海洋生物分类与系统演化实验室知识库

传统的微生物分离培养方法主要依赖于营养丰富的培养基，但这种方法可能会阻碍对海洋生态系统中寡营养细菌的发现。本研究以中国南海珍贝海山区水体样品为研究对象，采用寡营养条件分离培养到兼性寡营养细菌，并对其进行了多样性及其参与氮、硫和碳等元素循环的代谢分析。

1. 中国南海珍贝海山区可培养的兼性寡营养细菌多样性

南海珍贝海山区有机物浓度低，是典型的寡营养海域。本研究从南海珍贝海山21个站位的不同水层收集了150个水样，在寡营养条件下富集培养，再分别采用寡营养培养基（以葡萄糖、丙酮酸钠和醋酸钠为唯一碳源（15 mg C/L））和营养丰富的培养基（2216E海水培养基）筛选寡营养细菌，最终分离得到隶属于3门4纲17科21属42种的648株兼性寡营养细菌，这些细菌表现出在寡营养培养基和2216E海水培养基上均能生长的特性。其中，99.39%（644/648）的细菌隶属于假单胞菌门，γ-变形菌纲和α-变形菌纲分别占73.3%（472/644）和26.9%（173/644）；厚壁菌门有3株，放线菌门有1株；绝大部分细菌属为假交替单胞菌属Pseudoalteromonas（181/648，27.9%）、弧菌属Vibrio（168/648，25.9%）、玫瑰色菌属Roseibium（60/648，9.3%）和盐懒惰菌属Salipiger（45/648，6.9%）。其中阿拉伯海假交替单胞菌（Pseudoalteromonas arabiensis）、新喀里多尼亚弧菌（Vibrio neocaledonicus）和团聚玫瑰色菌（Roseibium aggregatum）在所有水层和几乎所有站位均有发现，推测它们是珍贝海山主要的可培养兼性寡营养细菌类群。

通过与富营养的海州湾水体中的浮游细菌类群进行比较，发现太平洋拟游动细杆菌（Bacterioplanoides pacificum）、金宁硝酸盐还原菌（Nitratireductor kimnyeongensis）和植物内生普里斯特氏菌（Priestia endophytica）三种寡营养细菌仅在南海珍贝海山水体中分离得到，推测这三种菌为南海珍贝海山独特的兼性寡营养细菌。

2. 中国南海珍贝海山区可培养的兼性寡营养细菌参与氮、硫和碳元素代谢分析

选取42株不同种的兼性寡营养菌株进行全基因组测序。基于硝酸盐还原表型实验，发现50%的菌株（21/42）均具备硝酸盐还原特性，这与它们全基因组都编码异化硝酸盐还原（dissimilatory nitrate reduction）途径的关键基因（narGHI/napAB）相一致。其中17株细菌的全基因组编码异化硝酸盐还原为铵（dissimilatory nitrate reduction to ammonium，DNRA）途径的关键基因（napAB/narGHI, nirBD, nrfA）；6株兼性寡营养细菌，包括转化异源物食烷菌ZBS-261（Alcanivorax xenomutans ZBS-261）、航海海杆菌ZBS-94-1（Marinobacter nauticus ZBS-94-1）、胜利油田海杆菌ZBS-703（Marinobacter shengliensis ZBS-703）、肇东假单胞菌ZBN-608（Pseudomonas zhaodongensis ZBN-608）、团聚玫瑰色菌ZBS-8（Roseibium aggregatum ZBS-8）和仙河近海螺旋菌ZBS-5（Thalassospira xianhensis ZBS-5）的全基因组编码硝酸盐还原为N₂途径的关键基因（napAB/narGHI, nirS/nirK, norBC, nosZ），这与它们能将硝酸盐还原为气体的表型特征相一致。

硫代硫酸盐是海洋硫元素循环的关键中间产物，我们从基因组水平分析了兼性寡营养细菌参与硫代硫酸盐的还原、氧化和歧化反应的代谢潜力。39株兼性寡营养细菌含有同化硫酸盐还原（assimilatory sulfate reduction，ASR）途径相关基因。6株菌，包括海角副球菌ZBS-68（Paracoccus homiensis ZBS-68）、团聚玫瑰色菌ZBS-8（Roseibium aggregatum ZBS-8）、大西洋玫瑰变色菌ZBS-192（Roseovarius atlanticus ZBS-192）、抑制云层玫瑰变色菌ZBS-326-2（Roseovarius nubinhibens ZBS-326-2）、百慕大盐懒惰菌ZBS-10（Salipiger bermudensis ZBS-10）和硫氧化盐懒惰菌ZBS-95（Salipiger thiooxidans ZBS-95）的全基因组编码硫氧化（sulfur oxidation，SOX）关键基因（soxA，soxB，soxC，soxX，soxY和soxZ），与其能氧化硫代硫酸盐导致pH值降低的表型实验相一致。我们发现河豚毒素假交替单胞菌ZBN-167（Pseudoalteromonas tetraodonis ZBN-167）、天蓝色弧菌ZBN-312（Vibrio azureus ZBN-312）、转化异源物食烷菌ZBS-261（Alcanivorax xenomutans ZBS-261）、热液口盐单胞菌ZBS-735-2（Halomonas hydrothermalis ZBS-735-2）、航海海杆菌ZBS-94-1（Marinobacter nauticus ZBS-94-1）和解藻酸弧菌ZBS-471（Vibrio alginolyticus ZBS-471）6株菌的基因组编码连四硫酸盐（Tetrathionate intermediate）（S₄I）途径的关键酶硫代硫酸根脱氢酶（thiosulfate dehydrogenase，TsdA）基因，推测它们能通过S₄I途径氧化硫代硫酸盐。我们发现解脂假交替单胞菌ZBS-149（Pseudoalteromonas lipolytica ZBS-149）、运动特里顿杆菌ZBS-29（Tritonibacter mobilis ZBS-29）、海绵假交替单胞菌ZBN-172（Pseudoalteromonas spongiae ZBN-172）、河豚毒素假交替单胞菌ZBN-167（Pseudoalteromonas tetraodonis ZBN-167）、俄罗斯假交替单胞菌ZBN-534（Pseudoalteromonas ruthenica ZBN-534）、天蓝色弧菌ZBN-312（Vibrio azureus ZBN-312）、副凝聚小短杆菌ZBN-604（Brachybacterium paraconglomeratum ZBN-604）和团聚玫瑰色ZBS-8（Roseibium aggregatum ZBS-8）8个菌株的基因组编码硫代硫酸盐歧化反应（Thiosulfate disproportionation）的关键酶3-巯基丙酮酸硫转移酶（3-mercaptopyruvate sulfurtransferase，sseA）和硫代硫酸盐硫转移酶（thiosulfate sulfurtransferase，glpE）的基因，推测它们能参与硫代硫酸盐的歧化反应途径。3株兼性寡营养细菌，包括罗伊赫海源菌ZBN-478（Idiomarina loihiensis ZBN-478）、肇东假单胞菌ZBN-608（Pseudomonas zhaodongensis ZBN-608）和金宁硝酸盐还原菌ZBN-30（Nitratireductor kimnyeongensis ZBN-30）编码dmdB和dmdC基因，推测其可能参与海水中二甲基巯基丙酸内盐（dimethylsulphoniopropionate, DMSP）的去甲基化代谢。

对42种兼性寡营养细菌全基因组进行碳水化合物酶（CAZymes）预测分析，共鉴定出6个CAZyme超家族的3836个基因，分别对应糖基转移酶（GT）（40%）、糖苷水解酶（GH）（36%）、碳水化合物结合模块（CBMs）（13%）、碳水化合物酯酶（CE）（5.1%）辅助活性（AA）（4.7%）和多糖裂解酶（PL）（4.7%）。其中，GH23、CBM50、GT4、GT2和GT51是主要的CAZymes。几丁质降解酶中，GT2（几丁质转糖基酶）丰度最高，其次是GH23（几丁质酶）和CBM5（几丁质结合蛋白）。我们发现兼性寡营养细菌基因组编码多个几丁质降解基因，推测几丁质可能是兼性寡营养细菌的一种重要碳源和氮源。

The traditional approach to cultivating bacteria from ocean ecosystems uses nutrient-rich medium, but this method may prevent the discovery of oligotrophic bacteria. In this study, we used oligotrophic conditions to isolate facultative oligotrophic bacteria from water samples collected in the South China Sea's Zhenbei Seamount area. The diversity of these culturable facultative oligotrophic bacteria was analyzed, along with their metabolic analysis in nitrogen, sulfur, carbon, and other element cycles.

1. Diversity of culturable facultative oligotrophic bacteria in the Zhenbei Seamount Area of the South China Sea

The Zhenbei Seamount area in the South China Sea is characterized by low organic matter concentration, making it a typical oligotrophic marine region. In this study, 150 water samples were collected from different water layers at 21 stations in the Zhenbei Seamount area. After enrichment culture under oligotrophic conditions, the oligotrophic bacteria were further selected using oligotrophic media containing glucose, sodium pyruvate, and sodium acetate as the sole carbon source (15 mg C/L), as well as nutrient-rich 2216E medium. Eventually, 648 strains of facultative oligotrophic bacteria were isolated, capable of growing on both oligotrophic and 2216E seawater media. These bacteria belonged to 3 phyla, 4 classes, 17 families, 21 genera, and 42 species. Among them, 99.39% (644/648) of the bacteria belonged to the phylum Proteobacteria, with 73.3% (472/644) and 26.9% (173/644) belonging to the classes Gammaproteobacteria and Alphaproteobacteria, respectively. There were three strains from the phylum Bacillota and one strain from the phylum Actinomycetota. Pseudoalteromonas (181/648, 27.9%), Vibrio (168/648, 25.9%), Roseibium (60/648, 9.3%) and Salipiger (45/648, 6.9%) were the most common genera. Pseudoalteromonas arabiensis, Vibrio neocaledonicus, and Roseibium aggregatum were found in all water layers and almost all stations, indicating that they are the dominant culturable oligotrophic bacterial groups in the Zhenbei Seamount.

By comparing to planktonic bacterial taxa in eutrophic environments in Haizhou Bay, Bacterioplanoides pacificum, Nitratireductor kimnyeongensis, and Priestia plantarum endophyta were only isolated from the water of the Zhenbei Seamount in the South China Sea, suggesting that these three species were unique facultative oligotrophic bacteria in this area.

2. Culturable facultative oligotrophic bacteria participating in nitrogen, sulfur and carbon metabolism in Zhenbei Seamount of the South China Sea

A total of 42 representative strains of facultative oligotrophic bacteria were selected for whole-genome sequencing. Based on phenotypic experiments on nitrate reduction, it was found that 50% (21/42) of the strains exhibited nitrate reduction characteristics. This is consistent with the fact that their genomes encode key genes (narGHI/napAB) involved in dissimilatory nitrate reduction. Among them, the genomes of 17 strains of bacteria also encode key genes (napAB/narGHI, nirBD, nrfA) involved in dissimilatory nitrate reduction to ammonium (DNRA) pathway. The genomes of 6 facultative oligotrophic bacteria, including Alcanivorax xenomutans ZBS-261, Marinobacter nauticus ZBS-94-1, Marinobacter shengliensis ZBS-703, Pseudomonas zhaodongensis ZBN-608, Roseibium aggregatum ZBS-8, and Thalassospira xianhensis ZBS-5, encode key genes (napAB/narGHI, nirS/nirK, norBC, nosZ) involved in the pathway of nitrate reduction to nitrogen gas. This is consistent with their phenotype of reducing nitrate to gas.

Thiosulfate is a key intermediate product in the marine sulfur element cycle. We analyzed the potential of oligotrophic bacteria to participate in the reduction, oxidation, and disproportionation reactions of thiosulfate at the genomic level. There are 39 strains of oligotrophic bacteria contain genes related to the assimilatory sulfate reduction (ASR) pathway. The genomes of 6 bacteria, including Paracoccus homiensis ZBS-68, Roseibium aggregatum ZBS-8, Roseovarius atlanticus ZBS-192, Roseovarius nubinhibens ZBS-326-2, Salipiger bermudensis ZBS-10, and Salipiger thiooxidans ZBS-95, encode key genes (soxA, soxB, soxC, soxX, soxY, and soxZ) involved in sulfur oxidation (SOX), consistent with their phenotype of acidic pH reduction caused by thiosulfate oxidation. The genomes of 6 bacteria, including Pseudoalteromonas tetraodonis ZBN-167, Vibrio azureus ZBN-312, Alcanivorax xenomutans ZBS-261, Halomonas hydrothermalis ZBS-735-2, Marinobacter nauticus ZBS-94-1, and Vibrio alginolyticus ZBS-471, encode the key enzyme thiosulfate dehydrogenase (TsdA) gene involved in the tetrathionate intermediate (S₄I) pathway, suggesting their ability to oxidize thiosulfate through the S₄I pathway. Eight strains of bacteria, including Pseudoalteromonas lipolytica ZBS-149, Tritonibacter mobilis ZBS-29, Pseudoalteromonas spongiae ZBN-172, Pseudoalteromonas tetraodonis ZBN-167, Pseudoalteromonas ruthenica ZBN-534, Vibrio azureus ZBN-312, Brachybacterium paraconglomeratum ZBN-604, and Roseibium aggregatum ZBS-8, encode the key enzymes 3-mercaptopyruvate sulfurtransferase (sseA) and thiosulfate sulfurtransferase (glpE) involved in the thiosulfate disproportionation reaction, suggesting their involvement in the thiosulfate disproportionation pathway. Dimethylsulphoniopropionate (DMSP) is one of the most abundant organic sulfur molecules on Earth. Three strains of facultative oligotrophic bacteria, including Idiomarina loihiensis ZBN-478, Pseudomonas zhaodongensis ZBN-608, and Nitratireductor kimnyeongensis ZBN-30, encode the dmdB and dmdC genes, suggesting their potential involvement in the demethylation metabolism of DMSP in seawater.

Based on the whole-genome analysis of carbohydrate-active enzymes (CAZymes), a total of 3836 genes belonging to 6 CAZyme superfamilies were identified. These included glycosyl transferases (GT) (40%), glycoside hydrolases (GH) (36%), carbohydrate-binding modules (CBMs) (13%), carbohydrate esterases (CE) (5.1%), auxiliary activities (AA) (4.7%), and polysaccharide lyases (PL) (4.7%). Among them, GH23, CBM50, GT4, GT2, and GT51 were the major CAZymes. In the chitin-degrading enzymes, the abundance of GT2 (chitin transferase) was the highest, followed by GH23 (chitinase) and CBM5 (chitin-binding protein). The genomes of oligotrophic bacteria encode multiple chitin-degrading genes, which may enable them to utilize chitin as an important nitrogen and carbon source to sustain their survival in the ocean.

第1章 绪论      1

1.1 研究背景      1

1.1.1 南海微生物多样性研究进展   1

1.1.2 寡营养细菌      2

1.1.3 微生物参与的关键元素循环   5

1.2 立题依据、研究内容和意义      12

1.2.1 立题依据   12

1.2.2 研究内容   13

1.2.3 研究意义   14

第2章 珍贝海山兼性寡营养细菌分离与鉴定      15

2.1 引言      15

2.2 材料和方法  15

2.2.1 实验所用试剂和仪器设备      15

2.2.2 主要培养基      16

2.2.3 样品采集   17

2.2.4 海水样品中寡营养细菌的分离培养与保藏   19

2.2.5 兼性寡营养细菌的分类地位   20

2.2.6 系统发育树的构建   20

2.3 实验结果分析      20

2.3.1 南海珍贝海山水体中兼性寡营养细菌筛选   20

2.3.2 南海珍贝海山水体中兼性寡营养细菌分布与系统发育分析       22

2.3.3 海州湾赤潮高发区富营养环境细菌多样性   24

2.4 贫营养与富营养环境中细菌多样性的比较      29

2.5 小结与讨论  30

第3章 珍贝海山兼性寡营养细菌碳、氮、硫元素代谢研究      32

3.1 引言      32

3.2 实验材料与试剂  32

3.2.1 主要仪器与设备      32

3.2.2 主要试剂   32

3.2.3 菌株与培养基   32

3.3 实验方法      32

3.3.1 兼性寡营养细菌的碳、氮、硫代谢能力检测      32

3.3.2 全基因组的碳、氮、硫循环关键基因的注释      33

3.4 结果分析      34

3.4.1 兼性寡营养细菌的氮元素代谢分析      34

3.4.2 兼性寡营养细菌中硫代谢分析      36

3.4.3 兼性寡营养细菌中碳水化合物代谢分析      38

3.5 小结与讨论  43

第4章 结论与展望   46

4.1 结论      46

4.2 研究展望和不足  47

参考文献     48

致  谢 60

作者简历及攻读学位期间发表的学术论文与其他相关学术成果 62