Analysis Of Symbiotic Microbial Diversity Of Styela Clava And Hemicentrotus Pulcherrimus And Classification And Identification Of Three Marine Bacteria

Associated bacteria were isolated from Styela clava and Hemicentrotus pulcherrimus collected from the coast of Weihai. They were cultured by using conventional culture-dependent method and investigated by using phylogenetic analysis based on16S rRNA gene sequence comparisons.78and113isolates were from the sample on marine agar2216(Difco), respectively.Based on morphological characters and weather growing on TCBS agar, we selected30strains from Styela clava and31strains from the Hemicentrotus pulcherrimus for molecular phylogenetic study using16S rRNA gene sequences. The results showed that30isolates from Styela clava were members of thirteen genera of thirteen families in four major phylogenetic groups(Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacterid). The lower similarity of16S rRNA gene sequence suggested that at least5strains (HQW7-. HQYD1、HQWD4、HQE1、HQE2) represented new species.31isolates from Hemicentrotus pulcherrimus were members of fourteen genera of thirteen families in four major phylogenetic groups (Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacterid). The isolates HD4、HD13、YD24may represent new species. Gamma-Proteobacteria bacteria were the dominant strains of with more than half of the associated. The results presented above showed there was abundant species diversity and phylogenetic diversity of bacteria isolated from Styela clava and Hemicentrotus pulcherrimus.During our study of the microbial diversity associated with Hemicentrotus pulcherrimus, a novel strain HD4T was isolated from its coelomic fluid, which is related to the genus Salegentibacter. The morphological, physiological, biochemical characteristics study showed that HD4was Gram-negative, strictly aerobic, heterotrophic, non-motile and formed yellow-pigmented colonies on marine agar2216(Difco). Optimum growth of the strain was observed at28-30℃and pH6.8-7.3, with3-5%(w/v) NaCl. The DNA (G+C) mol%content of strain HD4T was37.0mol%. Strain HD4T possessed iso-C15:0(12.44%), iso-C17:03-OH (12.08%) and summed feature3(comprising iso-C15:02OH/C16:1ω7c;22.63%) as the major cellular fatty acids. Based on the study, strain HD4T should be classified as a novel species within the genus Salegentibacter, for which the name Salegentibacter echinurum sp. nov. is proposed. The type strain is HD4T (=CICC10466T=NRRL B-59666T). The GenBank accession number for the16S rRNA gene sequence of Salegentibacter echinorum HD4T is JN040280.According to the same methods for identification, we studied the strain J221and H5. The strain J221was Gram-negative, oxidase-positive, aerobic, rod-shaped cell and motile with one to several polar flagella. Optimum growth of strain J221T was observed at25-28℃and pH7.5-8.0with2-3%(w/v) NaCl. The dominant fatty acids were summed feature3(comprising C16:1ω7c and/or C16:1ω6c), C16:0and C18:1ω7c The DNA (G+C) mol%content was46.8mol%. Phylogenetic analysis based on the16S rRNA gene sequences showed that strain J221T was a member of the Gamma-Proteobacteria. It formed a distinct phyletic line with less than91%sequence identity to any species within previously recognized genera. On the basis of the study, strain J221T should be classified in a novel genus and species, for which the name Neiella marinum gen. nov. sp. nov. is proposed, with strain J221T (=CGMCC1.10130T=NRRL B-513191) as the type strain. The GenBank accession number for the16S rRNA gene sequence of Neiella marinum J221T is EU513001.Strain H5T was Gram-positive, non-motile, straight or curved rods. Optimum growth occurred in the temperature range28-30℃and pH range7.5-8.0with0-1%(w/v) NaCl. Strain H5T had MK-9(14%), MK-9(H2)(9%) and MK-9(H4)(67%) as the major menaquinone and C18:1ω9c, C16:0, C14:0, C18:0and C16.1ω9c as major fatty acids. The cell-wall peptidoglycan type was A5alpha (L-Lys-L-Ala-L-Lys-D-Glu). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside. The genomic DNA (G+C) mol%content was61.8mol%. Phylogenetic analysis based on16S rRNA gene homo logy showed that strain H5T represents a new lineage in the family Actinomycetaceae. On the basis of the phenotypic, physiological and molecular characteristics of the novel isolates, it is proposed that it should be classified in a novel genus:Flaviflexus huanghaiensis gen. nov. sp. nov. with H5T (=DSM24315T=CICC10486T) as the type strain.The GenBank accession number for the16S rRNA gene sequence of Flaviflexus huanghaiensis H5T is JN815236.