Diversity Of Cultivable Bacteria In The Yellow Sea Cold Water Mass And Taxonomic Analysis Of Two Novel Marine Bacteria

The Yellow Sea Cold Water Mass (YSCWM) is an important phenomenon in the shelf sea of China. It is prominent in summer and autumn, and renewing in winter every year. The study of YSCWM in the past focused on physical oceanography but rarely on microbiology. Microorganisms play a major role in the biogeochemical cycling. However, phylogenetic diversity of cultivable bacteria in this area has not been reported yet.Phylogenetic diversity of cultivable bacteria from the inter of YSCWM area was studied in July 2008, and was compared with that from the exterior of YSCWM area. Sea water sample was collected in five stations 3500-5,3600-6, 3600-8,3400-6 and 3800-2 from the inter of YSCWM, and two stations 3200-3 and 3850-5 from the exterior of YSCWM. From the colony count method with 2216E plates, the abundance of the heterotrophic bacteria of sea water was 0.56×103～2.2×103cfu/ml; according to the AODC and DVC results, the total bacterial number and the total number of viable cells of the marine water samples were 1.3×105～4.8×105/ml and 6.0×104～1.6×105/ml, respectively.475 bacterial strains were isolated from 2216E plates, and the PCR-ARDRA technique was performed on these strains for cluster analysis, with fragments of 16S rDNA digested by restriction enzyme HhaⅠ. Samples from the inter of YSCWM stations showed as many RFLP patterns as those from exterior of YSCWM. The 16S rDNA of all major RFLP patterns were sequenced. Phylogenetic analysis of 114 bacterial strains showed that bacterial phylotypes from the inter of YSCWM were made up of four phyla bacteria, i.e. Proteobacteria, Actinobacteria, Firmicutes and Flavobacteria, including 24 genera and from the exterior were made up of the same four taxon, including 15 genera.α-,β- andγ- Proteobacteria subdivisions of the Proteobacteria were discovered from the inter of YSCWM. Howeverβ-Proteobacteria was not discovered from the exterior of YSCWM.γ-Proteobacteria represented the vast majority of bacterial isolates at every depth. By comparing,α-Proteobacteria represented the higher proportion with percentage of 26.8% inter of YSCWM than the proportion of exterior of YSCWM with percentage of 15.6%. It indicated that samples from the inter of YSCWM and the exterior of YSCWM showed the same high bacterial diversity, but bacterial community composition, as well as the predominant bacterial groups from the inter of YSCWM was different from the exterior of YSCWM.This study also aimed to identify two novel marine bacteria discovered in YSCWM and in Qingdao offshore during the massive green algae bloom. After sequencing the 16S rDNA, the sequences was blasted in Genbank database. Two strains WH134T and ZXM137 T were found to have low 16S rDNA sequences similarity to V. rumoiensis DSM 19141T and Jannaschia pohangensis with 96.6% and 95.3%. Further identification had been made by using classical morphologic identification methods including Gram-staining, flagella-staining, TEM, tranditional physiological and biochemical methods (such as temperature, pH and salty for growth, drug sensitivity, amylase activity, etc.), GC content, polar lipid composition, and the rapid identification systems, i.e. API 20E, API 20NE, API 50CH, API ZYM and BIOLOG. WH134Twas Gram-negative, cells of strain WH134T were slightly curved rods that were motile by means of a polar flagellum, facultatively anaerobic bacterial strain, negative for luminescence and swarming. The G+C content was 42.5 mol%, within the range of Vibrio 40～50%. Phylogenetic analysis based on gyrB, topA, recA, ftsZ, mreB, gap A and rpoA gene sequences revealed that strain WH134T belongs to the genus Vibrio, and showed similarities of 75.7,74.6,83.6,78.9,82.9,86.0 and 89.4% with the most closely relative Vibrio rumoiensis DSM 19141T. On the basis of phenotypic, phylogenetic and genetic evidence, strain WH134T should be classified as a novel Vibrio species, for which the name Vibrio marisflavi sp. nov. is proposed, with the type strain WH134T(=CGMCC 1.8994T=LMG 25284T=DSM 23086T).ZXM137T was isolated from seawater collected from a coastal region of Qingdao, P.R. China, when the massive green algae (Enteromorpha prolifera) bloomed. According to the same methods for identification, strain ZXM137T is Gram-negative, non-motile bacterium, strictly aerobic. The cells aggregated together when incubated at 28℃for more than 24h in marine 2216E broth. Growth occurred with 11%(w/v) NaCl. The major cellular fatty acid of strain ZXM137T was C18:1ω7c The G+C content of DNA was 60.1 mol%. It contained Q-10 as the predominant ubiquinone and the major polar lipids were phosphatidylglycerol, unidentified aminolipid and lipid. A phylogenetic analysis based on 16S rRNA gene sequences indicated that strain ZXM137T formed a distinct evolutionary lineage within the family Rhodobacteraceae of the phylum a-Proteobacteria. On the basis of phenotypic, chemotaxonomic and phylogenetic evidences, strain ZXM137T was considered to represent a novel genus and species, for which the name Huaishuia halophilus gen. nov. sp. nov. was proposed. The type strain was ZXM137T(=CGMCC 1.8891T= LMG 24854 T).