Animal development and physiology depend on beneficial interactions with microbial symbionts. In many cases, the microbial symbionts are horizontally transmitted among hosts, thereby making the acquisition of these microbes from the environment an important event within the life history of each host. The light organ symbiosis established between the Hawaiian squid Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri is a model system for examining how hosts acquire horizontally transmitted microbial symbionts. Recent studies have revealed that the light organ of wild-caught E. scolopes squid contains polyclonal populations of V. fischeri bacteria; however, the function and development of such strain diversity in the symbiosis are unknown. Here, we report our phenotypic and phylogenetic characterizations of FQ-A001, which is a V. fischeri strain isolated directly from the light organ of an E. scolopes individual. Relative to the type strain ES114, FQ-A001 exhibits similar growth in rich medium but displays increased bioluminescence and decreased motility in soft agar. FQ-A001 outcompetes ES114 in colonizing the crypt spaces of the light organs. Remarkably, we find that animals cocolonized with FQ-A001 and ES114 harbor singly colonized crypts, in contrast to the cocolonized crypts observed from competition experiments involving single genotypes. The results with our two-strain system suggest that strain diversity within the squid light organ is a consequence of diversity in the single-strain colonization of individual crypt spaces. IMPORTANCEThe developmental programs and overall physiologies of most animals depend on diverse microbial symbionts that are acquired from the environment. However, the basic principles underlying how microbes colonize their hosts remain poorly understood. Here, we report our findings of bacterial strain competition within the coevolved animal-microbe symbiosis composed of the Hawaiian squid and bioluminescent bacterium Vibrio fischeri. Using fluorescent proteins to differentially label two distinct V. fischeri strains, we find that the strains are unable to coexist in the same niche within the host. Our results suggest that strain competition for distinct colonization sites dictates the strain diversity associated with the host. Our study provides a platform for studying how strain diversity develops within a host. Microbes directly contribute to the physiology, development, and evolution of metazoans (1). Many metazoan-microbe symbioses are established through horizontal transmission, i.e., animals acquire microbial symbionts from their environment (2). Coevolution of host-microbe pairs can result in genetic factors that promote remarkably high specificity between the partners, thereby assisting in the acquisition of symbionts from typically unpredictable environments (3). An important but understudied topic in coevolved metazoan-microbe symbioses is how strain diversity impacts the establishment of these associations.A particularly powerful model system to explo...

Summary 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 coordinating 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.

Bacteria often use transcription factors to regulate the expression of metabolic genes in accordance to available nutrients. NagC is a repressor conserved among γ-proteobacteria that regulates expression of enzymes involved in the metabolism of N-acetyl-glucosamine (GlcNAc). The polymeric form of GlcNAc, known as chitin, has been shown to play roles in chemotactic signaling and nutrition within the light organ symbiosis established between the marine bacterium Vibrio fischeri and the Hawaiian squid Euprymna scolopes. Here, we investigate the impact of NagC regulation on the physiology of V. fischeri. We find that NagC repression contributes to the fitness of V. fischeri in the absence of GlcNAc. In addition, the inability to de-repress expression of NagC-regulated genes reduces the fitness of V. fischeri in the presence of GlcNAc. We find that chemotaxis toward GlcNAc or chitobiose, a dimeric form of GlcNAc, is independent of NagC regulation. Finally, we show that NagC represses gene expression during the early stages of symbiosis. Our data suggest that the ability to regulate gene expression with NagC contributes to the overall fitness of V. fischeri in environments that vary in levels of GlcNAc. Furthermore, our finding that NagC represses gene expression within the squid light organ during an early stage of symbiosis supports the notion that the ability of the squid to provide a source of GlcNAc emerges later in host development.

Either Tabasco® sauce or a horseradish-based seafood cocktail sauce was placed on freshly shucked oysters which were incubated for 10 min on the half shell. Oysters were then assayed for numbers of Vibrio vulnificus cells present on the surface and within the oyster tissue, and the results compared to control oysters to which no sauce was added. Results indicated that Tabasco® sauce, but not the cocktail sauce, was highly effective in reducing the number of V. vulnificus cells present on the oyster meat surface. However, little reduction in the numbers of V. vulnificus cells present within the oysters was observed with either sauce. Our results suggest that hot sauces are not capable of significantly reducing the overall numbers of V. vulnificus cells associated with oysters, and that persons who are at risk for infection with this bacterium should continue to avoid the consumption of raw seafood, especially raw oysters.

Twenty-eight samples, comprising a total of 224 oysters, were examined for the presence of Vibrio vulnificus. Oyster homogenates were plated onto cellobiose—polymyxin B–colistin (CPC) agar or V. vulnificus enumeration (VVE) agar, with subsequent hybridization with a gene probe specific for this pathogen. Of over 3,500 cellobiose-positive colonies initially tested from CPC agar, 28.7% could be identified as V. vulnificus on the basis of probe hybridization. Of the 19,000 colonies developing on VVE agar, only 2.8% were identified as this species. When in subsequent CPC agar studies colony morphology as well as color was considered, 81.6% of over 1,000 colonies probed proved to be V. vulnificus. We conclude that CPC agar is highly selective for this pathogen, and may be effectively employed in monitoring studies to determine levels of this bacterium in molluscan shellfish.