Study On The Diversity And Ecology Of Culturable Bacteria In Four U.S. Great Basin Hot Springs

Since terrestrial hot spring is a kind of typical and special extreme environment, it may harbor many thermophilic microorganisms with special metabolic and physiological characteristics. The aim of this study was to investigate the diversity of culturable thermophilic bacteria in four U.S. Great Basin Hot Springs, to obtain valuable and (or) novel microbial resources. In addition, the physiological characters of Thermus strains were investigated to help in understanding the potential ecological functions of this group in hot spring ecosystem and the ecological distribution of previously uncultivated class TK17 of the the phylum Chloroflexi were studied.In this dissertation, a total of 486 strains were isolated from 19 different samples collected from four hot springs of Great Basin geothermal system using the serial dilution technique. Five isolation media and six different temperature gradients method were employed for isolation work. A total of 364 strains were selected for 16S rRNA gene sequencing according to the strains morphological characters and isolation source. BLAST and phylogenetic analyses based on 16S rRNA gene sequences showed that those isolates should be classified into 31 different genera of 20 families,10 classes of 5 phyla, including the genera Thermus, Thermobacillus, Caldimonas, Chloroflexus, Rhodothermus, Roseomonas, Rubellimicrobium, Elioraea, Anoxybacillus, Aquimonas, Arenimonas, Bacillus, Geobacillus, Meiothermus, Methyloversatilis, Paenibacillus, Phreatobacter, Piscinibacter, Porphyrobacter, Rehaibacterium, Rhodobacter, Schleiferia, Silanimonas, Tepidamorphus, Tepidicella, Tepidimonas, Thermoactinomyces, and four potential novel genera. The most abundant group was Thermus spp. accounting for 52.47%of sequenced strains. The identities of 16S rRNA gene sequences from 36.8%of sequenced strains were lower than 98%to described type strains, may present novel taxons, including one potential novel class, four potential novel genera, and 19 potential novel species.A total of 191 Thermus strains retrieved from Great Basin geothermal system could be classified into 65 phylotype, a limited geographical distribution of Thermus strains were observed among Great Boiling Spring (GBS) and Little Hot Creek (LHC) hot springs. Thermus strains harbored in GBS hot spring were more diverse than in LHC, this may be caused by different hot spring water residence time among two sites. Twenty-two strains were selected for physiological characterization experiments, including oxidation of thiosulfate and arsenite as well as reduction of ferric iron, nitrate, and polysulfide. The results demonstrated substantial phenotypic diversity and broad metabolic capability among each Thermus species. They may involve the biogeochemical cycle of nitrogen, iron, sulfur, and arsenic and play important roles.Two novel Thermus strains L198T and L423-2 isolated from LHC hot spring were subjected to polyphasic taxonomy work including analysis of physiological and biochemical tests, morphological characteristics, chemotaxondmic properties, and phylogenetic analysis based on 16S rRNA gene sequences. The two strains represent as members of the novel species of the genus Thermus, the name Thermus sediminis sp. nov. was proposed and the type strain is L198T. The genome of strain L198T is 2,160,271 bp long and consists of 4 scaffolds with 68.2%average GC content. A total of 2,308 genes were predicted, comprising 2,251 protein-coding and 57 RNA genes. 1868 protein-coding genes were predicted with function and 1618 protein-coding genes were distributed into COG functional categories. The genome is predicted to encode a complete glycolysis, gluconeogenesis, pentose phosphate pathway, and tricarboxylic acid (TCA) cycle. Additionally, the large number of transporters and enzymes for heterotrophy highlight the broad hetertrophic lifestyle of this organism. A sulfur oxidation gene cluster (soxDCAXBXAZY) was identified, consistent with the sulfur oxidation phenotype of mis strain.Novel thermophilic strains apparently constitute a previously uncultivated class TK17 of the the phylum Chloroflexi were isolated from GBS. The physiology, genomics characters and the ecological distribution of class TK17 were studied. Strain G233 had cells of 0.2-0.4 um X 2.0-3.0 urn. The optimum temperature for growth was 55-60 ℃. The genome of strain G233 is 2.74 Mbp long and consists of 1 scaffolds with 69.37%average GC content. A total of 2,707 genes were predicted, comprising 2,656 protein-coding and 51 RNA genes.2172 protein-coding genes were predicted with function accounting for 80.24%of total genes. Chloroflexi class TK17 are globally distributed and may have a higher abundance in marine environment. Phylogenetic analyses indicated class TK17 include members of four different order lineages and strain G233 represent the order TK17_O_mlel-48. Class TK17 is a global inhabitant of geothermal springs but with a lower abundance.This study expands our knowledge about the phylogenetic diversity of culturable thermophilic bacteria harbored in the Great Basin geothermal system, especially the diversity and physiology of the genus Thermus and Chloroflexi class TK17 and provides a basis for understanding the potential ecological functions of Thermus spp. and ecological distribution of Chloroflexi class TK17 in hot springs ecosystem.