*3.2.1 Global genetic regulation of LuxIR and AI-2/Lsr systems*

The dawn of genomic profiling has unveiled that quorum sensing, in many bacteria, controls gene expression in a global manner. QS-mutants of *S. pneumoniae*

#### **Figure 2.**

*Regulatory mechanisms of the lsr/AI-2 circuit in E. coli. AI-2 is imported by the Lsr transporter (LsrACDB) and in turn, is processed by LsrK, transforming to its phosphorylated form (AI-2P). As AI-2P binds LsrR, it relieves the repression of LsrR on the Lsr genes and accelerates AI-2 intake. LuxS produces DPD, the precursor of AI-2. The autoinducer is then transported out of the bacteria by YdgG (TqsA), a putative transporter belonging to the exporter superfamily [40].*

**173**

*Repurposing E. coli by Engineering Quorum Sensing and Redox Genetic Circuits*

and related *Streptococci* show defects in multiple pathways, including biofilm formation, acid tolerance, bacteriocin production, and virulence [31]. *E. coli*, too, has been reported to elicit broad QS activities. For example, the quantity and architecture of biofilms are regulated by *lsrR/K* through motility QS regulator (MqsR, B3022), as well as the generation of several small RNAs [36, 41]. Together, these and other reports suggest that QS systems control many aspects of the whole genome rather than just one key gene locus. Further evidence that quorum sensing coordinates the control of a large subset of genes comes from transcriptome analyses of an *E. coli luxS* mutant, which showed that 242 genes (5.6% of the whole genome) exhibited significant transcriptional changes upon a 300-fold AI-2 signaling dif-

Interestingly, AI-2 synthesis and signaling levels are linked to the accumulation of protein product expressed from plasmid-encoded genes [44]. This suggests that recombinant *E. coli* are able to communicate the burden of overexpressing heterologous protein through AI-2 QS pathways. Most recently, the sugar metabolism of *E. coli* was found to be directly connected to the LuxS/AI-2 QS system. That is, HPr, a phosphocarrier protein central to the sugar phosphotransferase system, was recently reported to copurify with LsrK such that the activity of LsrK was inhibited when bound to HPr [45]. In sum, these finding shed new light on how bacteria respond to changing nutrient levels on a population scale. The intentional manipulation of the QS signaling processes, therefore, has become an interesting target for heterologous gene expression in *E. coli* among many other applications [46].

*De novo* engineering of gene circuits inside cells is proven to be difficult, in large part due to connectivity to non-targeted pathways and genes [47, 48]. QS regulons, coupling intraspecies communication and global genome regulation, can serve as excellent platforms for many technologies to be built upon, particularly if one understands the regulatory "reach" of the genetic circuits. Attempting to eliminate the variation in phenotype between cells, You et al*.* coupled gene expression to cell survival and death using the LuxIR QS system [49]. With the 'population control' gene circuit, they successfully regulated the density of an *E. coli* population autonomously and were able to program the dynamics of an entire population despite behavioral variability between individual cells. Based on the same LuxIR system of *V. fischeri* and the QS system of *Bacillus thuringiensis*, a synchronized genetic clock was engineered [50]. This novel gene network with global intercellular coupling can generate synchronized oscillations in a growing population of cells. In biology, synchronized oscillation holds the same importance as in physics and engineering, where it governs many fundamental physiological processes such as cardiac function and circadian rhythm [51]. These studies have set the stage for future development of using microbes as macroscopic biosensors with oscillatory output, as the colony-level synchronized oscillation could diminish single-cell variability in most synthetic gene networks and increase the sensitivity and robustness of response to

On top of employing the LuxIR system as a platform for innovative genetic and population regulators, intentional rewiring of *E. coli*'s native QS networks can also benefit biotechnological applications. For example, in [52], autonomous induction of recombinant proteins is realized through minimal rewiring of the AI-2/Lsr system. Since the QS network is capable of 'reporting' the metabolic state of a bacterial population and the metabolic burden is self-indicated by this network [44], Tsao et al*.* made it possible to achieve metabolically-balanced coordination of the entire culture for a user-specified purpose through minimal rewiring of the QS network

*DOI: http://dx.doi.org/10.5772/intechopen.81245*

ferential [42–44].

*3.2.2 Applications*

external signals.

#### *Repurposing E. coli by Engineering Quorum Sensing and Redox Genetic Circuits DOI: http://dx.doi.org/10.5772/intechopen.81245*

and related *Streptococci* show defects in multiple pathways, including biofilm formation, acid tolerance, bacteriocin production, and virulence [31]. *E. coli*, too, has been reported to elicit broad QS activities. For example, the quantity and architecture of biofilms are regulated by *lsrR/K* through motility QS regulator (MqsR, B3022), as well as the generation of several small RNAs [36, 41]. Together, these and other reports suggest that QS systems control many aspects of the whole genome rather than just one key gene locus. Further evidence that quorum sensing coordinates the control of a large subset of genes comes from transcriptome analyses of an *E. coli luxS* mutant, which showed that 242 genes (5.6% of the whole genome) exhibited significant transcriptional changes upon a 300-fold AI-2 signaling differential [42–44].

Interestingly, AI-2 synthesis and signaling levels are linked to the accumulation of protein product expressed from plasmid-encoded genes [44]. This suggests that recombinant *E. coli* are able to communicate the burden of overexpressing heterologous protein through AI-2 QS pathways. Most recently, the sugar metabolism of *E. coli* was found to be directly connected to the LuxS/AI-2 QS system. That is, HPr, a phosphocarrier protein central to the sugar phosphotransferase system, was recently reported to copurify with LsrK such that the activity of LsrK was inhibited when bound to HPr [45]. In sum, these finding shed new light on how bacteria respond to changing nutrient levels on a population scale. The intentional manipulation of the QS signaling processes, therefore, has become an interesting target for heterologous gene expression in *E. coli* among many other applications [46].

## *3.2.2 Applications*

*Gene Expression and Control*

mathematical model [39].

**3.2 Global quorum sensing regulons**

*3.2.1 Global genetic regulation of LuxIR and AI-2/Lsr systems*

The dawn of genomic profiling has unveiled that quorum sensing, in many bacteria, controls gene expression in a global manner. QS-mutants of *S. pneumoniae*

*Regulatory mechanisms of the lsr/AI-2 circuit in E. coli. AI-2 is imported by the Lsr transporter (LsrACDB) and in turn, is processed by LsrK, transforming to its phosphorylated form (AI-2P). As AI-2P binds LsrR, it relieves the repression of LsrR on the Lsr genes and accelerates AI-2 intake. LuxS produces DPD, the precursor of AI-2. The autoinducer is then transported out of the bacteria by YdgG (TqsA), a putative transporter* 

researchers turned toward the signal recognition motif. Thus, another component of the system: the *luxS*-regulated (Lsr) transporter that intakes the extracellular AI-2 was later discovered to be the reason behind the decrease in extracellular AI-2, and not LuxS protein, during stationary phase. As a part of the *lsr* operon, this ATP-binding cassette (ABC) transporter is regulated by both cyclic AMP/cyclic AMP receptor protein and LsrK/LsrR proteins that are transcribed in its own *lsrRK* operon located upstream of *lsr* [36]. The fact that AI-2 intake requires a separate transporter (LsrACDB) is backed up by [37]. Comparing to AI-1 (AHL, 3OC6 homoserine lactone), AI-2 (4,5-dihydroxy-2,3-pentanedione, DPD) is found to be less membrane active and does not intercalate into the bacterial membrane. After modification with carbon chains, products (especially heptyl AI-2) display strong surface activity. These results indicate that AI-2, a more hydrophilic entity, shows less affinity to lipids and thus requires a transportation system. **Figure 2** provides a schematic illustration of the *lsr* circuit comprising of *lsrACDB* (encoding the Lsr transporter), *lsrR* (encodes the transcriptional repressor)*, lsrK* (encodes the AI-2 kinase), and *lsrFG* (encodes phosphorylated AI-2 (AI-2P) degradation enzymes) which are all directly regulated by AI-2. A recent mathematical model of this system was provided by Graff and Bentley [38], which helps to discriminate among hypothetical Lsr regulatory mechanisms and points to the importance of repressor LsrR dimer formation and binding on genetic regulation. Desynchronization of Lsr QS system, unlike the LuxIR system where its topology only consists of positive feedback, can display bimodal Lsr signaling and fractional induction. This phenomenon has been both observed in experiments and was also simulated with a

**172**

**Figure 2.**

*belonging to the exporter superfamily [40].*

*De novo* engineering of gene circuits inside cells is proven to be difficult, in large part due to connectivity to non-targeted pathways and genes [47, 48]. QS regulons, coupling intraspecies communication and global genome regulation, can serve as excellent platforms for many technologies to be built upon, particularly if one understands the regulatory "reach" of the genetic circuits. Attempting to eliminate the variation in phenotype between cells, You et al*.* coupled gene expression to cell survival and death using the LuxIR QS system [49]. With the 'population control' gene circuit, they successfully regulated the density of an *E. coli* population autonomously and were able to program the dynamics of an entire population despite behavioral variability between individual cells. Based on the same LuxIR system of *V. fischeri* and the QS system of *Bacillus thuringiensis*, a synchronized genetic clock was engineered [50]. This novel gene network with global intercellular coupling can generate synchronized oscillations in a growing population of cells. In biology, synchronized oscillation holds the same importance as in physics and engineering, where it governs many fundamental physiological processes such as cardiac function and circadian rhythm [51]. These studies have set the stage for future development of using microbes as macroscopic biosensors with oscillatory output, as the colony-level synchronized oscillation could diminish single-cell variability in most synthetic gene networks and increase the sensitivity and robustness of response to external signals.

On top of employing the LuxIR system as a platform for innovative genetic and population regulators, intentional rewiring of *E. coli*'s native QS networks can also benefit biotechnological applications. For example, in [52], autonomous induction of recombinant proteins is realized through minimal rewiring of the AI-2/Lsr system. Since the QS network is capable of 'reporting' the metabolic state of a bacterial population and the metabolic burden is self-indicated by this network [44], Tsao et al*.* made it possible to achieve metabolically-balanced coordination of the entire culture for a user-specified purpose through minimal rewiring of the QS network

and signal amplification by the T7 RNA polymerase [53, 54]. This study demonstrated one cell population was able to guide protein synthesis process of another by guiding intraspecies communication. Moreover, it was reported in [55] that by simply adding conditioned medium (containing a high amount of AI-2) during recombinant protein induction, one can double the yield of active product. Also, by altering the coincident *luxS* expression to control the AI-2 concentration while also inducing heterologous protein expression, they found an optimal condition where protein yield is dramatically increased. The authors further elucidated the mechanism behind this phenomenon: chaperone GroEL was shown to be coincidentally upregulated post-transcriptionally by AI-2. Because of its native role as a stabilizer of heterologous protein and its role in folding, the upregulation of GroEL might be the reason behind the higher product yield.

More endeavors have been made [56] to increase protein yield in this autonomous system through a different approach. With the same intention in mind [49, 50], a new study showed that reduced heterogeneity between independent cells could be achieved by inserting an enhanced feedback loop to the *E. coli*'s native AI-2 QS system. Upon activation of the engineered system, not only does the foreign pET plasmid concurrently express more sfGFP signal, but it also transcribed more LsrACDB and LsrK than the native *lsr* operon [57]. This overexpression resulted in increased uptake of AI-2, leading to amplified system response and minimized heterogeneity. Heterogeneity, on the other hand, could also be leveraged. In [58], quantized *E. coli* quorums were intentionally assembled through independent engineering of the AI-2 transduction cascade increasing the sensitivity of detector cells. Upon encountering a particular AI-2 level, a discretized sub-population of cells emerge with the desired phenotype. This sensitive, robust detection process could pave the way for future cell-based biosensors for AI-2 and subsequent programmed cell function.

That is, in [59] and as shown in **Figure 3**, *E. coli* were modified to enable programmed motility, sensing and actuation based on the density of user-selected features on nearby surfaces. These 'smart' bacteria can then express marker proteins to indicate phenotypic response based on calculated feature density displayed on the surfaces of nearby eukaryotic cells. Specifically, the AI-2/Lsr signaling pathway was rewired an introduced onto the eukaryotic cells as a 'nanofactory' to direct *E. coli* to swim toward a cancer cell line (SCCHN), where they then initiated synthesis of a drug surrogate based on a threshold density of epidermal growth factor receptor (EGFR). This novel technology represented a new type of targeted drug synthesis and delivery and a new area-based switch that could serve multiple purposes within in the field of synthetic biology.

#### **3.3 Interspecies communication**

#### *3.3.1 Universal autoinducer AI-2*

Beyond controlling genetic expression on a global scale, quorum sensing allows bacteria to communicate within and between species. This notion arose with the study and discovery of the aforementioned autoinducer AI-2. Derived from SAM as a part of bacterial 1-carbon metabolism, AI-2 is a general term for a family of cyclic furanones utilized in interspecies communication [60]. In LuxS-containing bacteria, SAM is converted into SAH and then broken down by enzymes Pfs and LuxS sequentially into signaling molecule DPD and other byproducts. Due to the high reactivity of DPD, many distinct but related products could be recognized by different bacterial species as AI-2. Though it is postulated that small molecules of similar structure as AI-2 could serve as potential antagonists that halt the bacterial conversation, only a handful are found (compared to a large number of AI-1 inhibitors). In [61], C-1

**175**

*3.3.2 Applications*

**Figure 3.**

*expression (adapted from Wu et al. [59]).*

*Repurposing E. coli by Engineering Quorum Sensing and Redox Genetic Circuits*

alkyl analogs of AI-2 that quench QS responses in multiple bacterial species simultaneously were developed and synthesized. Interestingly, addition of a single carbon to the C1-alkyl chain of the analog plays a critical role in determining the effect on quenching the QS response. This analog, isobutyl-DPD, was later used to inhibit maturation of *E. coli* biofilms [62]. An expanded and diverse array of AI-2 analogs, including aromatic and cyclic C1-alkyl analogs are synthesized in [63]. Some were identified as species-specific QS disruptors for *E. coli* and *Salmonella typhimurium*, and so were QS quenchers for *Pseudomonas aeruginosa*. Remarkably, these synthetic analogs selectively antagonized quorum sensing among individual bacterial strains

*Biological nanofactories that synthesize AI-2 are targeted to EGFR on the surface of SCCHN cells. AI-2 is emitted from the cell surface and recognized by reprogrammed bacteria, which swim to the site of signal generation and decide, based on AI-2 level (proportional to the EGFR surface density), whether to initiate gene* 

AI-2 is also one of the several signals used by marine bacteria *V. harveyi.* Specifically, AI-2 encoding *luxS* has been found in roughly half of all sequenced bacterial genomes. AI-2 production has been verified in over 80 species, and AI-2 controls gene expression in a variety of bacteria. By using Local Modular Network Alignment Similarity Tool (LMNAST) to study gene order and generate homologous loci, the AI-2/Lsr system was reported to be phylogenetically more dispersed than the well-studied *lac* operon, while its distribution remained densest among gammaproteobacteria [64]. These findings together reinforced the hypothesis that

Interkingdom communication was also shown to be mediated by AI-2. In [66], transcriptomic effects of bacterial secretions from two nonpathogenic *E. coli* strains (BL21 and W3110) on the human colonic cell line HCT-8 were explored using RNA-Seq. Expression of inflammatory cytokine interleukin 8 (IL-8) in HCT-8 cells was found to respond to AI-2 with a pattern of rapid upregulation followed by a subsequent downregulation after 24 h. This discovery helps provide a deeper understanding of the relationship of microbiome and the host, which is of signifi-

This discovery suggests that AI-2 QS manipulation might find application in guiding human physiology and that 'smart' bacteria, those making heterologous proteins such as drugs or essential nutrients and that otherwise serve as decision makers, might find application in a variety of other fields. As an extension, Lentini et al*.* [67] engineered minimal 'artificial' cells capable of expressing AI-2 synthesizing fusion protein His6-LuxS-Pfs-Tyr5 (HLPT) [68] wherein newly synthesized

within a physiologically relevant polymicrobial culture.

bacteria use AI-2 to communicate between species [31, 65].

cant importance in maintaining human health.

*DOI: http://dx.doi.org/10.5772/intechopen.81245*

*Repurposing E. coli by Engineering Quorum Sensing and Redox Genetic Circuits DOI: http://dx.doi.org/10.5772/intechopen.81245*

#### **Figure 3.**

*Gene Expression and Control*

the reason behind the higher product yield.

in the field of synthetic biology.

**3.3 Interspecies communication**

*3.3.1 Universal autoinducer AI-2*

and signal amplification by the T7 RNA polymerase [53, 54]. This study demonstrated one cell population was able to guide protein synthesis process of another by guiding intraspecies communication. Moreover, it was reported in [55] that by simply adding conditioned medium (containing a high amount of AI-2) during recombinant protein induction, one can double the yield of active product. Also, by altering the coincident *luxS* expression to control the AI-2 concentration while also inducing heterologous protein expression, they found an optimal condition where protein yield is dramatically increased. The authors further elucidated the mechanism behind this phenomenon: chaperone GroEL was shown to be coincidentally upregulated post-transcriptionally by AI-2. Because of its native role as a stabilizer of heterologous protein and its role in folding, the upregulation of GroEL might be

More endeavors have been made [56] to increase protein yield in this autonomous system through a different approach. With the same intention in mind [49, 50], a new study showed that reduced heterogeneity between independent cells could be achieved by inserting an enhanced feedback loop to the *E. coli*'s native AI-2 QS system. Upon activation of the engineered system, not only does the foreign pET plasmid concurrently express more sfGFP signal, but it also transcribed more LsrACDB and LsrK than the native *lsr* operon [57]. This overexpression resulted in increased uptake of AI-2, leading to amplified system response and minimized heterogeneity. Heterogeneity, on the other hand, could also be leveraged. In [58], quantized *E. coli* quorums were intentionally assembled through independent engineering of the AI-2 transduction cascade increasing the sensitivity of detector cells. Upon encountering a particular AI-2 level, a discretized sub-population of cells emerge with the desired phenotype. This sensitive, robust detection process could pave the way for future

cell-based biosensors for AI-2 and subsequent programmed cell function.

That is, in [59] and as shown in **Figure 3**, *E. coli* were modified to enable programmed motility, sensing and actuation based on the density of user-selected features on nearby surfaces. These 'smart' bacteria can then express marker proteins to indicate phenotypic response based on calculated feature density displayed on the surfaces of nearby eukaryotic cells. Specifically, the AI-2/Lsr signaling pathway was rewired an introduced onto the eukaryotic cells as a 'nanofactory' to direct *E. coli* to swim toward a cancer cell line (SCCHN), where they then initiated synthesis of a drug surrogate based on a threshold density of epidermal growth factor receptor (EGFR). This novel technology represented a new type of targeted drug synthesis and delivery and a new area-based switch that could serve multiple purposes within

Beyond controlling genetic expression on a global scale, quorum sensing allows bacteria to communicate within and between species. This notion arose with the study and discovery of the aforementioned autoinducer AI-2. Derived from SAM as a part of bacterial 1-carbon metabolism, AI-2 is a general term for a family of cyclic furanones utilized in interspecies communication [60]. In LuxS-containing bacteria, SAM is converted into SAH and then broken down by enzymes Pfs and LuxS sequentially into signaling molecule DPD and other byproducts. Due to the high reactivity of DPD, many distinct but related products could be recognized by different bacterial species as AI-2. Though it is postulated that small molecules of similar structure as AI-2 could serve as potential antagonists that halt the bacterial conversation, only a handful are found (compared to a large number of AI-1 inhibitors). In [61], C-1

**174**

*Biological nanofactories that synthesize AI-2 are targeted to EGFR on the surface of SCCHN cells. AI-2 is emitted from the cell surface and recognized by reprogrammed bacteria, which swim to the site of signal generation and decide, based on AI-2 level (proportional to the EGFR surface density), whether to initiate gene expression (adapted from Wu et al. [59]).*

alkyl analogs of AI-2 that quench QS responses in multiple bacterial species simultaneously were developed and synthesized. Interestingly, addition of a single carbon to the C1-alkyl chain of the analog plays a critical role in determining the effect on quenching the QS response. This analog, isobutyl-DPD, was later used to inhibit maturation of *E. coli* biofilms [62]. An expanded and diverse array of AI-2 analogs, including aromatic and cyclic C1-alkyl analogs are synthesized in [63]. Some were identified as species-specific QS disruptors for *E. coli* and *Salmonella typhimurium*, and so were QS quenchers for *Pseudomonas aeruginosa*. Remarkably, these synthetic analogs selectively antagonized quorum sensing among individual bacterial strains within a physiologically relevant polymicrobial culture.

AI-2 is also one of the several signals used by marine bacteria *V. harveyi.* Specifically, AI-2 encoding *luxS* has been found in roughly half of all sequenced bacterial genomes. AI-2 production has been verified in over 80 species, and AI-2 controls gene expression in a variety of bacteria. By using Local Modular Network Alignment Similarity Tool (LMNAST) to study gene order and generate homologous loci, the AI-2/Lsr system was reported to be phylogenetically more dispersed than the well-studied *lac* operon, while its distribution remained densest among gammaproteobacteria [64]. These findings together reinforced the hypothesis that bacteria use AI-2 to communicate between species [31, 65].

Interkingdom communication was also shown to be mediated by AI-2. In [66], transcriptomic effects of bacterial secretions from two nonpathogenic *E. coli* strains (BL21 and W3110) on the human colonic cell line HCT-8 were explored using RNA-Seq. Expression of inflammatory cytokine interleukin 8 (IL-8) in HCT-8 cells was found to respond to AI-2 with a pattern of rapid upregulation followed by a subsequent downregulation after 24 h. This discovery helps provide a deeper understanding of the relationship of microbiome and the host, which is of significant importance in maintaining human health.

#### *3.3.2 Applications*

This discovery suggests that AI-2 QS manipulation might find application in guiding human physiology and that 'smart' bacteria, those making heterologous proteins such as drugs or essential nutrients and that otherwise serve as decision makers, might find application in a variety of other fields. As an extension, Lentini et al*.* [67] engineered minimal 'artificial' cells capable of expressing AI-2 synthesizing fusion protein His6-LuxS-Pfs-Tyr5 (HLPT) [68] wherein newly synthesized

AI-2 was proven to induce luminescence in nearby cells, particularly an AI-2 reporter strain of *V. harveyi*. This not only demonstrates QS-mediate communication between cells and non-biological, artificial cell mimics, but presents a new technique to alter the complex networks of natural cells without tampering with the original genetic makeup.

Developing, silencing, or intervening with the communication between cells has revolutionized the way we control gene expression. In [69], communication between cells is developed further by modifying the biological 'nanofactories' proposed by LeDuc et al*.* [70] to trigger QS responses in the absence of autoinducers. They are self-assembled and comprised of four functional modules: a targeting module (an antibody), a material sensing module, an assembly module, and a synthesis module (fusion protein His6-Protein G-LuxS-Pfs-Tyr5, HGLPT, (**Figure 4**). Protein G (assembly module) allows the chimeric enzyme to attach to a targeting antibody *ex vivo*, and LuxS and Pfs together convert raw material, SAH, into autoinducer AI-2. The targeting antibody is proven to successfully attach onto targeted *S. typhimurium* in a mixed culture that also includes *E. coli*. Remarkably, this study built up interspecies 'conversation' between cells that do not usually communicate with each other. After *E. coli*-targeted nanofactories were added to non-QS *E. coli* to 'unmute' the null *E. coli*, the activated *E. coli* are co-cultured with reporter *luxS* null *S. typhimurium*. As the levels of activated *E. coli* increased, *S. typhimurium* begin to 'respond' as they received the AI-2 produced by activated *E. coli* and initiate the expression of their own reporter gene. Interkingdom communication between *E. coli* and human intestine epithelial (Caco-2) cells was also developed using this technique [71]. This tool may be very useful for interrogating and interpreting signaling events in human GI tract.

Perhaps next generation antimicrobials can be created by intercepting bacterial communication and creating 'smart' bacteria. Instead of targeting the viability of pathogenic strains, interruption of their communication is proposed, as it is hypothesized that there will be less selective pressure to develop resistance if instead one targets the mechanisms keyed to pathogenicity [72]. As a global autoinducer, inhibition of the signal AI-2 could possibly lead to decreased virulence in a variety of bacterial species. Many parts of the AI-2/LuxS system, from signal generators (Pfs and LuxS) to signal receptors are all likely targets for inhibition, especially as there are many synthesized AI-2 analogs that are available for quorum quenching [61–63, 73]. In another case [74], probiotic *E. coli* were themselves, engineered to eliminate and prevent *P. aeruginosa* gut infection by reducing biofilm formation. However, it was the *P. aeruginosa*-secreted, speciesspecific autoinducer AHL (3OC12HSL) secreted detected by the probiotic *E. coli* and served as the trigger the for the expression of an anti-biofilm enzyme dispersin B (DspB) and a *P. aeruginosa* toxin, pyocin.

In addition to potential for therapeutic synthesis and delivery, *E. coli* cells can be rewired to serve in networks that provide molecular information about their surroundings or as cell sensors or 'sentinels'. For example in [75], engineered *E. coli* sentinels are made to recognize and move toward hydrogen peroxide, a non-native chemoattractant and potential toxin. Similarly, commensal gastrointestinal strain *E. coli* Nissle 1917 are engineered to recognize gastrointestinal dysfunction biomarker nitric oxide (NO) [76]. These 'smart' bacterial sensors can generate strong fluorescent response upon NO recognition and may serve as simple diagnostic tool for diseases like Crohn's Disease and ulcerative colitis. In [77], nano-guided cell networks that serve as conveyors of molecular communication are developed (**Figure 5**). This system interprets molecular information by intercepting diverse molecular inputs, processes them autonomously through independent cell units within the system and refines output to include positive responders that are viewed via orthogonal, simple optical means. That is,

**177**

by the engineered cell.

**systems**

**Figure 4.**

**Figure 5.**

*Repurposing E. coli by Engineering Quorum Sensing and Redox Genetic Circuits*

in the preceding sections we have described how engineering cells and the signaling processes that guide their behavior can be used to enhance the overall expression of proteins, but also that when coupled with more advanced functions, cells can serve as their own autonomous factories or surveyors of various microenvironments. A key to performing these functions in an optimal manner is the control of the signaling process, the signal itself, its positioning, its strength or frequency, and its recognition

*Schematic of a cell population and nanomaterial-based network. This conceptual system describes cells and magnetic nanoparticle networks that intercept diverse molecular inputs, process them autonomously through independent cell units, and refines output to include positive responders that are viewed via visual classification* 

*Biological nanofactory induced interspecies communication. SAH (blue circle) is converted into AI-2 (yellow circle) by the nanofactory fusion protein anchored onto E. coli. AI-2 thus activated QS gene expression in* 

**4. Bridging the bio-electro interface: Redox signaling and electrogenetic** 

In addition to quorum sensing, bacteria use numerous other small chemical molecules to build up conversations between themselves and with the environment.

*DOI: http://dx.doi.org/10.5772/intechopen.81245*

*reporter cells (adapted from Fernandes et al. [69]).*

*(red or red and green, adapted from Terrell et al. [77]).*

*Repurposing E. coli by Engineering Quorum Sensing and Redox Genetic Circuits DOI: http://dx.doi.org/10.5772/intechopen.81245*

#### **Figure 4.**

*Gene Expression and Control*

original genetic makeup.

AI-2 was proven to induce luminescence in nearby cells, particularly an AI-2 reporter strain of *V. harveyi*. This not only demonstrates QS-mediate communication between cells and non-biological, artificial cell mimics, but presents a new technique to alter the complex networks of natural cells without tampering with the

Developing, silencing, or intervening with the communication between cells has revolutionized the way we control gene expression. In [69], communication between cells is developed further by modifying the biological 'nanofactories' proposed by LeDuc et al*.* [70] to trigger QS responses in the absence of autoinducers. They are self-assembled and comprised of four functional modules: a targeting module (an antibody), a material sensing module, an assembly module, and a synthesis module (fusion protein His6-Protein G-LuxS-Pfs-Tyr5, HGLPT, (**Figure 4**). Protein G (assembly module) allows the chimeric enzyme to attach to a targeting antibody *ex vivo*, and LuxS and Pfs together convert raw material, SAH, into autoinducer AI-2. The targeting antibody is proven to successfully attach onto targeted *S. typhimurium* in a mixed culture that also includes *E. coli*. Remarkably, this study built up interspecies 'conversation' between cells that do not usually communicate with each other. After *E. coli*-targeted nanofactories were added to non-QS *E. coli* to 'unmute' the null *E. coli*, the activated *E. coli* are co-cultured with reporter *luxS* null *S. typhimurium*. As the levels of activated *E. coli* increased, *S. typhimurium* begin to 'respond' as they received the AI-2 produced by activated *E. coli* and initiate the expression of their own reporter gene. Interkingdom communication between *E. coli* and human intestine epithelial (Caco-2) cells was also developed using this technique [71]. This tool may be very useful for interrogating and interpreting signaling events in human GI tract. Perhaps next generation antimicrobials can be created by intercepting bacterial communication and creating 'smart' bacteria. Instead of targeting the viability of pathogenic strains, interruption of their communication is proposed, as it is hypothesized that there will be less selective pressure to develop resistance if instead one targets the mechanisms keyed to pathogenicity [72]. As a global autoinducer, inhibition of the signal AI-2 could possibly lead to decreased virulence in a variety of bacterial species. Many parts of the AI-2/LuxS system, from signal generators (Pfs and LuxS) to signal receptors are all likely targets for inhibition, especially as there are many synthesized AI-2 analogs that are available for quorum quenching [61–63, 73]. In another case [74], probiotic *E. coli* were themselves, engineered to eliminate and prevent *P. aeruginosa* gut infection by reducing biofilm formation. However, it was the *P. aeruginosa*-secreted, speciesspecific autoinducer AHL (3OC12HSL) secreted detected by the probiotic *E. coli* and served as the trigger the for the expression of an anti-biofilm enzyme disper-

**176**

sin B (DspB) and a *P. aeruginosa* toxin, pyocin.

In addition to potential for therapeutic synthesis and delivery, *E. coli* cells can be rewired to serve in networks that provide molecular information about their surroundings or as cell sensors or 'sentinels'. For example in [75], engineered *E. coli* sentinels are made to recognize and move toward hydrogen peroxide, a non-native chemoattractant and potential toxin. Similarly, commensal gastrointestinal strain *E. coli* Nissle 1917 are engineered to recognize gastrointestinal dysfunction biomarker nitric oxide (NO) [76]. These 'smart' bacterial sensors can generate strong fluorescent response upon NO recognition and may serve as simple diagnostic tool for diseases like Crohn's Disease and ulcerative colitis. In [77], nano-guided cell networks that serve as conveyors of molecular communication are developed (**Figure 5**). This system interprets molecular information by intercepting diverse molecular inputs, processes them autonomously through independent cell units within the system and refines output to include positive responders that are viewed via orthogonal, simple optical means. That is,

*Biological nanofactory induced interspecies communication. SAH (blue circle) is converted into AI-2 (yellow circle) by the nanofactory fusion protein anchored onto E. coli. AI-2 thus activated QS gene expression in reporter cells (adapted from Fernandes et al. [69]).*

#### **Figure 5.**

*Schematic of a cell population and nanomaterial-based network. This conceptual system describes cells and magnetic nanoparticle networks that intercept diverse molecular inputs, process them autonomously through independent cell units, and refines output to include positive responders that are viewed via visual classification (red or red and green, adapted from Terrell et al. [77]).*

in the preceding sections we have described how engineering cells and the signaling processes that guide their behavior can be used to enhance the overall expression of proteins, but also that when coupled with more advanced functions, cells can serve as their own autonomous factories or surveyors of various microenvironments. A key to performing these functions in an optimal manner is the control of the signaling process, the signal itself, its positioning, its strength or frequency, and its recognition by the engineered cell.
