The coral pathogen Vibrio coralliilyticus uses a T6SS to secrete a group of novel anti-eukaryotic effectors that contribute to virulence.

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.

Evaluating the coral microbiome during cryopreservation.

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.

Genome-based taxonomic analysis of the genus Pseudoalteromonas reveals heterotypic synonyms.

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.

Osmotic stress response of the coral and oyster pathogen Vibrio coralliilyticus: acquisition of catabolism gene clusters for the compatible solute and signaling molecule myo-inositol.

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.IMPORTANCEHost associated bacteria such as Vibrio 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.

The dynamic hypoosmotic response of Vibrio cholerae relies on the mechanosensitive channel MscS.

Vibrio cholerae adapts to osmotic down-shifts by releasing metabolites through two mechanosensitive (MS) channels, low-threshold MscS and high-threshold MscL. To investigate each channel's contribution to the osmotic response, we generated ΔmscS, ΔmscL, and double ΔmscL ΔmscS mutants in V. cholerae O395. We characterized their tension-dependent activation in patch-clamp, and the millisecond-scale osmolyte release kinetics using a stopped-flow light scattering technique. We additionally generated numerical models describing osmolyte and water fluxes. We illustrate the sequence of events and define the parameters that characterize discrete phases of the osmotic response. Survival is correlated to the extent of cell swelling, the rate of osmolyte release, and the completeness of post-shock membrane resealing. Not only do the two channels interact functionally, but there is also an up-regulation of MscS in the ΔmscL strain, suggesting transcriptional crosstalk. The data reveal the role of MscS in the termination of the osmotic permeability response in V. cholerae.

Osmotic stress response of the coral and oyster pathogen Vibrio coralliilyticus : acquisition of catabolism gene clusters for the compatible solute and signaling molecule myo -inositol.

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.

Fluctibacter corallii gen. nov., sp. nov., isolated from the coral Montipora capitata on a reef in Kane'ohe Bay, O'ahu, Hawai'i, reclassification of Aestuariibacter halophilus as Fluctibacter halophilus comb. nov., and Paraglaciecola oceanifecundans as a later heterotypic synonym of Paraglaciecola agarilytica.

Strain AA17T was isolated from an apparently healthy fragment of Montipora capitata coral from the reef surrounding Moku o Lo'e in Kane'ohe Bay, O'ahu, Hawai'i, USA, and was taxonomically evaluated using a polyphasic approach. Comparison of a partial 16S rRNA gene sequence found that strain AA17T shared the greatest similarity with Aestuariibacter halophilus JC2043T (96.6%), and phylogenies based on 16S rRNA gene sequences grouped strain AA17T with members of the Aliiglaciecola, Aestuariibacter, Lacimicrobium, Marisediminitalea, Planctobacterium, and Saliniradius genera. To more precisely infer the taxonomy of strain AA17T, a phylogenomic analysis was conducted and indicated that strain AA17T formed a monophyletic clade with A. halophilus JC2043T, divergent from Aestuariibacter salexigens JC2042T and other related genera. As a result of monophyly and multiple genomic metrics of genus demarcation, strain AA17T and A. halophilus JC2043T comprise a distinct genus for which the name Fluctibacter gen. nov. is proposed. Based on a polyphasic characterisation and identifying differences in genomic and taxonomic data, strain AA17T represents a novel species, for which the name Fluctibacter corallii sp. nov. is proposed. The type strain is AA17T (= LMG 32603 T = NCTC 14664T). This work also supports the reclassification of A. halophilus as Fluctibacter halophilus comb. nov., which is the type species of the Fluctibacter genus. Genomic analyses also support the reclassification of Paraglaciecola oceanifecundans as a later heterotypic synonym of Paraglaciecola agarilytica.

Genome sequence of Alteromonas macleodii strain OCN004 isolated from the extracellular mucus of an apparently healthy rice coral (Montipora capitata).

Alteromonas macleodii strain OCN004, a marine gammaproteobacterium in the Alteromonadaceae family, has primarily been studied as a non-pathogenic negative control bacterium during laboratory infection trials to test the virulence of bacterial coral pathogens. The draft genome sequence of A. macleodii strain OCN004 is presented here.

Horizon scanning the application of probiotics for wildlife.

The provision of probiotics benefits the health of a wide range of organisms, from humans to animals and plants. Probiotics can enhance stress resilience of endangered organisms, many of which are critically threatened by anthropogenic impacts. The use of so-called 'probiotics for wildlife' is a nascent application, and the field needs to reflect on standards for its development, testing, validation, risk assessment, and deployment. Here, we identify the main challenges of this emerging intervention and provide a roadmap to validate the effectiveness of wildlife probiotics. We cover the essential use of inert negative controls in trials and the investigation of the probiotic mechanisms of action. We also suggest alternative microbial therapies that could be tested in parallel with the probiotic application. Our recommendations align approaches used for humans, aquaculture, and plants to the emerging concept and use of probiotics for wildlife.

Investigating microbial size classes associated with the transmission of stony coral tissue loss disease (SCTLD).

Effective treatment and prevention of any disease necessitates knowledge of the causative agent, yet the causative agents of most coral diseases remain unknown, in part due to the difficulty of distinguishing the pathogenic microbe(s) among the complex microbial backdrop of coral hosts. Stony coral tissue loss disease (SCTLD) is a particularly destructive disease of unknown etiology, capable of transmitting through the water column and killing entire colonies within a matter of weeks. Here we used a previously described method to (i) isolate diseased and apparently healthy coral colonies within individual mesocosms containing filtered seawater with low microbial background levels; (ii) incubate for several days to enrich the water with coral-shed microbes; (iii) use tangential-flow filtration to concentrate the microbial community in the mesocosm water; and then (iv) filter the resulting concentrate through a sequential series of different pore-sized filters. To investigate the size class of microorganism(s) associated with SCTLD transmission, we used 0.8 µm pore size filters to capture microeukaryotes and expelled zooxanthellae, 0.22 µm pore size filters to capture bacteria and large viruses, and 0.025 µm pore size filters to capture smaller viruses. In an attempt to further refine which size fraction(s) contained the transmissible element of SCTLD, we then applied these filters to healthy "receiver" coral fragments and monitored them for the onset of SCTLD signs over three separate experimental runs. However, several factors outside of our control confounded the transmission results, rendering them inconclusive. As the bulk of prior studies of SCTLD in coral tissues have primarily investigated the associated bacterial community, we chose to characterize the prokaryotic community associated with all mesocosm 0.22 µm pore size filters using Illumina sequencing of the V4 region of the 16S rRNA gene. We identified overlaps with prior SCTLD studies, including the presence of numerous previously identified SCTLD bioindicators within our mesocosms. The identification in our mesocosms of specific bacterial amplicon sequence variants that also appear across prior studies spanning different collection years, geographic regions, source material, and coral species, suggests that bacteria may play some role in the disease.