海洋生物分类与系统演化实验室知识库

海山因其独特的地形构造和特殊的水文环境已经逐渐成为海洋生态系统的重要组成部分。海山为海洋微生物分布、进化和生态学的研究提供了一个理想的“天然实验室”，但是目前对海山区脱氮细菌多样性的研究还是比较有限。本论文通过涂布平板法和16S rRNA基因序列分析，对西太平洋卡罗琳海山区14个站位水体样品和1个生物样品（海绵）中可培养脱氮细菌多样性进行了初步研究。此外，采用多相分类的手段对4株潜在的海洋细菌新种进行分类鉴定。

从卡罗琳海山区14个站位水体样品分离到343株脱氮细菌，从1个生物样品（海绵）中分离到9株脱氮细菌，这些菌株隶属于3个门（变形菌门、放线菌门和拟杆菌门），38个属，62个种。其中变形菌门（Proteobacteria）331株，占分离总菌株的94%，分布在α-变形菌纲（138/331，42%）和γ-变形菌纲（193/331，58%）。根据海水样品采样深度不同，对不同生物区脱氮细菌分布分析，上层区（0 m≤水深≤200 m）162株、中层区（200 m＜水深≤1000 m）125株、深海区（1000 m＜水深≤4000 m）51株、深渊区（水深＞4000 m）5株。其中盐单胞菌属（Halomonas）、拉布伦茨氏菌属（Labrenzia）和海旋菌属（Thalassospira）菌株在四个生物区都有分布，甚至可以在水深5603 m的环境中生存，表明这三个属菌株对海洋环境具有很强的适应性。

对4株海洋新细菌的分类鉴定结果如下：

(1) TP462^T菌株为海默氏菌属（Rheinheimera）的一个新种，属于革兰氏阴性（G^-）菌，杆状，且端生鞭毛，具有运动性，生长温度范围为4-47℃，最适生长温度为25-30℃；生长盐浓度范围为0-4%（w/v）NaCl，最适盐度为2-3%。细胞主要脂肪酸为C_17:1 ω8c、 summed feature 3（包括iso-C_15:0 2-OH和/或C_16:1 ω7c）和C_16:0；极性脂包含磷脂酰甘油（Phosphatidylglycerol，PG）、磷脂酰乙醇胺（Phosphatidylethanolamine，PE）和两种未知的脂（Lipid，L1-2）。通过16S rRNA基因序列比对，与海默氏菌属已发表种Rheinheimera tangshanensis JA3-B52^T相似度最高为96.8%，DNA G+C含量为48.7 mol%。命名为Rheinheimera marina。

(2) S1-10^T菌株为栖海面菌属（Aequorivita）的一个新种，属于G^-菌，杆状，没有运动性，兼性厌氧，氧化酶呈阴性，过氧化氢酶呈阳性，生长最适温度为30-35℃；最适pH为9.0；最适盐浓度为1%（w/v）NaCl。极性脂包含磷脂酰乙醇胺（Phosphatidylethanolamine，PE）、两种氨基磷脂（Aminolipid，AL1-2）、两种糖脂（Glycolipid，GL1-2）、一种磷酸糖脂（Phosphoglycolipid，PGL）和三种未知的极性脂（Lipid，L1-3）；主要呼吸醌类型为甲基萘醌6（MK-6）。通过16S rRNA基因序列比对，与栖海面菌属已发表种Aequorivita viscosa 8-1b^T相似度最高为97.7%，DNA G+C含量为34.6 mol%，与该属亲缘关系最近的菌株Aequorivita viscosa CGMCC 1.11023^T的isDDH值和ANI值分别为15.4%和75.7%。命名为Aequorivita sinensis。此外，对Vitellibacter属的两个种V. todarodis和V. aquimaris的分类地位重新进行了厘定，将它们重新归为栖海面菌属，命名为Aequorivita todarodis和Aequorivita aquimaris。

(3) TP390^T菌株是黄杆菌属（Flavobacterium）的一个新种，属于G^-菌，杆状，兼性厌氧，滑动运动，生长最适温度为25℃，最适盐浓度为1%（w/v）NaCl，最适pH为6.0-7.0。细胞主要脂肪酸为iso-C_15:0和iso-C_15:1 G；主要呼吸醌类型为MK-6，极性脂包含一种糖脂（Glycolipid，GL）、磷脂酰乙醇胺（Phosphatidylethanolamine，PE）、四种未知的氨基磷脂（Aminolipid，AL1-4）和六种未知的极性脂（Lipid，L1-6）。通过16S rRNA基因序列比对，与黄杆菌属已发表种Flavobacterium jejuense EC11^T相似度最高为97.8%，DNA G+C含量为31.2 mol%，与该属亲缘关系最近的菌株F. jejuense KCTC 42149^T的isDDH和ANI值分别为22.60%和80.01%。命名为Flavobacterium profundi。

(4) TP-CH-4^T菌株是黄杆菌科（Flavobacteriaceae）的一个新属，属于G^-菌，不产孢子，没有运动性，严格好氧，在2216E培养基上菌落呈橘色。生长温度范围为4-37℃、盐浓度范围为1-4%（w/v）NaCl，并且生长需要海盐。细胞主要脂肪酸为iso-C_15:0和summed feature 1（包括C_13:0 3-OH和/或iso-C_15:1 H）；主要呼吸醌类型为MK-6。通过16S rRNA基因序列比对，与黄杆菌科假卓贝尔属模式种Pseudozobellia thermophila KMM 3531^T相似度最高为95.1%，其次与鞭毛单胞属模式种Flagellimonas flava A11^T菌株相似度为93.9%，DNA G+C含量为44.5 mol%。提出新属名为Pelagihabitans，该新属模式种命名为Pelagihabitans pacificus。

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 rRNA 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), 38 genera 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 5 denitrifying 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) NaCl (optimum, 2-3%). The predominant cellular fatty acids were C_17:1 ω8c, summed feature 3 (composed of iso-C_15:0 2-OH and/or C_16:1 ω7c) and C_16:0. The polar lipid profile contained phosphatidylglycerol, phosphatidylethanolamine and two unidentified lipids. After 16S rRNA 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 pH 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 rRNA 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 (isDDH) 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 pH 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 rRNA gene showed that the highest similarity with the published species Flavobacterium jejuense EC11^T was 97.8%. The genomic DNA G+C content of strain TP390^T was 31.2 mol%. The maximum values of in silico DNA-DNA hybridization (isDDH) and average nucleotide identity (ANI) between strain TP390^T with F. jejuense KCTC 42149^T were 22.60 and 80.01%, 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) NaCl, 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.