerases can affect DNA strand exchanges [27]. This raises an interesting question as to whether protelomerases, under the correct conditions, may also be able to exhibit topoisomerase activity [27].

#### **8. Applications in biotechnology**

The unique properties of protelomerases, and the DNA structures they produce, makes them a valuable class of protein that have important applications in synthetic biology and biotechnology. Linear DNA does not exhibit the supercoiling associated with plasmids, as the ends are free to rotate. This makes them stable vectors invaluable for cloning difficult sequences. DNA that is rich in adenine and thymidine, or contains lots of short tandem repeats is typically hard, if not impossible, to clone into circular vectors [10]. A commercially available cloning vector, pJAZZ from Lucigen, is based on the linear N15 phage genome. pJAZZ is sold as part of a cloning system (BigEasy Kit), enabling the insertion of otherwise unclonable sequences into the vector for the creation of viable linear plasmids that can be transformed into cells. pJAZZ vectors encode RepA and TelN, essential for bidirectional replication and telomere resolution. Transcriptional terminators flank the cloning site, thus minimizing interference and preventing transcription into and out of this region. These modifications extend the cloning possibilities and allow for the insertion of large cDNAs or operons [10].

The pJAZZ system has been further modified to specifically enhance its efficiency for the production of in vitro transcribed (IVT) mRNA. IVT mRNA is a powerful therapeutic tool, enabling the transient expression of heterologous proteins [61, 62]. However, in order to optimize translation efficiency and mRNA stability, the poly(A) tail length of the mRNA needs to be defined and optimized [63]. In particular, mRNA with poly(A) tails >300 nucleotides and purines at the 3′ end have been demonstrated as highly effective and appear to exhibit improved translation properties compared to those with shorter poly(A) tracts [64]. Due to its linear structure, a pJAZZ derived plasmid, called p(Extended Variable Length) (pEVL), can create poly(A) tracts of up to 500 bps in length. Furthermore, the residues at the 3′ end can be defined as either adenine or guanine. This has a significant advantage over conventional circular DNA, which cannot incorporate more than 174 bp of poly(A) tract without conferring extreme instability [64].

Mediphage Bioceuticals is a genetic medicine company that has also utilized the unique DNA processing capability of protelomerases for their technology. They have developed a one-step in vivo platform to produce linear covalently closed constructs, called ministring DNA [65]. These constructs are produced in *E. coli* cells that have been engineered to express the PY54 protelomerase under the control of a heat-inducible promoter. Following induction, the protelomerase processes precursor plasmids in the cell, in doing so it effectively separates the desired expression cassette from the bacterial plasmid backbone. The ministring DNA can then be purified and used as a vector for gene or cell therapies and gene editing. Although ministring production is reliant on large scale bacterial fermentation, the absence of bacterial DNA elements render these constructs preferential to plasmid DNA for medicinal applications. Furthermore, the constructs are typically smaller than plasmids, this enhances their transfection efficiency thus making them less toxic, as fewer transfection reagents are required [12]. They are also more resistant to the shear-induced degradation that large plasmids are highly susceptible to [66].

Another in vivo application of protelomerases has been explored by Katzen at colleagues, who used TelN to fragment an *E. coli* chromosome into smaller, autonomous units [67]. These proof of concept experiments were designed as a solution to the considerable difficulties associated with synthesizing and manipulating large, stable genetic elements. In splitting the chromosome into two smaller units, which together contain the essential components required for cell viability, they significantly simplified the problem. Not only are the smaller units of genetic material easier to manipulate, but each episome is of a size that it can be assembled without the need for an assembly host. This work may be extended to fragment and linearize other genomic elements of interest, in particular for the study of large units, >2 Mbp, which at present cannot be assembled and maintained in any biological platform [67].

Touchlight Genetics Ltd. has also utilized protelomerases for their technology. The platform they have developed is a purely in vitro DNA production process that eliminates all the major problems associated with using bacterial fermentation for DNA amplification. Their cell-free technology uses a phage DNA polymerase from Phi29 to produce large amounts of DNA concatemers from small amounts of starting plasmid DNA template. DNA concatemers are processed by the protelomerase TelN, to create linear covalently closed constructs that are marketed as dbDNA. The closed ended linear DNA construct is able to encode long, difficult DNA sequences which are not tolerated in high yield production of plasmid DNA due to selection pressures. The in vitro amplification technology produces DNA containing no bacterial origin of replication sequences or antibiotic selection marker. These vectors are capable of immunizing against influenza infection, with a response comparable to that of plasmid DNA [68], as well as improving tumor growth control in a Human papillomavirus (HPV) driven head and neck cancer model by delivering a therapeutic vaccine encoding for HPV16 E6 and E7 antigens [69]. Furthermore, dbDNA constructs have been used to generate functional lentiviral vectors [14] and are promising candidates for the production of recombinant adeno-associated virus (AAV) vectors [13]. This platform could therefore be important for gene therapy [70, 71] and this synthetic process for DNA production will have a broad range of application within the wider synthetic biology field.

**69**

*The Unusual Linear Plasmid Generating Systems of Prokaryotes*

mechanism and evolutionary history remain unanswered.

into how plasmid and phage can interact and evolve.

**Appendices and nomenclature**

bp base pair

**Conflict of interest**

dbDNA doggybone DNA

Cos cohesive end sites Ori origin of replication IVT in vitro transcribed

Tos Telomerase occupancy site

pEVL p(Extended Variable Length) AAV adeno-associated virus HPV Human papillomavirus

open up opportunities to produce variants with altered activities.

Protelomerases are an interesting and unique class of protein. In forming telomeric structures at the ends of linear plasmids, they protect the genetic material from degradation and provide a novel solution to the end replication problem. They have also been implemented as an essential component of certain human pathogens and have important applications in synthetic biology. Despite this, protelomerases remain poorly characterised and many questions about their structure, function,

Much of our current knowledge has been obtained by combining information from crystal structures, analysing sequences and performing in vitro assays with protein and substrate variations. The results of these studies have enabled us to compare the protelomerases from different systems and it is clear that there is much variety within this protein family. In particular, ResT has been identified as having additional, largely unexplainable, functionality aside from telomere resolution. Potentially further characterization of the other protelomerases will lead to similar

Biochemical analysis of protelomerases from ΦHAP1, PY54 and VP58.2, will shed further light on the underlying properties that differentiate these telomere resolving enzymes. Such work could also explore the evolution of protelomerases and prokaryotes with linear plasmids. Similarities in the genome organization of telomere phage suggest a common ancestor [43] and whether the bacterial proteins originated from these is currently unknown. Introducing different phage into the same cell and determining their compatibility can give insight into evolutionary background [72]. An understanding of the relationship between phage lambda and telomere phage, in particular N15, will provide an interesting and important insight

Crystal structures of TelK and TelA provide a solid starting point for research aiming to solve structures of protelomerases. These can also be used for homology modeling to infer information about proteins with high sequence similarities. Advances in the field of synthetic biology and protein engineering make detailed knowledge of these proteins even more valuable. An increased understanding of how they operate, and which parts are responsible for specific functionalities, will

SK has recently started a PhD jointly between Touchlight Genetics and Renos Savva; however, this has not relatively biased or affected the content of this chapter.

*DOI: http://dx.doi.org/10.5772/intechopen.86882*

**9. Conclusion**

revelations.

### **9. Conclusion**

*Bacteriophages - Perspectives and Future*

highly susceptible to [66].

platform [67].

(pEVL), can create poly(A) tracts of up to 500 bps in length. Furthermore, the residues at the 3′ end can be defined as either adenine or guanine. This has a significant advantage over conventional circular DNA, which cannot incorporate more than

Mediphage Bioceuticals is a genetic medicine company that has also utilized the unique DNA processing capability of protelomerases for their technology. They have developed a one-step in vivo platform to produce linear covalently closed constructs, called ministring DNA [65]. These constructs are produced in *E. coli* cells that have been engineered to express the PY54 protelomerase under the control of a heat-inducible promoter. Following induction, the protelomerase processes precursor plasmids in the cell, in doing so it effectively separates the desired expression cassette from the bacterial plasmid backbone. The ministring DNA can then be purified and used as a vector for gene or cell therapies and gene editing. Although ministring production is reliant on large scale bacterial fermentation, the absence of bacterial DNA elements render these constructs preferential to plasmid DNA for medicinal applications. Furthermore, the constructs are typically smaller than plasmids, this enhances their transfection efficiency thus making them less toxic, as fewer transfection reagents are required [12]. They are also more resistant to the shear-induced degradation that large plasmids are

Another in vivo application of protelomerases has been explored by Katzen at colleagues, who used TelN to fragment an *E. coli* chromosome into smaller, autonomous units [67]. These proof of concept experiments were designed as a solution to the considerable difficulties associated with synthesizing and manipulating large, stable genetic elements. In splitting the chromosome into two smaller units, which together contain the essential components required for cell viability, they significantly simplified the problem. Not only are the smaller units of genetic material easier to manipulate, but each episome is of a size that it can be assembled without the need for an assembly host. This work may be extended to fragment and linearize other genomic elements of interest, in particular for the study of large units, >2 Mbp, which at present cannot be assembled and maintained in any biological

Touchlight Genetics Ltd. has also utilized protelomerases for their technology. The platform they have developed is a purely in vitro DNA production process that eliminates all the major problems associated with using bacterial fermentation for DNA amplification. Their cell-free technology uses a phage DNA polymerase from Phi29 to produce large amounts of DNA concatemers from small amounts of starting plasmid DNA template. DNA concatemers are processed by the protelomerase TelN, to create linear covalently closed constructs that are marketed as dbDNA. The closed ended linear DNA construct is able to encode long, difficult DNA sequences which are not tolerated in high yield production of plasmid DNA due to selection pressures. The in vitro amplification technology produces DNA containing no bacterial origin of replication sequences or antibiotic selection marker. These vectors are capable of immunizing against influenza infection, with a response comparable to that of plasmid DNA [68], as well as improving tumor growth control in a Human papillomavirus (HPV) driven head and neck cancer model by delivering a therapeutic vaccine encoding for HPV16 E6 and E7 antigens [69]. Furthermore, dbDNA constructs have been used to generate functional lentiviral vectors [14] and are promising candidates for the production of recombinant adeno-associated virus (AAV) vectors [13]. This platform could therefore be important for gene therapy [70, 71] and this synthetic process for DNA production will have a broad range of

174 bp of poly(A) tract without conferring extreme instability [64].

**68**

application within the wider synthetic biology field.

Protelomerases are an interesting and unique class of protein. In forming telomeric structures at the ends of linear plasmids, they protect the genetic material from degradation and provide a novel solution to the end replication problem. They have also been implemented as an essential component of certain human pathogens and have important applications in synthetic biology. Despite this, protelomerases remain poorly characterised and many questions about their structure, function, mechanism and evolutionary history remain unanswered.

Much of our current knowledge has been obtained by combining information from crystal structures, analysing sequences and performing in vitro assays with protein and substrate variations. The results of these studies have enabled us to compare the protelomerases from different systems and it is clear that there is much variety within this protein family. In particular, ResT has been identified as having additional, largely unexplainable, functionality aside from telomere resolution. Potentially further characterization of the other protelomerases will lead to similar revelations.

Biochemical analysis of protelomerases from ΦHAP1, PY54 and VP58.2, will shed further light on the underlying properties that differentiate these telomere resolving enzymes. Such work could also explore the evolution of protelomerases and prokaryotes with linear plasmids. Similarities in the genome organization of telomere phage suggest a common ancestor [43] and whether the bacterial proteins originated from these is currently unknown. Introducing different phage into the same cell and determining their compatibility can give insight into evolutionary background [72]. An understanding of the relationship between phage lambda and telomere phage, in particular N15, will provide an interesting and important insight into how plasmid and phage can interact and evolve.

Crystal structures of TelK and TelA provide a solid starting point for research aiming to solve structures of protelomerases. These can also be used for homology modeling to infer information about proteins with high sequence similarities. Advances in the field of synthetic biology and protein engineering make detailed knowledge of these proteins even more valuable. An increased understanding of how they operate, and which parts are responsible for specific functionalities, will open up opportunities to produce variants with altered activities.

#### **Appendices and nomenclature**


#### **Conflict of interest**

SK has recently started a PhD jointly between Touchlight Genetics and Renos Savva; however, this has not relatively biased or affected the content of this chapter. *Bacteriophages - Perspectives and Future*

### **Author details**

Sophie E. Knott, Sarah A. Milsom and Paul J. Rothwell\* Touchlight Genetics, Hampton, UK

\*Address all correspondence to: paul.rothwell@touchlight.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**71**

*The Unusual Linear Plasmid Generating Systems of Prokaryotes*

of the National Academy of Sciences of the United States of America. 2013;**41**(22):10438-10448

[9] Huang SH, Cozart MR, Hart MA, Kobryn K. The *Borrelia burgdorferi* telomere resolvase, ResT, possesses ATP-dependent DNA unwinding activity. Nucleic Acids Research.

[10] Godiska R, Mead D, Dhodda V, Wu C, Hochstein R, Karsi A, et al. Linear plasmid vector for cloning of repetitive or unstable sequences in *Escherichia coli*. Nucleic Acids Research.

[11] Godiska R, Ravin N, Mead D. Linear plasmid vector for cloning "Unclonable" DNA. BioTechniques.

[12] Nafissi N, Slavcev R. Construction and characterization of an in-vivo linear covalently closed DNA vector production system. Microbial Cell Factories. 2012;**11**:154. DOI: 10.1186/1475-2859-11-154

[13] Karbowniczek K, Rothwell P, Extance J, Milsom S, Lukashchuk V, Bowes K, et al. Doggybone™ DNA: An advanced platform for AAV production.

Cell & Gene Therapy Insights.

Karbowniczek K, Caproni LJ, Tite JP, Waddington SN. Production of lentiviral vectors using novel, enzymatically produced, linear DNA. Gene Therapy. 2019;**26**:86-92

[15] Tourand Y, Kobryn K, Chaconas G. Sequence-specific recognition but position-dependent cleavage of two distinct telomeres by the *Borrelia burgdorferi* telomere resolvase, ResT. Molecular Microbiology.

[14] Karda R, Counsell JR,

2017;**45**(3):1319-1329

2010;**38**(6):e88

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*DOI: http://dx.doi.org/10.5772/intechopen.86882*

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[2] Lan SF, Huang CH, Chang CH, Liao WC, Lin IH, Jian WN, et al. Characterization of a new plasmidlike prophage in a pandemic *Vibrio parahaemolyticus* O3:K6 strain. Applied and Environmental Microbiology.

[3] Kobryn K, Chaconas G. ResT, a telomere resolvase encoded by the Lyme disease spirochete. Molecular Cell.

[4] Chaconas G, Kobryn K. Structure, function, and evolution of linear replicons in *Borrelia*. Annual Review of Microbiology. 2010;**64**(1):185-202

[5] Kobryn K, Chaconas G. Hairpin telomere resolvases. Microbiology Spectrum. 2014;**2**(6):273-287

[6] Loh T, Elvitigala T, Wang C, Wollam A, Welsh EA, Liberton M, et al. The genome of Cyanothece 51142, a unicellular diazotrophic cyanobacterium important in the marine nitrogen cycle. Proceedings of the National Academy of Sciences of the United States of America.

[7] Huang WM, DaGloria J, Fox H, Ruan Q, Tillou J, Shi K, et al. Linear chromosome-generating system of *Agrobacterium tumefaciens* C58: Protelomerase generates and protects hairpin ends. The Journal of Biological Chemistry. 2012;**287**(30):25551-25563

[8] Mir T, Huang SH, Kobryn K. The telomere resolvase of the Lyme disease spirochete, *Borrelia burgdorferi*,

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*The Unusual Linear Plasmid Generating Systems of Prokaryotes DOI: http://dx.doi.org/10.5772/intechopen.86882*

#### **References**

*Bacteriophages - Perspectives and Future*

**70**

**Author details**

Sophie E. Knott, Sarah A. Milsom and Paul J. Rothwell\*

\*Address all correspondence to: paul.rothwell@touchlight.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Touchlight Genetics, Hampton, UK

provided the original work is properly cited.

[1] Ravin V, Ravin N, Casjens S, Ford ME, Hatfull GF, Hendrix RW. Genomic sequence and analysis of the atypical temperate bacteriophage N15. Journal of Molecular Biology. 2000;**299**(1):53-73

[2] Lan SF, Huang CH, Chang CH, Liao WC, Lin IH, Jian WN, et al. Characterization of a new plasmidlike prophage in a pandemic *Vibrio parahaemolyticus* O3:K6 strain. Applied and Environmental Microbiology. 2009;**75**(9):2659-2667

[3] Kobryn K, Chaconas G. ResT, a telomere resolvase encoded by the Lyme disease spirochete. Molecular Cell. 2002;**9**:195-201

[4] Chaconas G, Kobryn K. Structure, function, and evolution of linear replicons in *Borrelia*. Annual Review of Microbiology. 2010;**64**(1):185-202

[5] Kobryn K, Chaconas G. Hairpin telomere resolvases. Microbiology Spectrum. 2014;**2**(6):273-287

[6] Loh T, Elvitigala T, Wang C, Wollam A, Welsh EA, Liberton M, et al. The genome of Cyanothece 51142, a unicellular diazotrophic cyanobacterium important in the marine nitrogen cycle. Proceedings of the National Academy of Sciences of the United States of America. 2008;**75**(9):2659-2667

[7] Huang WM, DaGloria J, Fox H, Ruan Q, Tillou J, Shi K, et al. Linear chromosome-generating system of *Agrobacterium tumefaciens* C58: Protelomerase generates and protects hairpin ends. The Journal of Biological Chemistry. 2012;**287**(30):25551-25563

[8] Mir T, Huang SH, Kobryn K. The telomere resolvase of the Lyme disease spirochete, *Borrelia burgdorferi*, promotes DNA single-strand annealing and strand exchange. Proceedings

of the National Academy of Sciences of the United States of America. 2013;**41**(22):10438-10448

[9] Huang SH, Cozart MR, Hart MA, Kobryn K. The *Borrelia burgdorferi* telomere resolvase, ResT, possesses ATP-dependent DNA unwinding activity. Nucleic Acids Research. 2017;**45**(3):1319-1329

[10] Godiska R, Mead D, Dhodda V, Wu C, Hochstein R, Karsi A, et al. Linear plasmid vector for cloning of repetitive or unstable sequences in *Escherichia coli*. Nucleic Acids Research. 2010;**38**(6):e88

[11] Godiska R, Ravin N, Mead D. Linear plasmid vector for cloning "Unclonable" DNA. BioTechniques. 2008;**45**(5):592

[12] Nafissi N, Slavcev R. Construction and characterization of an in-vivo linear covalently closed DNA vector production system. Microbial Cell Factories. 2012;**11**:154. DOI: 10.1186/1475-2859-11-154

[13] Karbowniczek K, Rothwell P, Extance J, Milsom S, Lukashchuk V, Bowes K, et al. Doggybone™ DNA: An advanced platform for AAV production. Cell & Gene Therapy Insights. 2017:731-738

[14] Karda R, Counsell JR, Karbowniczek K, Caproni LJ, Tite JP, Waddington SN. Production of lentiviral vectors using novel, enzymatically produced, linear DNA. Gene Therapy. 2019;**26**:86-92

[15] Tourand Y, Kobryn K, Chaconas G. Sequence-specific recognition but position-dependent cleavage of two distinct telomeres by the *Borrelia burgdorferi* telomere resolvase, ResT. Molecular Microbiology. 2003;**48**:901-911

[16] Deneke J, Ziegelin G, Lurz R, Lanka E. The protelomerase of temperate *Escherichia coli* phage N15 has cleaving-joining activity. Proceedings of the National Academy of Sciences of the United States of America. 2000;**97**(14):7721-7726

[17] Landry MP, Zou X, Wang L, Huang WM, Schulten K, Chemla YR. DNA target sequence identification mechanism for dimer-active protein complexes. Nucleic Acids Research. 2013;**41**(4):2416-2427

[18] Ravin NV. Mechanisms of replication and telomere resolution of the linear plasmid prophage N15. FEMS Microbiology Letters. 2003;**221**(1):1-6

[19] Lucyshyn D, Huang SH, Kobryn K. Spring loading a pre-cleavage intermediate for hairpin telomere formation. Nucleic Acids Research. 2015;**43**(12):6062-6074

[20] Chou S, Chin K, Wang AH. Unusual DNA duplex and hairpin motifs. Nucleic Acids Research. 2003;**31**(10):2461-2474

[21] Aihara H, Huang WM, Ellenberger T. An interlocked dimer of the protelomerase TelK distorts DNA structure for the formation of hairpin telomeres. Molecular Cell. 2007;**27**(6):901-913

[22] Shi K, Huang WM, Aihara H. An enzyme-catalyzed multistep DNA refolding mechanism in hairpin telomere formation. PLoS Biology. 2013;**11**(1):e1001472

[23] Bankhead T, Chaconas G. Mixing active-site components: A recipe for the unique enzymatic activity of a telomere resolvase. Proceedings of the National Academy of Sciences. 2004;**101**(38):13768-13773

[24] Briffotaux J, Kobryn K. Preventing broken *Borrelia* telomeres ResT

couples dual hairpin telomere formation with product release. The Journal of Biological Chemistry. 2010;**285**(52):41010-41018

[25] Casjens S, Murphy M, Sampson L. Telomeres of the linear chromosomes of Lyme disease spirochaetes: Nucleotide sequence and possible exchange with linear plasmid telomeres. Molecular Microbiology. 1997;**26**:581-596

[26] Deneke J, Lurz R, Lanka E. Phage N15 telomere resolution. The Journal of Biological Chemistry. 2002;**277**(12):10410-10419

[27] Huang WM, Joss L, Hsieh T, Casjens S. Protelomerase uses a topoisomerase IB/Y-recombinase type mechanism to generate DNA hairpin ends. Journal of Molecular Biology. 2004;**337**(1):77-92

[28] Kobryn K, Burgin AB, Chaconas G. Uncoupling the chemical steps of telomere resolution by ResT. The Journal of Biological Chemistry. 2005;**280**(29):26788-26795

[29] Zabala B, Hammerl JA, Espejo RT, Hertwig S. The linear plasmid prophage Vp58. 5 of *Vibrio parahaemolyticus* is closely related to the integrating phage VHML and constitutes a new incompatibility group of telomere phages. Journal of Virology. 2009;**83**(18):9313-9320

[30] Hertwig S, Klein I, Lurz R, Lanka E, Appel B. PY54, a linear plasmid prophage of *Yersinia enterocolitica* with covalently closed ends. Molecular Microbiology. 2003;**48**(4):989-1003

[31] Mobberley JM, Authement RN, Segall AM, Paul JH. The temperate marine phage HAP-1 of *Halomonas aquamarina* possesses a linear plasmidlike prophage genome. Journal of Virology. 2008;**82**(13):6618-6630

**73**

*The Unusual Linear Plasmid Generating Systems of Prokaryotes*

cohesive end mismatch. PLoS ONE.

[42] Papagiannis CV, Sam MD, Abbani MA, Yoo D, Cascio D, Clubb RT, et al. Fis targets assembly of the Xis nucleoprotein filament to promote excisive recombination by phage lambda. Journal of Molecular Biology.

[43] Replication RNV. Maintenance of linear phage-plasmid N15. Microbiology

[44] Baril C, Richaud C, Baranton G. Linear chromosome of *Borrelia burgdorferi*. Research in Microbiology.

[45] Picardeau M, Lobry JR, Joseph B, Bernard C. Physical mapping of an origin of bidirectional replication at the centre of the *Borrelia burgdorferi* linear chromosome. Molecular Microbiology.

[46] Chaconas G, Stewart PE, Tilly K, Bono JL, Rosa P. Telomere resolution in the Lyme disease spirochete. The EMBO

[47] Byram R, Stewart PE, Rosa P. The essential nature of the ubiquitous 26-kilobase circular replicon of *Borrelia burgdorferi*. Journal of Bacteriology.

[48] Yang W, Mizuuchi K. Site-specific recombination in plane view. Structure.

[49] Cheng C, Kussie P, Pavletich N, Shuman S. Conservation of structure and mechanism between eukaryotic topoisomerase I and site-specific recombinases. Cell. 1998;**92**:841-850

poxvirus-like type IB topoisomerase family in bacteria. Proceedings of the National Academy of Sciences.

[50] Krogh BO, Shuman S. A

2002;**99**(4):1853-1858

Journal. 2001;**20**(12):3229-3237

2004;**186**(11):3561-3569

1997;**5**(11):1401-1406

2015;**10**(12):e0141934

2007;**367**(2):328-343

1989;**140**(8):507-516

1999;**32**(2):437-445

Spectrum. 2015;**3**(1):1-12

*DOI: http://dx.doi.org/10.5772/intechopen.86882*

[32] Black L. DNA packaging in dsDNA. Annual Review of Microbiology. 1989;**43**:267-292

[33] Casjens S. Evolution of the linear DNA replicons of the *Borrelia* spirochetes. Molecular Microbiology.

[34] Ravin NV. N15: The linear phageplasmid. Plasmid. 2011;**65**(2):102-109

[35] Mardanov AV, Ravin NV. Conversion of linear DNA with hairpin telomeres into a circular molecule in the course of phage N15 lytic replication. Journal of Molecular Biology. 2009;**391**(2):261-268

[36] Ziegelin G, Scherzinger E, Lurz R, Lanka E. Phage P4 alpha protein is multifunctional with origin recognition, helicase and primase activities. The EMBO Journal. 1993;**12**(9):3703-3708

[37] Orejas RD, Ziegelin G, Lurz R, Lanka E, Genetik MM, Schuster A.

[38] Rybchin VN, Svarchevsky AN. The plasmid prophage N15: A linear DNA with covalently closed ends. Molecular

[40] Ravin NV, Strakhova TS, Kuprianov V. The protelomerase of the phageplasmid N15 is responsible for its maintenance in linear form. Journal of Molecular Biology. 2001;**312**(5):899-906

[41] Feiss M, Young J, Sultana S, Patel P, Sippy JDNA. Packaging specificity of bacteriophage N15 with an excursion into the genetics of a

Microbiology. 1999;**33**:895-903

[39] Landschulz WH, Johnson PF, Mcknight SL, Landschulz WH, Johnson PF, Mcknight SL. Structure common to a new class of the leucine zipper: A hypothetical DNA binding proteins. Science. 1988;**240**(4860):1759-1764

Phage P4 DNA replication in vitro. Nucleic Acids Research.

1994;**22**(11):2065-2070

1999;**2**(5):529-534

*The Unusual Linear Plasmid Generating Systems of Prokaryotes DOI: http://dx.doi.org/10.5772/intechopen.86882*

[32] Black L. DNA packaging in dsDNA. Annual Review of Microbiology. 1989;**43**:267-292

*Bacteriophages - Perspectives and Future*

[16] Deneke J, Ziegelin G, Lurz R, Lanka E. The protelomerase of

2000;**97**(14):7721-7726

2013;**41**(4):2416-2427

WM, Schulten K, Chemla YR. DNA target sequence identification mechanism for dimer-active protein complexes. Nucleic Acids Research.

[18] Ravin NV. Mechanisms of

Spring loading a pre-cleavage intermediate for hairpin telomere formation. Nucleic Acids Research.

2015;**43**(12):6062-6074

replication and telomere resolution of the linear plasmid prophage N15. FEMS Microbiology Letters. 2003;**221**(1):1-6

[19] Lucyshyn D, Huang SH, Kobryn K.

[20] Chou S, Chin K, Wang AH. Unusual DNA duplex and hairpin motifs. Nucleic Acids Research. 2003;**31**(10):2461-2474

[21] Aihara H, Huang WM, Ellenberger

[22] Shi K, Huang WM, Aihara H. An enzyme-catalyzed multistep DNA refolding mechanism in hairpin telomere formation. PLoS Biology.

[23] Bankhead T, Chaconas G. Mixing active-site components: A recipe for the unique enzymatic activity of a telomere resolvase. Proceedings of the National Academy of Sciences.

[24] Briffotaux J, Kobryn K. Preventing

T. An interlocked dimer of the protelomerase TelK distorts DNA structure for the formation of hairpin telomeres. Molecular Cell.

2007;**27**(6):901-913

2013;**11**(1):e1001472

2004;**101**(38):13768-13773

broken *Borrelia* telomeres ResT

temperate *Escherichia coli* phage N15 has cleaving-joining activity. Proceedings of the National Academy of Sciences of the United States of America.

couples dual hairpin telomere formation with product release. The Journal of Biological Chemistry. 2010;**285**(52):41010-41018

of Lyme disease spirochaetes: Nucleotide sequence and possible exchange with linear plasmid telomeres. Molecular Microbiology.

[26] Deneke J, Lurz R, Lanka E. Phage N15 telomere resolution. The Journal of Biological Chemistry. 2002;**277**(12):10410-10419

[27] Huang WM, Joss L, Hsieh T, Casjens S. Protelomerase uses a topoisomerase IB/Y-recombinase type mechanism to generate DNA hairpin ends. Journal of Molecular Biology.

[28] Kobryn K, Burgin AB, Chaconas G. Uncoupling the chemical steps of telomere resolution by ResT. The Journal of Biological Chemistry. 2005;**280**(29):26788-26795

[29] Zabala B, Hammerl JA, Espejo RT, Hertwig S. The linear plasmid prophage Vp58. 5 of *Vibrio parahaemolyticus* is closely related to the integrating phage VHML and constitutes a new incompatibility group of

telomere phages. Journal of Virology.

[30] Hertwig S, Klein I, Lurz R, Lanka E, Appel B. PY54, a linear plasmid prophage of *Yersinia enterocolitica* with covalently closed ends. Molecular Microbiology. 2003;**48**(4):989-1003

[31] Mobberley JM, Authement RN, Segall AM, Paul JH. The temperate marine phage HAP-1 of *Halomonas aquamarina* possesses a linear plasmidlike prophage genome. Journal of Virology. 2008;**82**(13):6618-6630

2009;**83**(18):9313-9320

1997;**26**:581-596

2004;**337**(1):77-92

[25] Casjens S, Murphy M, Sampson L. Telomeres of the linear chromosomes

[17] Landry MP, Zou X, Wang L, Huang

**72**

[33] Casjens S. Evolution of the linear DNA replicons of the *Borrelia* spirochetes. Molecular Microbiology. 1999;**2**(5):529-534

[34] Ravin NV. N15: The linear phageplasmid. Plasmid. 2011;**65**(2):102-109

[35] Mardanov AV, Ravin NV. Conversion of linear DNA with hairpin telomeres into a circular molecule in the course of phage N15 lytic replication. Journal of Molecular Biology. 2009;**391**(2):261-268

[36] Ziegelin G, Scherzinger E, Lurz R, Lanka E. Phage P4 alpha protein is multifunctional with origin recognition, helicase and primase activities. The EMBO Journal. 1993;**12**(9):3703-3708

[37] Orejas RD, Ziegelin G, Lurz R, Lanka E, Genetik MM, Schuster A. Phage P4 DNA replication in vitro. Nucleic Acids Research. 1994;**22**(11):2065-2070

[38] Rybchin VN, Svarchevsky AN. The plasmid prophage N15: A linear DNA with covalently closed ends. Molecular Microbiology. 1999;**33**:895-903

[39] Landschulz WH, Johnson PF, Mcknight SL, Landschulz WH, Johnson PF, Mcknight SL. Structure common to a new class of the leucine zipper: A hypothetical DNA binding proteins. Science. 1988;**240**(4860):1759-1764

[40] Ravin NV, Strakhova TS, Kuprianov V. The protelomerase of the phageplasmid N15 is responsible for its maintenance in linear form. Journal of Molecular Biology. 2001;**312**(5):899-906

[41] Feiss M, Young J, Sultana S, Patel P, Sippy JDNA. Packaging specificity of bacteriophage N15 with an excursion into the genetics of a

cohesive end mismatch. PLoS ONE. 2015;**10**(12):e0141934

[42] Papagiannis CV, Sam MD, Abbani MA, Yoo D, Cascio D, Clubb RT, et al. Fis targets assembly of the Xis nucleoprotein filament to promote excisive recombination by phage lambda. Journal of Molecular Biology. 2007;**367**(2):328-343

[43] Replication RNV. Maintenance of linear phage-plasmid N15. Microbiology Spectrum. 2015;**3**(1):1-12

[44] Baril C, Richaud C, Baranton G. Linear chromosome of *Borrelia burgdorferi*. Research in Microbiology. 1989;**140**(8):507-516

[45] Picardeau M, Lobry JR, Joseph B, Bernard C. Physical mapping of an origin of bidirectional replication at the centre of the *Borrelia burgdorferi* linear chromosome. Molecular Microbiology. 1999;**32**(2):437-445

[46] Chaconas G, Stewart PE, Tilly K, Bono JL, Rosa P. Telomere resolution in the Lyme disease spirochete. The EMBO Journal. 2001;**20**(12):3229-3237

[47] Byram R, Stewart PE, Rosa P. The essential nature of the ubiquitous 26-kilobase circular replicon of *Borrelia burgdorferi*. Journal of Bacteriology. 2004;**186**(11):3561-3569

[48] Yang W, Mizuuchi K. Site-specific recombination in plane view. Structure. 1997;**5**(11):1401-1406

[49] Cheng C, Kussie P, Pavletich N, Shuman S. Conservation of structure and mechanism between eukaryotic topoisomerase I and site-specific recombinases. Cell. 1998;**92**:841-850

[50] Krogh BO, Shuman S. A poxvirus-like type IB topoisomerase family in bacteria. Proceedings of the National Academy of Sciences. 2002;**99**(4):1853-1858

[51] Kobryn K, Briffotaux J, Karpov V. Holliday junction formation by the *Borrelia burgdorferi* telomere resolvase, ResT: Implications for the origin of genome linearity. Molecular Microbiology. 2009;**71**(5):1117-1130

[52] Grainge I, Jayaram M. The integrase family of recombinases: Organization and function of the active site. Molecular Microbiology. 1999;**33**:449-456

[53] Esposito D, Scocca JJ. The integrase family of tyrosine recombinases: Evolution of a conserved active site domain. Nucelic Acids Research. 1997;**25**(18):3605-3614

[54] Tourand Y, Lee L, Chaconas G. Telomere resolution by *Borrelia burgdorferi* ResT through the collaborative efforts of tethered DNA binding domains. Molecular Microbiology. 2007;**64**:580-590

[55] Lee J, Tonozuka T, Jayaram M. Mechanism of active site exclusion in a site-specific recombinase: Role of the DNA substrate in conferring half-of-the-sites activity. Genes & Development. 1997;**11**:3061-3071

[56] Nash HA, Robertson CA. Heterduplex substrates for bacteriophage lambda site-specific recombination: Cleavage and strand transfer products. The EMBO Journal. 1989;**8**(11):3523-3533

[57] Deneke J, Burgin AB, Wilson SL, Chaconas G. Catalytic residues of the telomere resolvase ResT. The Journal of Biological Chemistry. 2004;**279**(51):53699-53706

[58] Chen Y, Rice PA. The role of the conserved Trp 330 in Flp-mediated recombination. The Journal of Biological Chemistry. 2003;**278**(27):24800-24807

[59] Nunes-düby SE, Kwon HJ, Tirumalai RS, Ellenberger T, Landy A. Similarities

and differences among 105 members of the Int family of site-specific recombinases. Nucleic Acids Research. 1998;**26**(2):391-406

[60] Wang JC. DNA topoisomerases. Annual Review of Biochemistry. 1996;**65**:635-692

[61] Kormann MSD, Hasenpusch G, Aneja MK, Nica G, Flemmer AW, Herber-jonat S, et al. Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nature Biotechnology. 2011;**29**(2):154-157

[62] Bangel-ruland N, Fernández EF, Leier G, Leciejewski B, Rudolph C, Rosenecker J, et al. Cystic fibrosis transmembrane conductance regulatormRNA delivery: A novel alternative for cystic fibrosis gene therapy. The Journal of Gene Medicine. 2013;**15**:414-426

[63] Gallie D. The cap and poly (A) tail function synergistically to regulate mRNA translational efficiency. Genes & Development. 1991;**5**:2108-2116

[64] Grier AE, Burleigh S, Sahni J, Clough CA, Cardot V, Choe DC, et al. pEVL: A linear plasmid for generating mRNA IVT templates with extended encoded poly(A) sequences. Molecular Therapy--Nucleic Acids. 2016;**5**(4):e306

[65] Wong S, Lam P, Nafissi N, Denniss S, Slavcev R. Production of doublestranded DNA ministrings. Journal of Visualized Experiments. 2016;**108**:3-9

[66] Catanese DJ, Fogg JM, Schrock DE, Gilbert BE, Zechiedrich L. Supercoiled minivector DNA resists shear forces associated with gene therapy delivery. Gene Therapy. 2011;**19**(1):94-100

[67] Liang X, Baek C, Katzen F. *Escherichia coli* with two linear chromosomes. ACS Synthetic Biology. 2013;**2**(12):734-740

**75**

*The Unusual Linear Plasmid Generating Systems of Prokaryotes*

*DOI: http://dx.doi.org/10.5772/intechopen.86882*

[68] Scott VL, Patel A, Villarreal DO, Hensley SE, Ragwan E, Yan J, et al. Novel synthetic plasmid and doggybone DNA vaccines induce neutralizing antibodies and provide protection from lethal influenza challenge in mice. Human Vaccines & Immunotherapeutics.

[69] Allen A, Wang C, Caproni LJ, Sugiyarto G, Harden E, Douglas LR, et al. Linear doggybone DNA vaccine induces similar immunological

responses to conventional plasmid DNA independently of immune recognition by TLR9 in a pre-clinical model. Cancer Immunology, Immunotherapy.

[70] Daya S, Berns KI. Gene therapy using adeno-associated virus vectors. Clinical Microbiology Reviews.

[71] Naso MF, Tomkowicz B, Perry WL, Strogl WR. Adeno-associated virus (AAV) as a vector for gene therapy. BioDrugs. 2017;**31**(4):315-332

[72] Hammerl JA, Klein I, Appel B, Hertwig S. Interplay between the temperate phages PY54 and N15, linear plasmid prophages with covalently closed ends. Journal of Bacteriology.

2007;**189**(22):8366-8370

2015;**11**(8):1972-1982

2018;**67**(4):627-638

2008;**21**(4):583-593

*The Unusual Linear Plasmid Generating Systems of Prokaryotes DOI: http://dx.doi.org/10.5772/intechopen.86882*

[68] Scott VL, Patel A, Villarreal DO, Hensley SE, Ragwan E, Yan J, et al. Novel synthetic plasmid and doggybone DNA vaccines induce neutralizing antibodies and provide protection from lethal influenza challenge in mice. Human Vaccines & Immunotherapeutics. 2015;**11**(8):1972-1982

*Bacteriophages - Perspectives and Future*

[52] Grainge I, Jayaram M. The integrase family of recombinases: Organization and function of the active site. Molecular Microbiology.

[53] Esposito D, Scocca JJ. The integrase family of tyrosine recombinases: Evolution of a conserved active site domain. Nucelic Acids Research.

[54] Tourand Y, Lee L, Chaconas G. Telomere resolution by *Borrelia burgdorferi* ResT through the collaborative efforts of tethered DNA binding domains. Molecular Microbiology. 2007;**64**:580-590

[55] Lee J, Tonozuka T, Jayaram M. Mechanism of active site exclusion in a site-specific recombinase: Role of the DNA substrate in conferring half-of-the-sites activity. Genes & Development. 1997;**11**:3061-3071

[56] Nash HA, Robertson CA. Heterduplex substrates for

1989;**8**(11):3523-3533

bacteriophage lambda site-specific recombination: Cleavage and strand transfer products. The EMBO Journal.

[57] Deneke J, Burgin AB, Wilson SL, Chaconas G. Catalytic residues of the telomere resolvase ResT. The Journal of Biological Chemistry. 2004;**279**(51):53699-53706

[58] Chen Y, Rice PA. The role of the conserved Trp 330 in Flp-mediated recombination. The Journal of Biological Chemistry. 2003;**278**(27):24800-24807

[59] Nunes-düby SE, Kwon HJ, Tirumalai RS, Ellenberger T, Landy A. Similarities

1999;**33**:449-456

1997;**25**(18):3605-3614

[51] Kobryn K, Briffotaux J, Karpov V. Holliday junction formation by the *Borrelia burgdorferi* telomere resolvase, ResT: Implications for the origin of genome linearity. Molecular Microbiology. 2009;**71**(5):1117-1130

and differences among 105 members of the Int family of site-specific recombinases. Nucleic Acids Research.

[60] Wang JC. DNA topoisomerases. Annual Review of Biochemistry.

[61] Kormann MSD, Hasenpusch G, Aneja MK, Nica G, Flemmer AW, Herber-jonat S, et al. Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nature Biotechnology.

[62] Bangel-ruland N, Fernández EF, Leier G, Leciejewski B, Rudolph C, Rosenecker J, et al. Cystic fibrosis transmembrane conductance regulatormRNA delivery: A novel alternative for cystic fibrosis gene therapy. The Journal of Gene Medicine. 2013;**15**:414-426

[63] Gallie D. The cap and poly (A) tail function synergistically to regulate mRNA translational efficiency. Genes &

Development. 1991;**5**:2108-2116

2016;**5**(4):e306

[64] Grier AE, Burleigh S, Sahni J, Clough CA, Cardot V, Choe DC, et al. pEVL: A linear plasmid for generating mRNA IVT templates with extended encoded poly(A) sequences. Molecular Therapy--Nucleic Acids.

[65] Wong S, Lam P, Nafissi N, Denniss S, Slavcev R. Production of doublestranded DNA ministrings. Journal of Visualized Experiments. 2016;**108**:3-9

[66] Catanese DJ, Fogg JM, Schrock DE, Gilbert BE, Zechiedrich L. Supercoiled minivector DNA resists shear forces associated with gene therapy delivery. Gene Therapy. 2011;**19**(1):94-100

[67] Liang X, Baek C, Katzen F. *Escherichia coli* with two linear

2013;**2**(12):734-740

chromosomes. ACS Synthetic Biology.

1998;**26**(2):391-406

1996;**65**:635-692

2011;**29**(2):154-157

**74**

[69] Allen A, Wang C, Caproni LJ, Sugiyarto G, Harden E, Douglas LR, et al. Linear doggybone DNA vaccine induces similar immunological responses to conventional plasmid DNA independently of immune recognition by TLR9 in a pre-clinical model. Cancer Immunology, Immunotherapy. 2018;**67**(4):627-638

[70] Daya S, Berns KI. Gene therapy using adeno-associated virus vectors. Clinical Microbiology Reviews. 2008;**21**(4):583-593

[71] Naso MF, Tomkowicz B, Perry WL, Strogl WR. Adeno-associated virus (AAV) as a vector for gene therapy. BioDrugs. 2017;**31**(4):315-332

[72] Hammerl JA, Klein I, Appel B, Hertwig S. Interplay between the temperate phages PY54 and N15, linear plasmid prophages with covalently closed ends. Journal of Bacteriology. 2007;**189**(22):8366-8370

**77**

**Chapter 5**

**Abstract**

Phages

*and Eli Keshavarz-Moore*

Scale-Up and Bioprocessing of

*John Maxim Ward, Steven Branston, Emma Stanley* 

of the *E. coli* inoculum and phage precipitation methods.

phage diagnostics, phage laser, fermenter

**1. Introduction**

**Keywords:** phage, PEG precipitation, nuclease, filamentous phage, lambda,

Bacteriophages, often shortened to just phages, are viruses that infect bacteria. Their discovery and characterisation in the early days of bacterial molecular biology has led to certain phages being very well understood in terms of their life cycle, and several phages that infect *Escherichia coli* have become tools in molecular biology techniques such as cloning [1–3]. There has been a resurgence recently in the use of bacteriophages as therapeutics, as vectors for the delivery of vaccines [4], for the killing of pathogenic bacteria as an alternative to antibiotics [5] and for gene therapy to transfer DNA to target human or animal cells [6]. Some of these uses would need the production of many millions of doses of a vaccine, for example, or very large quantities for use as an antibacterial. This has increased demand for investigation into the large-scale production of bacteriophage which would necessitate volumes from hundreds to thousands of litres. The use of phage as biotherapeutics such as vaccines or for gene therapy may be advantageous as phage is considered cheap to manufacture, with large quantities of the product being rapidly produced. But the large-scale production of wild type or genetically modified bacteriophages

A profusion of new applications for phage technologies has been developed within the last few years, stimulating investigations into the large-scale production of different phages. Applications such as antibiotic replacement, phages as gene therapy vectors, phages as vaccines, diagnostics using filamentous phages and novel optical applications such as the phage laser may need grams to kilogrammes of phage in the future. However, many of the techniques that are used for the growth and purification of bacteriophage at small scale are not transferable to large-scale production facilities of phage in industrial processes. In this chapter, the stages of production that need to be carried out at scale are examined for the efficient large-scale fermentation of the filamentous phage M13 and the *Siphoviridae* phage lambda (λ). A number of parameters are discussed: the multiplicity of infection (MOI) of phage to host cells, the impact of agitation on the initial infection stages, the co-growth with phage rather than static attachment, the use of engineered host cells expressing nuclease, the optimisation of both the quantity and the physiology

#### **Chapter 5**
