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The Type VI Secretion System (T6SS) is a widespread and highly effective mechanism of microbial warfare; it confers the ability to efficiently kill susceptible cells within close proximity. Due to its large physical size, complexity, and ballistic basis for intoxication it has widely been assumed to incur significant growth costs in the absence of improved competitive outcomes. In this study, we precisely examine the fitness costs of constitutive T6SS firing in the bacteriumVibrio cholerae. We find that, contrary to expectations, constitutive use of T6SS has a negligible impact on growth, reducing growth fitness by 0.025 ± 0.5% (95% CI) relative to a T6SS-control. Mathematical modeling of microbial populations demonstrates that, due to clonal interference, constitutive expression of the T6SS will often be neutral, with little impact on evolutionary outcomes. Our findings underscore the importance of precisely measuring the fitness costs of microbial social behaviors, and help explain the prevalence of the T6SS across Gram negative bacteria.

During the biofilm life cycle, bacteria attach to a surface then reproduce, forming crowded, growing communities. Many theoretical models of biofilm growth dynamics have been proposed; however, difficulties in measuring biofilm height accurately across relevant time and length scales have prevented testing these models or their biophysical underpinnings empirically. Using white light interferometry, we measure the heights of microbial colonies with nanometer precision from inoculation to their final equilibrium height, producing a novel and detailed empirical characterization of vertical growth dynamics. We show that models relying on logistic growth or nutrient depletion fail to capture biofilm height dynamics on short and long time scales. Our empirical results support a simple model inspired by the fact that biofilms only interact with the environment through their interfaces. This interface model captures the growth dynamics from short to long time scales (10 minutes to 14 days) of diverse microorganisms, including prokaryotes like gram-negative and gram-positive bacteria and eukaryotes like aerobic and anaerobic yeast. This model provides heuristic value, highlighting the biophysical constraints that limit vertical growth as well as establishing a quantitative model for biofilm development.

Evolutionary arms races are broadly prevalent among organisms including bacteria, which have evolved defensive strategies against various attackers. A common microbial aggression mechanism is the type VI secretion system (T6SS), a contact-dependent bacterial weapon used to deliver toxic effector proteins into adjacent target cells. Sibling cells constitutively express immunity proteins that neutralize effectors. However, less is known about factors that protect non-sibling bacteria from T6SS attacks independently of cognate immunity proteins. In this study, we observe that human Escherichia coli commensal strains sensitive to T6SS attacks from Vibrio cholerae are protected when co-cultured with glucose. We confirm that glucose does not impair V. cholerae T6SS activity. Instead, we find that cells lacking the cAMP receptor protein (CRP), which regulates expression of hundreds of genes in response to glucose, survive significantly better against V. cholerae T6SS attacks even in the absence of glucose. Finally, we show that the glucose-mediated T6SS protection varies with different targets and killers. Our findings highlight the first example of an extracellular small molecule modulating a genetically controlled response for protection against T6SS attacks. This discovery may have major implications for microbial interactions during pathogen-host colonization and survival of bacteria in environmental communities.

Mutations in regulatory mechanisms that control gene expression contribute to phenotypic diversity and thus facilitate the adaptation of microbes and other organisms to new niches. Comparative genomics can be used to infer rewiring of regulatory architecture based on large effect mutations like loss or acquisition of transcription factors but may be insufficient to identify small changes in noncoding, intergenic DNA sequence of regulatory elements that drive phenotypic divergence. In human-derived Vibrio cholerae, the response to distinct chemical cues triggers production of multiple transcription factors that can regulate the type VI secretion system (T6), a broadly distributed weapon for interbacterial competition. However, to date, the signaling network remains poorly understood because no regulatory element has been identified for the major T6 locus. Here we identify a conserved

Vibrio cholerae, the causative agent of cholera, is commonly isolated, along with other bacterial species, from chironomid insects (Diptera: Chironomidae). Nevertheless, its prevalence in the chironomid egg masses' microbiota is less than 0.5%. V. cholerae secretes haemagglutinin/protease (HAP) that degrades the gelatinous matrix of chironomid egg masses and prevents hatching. Quorum sensing (QS) activates HAP production in response to accumulation of bacterial autoinducers (AIs). Our aim was to define the impact of chironomid microbiota on HAP production by V. cholerae. To study QS signaling, we used V. cholerae bioluminescence reporter strains (QS-proficient O1 El-Tor wild-type and QS-deficient mutants) and different bacterial species that we isolated from chironomid egg masses. These egg mass isolates, as well as a synthetic AI-2, caused an enhancement in lux expression by a V. cholerae QS-deficient mutant. The addition of the egg mass bacterial isolate supernatant to the QS-deficient mutant also enhanced HAP production and egg mass degradation activities. Moreover, the V. cholerae wild-type strain was able to proliferate using egg masses as their sole carbon source, while the QS-deficient was not. The results demonstrate that members of the chironomid bacterial consortium produce external chemical cues that, like AI-2, induce expression of the hapA gene in V. cholerae. Understanding the interactions between V. cholerae and the insects' microbiota may help uncover the interactions between this pathogen and the human gut microbiota.

Background Like many bacteria, Vibrio cholerae deploys a harpoon-like type VI secretion system (T6SS) to compete against other microbes in environmental and host settings. The T6SS punctures adjacent cells and delivers toxic effector proteins that are harmless to bacteria carrying cognate immunity factors. Only four effector/immunity pairs encoded on one large and three auxiliary gene clusters have been characterized from largely clonal, patient-derived strains of V. cholerae. Results We sequence two dozen V. cholerae strain genomes from diverse sources and develop a novel and adaptable bioinformatics tool based on hidden Markov models. We identify two new T6SS auxiliary gene clusters and describe Aux 5 here. Four Aux 5 loci are present in the host strain, each with an atypical effector/immunity gene organization. Structural prediction of the putative effector indicates it is a lipase, which we name TleV1 (type VI lipase effector Vibrio). Ectopic TleV1 expression induces toxicity in Escherichia coli, which is rescued by co-expression of the TliV1a immunity factor. A clinical V. cholerae reference strain expressing the Aux 5 cluster uses TleV1 to lyse its parental strain upon contact via its T6SS but is unable to kill parental cells expressing the TliV1a immunity factor. Conclusion We develop a novel bioinformatics method and identify new T6SS gene clusters in V. cholerae. We also show the TleV1 toxin is delivered in a T6SS manner by V. cholerae and can lyse other bacterial cells. Our web-based tool can be modified to identify additional novel T6SS genomic loci in diverse bacterial species. Electronic supplementary material The online version of this article (10.1186/s13059-019-1765-5) contains supplementary material, which is available to authorized users.

Vibrio cholerae is an aquatic Gram-negative bacterium that causes severe diarrheal cholera disease when ingested by humans. To eliminate competitor cells in both the external environment and inside hosts, V. cholerae uses the Type VI Secretion System (T6SS). The T6SS is a macromolecular weapon employed by many Gram-negative bacteria to deliver cytotoxic proteins into adjacent cells. In addition to canonical T6SS gene clusters encoded by all sequenced V. cholerae isolates, strain BGT49 encodes an additional locus, which we named auxiliary cluster 4 (Aux 4). The Aux 4 cluster is located on a mobile genetic element and can be used by killer cells to eliminate both V. cholerae and Escherichia coli cells in a T6SS-dependent manner. A putative toxin encoded in the cluster, which we name TpeV (Type VI Permeabilizing Effector Vibrio), shares no homology to known proteins and does not contain motifs or domains indicative of function. Ectopic expression of TpeV in the periplasm of E. coli permeabilizes cells and disrupts the membrane potential. Using confocal microscopy, we confirm that susceptible target cells become permeabilized when competed with killer cells harboring the Aux 4 cluster. We also determine that tpiV, the gene located immediately downstream of tpeV, encodes an immunity protein that neutralizes the toxicity of TpeV. Finally, we show that TpeV homologs are broadly distributed across important animal and plant pathogens and are localized in proximity to other T6SS genes. Our results suggest that TpeV is a toxin that belongs to a large family of T6SS proteins.IMPORTANCEBacteria live in polymicrobial communities where competition for resources and space is essential for survival. Proteobacteria use the T6SS to eliminate neighboring cells and cause disease. However, the mechanisms by which many T6SS toxins kill or inhibit susceptible target cells are poorly understood. The sequence of the TpeV toxin we describe here is unlike any previously described protein. We demonstrate that it has antimicrobial activity by permeabilizing cells, eliminating membrane potentials and causing severe cytotoxicity. TpeV homologs are found near known T6SS genes in human, animal and plant bacterial pathogens, indicating that the toxin is a representative member of a broadly distributed protein family. We propose that TpeV-like toxins contribute to the fitness and pathogenicity of many bacteria. Finally, since antibiotic resistance is a critical global health threat, the discovery of new antimicrobial mechanisms could lead to the development of new treatments against resistant strains.

Biofilms are highly structured, densely packed bacterial consortia where many different species can coexist. During biofilm development and growth, the different species often form spatial distribution patterns that govern biofilm composition and function. In some cases, emerging structures have been explained as the result of social interactions between bacteria, e.g. cooperation and competition. Others emphasize the role of local mechanics, where spatial structuring arises from forces exerted between cells or between cells and their environment. Typically, these two lines of argumentation are treated separately. Here, we show that mechanics and social interactions can be strongly interrelated and their combination can crucially impact biofilm formation and dynamics. Using confocal microscopy and bacterial co-culture assays, we examine how bacterial antagonism impacts biofilm mechanics, and vice versa. We study competing Vibrio cholerae strains that kill on contact using the Type 6 secretion system. In case of mutual killers, i.e. two V. cholerae strains that can kill each other on contact, this social interaction leads to the formation of clonal domains of the competing strains (Mc Nally et al., Nat Commun, 2017). Intuitively, an unequal fight may enable a superior killer to invade and quickly eliminate a much weaker competitor. However, we observe that killer cells can coexist with killing-deficient target cells for very long times, and find that this results from the mechanical consequences of the deadly competition. Killing produces dead cells, which accumulates between domains of competing cells and prevents subsequent killing. Counterintuitively, our results suggest that antagonistic interactions stabilize coexistence in diverse communities. The findings demonstrate that the impact of social interactions in bacterial consortia is complex, requiring the understanding of the structural and the statistical-mechanical processes in biofilms.

Evolutionary arms races among organisms are broadly prevalent and bacteria have evolved defensive strategies against various attackers. A common microbial aggression mechanism is the Type VI Secretion System (T6SS), a contact-dependent bacterial weapon used to deliver toxic effector proteins into adjacent target cells. Sibling cells constitutively express immunity proteins that neutralize effectors. However, less is known about mechanisms that allow non-sibling bacteria to respond to external cues and survive T6SS attacks independently of immunity proteins. In this study, we show that resistance to T6SS attacks is promoted by a genetically controlled response to exogenous glucose. We observe that multiple human Escherichia coli commensal strains lacking immunity proteins are sensitive to T6SS attacks from pandemic Vibrio cholerae on nutrient-rich media. By contrast, E. coli cells become resistant to attacks when co-cultured on the same media with glucose. We confirm that glucose does not impair V. cholerae T6SS activity. Instead, we find that cAMP receptor protein (CRP), which alters expression of hundreds of genes in response to glucose, controls resistance to T6SS attacks in E. coli cells. Consistent with the observed resistance on media with glucose, an E. coli crp disruption mutant survives significantly better against V. cholerae T6SS attacks even in the absence of glucose. Finally, we also show that resistance to T6SS attacks depends on the pH of the medium and varies based on the target and killer strains.IMPORTANCEMany Gram-negative bacteria, including important pathogens, encode T6SS genes to deliver toxic effectors and eliminate competitors. Our results uncover a novel defense mechanism against T6SS attacks that is triggered by an external stimulus and mediated by a metabolic response in non-kin target cells. In microbiomes such as those in gastrointestinal tracts where T6SS activity is known to occur, signaling by metabolites like glucose may affect the efficacy of T6SS attacks and alter microbial community composition. Our findings could have vast implications for microbial interactions during pathogen colonization of hosts and survival of bacterial cells in environmental communities. Furthermore, the glucose-mediated resistance observed here might provide a novel example of an evolutionary arms race between killer T6SS cells and target bacteria.

Bacterial communities are governed by a wide variety of social interactions, some of which are antagonistic with potential significance for bacterial warfare. Several antagonistic mechanisms, such as killing via the type VI secretion system (T6SS), require killer cells to directly contact target cells. The T6SS is hypothesized to be a highly potent weapon, capable of facilitating the invasion and defence of bacterial populations. However, we find that the efficacy of contact killing is severely limited by the material consequences of cell death. Through experiments withVibrio choleraestrains that kill via the T6SS, we show that dead cell debris quickly accumulates at the interface that forms between competing strains, preventing physical contact and thus preventing killing. While previous experiments have shown that T6SS killing can reduce a population of target cells by as much as 106-fold, we find that, as a result of the formation of dead cell debris barriers, the impact of contact killing depends sensitively on the initial concentration of killer cells. Killer cells are incapable of invading or eliminating competitors on a community level. Instead, bacterial warfare itself can facilitate coexistence between nominally antagonistic strains. While a variety of defensive strategies against microbial warfare exist, the material consequences of cell death provide target cells with their first line of defence.

In a communication network with extremely high processing delays, an efficient addressing and multiple-access control mechanism to improve the throughput performance of the system is a necessity. This paper focuses on source addressing in multiple-source single-receiver bacterial communication networks. We propose amplitude-division multiple access (ADMA), a method that assigns the amplitude of the transmitted signal as the address of the source. We demonstrate using genetically engineered Escherichia coli bacteria in a microfluidic device that using amplitude for addressing is feasible. We analyze the performance of the network with several addressing mechanisms and propose an amplitude sequence and a low-complexity receiver design that minimizes error in resolving the source addresses in the presence of collisions. Finally, we demonstrate that ADMA implicitly solves the problem of multiple-access control.

The type VI secretion system (T6SS) is a proteinaceous weapon used by many Gram-negative bacteria to deliver toxins into adjacent target cells. Vibrio cholerae, the bacterium responsible for the fatal water-borne cholera disease, uses the T6SS to evade phagocytic eukaryotes, cause intestinal inflammation, and compete against other bacteria with toxins that disrupt lipid membranes, cell walls and actin cytoskeletons. The control of T6SS genes varies among V. cholerae strains and typically includes inputs from external signals and cues, such as quorum sensing and chitin availability. In the following review, we highlight the repertoire of toxic T6SS effectors and the diverse genetic regulation networks among different isolates of V. cholerae. Finally, we discuss the roles played by the T6SS of V. cholerae in both natural environments and hosts.

BackgroundLike many bacteria, Vibrio cholerae, which causes fatal cholera, deploys a harpoon-like Type VI Secretion System (T6SS) to compete against other microbes in environmental and host settings. The T6SS punctures adjacent cells and delivers toxic effector proteins that are harmless to bacteria carrying cognate immunity factors. Only four effector/immunity pairs encoded on one large and three auxiliary gene clusters have been characterized from largely clonal, patient-derived strains of V. cholerae.ResultsWe sequenced two dozen V. cholerae strain genomes from diverse sources and developed a novel and adaptable bioinformatic tool based on Hidden Markov Models. We identified two new T6SS auxiliary gene clusters; one, Aux 5, is described here. Four Aux 5 loci are present in the host strain, each with an atypical effector/immunity gene organization. Structural prediction of the putative effector indicated it is a lipase, which we name TleV1 (Type VI lipase effector Vibrio, TleV1). Ectopic TleV1 expression induced toxicity in E. coli, which was rescued by co-expression of the TleV1 immunity factor. A clinical V. cholerae reference strain expressing the Aux 5 cluster used TleV1 to lyse its parental strain upon contact via its T6SS but was unable to kill parental cells expressing TleV1’s immunity factor.ConclusionWe developed a novel bioinformatic method and identified new T6SS gene clusters in V. cholerae. We also showed the TleV1 toxin is delivered in a T6SS-manner by V. cholerae and can lyse other bacterial cells. Our web-based tool may be modified to identify additional novel T6SS genomic loci in diverse bacterial species.

The 6th American Society for Microbiology Conference on Cell-Cell Communication in Bacteria convened from 16 to 19 October 2017 in Athens, GA. In this minireview, we highlight some of the research presented at that meeting that addresses central questions emerging in the field, including the following questions. How are cell-cell communication circuits designed to generate responses? Where are bacteria communicating? Finally, why are bacteria engaging in such behaviors?

Michele Swanson credits Title IX for providing her the opportunity for a university education, during which she began the scientific training that has led to her current position as president-elect of the American Society for Microbiology. Michele’s story is inspirational, for she has charted a unique, rewarding, and balanced path. Michele has contributed in many significant ways to the field of microbiology, recognized and seized opportunities, and done so by making wise professional and personal choices. This chapter, by Brian Hammer, is a reflection on his Ph.D. mentor during her early career as an assistant professor, looking at her scientific influences prior to and her accomplishments since that time.

Significance Vibrio cholerae , the causative agent of the diarrheal disease cholera, uses the syringe-like type VI secretion system (T6SS) to pierce adjacent cells. To investigate the role of the T6SS in invasion of intestines already occupied by symbiotic microbes, we genetically engineered V. cholerae strains and performed live 3D imaging in zebrafish to find that V. cholerae can expel a resident bacterial species in a T6SS-dependent manner. Surprisingly, the T6SS acts primarily to increase the strength of gut contractions, rather than directly killing the bacterial competitor. Deletion of an actin cross-linking domain from the T6SS returned gut activity to normal and eliminated V. cholerae ’ s competitive advantage. These findings reveal a strategy by which pathogens can manipulate host biomechanics to redefine gut communities.

Host-associated microbiota help defend against bacterial pathogens; the mechanisms that pathogens possess to overcome this defense, however, remain largely unknown. We developed a zebrafish model and used live imaging to directly study how the human pathogenVibrio choleraeinvades the intestine. The gut microbiota of fish mono-colonized by commensal strainAeromonas veroniiwas displaced byV. choleraeexpressing its Type VI Secretion System (T6SS), a syringe-like apparatus that deploys effector proteins into target cells. Surprisingly, displacement was independent of T6SS-mediated killing ofAeromonas, driven instead by T6SS-induced enhancement of zebrafish intestinal movements that led to expulsion of the resident commensal by the host. Deleting an actin crosslinking domain from the T6SS apparatus returned intestinal motility to normal and thwarted expulsion, without weakeningV. cholerae′sability to killAeromonas in vitro. Our finding that bacteria can manipulate host physiology to influence inter-microbial competition has implications for both pathogenesis and microbiome engineering.

Genetically engineered bacteria can be used for a wide range of applications, from monitoring environmental toxins to studying complex communication networks in the human digestive system. Although great strides have been made in studying single strains of bacteria in well-controlled microfluidic environments, there remains a need for tools to reliably control and measure communication between multiple discrete bacterial populations. Stable long-term experiments (e.g., days) with controlled population sizes and regulated input (e.g., concentration) and output measurements can reveal fundamental limits of cell-to-cell communication. In this work, we developed a microfluidic platform that utilizes a porous monolith to reliably and stably partition adjacent strains of bacteria while allowing molecular communication between them for several days. We measured small molecule production by the bacterial populations in response to stimuli using analytical chemistry methods and measured fluorescent output. The results are compared with communication and diffusion delay models. This porous monolith microfluidic system enables bacterial cell-to-cell communication assays with dynamic control of inputs, relatively long-term experimentation with no cross contamination, and stable bacterial population size. This system can serve as a valuable tool in understanding bacterial communication and improving biosensor design capabilities.

Bacterial populations housed in microfluidic environments can serve as transceivers for molecular communication, but the data-rates are extremely low (e.g., 10-5 bits per second.). In this work, genetically engineered Escherichia coli bacteria were maintained in a microfluidic device where their response to a chemical stimulus was examined over time. The bacteria serve as a communication receiver where a simple modulation such as on-off keying (OOK) is achievable, although it suffers from very poor data-rates. We explore an alternative communication strategy called time-elapse communication (TEC) that uses the time period between signals to encode information. We identify the limitations of TEC under practical non-zero error conditions and propose an advanced communication strategy called smart time-elapse communication (TEC-SMART) that achieves over a 10x improvement in data-rate over OOK. We derive the capacity of TEC and provide a theoretical maximum data-rate that can be achieved.

Predictive models are beneficial tools for researchers to use in prioritizing nanoparticles (NPs) for toxicological tests, but experimental evaluation can be time-consuming and expensive, and thus, priority should be given to tests that identify the NPs most likely to be harmful. For characterization of NPs, the physical binding of NPs to DNA molecules is important to measure, as interference with DNA function may be one cause of toxicity. Here, we determined the interaction energy between 12 types of NPs and DNA based on the Derjaguin-Landau-Verwey-Overbeek (DLVO) model and then predicted the affinity of the NPs for DNA. Using the single-molecule imaging technique known as atomic force microscopy (AFM), we experimentally determined the binding affinity of those NPs for DNA. Theoretical predictions and experimental observations of the binding affinity agreed well. Furthermore, the effect of NPs on DNA replication in vitro was investigated with the polymerase chain reaction (PCR) technique. The results showed that NPs with a high affinity for DNA strongly inhibited DNA replication, whereas NPs with low affinity had no or minimal effects on DNA replication. The methodology here is expected to benefit the genotoxicological testing of NPs as well as the design of safe NPs.

Summary Biofilms promote attachment of Vibrio cholerae in aquatic ecosystems and aid in transmission. Intracellular c-di-GMP levels that control biofilm development positively correlate with expression of Qrr sRNAs, which are transcribed when quorum sensing (QS) autoinducer levels are low. The Qrr sRNAs base-pair with and repress translation of hapR encoding the QS ‘master regulator’, hence increased c-di-GMP and biofilm development at low density were believed to be solely a consequence of Qrr/hapR pairing. We show that Qrr sRNAs also base-pair with and activate translation of the mRNA of a diguanylate cyclase (DGC), Vca0939; relieving an inhibitory structure in vca0939 that occludes the ribosome binding site. A nucleotide substitution in vca0939 disrupted sRNA/mRNA base-pairing and prevented vca0939 translation, while a compensating Qrr sRNA substitution restored pairing and Vca0939 levels. Qrr-dependent DGC activation led to c-di-GMP accumulation and biofilm development in V. cholerae. This represents the first description of (1) a DGC post-transcriptionally activated by direct pairing with an Hfq-dependent sRNA, and (2) control of a V. cholerae QS phenotype, independent of HapR. Thus, direct interactions of the same sRNAs with two mRNAs promote c-di-GMP-dependent biofilm formation by complementary mechanisms in V. cholerae; by negatively regulating HapR, and positively regulating the DGC Vca0939.

Natural transformation is a major mechanism of horizontal gene transfer in bacteria. By incorporating exogenous DNA elements into chromosomes, bacteria are able to acquire new traits that can enhance their fitness in different environments. Within the past decade, numerous studies have revealed that natural transformation is prevalent among members of the Vibrionaceae, including the pathogen Vibrio cholerae. Four environmental factors: (i) nutrient limitation, (ii) availability of extracellular nucleosides, (iii) high cell density and (iv) the presence of chitin, promote genetic competence and natural transformation in Vibrio cholerae by co-ordinating expression of the regulators CRP, CytR, HapR and TfoX respectively. Studies of other Vibrionaceae members highlight the general importance of natural transformation within this bacterial family.

The human pathogen Vibrio cholerae employs several adaptive mechanisms for environmental persistence, including natural transformation and type VI secretion, creating a reservoir for the spread of disease. Here, we report whole-genome sequences of 26 diverse V. cholerae isolates, significantly increasing the sequence diversity of publicly available V. cholerae genomes.

SummaryBy nature of their small size, dense growth and frequent need for extracellular metabolism, microbes face persistent public goods dilemmas1–5. Spatial assortment can act as a general solution to social conflict by allowing extracellular goods to be utilized preferentially by productive genotypes1,6,7. Established mechanisms that generate microbial assortment depend on the availability of free space8–14; however, microbes often live in densely-packed environments, wherein these mechanisms are ineffective. Here, we describe a novel class of self-organized pattern formation that facilitates the development of spatial structure within densely-packed bacterial colonies. Contact-mediated killing through the Type VI secretion system (T6SS) drives high levels of assortment by precipitating phase separation, even in initially well-mixed populations that do not necessarily exhibit net growth. We examine these dynamics using three different classes of mathematical models and experiments with mutually antagonistic strains of Vibrio cholerae growing on solid media, and find that all appear to de-mix via the same ‘Model A’ universality class of order-disorder transition. We mathematically demonstrate that contact killing should favour the evolution of public goods cooperation, and empirically examine the relationship between T6SSs and potential cooperation through phylogenetic analysis. Across 26 genera of Proteobacteria and Bacteroidetes, the proportion of a strain’s genome that codes for potentially-exploitable secreted proteins increases significantly with boththe number of Type 6 secretion systems and the number of T6SS effectors that it possesses. This work demonstrates how antagonistic traits—likely evolved for the purpose of killing competitors—can indirectlylead to the evolution of cooperation by driving genetic phase separation.

The bacterial pathogen Vibrio cholerae can occupy both the human gut and aquatic reservoirs, where it may colonize chitinous surfaces that induce the expression of factors for three phenotypes: chitin utilization, DNA uptake by natural transformation, and contact-dependent bacterial killing via a type VI secretion system (T6SS). In this study, we surveyed a diverse set of 53 isolates from different geographic locales collected over the past century from human clinical and environmental specimens for each phenotype outlined above. The set included pandemic isolates of serogroup O1, as well as several serogroup O139 and non-O1/non-O139 strains. We found that while chitin utilization was common, only 22.6% of the isolates tested were proficient at chitin-induced natural transformation, suggesting that transformation is expendable. Constitutive contact-dependent killing of Escherichia coli prey, which is indicative of a functional T6SS, was rare among clinical isolates (only 4 of 29) but common among environmental isolates (22 of 24). These results bolster the pathoadaptive model in which tight regulation of T6SS-mediated bacterial killing is beneficial in a human host, whereas constitutive killing by environmental isolates may give a competitive advantage in natural settings. Future sequence analysis of this set of diverse isolates may identify previously unknown regulators and structural components for both natural transformation and T6SS.

Summary Competence for genetic transformation in Vibrio cholerae is triggered by chitin-induced transcription factor TfoX and quorum sensing (QS) regulator HapR. Transformation requires expression of ComEA, described as a DNA receptor in other competent bacteria. A screen for mutants that poorly expressed a comEA–luciferase fusion identified cytR, encoding the nucleoside scavenging cytidine repressor, previously shown in V. cholerae to be a biofilm repressor and positively regulated by TfoX, but not linked to transformation. A ΔcytR mutant was non-transformable and defective in expression of comEA and additional TfoX-induced genes, including pilA (transformation pseudopilus) and chiA-1 (chitinase). In Escherichia coli, ‘anti-activation’ of nucleoside metabolism genes, via protein–protein interactions between critical residues in CytR and CRP (cAMP receptor protein), is disrupted by exogenous cytidine. Amino acid substitutions of the corresponding V. cholerae CytR residues impaired expression of comEA, pilA and chiA-1, and halted DNA uptake; while exogenous cytidine hampered comEA expression levels and prevented transformation. Our results support a speculative model that when V. cholerae reaches high density on chitin, CytR–CRP interactions ‘anti-activate’ multiple genes, including a possible factor that negatively controls DNA uptake. Thus, a nucleoside scavenging mechanism couples nutrient stress and cell–cell signalling to natural transformation in V. cholerae as described in other bacterial pathogens.

This article presents a branch of research where the use of molecules to encode and transmit information among nanoscale devices (nanomachines) is investigated as a bio-inspired viable solution to realize nano-communication networks. Unlike traditional technologies, molecular communication is a radically new paradigm, which demands novel solutions, including the identification of naturally existing molecular communication mechanisms, the establishment of the foundations of a molecular information theory, or the development of architectures and networking protocols for nanomachines. The tight connection of this cutting edge engineering research field with biology will ultimately enable both the bio-inspired study of molecular nanonetwork architectures and their realization with tools already available in nature. The testbed described in this article, which is based on a microfluidic device hosting intercommunicating populations of genetically engineered bacteria, is a clear example of this research direction.

Vibrio cholerae, the causative agent of cholera and a natural inhabitant of aquatic environments, regulates numerous behaviors using a quorum-sensing (QS) system conserved among many members of the marine genus Vibrio. The Vibrio QS response is mediated by two extracellular autoinducer (AI) molecules: CAI-I, which is produced only by Vibrios, and AI-2, which is produced by many bacteria. In marine biofilms on chitinous surfaces, QS-proficient V. cholerae become naturally competent to take up extracellular DNA. Because the direct role of AIs in this environmental behavior had not been determined, we sought to define the contribution of CAI-1 and AI-2 in controlling transcription of the competence gene, comEA, and in DNA uptake. In this study we demonstrated that comEA transcription and the horizontal acquisition of DNA by V. cholerae are induced in response to purified CAI-1 and AI-2, and also by autoinducers derived from other Vibrios co-cultured with V. cholerae within a mixed-species biofilm. These results suggest that autoinducer communication within a consortium may promote DNA exchange among Vibrios, perhaps contributing to the evolution of these bacterial pathogens.

Summary Vibrio cholerae quorum sensing controls expression of four redundant sRNAs, Qrr1-4. The Qrr sRNAs are predicted to alter the translation of several mRNAs, including, hapR, which encodes a transcription factor that controls genes for virulence factors, biofilm formation, protease production and DNA uptake. Each Qrr contains a 21 nucleotide region absolutely conserved among pathogenic Vibrios, and predicted to base pair with mRNA targets, like hapR, aided by the RNA chaperone Hfq. This molecular mechanism was not experimentally tested previously, and we provide here both in vivo and in vitro evidence to validate this model. In Escherichia coli, Qrr expression repressed a HapR-GFP translational fusion, and a specific nucleotide substitution in the 21 nucleotide region eliminated HapR control, while a compensatory mutation in hapR restored it. In V. cholerae, the identical mutations also deregulated HapR-dependent gene expression and corresponding QS phenotypes by altering HapR protein levels. We calculated in vitro binding affinities of a Qrr/hapR complex and show that Hfq stabilizes complex formation. Finally, the Qrr mutation with in vivo defects also prevented Qrr/hapR binding, while the compensatory hapR mutation restored binding. These results demonstrate that the V. cholerae QS response is mediated by base pairing interactions between Qrr sRNAs and hapR mRNA.

Using a process called quorum sensing (QS), bacteria communicate with extracellular signal molecules called autoinducers (AIs). Response to AIs allows bacteria to coordinate gene expression on a population-wide scale and thereby carry out particular behaviors in unison, much like multicellular organisms. In Vibrio cholerae El Tor, the etiological agent of the current cholera pandemic, AI information is transduced internally through a phosphorelay circuit that impinges on the transcription of multiple small regulatory RNAs (sRNAs). These RNAs base-pair with, and repress the translation of, the mRNA encoding the master transcriptional regulator HapR. In V. cholerae , HapR controls virulence factor expression and biofilm formation. Here we identify a sRNA-dependent, HapR-independent QS pathway in which the sRNAs base-pair with a new target mRNA and activate translation by preventing formation of a translation-inhibiting stem-loop structure. We show that the classical V. cholerae strain, which caused previous pandemics and is reportedly incapable of QS because of a nonfunctional HapR, nonetheless exhibits QS-controlled gene expression through this new HapR-independent pathway.

Many Gram-positive and Gram-negative bacteria can become naturally competent to take up extracellular DNA from the environment via a dedicated uptake apparatus. The genetic material that is acquired can (i) be used for nutrients, (ii) aid in genome repair, and (iii) promote horizontal gene transfer when incorporated onto the genome by homologous recombination, the process of “transformation.” Recent studies have identified multiple environmental cues sufficient to induce natural transformation in Vibrio cholerae and several other Vibrio species. In V. cholerae , nutrient limitation activates the cAMP receptor protein regulator, quorum-sensing signals promote synthesis of HapR-controlled QstR, chitin stimulates production of TfoX, and low extracellular nucleosides allow CytR to serve as an additional positive regulator. The network of signaling systems that trigger expression of each of these required regulators is well described, but the mechanisms by which each in turn controls competence apparatus genes is poorly understood. Recent work has defined a minimal set of genes that encode apparatus components and begun to characterize the architecture of the machinery by fluorescence microscopy. While studies with a small set of V. cholerae reference isolates have identified regulatory and competence genes required for DNA uptake, future studies may identify additional genes and regulatory connections, as well as revealing how common natural competence is among diverse V. cholerae isolates and other Vibrio species.

Summary Multiple quorum-sensing circuits function in parallel to control virulence and biofilm formation in Vibrio cholerae. In contrast to other bacterial pathogens that induce virulence factor production and/or biofilm formation at high cell density in the presence of quorum-sensing autoinducers, V. cholerae represses these behaviours at high cell density. Consistent with this, we show here that V. cholerae strains ‘locked’ in the regulatory state mimicking low cell density are enhanced for biofilm production whereas mutants ‘locked’ in the regulatory state mimicking high cell density are incapable of producing biofilms. The quorum-sensing cascade we have identified in V. cholerae regulates the transcription of genes involved in exopolysaccharide production (EPS), and variants that produce EPS and form biofilms arise at high frequency from non-EPS, non-biofilm producing strains. Our data show that spontaneous mutation of the transcriptional regulator hapR is responsible for this effect. Several toxigenic strains of V. cholerae possess a naturally occurring frameshift mutation in hapR. Thus, the distinct environments occupied by this aquatic pathogen presumably include niches where cell-cell communication is crucial, as well as ones where loss of quorum sensing via hapR mutation confers a selective advantage. Bacterial biofilms could represent a complex habitat where such differentiation occurs.

Pathogenic Legionella pneumophila evolved as a parasite of aquatic amoebae. To persist in the environment, the microbe must be proficient at both replication and transmission. In laboratory cultures, as nutrients become scarce a stringent response-like pathway coordinates exit from the exponential growth phase with induction of traits correlated with virulence, including motility. A screen for mutants that express the flagellin gene poorly identified five activators of virulence: LetA/LetS, a two-component regulator homologous to GacA/GacS of Pseudomonas and SirA/BarA of Salmonella; the stationary-phase sigma factor RpoS; the flagellar sigma factor FliA; and a new locus, letE. Unlike wild type, post-exponential-phase letA and letS mutants were not motile, cytotoxic, sodium sensitive or proficient at infecting macrophages. L. pneumophila also required fliA to become motile, cytotoxic and to infect macrophages efficiently and letE to express sodium sensitivity and maximal motility and cytotoxicity. When induced to express RelA, all of the strains exited the exponential phase, but only wild type converted to the fully virulent form. In contrast, intracellular replication was independent of letA, letS, letE or fliA. Together, the data indicate that, as the nutrient supply wanes, ppGpp triggers a regulatory cascade mediated by LetA/ LetS, RpoS, FliA and letE that coordinates differentiation of replicating L. pneumophila to a transmissible form.

Many bacteria can become naturally competent to take up extracellular DNA across their outer and inner membranes by a dedicated competence apparatus. Whereas some studies show that the DNA delivered to the cytoplasm may be used for genome repair or for nutrition, it can also be recombined onto the chromosome by homologous recombination: a process called natural transformation. Along with conjugation and transduction, natural transformation represents a mechanism for horizontal transfer of genetic material, e.g., antibiotic resistance genes, which can confer new beneficial characteristics onto the recipient bacteria. Described here are protocols for quantifying the frequency of transformation for the human pathogen Vibrio cholerae, one of several Vibrio species recently shown to be capable of natural transformation.

Biosensors exploiting communication within genetically engineered bacteria are becoming increasingly important for monitoring environmental changes. Currently, there are a variety of mathematical models for understanding and predicting how genetically engineered bacteria respond to molecular stimuli in these environments, but as sensors have miniaturized towards microfluidics and are subjected to complex time-varying inputs, the shortcomings of these models have become apparent. The effects of microfluidic environments such as low oxygen concentration, increased biofilm encapsulation, diffusion limited molecular distribution, and higher population densities strongly affect rate constants for gene expression not accounted for in previous models. We report a mathematical model that accurately predicts the biological response of the autoinducer N-acyl homoserine lactone-mediated green fluorescent protein expression in reporter bacteria in microfluidic environments by accommodating these rate constants. This generalized mass action model considers a chain of biomolecular events from input autoinducer chemical to fluorescent protein expression through a series of six chemical species. We have validated this model against experimental data from our own apparatus as well as prior published experimental results. Results indicate accurate prediction of dynamics (e.g., 14% peak time error from a pulse input) and with reduced mean-squared error with pulse or step inputs for a range of concentrations (10 μM–30 μM). This model can help advance the design of genetically engineered bacteria sensors and molecular communication devices.