A Multiphasic Study of Patterns in Diversity and Structure Within a Coastal Vibrio Cholerae Population

While discovery of Vibrio cholerae as the causative agent of Asiatic cholera dates back to the 19th century, the ecology of this bacterium in environmental systems remains incompletely understood. To further our understanding of V. cholerae in the environment, patterns in gene content distribution, spatial and temporal occurrence of similar genotypes, and population structure of a coastal V. cholerae population were analyzed using several molecular tools. First, genomic and metabolic profiles of 45 V. cholerae isolates were used to characterize the core genome and metabolome of this species. Comparative genome hybridization using microarrays identified 2787 core genes that were almost universally present in strains with widely different niches, suggesting that these genes are essential for persistence in diverse aquatic environments. In contrast, the presence of certain dispensable genes and phenotypic traits identified in this study (e.g. utilization of mannose, sialic acid, citrate, and chitosan oligosaccharides) were correlated with environmental factors and should provide increased fitness in specific niche environments. Next, the genomic fingerprinting technique ERIC-PCR was used to profile 835 environmental Vibrio isolates from waters and sediments at 9 sites along the central California coast. We identified 115 genotypes from 998 fingerprints and assessed biogeographic patterns in genotype occurrence using numerous methods. Partial Mantel tests established that genotypic similarity of isolates across all sampling events was correlated with environmental similarity, temporal proximity and geographic distance. A neutral community model based on stochastic processes alone explained 61% of the variation in genotype abundance among all sampling events. Co-occurrence indices (C-score, C-board, Combo) were significantly different than expected by chance, suggesting that the V. cholerae population may have competitive structure, especially at the regional scale. Finally, we characterized recombination and structure among 156 environmental V. cholerae isolates and 13 other putative Vibrios using multi-locus sequence analysis of 7 housekeeping genes. Recombinant regions were detected in 88% of our isolates, and divergence of alleles in clonal genotypes was at least 4 times as likely to result from recombination as from mutation. Despite frequent recombination, significant linkage disequilibrium was still detected among the V. cholerae sequence types. Incongruent, but nonrandom associations were observed for maximum likelihood topologies from the individual loci. Overall, recombination rates in environmental V. cholerae were similar to other sexual bacteria and appear frequent enough to restrict selection from purging much of the neutral intraspecies diversity. Taken together, these results suggest a significant, albeit small, role of deterministic factors such as niche selection and competition in shaping V. cholerae population structure in this system. Our data refute the clonality of V. cholerae and are consistent with a single cohesive species cluster maintained by frequent intraspecific recombination. These findings not only provide valuable insight into the genetic characteristics of environmental V. cholerae, but also inform future research and debate in the areas of microbial ecology, evolution of pathogens, anthropogenic impacts on coastal ecosystems, and effects of diversity in environmental or engineered systems.