Electrostatic Interactions Are the Primary Determinant of the Binding Affinity of SARS-CoV-2 Spike RBD to ACE2: A Computational Case Study of Omicron Variants.

To explore the mechanistic origin that determines the binding affinity of SARS-CoV-2 spike receptor binding domain (RBD) to human angiotensin converting enzyme 2 (ACE2), we constructed the homology models of RBD-ACE2 complexes of four Omicron subvariants (BA.1, BA.2, BA.3 and BA.4/5), and compared them with wild type complex (RBDWT-ACE2) in terms of various structural dynamic properties by molecular dynamics (MD) simulations and binding free energy (BFE) calculations. The results of MD simulations suggest that the RBDs of all the Omicron subvariants (RBDOMIs) feature increased global structural fluctuations when compared with RBDWT. Detailed comparison of BFE components reveals that the enhanced electrostatic attractive interactions are the main determinant of the higher ACE2-binding affinity of RBDOMIs than RBDWT, while the weakened electrostatic attractive interactions determine RBD of BA.4/5 subvariant (RBDBA.4/5) lowest ACE2-binding affinity among all Omicron subvariants. The per-residue BFE decompositions and the hydrogen bond (HB) networks analyses indicate that the enhanced electrostatic attractive interactions are mainly through gain/loss of the positively/negatively charged residues, and the formation or destruction of the interfacial HBs and salt bridges can also largely affect the ACE2-binding affinity of RBD. It is worth pointing out that since Q493R plays the most important positive contribution in enhancing binding affinity, the absence of this mutation in RBDBA.4/5 results in a significantly weaker binding affinity to ACE2 than other Omicron subvariants. Our results provide insight into the role of electrostatic interactions in determining of the binding affinity of SARS-CoV-2 RBD to human ACE2.

Characterization of a Cu2+, SDS, alcohol and glucose tolerant GH1 ß-glucosidase from Bacillus sp. CGMCC 1.16541.

A ß-glucosidase gene (bsbgl1a) from Bacillus sp. CGMCC 1.16541 was expressed in Escherichia coli BL21 and subsequently characterized. The amino acid sequence shared 83.64% identity with ß-glucosidase (WP_066390903.1) from Fictibacillus phosphorivorans. The recombinant ß-glucosidase (BsBgl1A) had a molecular weight of 52.2 kDa and could hydrolyze cellobiose, cellotriose, cellotetrose, p-nitrophenyl-ß-D-glucopyranoside (pNPG), and p-nitrophenyl-ß-D-xylopyranoside (pNPX). Optimal activity for BsBgl1A was recorded at 45 °C with a pH between 5.6 and 7.6, and 100% of its activity was maintained after a 24 h incubation between pH 4 and 9. Kinetic characterization revealed an enzymatic turnover (Kcat) of 616 ± 2 s-1 (with cellobiose) and 3.5 ± 0.1 s-1 (with p-nitrophenyl-ß-D-glucopyranoside). Interestingly, the recombinant enzyme showed cupric ion (Cu2+), sodium dodecyl sulfate (SDS) and alcohol tolerance at 10 mM for Cu2+ and 10% for both SDS and alcohol. Additionally, BsBgl1A had high tolerance for glucose (Ki = 2095 mM), which is an extremely desirable feature for industrial applications. Following the addition of BsBgl1A (0.05 mg/ml) to a commercial cellulase reaction system, glucose yields from sugarcane bagasse increased 100% after 1 day at 45 °C. This work identifies a Cu2+, SDS, alcohol, and glucose tolerant GH1 ß-glucosidase with potential applications in the hydrolysis of cellulose for the bioenergy industry.

Novosphingobium meiothermophilum sp. nov., isolated from a hot spring.

A moderately thermophilic, aerobic, Gram-stain-negative, non-spore-forming, rod-shaped and yellow-pigmented bacterium, designated strain SYSU G00007T, 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 G00007T and other Novosphingobium species showed sequence similarities ranging from 93.7 to 97.9 %. Strain SYSU G00007T showed highest sequence identity to Novosphingobium subterraneum DSM 12447T (97.9 %). The average nucleotide identities and digital DNA-DNA hybridization values between strain SYSU G00007T and its closely related phylogenetic neighbours were below 81 and 31 %, respectively, indicating that strain SYSU G00007T represented a novel species of the genus Novosphingobium. The DNA G+C content of strain SYSU G00007T 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 G00007T represents a novel species of the genus Novosphingobium, for which we proposed the name Novosphingobiummeiothermophilum sp. nov. The type strain is SYSU G00007T (=KCTC 52672T=CCTCC AB2017010T).

Thermus caldilimi sp. nov., a thermophilic bacterium isolated from a geothermal area.

A Gram-stain negative, aerobic bacterium, designated strain YIM 78456T, 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 78456T and other members of the genus Thermus showed sequence similarities ranging from 90.3 to 97.3%, with strain YIM 78456T showing close sequence similarity to Thermus caliditerrae YIM 77925T (97.3%). The phylogenetic trees based on 16S rRNA gene sequences showed that strain YIM 78456T forms a distinct clade with T. caliditerrae YIM 77925T. 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-C15:0, anteiso-C15:0 and iso-C17:0. 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 78456T represents a novel species of the genus Thermus, for which the name Thermus caldilimi sp. nov. is proposed. The type strain is YIM 78456T (= KCTC 52948T = NBRC 113036T).

Expression and characterisation of a pH and salt tolerant, thermostable and xylose tolerant recombinant GH43 ß-xylosidase from Thermobifida halotolerans YIM 90462T for promoting hemicellulose degradation.

A gene encoding a ß-xylosidase (designated as Thxyl43A) was cloned from strain Thermobifida halotolerans YIM 90462T. The open reading frame of this gene encodes 550 amino acid residues. The gene was over-expressed in Escherichia coli and the recombinant protein was purified. The monomeric Thxyl43A protein presented a molecular mass of 61.5 kDa. When p-nitrophenyl-ß-d-xylopyranoside was used as the substrate, recombinant Thxyl43A exhibited optimal activity at 55 °C and pH 4.0 to 7.0, being thermostable by maintaining 47% of its activity after 30 h incubation at 55 °C. The recombinant enzyme retained more than 80% residual activity after incubation at pH range of 4.0 to 12.0 for 24 h, respectively, which indicated notable thermostability and pH stability of Thxyl43A. Moreover, Thxyl43A displayed high catalytic activity (> 60%) in presence of 5-35% NaCl (w/v) or 1-20% ionic liquid (w/v) or 1-50 mM xylose. These properties suggest that Thxyl43A has potential for promoting hemicellulose degradation and other industrial applications.

Characterization of a neutral recombinant xylanase from Thermoactinospora rubra YIM 77501T.

A xylanase gene (TrXyn10) from Thermoactinospora rubra YIM 77501T was cloned and expressed in Escherichia coli. The amino acid sequence displayed 78% homology with Microbispora mesophila xylanase (WP_062413927.1). The recombinant xylanase (TrXyn10), with MW 46.1 kDa, could hydrolyse beechwood, birchwood and oatspelt xylan. Based on the sequence, enzymatic properties and tertiary structure of the protein, TrXyn10 belongs to glycoside hydrolase family 10 (GH10). The optimal pH and temperature for the recombinant enzyme were determined to be 7.0 and 55 °C, respectively. TrXyn10 was stable over a wide pH range, and it retained more than 45% of the total activity at pH 6.0-12.0 for 12 h. In addition, the activity was greatly promoted, by approximately 200% of the initial activity, after incubation at pH 6.0 and 7.0 for 12 h. Based on enzymatic properties and product analysis, we showed that TrXyn10 is a neutral endoxylanase.

Crenalkalicoccus roseus gen. nov., sp. nov., a thermophilic bacterium isolated from alkaline hot springs.

Two closely related thermophilic bacterial strains, designated YIM 78023T and YIM 78058, were isolated from samples collected from two alkaline hot springs in Tengchong county, Yunnan province, south-west China. The novel isolates were Gram-stain-negative, non-motile, aerobic ovoid- to coccoid-shaped and non-spore-forming. Strain YIM 78023T grew at 20-60 ºC and pH 6.0-9.0 with optimal growth observed at 40-50 ºC and pH 8.0, while strain YIM 78058 grew at 25-60 ºC and pH 6.0-10.0 with optimal growth at 45-50 ºC and pH 8.0. Phylogenetic analysis based on 16S rRNA gene sequences affiliated these two isolates within the family Acetobacteraceae with high sequence similarities to members of the genera Roseomonas and Belnapia (all sequence similarities <94.5 %). In addition to the above two genera, these strains also clustered with the genera Craurococcus and Paracraurococcus (having sequence similarities <93.3 %) in the phylogenetic tree, but with a distinct lineage within the family Acetobacteraceae. The major ubiquinone was Q-10 and the major fatty acids observed were C18:1ω7c, summed feature 4 and C16:0. The genomic DNA G+C contents observed for strains YIM 78023T and YIM 78058 were 74.3 and 74.0 mol%, respectively. Morphological, phylogenetic and chemotaxonomic results suggest that strains YIM 78023T and YIM 78058 are representatives of a novel species of a new genus within the family Acetobacteraceae, for which the name Crenalkalicoccus roseus gen. nov., sp. nov. is proposed. The type strain of Crenalkalicoccus roseus is YIM 78023T (=JCM 19657T=KACC 17825T).

Brevibacillus sediminis sp. nov., isolated from a hot spring.

Strain YIM 78300T, a novel Gram-stain-positive, moderately thermophilic, endospore-forming, rod-shaped, motile bacterium, was recovered from the sediment of a hot spring in the Tagejia Geothermal Field, Angren, Tibet province, western China. Optimum growth was observed at 50-55 °C, at pH 7.0 and with 0-1.5 % (w/v) NaCl. Phylogenetic analysis of the 16S rRNA gene sequence of strain YIM 78300T indicated that it belongs to the genus Brevibacillus. Similarity levels between the 16S rRNA gene sequences of the new isolate and those of the type strains of Brevibacillus members were 96.9-96.3 %; highest sequence similarity was with Brevibacillus thermoruber DSM 7064T. The predominant menaquinone was MK-7 and the major cellular fatty acids were iso-C15 : 0 and iso-C17 : 0. The major polar lipids were phosphatidyl-N-methylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, two unidentified phospholipids, an unidentified aminophospholipid and two unidentified polar lipids. The G+C content of the genomic DNA of strain YIM 78300T was 57.9 mol%. Based on phylogenetic analyses, and physiological and biochemical characteristics, strain YIM 78300T is considered to represent a novel species of the genus Brevibacillus, for which the name Brevibacillus sediminis sp. nov. is proposed. The type strain is YIM 78300T ( = DSM 29928T = CPCC 100738T).

Diversity of Culturable Thermophilic Actinobacteria in Hot Springs in Tengchong, China and Studies of their Biosynthetic Gene Profiles.

The class Actinobacteria has been a goldmine for the discovery of antibiotics and has attracted interest from both academics and industries. However, an absence of novel approaches during the last few decades has limited the discovery of new microbial natural products useful for industries. Scientists are now focusing on the ecological aspects of diverse environments including unexplored or underexplored habitats and extreme environments in the search for new metabolites. This paper reports on the diversity of culturable actinobacteria associated with hot springs located in Tengchong County, Yunnan Province, southwestern China. A total of 58 thermophilic actinobacterial strains were isolated from the samples collected from ten hot springs distributed over three geothermal fields (e.g., Hehua, Rehai, and Ruidian). Phylogenetic positions and their biosynthetic profiles were analyzed by sequencing 16S rRNA gene and three biosynthetic gene clusters (KS domain of PKS-I, KSα domain of PKS-II and A domain of NRPS). On the basis of 16S rRNA gene phylogenetic analysis, the 58 strains were affiliated with 12 actinobacterial genera: Actinomadura Micromonospora, Microbispora, Micrococcus, Nocardiopsis, Nonomuraea, Promicromonospora, Pseudonocardia, Streptomyces, Thermoactinospora, Thermocatellispora, and Verrucosispora, of which the two novel genera Thermoactinospora and Thermocatellisopora were recently described from among these strains. Considering the biosynthetic potential of these actinobacterial strains, 22 were positive for PCR amplification of at least one of the three biosynthetic gene clusters (PKS-I, PKS-II, and NRPS). These actinobacteria were further subjected to antimicrobial assay against five opportunistic human pathogens (Acinetobacter baumannii, Escherichia coli, Micrococcus luteus, Staphylococcus aureus and Streptococcus faecalis). All of the 22 strains that were positive for PCR amplification of at least one of the biosynthetic gene domains exhibited antimicrobial activities against at least one of the five test organisms. Among the remaining 36 actinobacteria that are negative for PCR amplification of the domains for the biosynthetic genes, 33 strains showed antimicrobial activities against at least one of the five test pathogens. In summary, the findings presented in this study emphasized the importance of underexplored habitats such as Tengchong hot springs as potential sources for search of bioactive molecules.

Crenobacter luteus gen. nov., sp. nov., isolated from a hot spring.

A slightly thermophilic, Gram-staining-negative and strictly aerobic bacteria, designated strain YIM 78141(T), was isolated from a sediment sample collected at Hehua hot spring, Tengchong, Yunnan province, south-west China. Cells of the strain were short-rod-shaped and colonies were yellowish and circular. The strain grew at pH 6.0-10.0 (optimum, pH 8.0-9.0) and 10-55 °C (optimum, 40-50 °C). Phylogenetic analyses based on 16S rRNA gene sequence comparison demonstrated that strain YIM 78141(T) belongs to the family Neisseriaceae, and strain YIM 78141(T) also showed low levels of 16S rRNA gene sequence similarity (below 93.4%) with all other genera in this family. The only quinone was ubiquinone 8 and the genomic DNA G+C content was 67.3 mol%. Major fatty acids (>5%) were C12:0, C16:0, C18:1ω7c and summed feature 3. The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phospholipids of unknown structure containing aminoglycophospholipid and three unidentified polar lipids. On the basis of the morphological, physiological and biochemical characteristics as well as genotypic data, this strain should be classified as a representative of a novel genus and species of the family Neisseriaceae, for which the name Crenobacter luteus gen. nov., sp. nov. is proposed. The type strain is YIM 78141(T) ( =BCRC 80650(T) =KCTC 32558(T) =DSM 27258(T)).

Hymenobacter mucosus sp. nov., isolated from a karst cave soil sample.

A novel Gram-stain-negative, non-motile, rod-shaped and watermelon-red-pigmented aerobic bacterial strain, designated YIM 77969T, was isolated from a soil sample of Jiuxiang cave, a tourism cave located in Yiliang county, Yunnan province, south-west China. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain YIM 77969T belongs to the genus Hymenobacter, and was closely related to Hymenobacter tibetensis XTM003T (96.58 %), Hymenobacter gelipurpurascens Txg1T (96.02 %) and Hymenobacter xinjiangensis X2-1gT (95.80 %). Growth of strain YIM 77969T occurred at 5-35 °C, at pH 5.0-9.0 and in the presence of 0-1 % (w/v) NaCl. The predominant menaquinone was MK-7. The major fatty acids were iso-C15 : 0, C16 : 1ω5c and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The polar lipid profiles consisted of the major compound phosphatidylethanolamine, two unknown aminolipids, three unknown aminophospholipids, one glycolipid and one unknown polar lipid. Pigment analysis showed that the pigment belonged to the plectaniaxanthin series of carotenoid pigments. The genomic DNA G+C content was 55.2 mol%. On the basis of phylogenetic, phenotypic and chemotaxonomic characteristics, strain YIM 77969T is considered to represent a novel species of the genus Hymenobacter, for which the name Hymenobacter mucosus sp. nov. is proposed. The type strain is YIM 77969T ( = KCTC 32567T = DSM 28041T).

Cecembia rubra sp. nov., a thermophilic bacterium isolated from a hot spring sediment.

A Gram-staining negative, rod-shaped bacterium, designated strain YIM 78110(T), was isolated from a sediment sample collected from Hehua hot spring in Tengchong, Yunnan province, south-west China. The taxonomic status of strain YIM 78110(T) was confirmed by a polyphasic approach. 16S rRNA gene sequence analysis indicated that strain YIM 78110(T) belongs to the genus Cecembia, displaying 96.8% and 94.7% sequence similarity with the two most closely related type strains, Cecembia calidifontis RQ-33(T) and Cecembia lonarensis LW9T, respectively. The low value of DNA-DNA hybridization (52.3 ± 2.3%) between strain YIM 78110(T) and its closest neighbour, Cecembia calidifontis RQ-33(T), indicated that this new isolate represented a different genomic species in the genus Cecembia. The temperature for growth ranged from 30 to 50 °C. The pH for growth ranged from pH 4.0 to 10.0, with NaCl tolerance of 0.5-6.0% (w/v). The predominant menaquinone of strain YIM 78110(T) was MK-7 and the major polar lipid was phosphatidylethanolamine. The major fatty acids were iso-C15:0 and C15:0. The DNA G+C content was 47.1 mol%. On the basis of physiological, biochemical and phylogenetic analyses, it is proposed that strain YIM 78110(T) represents a novel species of the genus Cecembia, for which the name Cecembia rubra sp. nov. is proposed. The type strain is YIM 78110(T) ( = CCTCC AB2013287(T) = DSM 28057(T)).

Meiothermus roseus sp. nov., a thermophilic bacterium isolated from a geothermal area.

Two closely related thermophilic bacterial strains, designated YIM 71031(T) and YIM 71039, were isolated from a hot spring in Tengchong county, Yunnan province, south-western China. The novel isolates were observed to be Gram-negative, aerobic, rod-shaped and yellow-pigmented bacteria. The strains were found to be able to grow at 37-65 °C, pH 6.0-9.0 and with a NaCl tolerance up to 1.0 % (w/v). Phylogenetic analysis based on 16S rRNA gene sequences placed these two isolates in the genus Meiothermus. They were found to be closely related to Meiothermus timidus DSM 17022(T) (98.6 % similarity), and formed a cluster with this species. The predominant menaquinone was identified as MK-8 and the major fatty acids (>10 %) as anteiso-C15:0, iso-C15:0, anteiso-C17:0, iso-C16:0 and C16:0. The genomic DNA G+C contents of strains YIM 71031(T) and YIM 71039 were determined to be 64.0 and 65.4 mol%, respectively. DNA-DNA hybridizations showed low values between strains YIM 71031(T) and YIM 71039 and their closely related neighbour M. timidus DSM 17022(T). Morphological phylogenetic and chemotaxonomic results suggest that strains YIM 71031(T) and YIM 71039 are representatives of a new species within the genus Meiothermus, for which the name Meiothermus roseus sp. nov. is proposed. The type strain is YIM 71031(T) (=KCTC 42495(T) =NBRC 110900(T)).

Heterologous expression and characterization of a novel halotolerant, thermostable, and alkali-stable GH6 endoglucanase from Thermobifida halotolerans.

A novel endoglucanase gene was cloned from Thermobifida halotolerans YIM 90462(T), designated as thcel6A for being a member of glycoside hydrolase family 6. The gene was 1332 bp long and encoded a 443-amino-acid protein with a molecular mass of 45.9 kDa. The purified recombinant endoglucanase had optimal activity at 55 °C and pH 8.5. Thcel6A showed high hydrolytic activities at 25-55 °C and retained 58% of initial activity after incubation at 90 °C for 1 h. It retained more than 80% of activity after incubation for 12 h at pH values from 4 to 12. Thcel6A displayed higher hydrolytic activities in 5-15% NaCl (w/v) than at 0% NaCl. Activity increased 2.5-fold after incubation with 20% (w/v) NaCl at 37 °C for 10 min. These properties suggest that this novel endoglucanase has potential for specific industrial application.

Thermus caliditerrae sp. nov., a novel thermophilic species isolated from a geothermal area.

Two thermophilic bacterial strains, designated YIM 77925(T) and YIM 77777, were isolated from two hot springs, one in the Hydrothermal Explosion (Shuirebaozhaqu) area and Frog Mouth Spring in Tengchong county, Yunnan province, south-western China. The taxonomic positions of the two isolates were investigated by a polyphasic approach. Cells of the two strains were Gram-stain-negative, aerobic and rod-shaped. They were able to grow at 50-70 °C, pH 6.0-8.0 and with a NaCl tolerance up to 0.5% (w/v). Colonies are circular, convex, non-transparent and produce yellow pigment. Phylogenetic analyses based on 16S rRNA gene sequences comparison clearly demonstrated that strains YIM 77925(T) and YIM 77777 represent members of the genus Thermus, and they also detected low-level similarities of 16S rRNA gene sequences (below 97%) compared with all other species in this genus. Their predominant menaquinone was MK-8. The genomic DNA G+C contents of strains YIM 77925(T) and YIM 77777 were 65.6 mol% and 67.2 mol%, respectively. Based on the results of physiological and biochemical tests and phylogenetic analyses, strains YIM 77925(T) and YIM 77777 could not be classified as representing any species of the genus Thermus with a validly published name. Thus the two strains are considered to represent a novel species of the genus Thermus, for which the name Thermus caliditerrae sp. nov. is proposed. The type strain is YIM 77925(T) ( = DSM 25901(T) = CCTCC 2012061(T)).

Geothermomicrobium terrae gen. nov., sp. nov., a novel member of the family Thermoactinomycetaceae.

Strains YIM 77562(T) and YIM 77580, two novel Gram-staining-positive, filamentous bacterial isolates, were recovered from the Rehai geothermal field, Tengchong, Yunnan province, south-west China. Good growth was observed at 50-55 °C and pH 7.0. Aerial mycelium was absent on all media tested. Substrate mycelium was well-developed, long and moderately flexuous, and formed abundant, single, warty, ornamented endospores. Phylogenetic analysis of the 16S rRNA gene sequences of the two strains indicated that they belong to the family Thermoactinomycetaceae. Similarity levels between the 16S rRNA gene sequences of the two strains and those of type strains of members of the Thermoactinomycetaceae were 88.33-93.24 %; the highest sequence similarity was with Hazenella coriacea DSM 45707(T). In both strains, the predominant menaquinone was MK-7, the diagnostic diamino acid was meso-diaminopimelic acid and the major cellular fatty acids were iso-C14 : 0, iso-C15 : 0 and iso-C16 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, unidentified polar lipids and unidentified phospholipids. The genomic DNA G+C contents of strains YIM 77562(T) and YIM 77580 were 45.5 and 44.2 mol%, respectively. DNA-DNA relatedness data suggest that the two isolates represent a single species. Based on phylogenetic analyses and physiological and biochemical characteristics, it is proposed that the two strains represent a single novel species in a new genus, Geothermomicrobium terrae gen. nov., sp. nov. The type strain of Geothermomicrobium terrae is YIM 77562(T) ( = CCTCC AA 2011022(T) = JCM 18057(T)).

Meiothermus terrae sp. nov., isolated from a geothermally heated soil sample.

A Gram-negative, aerobic bacterium, designated strain YIM 77755(T), was isolated from a geothermally heated soil sample collected at Rehai National Park, Tengchong, Yunnan province, south-west China. Cells of the strain were rod-shaped and colonies were yellow and circular. Growth occurred in 0-1 % (w/v) NaCl, at pH 6.0-8.0 (optimum, pH 7.0) and at 35-55 °C (optimum, 50 °C). The predominant menaquinone was MK-8 and the DNA G+C content was 68.9 mol%. Major fatty acids (>10 %) were anteiso-C15 : 0 and iso-C15 : 0. The polar lipids consisted of an uncharacterized phospholipid and four glycolipids. Based on 16S rRNA gene sequence analysis, strain YIM 77755(T) formed a cluster with Meiothermus chliarophilus ALT-8(T) and showed the highest 16S rRNA gene sequence similarity to M. chliarophilus ALT-8(T) (98.23 %). DNA-DNA relatedness between YIM 77755(T) and M. chliarophilus DSM 9957(T) was 54.9±4.1 %. On the basis of the morphological and chemotaxonomic characteristics as well as genotypic data, it is proposed that strain YIM 77755(T) represents a novel species of the genus Meiothermus, named Meiothermus terrae sp. nov. The type strain is YIM 77755(T) ( = DSM 26712(T) = CCTCC AB 2012942(T)).

Zhizhongheella caldifontis gen. nov., sp. nov., a novel member of the family Comamonadaceae.

An alkalitolerant, thermotolerant, strictly aerobic and Gram-staining negative bacterial strain, designated YIM 78140(T), was isolated from a water sample in Hehua hot spring, Tengchong, Yunnan province, south-west China. The colonies were light brown, convex and circular. Phylogenetic analysis of the 16S rRNA gene sequence of strain YIM 78140(T) indicated that it was clustered with members of ß-Proteobacteria (with the similarity from 96.9 to 93.6 %). Good growth occurred at 40-50 °C, pH 8.0-9.0 and in the presence of 0-3 % (w/v) NaCl. The predominant ubiquinones were Q-8 and Q-9. The major fatty acids were C16:0, C17:0 cyclo, C18:1 ω7c and summed feature 3. The G+C content of genomic DNA was 70.8 mol%. The results of physiological and biochemical characteristics, phylogenetic analysis allowed the phenotypic and genotypic differentiation of strain YIM 78140(T) from its closest phylogenetic neighbours. Therefore, the strain YIM 78140(T) represents a novel genus of the family Comamonadaceae, for which the name Zhizhongheella caldifontis gen. nov., sp. nov. is proposed. The type strain is YIM 78140(T) (= BCRC 80649(T) = KCTC 32557(T)).

Roseomonas alkaliterrae sp. nov., isolated from an alkali geothermal soil sample in Tengchong, Yunnan, South-West China.

An alkalitolerant, thermotolerant and Gram-stain negative bacterium, designated strain YIM 78007(T), was isolated from an alkaline geothermal soil sample from Hehua hot spring, Tengchong, Yunnan province, south-west China. Cells of strain YIM 78007(T) were observed to be aerobic and short rod-shaped. The colonies were observed to be orange-red, convex and circular. 16S rRNA gene sequence-based phylogenetic analysis showed that strain YIM 78007(T) clustered with members of the genus Roseomonas (with similarities from 97.2 to 92.2 %). Optimal growth of strain YIM 78007 occurs at 40-50 °C and pH 8.0-10.0. The predominant ubiquinone was identified as Q-10 and the major fatty acids were identified as C18:1 ω7c and C16:0. The polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, two unidentified aminolipids and one unknown phospholipid. The G + C content of the genomic DNA was determined to be 63 mol %. The levels of DNA-DNA hybridization relatedness between strain YIM 78007(T) and its closet neighbours (Roseomonas lacus JCM 13283(T) and Roseomonas terrae JCM 14592(T)) were well below the threshold required for the proposal of a novel species. The results of physiological and biochemical characteristics, the phylogenetic analysis, as well as low DNA-DNA hybridization values, allowed the phenotypic and genotypic differentiation of strain YIM 78007(T) from its closest phylogenetic neighbours. Therefore, strain YIM 78007(T) is considered to represent a novel species of the genus Roseomonas, for which the name Roseomonas alkaliterrae sp. nov. is proposed. The type strain is YIM 78007(T) (=BCRC 80644(T) = JCM 19656(T)).

Streptomyces calidiresistens sp. nov., isolated from a hot spring sediment.

A Streptomyces-like actinomycete strain, designated as YIM 78087(T), was isolated from a sediment sample collected from Hehua hot spring in Tengchong, Yunnan province, south-west China. The taxonomic position of strain YIM 78087(T) was investigated by a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain YIM 78087(T) belongs to the genus Streptomyces and is closely related to Streptomyces fimbriatus DSM 40942(T), Streptomyces marinus DSM 41968(T) and Streptomyces qinglanensis DSM 42035(T) (97.18, 97.05 and 97.1 % similarity, respectively). Combined with the low values of DNA-DNA hybridization between strain YIM 78087(T) and its closest neighbours, these analyses indicated that this new isolate represents a different genomic species in the genus Streptomyces. The predominant menaquinones of strain YIM 78087(T) were identified as MK-9 (H4) and MK-9 (H6). The major fatty acids were identified as anteiso-C15:0 (28.4 %), anteiso-C17:0 (23.0 %) and iso-C16:0 (15.1 %). The whole-cell hydrolysates found to contain glucose, mannose and ribose. The DNA G+C content was determined to be 73.0 mol%. Based on the comparative analysis of phenotypic and genotypic characteristics, it is proposed that strain YIM 78087(T) represents a novel species of the genus Streptomyces, for which the name Streptomyces calidiresistens sp. nov., is proposed. The type strain is YIM 78087(T) (=BCRC 16955(T)=DSM 42108(T)=JCM 19629(T)).