Classification,Identification And Sulfur Oxidation Mechanisms Of Marine Sulfur-oxidizing Bacterium Thioclava

With the rapid development of our economic society,sulfur pollution represented by sulfide/hydrogen sulfide has gone from bad to worse.In recent years,the biological treatment method for sulfur pollution has received extensive attention due to its environmental-friendly,highly-efficient,and cost-effective advantages.However,there are two prominent problems in the current application:the lack of sulfur-oxidizing bacteria?SOB?and weak understanding of sulfur oxidation metabolism.This not only fails to meet the needs of different types of sulfur pollution treatment,but also limits the purposeful optimization of conditions and genetic improvement of SOB.Therefore,it is particularly of importance to obtain more outstanding SOB and to discern further their sulfur oxidation characteristics and metabolic mechanisms.The previous research from our team found that the Thioclava bacteria,which were widely distributed in the marine environments,showed excellent capacity in sulfur oxidation,but their classification,identification,and sulfur oxidation mechanism are still unclear.Therefore,the current study has focused on the classification,new species identification,and sulfur oxidation pathway of the Thioclava bacteria,which will provide a theoretical foundation for the subsequent mining and applications of SOB.This paper established a multilocus sequence analysis?MLSA?method based on five housekeeping genes?In order:gyrB,rpoD,dnaK,trpB and recA?by concatenated artificially for the classification and identification of the Thioclava bacteria.The 16S rRNA gene sequences of 23 Thioclava strains divided all strains into six taxa,but could not completely distinguish the intra-taxa strains.In contrast,MLSA sequences accurately divided these strains into seven taxa including the three new taxa,and could accurately distinguish the intra-taxa strains.The digital DNA-DNA hybridization?dDDH?and average nucleotide identity?ANI?analyses based on genomic sequences of the fifteen representative strains confirmed the seven taxa from MLSA and nine species including the five putative novel species.The correlation analyses of dDDH-MLSA and dDDH-ANI showed that both 97.3%of MLSA similarity and 96.07%of ANI corresponding to 70%of dDDH for species definition could be used as thresholds of species description.In addition,the rpoD gene was proposed as a reliable marker for rapid classification of the Thioclava strains.This paper identified the three putative new species discovered by MLSA using the polyphasic taxonomic approach.The three type strains of the three new species were Gram-staining-negative,and the cells were short-rod-shaped,aerobic,and similar in various enzymes activities.The main fatty acids of them was Summed Feature 8;the main polar lipids were phosphatidylglycerol,phosphatidylethanolamine,and glycolipid;the isoprenoid quinone was Q-10.The DNA G+C contents of the three type strains were64.0 mol%,63.8 mol%,and 64.1 mol%,respectively.The three type strains presented significant differences in the colonial morphology,cell size,presence or absence of flagellum,enzymes activities,fatty acid contents,etc..Meanwhile,multiple genotypic characteristics including 16S rRNA gene,MLSA,dDDH,and ANI also supported the establishment of the three new species.The three new species were named as Thioclava sediminum,Thioclava nitratireducens,and Thioclava marina,respectively.The type strains of the three species were MCCC 1A10143^T?MCCC 1A07302^T and MCCC1A03502^T,respectively.The representative strain T.nitratireducens MCCC 1A07302^T was selected for investigation of its sulfur oxidation mechanism.In the case of obtaining its complete genomic sequence,the genes and pathways of sulfur oxidation for strain MCCC1A07302^T were initially identified by using the BLAST,sequence characteristics,and phylogenetic analyses,and further determined by using the real-time PCR and transcriptome sequencing;on this basis,the complete sulfur oxidation model of the strain was constructed as follow:Sqr was responsible for sulfide oxidation,SqrR negatively regulated the transcription of sqr gene;Pdo2?AQS46486.1?was responsible to oxidize elemental sulfur to sulfite,Rho catalyzed the reaction of glutathione persulfide with sulfite to thiosulfate;Sox oxidized thiosulfate to sulfate under alkaline and neutral conditions,while Tsd could also catalyze the oxidation of thiosulfate to tetrathionate under the acidic condition;Sox or other new genes/enzymes were responsible for the sulfite oxidation.Strain MCCC 1A07302^T was an outstanding SOB based on the analysis of sulfur oxidation characteristics;its BMG03₁5715 should be specific to transport thiosulfate and sulfite.Strain MCCC 1A07302^T possessed diverse metabolic pathway of carbon,nitrogen,and sulfur,and intact electron transport chain.In addition,the sulfur oxidation of strain MCCC 1A07302^T was closely related to assimilatory sulfate reduction,denitrification and electron transport.In summary,this paper carried out the classification of the genus Thioclava bacteria,improved the taxonomic system,and determined the sulfur oxidation pathway,and explored the relationship between sulfur oxidation and other metabolism.The study will provide a theoretical basis for the subsequent exploration for the metabolic regulation of sulfur oxidation,development,and applications of sulfur-oxidizing bacteria.