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TRAF (Tumor necrosis factor receptor-associated factor) proteins are key mediators of signal transduction in cell signaling and immune regulation within the toll-like receptor (TLR) and tumor necrosis factor (TNFR) superfamily. Despite the importance of TRAF genes in teleost innate immunity, study on their functions in C. altivelis is limited. This study utilized bioinformatics methods to identify and named eight TRAF genes (CaTRAF2a, CaTRAF2a-like, CaTRAF2b, CaTRAF3, CaTRAF4a, CaTRAF5, CaTRAF6 and CaTRAF7) in C. altivelis. Phylogenetic, syntenic and molecular evolution revealed that all CaTRAF members were evolutionarily conserved in teleost. Domain analysis indicated the presence of a conserved N-terminal RING finger domain in all CaTRAF proteins. Most CaTRAF proteins also featured a MATH domain at the C-terminal, with the exception of CaTRAF7 which contained seven repeat WD40 domains. In addition, qRT-PCR was used to detect the expression patterns of nine different tissues and eight different embryonic development stages of healthy fish, and it was found that there were spatial and tissue specificities among the members. HE staining revealed evident pathological lesions in the tissues post V. harveyi infection. Atrophy and significant bending of the gill lamellae were observed in the gills, while irregular cell shapes, increased fat vacuoles, and enlarged cell volume were noted in the liver. Intestinal tissues displayed thickening of the muscle layer, elongation of intestinal villi, and increased folds. Moreover, the expression of TRAF gene changed significantly after V. harveyi infection. These results would help to clarify the molecular role of CaTRAF gene in the regulation of immune and inflammatory responses in C. altivelis., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Vibrio spp. are major pathogens responsible for mortality and disease in various marine aquaculture organisms. Effective disease control and genetic breeding strategies rely heavily on understanding host vibriosis resistance mechanisms. The Chinese tongue sole (Cynoglossus semilaevis) is economically vital but suffers from substantial mortalities due to vibriosis. Through continuous selective breeding, we have successfully obtained vibriosis-resistant families of this species. In this study, we conducted RNA-seq analysis on three organs, including liver, spleen and intestine from selected resistant and susceptible tongue soles. Additionally, we integrated these data with our previously published RNA-seq datasets of skin and gill, enabling the construction of organ-specific transcriptional profiles and a comprehensive gene co-expression network elucidating the differences in vibriosis resistance. Furthermore, we identified 12 modules with organ-specific functional implications. Overall, our findings provide a valuable resource for investigating the molecular basis of vibriosis resistance in fish, offering insights into target genes and pathways essential for molecular selection and genetic manipulation to enhance vibriosis resistance in fish breeding programs., (© 2024. The Author(s).)

C-type lectins (CTLs) are glycan-binding pattern recognition receptors (PRRs) that can bind to carbohydrates on pathogen surfaces, triggering immune responses in shrimp innate immunity. In this study, a unique Ca 2+ -inhibited CTL named FcLec was identified and characterized in Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA sequence of FcLec was 976 bp (GenBank accession number KU361826), with a 615 bp open reading frame (ORF) encoding 204 amino acids. FcLec possesses a C-type lectin-like domain (CTLD) containing four conserved cysteines (Cys105, Cys174, Cys192, and Cys200) and two sugar-binding site structures (QPD and LNP). The tertiary structure of FcLec deduced revealed three α-helices and eight β-pleated sheets. The mRNA expression levels of FcLec in hemocytes and the hepatopancreas were markedly elevated after stimulation with Vibrio anguillarum and white spot syndrome virus (WSSV). The recombinant FcLec protein exhibited Ca 2+ -independent hemagglutination and bacterial agglutination, but these activities were observed only in the presence of EDTA to chelate metal ions. These findings suggest that FcLec plays important and functionally distinct roles in the shrimp's innate immune response to bacteria and viruses, enriching the current understanding of the relationship between CTL activity and Ca 2+ in invertebrates., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

MicroRNAs are increasingly recognized for their pivotal role in the immune system, yet the specific regulatory functions of fish-derived microRNAs remain largely unexplored. In this research, we discovered a novel miRNA, Cse-miR-144, in the Chinese tongue sole (Cynoglossus semilaevis), characterized by a 73-base pair precursor and a 21-nucleotide mature sequence. Our findings revealed that the expression of Cse-miR-144 was notably inhibited by various Vibrio species. Utilizing bioinformatics and dual-luciferase assay techniques, we established that the pro-inflammatory cytokine gene CsMAPK6 is a direct target of Cse-miR-144. Subsequent in vitro and in vivo western blotting analyses confirmed that Cse-miR-144 can effectively reduce the protein levels of CsMAPK6 post-transcriptionally. Moreover, CsMAPK6 is known to be involved in the activation of the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kB). Additional investigations using qPCR and ELISA demonstrated that suppression of Cse-miR-144 leads to an upsurge in the liver mRNA levels of various immune genes (including MYD88, TRAF6, NF-κB, TRAF2, TRAF3, and TNF), alongside a marked increase in the production and secretion of pro-inflammatory cytokines (IL-1β, IL-6, and IL-8) in the bloodstream of C. semilaevis. These findings collectively underscore the potential of Cse-miR-144 as a key inhibitor of CsMAPK and its crucial role in modulating the immune and inflammatory responses in teleost fish. Compared to the siRNA, miRNA is a better tool in controlling the expression of target gene with a lower cost., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Alginate oligosaccharides (AOS), products of alginate degradation by endotype alginate lyases, possess favorable biological activities and have broad applications. Although many have been reported, alginate lyases with homogeneous AOS products and secretory production by an engineered host are scarce. Herein, the alginate lyase AlyC7 from Vibrio sp. C42 was characterized as a trisaccharide-producing lyase exhibiting high activity and broad substrate specificity. With PelB as the signal peptide and 500 mM glycine as the additive, the extracellular production of AlyC7 in Escherichia coli reached 1122.8 U/mL after 27 h cultivation in Luria-Bertani medium. The yield of trisaccharides from sodium alginate degradation by the produced AlyC7 reached 758.6 mg/g, with a purity of 85.1%. The prepared AOS at 20 μg/mL increased the root length of lettuce, tomato, wheat, and maize by 27.5%, 25.7%, 9.7%, and 11.1%, respectively. This study establishes a robust foundation for the industrial and agricultural applications of AlyC7.

Vibrio is a prevalent bacterial genus in aquatic environments and exhibits diverse metabolic capabilities, playing a vital role in marine biogeochemical cycles. This study isolated a novel virus infecting Vibrio cyclitrophicus, vB_VviC_ZQ26, from coastal waters near Qingdao, China. The vB_VviC_ZQ26 comprises a linear double-stranded DNA genome with a length of 42,982 bp and a G + C content of 43.21 %, encoding 72 putative open reading frames (ORFs). Transmission electron microscope characterization indicates a siphoviral-morphology of vB_VviC_ZQ26. Nucleic-acids-wide analysis indicates a tetranucleotide frequency deviation for genomic segments encoding putative gene transfer agent protein (GTA) and coil-containing protein, implying divergent origins occurred in different parts of viral genomes. Phylogenetic and genome-content-based analysis suggest that vB_VviC_ZQ26 represents a novel vibriophage-specific family designated as Coheviridae. From the result of biogeographic analysis, Coheviridae is mainly colonized in the temperate and tropical epipelagic zones. This study describes a novel vibriophage infecting V. cyclitrophicus, shedding light on the evolutionary divergence of different parts of the viral genome and its ecological footprint in marine environments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

A Gram-stain-negative, facultative anaerobic, methylphosphonate-decomposing, motile by a polar flagellum and rod-shaped marine bacterium, designated S4B1 T , was isolated from the surface seawater collected from the Yongle Atoll (Xisha Islands, PR China). The pairwise alignment showed the highest sequence similarity of 97.5 and 96.6 % to Vibrio aestuarianus subsp. cardii 12_122_3T3 T and Vibrio atypicus HHS02 T , respectively. Phylogenetic analysis based on 16S rRNA gene and the phylogenomic analysis of single-copy genes showed that strain S4B1 T belonged to the genus Vibrio and formed a close branch with Vibrio qingdaonensis ZSDZ65 T . Growth of strain S4B1 T occurred at 4-30 °C (optimum, 28 °C), at pH 6.0-8.0 (optimum, pH 7.0) and in the presence of 2-7 % (w/v) NaCl (optimum, 3 %). The predominant fatty acids (>10 %) were C16 : 0, iso-C 16 : 0 and summed feature 3 (C 16 : 1  ω 7 c or/and C 16 : 1 ). The DNA G+C content of the assembled genomic sequence was 44.3 mol%. Average nucleotide identity (ANI) values between S4B1  ω 6 c ). The DNA G+C content of the assembled genomic sequence was 44.3 mol%. Average nucleotide identity (ANI) values between S4B1 T and its reference species were lower than the threshold for species delineation (95-96 %), in which its highest ANI value with V. qingdaonensis ZSDZ65 T had less than 70 % similarity to its relatives. On the basis of the polyphasic evidence, strain S4B1 In silico is proposed to represent a novel species of the genus T had less than 70 % similarity to its relatives. On the basis of the polyphasic evidence, strain S4B1 T is proposed to represent a novel species of the genus Vibrio , for which the name Vibrio methylphosphonaticus sp. nov. is proposed. The type strain is S4B1 T (=KCTC 92311 T =MCCC 1K06168 T ).

Coral reefs are among the most biodiverse ecosystems, providing habitats for various organisms. Studies on coral bleaching have been increasing recently, but little is known about the distribution and community assembly of coral pathogenic bacteria (e.g., several Vibrio species). We elucidated the distribution pattern and interaction relationships of total bacteria and Vibrio spp. in sediments from the Xisha Islands, which are characterized by their high coverage and diversity of coral resources. Vibrio spp. showed significantly higher relative abundance values in the Xisha Islands (1.00 × 10 8 copies/g) than in other areas (approximately 1 × 10 4 to 9.04 × 10 5 copies/g), indicating that the coral bleaching event of 2020 may have promoted the bloom of vibrios. A spatial shift in community composition was observed between the northern (Photobacterium rosenbergii and Vibrio ponticus) and southern (Vibrio ishigakensis and Vibrio natriegens) sites, accompanied by a clear distance-decay pattern. The spatial distance and coral species (e.g., Acroporidae and Fungiidae ) had much greater correlations with the Vibrio community than did environmental factors. However, complex mechanisms may exist in the community assembly of Vibrio spp. due to the large proportion of unexplained variation. Stochastic processes may play an important role, as shown by the neutral model. Vibrio harveyi had the highest relative abundance (77.56%) and niche breadth, compared to other species, and it was negatively correlated with Acroporidae, likely reflecting its strong competitive ability and adverse effects on specific corals. Our study provides insights into the bloom and underlying assembly mechanisms of sedimentary vibrios in the Xisha Islands, thereby contributing to identify the potential indicator of coral bleaching and provide inspiration for the environmental management of coral reef areas. IMPORTANCE Coral reefs exert important roles in maintaining the sustainability of marine ecosystems but decline worldwide due to various drivers, especially pathogenic microorganisms. Here, we investigated the distribution pattern and interactions of total bacteria and Vibrio spp. in the sediments from Xisha Islands during the coral bleaching event of 2020. Our results showed that the abundances of Vibrio (1.00 × 10 8 copies/g) were high across the whole sites, indicating the bloom of sedimentary Vibrio spp. Coral pathogenic Vibrio species were abundant in the sediments, likely reflecting adverse effects on several kinds of corals. The compositions of the Vibrio spp. were separated by geographical location, which was mainly attributable to the spatial distance and coral species. Overall, this work contributes by providing evidence for the outbreak of coral pathogenic vibrios. The pathogenic mechanism of the dominant species (especially V. harveyi) should be comprehensively considered by laboratory infection experiments in the future., Competing Interests: The authors declare no conflict of interest.

Two Gram-stain-negative, facultatively anaerobic, motile, rod-shaped and flagellated marine bacteria, designated strains DBSS07 T and ZSDZ65 T , were isolated from the surface sediments of the Bohai sea and Qingdao coastal seawater, respectively. Phylogenetic analysis based on 16S rRNA genes, multilocus sequence analysis (MLSA), phylogenomic analysis of single-copy gene families and whole-genome data placed DBSS07 T and ZSDZ65 T within the genus Vibrio . DBSS07 T was most closely related to Vibrio aestivus M22 T , with 97.51 % sequence similarity, whereas ZSDZ65 T was most closely related to Vibrio variabilis R-40492 T with 97.58 % sequence similarity. DBSS07 T grew with 1-7 % (w/v) NaCl (optimum 3 %), at 16-37 °C (optimum 28 °C) and at pH 6.0-9.0 (optimum pH 7.0); whereas ZSDZ65 T grew with 1-5 % (w/v) NaCl (optimum 2 %), at 16-32 °C (optimum 28 °C) and at pH 6.0-9.0 (optimum pH 8.0). Both strains shared the same major fatty acid components (more than 10 % of total fatty acids) of summed feature 3 (C 16 : 1 ω7 c or/and C 16 : 1 ω6 c ), with different proportions. The DNA G+C contents of DBSS07 T and ZSDZ65 T were 44.7 and 44.3 %, respectively. On the basis of the results of polyphasic analysis, DBSS07 T and ZSDZ65 T are considered to represent novel species within the genus Vibrio , for which the names V. paucivorans sp. nov. (type strain, DBSS07 T = KCTC 82896 T = MCCC 1K06284 T ) and V. qingdaonensis sp. nov. (type strain, ZSDZ65 T = KCTC 82893 T = MCCC 1K06289 T ) are proposed, respectively.

Two Gram-stain-negative, oxidase-positive, facultative anaerobic and rod-shaped motile bacteria, designated strains ZSDZ34 and ZSDE26, were isolated from offshore surface seawater collected near Qingdao. Phylogenetic analysis based on 16S rRNA gene sequences placed ZSDE26 T and ZSDZ34 T within the genus Vibrio , family Vibrionaceae , class Gammaproteobacteria . Strain ZSDE26 T was most closely related to Vibrio gallaecicus VB 8.9 T with 97.3 % sequence similarity, whereas ZSDZ34 T was most closely related to Vibrio aestuarianus subsp. cardii DSM 109723 T with 97.8 % sequence similarity. Strain ZSDE26 T grew with 1-5 % (w/v) NaCl (optimum, 4 %), at 16-28 °C (optimum, 28 °C) and at pH 6.0-9.0 (optimum, pH 7.0). Growth of strain ZSDZ34 T occurred with 1-6 % (w/v) NaCl (optimum, 3 %), at 16-37 °C (optimum, 28 °C) and at pH 6.0-9.0 (optimum, pH 7.0). Both strains shared the same major fatty acid components (more than 10 % of total fatty acids) of summed feature 3 (C 16 : 1 ω 7 c and/or C 16 : 1 ω 6 c ), summed feature 8 (C 18 : 1 ω 7 c and/or C 18 : 1 contained a higher proportion of iso-C ω . The DNA G+C contents of strains ZSDE26 c ) and C 16 : 0 were 42.8 and 44.5 mol%, respectively. On the basis of the results of polyphasic analysis, ZSDE26 T contained a higher proportion of iso-C 16 : 0 . The DNA G+C contents of strains ZSDE26 T and ZSDZ34 T were 42.8 and 44.5 mol%, respectively. On the basis of the results of polyphasic analysis, ZSDE26 T and ZSDZ34 T are considered to represent novel species within the genus Vibrio , for which the names Vibrio amylolyticus sp. nov. (type strain, ZSDE26 T =KCTC 82890 T =MCCC 1K06290 T ) and Vibrio gelatinilyticus sp. nov. (type strain, ZSDZ34 T =KCTC 82888 T =MCCC 1K06292 T ) are proposed, respectively.

A Gram-straining-negative, facultatively anaerobic, motile by means of a polar flagellum and rod-shaped marine bacterium, designated S4M6 T , was isolated from surface seawater collected in Dongshan Bay (Fujian, PR China). Phylogenetic analysis based on 16S rRNA genes, phylogenomic analysis of single-copy gene families and whole genome data indicated that S4M6 T represented a member of the genus Vibrio . The closest phylogenetic relatives of S4M6 T were Vibrio marisflavi CGMCC 1.8994 T (97.8 % 16S rRNA gene sequence pairwise similarity), Vibrio variabilis LMG 25438 T (96.9 %), Vibrio gangliei SZDIS-1 T (96.2 %) and Vibrio aestivus M22 T (96.1 %). The growth of S4M6 T occurred at 15-35 °C (optimum 28 °C), pH 4.0-9.0 (optimum 5.0-7.0) and in the presence of 2-5 % (w/v) NaCl (optimum 3 %). The predominant fatty acids (>10 %) are C 16 : 0 , summed feature 3 (C 16 : 1 ω7 c and/or C 16 :   1 ω6c) and summed feature 8 (C 18 : 1 ω7 c and/or C 18 :   1 ω6 c ). The DNA G+C content of the assembled genomic sequences was 43.4 % for S4M6 T . Average nucleotide identity (ANI) values between S4M6 T and the reference species were lower than the threshold for species delineation (95-96 %); in silico DNA-DNA hybridization further indicated that S4M6 T had less than 70 % similarity to its relatives. On the basis of the polyphasic evidence, strain S4M6 T is proposed to represent a novel species of the genus Vibrio , for which the name Vibrio sinus sp. nov. is proposed. The type strain is S4M6 T (= KCTC 92312 T = MCCC 1K06167 T ).

Vibrio is ubiquitous in marine environments with high metabolism flexibility and genome plasticity. Studies have investigated the ecological distribution of Vibrio spp. in several narrow zones, but a broad scale pattern of distribution and community assembly is still lacking. Here, we elucidated the distribution of Vibrio spp. in seawater along the Chinese marginal seas with a high spatial range. Comparison of Vibrio abundance between 3- and 0.2-μm-pore-size membranes showed distinction in preferential lifestyle. Vibrio spp. in the Yellow Sea (YS) was low in abundance and adopted a particle-associated lifestyle, whereas that in the East China Sea (ECS) and South China Sea (SCS) was more abundant and was likely in a temporary free-living state as a strategy to cope with nutrient limitation. Vibrio community compositions were also separated by sampling area, with different dominant groups in YS (Vibrio chagasii and Vibrio harveyi), ECS and SCS (Vibrio japonicus and V. chagasii ). The community niche breadth was significantly wider in ECS and SCS than that of YS. Among species, V. chagasii and V. harveyi had the largest niche breadths likely reflecting strong competitive positions. Stochastic processes played important roles in shaping the geographical pattern of the vibrionic community. Environmental selection (e.g., temperature, salinity, and dissolved oxygen) had a much greater impact on the community in surface than in bottom water. The large proportions of unexplained variations (78.9%) imply complex mechanisms in their community assembly. Our study provides insights into the spatial distribution patterns and underlying assembly mechanisms of Vibrio at a broad spatial scale. IMPORTANCE spp. may exert large impacts on biogeochemical cycling in coastal habitats, and their ecological importance has drawn increasing attention. Here, we investigated the spatial distribution pattern and community assembly of Vibrio populations along the Chinese marginal seas, spanning a wide spatial scale. Our results showed that the abundances of the Vibrio population increased with decreasing latitude and their preferential lifestyle differed among adjacent coastal areas. The compositions of Vibrio spp. were also separated by geographical location, which was mainly attributable to stochastic processes. Overall, this work contributes to the understanding of the ecological distribution patterns and the community assembly mechanisms of marine vibrios at a high spatial range. The large proportion of unexplained variations indicates the existence of complex mechanisms in the assembly of vibrionic community which should be considered comprehensively in future.Vibrio spp. were also separated by geographical location, which was mainly attributable to stochastic processes. Overall, this work contributes to the understanding of the ecological distribution patterns and the community assembly mechanisms of marine vibrios at a high spatial range. The large proportion of unexplained variations indicates the existence of complex mechanisms in the assembly of vibrionic community which should be considered comprehensively in future.

Alginate is an important polysaccharide in the ocean that supports the growth of marine microorganisms. Many widespread Vibrio species possess alginate lyases and can utilize alginate as a carbon source, but the detailed alginate degradation mechanism in Vibrio remains to be further explored. In this study, we obtained a highly efficient alginate-degrading strain, Vibrio pelagius WXL662, with 11 alginate lyases (VpAly-I to -XI) and further elucidated its molecular mechanism of alginate degradation. Three alginate utilization loci (AUL) were identified in different parts of WXL662's genome, comprising six alginate lyases (VpAly-I, -II, -VIII, -IX, -X, and -XI) and other genes related to alginate degradation. Most of the alginate-degrading genes are strongly induced when alginate is provided as the sole carbon source. Ten alginate lyases (VpAly-I to -X) had been purified and characterized, including six from polysaccharide lyase family 7 (PL7), three from PL17, and one from PL6. These recombinant alginate lyases existing in different cellular locations were active at a wide temperature (10 to 50°C) and pH (4.0 to 9.0) range, with different substrate preferences and diverse degradation products, enabling WXL662 to efficiently utilize alginate in a changing marine environment. Importantly, outer membrane vesicles (OMVs) can act as vectors for alginate lyases (VpAly-II, -V, and -VI) in WXL662. Further investigations of public Vibrio genomes revealed that most alginate-degrading vibrios possess one AUL instead of previously reported "scattered" system. These results emphasize the specific alginate degradation strategy in Vibrio pelagius WXL662, which can be used as a model strain to study the ecological importance of effective alginate-degrading vibrios in the ocean. IMPORTANCE Alginate is an important carbon source in the marine environment, and vibrios are major alginate utilizers. Previous studies focused only on the characteristics of individual alginate lyases in vibrios, but few of them discussed the comprehensive alginate-degrading strategy. Here, we depicted the alginate utilization mechanism and its ecological implications of a highly efficient alginate-degrading Vibrio strain, WXL662, which contained 11 alginate lyases with distinct enzymatic characteristics. Importantly, unlike other vibrios with only one alginate utilization locus (AUL) or the previously reported "scattered" system, three AUL were identified in WXL662. Additionally, the involvement of outer membrane vesicles (OMVs) in the secretion of alginate lyases is proposed for the first time.

Alginate is abundant in the cell walls of brown algae. Alginate lyases can degrade alginate, and thus play an important role in the marine carbon cycle and industrial production. Currently, most reported alginate lyases contain only one functional alginate lyase domain. AlyC8 is a putative alginate lyase with two alginate lyase domains (CD1 and CD2) from the marine alginate-degrading strain Vibrio sp. C42. To characterize AlyC8 and its two catalytic domains, AlyC8 and its two catalytic domain-deleted mutants, AlyC8-CD1 and AlyC8-CD2, were expressed in Escherichia coli . All three proteins have noticeable activity toward sodium alginate and exhibit optimal activities at pH 8.0-9.0 and at 30-40 °C, demonstrating that both CD1 and CD2 are functional. However, CD1 and CD2 showed opposite substrate specificity. The differences in substrate specificity and degradation products of alginate between the mutants and AlyC8 demonstrate that CD1 and CD2 can act synergistically to enable AlyC8 to degrade various alginate substrates into smaller oligomeric products. Moreover, kinetic analysis indicated that AlyC8-CD1 plays a major role in the degradation of alginate by AlyC8. These results demonstrate that AlyC8 is a novel alginate lyase with two functional catalytic domains that are synergistic in alginate degradation, which is helpful for a better understanding of alginate lyases and alginate degradation.

The genus Vibrio is characterized by high metabolic flexibility and genome plasticity and is widely distributed in the ocean from euphotic layers to deep-sea environments. The relationship between genome features and environmental adaptation strategies of Vibrio has been extensively investigated in coastal environments, yet very little is known about their survival strategies in oligotrophic deep-sea. In this study, we compared genomes of five Vibrio campbellii strains isolated from the Mariana and Yap Trenches at different water depths, including two epipelagic strains and three hadopelagic strains, to identify genomic characteristics that facilitate survival in the deep sea. Genome streamlining is found in pelagic strains, such as smaller genome sizes, lower G+C contents, and higher gene densities, which might be caused by long-term residence in an oligotrophic environment. Phylogenetic results showed that these five Vibrio strains are clustered into two clades according to their collection depth. Indeed, hadopelagic isolates harbor more genes involved in amino acid metabolism and transport, cell wall/membrane/envelope biogenesis, and inorganic ion transport and metabolism through comparative genomics analysis. Specific macrolide export gene and more tellurite resistance genes present in hadopelagic strains by the annotation of antibiotic and metal resistance genes. In addition, several genes related to substrate degradation are enriched in hadopelagic strains, such as chitinase genes, neopullulanase genes, and biopolymer transporter genes. In contrast, epipelagic strains are unique in their capacity for assimilatory nitrate reduction. The genomic characteristics investigated here provide insights into how Vibrio adapts to the deep-sea environment through genomic evolution. IMPORTANCE With the development of deep-sea sampling technology, an increasing number of deep-sea Vibrio strains have been isolated, but the adaptation mechanism of these eutrophic Vibrio strains to the deep-sea environment is unclear. Here, our results show that the genome of pelagic Vibrio is streamlined to adapt to a long-term oligotrophic environment. Through a phylogenomic analysis, we find that genomic changes in marine Vibrio campbellii strains are related to water depth. Our data suggest that an increase in genes related to antibiotic resistance, degradation of macromolecular and refractory substrates, and utilization of rare ions is related to the adaptation of V. campbellii strains to adapt to hadal environments, and most of the increased genes were acquired by horizontal gene transfer. These findings may deepen our understanding of adaptation strategies of marine bacteria to the extreme environment in hadal zones.

The diseases triggered by Vibrio anguillarum infection have created huge economic losses to the turbot (Scophthalmus maximus) farming industry. However, the immune mechanism of turbot to V. anguillarum infection has not been deeply investigated. To better understand the immune response of turbot to V. anguillarum infection, transcriptome analysis of the head kidney and liver of turbot was performed. A total of 15,948 and 11,494 differentially expressed genes (DEGs) were obtained from the turbot head kidney and liver, respectively. Transcriptome analysis revealed that the head kidney and liver of turbot have some differences in the gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the DEGs for the different functions of these two organs. Although there are many uncertain factors in this immune process, such as the occurrence of alternative splicing (AS) events and the differences in the protein structure of the DEGs, the NFκB signalling pathway, MKK-dependent AP-1 activation, JAK-STAT signalling pathway, the signal transmission of MHC Ⅰ and a series of DEGs including HSP90 driving NLRP3 to produce inflammatory factors (IL-1β, IL-8, TNFα, etc.) were possible important immune response pathways for turbot to V. anguillarum infection. Overall, our research has conducted a preliminary exploration of the immune mechanism of turbot in response to V. anguillarum infection., (© 2022 John Wiley & Sons Ltd.)

Vibrio collagenases of the M9A subfamily are closely related to Vibrio pathogenesis for their role in collagen degradation during host invasion. Although some Vibrio collagenases have been characterized, the collagen degradation mechanism of Vibrio collagenase is still largely unknown. Here, an M9A collagenase, VP397, from marine Vibrio pomeroyi strain 12613 was characterized, and its fragmentation pattern on insoluble type I collagen fibers was studied. VP397 is a typical Vibrio collagenase composed of a catalytic module featuring a peptidase M9N domain and a peptidase M9 domain and two accessory bacterial prepeptidase C-terminal domains (PPC domains). It can hydrolyze various collagenous substrates, including fish collagen, mammalian collagens of types I to V, triple-helical peptide [(POG) 10 ] 3 , gelatin, and 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-o-Arg (Pz-peptide). Atomic force microscopy (AFM) observation and biochemical analyses revealed that VP397 first assaults the C-telopeptide region to dismantle the compact structure of collagen and dissociate tropocollagen fragments, which are further digested into peptides and amino acids by VP397 mainly at the Y-Gly bonds in the repeating Gly-X-Y triplets. In addition, domain deletion mutagenesis showed that the catalytic module of VP397 alone is capable of hydrolyzing type I collagen fibers and that its C-terminal PPC2 domain functions as a collagen-binding domain during collagenolysis. Based on our results, a model for the collagenolytic mechanism of VP397 is proposed. This study sheds light on the mechanism of collagen degradation by Vibrio collagenase, offering a better understanding of the pathogenesis of Vibrio and helping in developing the potential applications of Vibrio collagenase in industrial and medical areas. IMPORTANCE Many Vibrio species are pathogens and cause serious diseases in humans and aquatic animals. The collagenases produced by pathogenic Vibrio species have been regarded as important virulence factors, which occasionally exhibit direct pathogenicity to the infected host or facilitate other toxins' diffusion through the digestion of host collagen. However, our knowledge concerning the collagen degradation mechanism of Vibrio collagenase is still limited. This study reveals the degradation strategy of Vibrio collagenase VP397 on type I collagen. VP397 binds on collagen fibrils via its C-terminal PPC2 domain, and its catalytic module first assaults the C-telopeptide region and then attacks the Y-Gly bonds in the dissociated tropocollagen fragments to release peptides and amino acids. This study offers new knowledge regarding the collagenolytic mechanism of Vibrio collagenase, which is helpful for better understanding the role of collagenase in Vibrio pathogenesis and for developing its industrial and medical applications.

The collagenases of Vibrio species, many of which are pathogens, have been regarded as an important virulence factor. However, there is little information on the structure and collagenolytic mechanism of Vibrio collagenase. Here, we report the crystal structure of the collagenase module (CM) of Vibrio collagenase VhaC and the conformation of VhaC in solution. Structural and biochemical analyses and molecular dynamics studies reveal that triple-helical collagen is initially recognized by the activator domain, followed by subsequent cleavage by the peptidase domain along with the closing movement of CM. This is different from the peptidolytic mode or the proposed collagenolysis of Clostridium collagenase. We propose a model for the integrated collagenolytic mechanism of VhaC, integrating the functions of VhaC accessory domains and its collagen degradation pattern. This study provides insight into the mechanism of bacterial collagenolysis and helps in structure-based drug design targeting of the Vibrio collagenase., (© 2022. The Author(s).)

A Gram-strain-negative, facultatively anaerobic, motile, rod-shaped and flagellated marine bacterium, designated SM6 T , was isolated from surface seawater collected in Daya Bay (Guangdong, China). Phylogenetic analysis based on 16S rRNA gene sequences, multilocus sequence analysis, phylogenomic analysis of single-copy gene families and whole genome data showed that strain SM6 T belonged to the genus Vibrio . The closest phylogenetic relatives of SM6 T were Vibrio plantisponsor MSSRF60 T (97.38 % 16S rRNA gene sequence pairwise similarity), Vibrio variabilis R-40492 T (97.27 %), Vibrio aestuarianus ATCC 35048 T (97.21 %) and Vibrio sagamiensis LC2-047 T (97.3 %). Growth of strain SM6 T occurred at 10-45 °C (optimum 30 °C), at pH 6.0-9.0 (optimum 6.0) and in the presence of 0-10 % (w/v) NaCl (optimum 3-8 %). The predominant fatty acids (>10 %) were summed feature 3 (C 16 : 1 ω 7 c or/and C 16 : 1 ω 6 c ), C 16 : 0 and summed feature 8 (C 18 : 1 ω ). The DNA G+C content of the assembled genomic sequences was 47.37 % for strain SM6 c or/and C 18 : 1 ω DNA-DNA hybridization further showed that the strains shared less than 70 % similarity. On the basis of evidence from the present polyphasic study, strain SM6 c is considered to represent a novel species of the genus T . Average nucleotide identity values between SM6 T and its reference species were lower than the threshold for species delineation (95-96 %); in silico DNA-DNA hybridization further showed that the strains shared less than 70 % similarity. On the basis of evidence from the present polyphasic study, strain SM6 T is considered to represent a novel species of the genus Vibrio , for which the name Vibrio agarilyticus sp. nov. is proposed. The type strain is SM6 T (=KCTC 82076 T =MCCC 1K04327 T ).

Vibrio is ubiquitously distributed in marine environments and is the most extensively characterized group within Gammaproteobacteria. Studies have investigated Vibrio spp. worldwide, but mostly focused on pathogenic vibrios and based on cultivation methods. Here, using a combination of molecular and culturing methods, we investigated the dynamics of the total and active Vibrio spp. throughout the Changjiang estuary in China. The total Vibrio abundance was higher in summer (~6.59 × 10 3  copies ml -1 ) than in winter (~1.85 × 10 3  copies ml -1 ) and increased from freshwater to saltwater (e.g. 8.04 × 10 1 to 9.39 × 10 3  copies ml -1 in summer). The ratio of active to total Vibrio (Va/Vt) revealed a high activity of vibrios, with remarkable differences between freshwater and saltwater (p < 0.05). Based on the community compositions of the culturable, total and active Vibrio, Vibrio atlanticus and Vibrio owensii were the dominant and active species in winter and summer, respectively. The distribution of Vibrio was governed by the effects of diverse environmental factors, such as temperature, salinity, pH, dissolved oxygen and SiO 3 . Our study clearly demonstrates the spatiotemporal dynamics of total and active Vibrio spp. and lays a foundation for fully understanding the ecological roles of marine Vibrio.2- . Our study clearly demonstrates the spatiotemporal dynamics of total and active Vibrio spp. and lays a foundation for fully understanding the ecological roles of marine Vibrio., (© 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

A Gram-stain-negative, oxidase- and catalase-positive, facultative anaerobic and rod-shaped bacterium, designated strain SM1977 T , was isolated from the surface of coralline algae collected from the intertidal zone at Qingdao, PR China. The strain grew at 10-35 °C, pH 4.5-8.5 and with 1-8.5% (w/v) NaCl. It reduced nitrate to nitrite and hydrolysed Tween 20 and DNA. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SM1977 T was affiliated with the genus Vibrio , having the highest sequence similarity (97.6 %) to the type strain of Vibrio casei , followed by those of another five species (95.6-97.6 %) in the Rumoiensis clade of the genus Vibrio . However, the in silico DNA-DNA hybridization (75.3-75.9 %) and average nucleotide identity (21.6-22.8 %) values of SM1977 T against these close relatives were all below the corresponding thresholds to discriminate bacterial species. The major fatty acids were summed feature 3 (C 16:1  ω 7 c and/or C 16:1  ω 6 c ), C 16:0 and summed feature 8 (C 18:1  ω ). The predominant polar lipids were phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. The sole respiratory quinone was Q-8. The genomic DNA G+C content of strain SM1977 c , determined from the obtained whole genomic sequence, was 42.3 mol%. On the basis of the polyphasic results obtained in this study, strain SM1977 18:1  ω 7 c ). The predominant polar lipids were phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. The sole respiratory quinone was Q-8. The genomic DNA G+C content of strain SM1977 T , determined from the obtained whole genomic sequence, was 42.3 mol%. On the basis of the polyphasic results obtained in this study, strain SM1977 T is considered to represent a novel species within the genus Vibrio , for which the name Vibrio algicola sp. nov. is proposed. The type strain is SM1977 T (=MCCC 1K04351 T =KCTC 72847 T ).

3,6-anhydro-α-L-galactose (L-AHG) is one of the main monosaccharide constituents of red macroalgae. In the recently discovered bacterial L-AHG catabolic pathway, L-AHG is first oxidized by a NAD(P) + -dependent dehydrogenase (AHGD), which is a key step of this pathway. However, the catalytic mechanism(s) of AHGDs is still unclear. Here, we identified and characterized an AHGD from marine bacterium Vibrio variabilis JCM 19239 (VvAHGD). The NADP + -dependent VvAHGD could efficiently oxidize L-AHG. Phylogenetic analysis suggested that VvAHGD and its homologs represent a new aldehyde dehydrogenase (ALDH) family with different substrate preferences from reported ALDH families, named the L-AHGDH family. To explain the catalytic mechanism of VvAHGD, we solved the structures of VvAHGD in the apo form and complex with NADP + and modeled its structure with L-AHG. Based on structural, mutational, and biochemical analyses, the cofactor channel and the substrate channel of VvAHGD are identified, and the key residues involved in the binding of NADP + and L-AHG and the catalysis are revealed. VvAHGD performs catalysis by controlling the consecutive connection and interruption of the cofactor channel and the substrate channel via the conformational changes of its two catalytic residues Cys282 and Glu248. Comparative analyses of structures and enzyme kinetics revealed that differences in the substrate channels (in shape, size, electrostatic surface, and residue composition) lead to the different substrate preferences of VvAHGD from other ALDHs. This study on VvAHGD sheds light on the diversified catalytic mechanisms and evolution of NAD(P) + -dependent ALDHs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Chitinase, as one of the most important extracellular enzymes in the marine environment, has great ecological and applied values. In this study, two chitinases (Chi1557 and Chi4668) with 97.33% amino acid sequences identity were individually found in Vibrio rotiferianus and Vibrio harveyi . They both were encoding by 561 amino acids, but differed in 15 amino acids and showed different enzymatic properties. The optimal temperature and pH ranges were 45-50 °C and pH 5.0-7.0 for Chi1557, while ~50 °C and pH 3.0-6.0 for Chi4668. K + , Mg 2+ , and EDTA increased the enzymatic activity of Chi4668 significantly, yet these factors were inhibitory to Chi1557. Moreover, Chi1557 degraded colloidal chitin to produce (GlcNAc) 2 and minor GlcNAc, whereas Chi4668 produce (GlcNAc) 2 with minor (GlcNAc) 3 and (GlcNAc) 4 . The K cat/ K m of Chi4668 was ~4.7 times higher than that of Chi1557, indicating that Chi4668 had stronger catalytic activity than Chi1557. Furthermore, site-directed mutagenesis was performed on Chi1557 focusing on seven conserved amino acid residues of family GH18 chitinases. Chi1557 was almost completely inactive after Glu154, Gln219, Tyr221, or Trp312 was individually mutated, retained ~50% activity after Tyr37 was mutated, and increased two times activity after Asp152 was mutated, indicating that these six amino acids were key sites for Chi1557.

Two Gram-staining-negative, facultatively anaerobic, motile, short clavate and flagellated marine bacteria, designated strains BEI233 T and LJC006 T , were isolated from the East China Sea. On the basis of the results of 16S rRNA gene sequencing and multilocus sequence analysis, BEI233 T and LJC006 T should be assigned to the genus Vibrio . The closest phylogenetic relatives of BEI233 T are Vibrio scophthalmi LMG 19158 T (98.7 % 16S rRNA gene sequence pairwise similarity), Vibrio ichthyoenteri DSM 14397 T (98.5 %), Vibrio renipiscarius KCTC 42287 T (97.7 %), Vibrio aestuarianus ATCC 35048 T (97.3 %) and Vibrio thalassae MD16 T (96.5 %), whereas for LJC006 T they were Vibrio furnissii CAIM 518 T (97.1 %), Vibrio fluvialis LMG 7894 T (97.0%), Vibrio tritonius JCM 16456 T (96.9 %) and Vibrio xuii LMG 21346 T (96.1 %). The growth of BEI233 T occurred at 10-37 °C, pH 5.0-8.0 and with 1-7 % (w/v) NaCl, while the growth of LJC006 T occurred at 10-37 °C, pH 6.0-9.0, and 0-8 % (w/v) NaCl. The predominant fatty acids (>10 %) were summed feature 3 (C 16 : 1 ω7 c or/and C 16 : 1 ω6 c ), C 16 : 0 and summed feature 8 (C 18 : 1 ω7 c or/and C 18 : 1 ω6 c ), with different proportions. The DNA G+C contents of BEI233 T and LJC006 T are 42.41 mol% and 41.88 mol%, respectively. On the basis of the results of polyphasic analysis, BEI233 T and LJC006 T are considered to represent novel species of the genus Vibrio, for which the names Vibrio sinensis sp. nov. and Vibrio viridaestus sp. nov. are proposed. The type strains are BEI233 T (=JCM 32692 T =KCTC 62618 T ) and LJC006 T (=JCM 32693 T =KCTC 62620 T ), respectively.

Two Gram-stain-negative, catalase- and oxidase-positive, facultative anaerobic and rod-shaped motile bacteria, designated strains BEI176 T and BEI207 T , were isolated from seawater collected in the East China Sea. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains BEI176 T and BEI207 T belonged to the genus Vibrio and were closely related to each other with 98.18 % similarity. The closest phylogenetic relatives of strain BEI176 T were Vibrio alginolyticus LMG 4409 T (98.85 %) and Vibrio campbellii LMG 11216 T (98.81 %), whereas the closest relative of strain BEI207 T was Vibrio hepatarius LMG 20362 T (98.64 %). The two strains showed growth at different conditions; while strain BEI176 T grew at 16-37 °C, pH 5.0-9.5 and 0-7.0 % (w/v) NaCl, the growth of strain BEI207 T occurred at 10-37 °C, pH 6.0-9.5 and 1.0-7.0 % (w/v) NaCl. Both strains shared the same major fatty acid components of summed feature 3 (C 16 : 1 ω7 c or C 16 : 1 ω6 c ), C 16 : 0 and summed feature 8 (C 18 : 1 ω6 c or C 18 : 1 ω7 c ). The DNA G+C contents of the assembled genomic sequences were 44.73 and 45.06 mol% for strains BEI176 T and BEI207 T , respectively. Average nucleotide identity values between the two strains and their reference species were lower than the threshold for species delineation (95-96 %); in silico DNA-DNA hybridization further showed that the two strains had less than 70 % similarity to their relatives. Therefore, two novel Vibrio species are proposed to accommodate them: Vibrioouci sp. nov. (type strain, BEI176 T =MCCC 1K03515 T =JCM 32690 T = KCTC 62616 T ) and Vibrioaquaticus sp. nov. (type strain, BEI207 T =MCCC 1K03516 T =JCM 32691 T =KCTC 62617 T ).

Two novel Vibrio phages, LP.1 and LP.2 that infected Vibrio maritimus R-40493, were isolated from surface seawater in Qingdao coastal area by the double-agar layer method. Morphological analysis by transmission electron microscope showed that the two phages displayed head-tail structures with icosahedral heads of 62.37 and 54.00 nm in diameter and long non-contractile tails of 119.00 and 105.20 nm in length, respectively, and can be grouped into the Siphoviridae family. Thermal and pH sensitivity tests exhibited that LP.1 was stable at temperature ranging from - 20 to 65 °C and at pH ranging from 5 to 12, and LP.2 showed vitality over a wider range of temperature (- 20-75 °C) and pH (3-12). Both LP.1 and LP.2 contained linear and double-stranded DNA genomes with a length of 46,791-bp and 37,128-bp, respectively. The genome of both phages can be classified into four functional groups, including DNA replication and regulation, phage packaging, phage structure, and additional function. The bioinformatic analysis demonstrated that the Vibrio phages LP.1 and LP.2 are novel phages. By conducting morphological, biochemical, and genomic analysis, our study provides useful information for further research on the interaction between Vibrio phages and their host.

Vibrio spp. are ubiquitous marine bacteria with high metabolism flexibility and genome plasticity. Previous studies have revealed the dynamics of planktonic vibrios in relation to environmental forces, such as temperature and salinity. However, little is known about Vibrio ecology in benthic environments. Here, we elucidate the abundance, diversity, and spatial distribution of Vibrio spp. in sediments of the Chinese marginal seas, with a wide spatial range from north to south covering the Yellow Sea (YS), East China Sea (ECS), and South China Sea (SCS). Quantitative analysis showed that Vibrio spp. were most abundant in the SCS (∼9.04 × 10 5 copies/g) compared to the YS (∼1.00 × 10 5 copies/g) and ECS (∼8.86 × 10 5 copies/g). Vibrio community compositions derived from Illumina sequencing of Vibrio -specific 16S rRNA genes varied significantly between sampling areas, which was reflected by a strong distance-decay pattern. The spatial distribution of Vibrio was governed by a joint effect of spatial and environmental factors (especially temperature, salinity, and SiO 3 ), and their respective pure effects explained only a small fraction of the community variation. Moreover, we identified the most prominent operational taxonomic units (OTUs) that were partitioned in these sea areas. Whereas 2- ), and their respective pure effects explained only a small fraction of the community variation. Moreover, we identified the most prominent operational taxonomic units (OTUs) that were partitioned in these sea areas. Whereas Vibrionaceae OTU20 and Photobacterium lipolyticum were prevalent in the YS, Vibrio gigantis and Photobacterium piscicola , and P. piscicola , Photobacterium lutimaris were prevalent in the ECS and SCS, respectively. Our study demonstrated clear spatial heterogeneity of Photobacterium alginatilyticum spp. in sediments of the Chinese marginal seas, laying a foundation for fully understanding the marine Vibrio ecology and the ecological roles of the species. Vibrio is an important component of natural marine microbial populations in terms of pathogenicity and roles in carbon cycling. Compared to the marine pelagic environment, our knowledge of the diversity and distribution pattern of IMPORTANCE Vibrio abundance in Chinese marginal seas than in other studied sediments. There was a clear spatial differentiation of Vibrio spp. in sediment is limited. Here, we show higher Vibrio community displayed a strong distance-decay pattern across a wide spatial range, which was formed under the combined effects of spatial and environmental factors. These results provide deep insights into the ecological dynamics of Vibrio and its environmental controls, facilitating a more comprehensive understanding of the marine Vibrio community displayed a strong distance-decay pattern across a wide spatial range, which was formed under the combined effects of spatial and environmental factors. These results provide deep insights into the ecological dynamics of Vibrio and its environmental controls, facilitating a more comprehensive understanding of the marine Vibrio ecology., (Copyright © 2019 American Society for Microbiology.)

Vibrio species are associated with human health and play important roles in the carbon cycle. The interest in the Vibrio ecology in marine pelagic environments has increased in recent years, and the correlations between the Vibrio community structure and various environmental factors have been demonstrated. However, the identification of planktonic Vibrio species and their relationship with particulate matter are unclear. Here, we elucidated the spatiotemporal dynamics of Vibrio diversity and in relation to environmental factors in the northern Chinese marginal seas, which feature complex and ever-changing environmental conditions. Vibrio abundance derived from quantitative PCR analysis was higher in summer (∼1.4 × 10 6 copies liter -1 ) than in winter (∼1.9 × 10 5 copies liter -1 ). Interestingly, the average amount of free-living (on a 0.22-μm-pore-size filter membrane) Vibrio was higher (∼3.89 times) than that of particle-associated Vibrio (on a 3-μm-pore-size filter membrane), making it likely that the preferential lifestyle of the planktonic Vibrio community was free living. Shifts in Vibrio community composition identified by high-throughput amplicon sequencing of the Vibrio -specific 16S rRNA gene were observed at both spatial and temporal scales, which were mainly driven by temperature, dissolved oxygen, ammonium, salinity, nitrite, and phosphate. The most prominent operational taxonomic units in summer were closely related to Vibrio campbellii and Vibrio caribbeanicus and shifted to those affiliated with Vibrio atlanticus in winter. Our study demonstrated abundant and diverse Vibrio populations in the northern Chinese marginal seas, contributing to a better understanding of their potential ecological roles in these ecosystems. IMPORTANCE The dynamics of Vibrio communities have been shown in many marine habitats that are close to land, including estuary or harbor areas. Here, we investigated the spatiotemporal dynamics of Vibrio populations in the northern Chinese marginal seas, spanning a wide spatial scale. We showed that the abundances of the Vibrio population in the present study were higher than those in most previously studied areas and that Vibrio species are more likely to adopt a free-living lifestyle. Moreover, our results expanded upon previous results by showing a clear shift in the dominant Vibrio species from summer to winter, which was mainly attributable to the reduction in the abundance of dominant species in summer. Overall, this work contributes to the understanding of the ecology of the Vibrio communities in the marginal seas., (Copyright © 2019 American Society for Microbiology.)

Noncatalytic carbohydrate binding modules (CBMs) have been demonstrated to play various roles with cognate catalytic domains. However, for polysaccharide lyases (PLs), the roles of CBMs remain mostly unknown. AlyB is a multidomain alginate lyase that contains CBM32 and a PL7 catalytic domain. The AlyB structure determined herein reveals a noncanonical alpha helix linker between CBM32 and the catalytic domain. More interestingly, CBM32 and the linker does not significantly enhance the catalytic activity but rather specifies that trisaccharides are predominant in the degradation products. Detailed mutagenesis, biochemical and cocrystallization analyses show "weak but important" CBM32 interactions with alginate oligosaccharides. In combination with molecular modeling, we propose that the CBM32 domain serves as a "pivot point" during the trisaccharide release process. Collectively, this work demonstrates a novel role of CBMs in the activity of the appended PL domain and provides a new avenue for the well-defined generation of alginate oligosaccharides by taking advantage of associated CBMs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Alginate lyases are enzymes that degrade alginate into oligosaccharides which possess a variety of biological activities. Discovering and characterizing novel alginate lyases has great significance for industrial and medical applications. In this study, we reported a novel alginate lyase, AlyA-OU02, derived from the marine Vibrio splendidus OU02. The BLASTP searches showed that AlyA-OU02 belonged to polysaccharide lyase family 7 (PL7) and contained two consecutive PL7 domains, which was rare among the alginate lyases in PL7 family. Both the two domains, AlyA a and AlyA b , had lyase activities, while AlyA a exhibited polyM preference, and AlyA b was polyG-preferred. In addition, the enzyme activity of AlyA a was much higher than AlyA b at 25 °C. The full-length enzyme of AlyA-OU02 showed polyM preference, which was the same as AlyA a . AlyA a degraded alginate into di-, tri-, and tetra-alginate oligosaccharides, while AlyA b degraded alginate into tri-, tetra-, and penta-alginate oligosaccharides. The degraded products of AlyA-OU02 were similar to AlyA a . Our work provided a potential candidate in the application of alginate oligosaccharide production and the characterization of the two domains might provide insights into the use of alginate of this organism.

Background: Vibrios are among the most diverse and ecologically important marine bacteria, which have evolved many characteristics and lifestyles to occupy various niches. The relationship between genome features and environmental adaptation strategies is an essential part for understanding the ecological functions of vibrios in the marine system. The advent of complete genome sequencing technology has provided an important method of examining the genetic characteristics of vibrios on the genomic level., Results: Two Vibrio genomes were sequenced and found to occupy many unique orthologues families which absent from the previously genes pool of the complete genomes of vibrios. Comparative genomics analysis found vibrios encompass a steady core-genome and tremendous pan-genome with substantial gene gain and horizontal gene transfer events in the evolutionary history. Evolutionary analysis based on the core-genome tree suggested that V. fischeri emerged ~ 385 million years ago, along with the occurrence of cephalopods and the flourish of fish. The relatively large genomes, the high number of 16S rRNA gene copies, and the presence of R-M systems and CRISPR system help vibrios live in various marine environments. Chitin-degrading related genes are carried in nearly all the Vibrio genomes. The number of chitinase genes in vibrios has been extremely expanded compared to which in the most recent ancestor of the genus. The chitinase A genes were estimated to have evolved along with the genus, and have undergone significant purifying selective force to conserve the ancestral state., Conclusions: Vibrios have experienced extremely genome expansion events during their evolutionary history, allowing them to develop various functions to spread globally. Despite their close phylogenetic relationships, vibrios were found to have a tremendous pan-genome with a steady core-genome, which indicates the highly plastic genome of the genus. Additionally, the existence of various chitin-degrading related genes and the expansion of chitinase A in the genus demonstrate the importance of the chitin utilization for vibrios. Defensive systems in the Vibrio genomes may protect them from the invasion of external DNA. These genomic features investigated here provide a better knowledge of how the evolutionary process has forged Vibrio genomes to occupy various niches.

The type VI secretion system (T6SS) was recently shown to modulate quorum sensing and the stress response in Vibrio anguillarum serotype O1 strain NB10. It is not known whether there is a functionally active T6SS in other serotypes of V. anguillarum. Here, homologues to T6SS cluster VtsEFGH and hemolysin-coregulated protein (Hcp)-encoding genes were found to be prevalent and conserved in clinical isolates of V. anguillarum from fish, including four O1 and five non-O1 serotype strains. Unexpectedly, only the non-O1 serotype strains expressed VtsEFGH and Hcp under laboratory and marine-like conditions, in contrast to the serotype O1 strains. This suggested that the V. anguillarum non-O1 serotype strains tested have constitutive expression of T6SS. Examination of a representative non-O1 strain, MHK3, showed that Hcp production was growth phase dependent and that maximum Hcp production was observed in the exponential growth phase. Moreover, Hcp production by MHK3 was most active under warm marine-like conditions. Further examination revealed a correlation of the constitutive expression of T6SS with bactericidal activity against Escherichia coli and Edwardsiella tarda. The work presented here suggests that the constitutive expression of T6SS provides V. anguillarum with advantage in microbial competition in marine environments.

Tumor necrosis factors receptors (TNFRs) comprise a superfamily of proteins characterized by a unique cysteine-rich domain (CRD) and play important roles in diverse physiological and pathological processes in the innate immune system, including inflammation, apoptosis, autoimmunity and organogenesis. Although significant effects of TNFRs on immunity have been reported in most vertebrates as well as some invertebrates, the complete TNFR superfamily has not been systematically characterized in scallops. In this study, two different types of TNFR-like genes, including PyTNFR1 and PyTNFR2 genes were identified from Yesso scallop (Patinopecten yessoensis, Jay, 1857) through whole-genome scanning. Phylogenetic and protein structural analyses were carried out to determine the identities and evolutionary relationships of the two genes. The expression profiling of PyTNFRs was performed at different development stages, in healthy adult tissues and in hemocytes after bacterial infection and heat stress. Expression analysis revealed that both PyTNFRs were significantly induced during the acute phase (3 h) after infection with Gram-positive (Micrococcus luteus) and Gram-negative (Vibrio anguillarum) bacteria, though much more dramatic chronic-phase (24 h) changes were observed after V. anguillarum challenge. For heat stress, only PyTNFR2 displayed significant elevation at 12 h and 24 h, which suggests a functional difference in the two PyTNFRs. Collectively, this study provides novel insight into the PyTNFRs and the specific role and response of TNFR-involved pathways in host immune responses against different bacterial pathogens and heat stress in bivalves., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Phage H188, a novel Vibrio kanaloae phage, was isolated from the surface water of Yellow Sea. Morphological analysis by transmission electron microscopy reveals that it belongs to the family Myoviridae. Present result suggests that the phage is stable at pH between 4.0 and 12.0. No significant difference in phage titers is noted at temperature 30-50 °C. A latent period of approximately 96 mins is indicated by the one-step growth curve. And, the burst size is about three virions per cell. Furthermore, genomic analysis of H188 reveals a genome size of 50364 bp with 43.63 % G+C content, and 76 putative open reading frames. There is no obvious similarity between H188 and other known phages by genomic comparison. Moreover, the H188 genome includes modules for phage structure, phage packaging, DNA replication and regulation, and some additional functions.

The tumor necrosis factor (TNF) receptor associated factors (TRAFs) are the major signal transducers for the TNF receptor superfamily and the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily, which regulate a variety of cellular activities and innate immune responses. TRAF genes have been extensively studied in various species, including vertebrates and invertebrates. However, as one of the key component of NF-κB pathway, TRAF genes have not been systematically characterized in marine invertebrates. In this study, we identified and characterized five TRAF genes, PyTRAF2, PyTRAF3, PyTRAF4, PyTRAF6 and PyTRAF7, in the Yesso scallop (Patinopecten yessoensis). Phylogenetic and protein structural analyses were conducted to determine their identities and evolutionary relationships. In comparison with the TRAF genes from vertebrate species, the structural features were all relatively conserved in the PyTRAF genes. To gain insights into the roles of TRAF genes during scallop innate immune responses, quantitative real-time PCR was used to investigate the expression profiles in the different stages of scallop development, in the healthy adult tissues, and in the hemocytes after bacterial infection with Micrococcus luteus and Vibrio anguillarum. Based on the qRT-PCR analysis, the expression of most of the PyTRAFs was significantly induced in the acute phases (3-6 h) after infection with Gram-positive (M. luteus) and Gram-negative (V. anguillarum) bacteria, and many more dramatic changes in PyTRAFs expression were observed after V. anguillarum challenge. Notably, the strong response in the up-regulation of PyTRAF6 post-bacterial challenge was distinct from that previously reported in scallops and crabs but was similar to that of other shellfish, Echinodermata and even teleost fish. The high level expressions of PyTRAFs in the hemocytes and the gill, and their specific expression patterns after challenges provide insights into the versatile roles and responses of TRAFs in the innate immune system against Gram-negative bacterial pathogens in bivalves., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Mitogen-activated protein kinase kinases (MKK) are the essential components of the evolutionarily conserved MAPK signaling cascade, which regulates a variety of cellular activities and innate immune responses. Although MKK genes have been extensively studied in various vertebrate and invertebrate species, they have not been systematically characterized in bivalves. In this study, we identified and characterized five MKK genes (PyMKK1/2, PyMKK4, PyMKK5, PyMKK3/6 and PyMKK7) in the Yesso scallop (Patinopecten yessoensis). Phylogenetic and protein structural analyses were conducted to determine their identities and evolutionary relationships. To gain insights into the possible roles of MKK genes during scallop innate immune responses, quantitative real-time PCR (qRT-PCR) was used to investigate their expression profiles during different developmental stages in samples taken from healthy adult tissues and hemocytes after Micrococcus luteus and Vibrio anguillarum bacterial infections. The Yesso scallop MKKs (PyMKKs) were found to have highly conserved structural features compared to the MKK genes from other invertebrate species. Using qRT-PCR analysis, three distinct expression patterns were detected among the PyMKKs over the course of ten different developmental stages. In adult scallops, the majority of the PyMKKs were highly expressed in mantle, gill, muscle and hemocytes. The differential expression patterns of the five PyMKKs after M. luteus (Gram-positive) and V. anguillarum (Gram-negative) bacterial infections suggested their possible involvement in the innate immune response and provide the foundation and resource for the further study on innate immune response of MAPK signal pathway in mollusk., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

As a primary iron storage protein, ferritin plays a vital role in iron homeostasis and innate immunity. In this study, four ferritin subunits (PyFer1, PyFer2, PyFer3, and PyFer4) were cloned from the Yesso scallop, Patinopecten yessoensis, by rapid amplification of cDNA ends (RACE) following in silico transcriptome analysis. The full-length cDNAs of the four ferritins are 895, 920, 891, and 1400 bp in length, respectively, and each contains a putative iron response element (IRE) in its 5' UTR. Meanwhile, multiple A+U-destabilizing elements (TATT or ATTTA) are present in the 3' UTRs of PyFer2 and PyFer4. The open reading frames of the four ferritins are 522, 516, 516, and 519 bp, encoding 173, 171, 171, and 172 amino acids, respectively. These proteins have typical ferritin structures, with four long α-helices, one short α-helix and an L-loop. All of the predicted proteins possess both the ferroxidase center of mammalian H ferritins (E25, Y32, E59, E60, H63, E105, and Q139) and the iron nucleation site of mammalian L ferritins (H116, D129, and E132), and the recombinant proteins possess apparent ferroxidase activity. Quantitative real-time PCR analysis revealed that the expression of the four PyFers was significantly elevated at the D-shaped stage and was relatively high in the adult mantle and hepatopancreas. Furthermore, the four PyFers were significantly up-regulated by iron or bacterial challenge, and all four purified recombinant PyFers were able to inhibit the growth of the scallop pathogen Vibrio anguillarum. These results suggest that these PyFers are likely to play important roles in many fundamental biological processes in P. yessoensis, including immune defense, iron homeostasis, and shell development., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Aims: The main aims of this study were to clone and express a new outer membrane protein U (OmpU) from a pathogenic Vibrio harveyi SF-1 and investigate its immune efficiency as a vaccine candidate against V. harveyi infection in turbot (Scophthalmus maximus)., Methods and Results: In this study, a new gene, ompU was cloned from the genomic DNA of pathogenic V. harveyi SF-1. The ompU gene encoded a 35 kDa protein, which was purified by Ni-NTA His-Bind Resin column. A DNA vaccine was constructed by inserting ompU gene into pEGFP-N1 plasmid. Turbot were injected intramuscularly with the purified OmpU protein and the recombinant pEGFP-N1/ompU plasmid, respectively. The fish vaccinated with the purified OmpU protein were completely protected with a relative per cent of survival (RPS) of 100% against pathogenic V. harveyi infection. Efficient protection was also found in the pEGFP-N1/ompU vaccinated group, with a RPS of 51.4%. Significant specific antibody responses were detected in the vaccinated turbot by indirect enzyme-linked immunosorbent assay., Conclusions:  A new OmpU was cloned and expressed. Both OmpU protein vaccine and DNA vaccine showed good immune protections in turbot., Significance and Impact of the Study:  The OmpU was identified to be a new effective vaccine candidate and could be used as subunit vaccine and DNA vaccine for disease control caused by pathogenic V. harveyi., (© 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.)

Vibrio anguillarum, a halophilic Gram-negative bacterium, is the causative agent of vibriosis, which is a major problem for the aquaculture industry worldwide. Previously, a virulence-related gene fragment of V. anguillarum was obtained from a suppression subtractive hybridization (SSH) library. In this study, the complete gene sequence was obtained by long and accurate PCR (LA-PCR). After sequence analysis and homologous comparison, this new virulence-related gene was revealed to encode a putative membrane-bound lytic murein transglycosylase D (MltD), which consisted of 547 amino acids, and showed 34% identity to the MltD in Escherichia coli. An mltD mutant of pathogenic V. anguillarum CW-1 was constructed by homologous recombination. Production of extracellular gelatinase and protease of the mltD mutant decreased markedly compared with those of the wild-type strain, and the hemolytic activity was totally lost. Sodium chloride challenge and antibiotic sensitivity assay showed that the resistance of the mltD mutant to high concentrations of sodium chloride, and rocephin, fortun, cefobid, gentamicin, kanamycin and carbenicillin was enhanced. Most importantly, virulence of the mltD mutant was enhanced compared with that of the wild type when it was inoculated intraperitoneally into zebra fish; the LD₅₀ of the wild type and the mutant was 3.92 × 10³ CFU and 1.01 × 10² CFU fish⁻¹, respectively. The mltD was cloned and overexpressed in E. coli, and the recombinant MltD protein showed hemolytic, phospholipase, gelatinase and diastase activities. This is the first report that MltD possibly has a virulence-related function., (Copyright © 2010 Institut Pasteur. Published by Elsevier SAS. All rights reserved.)

Aims: The aim was to characterize the viable but nonculturable (VBNC) state of Vibrio cincinnatiensis and its resuscitation., Methods and Results: Vibrio cincinnatiensis VIB287 was cultured in sterilized seawater microcosms at 4 degrees C. Plate counts, direct viable counts and total counts were used. A large population of the V. cincinnatiensis became nonculturable after approx. 50 day at 4 degrees C. Electron microscopy revealed that the VBNC cells changed from rod to coccoid and decreased in size. Resuscitation of VBNC cells was achieved by temperature upshift in nutrition of yeast extract and peptone by addition of catalase or compound vitamin B. The VBNC and resuscitative cells were intraperitoneally injected into zebra fish separately. No death was observed in the group inoculated with the VBNC cells., Conclusions: Vibrio cincinnatiensis VIB287 could enter VBNC state in adverse environments. Resuscitation of VBNC cells occurred by addition of compound vitamin B or catalase to VBNC cells containing nutrient. The resuscitative cells might retain their pathogenicity., Significance and Impact of the Study: The study confirmed that V. cincinnatiensis could enter into VBNC state in seawater at low temperature and resuscitated. The resuscitative cells retained their pathogenicity, which may be important in future studies of ecology of V. cincinnatiensis.

Vibrio harveyi has been reported to enter into a viable but nonculturable (VBNC) state. Two clinical V. harveyi strains, SF1 and CW2, and the type strain, VIB295(T), were incubated in sterilized seawater at 4 degrees C. Plate counts in these strains declined to undetectable levels (<0.1 CFU mL(-1)) within 69, 67 and 65 days, respectively. The direct viable count (DVC) declined from 10(6) to 10(4) active cells mL(-1) and remained constant at this level by a DVC. VBNC cell numbers could be restored via a temperature upshift when grown in yeast extract with the addition of Tween 20 or compound vitamin B. Reverse transcriptase-PCR was used to monitor virulence gene expression within VBNC cells. No expression of the hemolysin gene was detected in VBNC cells. VBNC and resuscitative cells were intraperitoneally injected into zebra fish separately. No death was observed in the groups inoculated with VBNC cells. The fish inoculated with the resuscitative cells died within 7 days, the lethal dose 50% (LD(50)) being 2.85 x 10(4) CFU mL(-1), a value similar to that for groups inoculated with normal cells (2.28 x 10(4) CFU mL(-1)). This suggested that VBNC V. harveyi might retain pathogenic potential.

The extracellular zinc metalloprotease (EmpA) is a putative pathogenic factor involved in the invasive process of the fish pathogen Vibrio anguillarum. It is synthesized as a 611-amino-acid preproprotease. The gene encoding EmpA (empA) has already been cloned and sequenced. In this study, empA was inserted into pET24d(+) and expressed in Escherichia coli BL21(DE3). Recombinant EmpA with His-tag was purified in a single step with a His-binding Ni-affinity column to a purity >95%. In addition, proteolytic activity, cytotoxicity, fish pathogenicity, and solubility of the recombinant protein were determined.

The extracellular zinc metalloprotease, EmpA, is a putative virulence factor involved in pathogenicity of the fish pathogen Vibrio anguillarum. The 611-amino acid precursor of this enzyme is encoded by the empA gene. The residues His346, His350, Glu370, Glu347, His429, Tyr361 and Asp417 are highly conserved and putatively function together at the active site of the enzyme. In this study, empA was inserted into pET24d(+) and expressed in Escherichia coli strain BL21(DE3) as a 6 x His tagged protein (r-EmpA). All the conserved residues of EmpA mentioned above were individually mutated by site-directed mutagenesis and the mutants were also expressed (m-r-EmpAs). r-EmpA and m-r-EmpAs were purified, and assayed for their proteolytic activities with azocasein as the substrate and cytotoxicities on a flounder gill cell line. m-r-EmpAs that had been mutated at His346, His350, Glu370 and Glu347 almost completely lost their proteolytic activity and cytotoxicity, pointing towards the essential roles played by these residues. In contrast, those mutated at Tyr361, His429 and Asp417 still retained a partial proteolytic activity and cytotoxicity. Our results indicate that these conserved residues play important roles in enzymatic activity and that the proteolytic activity of the enzyme is involved in the pathogenesis of V. anguillarum

The taxonomic position of the fluorescent amplified fragment length polymorphism fingerprinting groups A46 (five isolates), A51 (six isolates), A52 (five isolates) and A53 (seven isolates) obtained in a previous study were further analysed through a polyphasic approach. The 23 isolates were phylogenetically related to Vibrio splendidus, but DNA-DNA hybridization experiments proved that they belong to three novel species. Chemotaxonomic and phenotypic analyses further disclosed several features that differentiate between the 23 isolates and known Vibrio species. The names Vibrio kanaloae sp. nov. (type strain LMG 20539(T) = CAIM 485(T); EMBL accession no. AJ316193; G + C content 44.7 mol%), Vibrio pomeroyi sp. nov. (type strain LMG 20537(T) = CAIM 578(T); EMBL accession no. AJ491290; G +C content 44.1 mol%) and Vibrio chagasii sp. nov. (type strain LMG 21353(T) = CAIM 431(T); EMBL accession no. AJ316199; G + C content 44.6 mol%) are respectively proposed to encompass the five isolates of A46, the six isolates of A51 and the 12 isolates of A52/A53. The three novel species can be distinguished from known Vibrio species by several phenotypic features, including utilization and fermentation of various carbon sources, beta-galactosidase activity and fatty acid content (particularly of 12 : 0, 14: 0, 14 : 0 iso and 16 : 0 iso).

The fluorescent amplified fragment length polymorphism (FAFLP) groups A5 (21 isolates), A8 (6 isolates) and A23 (3 isolates) distinguished in an earlier paper (Thompson et al., Syst Appl Microbiol 24, 520-538, 2001) were examined in more depth. These three groups were phylogenetically related to Vibrio tubiashii, but DNA-DNA hybridization experiments proved that the three AFLP groups are in fact novel species. Chemotaxonomic and phenotypic analyses further revealed several differences among the 30 isolates and known Vibrio species. It is proposed to accommodate these isolates in three novel species, namely Vibrio neptunius (type strain LMG 20536T; EMBL accession no. AJ316171; G +C content of the type strain 46.0 mol%), Vibrio brasiliensis (type strain LMG 20546T; EMBL accession no. AJ316172; G + C content of the type strain 45.9 mol%) and Vibrio xuii (type strain LMG 21346T; EMBL accession no. AJ316181; G +C content of the type strain 46.6 mol%). These species can be differentiated on the basis of phenotypic features, including fatty acid composition (particularly 14:0 iso, 14:0 iso 3-OH, 16:0 iso, 16:0, 17:0 and 17:1 omega8c), enzyme activities and utilization and fermentation of various carbon sources.