Enhanced electrochemical measurement of ß-galactosidase activity in whole cells by coexpression of lactose permease, LacY.

Whole-cell biosensing links the sensing and computing capabilities of microbes to the generation of a detectable reporter. Whole cells enable dynamic biological computation (filtered noise, amplified signals, logic gating etc.). Enzymatic reporters enable in situ signal amplification. Electrochemical measurements are easily quantified and work in turbid environments. In this work we show how the coexpression of the lactose permease, LacY, dramatically improves electrochemical sensing of ß-galactosidase (LacZ) expressed as a reporter in whole cells. The permease facilitates transport of the LacZ substrate, 4-aminophenyl ß-d-galactopyranoside, which is converted to redox active p-aminophenol, which, in turn, is detected via cyclic voltammetry or chronocoulometry. We show a greater than fourfold improvement enabled by lacY coexpression in cells engineered to respond to bacterial signal molecules, pyocyanin and quorum-sensing autoinducer-2.

Electrogenetic Signal Transmission and Propagation in Coculture to Guide Production of a Small Molecule, Tyrosine.

There are many strategies to actuate and control genetic circuits, including providing stimuli like exogenous chemical inducers, light, magnetic fields, and even applied voltage, that are orthogonal to metabolic activity. Their use enables actuation of gene expression for the production of small molecules and proteins in many contexts. Additionally, there are a growing number of reports wherein cocultures, consortia, or even complex microbiomes are employed for the production of biologics, taking advantage of an expanded array of biological function. Combining stimuli-responsive engineered cell populations enhances design space but increases complexity. In this work, we co-opt nature's redox networks and electrogenetically route control signals into a consortium of microbial cells engineered to produce a model small molecule, tyrosine. In particular, we show how electronically programmed short-lived signals (i.e., hydrogen peroxide) can be transformed by one population and propagated into sustained longer-distance signals that, in turn, guide tyrosine production in a second population building on bacterial quorum sensing that coordinates their collective behavior. Two design methodologies are demonstrated. First, we use electrogenetics to transform redox signals into the quorum sensing autoinducer, AI-1, that, in turn, induces a tyrosine biosynthesis pathway transformed into a second population. Second, we use the electrogenetically stimulated AI-1 to actuate expression of ptsH, boosting the growth rate of tyrosine-producing cells, augmenting both their number and metabolic activity. In both cases, we show how signal propagation within the coculture helps to ensure tyrosine production. We suggest that this work lays a foundation for employing electrochemical stimuli and engineered cocultures for production of molecular products in biomanufacturing environments.

Parsed synthesis of pyocyanin via co-culture enables context-dependent intercellular redox communication.

BACKGROUND: Microbial co-cultures and consortia are of interest in cell-based molecular production and even as "smart" therapeutics in that one can take advantage of division of labor and specialization to expand both the range of available functions and mechanisms for control. The development of tools that enable coordination and modulation of consortia will be crucial for future application of multi-population cultures. In particular, these systems would benefit from an expanded toolset that enables orthogonal inter-strain communication. RESULTS: We created a co-culture for the synthesis of a redox-active phenazine signaling molecule, pyocyanin (PYO), by dividing its synthesis into the generation of its intermediate, phenazine carboxylic acid (PCA) from the first strain, followed by consumption of PCA and generation of PYO in a second strain. Interestingly, both PCA and PYO can be used to actuate gene expression in cells engineered with the soxRS oxidative stress regulon, although importantly this signaling activity was found to depend on growth media. That is, like other signaling motifs in bacterial systems, the signaling activity is context dependent. We then used this co-culture's phenazine signals in a tri-culture to modulate gene expression and production of three model products: quorum sensing molecule autoinducer-1 and two fluorescent marker proteins, eGFP and DsRed. We also showed how these redox-based signals could be intermingled with other quorum-sensing (QS) signals which are more commonly used in synthetic biology, to control complex behaviors. To provide control over product synthesis in the tri-cultures, we also showed how a QS-induced growth control module could guide metabolic flux in one population and at the same time guide overall tri-culture function. Specifically, we showed that phenazine signal recognition, enabled through the oxidative stress response regulon soxRS, was dependent on media composition such that signal propagation within our parsed synthetic system could guide different desired outcomes based on the prevailing environment. In doing so, we expanded the range of signaling molecules available for coordination and the modes by which they can be utilized to influence overall function of a multi-population culture. CONCLUSIONS: Our results show that redox-based signaling can be intermingled with other quorum sensing signaling in ways that enable user-defined control of microbial consortia yielding various outcomes defined by culture medium. Further, we demonstrated the utility of our previously designed growth control module in influencing signal propagation and metabolic activity is unimpeded by orthogonal redox-based signaling. By exploring novel multi-modal strategies for guiding communication and consortia outcome, the concepts introduced here may prove to be useful for coordination of multiple populations within complex microbial systems.

Electronic signals are electrogenetically relayed to control cell growth and co-culture composition.

There is much to be gained by enabling electronic interrogation and control of biological function. While the benefits of bioelectronics that rely on potential-driven ionic flows are well known (electrocardiograms, defibrillators, neural prostheses, etc) there are relatively few advances targeting nonionic molecular networks, including genetic circuits. Redox activities combine connectivity to electronics with the potential for specific genetic control in cells. Here, electrode-generated hydrogen peroxide is used to actuate an electrogenetic "relay" cell population, which interprets the redox cue and synthesizes a bacterial signaling molecule (quorum sensing autoinducer AI-1) that, in turn, signals increased growth rate in a second population. The dramatically increased growth rate of the second population is enabled by expression of a phosphotransferase system protein, HPr, which is important for glucose transport. The potential to electronically modulate cell growth via direct genetic control will enable new opportunities in the treatment of disease and manufacture of biological therapeutics and other molecules.

Bioelectronic control of a microbial community using surface-assembled electrogenetic cells to route signals.

We developed a bioelectronic communication system that is enabled by a redox signal transduction modality to exchange information between a living cell-embedded bioelectronics interface and an engineered microbial network. A naturally communicating three-member microbial network is 'plugged into' an external electronic system that interrogates and controls biological function in real time. First, electrode-generated redox molecules are programmed to activate gene expression in an engineered population of electrode-attached bacterial cells, effectively creating a living transducer electrode. These cells interpret and translate electronic signals and then transmit this information biologically by producing quorum sensing molecules that are, in turn, interpreted by a planktonic coculture. The propagated molecular communication drives expression and secretion of a therapeutic peptide from one strain and simultaneously enables direct electronic feedback from the second strain, thus enabling real-time electronic verification of biological signal propagation. Overall, we show how this multifunctional bioelectronic platform, termed a BioLAN, reliably facilitates on-demand bioelectronic communication and concurrently performs programmed tasks.

Homologous Quorum Sensing Regulatory Circuit: A Dual-Input Genetic Controller for Modulating Quorum Sensing-Mediated Protein Expression in E. coli.

We developed a hybrid synthetic circuit that co-opts the genetic regulation of the native bacterial quorum sensing autoinducer-2 and imposes an extra external controller for maintaining tightly controlled gene expression. This dual-input genetic controller was mathematically modeled and, by design, can be operated in three modes: a constitutive mode that enables consistent and high levels of expression; a tightly repressed mode in which there is very little background expression; and an inducible mode in which concentrations of two signals (arabinose and autoinducer-2) determine the net amplification of the gene(s)-of-interest. We demonstrate the utility of the circuit for the controlled expression of human granulocyte macrophage colony stimulating factor in an engineered probiotic E. coli. This dual-input genetic controller is the first homologous AI-2 quorum sensing circuit that has the ability to be operated in three different modes. We believe it has the potential for wide-ranging biotechnological applications due its versatile features.

A redox-based electrogenetic CRISPR system to connect with and control biological information networks.

Electronic information can be transmitted to cells directly from microelectronics via electrode-activated redox mediators. These transmissions are decoded by redox-responsive promoters which enable user-specified control over biological function. Here, we build on this redox communication modality by establishing an electronic eCRISPR conduit of information exchange. This system acts as a biological signal processor, amplifying signal reception and filtering biological noise. We electronically amplify bacterial quorum sensing (QS) signaling by activating LasI, the autoinducer-1 synthase. Similarly, we filter out unintended noise by inhibiting the native SoxRS-mediated oxidative stress response regulon. We then construct an eCRISPR based redox conduit in both E. coli and Salmonella enterica. Finally, we display eCRISPR based information processing that allows transmission of spatiotemporal redox commands which are then decoded by gelatin-encapsulated E. coli. We anticipate that redox communication channels will enable biohybrid microelectronic devices that could transform our abilities to electronically interpret and control biological function.

Synthetic Biology for Manipulating Quorum Sensing in Microbial Consortia.

Bacteria exist as communities in diverse multispecies environments. Quorum sensing, a process for cell-cell communication, allows individual bacteria to glean information about their surroundings and coordinate activities with their neighbors. Recent studies indicate the importance of quorum sensing in microbiomes, but many questions remain regarding how quorum sensing may influence the composition and function of these communities. Synthetic biology, a field where scientists seek to design biological systems with predictable behavior, may provide tools to probe and manipulate quorum sensing behavior in natural consortia. In parallel, quorum sensing processes can be used as a tool in synthetic biology to construct synthetic cocultures with desired behavior. Here, we review recent synthetic biology strategies for manipulating quorum sensing processes in microbial consortia.

Bacterial co-culture with cell signaling translator and growth controller modules for autonomously regulated culture composition.

Synthetic biology and metabolic engineering have expanded the possibilities for engineered cell-based systems. The addition of non-native biosynthetic and regulatory components can, however, overburden the reprogrammed cells. In order to avoid metabolic overload, an emerging area of focus is on engineering consortia, wherein cell subpopulations work together to carry out a desired function. This strategy requires regulation of the cell populations. Here, we design a synthetic co-culture controller consisting of cell-based signal translator and growth-controller modules that, when implemented, provide for autonomous regulation of the consortia composition. The system co-opts the orthogonal autoinducer AI-1 and AI-2 cell-cell signaling mechanisms of bacterial quorum sensing (QS) to enable cross-talk between strains and a QS signal-controlled growth rate controller to modulate relative population densities. We further develop a simple mathematical model that enables cell and system design for autonomous closed-loop control of population trajectories.

Engineering Escherichia coli for enhanced sensitivity to the autoinducer-2 quorum sensing signal.

The autoinducer-2 (AI-2) quorum sensing system is involved in a range of population-based bacterial behaviors and has been engineered for cell-cell communication in synthetic biology systems. Investigation into the cellular mechanisms of AI-2 processing has determined that overexpression of uptake genes increases AI-2 uptake rate, and genomic deletions of degradation genes lowers the AI-2 level required for activation of reporter genes. Here, we combine these two strategies to engineer an Escherichia coli strain with enhanced ability to detect and respond to AI-2. In an E. coli strain that does not produce AI-2, we monitored AI-2 uptake and reporter protein expression in a strain that overproduced the AI-2 uptake or phosphorylation units LsrACDB or LsrK, a strain with the deletion of AI-2 degradation units LsrF and LsrG, and an "enhanced" strain with both overproduction of AI-2 uptake and deletion of AI-2 degradation elements. By adding up to 40 µM AI-2 to growing cell cultures, we determine that this "enhanced" AI-2 sensitive strain both uptakes AI-2 more rapidly and responds with increased reporter protein expression than the others. This work expands the toolbox for manipulating AI-2 quorum sensing processes both in native environments and for synthetic biology applications.

Bacteria Floc, but Do They Flock? Insights from Population Interaction Models of Quorum Sensing.

Quorum sensing (QS) enables coordinated, population-wide behavior. QS-active bacteria "communicate" their number density using autoinducers which they synthesize, collect, and interpret. Tangentially, chemotactic bacteria migrate, seeking out nutrients and other molecules. It has long been hypothesized that bacterial behaviors, such as chemotaxis, were the primordial progenitors of complex behaviors of higher-order organisms. Recently, QS was linked to chemotaxis, yet the notion that these behaviors can together contribute to higher-order behaviors has not been shown. Here, we mathematically link flocking behavior, commonly observed in fish and birds, to bacterial chemotaxis and QS by constructing a phenomenological model of population-scale QS-mediated phenomena. Specifically, we recast a previously developed mathematical model of flocking and found that simulated bacterial behaviors aligned well with well-known QS behaviors. This relatively simple system of ordinary differential equations affords analytical analysis of asymptotic behavior and describes cell position and velocity, QS-mediated protein expression, and the surrounding concentrations of an autoinducer. Further, heuristic explorations of the model revealed that the emergence of "migratory" subpopulations occurs only when chemotaxis is directly linked to QS. That is, behaviors were simulated when chemotaxis was coupled to QS and when not. When coupled, the bacterial flocking model predicts the formation of two distinct groups of cells migrating at different speeds in their journey toward an attractant. This is qualitatively similar to phenomena spotted in our Escherichiacoli chemotaxis experiments as well as in analogous work observed over 50 years ago.IMPORTANCE Our modeling efforts show how cell density can affect chemotaxis; they help to explain the roots of subgroup formation in bacterial populations. Our work also reinforces the notion that bacterial mechanisms are at times exhibited in higher-order organisms.

Evidence of link between quorum sensing and sugar metabolism in Escherichia coli revealed via cocrystal structures of LsrK and HPr.

Quorum sensing (QS), a bacterial process that regulates population-scale behavior, is mediated by small signaling molecules, called autoinducers (AIs), that are secreted and perceived, modulating a "collective" phenotype. Because the autoinducer AI-2 is secreted by a wide variety of bacterial species, its "perception" cues bacterial behavior. This response is mediated by the lsr (LuxS-regulated) operon that includes the AI-2 transporter LsrACDB and the kinase LsrK. We report that HPr, a phosphocarrier protein central to the sugar phosphotransferase system of Escherichia coli, copurifies with LsrK. Cocrystal structures of an LsrK/HPr complex were determined, and the effects of HPr and phosphorylated HPr on LsrK activity were assessed. LsrK activity is inhibited when bound to HPr, revealing new linkages between QS activity and sugar metabolism. These findings help shed new light on the abilities of bacteria to rapidly respond to changing nutrient levels at the population scale. They also suggest new means of manipulating QS activity among bacteria and within various niches.

Modification and Assembly of a Versatile Lactonase for Bacterial Quorum Quenching.

This work sets out to provide a self-assembled biopolymer capsule activated with a multi-functional enzyme for localized delivery. This enzyme, SsoPox, which is a lactonase and phosphotriesterase, provides a means of interrupting bacterial communication pathways that have been shown to mediate pathogenicity. Here we demonstrate the capability to express, purify and attach SsoPox to the natural biopolymer chitosan, preserving its activity to "neutralize" long-chain autoinducer-1 (AI-1) communication molecules. Attachment is shown via non-specific binding and by engineering tyrosine and glutamine affinity 'tags' at the C-terminus for covalent linkage. Subsequent degradation of AI-1, in this case N-(3-oxododecanoyl)-l-homoserine lactone (OdDHL), serves to "quench" bacterial quorum sensing (QS), silencing intraspecies communication. By attaching enzymes to pH-responsive chitosan that, in turn, can be assembled into various forms, we demonstrate device-based flexibility for enzyme delivery. Specifically, we have assembled quorum-quenching capsules consisting of an alginate inner core and an enzyme "decorated" chitosan shell that are shown to preclude bacterial QS crosstalk, minimizing QS mediated behaviors.

Insightful directed evolution of Escherichia coli quorum sensing promoter region of the lsrACDBFG operon: a tool for synthetic biology systems and protein expression.

Quorum sensing (QS) regulates many natural phenotypes (e.q. virulence, biofilm formation, antibiotic resistance), and its components, when incorporated into synthetic genetic circuits, enable user-directed phenotypes. We created a library of Escherichia coli lsr operon promoters using error-prone PCR (ePCR) and selected for promoters that provided E. coli with higher tetracycline resistance over the native promoter when placed upstream of the tet(C) gene. Among the fourteen clones identified, we found several mutations in the binding sites of QS repressor, LsrR. Using site-directed mutagenesis we restored all p-lsrR-box sites to the native sequence in order to maintain LsrR repression of the promoter, preserving the other mutations for analysis. Two promoter variants, EP01rec and EP14rec, were discovered exhibiting enhanced protein expression. In turn, these variants retained their ability to exhibit the LsrR-mediated QS switching activity. Their sequences suggest regulatory linkage between CytR (CRP repressor) and LsrR. These promoters improve upon the native system and exhibit advantages over synthetic QS promoters previously reported. Incorporation of these promoters will facilitate future applications of QS-regulation in synthetic biology and metabolic engineering.