Coral disease outbreaks pose a major threat to reef ecosystems, often leading to widespread mortality and declines in coral cover. A key factor predicted in disease susceptibility is the coral microbiome, which is thought to protect corals from pathogens like Vibrio coralliilyticus . However, this protective function has not been empirically demonstrated with live coral, nor is it well understood how these microbiome-pathogen interactions contribute to the observed variation in virulence among different V. coralliilyticus strains. This study investigated the role of the Hawaiian rice coral ( Montipora capitata ) microbiome in susceptibility to infection by endemic and non-endemic strains of V. coralliilyticus . Laboratory infection experiments revealed that antibiotic-induced dysbiosis generally increased host susceptibility, though infection rates varied between strains. Notably, the type of M. capitata colour morph did not influence infection outcomes, and induced dysbiosis did not affect susceptibility to another pathogen, Pseudoalteromonas piratica . Additionally, the quorum sensing genes vcpR and aphA were examined for their roles in V. coralliilyticus pathogenicity in relation to the host microbiome. These findings underscore the protective function of the coral microbiome and highlight the complexity of host-pathogen interactions, contributing to a deeper understanding of coral disease dynamics and informing future mitigation strategies.

Stony coral tissue loss disease (SCTLD) is a deadly, waterborne coral disease. Since 2014, SCTLD has spread throughout Florida's Coral Reef and is now confirmed in 28 Caribbean countries and territories. The causative agent(s) remain unknown; however, pathogenic bacteria are implicated in disease progression. In contrast, a beneficial microbe (a probiotic) with antibacterial activity isolated from a disease-resistant coral has arrested SCTLD transmission and progression during laboratory and field trials. Due to host and environmental specificity, more probiotics sourced from vulnerable coral species and different SCTLD-impacted regions are needed. Conventional methods used for probiotic discovery include plating samples for single colonies, streaking for purification, and then testing strains for antibacterial activity with drop culture assays. However, these manually intensive methods are low-throughput and the measurements for antibacterial activity can be subjective. Given these constraints, this study developed a platform to greatly increase the isolation and screening of coral probiotics. Using the new platform, microbial cells were isolated from a coral mucus sample using a microfluidic cell sorter, and isolates were then screened for inhibitory activity against target pathogenic bacteria modified to express yellow fluorescent protein (YFP) that allows growth quantification using a microplate reader. In a single run using the platform, 433 isolates were sorted then individually screened against two target pathogens within six days. The workflow described herein represents a proof of concept for high-throughput environmental probiotic discovery with potential to push forward treatment development for SCTLD and future disease outbreaks. ### Competing Interest Statement The authors have declared no competing interest. National Philanthropic Trust Oceankind Florida Department of Environmental Protection, https://ror.org/042v2q176, B7F150 University of North Carolina Wilmington, https://ror.org/02t0qr014

Coral disease outbreaks threaten reef ecosystems, often leading to widespread mortality and declines in coral cover. Outbreaks of tissue loss diseases like acute Montipora white syndrome (aMWS) have impacted coral populations that include the Hawaiian rice coral (Montipora capitata). Multiple strains of Vibrio coralliilyticus are known pathogens, and strain OCN008 has been demonstrated as an etiological agent of aMWS in Hawai'i. Recent work has demonstrated that probiotic bacterial strains can be used to directly treat or prevent transmission (prophylaxis) of coral diseases. Based on their production of zones of inhibition and isolation from disease-resistant corals, Pseudoalteromonas ardens R96, Pseudoalteromonas obscura P94, strain Y97 (the genomic similarity to Pseudoalteromonas piscicida is presented), Pseudoalteromonas umbrosa B95, and Vibrio tetraodonis subsp. pristinus OCN044 were assessed for their ability to impair V. coralliilyticus OCN008 infection of M. capitata during laboratory infection trials. Individual inoculation of each of the five aforementioned strains on M. capitata fragments for 48 h prior to V. coralliilyticus OCN008 inoculation resulted in up to a 93.75% reduction in mortality. These results indicate that strains of Pseudoalteromonas and Vibrio can act as prophylactics to prevent M. capitata mortality from V. coralliilyticus OCN008 infection and provide tools to improve disease resilience for Pacific corals.IMPORTANCECoral disease outbreaks are a growing threat to the continued health of coral reefs, which are already vulnerable ecosystems. Strains of the bacterium Vibrio coralliilyticus are known to infect various coral species worldwide, predominantly causing tissue loss and death of the coral animal. Previous research has indicated that constituents from healthy coral microbiomes can act as probiotics to treat or prevent coral infections, and the discovery of effective probiotics is important in the effort to further develop mitigation tools for disease outbreaks. This work provides a demonstration of probiotic species that can protect coral from tissue loss infections by a strain of Vibrio coralliilyticus and is an example of probiotics developed for coral species in Hawai'i. This work provides new tools for probiotic-based coral protection and evidence for this research as a viable avenue to protect coral in their native environments.

IntroductionMarine bivalve mortality events cause substantial economic losses in aquaculture and threaten global food security. While pathogenic Vibrio species are frequently implicated, growing evidence suggests that loss of beneficial microbes can increase host susceptibility to disease. We previously observed that Vibrio mediterranei was consistently isolated from healthy oysters but systematically disappeared prior to mortality events, coinciding with proliferation of pathogenic Vibrio species. Here, we test whether this pattern reflects a protective functional role.MethodsScreening eleven V. mediterranei strains against eastern oyster (Crassostrea virginica) larvae revealed three distinct virulence phenotypes – avirulent, pathogenic, and intermediate – that correspond to monophyletic phylogenetic clades.ResultsPre-colonization with the avirulent strain Vm02 increased larval survival from 10–19% (pathogen-only controls) to 94–97% when challenged with V. harveyi or V. coralliilyticus, representing near-complete protection maintained at both ambient (28 °C) and thermal stress (32 °C) temperatures. Protection was rapid, effective even under simultaneous co-inoculation with the pathogen, and durable through >96 hours post-exposure. Fluorescence microscopy confirmed persistent association of fluorescently tagged Vm02 with larval digestive tissues. Phylogenomic analysis of 33 V. mediterranei genomes placed the three phenotypic groups within well-supported clades separated by 97.1–97.8% average nucleotide identity, approaching species-level thresholds. Pangenome analysis revealed that protective-clade strains harbor 230 unique orthogroups encoding regulatory systems, stress tolerance, metabolic versatility, and a conserved bacteriocin biosynthetic operon. These same strains do not encode the Type I secretion system, Type VI secretion system variants, and TCP pathogenicity island found in pathogenic lineages.DiscussionTogether, these findings demonstrate that beneficial and pathogenic phenotypes are phylogenetically constrained within distinct V. mediterranei lineages, providing a framework for probiotic development and disease forecasting in shellfish aquaculture.

Lysogenic conversion, in which bacteriophages (viruses that infect bacteria) integrate into bacterial genomes and confer new phenotypic traits on their hosts, is a well-established mechanism for the emergence of pathogens. While this process underpins many bacterial diseases in humans and other animals, its role in coral disease remains largely unexplored. Here, we investigate the potential role of lysogenic conversion in stony coral tissue loss disease (SCTLD), which has caused unprecedented mortality of Caribbean corals since 2014 and for which the etiological agent remains unknown. Across 27 original coral samples and 174 publicly available metagenomes from the Florida Reef Tract spanning seven coral species, we detected a greater number of unique viral genomes harboring genomic features assoicated with lysogeny in diseased corals or visually healthy portions of diseased corals (DD and HD, respectively) compared to visually healthy (VH) corals. SCTLD-associated bacteriophages were primarily predicted to infect bacterial taxa previously implicated in the disease, such as Vibrionales, Rhodobacterales, and Flavobacteriia, and carried abundant and widespread virulence factors with the potential to enhance bacterial colonization, competition, or direct host damage, including homologs of Tse2, Zot, RTX, pneumolysin, and cytolytic delta-endotoxin. Although causal relationships remain unresolved, our findings indicate that phages have the genomic capacity to laterally transfer bacterial virulence genes in SCTLD-affected corals. The acquisition of genes via lysogenic conversion could contribute to bacterial virulence while maintaining community taxonomic profiles, helping to explain previous community profiling observations and providing a mechanistic framework for disease pathogenesis.

Coral disease outbreaks are an increasingly common threat to reefs. While coral disease research is expanding rapidly, there are still monumental challenges in diagnosing and differentiating among the different diseases on a reef. We used a collaborative multidisciplinary approach to characterize a potentially novel disease affecting Orbicella faveolata colonies in the Florida Keys. We tagged and fate-tracked individual lesions for progression and cessation rates, reanalyzed in situ photographs of previously monitored corals to determine prevalence and seasonality, and assessed the efficacy of amoxicillin treatments. Samples collected from lesions as well as unaffected and healthy controls were used to examine microbiomes, assess nine coral pathological parameters via histology, and measure 19 symbiont-specific physiological metrics using transmission electron microscopy (TEM). Across all analyses, we concluded that the observed disease is unlikely to be SCTLD. Many, but not all, metrics had similarities to previous descriptions of white plague, and so we additionally conducted preliminary histology on presumed white plague samples of O. franksi for comparison. However, the dearth of quantifiable histology and TEM studies on white plague did not allow us to conclusively confirm or refute comparisons to white plague. Using the recommended coral disease nomenclature, we define this specific outbreak as "Orbicella acute tissue loss disease" (OATLD). We provide unprecedented, quantified descriptions across numerous metrics of both diseased and control colonies. We suggest that these data lay the groundwork for future efforts on this disease as well as a comprehensive set of parameters against which other diseases can be compared. ### Competing Interest Statement The authors have declared no competing interest. Florida Department of Environmental Protection, C222EE, C20BE0, C3D89C, C21169, C40144

The bacterial pathogen Vibrio coralliilyticus causes disease in coral species worldwide. The mechanisms of V. coralliilyticus coral colonization, coral microbiome interactions, and virulence factor production are understudied. In other model Vibrio species, virulence factors like biofilm formation, toxin secretion, and protease production are controlled through a density-dependent communication system called quorum sensing (QS). Comparative genomics indicated that V. coralliilyticus genomes share high sequence identity for most of the QS signaling and regulatory components identified in other Vibrio species. Here, we identify an active QS signaling pathway in two V. coralliilyticus strains with distinct infection etiologies: type strain BAA-450 and coral isolate OCN008. In V. coralliilyticus, the inter-species AI-2 autoinducer signaling pathway in both strains controls expression of the master QS transcription factor and LuxR/HapR homolog VcpR to regulate >300 genes, including protease production, biofilm formation, and two conserved type VI secretion systems (T6SSs). Activation of T6SS1 by QS results in the secretion of effectors and enables interbacterial competition and killing of prey bacteria. We conclude that the QS system in V. coralliilyticus is functional and controls the expression of genes involved in relevant bacterial behaviors typically associated with host infection.IMPORTANCEVibrio coralliilyticus infects many marine organisms, including multiple species of corals, and is a primary causative agent of tissue loss diseases and bacterial-induced bleaching. Here, we investigated a common cell-cell communication mechanism called quorum sensing, which is known to be intimately connected to virulence in other Vibrio species. Our genetic and chemical studies of V. coralliilyticus quorum sensing uncovered an active pathway that directly regulates the following key virulence factors: proteases, biofilms, and secretion systems. These findings connect bacterial signaling in communities to the infection of corals, which may lead to novel treatments and earlier diagnoses of coral diseases in reefs.

Strains P94 T , B95 T and R96 T were isolated from apparently healthy fragments of the coral Montipora capitata , which were resistant to Vibrio coralliilyticus infection, from the reef surrounding Moku o Loʻe in Kāne‘ohe Bay, O‘ahu, Hawai‘i, USA, and were taxonomically evaluated using a polyphasic approach. Phylogenetic and phylogenomic analyses placed strains P94 T , B95 T and R96 T within the Pseudoalteromonas genus and most closely related to Pseudoalteromonas luteoviolacea and Pseudoalteromonas rubra . Following genome sequencing of strains P94 T , B95 T and R96 T , the average nt identity and in silico DNA–DNA hybridization comparisons with closely related strains resulted in values that fell below species-level cutoffs. Based on a polyphasic characterization and differences in genomic and taxonomic data, strains P94 T , B95 T and R96 T represent novel species, for which the names Pseudoalteromonas ardens sp. nov., Pseudoalteromonas obscura sp. nov. and Pseudoalteromonas umbrosa sp. nov. are proposed. The type strains are R96 T (=DSM 114998 T =LMG 32870 T ), P94 T (=DSM 114996 T =LMG 32871 T ) and B95 T (=DSM 114997 T =LMG 32872 T ), respectively.

Here, we announce the complete bacterial genome sequences of Vibrio coralliilyticus strains ATCC BAA-450 [3.48 Mb, 1.89 Mb, and pVC450 (381.33 kb)] and OCN014 [3.44 Mb, 1.86 Mb, and pVC14 (397.48 kb)] that comprise two chromosomes and one plasmid, respectively. This effort supports future genomic studies of V. coralliilyticus pathogenicity and host species.

Strains P94T, B95T and R96T were isolated from apparently healthy fragments of the coral Montipora capitata, which were resistant to Vibrio coralliilyticus infection, from the reef surrounding Moku o Lo`e in K & amacr;ne'ohe Bay, O'ahu, Hawai'i, USA, and were taxonomically evaluated using a polyphasic approach. Phylogenetic and phylogenomic analyses placed strains P94T, B95T and R96T within the Pseudoalteromonas genus and most closely related to Pseudoalteromonas luteoviolacea and Pseudoalteromonas rubra. Following genome sequencing of strains P94T, B95T and R96T, the average NT identity and in silico DNA-DNA hybridization comparisons with closely related strains resulted in values that fell below species- level cutoffs. Based on a polyphasic characterization and differences in genomic and taxonomic data, strains P94T, B95T and R96T represent novel species, for which the names Pseudoalteromonas ardens sp. nov., Pseudoalteromonas obscura sp. nov. and Pseudoalteromonas umbrosa sp. nov. are proposed. The type strains are R96T (=DSM 114998T=LMG 32870T), P94T (=DSM 114996T=LMG 32871T) and B95T (=DSM 114997T=LMG 32872T), respectively.

Stony coral tissue loss disease (SCTLD) has devastated numerous species of corals across the Western Atlantic but one reef coral, Siderastrea siderea, displays unusual tissue loss lesions. We examined the dynamics of lesions in S. siderea from the cellular to the ecological level and compared the disease with SCTLD in other coral species. We tagged and monitored six S. siderea colonies with bleached lesions in Fort Lauderdale and 17 S. siderea colonies with purple lesions in the Florida Keys for 18 months. Lesions on most colonies showed progressive tissue loss with an average change in healthy tissue of +5.5% in Fort Lauderdale (some bleached lesions resolved) and -51.1% in the Florida Keys. Case fatality rate was zero for colonies within Fort Lauderdale and 5.9% for colonies in the Florida Keys. The disease remained on S. siderea throughout the study in the Florida Keys but fluctuated through time in Fort Lauderdale. Lesion morphologies and disease pathogenesis differed between regions which could be due to different disease agents, environmental co-factors, intrinsic differences among colonies or different stages of the same disease. S. siderea is known to be a species complex which might also explain differences in lesion morphologies and disease pathogenesis. Aquaria studies found S. siderea with lesions transmitted disease to S. siderea and Orbicella faveolata and that S. siderea was also susceptible to SCTLD. Unlike SCTLD in other species, treatment with antibiotics did not stop lesion progression in S. siderea. Histology on lesions indicated a disease process regardless of lesion morphology and was consistent with SCTLD. We cannot completely rule out SCTLD but based on the other components of disease pathogenesis (rate of tissue loss, lesion morphology, colony mortality, response to antibiotics) we conclude this could be a different disease, which we term Siderastrea sidera chronic tissue loss disease, consistent with accepted disease nomenclature.

Stony coral tissue loss disease (SCTLD) has spread throughout Florida’s Coral Reef, causing extensive mortality of over 30 species of reef-building corals, and has rapidly spread to many other countries and territories throughout the Caribbean. Current treatments for SCTLD, including a proprietary paste mixed with the antibiotic amoxicillin, do not provide protection from future infections and may select for antibiotic-resistant pathogenic bacteria. In contrast, beneficial microorganisms (i.e., probiotics), may directly treat or act as prophylactics for corals exposed to SCTLD. This study investigated the use of the bacterium Pseudoalteromonas sp. McH1-7, previously isolated from a SCTLD-resistant fragment of Montastraea cavernosa, as a potential probiotic treatment for SCTLD-infected M. cavernosa colonies in the wild. We developed and tested two probiotic deployment methods: (1) the injection of a probiotic in seawater suspension into a weighted bag placed over the coral to treat the whole colony; and (2) a sodium alginate-based paste that was applied directly to each disease lesion. After treatment, the disease progression of each colony was routinely monitored using three-dimensional photogrammetry for 2.5 years. Slurries of tissue and mucus samples were taken from healthy and diseased colonies before treatment, two weeks after treatment, and three months after treatment to identify possible shifts in bacterial and archaeal communities. McH1–7 successfully slowed SCTLD lesion progression for 2.5 years following treatments when delivered using the whole-colony treatment technique. Our assessment of the microbiome following treatment showed that McH1–7 was effective without dominating bacterial communities among infected corals. In contrast, corals treated with the probiotic paste lost more tissue than corals treated with the control paste, indicating that the lesion-specific probiotic paste is not effective at stopping SCTLD. Probiotic inoculations via a whole-colony treatment technique may provide a path toward slowing the loss of reef-building corals due to SCTLD.

Strain TB1-E2-13 T was isolated from water collected from the Tidal Basin in Washington, D.C., USA, due to the bright purple colour of its colonies, and was taxonomically evaluated with a polyphasic approach. Comparison of a partial 16S rRNA gene sequence found that strain TB1-E2-13 T was most similar to species in the Janthinobacterium genus. For more precise taxonomic inference, a phylogenomic analysis was conducted and indicated that strain TB1-E2-13 T was most closely related to Janthinobacterium lividum , ‘ Janthinobacterium kumbetense ’, Janthinobacterium rivuli and Janthinobacterium violaceinigrum . Analyses of genomic indices found that pairwise comparisons between strain TB1-E2-13 T and other members of the Janthinobacterium genus returned values below the threshold of species novelty. Based on a polyphasic characterization and identifying differences in genomic and taxonomic data, strain TB1-E2-13 T represents a novel species, for which the name Janthinobacterium aestuarii sp. nov. is proposed. The type strain is TB1-E2-13 T (=ATCC TSD-339 T =JCM 36076 T ).

Coral reefs are threatened by various local and global stressors, including elevated ocean temperatures due to anthropogenic climate change. Coral cryopreservation could help secure the diversity of threatened corals. Recently, isochoric vitrification was used to demonstrate that coral fragments lived to 24 hr post-thaw; however, in this study, they were stressed post-thaw. The microbial portion of the coral holobiont has been shown to affect host fitness and the impact of cryopreservation treatment on coral microbiomes is unknown. Therefore, we examined the coral-associated bacterial communities pre- and post-cryopreservation treatments, with a view towards informing potential future stress reduction strategies. We characterized the microbiome of the Hawaiian finger coral, Porites compressa in the wild and at seven steps during the isochoric vitrification process. We observed significant changes in microbiome composition, including: 1) the natural wild microbiomes of P. compressa were dominated by Endozoicomonadaceae (76.5 % relative abundance) and consistent between samples, independent of collection location across Kane'ohe Bay; 2) Endozoicomonadaceae were reduced to <6.9 % in captivity, and further reduced to <0.5 % relative abundance after isochoric vitrification; and 3) Vibrionaceae dominated communities post-thaw (58.5-74.7 % abundance). Thus, the capture and cryopreservation processes, are implicated as possible causal agents of dysbiosis characterized by the loss of putatively beneficial symbionts (Endozoicomonadaceae) and overgrowth of potential pathogens (Vibrionaceae). Offsetting these changes with probiotic restoration treatments may alleviate cryopreservation stress and improve post-thaw husbandry.

Like other intestinal bacteria, the facultative pathogen Vibrio cholerae adapts to a wide range of osmotic environments. Under drastic osmotic down-shifts, Vibrio avoids mechanical rupture by rapidly releasing excessive metabolites through mechanosensitive (MS) channels that belong to two major types, low-threshold MscS and high-threshold MscL. To investigate each channel’s individual contribution to V. cholerae’s osmotic permeability response, we generated individual ΔmscS, ΔmscL, and double ΔmscL ΔmscS mutants in V. cholerae O395 and characterized their tension-dependent activation in patch-clamp experiments, as well as their millisecond-scale osmolyte release kinetics using a stopped-flow light scattering technique. We additionally generated numerical models reflecting the kinetic competition of osmolyte release with water influx. Both mutants lacking MscS exhibited delayed osmolyte release kinetics and decreased osmotic survival rates compared to WT. The ΔmscL mutant showed comparable release kinetics to WT, but a higher osmotic survival, while ΔmscS had low survival, comparable to the double ΔmscL ΔmscS mutant. By analyzing release kinetics following rapid medium dilution, we illustrate the sequence of events and define the set of parameters that characterize discrete phases of the osmotic response. Osmotic survival rates are directly correlated to the extent and duration of cell swelling, the rate of osmolyte release and the onset time, and the completeness of the post-shock membrane resealing. Not only do the two channels interact functionally during the resealing phase, but there is also a compensatory up-regulation of MscS in the ΔmscL strain suggesting some transcriptional crosstalk. The data reveal the advantage of the low-threshold MscS channel in curbing tension surges, without which MscL becomes toxic, and the role of MscS in the proper termination of the osmotic permeability response in Vibrio.

The Pseudoalteromonas genus comprises members that have been demonstrated to play significant ecological roles and produce enzymes, natural products, and activities that are beneficial to the environment and economy. A comprehensive evaluation of the genus revealed that the genomes of several Pseudoalteromonas species are highly similar to each other, exceeding species cutoff values. This evaluation involved determining and comparing the average nucleotide identity, in silico DNA-DNA hybridization, average amino acid identity, and the difference in G + C% between Pseudoalteromonas type strains with publicly available genomes. The genome of the Pseudoalteromonas elyakovii type strain was further assessed through additional sequencing and genomic comparisons to historical sequences. These findings suggest that six Pseudoalteromonas species, namely P. mariniglutinosa, P. donghaensis, P. maricaloris, P. elyakovii, P. profundi, and P. issachenkonii, should be reclassified as later heterotypic synonyms of the following validly published species: P. haloplanktis, P. lipolytica, P. flavipulchra, P. distincta, P. gelatinilytica, and P. tetraodonis. Furthermore, two names without valid standing, 'P. telluritireducens' and 'P. spiralis', should be associated with the validly published Pseudoalteromonas species P. agarivorans and P. tetraodonis, respectively.

Coral reefs are invaluable ecosystems that are under threat from various anthropogenic stressors. There has been a recent increase in the diagnostic tools utilized to understand how these threats impact coral reef health. Unfortunately, the application of diagnostic tools like transmission electron microscopy (TEM) is not as standardized or developed in coral research as in other research fields. Utilizing TEM in conjunction with other diagnostic methods can aid in understanding the impact of these stressors on the cellular level because TEM offers valuable insight into the structures and microsymbionts associated with coral tissue that cannot be obtained with a conventional light microscope. Additionally, a significant amount of coral tissue ultrastructure has not yet been extensively described, causing a considerable gap in our understanding of cellular structures that could relate to the immune response, cellular function, or symbioses. Moreover, additional standardization is needed for TEM in coral research to increase comparability and reproducibility of findings across studies. Here, we present standardized TEM sample fixation, embedding, and sectioning techniques for coral studies that ensure consistent ultrastructural preservation and minimize artifacts, enhancing the reliability and accuracy of TEM observations. We also demonstrate that these TEM protocols allow for the observation and quantification of bacterial and viral-like particles within the coral tissue as well as the endosymbiotic microalgae, potentially providing insight into their interactions within coral cells and how they relate to overall coral health and resilience. (c) 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Primary fixationBasic Protocol 2: DecalcificationBasic Protocol 3: Sample dissection, secondary fixation, dehydration, and embeddingBasic Protocol 4: Sectioning and grid stainingBasic Protocol 5: Imaging

Marine bacteria experience fluctuations in osmolarity that they must adapt to, and most bacteria respond to high osmolarity by accumulating compatible solutes also known as osmolytes. The osmotic stress response and compatible solutes used by the coral and oyster pathogen Vibrio coralliilyticus were unknown. In this study, we showed that to alleviate osmotic stress V. coralliilyticus biosynthesized glycine betaine (GB) and transported into the cell choline, GB, ectoine, dimethylglycine, and dimethylsulfoniopropionate, but not myo -inositol. Myo -inositol is a stress protectant and a signaling molecule that is biosynthesized and used by algae. Bioinformatics identified myo -inositol ( iol ) catabolism clusters in V. coralliilyticus and other Vibrio, Photobacterium, Grimontia, and Enterovibrio species. Growth pattern analysis demonstrated that V. coralliilyticus utilized myo -inositol as a sole carbon source, with a short lag time of 3 h. An iolG deletion mutant, which encodes an inositol dehydrogenase, was unable to grow on myo -inositol. Within the iol clusters were an MFS-type ( iolT1) and an ABC-type ( iolXYZ) transporter and analyses showed that both transported myo -inositol. IolG and IolA phylogeny among Vibrionaceae species showed different evolutionary histories indicating multiple acquisition events. Outside of Vibrionaceae , IolG was most closely related to IolG from a small group of Aeromonas fish and human pathogens and Providencia species. However, IolG from hypervirulent A. hydrophila strains clustered with IolG from Enterobacter, and divergently from Pectobacterium, Brenneria, and Dickeya plant pathogens. The iol cluster was also present within Aliiroseovarius, Burkholderia, Endozoicomonas, Halomonas, Labrenzia, Marinomonas, Marinobacterium, Cobetia, Pantoea, and Pseudomonas, of which many species were associated with marine flora and fauna. IMPORTANCE Host associated bacteria such as V. coralliilyticus encounter competition for nutrients and have evolved metabolic strategies to better compete for food. Emerging studies show that myo -inositol is exchanged in the coral-algae symbiosis, is likely involved in signaling, but is also an osmolyte in algae. The bacterial consumption of myo -inositol could contribute to a breakdown of the coral-algae symbiosis during thermal stress or disrupt the coral microbiome. Phylogenetic analyses showed that the evolutionary history of myo -inositol metabolism is complex, acquired multiple times in Vibrio, but acquired once in many bacterial plant pathogens. Further analysis also showed that a conserved iol cluster is prevalent among many marine species (commensals, mutualists, and pathogens) associated with marine flora and fauna, algae, sponges, corals, molluscs, crustaceans, and fish.

Vibrio coralliilyticus is a pathogen of coral and shellfish, leading to devastating economic and ecological consequences worldwide. Although rising ocean temperatures correlate with increased V. coralliilyticus pathogenicity, the specific molecular mechanisms and determinants contributing to virulence remain poorly understood. Here, we systematically analyzed the type VI secretion system (T6SS), a contact-dependent toxin delivery apparatus, in V. coralliilyticus. We identified 2 omnipresent T6SSs that are activated at temperatures in which V. coralliilyticus becomes virulent; T6SS1 is an antibacterial system mediating interbacterial competition, whereas T6SS2 mediates anti-eukaryotic toxicity and contributes to mortality during infection of an aquatic model organism, Artemia salina. Using comparative proteomics, we identified the T6SS1 and T6SS2 toxin arsenals of 3 V. coralliilyticus strains with distinct disease etiologies. Remarkably, T6SS2 secretes at least 9 novel anti-eukaryotic toxins comprising core and accessory repertoires. We propose that T6SSs differently contribute to V. coralliilyticus’s virulence: T6SS2 plays a direct role by targeting the host, while T6SS1 plays an indirect role by eliminating competitors.