*3.1.1 LuxIR system of V. fischeri*

Quorum sensing mechanisms vary from species to species, and hence here we introduce the first-described QS system of the bioluminescent marine bacterium *Vibrio fischeri* as a paradigm for most systems in Gram-negative bacteria [30]. Relevant differences for each organism will be provided as necessary, yet an excellent review by Waters [31] has described most known systems in detail. *V. fischeri* infects higher order organisms, such as luminescent Hawaiian squid *Euprymna scolopes*, within its light organ is completely occupied by the bacterium. When confined, the bacterial population density can reach up to 1011 cells per ml and at that point luminescence genes are expressed through a QS mechanism. The luminescence shed by the bacterial consortium can be used, presumably for counterillumination to mask the squid's shadow so that it avoids predation.

**Figure 1** illustrates the QS system of *V. fischeri*. Protein LuxI and LuxR control expression of the luciferase operon (*luxICDABE*) required for luminescence

**171**

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

production. *LuxI* encodes for an autoinducer synthase that produces the acylhomoserine lactone (AHL) autoinducer 3OC6-homoserine lactone. Following its production, the AHL will begin to accumulate - its concentration increasing as the cell density increases. Upon reaching a critical level, LuxR the cytoplasmic autoinducer receptor/DNA-binding transcriptional activator, will bind to AHL and this complex will initiate the expression of the luciferase operon. This actuates a positive feedback loop, as LuxI is encoded in the operon, and soon the environment will be flooded with AHL which, in turn, switches all bacteria nearby to the QS active, lightproducing mode [32]. The system observed in other *Vibrio* species is more complex, with additional sensing and phosphorylation components in the upstream of *luxR* [33]. In addition, small RNA (sRNA) have been shown to play a vital role in regulating the quorum circuits of *Vibrio harveyi* and *Vibrio cholerae* [34]. These LuxRI-type systems are mostly used for intraspecies communication, as extreme specificity exists between LuxR proteins and their cognate AHL autoinducer ligands.

*Quorum sensing in Vibrio fischeri green pentagons denote AHL autoinducer that LuxI produces (3OC6 homoserine lactone). Transcriptional regulator, LuxR, modulates expression of AHL synthase, LuxI, and the* 

While some of the *Vibrio* QS components are present in *E. coli* (and *Salmonella* strains), the QS system of both species has been found to be distinctively different than that of the *Vibrio*. Several interspecies signaling systems have been identified: those mediated by LuxR homolog SdiA; the LuxS/autoinducer 2 (AI-2) system; an AI-3 system; and a signaling system mediated by indole [35]. Remarkably, the LuxS/ AI-2 system possesses the unique feature of endowing cell-population-dependent behavior while interacting with central metabolism through the intracellular activated methyl cycle. LuxS intervenes in central metabolism by functioning in the pathway for metabolism of *S*-adenosylmethionine (SAM), the major cellular methyl donor. Transfer of the methyl moiety to various substrates produces the toxic by-product *S*-adenosylhomocysteine (SAH); while LuxS-containing bacteria have two enzymes (Pfs and LuxS) acting sequentially to convert SAH to adenine, homocysteine, and the signaling molecule DPD [31]. Together, LuxS/AI-2 system

The *luxS* gene, which has a wide range of functions between numerous species, is responsible for AI-2 synthesis in QS networks. However, it was noted that the *luxS* transcriptional profile was reportedly unsynchronized with the accumulation profile of extracellular AI-2 in bacterial supernatants. Confounding its interpretation,

has the potential to regulate both gene expression and the cell fitness.

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

*lux operon, leading to luciferase-mediated light emission.*

*3.1.2 LuxS/AI-2 system of E. coli*

**Figure 1.**

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

#### **Figure 1.**

*Gene Expression and Control*

in new platforms.

that follow, we describe efforts to minimally alter the native bacterial signaling processes of quorum sensing and oxidative stress to repurpose *E. coli* for application

Gene expression in bacteria can be regulated by a wide array of intra- and extracellular cues. On top of the common chemical inducers that are most often introduced manually to initiate protein overexpression, bacteria are actually capable of producing their own extracellular signals for intercellular communication. The term "quorum sensing (QS)" was coined by EP Greenberg and colleagues decades ago, to describe the phenomena where the secretion and perception of small signaling molecules are transduced to coordinate behavior of a minimal unit (quorum) of microorganisms. Since then, there's been an explosion in understanding how bacteria communicate with themselves. In this section, well-characterized quorumsensing systems and types of signals, receptors, mechanisms of signal transduction, and target outputs of each system are introduced. In addition, since quorum sensing in many bacteria is also shown to control gene expression in a global manner, several regulons will be introduced, again with the focus on *E. coli* and their potential application. Lastly, beyond controlling gene expression on a global scale, quorum sensing allows bacteria to communicate within and between species. Common pathways and inducers of interspecies communication will be introduced, and we will highlight some of the many applications built upon this ability to communicate not only between species, but also between kingdoms and non-biological substances. That is, by introducing QS phenomena, we develop its potential for keying

protein expression via genetic or other means to cue its signaling processes.

thus gaining the ability to function as a multicellular organism.

lumination to mask the squid's shadow so that it avoids predation.

Quorum sensing bacteria produce and release chemical signal molecules termed autoinducers, whose external concentration increases as a function of increasing cell-population density. Once the bacteria detect that autoinducers have reached a minimal threshold level of stimulatory concentration, they will respond by altering their gene expression and behavior. Autoinducers are the cues by which QS bacteria communicate and synchronize particular behaviors on a population-wide scale,

Quorum sensing mechanisms vary from species to species, and hence here we introduce the first-described QS system of the bioluminescent marine bacterium *Vibrio fischeri* as a paradigm for most systems in Gram-negative bacteria [30]. Relevant differences for each organism will be provided as necessary, yet an excellent review by Waters [31] has described most known systems in detail. *V. fischeri* infects higher order organisms, such as luminescent Hawaiian squid *Euprymna scolopes*, within its light organ is completely occupied by the bacterium. When confined, the bacterial population density can reach up to 1011 cells per ml and at that point luminescence genes are expressed through a QS mechanism. The luminescence shed by the bacterial consortium can be used, presumably for counteril-

**Figure 1** illustrates the QS system of *V. fischeri*. Protein LuxI and LuxR control

expression of the luciferase operon (*luxICDABE*) required for luminescence

**3.1 Quorum sensing and its networks**

*3.1.1 LuxIR system of V. fischeri*

**3. Decipher the bacterial dialog: quorum sensing**

**170**

*Quorum sensing in Vibrio fischeri green pentagons denote AHL autoinducer that LuxI produces (3OC6 homoserine lactone). Transcriptional regulator, LuxR, modulates expression of AHL synthase, LuxI, and the lux operon, leading to luciferase-mediated light emission.*

production. *LuxI* encodes for an autoinducer synthase that produces the acylhomoserine lactone (AHL) autoinducer 3OC6-homoserine lactone. Following its production, the AHL will begin to accumulate - its concentration increasing as the cell density increases. Upon reaching a critical level, LuxR the cytoplasmic autoinducer receptor/DNA-binding transcriptional activator, will bind to AHL and this complex will initiate the expression of the luciferase operon. This actuates a positive feedback loop, as LuxI is encoded in the operon, and soon the environment will be flooded with AHL which, in turn, switches all bacteria nearby to the QS active, lightproducing mode [32]. The system observed in other *Vibrio* species is more complex, with additional sensing and phosphorylation components in the upstream of *luxR* [33]. In addition, small RNA (sRNA) have been shown to play a vital role in regulating the quorum circuits of *Vibrio harveyi* and *Vibrio cholerae* [34]. These LuxRI-type systems are mostly used for intraspecies communication, as extreme specificity exists between LuxR proteins and their cognate AHL autoinducer ligands.

#### *3.1.2 LuxS/AI-2 system of E. coli*

While some of the *Vibrio* QS components are present in *E. coli* (and *Salmonella* strains), the QS system of both species has been found to be distinctively different than that of the *Vibrio*. Several interspecies signaling systems have been identified: those mediated by LuxR homolog SdiA; the LuxS/autoinducer 2 (AI-2) system; an AI-3 system; and a signaling system mediated by indole [35]. Remarkably, the LuxS/ AI-2 system possesses the unique feature of endowing cell-population-dependent behavior while interacting with central metabolism through the intracellular activated methyl cycle. LuxS intervenes in central metabolism by functioning in the pathway for metabolism of *S*-adenosylmethionine (SAM), the major cellular methyl donor. Transfer of the methyl moiety to various substrates produces the toxic by-product *S*-adenosylhomocysteine (SAH); while LuxS-containing bacteria have two enzymes (Pfs and LuxS) acting sequentially to convert SAH to adenine, homocysteine, and the signaling molecule DPD [31]. Together, LuxS/AI-2 system has the potential to regulate both gene expression and the cell fitness.

The *luxS* gene, which has a wide range of functions between numerous species, is responsible for AI-2 synthesis in QS networks. However, it was noted that the *luxS* transcriptional profile was reportedly unsynchronized with the accumulation profile of extracellular AI-2 in bacterial supernatants. Confounding its interpretation,

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 mathematical model [39].
