Studies On Microbial Diversity And Thermophiles Isolation From Hot Spring In Eryuan,Yunnan

The microbial community structures of hot springs are specific ecosystems based on environmental factors.There are Lots of unknown microorganisms in hot springs,and they are important gene resource pool of thermophilic enzyme.Four samples of hot springs were collected from Niujie township of Eryuan county.OUT（Operational Taxonomic Units）,α-diversity,β-diversity and LEfSe analysis（LDA Effect Size）were performed based on 16S rDNA V4 region.The microorganisms in the water samples were separated,purified and identified.The description of new species and genome analysis of strain YHS0011 were carried out.Thermophilic proteases of strain YHS0011 were selected based on genome annotation and homology modeling.Therefore,a thermophilic protease of strain YHS0011 was heterologous expressed through Escherichia coli.The main results are as follows:Gene sequence analysis of 16S rDNA showed that the species diversity of water samples is significantly higher than sediments samples,and those samples collected from frequent activity location showed higher diversity than those in natural condition.In water samples,Proteobacteria,Firmicutes and Deinococcota are dominant bacteria.In sediments samples,Acetothermia,Crenarchaeota,Proteobacteria,Chloroflexi and Aquificae are dominant bacteria.αdiversity analysis showed that Shanon index of community richness of samples water1,sediment1,water2 and sediment2 were 5.77,5.36,4.27 and 5.81 respectively.βdiversity analysis showed that dispersion of sediments samples was similar,while dispersion of water samples of water and sediments samples show obviously different.6 thermophilic bacilli were separated and purified.They were preliminarily identified based on morphological observation,16S rDNA similarity,DNA-DNA hybridization（DDH）and average nucleotide identity（ANI）.The results showed that the 16S rDNA similarity,DDH and ANI between strain YHS0010 and Thermus amyloliquefaciens YIM 77409 were 98.804%,96.70%and 99.06%respectively.The strain YHS0010 was preliminarily identified as T.amyloliquefaciens.The 16S rDNA similarity,DDH and ANI between strain YHS0012 and Geobacillus thermodenitrificans NG 80-2 were 97.906%,79.70%and 97.98%respectively.The strain YHS0012 was preliminarily identified as G.thermodenitrificans.Three strains YHS008,YHS009 and YHS0013 were aligned with G.kaustophilus HTA426DDH,16S rDNA similarity were 83.80%,80.50%and 80.80%respectively;DDH were83.80%,80.50%and 80.80%respectively;ANI were 98.42%,97.94%and 98.14%respectively.Those three strains were preliminarily identified as G.kaustophilus.On the basis of 16S r RNA sequence similarity,strain YHS0011 shown to belong to Geobacillus,being most closely related to G.kaustophilus HTA426 is（95.573%）.Strain YHS0011 represents a novel species based on 16S rDNA similarity of G.kaustophilus HTA426 less than 97%.The DDH and ANI values of YHS0011 and G.kaustophilus HTA426 were 54.40%and 94.07%respectively.The results of DDH and ANI with G.stearothermophilus ATCC 7953 were 55.10%and 89.84%respectively.The DDH and ANI results of G.vulcani PSS1 were 66.00%and 89.54%respectively.The DDH and ANI results of G.alihae SURF-189 were 59.20%and 94.95%respectively.YHS0011 could not be classified into those species.Cultured on T162solid medium,the pH range for YHS0011 growth was 5-9.5 with the optimum at pH6.5,the temperature was 30 to 75℃with the optimum at 55℃,and the NaCl concentration was 0-3.5%with the optimum at 0.5%.The API20A assay experiment showed that YHS0011 can produce reductase,heptylene hydrolase,gelatin hydrolaseβ-Galactosidase and aesculin hydrolase.The main fatty acids of YHS0011 are iso-C_15:0,iso-C_16:0,iso-C_17:0,antiiso-C_17:0,and C_16:0.Compared with G.kaustophilus（CGMCC 1.3655）on morphological,physiological,biochemical and gene analysis,the experimental strain YHS0011 was preliminarily classified as a new species belonging to Geobacillus.14 protease genes were mined and predicted from the genome of YHS0011.The structure of cysteine protease yhs0011-158 was predicted by Alphafold2 model and compared with the corresponding enzymes of Bacillus sp.FJAT-18017（A0A0M4GTY5 and A0A0M5JGV5）with PYMOL,and their RMSD results were1.722 and 1.960 respectively.The electrophoretic image after transformation verified that the expression vector pET-42b-yhs0011-158 was successfully constructed.However,the protease activity could not be detected.To sum up,The study conducted a metagenome of the hot water pond in Niujie Town,Eryuan County.Discovered a new species YHS0011 of Geobacillus and excavated a novel thermophilic protease.The results of this study Abstract also provide a reference for mining thermophilic enzymes with potential application value from hot springs.