Diversity Of Cultivable Denitrifying Bacteria Isolated From Caroline Seamount In The Western Pacific And Taxonomic Analysis Of Four Novel Marine Bacteria

Seamounts have gradually become an important part of marine ecosystem due to their unique topographic structures and special hydrological environment.Seamounts provide an ideal“natural laboratory”for the study of the distribution,evolution and ecology of marine microorganisms,but the research on the diversity of denitrifying bacteria in seamount area is still limited.In this study,microbial diversity of denitrifying bacteria from seawater and one biological sample(sponge)collected from the Caroline Seamount in the Western Pacific Ocean was examined by culture-dependent method and 16S r RNA gene sequence analysis.In addition,four potential novel marine bacteria were identified by the method of polyphasic taxonomy.343 denitrifying strains were isolated from water samples collected at 14 stations and 9 denitrifying strains were isolated from one biological sample(sponge).These strains belonged to 3 phyla(Proteobacteria,Bacteroidetes and Actinobacteria),38genera and 62 species.The majority of these strains(331/352;94%)belonged to the phylum Proteobacteria,with a predominance of the two classes Alphaproteobacteria(138/331,42%)and Gammaproteobacteria(193/331,58%).According to the depth of seawater sampling,these denitrifying strains were distributed in different biological zones.There are 162 denitrifying strains in the upper zone(0 m?water depth?200 m),125 denitrifying strains in the middle zone(200 m<water depth?1000 m),51 denitrifying strains in the deep sea zone(1000 m<water depth?4000 m)and 5denitrifying strains in the abyss zone(water depth>4000 m).Among them,Halomonas,Labrenzia,and Thalassospira strains were distributed in all four biological areas,and could even survive in a water depth of 5603 m,indicating that the stains in these three genera have strong adaptability to the marine environment.The results of taxonomic identification of 4 novel marine bacteria were as followings:(1)Strain TP462^T was identified as a novel species of Rheinheimera.Gram-negative,rod-shaped,and motile by means of a single polar flagellum.Growth occurred at 4-37°C(optimum,25-30°C),and with 0-4.0%(w/v)Na Cl(optimum,2-3%).The predominant cellular fatty acids were C_17:1?8c,summed feature 3(composed of iso-C15:0 2-OH and/or C16:1?7c)and C16:0.The polar lipid profile contained phosphatidylglycerol,phosphatidylethanolamine and two unidentified lipids.After 16S r RNA gene sequence alignment,the highest similarity to the published species Rheinheimera tangshanensis JA3-B52^T was 96.8%.The genomic DNA G+C content of strain TP462^T was 48.7 mol%.The name Rheinheimera marina was proposed.(2)Strain S1-10^T was identified as a novel species of Aequorivita.Gram-strain-negative,rod-shaped,facultatively anaerobic,oxidase-negative,catalase-positive,non-motile bacterial strain.The optimum growth temperature was 30-35?;the optimum p H was 9.0;the optimum salt concentration was 1%.The polar lipid profile contained two glycolipids,phosphatidylethanolamine,one phosphoglycolipid,two aminolipids,and three unidentified polar lipids.The main respiratory quinone was menaquinone 6(MK-6).After 16S r RNA gene sequence alignment showed that the highest similarity to Aequorivita published species Aequorivita viscosa 8-1b^T was 97.7%.The genomic DNA G+C content of strain S1-10^T was 34.6 mol%.In addition,the maximum values of in silico DNA-DNA hybridization(is DDH)and average nucleotide identity(ANI)between strain S1-10^T and A.viscosa CGMCC 1.11023^T were 15.4 and 75.7%,respectively.The name Aequorivita sinensis was proposed.In addition,Vitellibacter todarodis and Vitellibacter aquimaris were proposed to be transferred into genus Aequorivita and be named Aequorivita todarodis comb.nov.and Aequorivita aquimaris comb.nov.,respectively.(3)Strain TP390^T was identified as a novel species of Flavobacterium.Gram-stain-negative,rod-shaped,facultative anaerobic,motile bacterial strain.The optimum growth temperature was 25°C;the optimum salt concentration was 1%;the optimum p H was 6.0-7.0.The predominant cellular fatty acids were iso-C_15:0 and iso-C_15:1 G.The main respiratory quinone was menaquinone 6(MK-6)and the polar lipid profile contained one glycolipid,phosphatidylethanolamine,four unknown aminolipids,and six unknown polar lipids.The sequence alignment of 16S r RNA gene showed that the highest similarity with the published species Flavobacterium jejuense EC11^T was97.8%.The genomic DNA G+C content of strain TP390^T was 31.2 mol%.The maximum values of in silico DNA-DNA hybridization(is DDH)and average nucleotide identity(ANI)between strain TP390^T with F.jejuense KCTC 42149^T were 22.60 and80.01%,respectively.The name Flavobacterium profundi was proposed.(4)Strain TP-CH-4^T was identified as a novel genus of Flavobacteriaceae,Gram-negative,non-spore-forming,non-motile,strictly aerobic,on marine agar 2216,colonies are orange-pigmented.Growth occurred at 4-37°C,and in the presence of 1-4%(w/v)Na Cl,Na⁺alone did not support growth;sea salts were required for growth.The predominant cellular fatty acids were iso-C_15:0 and summed feature 1(composed of C_13:0 3-OH and/or iso-C_15:1 H).The main respiratory quinone was menaquinone 6(MK-6).The strain TP-CH-4^T displayed highest sequence similarities with Pseudozobellia thermophila KMM 3531^T(95.1%)and Flagellimonas flava A11^T(93.9%).The genomic DNA G+C content of strain TP-CH-4^T was 44.5 mol%.The name Pelagihabitans pacificus gen.nov.,sp.nov.was proposed.