Bacterial Diversity From Tengchong Hot Springs And Genomic Insights Into The Genus Tepidimonas And Previously Uncultivated Chloroflexi Class(Tk17)

Life is harmful to living organisms with respect to physicochemical properties such as temperature,pH,pressure,nutrients and saline concentration which are referred to as an extreme environmental condition.Among these extreme environments,thermal springs are of significant interest to researchers worldwide.Microbial communities present in such habitats make valuable sources for various biotechnology products based on their ecological role.In the present study,the culture-dependent method was used to evaluate the thermophilic bacterial diversity in hot springs of the Tengchong in China,using T5,R2A and modified R2A media,which were used to characterize novel isolates by using polyphasic taxonomy approach.In addition,the comparative genomic study on species/strains from the genus Tepidimonas was carried out to investigate the genomic characteristics,the metabolic diversity,and the phylogenetic relationships among the species of Tepidimonas.In the current study we also performed the identification and comparative genomics for the two novel thermophilic,bacterial strains,designated strain G233~T and YIM 72310~T,belonging to a novel class(TK17 group),isolated from sediments of Great Boiling Spring(GBS)in the US Great Basin,and Hamazui spring(HMZ)in Tengchong,China respectively.Total of 195 bacterial strains were isolated,which were composed of five phyla and20 genera,including seven novel candidates(one novel class,one new genus and five novel species).The most dominant phyla were Firmicutes(40%),Deinococcus-Thermus(30%),Proteobacteria(22%),Actinobacteria(4%)and Chloroflexi(4%)respectively.At the genus level,Tepidimonas was dominant followed by Thermus.The effect of modified R2A medium resulted in isolating many novel candidates.The genus Tepidimonas which belongs to the class betaproteobacteria,consists of seven bacterial species that were mainly isolated from water and sediment of hot springs.The genomic diversity of different species within the genus Tepidimonas and their relationships between genomic differentiation and to environmental factors have not yet been studied.In this study,six type species including,six strains of Tepidimonas sediminis and 3 strains of Tepidimonas alkaliphilus strains of two species(YIM 72259~T and YIM 72238~T)of genus Tepidimonas were sequenced to evaluate their genomic characteristics,metabolic diversity and phylogenetic relationships.The total genome sizes varied between 2.5(YIM 72259~T)and 3.1 megabases(YIM 72238~T),and the G+C content ranged from 68.4 to 71.7%.The genome dynamics analysis of the genus Tepidimonas revealed that Tepidimonas exhibits an open pan-genome structure,while the core genome size dropped off with thes equential addition at the genus and species group levels.A greater consistency was found between the phylogenetic trees based on the concatenated amino acid sequences of core genes and the phylogenetic tree obtained by16S ribosomal RNA(rRNA)genes,but the phylogenetic tree based on the concatenated amino acid sequences of core genes provided a more robust phylogenetic resolution than the 16S rRNA gene-based analysis.Comparative genomic study indicated the species of the genus Tepidimonas is diverse in genome size,gene content and genetic relationship,forming a separate clade isolated from different geographical locations which provides evidence for differentiation.Gene clusters for sulfur oxidation and metal resistance were found and highly conserved among all the genome of Tepidimonas genus.These results provide a quantitative basis for understanding the genomic and metabolic diversity within the genus Tepidimonas,placing the foundation for future studies on taxonomy and functional genomics.The two novel bacterial organisms(G233~T and YIM 72310~T)belonging to the phylum Chloroflexi,were distinct from its closest relative,Caldilinea aerophila DSM14535~T(85.8%nucleotide identity).Strikingly,both strains could motile by means of a polar flagellum,which is different from the most described members of this phylum except of gliding motility seen in the class Chloroflexi.The draft genome sequence of strains G233~T and YIM 72310~T are 2.74 and 2.76 M bp and with average G+C content of69.4%and 71.2%,respectively.Phylognetics inferred from the 16S rRNA gene sequence indicate that the two strains are the first cultivated representatives of the‘TK17’(Greengenes taxonomy),are a new class within the Phylum Chloroflexi.Metabolic reconstruction from the core genome suggests that they are strictly heterotrophic with a versatile metabolism.A full set of genes for flagella biosynthesis and chemotaxis are present.A urease gene cluster was only found in strain YIM 72310~T.In these genomes,Genes for enzymes implicated in the oxidation of recalcitrant organic compounds were found.These genes include multiple FMNO paralogs,P450 and enzymes predicted to function in aromatic compound catabolism.In the genome,the presence of genes encoding assimilatory sulfite reductase provides supporting evidence for the potential for sulfite reduction.On the basis of their unique phylogenetic placement and physiological character,we propose these organisms represent a novel class within the Chloroflexi.This study lays a foundation for understanding the physiology and ecological role of the phylum Chloroflexi,especially TK17 group.