实验海洋生物学重点实验室知识库

冷泉是继热液之后另一种特殊的深海环境。南海冷泉生态系统以甲烷流体、自生碳酸盐岩、化能自养微生物群落以及大量的单质硫为特点。本研究利用2017年和2018年“科学号”冷泉-热液航次获得的冷泉沉积物样品，通过宏基因组学对南海冷泉微生物多样性和功能分布进行了分析。进而对冷泉沉积物样品中参与硫元素循环的微生物进行了筛选，获得了一系列硫氧化微生物和潜在的新属新种。在此基础上，对赤杆菌氧化硫代硫酸钠产单质硫的分子机制进行了深入研究；分析了化能自养硫氧化微生物的基因组信息；同时对一株潜在的新属新种进行了多相分类学研究。

宏基因组学分析结果显示南海冷泉沉积物细菌主要类群为变形菌门、绿弯菌门和浮霉菌门；古菌主要类群为广古菌门和洛基古菌门。参与硫酸盐还原和甲烷厌氧氧化的微生物为冷泉沉积物环境中最主要的微生物类群。表层样品中异养微生物能量获得途径以三羧酸循环为主，深层样品中以糖酵解为主；表层样品中微生物固碳途径以还原性磷酸戊糖途径为主，而深层样品中以还原性三羧酸循环和WL（Wood-Ljungdahl）途径为主。参与硫氧化的功能基因主要分布在表层样品中，而参与硫酸盐还原的功能基因广泛分布在不同深度样品中。

为了进一步了解冷泉沉积物中参与硫元素循环微生物的多样性，我们采用选择性培养基对冷泉沉积物中微生物进行了富集培养，以期能够获得典型的或者特殊的参与硫元素代谢的微生物。本研究一共获得26种纯培养微生物，分布于变形菌门、厚壁菌门和放线菌门。其中包括典型的化能自养硫氧化细菌（Guyparkeria hydrothermalis SP-2）、化能异养硫氧化细菌（Citreicella thiooxidans），能够代谢硫代硫酸钠产生单质硫的赤杆菌（Erythrobacter flavus 21-3）以及三株潜在新属和新种。针对其中一株潜在新属NS-1进行了多相分类学研究。16S rRNA基因序列分析结果显示菌株NS-1属盐厌氧单胞菌科，与Halocella cellulosilytica相似性最高为92.52%。菌株NS-1是一株严格厌氧、发酵产氢、可降解纤维素的细菌。其最适生长温度为37 ^oC, 最适生长pH值为7，最适生长NaCl范围为25-75 g/L。可利用多种糖类进行发酵，发酵葡萄糖产物为乙酸、乙醇、乳酸、丁酸、二氧化碳和氢气。经过形态学观察、生理生化性质检测及遗传学分析等比较，菌株NS-1被认为是盐厌氧单胞菌科的新属新种。根据菌株NS-1的分离单位和样品来源，我们将其命名为Iocasia coldseepira，模式菌株为NS-1（=KCTC15988^T= MCCC 1K04439^T）。这些微生物菌株的获得是本论文后续研究工作的前提和基础。同时，微生物的分离培养丰富了冷泉微生物菌种资源，为挖掘有应用潜力的深海冷泉微生物提供了保障。

硫代硫酸盐是硫元素循环过程中重要的中间代谢产物，能够被多种微生物利用。我们从冷泉沉积物中获得了两株能够氧化硫代硫酸钠产生单质硫的细菌：赤杆菌（E. flavus 21-3）和硫氧化细菌G. hydrothermalis SP-2。结合南海冷泉区存在大量单质硫的事实，我们重点研究了这两株细菌代谢硫代硫酸钠产生单质硫的作用机制。

针对赤杆菌（E. flavus 21-3），我们深入研究了其代谢硫代硫酸钠产单质硫的分子机制。首先通过电子显微镜、能谱扫描和拉曼光谱确定E. flavus 21-3可以代谢硫代硫酸钠产生零价单质硫（zero-valent sulfur, ZVS）。结合蛋白质组和分子遗传学实验确定硫代硫酸根脱氢酶（thiosulfate dehydrogenase，TsdA）和硫氧化蛋白（thiosulfohydrolase，SoxB）是E. flavus 21-3代谢硫代硫酸钠产单质硫的关键蛋白。通过化学计量方法检测了不同的含硫中间代谢产物，进而确定TsdA负责将硫代硫酸根转化为连四硫酸根（^-O₃S-S-S-SO₃^-）；SoxB负责水解连四硫酸根的磺酸基形成单质硫；单质硫在单质硫双加氧酶（sulfur dioxygenases，SdoA/B）的作用下水解生成亚硫酸根。另外，TsdA、SoxB和SdoA/B同源序列广泛存在于包括赤杆菌在内的许多细菌基因组中。表明这种新型硫代硫酸钠氧化途径可能存在于多种微生物当中，并且在硫的生物地球化学循环过程中有重要作用。

针对化能自养硫氧化细菌G. hydrothermalis SP-2，结合能谱扫描、拉曼光谱和基因组测序，我们对其参与硫元素循环进行了初步分析。基因组测序结果显示，G. hydrothermalis Sp-2基因组大小为2.59 M，G+C含量为66.89%。G. hydrothermalis SP-2具有soxAXBYZC操纵子，通过经典的Sox多酶复合物途径氧化硫代硫酸钠。由于缺少soxD基因，G. hydrothermalis SP-2氧化硫代硫酸钠过程中形成单质硫中间代谢产物。另外，G. hydrothermalis SP-2通过磷酸戊糖途径固定二氧化碳，并利用羧酶体提高固碳效率。

Cold seep is another special deep sea environment in addition to hydrothermal vent. The cold seep ecosystem in the South China Sea is characterized by methane-rich migration, authigenic carbonate minerals, chemosynthetic communities and large amount of elemental sulfur. In this study, the microbial diversity and function distribution of the cold seep in the South China Sea were analyzed by metagenomics using the samples of cold seep sediments obtained from the “cold seep-hydrothermal” voyage of KEXUE in the years of 2017 and 2018. Furthermore, the microorganisms involved in sulfur cycling were screened, and a series of sulfur oxidizing microorganisms and potential new genera and species were obtained. On this basis, the molecular mechanism of the production of elemental sulfur by oxidation of thiosulfate by Erythrobacter flavus was studied, the genome sequence of chemoautotrophic sulfur oxidizing microorganism Guyparkeria hydrothermalis was analyzed, and the polyphasic taxonomy of a potential new genus and new species was studied.

The results of metagenomic analysis showed that the main group of bacteria in the cold seep sediments of the South China Sea were Proteobacteria, Chloroflexi, and Planctomycetes, while the main catagories of Archaea were Euryarchaeota and Lokiarchaeota. The main microflora in the sediment of cold seep were those involved in sulfate reduction and methane anaerobic oxidation. In the surface samples, the energy production by heterotrophic microorganisms was mainly derived from the tricarboxylic acid cycle, while in the deep samples, glycolysis was the main metabolic way adopted by microbes; in the surface samples, the way of microbial carbon fixation was mainly through reducing pentose phosphate pathway, while in the deep samples, the way of reducing tricarboxylic acid cycle and WL (wood ljungdahl) were the main metabolic ways adopted by microbes. The functional genes involved in sulfur oxidation were mainly distributed in surface samples, while those involved in sulfate reduction were widely distributed in samples of different depths.

To further understand the diversity of microorganisms involved in sulfur cycling in cold seep sediments, we used selective medium to enrich microorganisms in cold seep sediments in order to obtain typical or special microorganisms involved in sulfur metabolism. In this study, 26 kinds of pure culture microorganisms were obtained and distributed in Proteobacteria, Firmicutes and Actinobacteria. Among them, the typical chemoautotrophic sulfur oxidizing bacteria (Guyparkeria hydrothermalis SP-2), chemoheterotrophic sulfur oxidizing bacteria (Citreicella thiooxidans), Erythrobacter flavus 21-3, which can metabolize sodium thiosulfate to produce elemental sulfur, and three potential new genera and new species were included. In this study, polyphasic taxonomy of a potential novel genus and new species NS-1 was investigated. The phylogenetic analysis of 16S rRNA gene showed that strain NS-1 belonged to the family of Halanaerobiaceae, and the highest similarity between NS-1 and Halocella cellulosilytica was 92.52%. Strain NS-1 was a strict anaerobic, hydrogen producing and cellulose degrading bacterium. The optimum growth temperature was 37 ^oC, the optimum growth pH was 7, and the optimum growth range of NaCl was 25-75 g/L. Various substances can be used for fermentation. The products of glucose fermentation were acetic acid, ethanol, lactic acid, butyric acid, carbon dioxide and hydrogen. After morphological observation, physiological and biochemical properties test and genetic analysis, strain NS-1 was considered to be a new species and new genus of the family Halanaerobiaceae. According to the isolation institute and sample source of strain NS-1, we named it as Iocasia coldseepira, and the type strain was NS-1 (= KCTC15988^T = MCCC 1K04439^T). The acquisition of these microbial strains was the premise and basis of the follow-up research in this paper. At the same time, the isolation and cultivation of microorganisms enriched the resources of cold seep microorganisms, and provide a base for the exploration of deep-sea cold seep microorganisms with potential applications

Thiosulfate is an important intermediate metabolite in sulfur cycle, which can be utilized by many microorganisms. We obtained two strains that can oxidize sodium thiosulfate to produce elemental sulfur from cold seep sediments: E. flavus 21-3 and G. hydrothermalis SP-2. Based on the fact that there is a large amount of elemental sulfur in the cold seep area of the South China Sea, we investigated the mechanism of thiosulfate oxidizing to produce elemental sulfur by these two bacteria.

On the one hand, we studied the molecular mechanism of thiosulfate oxidation and sulfur production in E. flavus 21-3 isolated from the sediment of cold seep. Electronic microscopy, energy-dispersive and Raman spectra were used to confirm that E. flavus 21-3 effectively converts thiosulfate to zero-valent sulfur (ZVS). We next used a combined proteomic and genetic methods to identify thiosulfate dehydrogenase (TsdA) and thiosulfohydrolase (SoxB) playing key roles in the conversion of thiosulfate to ZVS. Stoichiometric results of different sulfur intermediates further clarified the function of TsdA in converting thiosulfate to tetrathionate (^-O₃S-S-S-SO₃^-), SoxB in liberating sulfone from tetrathionate to form ZVS and sulfur dioxygenases (SdoA/SdoB) in oxidizing ZVS to sulfite under some conditions. Notably, homologs of TsdA, SoxB and SdoA/SdoB widely exist across the bacteria including in Erythrobacter species derived from different environments. This strongly indicated that this novel thiosulfate oxidation pathway might be frequently used by microbes and played an important role in the biogeochemical sulfur cycle in nature.

On the other hand, G. hydrothermalis SP-2, a chemoautotrophic sulfur oxidizing bacteria, was analyzed for its role in sulfur cycle by combining energy spectrum scanning, Raman spectroscopy and genome sequencing. The results of genome sequencing showed that the size of G. hydrothermalis SP-2 was 2.59 M, and the content of G + C was 66.89%. G. Hydrothermalis SP-2 contained soxAXBYZC operon, which oxidizes thiosulfate through the classical Sox multienzyme complex. Due to the lack of SoxD gene, G. hydrothermalis SP-2 forms sulfur intermediate during the oxidation of thiosulfate. In addition, G. hydrothermalis SP-2 fixed carbon dioxide through pentose phosphate pathway, and improved carbon fixation efficiency by carboxygenase.