Sea surface temperatures (SST) are rising because of global climate change. As a result, pathogenic Vibrio species that infect humans and marine organisms during warmer summer months are of growing concern. Coral reefs, in particular, are already experiencing unprecedented degradation worldwide due in part to infectious disease outbreaks and bleaching episodes that are exacerbated by increasing SST. For example, Vibrio coralliilyticus, a globally distributed bacterium associated with multiple coral diseases, infects corals at temperatures above 27 1C. The mechanisms underlying this temperature-dependent pathogenicity, however, are unknown. In this study, we identify potential virulence mechanisms using whole genome sequencing of V. coralliilyticus ATCC (American Type Culture Collection) BAA-450. Furthermore, we demonstrate direct temperature regulation of numerous virulence factors using proteomic analysis and bioassays. Virulence factors involved in motility, host degradation, secretion, antimicrobial resistance and transcriptional regulation are upregulated at the higher virulent temperature of 27 1C, concurrent with phenotypic changes in motility, antibiotic resistance, hemolysis, cytotoxicity and bioluminescence. These results provide evidence that temperature regulates multiple virulence mechanisms in V. coralliilyticus, independent of abundance. The ecological and biological significance of this temperature-dependent virulence response is reinforced by climate change models that predict tropical SST to consistently exceed 27 1C during the spring, summer and fall seasons. We propose V. coralliilyticus as a model Gram-negative bacterium to study temperature-dependent pathogenicity in Vibrio-related diseases.

Coral-associated microbial communities, including protists, bacteria, archaea and viruses, are important components of the coral holobiont that influence the health of corals and coral reef ecosystems. Evidence suggests that the composition of these microbial communities is affected by numerous parameters; however, little is known about the confluence of these ecological and temporal effects. In this study, we used ribosomal RNA gene sequencing to identify the zooxanthellae, bacteria and archaea associated with healthy and yellow band diseased (YBD) colonies in the Media Luna reef of La Parguera, Puerto Rico, in order to examine the influence of YBD on the Montastraea faveolata microbiome. In addition, we evaluated the influence of season on the differences between healthy and YBD M. faveolata microbiomes by sampling from the same tagged colonies in both March and September of 2007. To the best of our knowledge, this is the first coral microbiome study to examine sequences from the zooxanthellar, bacterial and archaeal communities simultaneously from individual coral samples. Our results confirm differences in the M. faveolata zooxanthellar, bacterial and archaeal communities between healthy and YBD colonies in March; however, the September communities do not exhibit the same differences. Moreover, we provide evidence that the differences in the M. faveolata microbiomes between March and September are more significant than those observed between healthy and YBD. This data suggest that the entire coral microbiome, not just the bacterial community, is a dynamic environment where both disease and season play important roles.

A recently available transposition system was utilized to isolate a nonmotile mutant of the coral-bleaching pathogen Vibrio coralliilyticus. The mutation was localized to the fhlA gene, and the mutant lacked flagella. The flhA mutant was unable to exhibit chemotaxis toward coral mucus or to adhere to corals and subsequently cause infection.Coral reefs have been described as the rain forests of the sea due to their enormous biodiversity. Unfortunately, during the past few decades nearly 30% of the worldwide coral population has been severely damaged by various diseases (9). Coral bleaching is a disruption of the Symbiodinium-coral symbiosis and results in "whitening" of the coral due to the loss of the Symbiodinium symbiont or its pigment. On a global scale, bleaching is one of the major coral diseases (5) and tends to correlate with increased seawater temperatures (10). Thermal stress is the generally accepted hypothesis to explain the mechanism of the disease. In the last several years, bacterial bleaching of corals has been suggested as an alternative hypothesis to explain some coral bleaching episodes (21,22). Vibrio shiloi was the first bacterium shown to be a causative agent of coral bleaching in the Mediterranean coral Oculina patagonica (13,14). More recently, Vibrio coralliilyticus has been reported to be the causative agent of temperature-induced bleaching of Pocillopora damicornis (3, 4) and white syndrome in IndoPacific corals (25). Thus, infections by V. coralliilyticus could have an impact on global coral health.Chemotaxis and flagellum-mediated motility allow bacteria to pursue nutrients and to reach and maintain their preferred niches for colonization (7,8). Several Vibrio species (both pathogens and symbionts) require functional flagellum-mediated motility to invade their hosts and establish successful colonization (17,18,27,28).In this study, we utilized a recently available Tn5-based transposition system to isolate a nonmotile mutant of the coral-bleaching pathogen V. coralliilyticus. The mutation was localized to the gene flhA. Here we demonstrate that the flagellum is critical for chemotaxis toward coral mucus, adhesion to the corals, and infection by V. coralliilyticus. Transposon mutagenesis and isolation of a motility mutant.We used a Tn5 transposon system (15) that had been used with Vibrio parahaemolyticus previously (24) to perform mutagenesis in the wild type (WT) strain V. coralliilyticus YB2 (4). Equal volumes of exponentially growing Escherichia coli SM10 cells carrying the transposon delivery plasmid pRL27 (15) and V. coralliilyticus YB2 grown in heart infusion medium (Becton Dickinson, Sparks, MD) containing 1.5% NaCl (HIS) were mixed and spotted on HIS agar plates. After an incubation of 12 h at 30°C, cells from each mating plate were suspended in 1 ml of HIS and dilutions were spread on a Vibrio-selective thiosulfatecitrate-bile sucrose agar medium (TCBS) (Becton Dickinson, Sparks, MD) containing 200 g/ml kanamycin. Southern blot analysis of 11 transconjugants revealed that the ...

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