Sulfur Metabolism Characterization Of Sulfurimonas From Deep-sea Hydrothermal Vents

The deep-sea hydrothermal vent is a typical chemoautotrophic ecosystem.The chemoautotrophic sulfur-oxidizing bacteria inhabiting here occupy an important ecological niche.However,due to the difficulty of deep-sea sampling and the imperfection of microbial isolation and culture technology,there is a lack of research on microorganisms in hydrothermal areas based on pure culture,which limits the cognition of their ecological functions.Isolation and acquisition of pure cultures from extreme hydrothermal vents is very important for studying the mechanism of energy metabolism and the adaptation mechanism for extreme environments.It is also of great significance to further interpret the ecological contribution of chemoautotrophs.Sulfurimonas genus represent one of the most widespread and preponderant mesophilic bacteria in global deep-sea hydrothermal environments and in situ environments of all oceans.However,only 5 species have been reported in the world,two of which are from hydrothermal vents.Therefore,its resources and culture technology need to be urgently developed.In addition,it is not clear how Sulfurimonas in hydrothermal vents and non-hydrothermal vents differ in metabolic characteristics,environmental adaptation mechanism and life evolution relationship.The purpose of this paper is to isolate and obtain more Sulfurimonas bacteria with high abundance in in situ hydrothermal environments,so as to enrich the Sulfurimonas bacteria resource bank.Furthermore,the metabolic characteristics of carbon,hydrogen,sulfur and nitrogen of Sulfurimonas in hydrothermal vents and non-hydrothermal vents were compared at physiological,genomic and transcriptomic levels,as well as the molecular mechanism of adaptation to hydrothermal vents was investigated,which has important meaning for evaluating the in situ ecological contribution of Sulfurimonas in hydrothermal vents,and provide important theoretical support for the development and utilization of its resources.The main results are as follows:1.Discovery and classification of 6 new species from Sulfurimonas.In this study,six sulfur-oxidizing bacteria were isolated from different marine habitats,such as deep-sea hydrothermal vents,South China Sea and the offshore habitats.These 6 strains were identified as 6 new species of Sulfurimonas genus,and were published as model strains by polyphasic taxonomic methods,including morphological characteristics,physiological and biochemical characteristics,chemical composition analysis,16S r RNA gene sequence analysis,ANI,d DDH and UBCG core genome analysis.They were named as Sulfurimonas hydrogeniphila NW10^T,S.indica NW8N^T,S.sediminis S2-6^T,S.xiamenensis 1-1N^T,S.lithotrophicum GYSZ＿1^Tand S.aggregans B2^T.Among them,S.hydrogeniphila NW10 is a representative new species with high abundance isolated from the hydrothermal region of the Northwest Indian Ocean and is the main research object in the following study.Strain NW10 could grow at 4-45?C（optimum 33?C）,p H5.0-9.0（optimum p H 6.0）,2.0-4.0%（w/v）Na Cl concentration（optimum 3.0%）,and with 0-20%oxygen concentration（optimum 6.0%）.Obligate chemolithoautotrophic growth occurs with H₂,thiosulfate,sulfide,elemental sulfur as electron donors,and oxygen,nitrate,and S⁰could be utilized as an electron acceptor.At the same time,strain NW10 could quickly oxidize sulfide in acidic and neutral conditions in a short time to produce a large amount of extracellular elemental sulfur,which has a very important ecological significance.2.The environmental adaptability mechanism of Sulfurimonas in hydrothermal vents was analyzed.Sulfurimonas genus had an open pan-genome,suggesting that there are many new species resources to be discovered.The results of core genome,whole genome and collinearity analysis showed that strains in hydrothermal and non-hydrothermal habitats had obvious evolutionary characteristics,which suggested that environmental factors had a greater influence on genomic variation than spatial geographic factors.The analysis of sulfur metabolism mechanism showed that the hydrothermal strains had a unique type V sqr gene,and also contained multiple copies of Type II sqr gene,while other strains contained Type IV and Type VI sqr genes,which supported the fact that they were able to utilize a variety of sulfur compounds.Interestingly,strain NW10 had a complete Sox system,which indicated strain NW10 has the ability to completely oxidize thiosulfate to sulfate,but could generate extracellular elemental sulfur,suggesting that there may be new genes and pathways for sulfur metabolism.Besides Sox multienzyme complex and sqr genes,sor AB genes and incomplete assimilated sulfate reduction pathway genes,such as sat,cys C and cys DN,were also found.Analysis of carbon metabolism mechanism showed that Sulfurimonas had all enzymes required for complete r TCA cycle,including Acl,Oor and Por,which supported its chemoautotrophic growth characteristics.In addition,phylogenetic analysis showed Acl Bs from vent isolates clustered together,while non-vent Acl Bs formed a distinct cluster.Analysis of nitrogen metabolism mechanism showed that except for strain B2,genes encoding all components required for the complete reduction of nitrate to nitrogen gas,i.e.,nap,nir,nor and nos,were found in all Sulfurimonas species,which supported the physiological data that strains could utilize nitrate as electron acceptors.Phylogenetic analysis showed that Nap As from vent Sulfurimonas species clustered together,while those from non-vent but marine habitats clustered apart and were close to those from Arcobacter species.In addition,strain NW10 had the complete assimilatory nitrate reduction pathway,containing the genes encoded nitrate transporter（nas AB）and ferredoxin-nitrite reductase（nir A）.This gene cluster was also found in all other hydrothermal vent isolates,but absent in most non-vent strains.Hydrogen metabolism mechanism analysis:Hydrogen is one of the most important energy sources for hydrothermal strains,which is crucial for the survival of chemoautotrophs.The results showed that Sulfurimonas spp.contain three types of hydrogenases:Group 1,Group 2b and Group 4[Ni Fe]-Hydrogenases.Type II hydrogenase was found in hydrothermal Sulfurimonas strains for the first time,which was beneficial for Sulfurimonas to sense the high concentration of hydrogen in hydrothermal vents and make they more competitive in the changing in situ environments.Type IV hydrogenase could be divided into Hyc,Coo and Ech.It was firstly reported that Coo hydrogenase was found in hydrothermal Sulfurimonas strains and showed the ability to oxidize CO to produce carbon dioxide and hydrogen,which indicated its ecological significance in providing energy for other organisms in hydrothermal vents.It was also found that the strains in hydrothermal vents contained a higher proportion of sulfur transfer proteins,heat shock proteins,signal transduction proteins,a variety of heavy metal transporters and glycosyltransferases,and other related coding genes,which gave the bacteria a higher competitive advantage in hydrothermal environments.3.Sulfur reduction mechanism of strain NW10 from hydrothermal vents:strain NW10could obtain energy for growth by nitrate and elemental sulfur reduction coupled with hydrogen oxidation.The reduction of elemental sulfur coupled with hydrogen oxidation were first discovered in genus Sulfurimonas,which could be used as the main energy in in situ hydrothermal vents where oxygen is mostly unavailable while elemental sulfur is relatively abundant.Further analysis of transcriptome and RT-q PCR showed that there were two sulfur reduction pathways in strain NW10,which were mediated by periplasmic polysulfide reductase（Psr A₁B₁CDE）and cytoplasmic polysulfide reductase（Psr A₂B₂）.The existence of these two pathways made Sulfurimonas more adaptive to the hydrothermal environments.This results will lay a foundation for the regulation of sulfur oxidation or sulfur reduction in subsequent industrial applications.4.Sulfur elemental activation mechanism of hydrothermal strain NW10.The activation mechanism of extracellular sulfur in Sulfurimonas was studied for the first time.Strain NW10can oxidize and reduce elemental sulfur.But the mechanism by which solid sulfur interacts with bacteria is unclear.The results showed that there were many pits on the surface of elemental sulfur and a large number of extracellular substances were attached on the surface of elemental sulfur.FT-IR analysis was further proved that the surface of elemental sulfur was modified by some amphiphilic substances containing functional groups of aldehydes,ketones or amides.Furthermore,non-targeted metabolomics analysis showed that 84 different metabolites were found under elemental sulfur-reduction condition（EK）compared with the control group.Among them,S-Carboxymethyl-L-cysteine and S-Adenosylmethionine were enriched and significantly expressed,which has the potential in elemental sulfur activation.In conclusion,this study enriched the resources of marine chemolithoautotrophic bacteria.The metabolic diversity of Sulfurimonas in hydrothermal vents and the mechanism of its adaptability to hydrothermal environments were revealed.It was reported for the first time that Sulfurimonas had the characteristic of excreting elemental sulfur and the mechanism of sulfur reduction coupled with hydrogen oxidation.The results showed that Sulfurimonas had a very important ecological contribution in deep-sea hydrothermal vents,which provided the theoretical basis for the metallogenic process of marine organisms,and also provided the bacteria resources for development and utilization of extremophile resources.