Microbial Diversity Analysis Of Cold Seep In The South China Sea And Their Roles In The Key Process Of Sulfur Cycling

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 r RNA 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 p H was 7,and the optimum growth range of Na Cl 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 applicationsThiosulfate 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(Tsd A)and thiosulfohydrolase(Sox B)playing key roles in the conversion of thiosulfate to ZVS.Stoichiometric results of different sulfur intermediates further clarified the function of Tsd A in converting thiosulfate to tetrathionate(^-O₃S-S-S-SO₃^-),Sox B in liberating sulfone from tetrathionate to form ZVS and sulfur dioxygenases(Sdo A/Sdo B)in oxidizing ZVS to sulfite under some conditions.Notably,homologs of Tsd A,Sox B and Sdo A/Sdo B 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 sox AXBYZC operon,which oxidizes thiosulfate through the classical Sox multienzyme complex.Due to the lack of Sox D 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.