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
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.
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
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.
- NSF-PAR ID:
- 10360383
- Publisher / Repository:
- Springer Science + Business Media
- Date Published:
- Journal Name:
- Microbial Cell Factories
- Volume:
- 20
- Issue:
- 1
- ISSN:
- 1475-2859
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Abstract E. coli andSalmonella enterica . Finally, we display eCRISPR based information processing that allows transmission of spatiotemporal redox commands which are then decoded by gelatin-encapsulatedE. coli . We anticipate that redox communication channels will enable biohybrid microelectronic devices that could transform our abilities to electronically interpret and control biological function. -
Bondy-Denomy, Joseph (Ed.)more » « less
ABSTRACT Many bacterial histidine kinases work in two-component systems that combine into larger multi-kinase networks. NahK is one of the kinases in the GacS Multi-Kinase Network (MKN), which is the MKN that controls biofilm regulation in the opportunistic pathogen
Pseudomonas aeruginosa . This network has also been associated with regulating many virulence factorsP. aeruginosa secretes to cause disease. However, the individual role of each kinase is unknown. In this study, we identify NahK as a novel regulator of the phenazine pyocyanin (PYO). Deletion ofnahK leads to a fourfold increase in PYO production, almost exclusively through upregulation of phenazine operon two (phz2 ). We determined that this upregulation is due to mis-regulation of allP. aeruginosa quorum-sensing (QS) systems, with a large upregulation of thePseudomonas quinolone signal system and a decrease in production of the acyl-homoserine lactone-producing system,las . In addition, we see differences in expression of quorum-sensing inhibitor proteins that align with these changes. Together, these data contribute to understanding how the GacS MKN modulates QS and virulence and suggest a mechanism for cell density-independent regulation of quorum sensing.IMPORTANCE Pseudomonas aeruginosa is a Gram-negative bacterium that establishes biofilms as part of its pathogenicity.P. aeruginosa infections are associated with nosocomial infections. As the prevalence of multi-drug-resistantP. aeruginosa increases, it is essential to understand underlying virulence molecular mechanisms. Histidine kinase NahK is one of several kinases inP. aeruginosa implicated in biofilm formation and dispersal. Previous work has shown that the nitric oxide sensor, NosP, triggers biofilm dispersal by inhibiting NahK. The data presented here demonstrate that NahK plays additional important roles in theP. aeruginosa lifestyle, including regulating bacterial communication mechanisms such as quorum sensing. These effects have larger implications in infection as they affect toxin production and virulence. -
more » « less
Abstract Microelectronic devices can directly communicate with biology, as electronic information can be transmitted via redox reactions within biological systems. By engineering biology’s native redox networks, we enable electronic interrogation and control of biological systems at several hierarchical levels: proteins, cells, and cell consortia. First, electro-biofabrication facilitates on-device biological component assembly. Then, electrode-actuated redox data transmission and redox-linked synthetic biology allows programming of enzyme activity and closed-loop electrogenetic control of cellular function. Specifically, horseradish peroxidase is assembled onto interdigitated electrodes where electrode-generated hydrogen peroxide controls its activity.
E. coli ’s stress response regulon,oxyRS , is rewired to enable algorithm-based feedback control of gene expression, including an eCRISPR module that switches cell-cell quorum sensing communication from one autoinducer to another—creating an electronically controlled ‘bilingual’ cell. Then, these disparate redox-guided devices are wirelessly connected, enabling real-time communication and user-based control. We suggest these methodologies will help us to better understand and develop sophisticated control for biology. -
Quorum sensing (QS) is a molecular signaling modality that mediates molecular-based cell–cell communication. Prevalent in nature, QS networks provide bacteria with a method to gather information from the environment and make decisions based on the intel. With its ability to autonomously facilitate both inter- and intraspecies gene regulation, this process can be rewired to enable autonomously actuated, but molecularly programmed, genetic control. On the one hand, novel QS-based genetic circuits endow cells with smart functions that can be used in many fields of engineering, and on the other, repurposed QS circuitry promotes communication and aids in the development of synthetic microbial consortia. Furthermore, engineered QS systems can probe and intervene in interkingdom signaling between bacteria and their hosts. Lastly, QS is demonstrated to establish conversation with abiotic materials, especially by taking advantage of biological and even electronically induced assembly processes; such QS-incorporated biohybrid devices offer innovative ways to program cell behavior and biological function.more » « less
-
more » « less
Background Synthetic microbial consortia are conglomerations of genetically engineered microbes programmed to cooperatively bring about population‐level phenotypes. By coordinating their activity, the constituent strains can display emergent behaviors that are difficult to engineer into isogenic populations. To do so, strains are engineered to communicate with one another through intercellular signaling pathways that depend on cell density.
Methods Here, we used computational modeling to examine how the behavior of synthetic microbial consortia results from the interplay between population dynamics governed by cell growth and internal transcriptional dynamics governed by cell‐cell signaling. Specifically, we examined a synthetic microbial consortium in which two strains each produce signals that down‐regulate transcription in the other. Within a single strain this regulatory topology is called a “co‐repressive toggle switch” and can lead to bistability.
Results We found that in co‐repressive synthetic microbial consortia the existence and stability of different states depend on population‐level dynamics. As the two strains passively compete for space within the colony, their relative fractions fluctuate and thus alter the strengths of intercellular signals. These fluctuations drive the consortium to alternative equilibria. Additionally, if the growth rates of the strains depend on their transcriptional states, an additional feedback loop is created that can generate oscillations.
Conclusions Our findings demonstrate that the dynamics of microbial consortia cannot be predicted from their regulatory topologies alone, but are also determined by interactions between the strains. Therefore, when designing synthetic microbial consortia that use intercellular signaling, one must account for growth variations caused by the production of protein.
