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Biodegradation stands as an eco-friendly and effective approach for organic contaminant remediation. However, research on microorganisms degrading sodium benzoate contaminants in extreme environments remains limited. In this study, we report to display the isolation of a novel hot spring enriched cultures with sodium benzoate (400 mg/L) as the sole carbon source. The results revealed that the phylum Pseudomonadota was the potential sodium benzoate degrader and a novel genus within the family Geminicoccaceae of this phylum. The isolated strain was named Benzoatithermus flavus SYSU G07066 T and was isolated from HNT-2 hot spring samples. Genomic analysis revealed that SYSU G07066 T carried benABC genes and physiological experiments indicated the ability to utilize sodium benzoate as a sole carbon source for growth, which was further confirmed by transcriptomic data with expression of benABC. Phylogenetic analysis suggested that Horizontal Gene Transfer (HGT) plays a significant role in acquiring sodium benzoate degradation capability among prokaryotes, and SYSU G07066 T might have acquired benABC genes through HGT from the family Acetobacteraceae. The discovery of the first microorganism with sodium benzoate degradation function from a hot spring enhances our understanding of the diverse functions within the family Geminicoccaceae. This study unearths the first novel genus capable of efficiently degrading sodium benzoate and its evolution history at high temperatures, holding promising industrial applications, and provides a new perspective for further exploring the application potential of hot spring "microbial dark matter"., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Genus Thermus is the main focus of researcher among the thermophiles. Members of this genus are the inhabitants of both natural and artificial thermal environments. We performed phylogenomic analyses and comparative genomic studies to unravel the genomic diversity among the strains belonging to the genus Thermus in geographically different thermal springs. Sixteen Thermus strains were isolated and sequenced from hot springs, Qucai hot springs in Tibet and Tengchong hot springs in Yunnan, China. 16S rRNA gene based phylogeny and phylogenomic analyses based on concatenated set of 971 Orthologous Protein Families (supermatrix and gene content methods) revealed a mixed distribution of the Thermus strains. Whole genome based phylogenetic analysis showed, all 16 Thermus strains belong to five species; Thermus oshimai (YIM QC-2-109, YIM 1640, YIM 1627, 77359, 77923, 77838), Thermus antranikianii (YIM 73052, 77412, 77311, 71206), Thermus brokianus (YIM 73518, 71318, 72351), Thermus hydrothermalis (YIM 730264 and 77927) and one potential novel species 77420 forming clade with Thermus thalpophilus SYSU G00506 T . Although the genomes of different strains of Thermus of same species were highly similar in their metabolic pathways, but subtle differences were found. CRISPR loci were detected through genome-wide screening, which showed that Thermus isolates from two different thermal locations had well developed defense system against viruses and adopt similar strategy for survival. Additionally, comparative genome analysis screened competence loci across all the Thermus genomes which could be helpful to acquire DNA from environment. In the present study it was found that Thermus isolates use two mechanism of incomplete denitrification pathway, some Thermus strains produces nitric oxide while others nitrious oxide (dinitrogen oxide), which show the heterotrophic lifestyle of Thermus genus. All isolated organisms encoded complete pathways for glycolysis, tricarboxylic acid and pentose phosphate. Calvin Benson Bassham cycle genes were identified in genomes of T. oshimai and T. antranikianii strains, while genomes of all T. brokianus strains and organism 77420 were lacking. Arsenic, cadmium and cobalt-zinc-cadmium resistant genes were detected in genomes of all sequenced Thermus strains. Strains 77,420, 77,311, 73,518, 77,412 and 72,351 genomes were found harboring genes for siderophores production. Sox gene clusters were identified in all sequenced genomes, except strain YIM 730264, suggesting a mode of chemolithotrophy. Through the comparative genomic analysis, we also identified 77420 as the genome type species and its validity as novel organism was confirmed by whole genome sequences comparison. Although isolate 77420 had 99.0% 16S rRNA gene sequence similarity with T. thalpophilus SYSU G00506 T but based on ANI 95.86% (Jspecies) and digital DDH 68.80% (GGDC) values differentiate it as a potential novel species. Similarly, in the phylogenomic tree, the novel isolate 77,420 forming a separate branch with their closest reference type strain T. thalpophilus SYSU G00506 T ., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Terrestrial geothermal springs are physicochemically diverse and host abundant populations of Archaea. However, the diversity, functionality, and geological influences of these Archaea are not well understood. Here we explore the genomic diversity of Archaea in 152 metagenomes from 48 geothermal springs in Tengchong, China, collected from 2016 to 2021. Our dataset is comprised of 2949 archaeal metagenome-assembled genomes spanning 12 phyla and 392 newly identified species, which increases the known species diversity of Archaea by ~48.6%. The structures and potential functions of the archaeal communities are strongly influenced by temperature and pH, with high-temperature acidic and alkaline springs favoring archaeal abundance over Bacteria. Genome-resolved metagenomics and metatranscriptomics provide insights into the potential ecological niches of these Archaea and their potential roles in carbon, sulfur, nitrogen, and hydrogen metabolism. Furthermore, our findings illustrate the interplay of competition and cooperation among Archaea in biogeochemical cycles, possibly arising from overlapping functional niches and metabolic handoffs. Taken together, our study expands the genomic diversity of Archaea inhabiting geothermal springs and provides a foundation for more incisive study of biogeochemical processes mediated by Archaea in geothermal ecosystems., (© 2024. The Author(s).)

A Gram-stain-negative, non-endospore-forming, motile, short rod-shaped strain, designated SYSU G07232 T , was isolated from a hot spring microbial mat, sampled from Rehai National Park, Tengchong, Yunnan Province, south-western China. Strain SYSU G07232 T grew at 25-50 °C (optimum, 37 °C), at pH 5.5-9.0 (optimum, pH 6.0) and tolerated NaCl concentrations up to 1.0 % (w/v). Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain SYSU G07232 T showed closest genetic affinity with Chelatococcus daeguensis K106 T . The genomic features and taxonomic status of this strain were determined through whole-genome sequencing and a polyphasic approach. The predominant quinone of this strain was Q-10. Major cellular fatty acids comprised C 19 : 0 cyclo ω 8 c and summed feature 8. The whole-genome length of strain SYSU G07232 T was 4.02 Mbp, and the DNA G+C content was 69.26 mol%. The average nucleotide identity (ANIm ≤84.85 % and ANIb ≤76.08  %) and digital DNA-DNA hybridization (≤ 21.9 %) values between strain SYSU G07232 T and the reference species were lower than the threshold values recommended for distinguishing novel prokaryotic species. Thus, based on the provided phenotypic, phylogenetic, and genetic data, it is proposed that strain SYSU G07232 T (=KCTC 8141 T =GDMCC 1.4178 T ) be designated as representing a novel species within the genus Chelatococcus , named Chelatococcus albus sp. nov.

High-temperature geothermal springs host simplified microbial communities; however, the activities of individual microorganisms and their roles in the carbon cycle in nature are not well understood. Here, quantitative stable isotope probing (qSIP) was used to track the assimilation of 13 C-acetate and 13 C-aspartate into DNA in 74 °C sediments in Gongxiaoshe Hot Spring, Tengchong, China. This revealed a community-wide preference for aspartate and a tight coupling between aspartate incorporation into DNA and the proliferation of aspartate utilizers during labeling. Both 13 C incorporation into DNA and changes in the abundance of taxa during incubations indicated strong resource partitioning and a significant phylogenetic signal for aspartate incorporation. Of the active amplicon sequence variants (ASVs) identified by qSIP, most could be matched with genomes from Gongxiaoshe Hot Spring or nearby springs with an average nucleotide similarity of 99.4%. Genomes corresponding to aspartate primary utilizers were smaller, near-universally encoded polar amino acid ABC transporters, and had codon preferences indicative of faster growth rates. The most active ASVs assimilating both substrates were not abundant, suggesting an important role for the rare biosphere in the community response to organic carbon addition. The broad incorporation of aspartate into DNA over acetate by the hot spring community may reflect dynamic cycling of cell lysis products in situ or substrates delivered during monsoon rains and may reflect N limitation., (© 2023. The Author(s).)

A xylanase gene (named xyngmqa) was identified from the metagenomic data of the Gumingquan hot spring (92.5 °C, pH 9.2) in Tengchong City, Yunnan Province, southwest China. It showed the highest amino acid sequence identity (82.70%) to endo-1,4-beta-xylanase from Thermotoga caldifontis. A constitutive expression plasmid (denominated pSHY211) and double-layer plate (DLP) method were constructed for cloning, expression, and identification of the XynGMQA gene. The XynGMQA gene was synthesized and successfully expressed in Escherichia coli DH5α. XynGMQA exhibited optimal activity at 90 °C and pH 4.6, being thermostable by maintaining 100% of its activity after 2 h incubated at 80 °C. Interestingly, its enzyme activity was enhanced by high temperatures (70 and 80 °C) and low pH (3.0-6.0). About 150% enzyme activity was detected after incubation at 70 °C for 20 to 60 min or 80 °C for 10 to 40 min, and more than 140% enzyme activity after incubation at pH 3.0 to 6.0 for 12 h. Hydrolytic products of beechwood xylan with XynGMQA were xylooligosaccharides, including xylobiose (X2), xylotriose (X3), and xylotetraose (X4). These properties suggest that XynGMQA as an extremely thermophilic xylanase, may be exploited for biofuel and prebiotic production from lignocellulosic biomass., (© 2023. Springer Nature Limited.)

The recent discovery of Nitrospira species capable of complete ammonia oxidation (comammox) in non-marine natural and engineered ecosystems under mesothermal conditions has changed our understanding of microbial nitrification. However, little is known about the occurrence of comammox bacteria or their ability to survive in moderately thermal and/or hyperthermal habitats. Here, we report the wide distribution of comammox Nitrospira in five terrestrial hot springs at temperatures ranging from 36 to 80°C and provide metagenome-assembled genomes of 11 new comammox strains. Interestingly, the identification of dissimilatory nitrate reduction to ammonium (DNRA) in thermophilic comammox Nitrospira lineages suggests that they have versatile ecological functions as both sinks and sources of ammonia, in contrast to the described mesophilic comammox lineages, which lack the DNRA pathway. Furthermore, the in situ expression of key genes associated with nitrogen metabolism, thermal adaptation, and oxidative stress confirmed their ability to survive in the studied hot springs and their contribution to nitrification in these environments. Additionally, the smaller genome size and higher GC content, less polar and more charged amino acids in usage profiles, and the expression of a large number of heat shock proteins compared to mesophilic comammox strains presumably confer tolerance to thermal stress. These novel insights into the occurrence, metabolic activity, and adaptation of comammox Nitrospira in thermal habitats further expand our understanding of the global distribution of comammox Nitrospira and have significant implications for how these unique microorganisms have evolved thermal tolerance strategies., (© 2023. The Author(s).)

Recent discoveries of methyl-coenzyme M reductase-encoding genes ( mcr ) in uncultured archaea beyond traditional euryarchaeotal methanogens have reshaped our view of methanogenesis. However, whether any of these nontraditional archaea perform methanogenesis remains elusive. Here, we report field and microcosm experiments based on 13 C-tracer labeling and genome-resolved metagenomics and metatranscriptomics, revealing that nontraditional archaea are predominant active methane producers in two geothermal springs. Archaeoglobales performed methanogenesis from methanol and may exhibit adaptability in using methylotrophic and hydrogenotrophic pathways based on temperature/substrate availability. A five-year field survey found Candidatus Nezhaarchaeota to be the predominant mcr -containing archaea inhabiting the springs; genomic inference and mcr expression under methanogenic conditions strongly suggested that this lineage mediated hydrogenotrophic methanogenesis in situ. Methanogenesis was temperature-sensitive , with a preference for methylotrophic over hydrogenotrophic pathways when incubation temperatures increased from 65° to 75°C. This study demonstrates an anoxic ecosystem wherein methanogenesis is primarily driven by archaea beyond known methanogens, highlighting diverse nontraditional mcr -containing archaea as previously unrecognized methane sources.

An isolate, designated CFH 74404 T , was recovered from a hot spring in Tengchong, Yunnan province, PR China. Phylogenetic analysis indicated that the isolate belongs to the family Thermomicrobiaceae and showed the highest 16S rRNA gene sequence similarity to Thermorudis peleae KI4 T (93.6 %), Thermorudis pharmacophila WKT50.2 T (93.1 %), Thermomicrobium roseum DSM 5159 T (92.0 %) and Thermomicrobium carboxidum KI3 T (91.7 %). The average amino acid identity and average nucleotide identity values between strain CFH 74404 T and the closest relatives were 42.0-75.9 % and 67.0-77.3 %, respectively. Cells of strain CFH 74404 T stained Gram-positive and were aerobic, non-motile and short rod-shaped. Growth occurred at 20-65 °C (optimum, 55 °C), pH 6.0-8.0 (optimum, pH 7.0) and with up to 2.0 % (w/v) NaCl (optimum 0-1.0 %, w/v). The predominant respiratory quinone was MK-8. The major fatty acids (>10 %) were C 18 : 0 (50.8 %) and C 20 : 0 (16.8 %). The polar lipid profile of strain CFH 74404 T included diphosphatidylglycerol, four unidentified phosphoglycolipids, phosphatidylinositol and three unidentified glycolipids. The G+C content of the genomic DNA was determined to be 67.1 mol% based on the draft genome sequence. On the basis of phenotypic, phylogenetic and genotypic analyses, it is concluded that strain CFH 74404 T represents a new species of a novel genus Thermalbibacter of the family Thermomicrobiaceae , for which the name Thermalbibacter longus gen. nov., sp. nov. is proposed. The type strain is CFH 74404 T (=KCTC 62930 T =CGMCC 1.61585 T ).

The biology of Korarchaeia remains elusive due to the lack of genome representatives. Here, we reconstruct 10 closely related metagenome-assembled genomes from hot spring habitats and place them into a single species, proposed herein as Panguiarchaeum symbiosum. Functional investigation suggests that Panguiarchaeum symbiosum is strictly anaerobic and grows exclusively in thermal habitats by fermenting peptides coupled with sulfide and hydrogen production to dispose of electrons. Due to its inability to biosynthesize archaeal membranes, amino acids, and purines, this species likely exists in a symbiotic lifestyle similar to DPANN archaea. Population metagenomics and metatranscriptomic analyses demonstrated that genes associated with amino acid/peptide uptake and cell attachment exhibited positive selection and were highly expressed, supporting the proposed proteolytic catabolism and symbiotic lifestyle. Our study sheds light on the metabolism, evolution, and potential symbiotic lifestyle of Panguiarchaeum symbiosum, which may be a unique host-dependent archaeon within the TACK superphylum., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

A novel anaerobic bacterium, designated SYSU GA19001 T , was isolated from a hot spring sediment sample. Phylogenetic analysis indicated that the isolate belongs to the genus Clostridium , and showed the highest sequence similarity to Clostridium swellfunianum CICC 10730 T (96.63 %) and Clostridium prolinivorans PYR-10 T (96.11 %). Cells of strain SYSU GA19001 T were Gram-stain-positive, spore-forming, rod-shaped (0.6-0.8×2.6-4.0 µm) and motile. Growth was observed at pH 5.0-9.0 (optimum, pH 7.0), 37-55 °C (optimum, 45 °C) and in NaCl concentrations of 0-2.0 % (optimum, 0 %). The genomic DNA G+C content was 31.62 %. The major cellular fatty acids of strain SYSU GA19001 T were C 14 : 0 , iso-C 15 : 0 , C 16 : 0 and summed feature 8. The prominent polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol. Meso -diaminopimelic acid was the diamino acid in peptidoglycan. Based on the results of phylogenetic, chemotaxonomic and phenotypic analyses, strain SYSU GA19001 T represents a novel species of the genus Clostridium , for which the name Clostridium caldaquaticum sp. nov. is proposed. The type strain of the proposed novel species is SYSU GA19001 T (=NBRC 115040 T = CGMCC 1.17864 T ).

Two closely related, aerobic, Gram-stain-negative, motile, oval-shaped, non-endospore-forming, moderately thermophilic bacteria, designated strains SYSU G05006 T and SYSU G05005, were isolated from a bioreactor enrichment and the original sample was collected from Rehai National Park, Tengchong, Yunnan Province, PR China. The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that these two strains were closely related to Caldovatus sediminis YIM 72346 T (96.75 and 96.89 % sequence similarity, respectively). The whole genome size of strain SYSU G05006 T was 3.87 Mbp with a DNA G+C content of 75.33 mol%. The average nucleotide identity (ANI based on the MUMmer algorithm≤90.31 % and ANI based on blast≤89.36 %) and digital DNA-DNA hybridization (≤35.10 %) values between strain SYSU G05006 T and other members of the family Acetobacteraceae were all lower than the threshold values recommended for distinguishing novel prokaryotic species. Optimal growth of the strain was observed at 55 °C and pH 6.0. Ubiquinone-10 was the predominant respiratory lipoquinone. The major cellular fatty acids included iso-C 14 : 0 , C 16 : 1 ω5 c , summed feature 5 and summed feature 7. The major polar lipids consisted of diphosphatidylglycerol, one unidentified aminolipid, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, two unidentified phospholipids and two unidentified lipids. Based on results of phylogenetic analyses, comparative genomics and phenotypic characteristics, we describe a new species of the genus Caldovatus represented by strain SYSU G05006 T (=KCTC 82831 T =MCCC 1K06125 T ), for which we propose the name Caldovatus aquaticus sp. nov.

Biological denitrification is a significant process in nitrogen biogeochemical cycle of terrestrial geothermal environments, and Thermus species have been shown to be crucial heterotrophic denitrifier in hydrothermal system. Five Gram-stain negative, aerobic and rod-shaped thermophilic bacterial strains were isolated from hot spring sediments in Tibet, China. Phylogenetic analysis based on 16S rRNA gene and whole genome sequences indicated that these isolates should be assigned to the genus Thermus and were most closely related to Thermus caldifontis YIM 73026 T , and Thermus brockianus YS38 T . Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the five strains and the type strains of the genus Thermus were lower than the threshold values (95% and 70%, respectively) recommended for bacterial species, which clearly distinguished the five isolates from other species of the genus Thermus and indicated that they represent independent species. Colonies are circular, convex, non-transparent. Cell growth occurred at 37-80 °C (optimum, 60-65 °C), pH 6.0-8.0 (optimum, pH 7.0) and with 0-2.0% (w/v) NaCl (optimum, 0-0.5%). Denitrification genes (narG, nirK, nirS, and norB genes) detected in their genomes indicated their potential function in nitrogen metabolism. The obtained results combined with those of morphological, physiological, and chemotaxonomic characteristics, including the menaquinones, polar lipids, and cellular fatty acids showed that the isolates are proposed as representing five novel species of the genus Thermus, which are proposed as Thermus hydrothermalis sp. nov. SYSU G00291 T , Thermus neutrinimicus sp. nov. SYSU G00388 T , Thermus thalpophilus sp. nov. SYSU G00506 T , Thermus albus sp. nov. SYSU G00608 T , Thermus altitudinis sp. nov. SYSU G00630 T ., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

A few members of the bacterial genus Thermus have been shown to be incomplete denitrifiers, terminating with nitrite (NO 2 - ) or nitrous oxide (N 2 O). However, the denitrification abilities of the genus as a whole remain poorly characterized. Here, we describe diverse denitrification phenotypes and genotypes of a collection of 24 strains representing ten species, all isolated from a variety of geothermal systems in China. Confirmed terminal products of nitrate reduction were nitrite or N 2 O, while nitric oxide (NO) was inferred as the terminal product in some strains. Most strains produced N 2 O; complete denitrification was not observed. Denitrification phenotypes were largely consistent with the presence of denitrification genes, and strains of the same species often had the same denitrification phenotypes and largely syntenous denitrification gene clusters. Genes for nirS and nirK coexisted in three Thermus brockianus and three Thermus oshimai genomes, which is a unique hallmark of some denitrifying Thermus strains and may be ecologically important. These results show that incomplete denitrification phenotypes are prominent, but variable, within and between Thermus species. The incomplete denitrification phenotypes described here suggest Thermus species may play important roles in consortial denitrification in high-temperature terrestrial biotopes where sufficient supply of oxidized inorganic nitrogen exists., (© 2022. The Author(s).)

A thermophilic bacterium, designated strain SYSU G04325 T , was isolated from a hot spring sediment in Yunnan, China. Polyphasic taxonomic analyses and whole-genome sequencing were used to determine the taxonomic position of the strain. Phylogenetic analysis using 16S rRNA gene sequences indicated that strain SYSU G04325 T shows high sequence similarity to Thermoflexibacter ruber NBRC 16677 T (86.2%). The strain can be differentiated from other species of the family Thermoflexibacteraceae by its distinct phenotypic and genotypic characteristics. Cells of the strain SYSU G04325 T were observed to be aerobic, Gram-stain negative and filamentous. Growth was found to occur optimally at 45 ºC and pH 7.0. In addition, the respiratory quinone was identified as menaquinone-7, while the major fatty acids (> 10%) were identified as iso-C 15:0 , iso-C 17:0 and Summed Feature 9 (iso-C 17:1 ω9c). The polar lipids detected included phosphatidylethanolamine, three unidentified phospholipids, one unidentified glycolipid, five unidentified aminolipids and four unidentified polar lipids. The G + C content of the genomic DNA was determined to be 47.6% based on the draft genome sequence. On the basis of phenotypic, genotypic and phylogenetic data, strain SYSU G04325 T is concluded to represent a novel species of a novel genus in the family Thermoflexibacteraceae, for which the name Rhodoflexus caldus gen. nov., sp. nov. is proposed. The type strain of Rhodoflexus caldus is SYSU G04325 T (= MCCC 1K06127 T  = KCTC 82848 T )., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Two novel species, designated strains SYSU G04041 T and SYSU G04536 T , were isolated from hot spring sediments collected in Yunnan, PR China. Phenotypic and chemotaxonomic analyses, and whole-genome sequencing were used to determine the taxonomic positions of the candidate strains. Phylogenetic analysis using 16S rRNA gene sequence indicated that strain SYSU G04041 T showed the highest sequence similarity to Thermomonas haemolytica A50-7-3 T (97.5 %), and SYSU G04536 T showed the highest sequence similarity to Thermomonas hydrothermalis SGM-6 T (98.2 %). The strains could be differentiated from other species of the genus Thermomonas by their distinct phenotypic and genotypic characteristics. Cells of strains SYSU G04041 T and SYSU G04536 T were aerobic, motile and Gram-stain-negative. Growth both occurred optimally at 45 °C and pH 7.0 for SYSU G04041 T and SYSU G04536 T . In addition, the predominant respiratory quinone in both isolates was ubiquinone Q-8. The major fatty acids (>10 %) of strain SYSU G04041 T were C 16 : 0 , iso-C 15 : 0 and iso-C 16 : 0 , while the major fatty acids (>10 %) of strain SYSU G04536 T were iso-C 15 : 0 and iso-C 16 : 0 . The main detected polar lipids in strains SYSU G04041 T and SYSU G04536 T included phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. The G+C contents of the genomic DNA of strains SYSU G04041 T and SYSU G04536 T based on draft genomic sequences were 72.5 and 68.3 %, respectively. On the basis of phenotypic, genotypic and phylogenetic data, strains SYSU G04041 T and SYSU G04536 T represent two novel species of the genus Thermomonas , for which the names Thermomonas flagellata sp. nov. and Thermomonas alba sp. nov. are proposed, with the type strains SYSU G04041 T (=CGMCC 1.19366 T =KCTC 92228 T ) and SYSU G04536 T (=CGMCC 1.19367 T =KCTC 82839 T ), respectively.

A novel Gram-stain-negative, non-motile, short rod-shaped and aerobic bacterial strain, designated as CFH 74456  T , was isolated from sediment of a hot spring, Tengchong, Yunnan Province, south-western China. Growth occurred at 20-53 ºC (optimum 45 ºC), pH 7.0-9.0 (optimum pH 8.0) and up to 2.0% (w/v) NaCl (optimum 0-1.0%, w/v). The predominant respiratory quinone was ubiquinone 10 (Q-10). The major fatty acids (> 10%) were C 17:1 ω6c (17.9%) and summed feature 8 (38.6%). The polar lipid profile of strain CFH 74456  T was identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, sphingoglycolipid, three unidentified glycolipids and three unidentified polar lipids. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain CFH 74456  T belongs to the genus Qipengyuania, and was most closely related to Qipengyuania sediminis CGMCC 1.12928  T (95.7%). The draft genome size of the isolate was 2.29 Mb with G + C content of 68.5%. The amino acid identity, average nucleotide identity and the digital DNA-DNA hybridization values between strain CFH 74456  T and the closest relatives ranged from 67.0 to 67.9%, 73.0 to 74.2% and 18.2-19.3%, respectively. On the basis of phenotypic, phylogenetic and genotypic analyses, it is concluded that strain CFH 74456  T represents a new species of the genus Qipengyuania, for which the name Qipengyuania thermophila sp. nov. is proposed. The type strain is CFH 74456  T (= KCTC 62921  T  = CCTCC AB 2018237  T )., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Three closely related, facultative anaerobic, Gram-stain-negative, twitching motile, short rod-shaped, non-endospore-forming, moderately thermophilic bacteria, designated strains SYSU G05001 T , SYSU G05003 and SYSU G05004, were isolated from a hot spring microbial mat, collected from Rehai National Park, Tengchong, Yunnan Province, south-western China. The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that these three strains were closely related to Thermus scotoductus SE-1 T (97.97, 98.18, 97.90 % sequence similarity). Whole genome sequencing and polyphasic taxonomic approach were used to determine the genomic profile and taxonomic status of the novel strain SYSU G05001 T . Cell growth occurred at 37-80 °C (optimum, 55 °C), pH 6.0-8.0 (optimum, pH 7.0) and with 0-3.0 % (w/v) NaCl (optimum, 1%). Thiosulfate enhanced cell growth. MK-8 was the predominant menaquinone. The major cellular fatty acids included iso-C 15 : 0 , iso-C 17 : 0 and anteiso-C 15 : 0 . The major polar lipids were consisted of aminophospholipid, glycolipid and phospholipids. The whole genome of strain SYSU G05001 T consisted of 2.55 Mbp and the DNA G+C content was 64.94 mol%. The average nucleotide identity (≤94.95 %) and digital DNA-DNA hybridization (≤62.3 %) values between strain SYSU G05001 T and other members of the genus Thermus were all lower than the threshold values recommended for distinguishing novel prokaryotic species. On the basis of the presented polyphasic evidence and genotypic data, it is proposed that strain SYSU G05001 T (=KCTC 82627 T =MCCC 1K06118 T ) represents a novel species of the genus Thermus , for which the name Thermus brevis sp. nov. is proposed.

Two bacterial strains SYSU G02173 T and SYSU G03142 were isolated from hot springs in Tibet, China. Based on the results of nearly full-length 16S rRNA gene sequences and phylogenetic analyses, strains SYSU G02173 T and SYSU G03142 were assigned to the family Sphingosinicellaceae, and were closest to Sandaracinobacter sibiricus RB16-17  T (96.04% and 96.12% similarity, respectively). Cells of the both new strains were observed to be motile rod-shape, Gram-staining negative. Growth occurred at pH 6-8 (optimal: pH 7.0) and 37-55 °C (optimal: 45 °C) with 0-1.0% (w/v) NaCl in T4 broth. The cells were found to be positive for oxidase and catalase activities. The major respiratory ubiquinone was Q-8. The major fatty acids were identified as summed feature 8 (C 18:1 ω7c and/or C 18:1 ω6c), C 16:0 , C 14:0 2-OH. The major polar lipids were found to consist of sphingoglycolipid, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified phospholipid, one unidentified glycolipid, three unidentified aminolipids and two unidentified polar lipids. The DNA G + C contents of strains SYSU G02173 T and SYSU G03142 were 71.8%. The average nucleotide identity (ANI) value between strain SYSU G02173 T and SYSU G03142 was 99.98%. The amino acid identity (AAI) values between them and their closely related species were below 66.14%. The isolates are characterized by aerobic growth, a yellow endocellular pigment and a higher optimum growth temperature. The results showed that strains SYSU G02173 T and SYSU G03142 represent a novel species of a novel genus in the family Sphingomonadaceae, and thus the name Thermaurantiacus tibetensis (type strain SYSU G02173 T  = KCTC 72052  T  = CGMCC 1.16680  T ) is proposed.

Two anaerobic bacteria, designated strains SYSU GA16112 T and SYSU GA16107, were isolated from a hot spring in Tengchong County, Yunnan Province, south-west PR China. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains SYSU GA16112 T and SYSU GA16107 belong to the family Dysgonamonadaceae . Cells of strains SYSU GA16112 T and SYSU GA16107 were Gram-stain-negative, rod-shaped and non-motile. The major fatty acids (>10 %) of strains SYSU GA16112 T and SYSU GA16107 were identified as anteiso-C 15 : 0 and anteiso-C 17 : 0 3OH. The polar lipid profile of strain SYSU GA16112 T was found to consist of phosphatidylethanolamine, two unidentified aminophospholipids, two unidentified phosphoglycolipids, two unidentified aminolipids and one unidentified polar lipid, while that of strain SYSU GA16107 consisted of phosphatidylethanolamine, two unidentified polar lipids, three unidentified aminophospholipids, two unidentified phosphoglycolipids and one unidentified aminolipid. The genomic DNA G+C contents of strains SYSU GA16112 T and SYSU GA16107 were determined to be 41.90 and 41.89 %, respectively, and the average nucleotide identity value between them was 99.99 %. Based on their morphological and physiological properties, and results of phylogenetic analyses, strains SYSU GA16112 T and SYSU GA16107 are considered to represent a novel species of a novel genus, for which the name Seramator thermalis gen. nov., sp. nov. (type strain SYSU GA16112 T =CGMCC 1.5281 T =KCTC 15753 T ) is proposed.

Lignocellulose is considered a major source of renewable energy that serve as an alternative to the fossil fuels. Members of the genus Clostridium are some of the many microorganisms that have the ability to degrade lignocellulose efficiently to sugar, which can be further converted to biofuel. In this study, we isolated twelve Clostridium strains from hot spring samples of Yunnan and Tibet, of which isolates SYSU GA15002 T and SYSU GA17076 showed low 16S rRNA gene sequence identity profiles to any of the validly named Clostridium strains (<94.0%). Studies using a polyphasic taxonomy approach concluded that the two isolates represent one novel species of the genus Clostridium, for which we propose the name Clostridium thermarum sp. nov., with SYSU GA15002 T as the type strain of the species. Isolate SYSU GA15002 T has an optimum growth temperature at 45°C. Fermentation of the substrates cellobiose, cellulose, xylan and untreated straw powder by this strain results in the production of ethanol, along with acetate and formate. The complete pathways for the conversion of cellulose and xylan to ethanol is also predicted from the genome of isolate SYSU GA15002 T , which revealed a single step conversion of lignocellulosic biomass through consolidated bioprocessing. This paper is a comprehensive study encompassing isolation, polyphasic taxonomy, lignocellulose biodegradation and the genomic information of Clostridium in Yunnan-Tibet hot springs., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Two novel Gram-stain negative, moderately thermophilic, aerobic, rod-shaped strains, designated 3D203 T and 3D207, were isolated from hot spring sediment samples collected from Tibet, western China. Phylogenetic analyses based on 16S rRNA gene sequence similarities showed that two isolates belonged to the genus Microvirga and were most closely related to Microvirga makkahensis SV1470 T (98.5% and 98.4%, respectively) and two strains had 99.8% similarity to each other. The average nucleotide identity (ANI) based on whole genome sequences of two strains and M. makkahensis SV1470 T was 80.8% and 80.78%, respectively. Optimum growth was observed at 45 °C, pH 7.0 and 0.5% NaCl. They both could tolerate to high concentration arsenic. Ubiquinone 10 (Q10) was their predominant quinone. The differences of strains 3D203 T and 3D207 were phosphatidyl dimethyl ethanolamine, phosphatidyl-N-methylethanolamine, phosphatidylglycerol, unidentified glycolipids and unidentified lipids. The major fatty acids (> 5%) were identified C 18:1 ω7c and/or C 18:1 ω6c, C 18:0 and C 16:0 . The genomic DNA G + C contents of strain 3D203 T and 3D207 based on whole genome sequences were 64.8% and 64.7%, respectively. Phenotypic, chemotaxonomic, phylogenetic and genomic analyses suggested that two strains represent a novel species of the genus Microvirga, for which the name Microvirga arsenatis sp. nov. is proposed. The type strain is 3D203 T (= CGMCC 1.17691 T  = KCTC 72653 T ).

The perplexity of the complex multispecies community interactions is one of the many reasons why majority of the microorganisms are still uncultivated. We analyzed the entire co-occurrence networks between the OTUs of Tibet and Yunnan hot spring samples, and found that less abundant OTUs such as genus Tepidimonas (relative abundant <1%) had high-degree centricity (key nodes), while dominant OTUs particularly genus Chloroflexus (relative abundant, 13.9%) formed the peripheral vertexes. A preliminary growth-promotion assay determined that Tepidimonas sp. strain SYSU G00190W enhanced the growth of Chloroflexus sp. SYSU G00190R. Exploiting this result, an ameliorated isolation medium containing 10% spent-culture supernatant of Tepidimonas sp. strain SYSU G00190W was prepared for targeted isolation of Chloroflexi in the Tibet and Yunnan hot spring samples. 16S rRNA gene fingerprinting characterized majority of the colonies isolated from these media as previously uncultivated Chloroflexi, of which 36 are potential novel species (16S rRNA sequence identity <98.5%). Metabolomes studies indicated that the spent-culture supernatant comprises several low-molecular-weight organic substrates that can be utilized as potential nutrients for the growth of these bacteria. These findings suggested that limited knowledge on the interaction of microbes provide threshold to traditional isolation method.

A novel thermotolerant bacterial strain was isolated from a hot spring in a Tibetan geothermal field. Phylogenetic analysis of the 16S rRNA gene sequence of the novel strain showed that it belongs to a distinct lineage far from any known taxa. The new isolate shared the highest pairwise sequence identity with Phreatobacter cathodiphilus S-12 T (92.8 % similarity) according to the 16S rRNA gene sequences. Cells were Gram-stain-negative, aerobic, rod-shaped and formed white round colonies. The strain grew at the ranges of 28-45 °C (optimum, 37 °C), pH 5.0-7.0 (optimum, pH 6.0) and 0-2 % NaCl. The strain was positive for catalase and oxidase. The major respiratory quinone was ubiquinone Q-10. Polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine. The major fatty acids were summed feature 8 (C 18 : 1   ω7 c and/or C 18 : 1   ω6 c ). The DNA G+C content was 68.3 mol%. Based on these distinguishing properties, this strain is proposed to represent a new species of a new genus Rhabdaerophilum calidifontis gen. nov., sp. nov., within a new family Rhabdaerophilaceae fam. nov. The type strain of the type species of Rhabdaerophilum calidifontis is SYSU G02060 T (=KCTC 72351 T =CGMCC 1.17070 T ).

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas), such as Cas9 and Cpf1, are RNA-guided endonucleases that target and degrade nucleic acids, providing powerful genomic editing and molecular diagnostic tools. Cas12b enzymes are distinct effectors; however, their features and catalytic boundaries require further characterization. We identified BrCas12b from the thermophile bacterium Brevibacillus sp. SYSU G02855 as a novel ortholog of cas12b. Biochemical analyses revealed that BrCas12b is a dual-RNA-guided endonuclease with higher optimum reaction temperature than that of other reported members of Cas12b. The seed sequence of BrCas12b is only 4 nt in length, indicating that it has greater target mismatch tolerance than that of previously reported Cas effectors; however, it contains a compensatory effect at the position of the cleavage site. Using fluorescence-based detection method to evaluate target cleavage efficiency, we showed that BrCas12b has robust enzymatic cleavage activity (K cat /K m (s - 1  M - 1 ) = 8.80 × 10 11 ), which is significantly higher than that of AacCas12b (K cat /K m (s - 1  M - 1 ) = 7.56 × 10 8 ) from Alicyclobacillus acidoterrestris. The results increase our understanding of the catalytic mechanism of Cas12b family members and suggest that BrCas12b might be useful in the application of genomic editing and molecular diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The present study aimed to determine the taxonomic positions of strains designated R-5-52-3 T , R-5-33-5-1-2, R-5-48-2 and R-5-51-4 isolated from hot spring water samples. Cells of these strains were Gram-stain-negative, non-motile and rod-shaped. The strains shared highest 16S rRNA gene sequence similarity with Vulcaniibacterium thermophilum KCTC 32020 T (95.1%). Growth occurred at 28-55 °C, at pH 6-8 and with up to 3 % (w/v) NaCl. DNA fingerprinting, biochemical, phylogenetic and 16S rRNA gene sequence analyses suggested that R-5-52-3 T , R-5-33-5-1-2, R-5-48-2 and R-5-51-4 were different strains but belonged to the same species. Hence, R-5-52-3 T was chosen for further analysis and R-5-33-5-1-2, R-5-48-2 and R-5-51-4 were considered as additional strains of this species. R-5-52-3 T possessed Q-8 as the only quinone and iso-C 15:0 , iso-C 11:0 , C 16 : 0 and iso-C 17 : 0 as major fatty acids. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unidentified polar lipids and two unidentified phospholipids. The genomic G+C content was 71.6 mol%. Heat shock proteins (e.g. Hsp20, GroEL, DnaK and Clp ATPases) were noted in the R-5-52-3 T genome, which could suggest its protection in the hot spring environment. Pan-genome analysis showed the number of singleton gene clusters among Vulcaniibacterium members varied. Average nucleotide identity (ANI) values between R-5-52-3 T , Vulcaniibacterium tengchongense YIM 77520 T and V. thermophilum KCTC 32020 T were 80.1-85.8 %, which were below the cut-off level (95-96 %) recommended as the ANI criterion for interspecies identity. Thus, based on the above results, strain R-5-52-3 T represents a novel species of the genus Vulcaniibacterium , for which the name Vulcaniibacterium gelatinicum sp. nov. is proposed. The type strain is R-5-52-3 T (=KCTC 72061 T =CGMCC 1.16678 T ).

China is a hotspot for hot springs and during microbial diversity analysis of Tengchong hot spring, Yunnan province, south-west PR China, two strains designated SYSU G01001 T and SY-13 were isolated. SYSU G01001 T and SY-13 were Gram-stain-positive, motile and spore-forming. Colonies were white, circular, raised and punctiform. SYSU G01001 T and SY-13 grew at pH 6.0-9.0 (optimum pH 8.0) and at 23-37 °C (optimum 28 °C). The 16S rRNA gene sequence similarity between SYSU G01001 T and SY-13 was 99.6 % but these strains shared low sequence similarity with Paenibacillus azotifigens (97.5 %) indicating that they represented a novel species. On the basis of the results, SYSU G01001 T was selected for further investigations and SY-13 was considered to represent a second strain of the species. The cell wall peptidoglycan of SYSU G01001 T was meso -2,6-diaminopimelic acid and MK-7 was the only respiratory quinone. The polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), two unidentified aminolipids (AL), two unidentified amino phospholipids (APL), an unidentified phospholipid (PL) and an unidentified polar lipid (L). The G+C content of the genomic DNA was 53.9 mol%. The average nucleotide identity (ANIb and ANIm) values between SYSU G01001 T and Paenibacillus azotifigens LMG 29963 T were below the cut-off level (95-96 %) recommended as the average nucleotide identity (ANI) criterion for interspecies identity. On the basis of the above results strain SYSU G01001 T represents a novel species of the genus Paenibacillus , for which the name Paenibacillus tepidiphilus sp. nov. is proposed. The type strain is SYSU G01001 T (=KCTC 33952 T =CGMCC 1.13870 T ).

A thermophilic bacterium, designated SYSU G03088 T , was isolated from Moincer hot spring, Tibet, PR China. Polyphasic taxonomic analyses and whole-genome sequencing were used to determine the taxonomic position and genomic profiles of the strain. Phylogenetic analysis using 16S rRNA gene sequence indicated that SYSU G03088 T showed highest sequence similarity to Rhodobacter blasticus CGMCC 1.3365 T (96.0 % sequence identity). The strain could be differentiated from most recognized species of the genus Rhodobacter by its slightly purple colony colour, distinct phenotypic characters and low ANI values. Cells were Gram-staining negative, and oval-to-rod shaped. Poly-β-hydroxybutyrate and vesicular intracytoplasmic membrane structures were formed inside cells. Growth occurred optimally at 45 °C and pH 7.0. Ubiquinone 10 was the only respiratory quinone. The major fatty acids (>10 %) were C 18 : 0 , C 18 : 1 ω7 c 11-methyl and summed feature 8 (C 18 : 1 ω7 c and/or C 18 : 1 ω6 c ). The detected polar lipids of SYSU G03088 T included diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylmethylethanolamine. The DNA G+C content of SYSU G03088 T was 67.7 % (genome). On the basis of the differences in the phenotypic characteristics and phylogenetic analyses, SYSU G03088 T is considered to represent a novel species of the genus Rhodobacter , for which the name Rhodobacter flagellatus sp. nov. is proposed. The type strain is SYSU G03088 T (=CGMCC 1.16876 T =KCTC 72354 T ).

A Gram-staining-positive, aerobic, non-motile, non-spore-forming and coccoid-shaped actinobacterial strain, designated YIM 730233 T , was isolated from a sediment sample, collected from a hot spring in Tibet, China. Colonies were brownish, circular, smooth and convex. Strain YIM 730233 T was able to grow in the temperature range of 20-50 °C, pH 6.5-8.0 and in the presence of up to 1.0 % (w/v) NaCl. A comparison of the 16S rRNA gene sequence of strain YIM 730233 T with sequences of type strains of most closely related species of Marmoricola showed highest sequence similarities to Marmoricola bigeumensis MSL-05 T (98.3%) and Marmoricola pocheonensis Gsoil 818 T (98.1%). The draft genome of strain YIM 730233 T had a size of 4 806 234 bp with a DNA G+C content of 72.1 mol%. The major fatty acids (>10 %) of strain YIM 730233 T mainly consisted of iso-C 16 : 0 , anteiso-C 17 : 0 and C 18 : 1 ω 9c , typical of the genus Marmoricola . Strain YIM 730233 T had LL-2,6-diaminopimelic acid as the diagnostic diamino acid in the cell wall. The predominant isoprenoid quinone was MK-8(H 4 ). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, unidentified phospholipids and an unidentified lipid. DNA-DNA hybridizations between strain YIM 730233 T and type strains of Marmoricola bigeumensis MSL-05 T and Marmoricola pocheonensis Gsoil 818 T resulted in similarity values of 21 and 19% respectively. Based on DNA-DNA hybridization results, together with the differentiating biochemical and chemotaxonomic features, showed that strain YIM 730233 T represents a novel Marmoricola species, for which the name Marmoricola caldifontis sp. nov. (type strain YIM 730233 T =KCTC 49192 T =CGMCC 4.7521 T ), is proposed.

A Gram-staining-negative, aerobic, curved rod-shaped and thermophilic bacterial strain, designated YIM 72297 T , was isolated from a sediment sample collected from a hot spring in Tengchong county, Yunnan province, south-west China. Growth was observed at pH 5.0-9.0 with an optimum of pH 7.0-7.5, and at 45-60 °C with an optimum of 55 °C. Positive for catalase and oxidase. The 16S rRNA gene sequence comparison indicated that strain YIM 72297 T was most closely related to Elioraea tepidiphila DSM 17972 T (96.9 %) and showed <91 % sequence similarities to members of the order Rhodospirillales . Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain YIM 72297 T formed a distinct lineage within the genus Elioraea , and revealed that the genus Elioraea formed a novel family-level clade in the order Rhodospirillales . The ANI and the dDNA-DNA hybridization estimate values between strains YIM 72297 T and Elioraea tepidiphila DSM 17972 T were 70.8 and 20.1 %, respectively. Strain YIM 72297 T contained Q-10 as the predominant ubiquinone. The major fatty acids (>5 %) were summed C 18 : 0 (35.8 %), summed feature 8 (30.1 %), C 16 : 0 (12.6 %), C 18 : 1 2OH (5.6 %) and C 16 : 0 2OH (5.4 %). The polar lipids consisted of phosphatidylcholine, phosphatidylethanolamine, diphosphosphatidylglycerol and phosphatidylglycerol in addition to two unidentified aminolipids. The DNA G+C content of YIM 72297 T was 70.8 mol% (draft genome). On the basis of the polyphasic taxonomic evidence presented in this study, strain YIM 72297 T should be classified as representing a novel species of the genus Elioraea , for which the name Elioraea thermophila sp. nov. is proposed, with the type strain YIM 72297 T (=CCTCC AB 2017169 T =KCTC 62323 T ). In addition, a novel family, Elioraeaceae fam. nov., is proposed to accommodate the genus Elioraea .

Thermus species are thermophilic heterotrophs, with most capable of using a variety of organic and inorganic electron donors for respiration. Here, a combined cultivation-independent and -dependent approach was used to explore the diversity of Thermus in Great Boiling Spring (GBS) and Little Hot Creek (LHC) in the US Great Basin. A cultivation-independent 16S rRNA gene survey of ten LHC sites showed that Thermus made up 0-3.5% of sequences and were predominately Thermus thermophilus. 189 Thermus isolates from GBS and LHC were affiliated with T. aquaticus (73.0%), T. oshimai (25.4%), T. sediminis (1.1%), and T. thermophilus (0.5%), with T. aquaticus and T. oshimai forming biogeographic clusters. 22 strains were selected for characterization, including chemolithotrophic oxidation of thiosulfate and arsenite, and reduction of ferric iron, polysulfide, and nitrate, revealing phenotypic diversity and broad respiratory capability within each species. PCR demonstrated the wide distribution of aerobic arsenite oxidase genes. A GBS sediment metaproteome contained sulfite oxidase and Fe 3+ ABC transporter permease peptides, suggesting sulfur and iron transformations in situ. This study expands our knowledge of the physiological diversity of Thermus, suggesting widespread chemolithotrophic and anaerobic respiration phenotypes, and providing a foundation for better understanding the ecology of this genus in thermal ecosystems.

A Gram-stain negative, aerobic bacterium, designated strain YIM 78456 T , was isolated from a hot spring sediment, Ngamring county, Tibet, south-west China. The taxonomic position of the isolate was investigated by a polyphasic approach. The novel isolate was found to be aerobic and rod-shaped. Colonies were observed to be pale yellow and circular. The strain was found to grow at pH 7.0-8.0 (optimum, pH 7.0), 45-65 °C (optimum, 55 °C) and in the presence of up to 1.5% NaCl. Comparison of the 16S rRNA gene sequence of strain YIM 78456 T and other members of the genus Thermus showed sequence similarities ranging from 90.3 to 97.3%, with strain YIM 78456 T showing close sequence similarity to Thermus caliditerrae YIM 77925 T (97.3%). The phylogenetic trees based on 16S rRNA gene sequences showed that strain YIM 78456 T forms a distinct clade with T. caliditerrae YIM 77925 T . The predominant menaquinone was identified as MK-8 and the DNA G+C content was determined to be 65.1 mol%. The major cellular fatty acids (> 10%) were identified as iso-C 15:0 , anteiso-C 15:0 and iso-C 17:0 . The polar lipids were found to consist of an aminophospholipid, a phospholipid and glycolipids. On the basis of the morphological and chemotaxonomic characteristics, as well as genotypic data, it is proposed that strain YIM 78456 T represents a novel species of the genus Thermus, for which the name Thermus caldilimi sp. nov. is proposed. The type strain is YIM 78456 T (= KCTC 52948 T  = NBRC 113036 T ).

Several recent studies have shown the presence of genes for the key enzyme associated with archaeal methane/alkane metabolism, methyl-coenzyme M reductase (Mcr), in metagenome-assembled genomes (MAGs) divergent to existing archaeal lineages. Here, we study the mcr-containing archaeal MAGs from several hot springs, which reveal further expansion in the diversity of archaeal organisms performing methane/alkane metabolism. Significantly, an MAG basal to organisms from the phylum Thaumarchaeota that contains mcr genes, but not those for ammonia oxidation or aerobic metabolism, is identified. Together, our phylogenetic analyses and ancestral state reconstructions suggest a mostly vertical evolution of mcrABG genes among methanogens and methanotrophs, along with frequent horizontal gene transfer of mcr genes between alkanotrophs. Analysis of all mcr-containing archaeal MAGs/genomes suggests a hydrothermal origin for these microorganisms based on optimal growth temperature predictions. These results also suggest methane/alkane oxidation or methanogenesis at high temperature likely existed in a common archaeal ancestor.

A moderately thermophilic, aerobic, Gram-stain-negative, non-spore-forming, rod-shaped and yellow-pigmented bacterium, designated strain SYSU G00007 T , was isolated from a hot spring slurry sample. Optimum growth was observed at 37-45 °C and pH 7. Pairwise comparison of the 16S rRNA gene sequence of strain SYSU G00007 T and other Novosphingobium species showed sequence similarities ranging from 93.7 to 97.9 %. Strain SYSU G00007 T showed highest sequence identity to Novosphingobium subterraneum DSM 12447 T (97.9 %). The average nucleotide identities and digital DNA-DNA hybridization values between strain SYSU G00007 T and its closely related phylogenetic neighbours were below 81 and 31 %, respectively, indicating that strain SYSU G00007 T represented a novel species of the genus Novosphingobium. The DNA G+C content of strain SYSU G00007 T was 64.3 % (genome). The major respiratory quinone was ubiquinone Q-10. The polar lipid profile included diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, two sphingoglycolipids, two unidentified phospholipids, two unidentified aminophospholipids and two unidentified polar lipids. Spermidine was the only polyamine detected. The major fatty acids were C19 : 0cyclo ω8c, summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and C16 : 0. The results obtained from phylogenetic, chemotaxonomic and phenotypic analyses support the conclusion that strain SYSU G00007 T represents a novel species of the genus Novosphingobium, for which we proposed the name Novosphingobiummeiothermophilum sp. nov. The type strain is SYSU G00007 T (=KCTC 52672 T =CCTCC AB2017010 T ).

An ovoid to rod-shaped, phototrophic, purple non-sulfur bacterium was isolated from a sediment sample of a hot spring in Tibet, China. Cells of strain YIM 73036 T were Gram-stain negative, non-motile and multiplied by binary fission. Strain YIM 73036 T grew optimally at pH 7.0-7.5 at 37-45 °C. Growth occurred in 0.5-3.5% (w/v) NaCl. Vitamins were not required for growth. The presence of photosynthesis genes pufL and pufM were shown and photosynthesis pigments were formed. Bacteriochlorophyll α, the bacteriopheophytin and carotenoids were present as photosynthetic pigments. Internal cytoplasmic membranes were of the lamellar type. The organism YIM 73036 T was able to grow chemo-organoheterophically, chemo-lithoautotrophically and photo-organoheterotrophically but photo-lithoautotrophic and fermentative growth were not demonstrated. Phylogenetic analysis on the basis of 16S rRNA gene sequences showed that strain YIM 73036 T is closely related to Rhodobacter blasticus ATCC 33485 T (96.65% sequence similarity) and clustered with species of the genus Rhodobacter of the family Rhodobacteraceae. Whole-genome sequence analyses based on the average nucleotide BLAST identity (ANI < 82%) indicated that this isolate belongs to a novel species. The genomic DNA G+C content of organism YIM 73036 T was determined to be 66.0 mol%. Strain YIM 73036 T contained Q-10 as the predominant ubiquinone and C 18:1 ω7c, C 18:1 ω7c 11-methyl and C 18:0 as the major fatty acids. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and unidentified phospholipid. Differential phenotypic and chemotaxonomic properties, together with the phylogenetic distinctiveness, demonstrated that strain YIM 73036 T is distinguishable from other species of the genus Rhodobacter. On the basis of the data presented, strain YIM 73036 T is considered to represent a novel species of the genus Rhodobacter, for which the name Rhodobacter thermarum sp. nov. [type strain YIM 73036 T (= KCTC 52712 T  = CCTCC AB 2016298 T )] is proposed.

Two Gram-stain-negative and facultative anaerobic bacteria, designated strains SYSU G00088 T and YIM 73032, were isolated from sediment samples collected from hot springs in Tibet, China. Based on the analyses of the 16S rRNA gene sequences, the two isolates were observed to be a member of the family Comamonadaceae, sharing highest pairwise sequence identity with type strains of Piscinibacter defluvii SH-1 T (97.8  and 97.7 %, respectively). They were able to grow in the temperature range of 37-50 °C, pH 5.0-9.0 and in the presence of up to 0.5 % (w/v) NaCl. Both strains were positive for catalase and oxidase reactions. Cells of the novel isolates were short-rods and motile by means of a polar flagellum. The chemotaxonomic features of the two strains include ubiquinone 8 as the respiratory isoprenologue, diphosphatidylglycerol, phosphatidylethanolamine and phoshatidylglycerol as the known polar lipids, and C16 : 0 and C17 : 0cyclo as major fatty acids. The genomic DNA of strains SYSU G00088 T and YIM 73032 had G+C contents of 71.8 and 71.9%, respectively. Based on the analyses of the phylogenetic, chemotaxonomic, morphological and genomic data, the two isolates are considered to represent a novel species of a new genus, for which the name Calidifontimicrobium sediminis gen. nov., sp. nov. is proposed. The type strain of Calidifontimicrobium sediminis is SYSU G00088 T (=KCTC 52671 T =CGMCC 1.13597 T ).

A Gram-stain-negative, aerobic, non-spore-forming and rod-shaped bacterium, designated YIM 730274 T , was isolated from a sediment sample collected from a hot spring located in Tibet, PR China, and was characterized by using a polyphasic taxonomy approach. Cells were motile by means of a polar flagellum. The strain was oxidase- and catalase-positive, and contained polyalkanoates and polyphosphate as storage polymers. Growth occurred at 25-50 °C, at pH 6.0-8.5 and with 0.5-1.0 % NaCl. The major fatty acids (>10 %) were summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and C16 : 0. The known polar lipids comprised of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylserine. The isoprenoid quinone was Q-8. The G+C content of genomic DNA was 70.7 mol%. The results of phylogenetic analyses based on 16S rRNA gene sequences indicated that the strain forms a monophyletic branch at the periphery of the evolutionary radiation occupied by the genus Aquabacterium in the class Betaproteobacteria. The most closely related phylogenetic neighbours were Aquabacterium limnoticumABP-4 T (97.8 % 16S rRNA gene sequence identity) and Aquabacterium communeB8 T (97.2 % 16SrRNA gene sequence identity). DNA-DNA relatedness values between YIM 730274 T and A. limnoticum KCTC 23306 T (46.4±0.4 %) and A. commune DSM 11901 T (42.2±1.2 %) were well below the 70 % limit for species identification. YIM 730274 T was distinguishable from other members of the genus Aquabacterium by the differences in phenotypic, chemotaxonomic and genotypic characteristics. YIM 730274 T merits recognition as a representative of a novel species of the genus Aquabacterium. It is proposed that the isolate should be classified in the genus Aquabacterium as representing a novel species, Aquabacteriumtepidiphilum sp. nov. The type strain is YIM 730274 T (=KCTC 52716 T =CCTCC AB 2016295 T ).

A Gram-stain positive, moderately thermophilic, aerobic, spore-forming and rod-shaped bacterium, designated YIM 73012 T , was isolated from a sediment sample collected from a hot spring located in Tibet, China, and was characterized by using a polyphasic taxonomy approach. The strain is oxidase positive and catalase negative. Growth occurred at 37-65 °C (optimum, 45-50 °C), at pH 6.0-8.5 (optimum, pH 7.0-7.5) and with 0.5-3.5% NaCl (optimum, 0.5-1.0%, w/v). The major fatty acids were iso-C 15:0 , iso-C 16:0 and C 16:0 . The major polar lipids comprised of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine and phosphatidylglycerol. The cell wall peptidoglycan contained meso-diaminopimelic acid. The respiratory quinone was MK-7. The G+C content of genomic DNA was 43.6 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain YIM 73012 T forms a distinct lineage with respect to the genus Anoxybacillus in the family Bacillaceae. Based on 16S rRNA gene sequence identities the closely related phylogenetic neighbours are Anoxybacillus caldiproteolyticus DSM 15730 T (96.7%) and Saccharococcus thermophilus DSM 4749 T (96.6%). Strain YIM 73012 T was distinguishable from the closely related reference strains by the differences in phenotypic, chemotaxonomic and genotypic characteristics, and represents a novel species of the genus Anoxybacillus, for which the name Anoxybacillus sp. nov. is proposed. The type species is Anoxybacillus sediminis sp. nov., with the type strain YIM 73012 T (= KCTC 33884 T  = DSM 103835 T ).

Two closely related, thermophilic bacteria, designated strains YIM 76954 T and YIM 76947, were isolated from the Rehai Geothermal Field, Tengchong, Yunnan province, south-west China. Polyphasic approach and whole genome sequencing were used to determine the taxonomy status and genomic profiles of the novel strains. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the two isolates were closely related to Thermus scotoductus SE-1 T (97.1% sequence similarity), and T. amyloliquefaciens YIM 77409 T (96.6%). The strains could be differentiated from most recognized Thermus species by their whitish to slight reddish colony color, distinct DNA fingerprinting profiles and low ANI values. Cells stained Gram-negative, rod-shaped of diameter 0.2-0.5μm and length 1.5-5.0μm. Growth occurred at 50-75°C, pH 6.0-9.0 and in the presence of up to 1.0% (w/v) NaCl concentration. Thiosulfate was found to enhance cell growth, besides improving the intensity of its colony color. Oxygen, nitrate, sulfur, and Fe(III) could be used as terminal electron acceptors for growth. MK-8 was the major respiratory menaquinone. Major fatty acids were iso-C 17:0 , iso-C 15:0 , anteiso-C 17:0 , and anteiso-C 15:0 . The genome size was 2.26Mbp with 65.5% average GC content. A total of 2374 genes was predicted, comprising 2322 protein-coding and 52 RNA genes. On the basis of the polyphasic evidence presented, it is proposed that strain YIM 76954 T represents a novel species of the genus Thermus, for which the name Thermus tenuipuniceus sp. nov. is proposed. The type strain is YIM 76954 T (=JCM 30350 T =KCTC 4677 T )., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

The taxonomic positions of two moderately thermophilic bacteria, strains YIM 72259 T and YIM 72238 T , isolated from a hot spring sediment sample were determined using polyphasic taxonomic approaches. Pairwise comparison of the 16S rRNA gene sequences showed that strains YIM 72259 T and YIM 72238 T are closely related to Tepidimonas ignava SPS-1037 T (97.0 and 97.5%, respectively), Tepidimonas taiwanensis I1-1 T (96.5 and 97.3%), while sharing 98.0% sequence similarity with each other. The DNA-DNA relatedness values of strains YIM 72238 T with T. ignava SPS-1037 T and T. taiwanensis I1-1 T were determined to be 25.2 ± 1.2 and 24.6 ± 3.1%, respectively, while the two strains (YIM 72259 T and YIM 72238 T ) had a relatedness value of 38.3 ± 4.4% between themselves. The two strains were aerobic, Gram-stain negative, motile by means of a polar flagellum and formed non-pigmented colonies. The DNA G+C contents of strains YIM 72259 T and YIM 72238 T were determined to be 71.6 and 68.9 mol%, respectively. The major fatty acids in both strains were identified as (> 10%) C 16:0 , C 17:0 and summed feature 8, while the strain YIM 72238 T contained a moderately high amount of summed feature 3 as well. The polar lipid profiles for strains YIM 72259 T and YIM 72238 T consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminolipid and several unidentified phospholipids. Besides the morphological and chemotaxonomic characteristics, phylogenetic analyses of the 16S rRNA gene sequences and the biochemical characteristics indicated that the two strains represented two novel members of the genus Tepidimonas, for which the name Tepidimonas sediminis sp. nov. [type strain YIM 72259 T (= NBRC 112410 T  = CGMCC 1.15971 T ) and Tepidimonas alkaliphilus sp. nov. [type strain YIM 72238 T (= KCTC 52717 T  = CCTCC AB 2017168 T )] are proposed.

Fungal communities represent an indispensable part of the geothermal spring ecosystem; however, studies on fungal community within hot springs are still scant. Here, we used Illumina HiSeq 2500 sequencing to detect fungal community diversity in extremely acidic hot springs (pH < 4) and neutral and alkaline springs (pH > 6) of Tengchong-indicated by the presence of over 0.75 million valid reads. These sequences were phylogenetically assigned to 5 fungal phyla, 67 order, and 375 genera, indicating unexpected fungal diversity in the hot springs. The genera such as Penicillium, Entyloma, and Cladosporium dominated the fungal community in the acidic geothermal springs, while the groups such as Penicillium, Engyodontium, and Schizophyllum controlled the fungal assemblages in the alkaline hot springs. The alpha-diversity indices and the abundant fungal taxa were significantly correlated with physicochemical factors of the hot springs particularly pH, temperature, and concentrations of Fe 2+ , NH 4 + , NO 2 , suggesting that the diversity and distribution of fungal assemblages can be influenced by the complex environmental factors of hot springs.-, and S 2- , suggesting that the diversity and distribution of fungal assemblages can be influenced by the complex environmental factors of hot springs.

A novel Gram-stain-positive, obligately anaerobic and thermophilic bacterium, strain SYSU GA15001 T , was isolated from a sample collected from a hot spring located in Tengchong, China. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain SYSU GA15001 T belonged to the family Eubacteriaceae, and was closely related to 'Irregularibacter muris' 2PG-426-CC4.2 T (93.6 % similarity), Garciella nitratireducens DSM 15102 T (93.2 %) and Alkalibacter saccharofermentans Z-78920 T (89.7 %). Cells were rods and motile by means of a subpolar flagellum. Spores were formed in old cultures. Optimum growth was observed at 45 °C, at pH 7 and in the presence of 1.5 % (w/v) NaCl. The strain utilized d-arabinose, dulcitol, d-galactose, glucose, lactose, maltose, d-mannose, raffinose, l-rhamnose, d-sorbitol, xylitol, l-alanine, l-arginine, glycine, l-isoleucine, l-leucine, dl-methionine, NH4Cl, l-proline and l-threonine as sole carbon/nitrogen sources. Additionally, complex substrates including beef extract, peptone, starch and yeast extract can also be used as sole energy sources. The major fatty acid methyl esters were C14 : 0, anteiso-C15 : 0 and C18 : 0. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, an unidentified polar lipid, three unidentified phospholipids and an unidentified glycolipid. The DNA G+C content was determined to be 35.6 mol%. Strain SYSU GA15001 T was physiologically and phylogenetically distinguishable from its closely related genera, and merits assignment as representing the type species of a new genus Rhabdanaerobium, with the name Rhabdanaerobium thermarum gen. nov., sp. nov. The type strain of the type species is SYSU GA15001 T (=CGMCC 1.5219 T =KCTC 15623 T ).

A Gram-stain-negative, ovoid-shaped, aerobic, non-motile, catalase- and oxidase-positive, and moderately thermophilic bacterial strain, designated strain YIM 72346 T , was isolated from a sediment sample collected from a hot spring in Tengchong county, Yunnan province, south-west China. Growth occurred at 37-50 °C (optimum, 45 °C), at pH 6.0-9.0 (optimum, pH 6.5-7.0) and in the presence of 0.5-1.0 % (w/v) NaCl (optimum, 0.5 %). The major cellular fatty acids were C18 : 1ω7c, C16 : 0, C19 : 0cyclo ω8c,and C18 : 1 2-OH. The genomic DNA G+C content was determined to be 69.8 mol%. The predominant ubiquinone was Q-10. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, one unidentified aminolipid and two unidentified phospholipids. Bacteriochlorophyll α and carotenoic acids were not detected. Strain YIM 72346 T was not observed for the accumulation of poly-β-hydroxybutyrate. The strain shared highest 16S rRNA gene sequence identities with Crenalkalicoccus roseus YIM 78023 T (93.3 %) and Craurococcus roseus NS130 T (92.7 %), but formed a distinct lineage within the family Acetobacteraceae in the phylogenetic trees. On the basis of genotypic, phenotypic, chemotaxonomic and phylogenetic analyses, strain YIM 72346 T is considered to represent a novel genus and species of the family Acetobacteraceae, for which the name Caldovatus sediminis gen. nov., sp. nov. is proposed. The type strain of Caldovatus sediminis is YIM 72346 T (=KCTC 52714 T =CGMCC 1.16330 T ).

Two Gram-negative, moderately thermophilic, yellow-pigmented, rod-shaped and motile bacterial strains, designated YIM 75003 T and YIM 75004 T , were isolated from sediment samples collected from the Tagejia hot spring in Tibet, western China. The taxonomic affiliation of the two strains was investigated by a polyphasic approach. Pairwise comparison of the 16S rRNA gene sequences showed that strains YIM 75003 T and YIM 75004 T are closely related to Altererythrobacter buctense M0322 T (97.2 and 97.1% respectively), while sharing 99.9% sequence similarity against each other. Optimum growth of the two strains was observed at 37-45 °C, pH 8.0 and in the presence of 1-6% NaCl (w/v). Their predominant respiratory quinone was found to be ubiquinone 10. The major fatty acids in both the strains were identified as summed feature 8 (C 18:1 ω6c and/or C 18:1 ω7c) and summed feature 4 (C 17:1 anteiso B and/or iso I). Their major polar lipid profiles were found to be diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and sphingoglycolipid. The DNA G+C contents of strains YIM 75003 T and YIM 75004 T were determined to be 61.3 and 60.1 mol%, respectively. The DNA-DNA hybridization values between strains YIM 75003 T and YIM 75004 T , and between the two strains and their closely related phylogenetic neighbour A. buctense M0322 T (=CGMCC 1.12871 T ) were less than 70%. Based on the morphological and physiological properties, phylogenetic analyses, chemotaxonomic characteristics and DNA-DNA relatedness values, the two strains can be distinguished from each other and from their phylogenetically closely related strain. Strains YIM 75003 T and YIM 75004 T are, therefore, considered to represent two novel species of the genus Altererythrobacter, for which the names Altererythrobacter lauratis sp. nov. (type strain YIM 75003 T  = KCTC 52606 T  = CCTCC AB2016268 T ) and Altererythrobacter palmitatis sp. nov. (type strain YIM 75004 T  = KCTC 52607 T  = CCTCC AB2016270 T ) are proposed.

A novel themophilic bacterium, designated YIM 79486T, was isolated from a sediment sample collected from Jinze hot spring in Tengchong county, Yunnan province, south-west China. Phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that strain YIM 79486T should be assigned to the genus Laceyella and formed a monophyletic clade with the type strain Laceyella putida KCTC 3666T (98.7 % similarity). Strain YIM 79486T formed white aerial mycelium and brown substrate mycelium. Abundant endospores were produced on short sporophores. Cell-wall peptidoglycan contained meso-diaminopimelic acid. The predominant menaquinones were MK-9 and MK-8. The genomic DNA G+C content observed for strain YIM 79486T was 47.8 mol%. Based on low DNA-DNA hybridization data, chemotaxonomic characteristics and differential physiological properties, strain YIM 79486T is considered to represent a novel species within the genus Laceyella, for which the name Laceyella thermophila sp. nov. is proposed. The type strain is YIM 79486T (=CCTC AB 2015040T=NBRC 110772T).

A slightly thermophilic, aerobic bacterium, designated YIM 72257T, was isolated from a sediment sample taken from a hot spring in Tengchong county, Yunnan province, south-west China. The isolate was Gram-stain-negative, non-sporulating and forms non-motile rods, appearing in chains. The isolate grew at 50-65 °C, pH 6.0-9.0 and with 0.5-1 % NaCl (w/v). 16S rRNA gene sequence analysis showed that strain YIM 72257T was most closely related to Meiothermus cateniformans LY1T (95.6 %), Meiothermus ruber DSM 1279T (95.1 %) and Meiothermus taiwanensis WR-30T (94.6 %). The genomic DNA G+C content of strain YIM 72257T was 62.6 mol%. The main cellular fatty acids (>5 %) were anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0, iso-C17 : 0, iso-C15 : 0 and C16 : 0. The polar lipids consisted of an uncharacterized phospholipid and two glycolipids. Based on phenotypic, phylogenetic and chemotaxonomic characteristics, strain YIM 72257T is proposed to be a representative of a novel species of the genus Meiothermus, for which the name Meiothermus luteus sp. nov. is proposed. The type strain is YIM 72257T (=KCTC 52599T=CCTCC AB 2017100T).

A thermophilic bacterial strain, designated YIM 73026T was isolated from a sediment sample collected from a hot spring in Tibet, PR China. The taxonomic position of the novel isolate was investigated by a polyphasic approach. The novel isolate was Gram-stain-negative, aerobic and rod-shaped. Colonies were circular, convex, opaque and yellow. The strain grew at 50-70 °C (optimum, 60 °C), pH 6.0-8.0 (optimum, pH 7.0) and in the presence of up to 1.0 % NaCl (w/v). Comparison of the 16S rRNA gene sequences of YIM 73026T and those of other members of the genus Thermus showed sequence similarities ranging from 91.2 to 97.5 %, with YIM 73026T showing closest sequence similarity to Thermus scotoductus SE-1T (97.5 %). DNA-DNA hybridization results, however, revealed that DNA-DNA reassociation values between YIM 73026T and T. scotoductus DSM 8553T (37.6 %), Thermusamyloliquefaciens YIM 77409T (34.5 %), Thermusantranikianii DSM 12462T (30.3 %), Thermuscaliditerrae YIM 77925T (28.6 %) and Thermustengchongensis YIM 77924T (27.3 %) were well below the 70 % limit for species identification. YIM 73026T contained MK-8 as the respiratory quinone, and iso-C15 : 0, iso-C16 : 0, iso-C17 : 0 and anteiso-C15 : 0 as the major cellular fatty acids (>10 %). The polar lipids consisted of one aminophospholipid, one phospholipid and two glycolipids. The genomic DNA G+C content of YIM 73026T was 65.4 mol%. On the basis of morphological, chemotaxonomic and genotypic characteristics, it is proposed that the isolate represents a novel species of the genus Thermus, for which the name Thermus caldifontis sp. nov. is proposed. The type strain is YIM 73026T (=NBRC 112415T=CCTCC AB 2016305T).

Chilas and Hunza areas, located in the Main Mantle Thrust and Main Karakoram Thrust of the Himalayas, host a range of geochemically diverse hot springs. This Himalayan geothermal region encompassed hot springs ranging in temperature from 60 to 95 °C, in pH from 6.2 to 9.4, and in mineralogy from bicarbonates (Tato Field), sulfates (Tatta Pani) to mixed type (Murtazaabad). Microbial community structures in these geothermal springs remained largely unexplored to date. In this study, we report a comprehensive, culture-independent survey of microbial communities in nine samples from these geothermal fields by employing a bar-coded pyrosequencing technique. The bacterial phyla Proteobacteria and Chloroflexi were dominant in all samples from Tato Field, Tatta Pani, and Murtazaabad. The community structures however depended on temperature, pH, and physicochemical parameters of the geothermal sites. The Murtazaabad hot springs with relatively higher temperature (90-95 °C) favored the growth of phylum Thermotogae, whereas the Tatta Pani thermal spring site TP-H3-b (60 °C) favored the phylum Proteobacteria. At sites with low silica and high temperature, OTUs belonging to phylum Chloroflexi were dominant. Deep water areas of the Murtazaabad hot springs favored the sulfur-reducing bacteria. About 40% of the total OTUs obtained from these samples were unclassified or uncharacterized, suggesting the presence of many undiscovered and unexplored microbiota. This study has provided novel insights into the nature of ecological interactions among important taxa in these communities, which in turn will help in determining future study courses in these sites.

Two closely related thermophilic bacterial strains, designated YIM 73013T and YIM 73008, were isolated from a sediment sample collected from a hot spring in Tibet, western Tibet province, China. The taxonomic positions of the two isolates were investigated using a polyphasic approach. The novel isolates were Gram-stain-negative, aerobic, short-rod-shaped and motile by means of a polar flagellum. They were oxidase- and catalase-positive and were able to grow at 30-55 °C (optimum, 37-45 °C), at pH 6.0-8.0 (optimum, pH 7.0) and with NaCl tolerance up to 1 % (w/v). Phylogenetic analyses based on 16S rRNA gene sequences showed that strains YIM 73013T and YIM 73008 formed a distinct lineage with respect to closely related genera in the family Comamonadaceae and shared highest 16S rRNA gene sequences similarities with Acidovorax caeni R-24608T (96.3 and 96.4 %, respectively). The respiratory quinone was ubiquinone-8 (Q-8) and the major cellular fatty acids observed were C17 : 1ω6c, C16 : 0 and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The genomic DNA G+C contents of strains YIM 73013T and YIM 73008 were 68.7 and 68.3 mol%, respectively. Based on the morphological, phylogenetic and chemotaxonomic results, the two isolates represent a novel species in a new genus, for which the name Tibeticola sediminis gen. nov., sp. nov. is proposed. The type strain of Tibeticola sediminis is YIM 73013T (=DSM 101684T=KCTC 42873T).

A Gram-staining positive, non-motile, rod-shaped, catalase positive and oxidase negative bacterium, designated NCCP-1331 T , was isolated from a hot water spring soil collected from Tatta Pani, Kotli, Azad Jammu and Kashmir, Pakistan. The isolate grew at a temperature range of 18-40 °C (optimum 30 °C), pH 6.0-9.0 (optimum 7.0) and with 0-6 % NaCl (optimum 2 % NaCl (w/v)). The phylogenetic analysis based on 16S rRNA gene sequence revealed that strain NCCP-1331 T belonged to the genus Streptomyces and is closely related to Streptomyces brevispora BK160 T with 97.9 % nucleotide similarity, followed by Streptomyces drosdowiczii NRRL B-24297 T with 97.8 % nucleotide similarity. The DNA-DNA relatedness values of strain NCCP-1331 T with S. brevispora KACC 21093 T and S. drosdowiczii CBMAI 0498 T were 42.7 and 34.7 %, respectively. LL-DAP was detected as diagnostic amino acid along with alanine, glycine, leucine and glutamic acid. The isolate contained MK-9(H 8 ) as the predominant menaquinone. Major polar lipids detected in NCCP-1331 T were phosphatidylethanolamine, phosphatidylinositol and unidentified phospholipids. Major fatty acids were iso-C 16: 0 , summed feature 8 (18:1 ω7c/18:1 ω6c), anteiso-C 15:0 and C 16:0 . The genomic DNA G + C content was 69.8 mol %. On the basis of phylogenetic, phenotypic and chemotaxonomic analysis, it is concluded that strain NCCP-1331 T represents a novel species of the genus Streptomyces, for which the name Streptomyces caldifontis sp. nov. is proposed. The type strain is NCCP-1331 T (=KCTC 39537 T  = CPCC 204147 T ).