*4.1.2 OxyR: thiol-based, peroxide stress sensor*

The *E. coli* transcriptional activator, OxyR, is a member of the LysR family of transcriptional regulators. Although it is often cited as the model for bacterial redox sensors, the precise mechanism of thiol modification and the consequences for OxyR activity are the subject of ongoing controversy [80]. Like SoxR, OxyR acts as a repressor of *oxyS* RNA transcription in *E. coli*. Oxidation of cysteine residues in OxyR results in a dramatic secondary structure rearrangement, which leads to a change in the DNA-binding specificity of OxyR, recruitment of RNA polymerase to *OxyR/S* promoters, and the subsequent transcriptional activation of downstream genes such as *oxyS*. OxyS RNA, in turn, is a global oxidative stress regulator mediating the activation or repression of over 40 genes, including several detoxifying enzymes such as hydroperoxidase I (*katG*) and alkylhydroperoxide reductase (*ahpCF*) [75, 78]. Responses of *katG* and *ahpCF*, along with many genes in *SoxR/S* regulon (*sodA, zwf, fumC* and *acnA*) upon paraquat (superoxide ion regenerating redox reagent) insult have been revealed in [81].

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

**4.2 Redox capacitor and bio-electrode interface communication**

To probe bio-related redox reactions/signaling simply and readily, recently developed redox-capacitor films can serve as a bio-electrode interface. These are well-described and have been reviewed [82]. In brief, these electrochemical tools are capable of accepting, storing and donating electrons from mediators commonly used in electrochemistry and also in biology. Biofabricated from catechol and the polysaccharide chitosan, the former can be readily (and reversibly) oxidized. When catechol is oxidized, quinone is formed and it can be covalently grafted onto chitosan. In addition, chitosan can be easily 'electro-assembled' onto electrodes owing to its pH-responsive properties. That is, when a voltage is applied to an electrode submersed in an aqueous solution containing chitosan, the pH near the electrode can be controlled. When basic (above the pKa of chitosan, ~6.5), chitosan will form a hydrophobic network and assemble onto the electrode as a film or hydrogel, depending on the application of the electronic charge. When the catechol/quinone redox couple is integrated into the film, it can serve as a source or sink of electrons. Diffusible redox mediators can be added as they can exchange electrons ('charge/ discharge') with the redox-active films. Common biology-related mediators include molecules such as ascorbate and NADH, which can charge and discharge the film. Pyocyanin, a toxin secreted by *P. aeruginosa*, is also found to be able to donate electrons to catechol-chitosan film (charging). This metabolite is noted because it, like many other mediators, can also undergo redox-cycling in the film to amplify outputs and facilitate detection of is host cell. It can similarly carry electrons from electrodes directly to proteins or cells near the electrode where such transfer of

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

'information' can control biological processes.

but their activity was found to be tunable.

**4.3 Electrical process modulation and gene induction**

Many researchers have endeavored finding new ways to control cell processes. The use of optical means to regulate gene expression has garnered significant attention and resulted in an entire field of optogenetics [83]. Genetic switches that operate on optical signals (even small changes in wavelength or color) have been shown to be powerful exogenous controllers of cell function [84, 85]. More recently, researchers have turned to electronic devices to directly control biochemical reactions. In [86], a transistor-like device is engineered to control glucose metabolism of yeast (*S. cerevisiae*). Changes in gating voltage of the device are reported to bring about acceleration or deceleration of the depletion rate of glucose, and in turn the production rate of end-products (ATP and ethanol). Biofabrication and cell-based communication can also be enhanced through electrical control. In a nano-biosystem [87], electrical signals were used to assemble and tune an enzymatic pathway. The assembly comprised of electrodeposited chitosan film on top of a gold electrode, followed by the enzymatic and covalent grafting of a model enzyme HLPT [68] onto the chitosan scaffold. Through different electrical signals and with the help of diffusible redox mediators (pyocyanin), not only the amount of assembled enzymes

Even more recently, a synthetic, mammalian electro-genetic transcription circuit was created [88]. This was done by linking the electrochemical oxidation of ethanol to acetaldehyde, triggering an acetaldehyde-inducible gene expression circuit. While an indirect outcome of the applied voltage, this was the first study whereby specifically intended gene expression was induced by electronic means. A more direct methodology recently appeared [89] in which the engineered genetic circuit responds directly to the electrode-oxidized signal molecule, opening an entirely new modality for bioelectronic control (**Figure 6**). Again, pyocyanin was

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

#### **4.2 Redox capacitor and bio-electrode interface communication**

To probe bio-related redox reactions/signaling simply and readily, recently developed redox-capacitor films can serve as a bio-electrode interface. These are well-described and have been reviewed [82]. In brief, these electrochemical tools are capable of accepting, storing and donating electrons from mediators commonly used in electrochemistry and also in biology. Biofabricated from catechol and the polysaccharide chitosan, the former can be readily (and reversibly) oxidized. When catechol is oxidized, quinone is formed and it can be covalently grafted onto chitosan. In addition, chitosan can be easily 'electro-assembled' onto electrodes owing to its pH-responsive properties. That is, when a voltage is applied to an electrode submersed in an aqueous solution containing chitosan, the pH near the electrode can be controlled. When basic (above the pKa of chitosan, ~6.5), chitosan will form a hydrophobic network and assemble onto the electrode as a film or hydrogel, depending on the application of the electronic charge. When the catechol/quinone redox couple is integrated into the film, it can serve as a source or sink of electrons. Diffusible redox mediators can be added as they can exchange electrons ('charge/ discharge') with the redox-active films. Common biology-related mediators include molecules such as ascorbate and NADH, which can charge and discharge the film. Pyocyanin, a toxin secreted by *P. aeruginosa*, is also found to be able to donate electrons to catechol-chitosan film (charging). This metabolite is noted because it, like many other mediators, can also undergo redox-cycling in the film to amplify outputs and facilitate detection of is host cell. It can similarly carry electrons from electrodes directly to proteins or cells near the electrode where such transfer of 'information' can control biological processes.

#### **4.3 Electrical process modulation and gene induction**

Many researchers have endeavored finding new ways to control cell processes. The use of optical means to regulate gene expression has garnered significant attention and resulted in an entire field of optogenetics [83]. Genetic switches that operate on optical signals (even small changes in wavelength or color) have been shown to be powerful exogenous controllers of cell function [84, 85]. More recently, researchers have turned to electronic devices to directly control biochemical reactions. In [86], a transistor-like device is engineered to control glucose metabolism of yeast (*S. cerevisiae*). Changes in gating voltage of the device are reported to bring about acceleration or deceleration of the depletion rate of glucose, and in turn the production rate of end-products (ATP and ethanol). Biofabrication and cell-based communication can also be enhanced through electrical control. In a nano-biosystem [87], electrical signals were used to assemble and tune an enzymatic pathway. The assembly comprised of electrodeposited chitosan film on top of a gold electrode, followed by the enzymatic and covalent grafting of a model enzyme HLPT [68] onto the chitosan scaffold. Through different electrical signals and with the help of diffusible redox mediators (pyocyanin), not only the amount of assembled enzymes but their activity was found to be tunable.

Even more recently, a synthetic, mammalian electro-genetic transcription circuit was created [88]. This was done by linking the electrochemical oxidation of ethanol to acetaldehyde, triggering an acetaldehyde-inducible gene expression circuit. While an indirect outcome of the applied voltage, this was the first study whereby specifically intended gene expression was induced by electronic means. A more direct methodology recently appeared [89] in which the engineered genetic circuit responds directly to the electrode-oxidized signal molecule, opening an entirely new modality for bioelectronic control (**Figure 6**). Again, pyocyanin was

*Gene Expression and Control*

biological function.

expression in *E. coli*.

the promoter [78, 79].

*4.1.2 OxyR: thiol-based, peroxide stress sensor*

redox reagent) insult have been revealed in [81].

**4.1 Redox signaling in biological systems**

It is well known that redox reactions and redox based signaling pervade living cells and are extremely crucial to both anabolic and catabolic metabolism. Redox-based molecular systems, however, are also leveraged by bacteria for communication. Cells must detect a variety of oxidative stressors and quickly respond so as to avoid oxidative damage and maintain redox balance in order to survive. In this section, several redox signaling pathways will be introduced, yet emphasis will be on how redox signaling and electrochemistry help connect communication and information transfer between biological systems and electronic devices. In this way, redox molecules can serve as exogenous and electronically-programmed controllers of

In response to redox imbalance, new metabolic pathways are initiated, the repair

The *E. coli* SoxRS system enhances the production of ~45 proteins in response to superoxide exposure, including those in detoxification (*sodA*, manganese superoxide dismutase), DNA repair (*nfo*, endonuclease IV), maintaining cellular reducing power (*zwf*, glucose-6-phosphate dehydrogenase) and central metabolism (*fumC*, superoxide-stable fumarase C and *acnA*, aconitase A). The *E. coli* SoxR protein exists as a homodimer that contains one [2Fe–2S] cluster per subunit. During aerobic growth, up to 95% of SoxR are held in the reduced ([2Fe–2S]1+) state. Upon sensing conditions that promote the production of superoxide, SoxR is oxidized to ([2Fe–2S]2+) clusters and it leaves the *SoxR/S* promoter region (*psoxRS*) to activate the expression of transcription factor SoxS. SoxS, unlike SoxR, when bound to *psoxRS* initiates the expression of the proteins listed above located downstream of

The *E. coli* transcriptional activator, OxyR, is a member of the LysR family of transcriptional regulators. Although it is often cited as the model for bacterial redox sensors, the precise mechanism of thiol modification and the consequences for OxyR activity are the subject of ongoing controversy [80]. Like SoxR, OxyR acts as a repressor of *oxyS* RNA transcription in *E. coli*. Oxidation of cysteine residues in OxyR results in a dramatic secondary structure rearrangement, which leads to a change in the DNA-binding specificity of OxyR, recruitment of RNA polymerase to *OxyR/S* promoters, and the subsequent transcriptional activation of downstream genes such as *oxyS*. OxyS RNA, in turn, is a global oxidative stress regulator mediating the activation or repression of over 40 genes, including several detoxifying enzymes such as hydroperoxidase I (*katG*) and alkylhydroperoxide reductase (*ahpCF*) [75, 78]. Responses of *katG* and *ahpCF*, along with many genes in *SoxR/S* regulon (*sodA, zwf, fumC* and *acnA*) upon paraquat (superoxide ion regenerating

or bypassing of damaged cellular components is coordinated and systems that protect the cell from further damage are induced. Throughout the years, many studies have revealed a vast repertoire of elegant solutions that have evolved to allow bacteria to sense and respond to different redox signals [78]. Below, two oxidative stress sensors, SoxR and OxyR, and their corresponding signaling pathway will be introduced. These systems are later shown to enable electrical control of gene

*4.1.1 SoxR: [Fe-S]-cluster based, superoxide/nitric oxide stress sensor*

**178**

#### **Figure 6.**

*Electrogenetic induction system scheme. Pyo (O) initiates gene induction and Fcn (R/O), through interactions with respiratory machinery, allows electronic control of induction level. Fcn (R/O), ferro/ferricyanide; Pyo, pyocyanin. Encircled 'e<sup>−</sup>'and arrows indicate electron movement (adapted from Tschirhart et al. [89]).*

used in their system, it is responsible for translating electrical signals into a biochemical redox signal that, in turn, can be sensed by SoxR and in sequence, initiate the expression from *psoxS* promoter. Strikingly, gene expression controlled by this device is functionally reversible on relatively short time scales (30–45 min). It was also found to be quite robust, as oscillatory behaviors were shown over many cycles. Accordingly, both optogenetic and electrogenetic systems will require that an entirely new 'suite' of genetic elements be developed that respond to and coordinate these environmental cues. In the recent study, the expression of AHL-synthesizing enzyme LuxI was electronically actuated, resulting in electronic control of QS behavior of nearby cells. Analogously, motility regulator CheZ was also electronically stimulated demonstrating the electronic initiation of cell motility. This study is the first in which electronic signals guided engineered cells and those, in turn, guided others. While this chapter has focused on gene expression in *E. coli*, it also attempts to show how the simultaneous coordination of gene expression and of the host cells can result in interesting and new application areas.

#### **5. Conclusion**

Researchers in biotechnology are constantly seeking novel platforms or techniques from which to address problems: those that in a broad sense, have enhanced efficacy, while maintaining or intensifying specificity. In this chapter, innovative means that focus on controlling environmental cues to regulate gene expression are introduced. To optimize heterologous protein expression, methods seeking to repress stress responses and retain cells in a 'productive' state are carried out by carefully engineering host cells to respond to various cues that are either introduced exogenously or endogenously. QS systems have appeared that provide targets for controlling bacterial behavior. They are also shown to report on the prevailing metabolic state of a product-producing cell. Early methodologies such as RNAi, genetic mutation, product protein-directed evolution, all successful means to enhance yield, can be reexamined based on new understanding of how cells communicate with one another. That is, QS systems enable the rewiring of endogenous metabolism for the coordinated control of entire populations of cells. This ushers in a new way of viewing protein or product-producing cells as a cell 'collective' rather than as individual cells each identical to one another, responding to cues or inducers such as

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**Author details**

**Acknowledgements**

**Conflict of interest**

College Park, MD, USA

MD, USA

work.

Sally Wang1,2, Gregory F. Payne2

provided the original work is properly cited.

\*Address all correspondence to: bentley@umd.edu

© 2018 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,

and William E. Bentley1,2\*

1 Fischell Department of Bioengineering, University of Maryland, College Park,

2 Institute for Bioscience and Biotechnology Research, University of Maryland,

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

applications in fields such as biotechnology and biosensing.

#1435957) and by the National Institutes of Health (R21EB024102).

IPTG for the controlled overexpression of heterologous proteins. QS systems enable autonomous global gene regulation based on cell density. That is, instead of direct interrogation and control of genetic circuits, QS-based cell-cell communication allows indirect gene regulation through self-secretion and uptake of small signaling molecules. Further, exogenous and orthogonal signals, such as those provided by optical and electrical means can be interfaced with cells, providing exquisite control of gene expression. Importantly, in host cells were synthetic components contribute minimal perturbation to native systems, exogenously signaled protein expression can be coupled with exogenously controlled cell behavior (e.g., swimming or decision making). Electrochemistry, along with the invention of redox capacitors, thusly opens a new niche for genetic induction. That is, by leveraging the ability of mediators to translate electrical signals into chemical cues, researchers can cue changes in environmental electrical state that, in turn, are capable of inducing gene expression. These innovative methods will no doubt continue to generate impactful

This work was supported by DTRA (HDTRA1-13-0037) and NSF (DMREF

We declare that we have no conflicts of interest associated with the submitted

*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*

IPTG for the controlled overexpression of heterologous proteins. QS systems enable autonomous global gene regulation based on cell density. That is, instead of direct interrogation and control of genetic circuits, QS-based cell-cell communication allows indirect gene regulation through self-secretion and uptake of small signaling molecules. Further, exogenous and orthogonal signals, such as those provided by optical and electrical means can be interfaced with cells, providing exquisite control of gene expression. Importantly, in host cells were synthetic components contribute minimal perturbation to native systems, exogenously signaled protein expression can be coupled with exogenously controlled cell behavior (e.g., swimming or decision making). Electrochemistry, along with the invention of redox capacitors, thusly opens a new niche for genetic induction. That is, by leveraging the ability of mediators to translate electrical signals into chemical cues, researchers can cue changes in environmental electrical state that, in turn, are capable of inducing gene expression. These innovative methods will no doubt continue to generate impactful applications in fields such as biotechnology and biosensing.
