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ABSTRACT 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 cis-acting single nucleotide polymorphism (SNP) controlling T6 transcription and activity. Sequence alignment of the T6 regulatory region from diverse V. cholerae strains revealed conservation of the SNP that we rewired to interconvert V. cholerae T6 activity between chitin-inducible and constitutive states. This study supports a model of pathogen evolution through a noncoding cis-regulatory mutation and preexisting, active transcription factors that confers a different fitness advantage to tightly regulated strains inside a human host and unfettered strains adapted to environmental niches. IMPORTANCE Organisms sense external cues with regulatory circuits that trigger the production of transcription factors, which bind specific DNA sequences at promoters (“cis” regulatory elements) to activate target genes. Mutations of transcription factors or their regulatory elements create phenotypic diversity, allowing exploitation of new niches. Waterborne pathogen Vibrio cholerae encodes the type VI secretion system “nanoweapon” to kill competitor cells when activated. Despite identification of several transcription factors, no regulatory element has been identified in the promoter of the major type VI locus, to date. Combining phenotypic, genetic, and genomic analysis of diverse V. cholerae strains, we discovered a single nucleotide polymorphism in the type VI promoter that switches its killing activity between a constitutive state beneficial outside hosts and an inducible state for constraint in a host. Our results support a role for noncoding DNA in adaptation of this pathogen.

Bacteria 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.

ABSTRACT Horizontal gene transfer (HGT) can have profound effects on bacterial evolution by allowing individuals to rapidly acquire adaptive traits that shape their strategies for competition. One strategy for intermicrobial antagonism often used by Proteobacteria is the genetically encoded contact-dependent type VI secretion system (T6SS), a weapon used to kill heteroclonal neighbors by direct injection of toxic effectors. Here, we experimentally demonstrate that Vibrio cholerae can acquire new T6SS effector genes via horizontal transfer and utilize them to kill neighboring cells. Replacement of one or more parental alleles with novel effectors allows the recombinant strain to dramatically outcompete its parent. Using spatially explicit modeling, we examine how this process could affect the ecology and evolution of surface-attached microbial populations. HGT of T6SS effector-immunity pairs is risky: transformation brings a cell into conflict with its former clone mates but can be adaptive when superior T6SS alleles are acquired. More generally, we find that these costs and benefits are not symmetric and that high rates of HGT can act as a hedge against competitors with unpredictable T6SS efficacy. We conclude that antagonism and horizontal transfer drive successive rounds of weapon optimization and selective sweeps, dynamically shaping the composition of microbial communities. IMPORTANCE The contact-dependent type VI secretion system (T6SS) is frequently used by Proteobacteria to kill adjacent competitors. While DNA released by T6 killing can be horizontally acquired, it remains untested whether T6 genes themselves can be horizontally acquired and then utilized to compete with neighboring cells. Using naturally transformable Vibrio cholerae, we provide the first direct empirical support for the hypothesis that T6 genes are exchanged horizontally (e.g., from dead competitors) and functionally deployed to compete with neighboring cells. Using computational simulations, we also demonstrate that high rates of HGT can be adaptive, allowing V. cholerae to improve upon existing T6 weaponry and survive direct encounters with otherwise superior competitors. We anticipate that our evolutionary results are of broad microbiological relevance, applying to many bacteria capable of HGT that utilize the T6SS or similar antagonistic systems, and highlight the profound impact of HGT in shaping microbial community structure.

ABSTRACT Prior to the epidemic that emerged in Haiti in October of 2010, cholera had not been documented in this country. After its introduction, a strain of Vibrio cholerae O1 spread rapidly throughout Haiti, where it caused over 600,000 cases of disease and >7,500 deaths in the first two years of the epidemic. We applied whole-genome sequencing to a temporal series of V. cholerae isolates from Haiti to gain insight into the mode and tempo of evolution in this isolated population of V. cholerae O1. Phylogenetic and Bayesian analyses supported the hypothesis that all isolates in the sample set diverged from a common ancestor within a time frame that is consistent with epidemiological observations. A pangenome analysis showed nearly homogeneous genomic content, with no evidence of gene acquisition among Haiti isolates. Nine nearly closed genomes assembled from continuous-long-read data showed evidence of genome rearrangements and supported the observation of no gene acquisition among isolates. Thus, intrinsic mutational processes can account for virtually all of the observed genetic polymorphism, with no demonstrable contribution from horizontal gene transfer (HGT). Consistent with this, the 12 Haiti isolates tested by laboratory HGT assays were severely impaired for transformation, although unlike previously characterized noncompetent V. cholerae isolates, each expressed hapR and possessed a functional quorum-sensing system. Continued monitoring of V. cholerae in Haiti will illuminate the processes influencing the origin and fate of genome variants, which will facilitate interpretation of genetic variation in future epidemics. IMPORTANCE Vibrio cholerae is the cause of substantial morbidity and mortality worldwide, with over three million cases of disease each year. An understanding of the mode and rate of evolutionary change is critical for proper interpretation of genome sequence data and attribution of outbreak sources. The Haiti epidemic provides an unprecedented opportunity to study an isolated, single-source outbreak of Vibrio cholerae O1 over an established time frame. By using multiple approaches to assay genetic variation, we found no evidence that the Haiti strain has acquired any genes by horizontal gene transfer, an observation that led us to discover that it is also poorly transformable. We have found no evidence that environmental strains have played a role in the evolution of the outbreak strain.