Characterization of a GH10 extremely thermophilic xylanase from the metagenome of hot spring for prebiotic production.

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.

Cloning, Heterologous Expression, and Characterization of a Neutral Uricase from Arthrobacter sp. CSAJ-16 in Cangshan Mountain.

Uricase (or Urate oxidase), a key enzyme involved in purine metabolism, is commonly used in treating conditions such as gout, hyperuricemia, and tumor lysis syndrome. In this study, a uricase-producing strain (named CSAJ-16) was isolated from the soil sample of Cangshan Mountain, Yunnan Province, China. This strain was identified as Arthrobacter sp. CSAJ-16. Based on the gene sequence alignment, the uricase gene (named aruox) of Arthrobacter sp. CSAJ-16 was amplified and heterologously expressed. The recombinant uricase (ArUOX) was about 32 kDa. The optimal pH and temperature of ArUOX were pH 7 and 20°C, respectively. The ArUOX remained above 50% relative activity after incubation at 37°C for 100 min or at pH 6.0-8.6 for 24 h. Moreover, metal ions such as K+, Mg2+, Ca2+, Ba2+ and Pb2+ can significantly enhance the activity of ArUOX (> 200%). These enzymatic properties indicate that ArUOX has potential applications in pharmaceutical enzymes and uric acid detection kits.

Characterization of a thermophilic and glucose-tolerant GH1 ß-glucosidase from hot springs and its prospective application in corn stover degradation.

Introduction: ß-Glucosidase serves as the pivotal rate-limiting enzyme in the cellulose degradation process, facilitating the hydrolysis of cellobiose and cellooligosaccharides into glucose. However, the widespread application of numerous ß-glucosidases is hindered by their limited thermostability and low glucose tolerance, particularly in elevated-temperature and high-glucose environments. Methods: This study presents an analysis of a ß-glucosidase gene belonging to the GH1 family, denoted lqbg8, which was isolated from the metagenomic repository of Hehua hot spring located in Tengchong, China. Subsequently, the gene was cloned and heterologously expressed in Escherichia coli BL21(DE3). Post expression, the recombinant ß-glucosidase (LQBG8) underwent purification through a Ni affinity chromatography column, thereby enabling the in-depth exploration of its enzymatic properties. Results: LQBG8 had an optimal temperature of 70°C and an optimum pH of 5.6. LQBG8 retained 100 and 70% of its maximum activity after 2-h incubation periods at 65°C and 70°C, respectively. Moreover, even following exposure to pH ranges of 3.0-10.0 for 24 h, LQBG8 retained approximately 80% of its initial activity. Notably, the enzymatic prowess of LQBG8 remained substantial at glucose concentrations of up to 3 M, with a retention of over 60% relative activity. The kinetic parameters of LQBG8 were characterized using cellobiose as substrate, with Km and Vmax values of 28 ± 1.9 mg/mL and 55 ± 3.2 µmol/min/mg, respectively. Furthermore, the introduction of LQBG8 (at a concentration of 0.03 mg/mL) into a conventional cellulase reaction system led to an impressive 43.7% augmentation in glucose yield from corn stover over a 24-h period. Molecular dynamics simulations offered valuable insights into LQBG8's thermophilic nature, attributing its robust stability to reduced fluctuations, conformational changes, and heightened structural rigidity in comparison to mesophilic ß-glucosidases. Discussion: In summation, its thermophilic, thermostable, and glucose-tolerant attributes, render LQBG8 ripe for potential applications across diverse domains encompassing food, feed, and the production of lignocellulosic ethanol.

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.

Clinical significance of herpes virus entry mediator expression in hepatitis B virus-related hepatocellular carcinoma.

Herpes virus entry mediator (HVEM) is overexpressed in several malignancies, including hepatocellular carcinoma (HCC). However, to the best of our knowledge, the clinical significance of HVEM in hepatitis B virus (HBV)-related HCC remains unclear. Thus, the present study aimed to explore the clinical significance of HVEM in HBV-related HCC. In the present study, HVEM expression was evaluated in HCC cell lines and HCC frozen samples. The prognostic value of HVEM was assessed in a cohort of 221 patients with HBV-related HCC, following radical resection. B- and T-lymphocyte attenuator (BTLA) expression in subsets of CD8+ T cells was determined via flow cytometry analysis. The results demonstrated high HVEM expression in HCC cell lines, and in HCC tissues compared with paired non-cancerous liver tissues. HVEM expression was demonstrated to be significantly associated with tumor encapsulation and vascular invasion. Furthermore, tumor HVEM status was significantly associated with infiltration of regulatory T cells, but not with CD8+ T cells. Notably, high HVEM expression in HCC was determined to be an independent predictor of an unfavorable outcome of patients with HCC following radical resection. Higher BTLA expression (the receptor of HVEM) was observed in both HCC-infiltrating CD8+ effector memory (CCR7- CD45RA-) and CD45RA+ effector memory (CCR7- CD45RA+) T cells in HCC tissues and blood compared with those in paired peritumor tissues or peripheral blood. Taken together, the results of the present study suggest that HVEM may serve a critical role in HBV-related HCC, most likely by promoting tumor progression and tumor immune evasion, thus the HVEM/BLTA signaling pathway may be a potential target in tumor immunotherapy.

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.

Network-directed efficient isolation of previously uncultivated Chloroflexi and related bacteria in hot spring microbial mats.

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.

Insight derived from molecular dynamics simulation into cold-adaptation mechanism of trypsins.

Communicated by Ramaswamy H. Sarma.

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).

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).

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.

Physiological and genomic properties of Thermus tenuipuniceus sp. nov., a novel slight reddish color member isolated from a terrestrial geothermal spring.

Two closely related, thermophilic bacteria, designated strains YIM 76954T 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-1T (97.1% sequence similarity), and T. amyloliquefaciens YIM 77409T (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-C17:0, iso-C15:0, anteiso-C17:0, and anteiso-C15:0. 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 76954T represents a novel species of the genus Thermus, for which the name Thermus tenuipuniceus sp. nov. is proposed. The type strain is YIM 76954T (=JCM 30350T=KCTC 4677T).

Integrated analysis of the impact of age on genetic and clinical aspects of hepatocellular carcinoma.

Despite the rapid growing and aging of populations worldwide, our knowledge on hepatocellular carcinoma (HCC) is still age-standardized rather than age-specific, with only few studies exploring the topic from a genetic point of view. Here, we analyze clinical and genetic aspects of HCC in patients of different age groups with the major attention directed to children (≤20 y) and elderly groups (≥80 y). A number of significant differences were found in elderly patients compared to children group, including smaller tumor size (P=0.001) and improved survival rates (P=0.002). Differences in gene mutations, copy number variants, and mRNA expressions were identified between the groups, with alteration rates for some genes like AKR1B10 increasing significantly with the age of patients. Immunohistochemistry testing of AKR1B10 showed a significant difference in expression levels at the age of 40 (30.77% high expression rate in patients younger than 40 compared to 51.57% in older patients) (P=0.043). Expression levels also differed between HCC tissues (49.64%) and near-tumor tissues (6.58%) (P<0.001). These findings contribute to the limited data available regarding the age-specific aspects of HCC patients, and support the need to address potential differences in the diagnosis, treatment, and prevention strategies of HCC.

Genomic inference of the metabolism and evolution of the archaeal phylum Aigarchaeota.

Microbes of the phylum Aigarchaeota are widely distributed in geothermal environments, but their physiological and ecological roles are poorly understood. Here we analyze six Aigarchaeota metagenomic bins from two circumneutral hot springs in Tengchong, China, to reveal that they are either strict or facultative anaerobes, and most are chemolithotrophs that can perform sulfide oxidation. Applying comparative genomics to the Thaumarchaeota and Aigarchaeota, we find that they both originated from thermal habitats, sharing 1154 genes with their common ancestor. Horizontal gene transfer played a crucial role in shaping genetic diversity of Aigarchaeota and led to functional partitioning and ecological divergence among sympatric microbes, as several key functional innovations were endowed by Bacteria, including dissimilatory sulfite reduction and possibly carbon monoxide oxidation. Our study expands our knowledge of the possible ecological roles of the Aigarchaeota and clarifies their evolutionary relationship to their sister lineage Thaumarchaeota.

Expression and Characteristics of Two Glucose-Tolerant GH1 ß-glucosidases From Actinomadura amylolytica YIM 77502T for Promoting Cellulose Degradation.

The bioconversion of lignocellulose in various industrial processes, such as biofuel production, requires the degradation of cellulose. Actinomadura amylolytica YIM 77502T is an aerobic, Gram-positive actinomycete that can efficiently degrade crystalline cellulose by extracellular cellulases. Genomic analysis of A. amylolytica identified 9 cellulase and 11 ß-glucosidase genes that could potentially encode proteins that digest cellulose. Extracellular proteome characterization of A. amylolytica cell-free culture supernatant by liquid chromatography tandem mass spectrometry analysis revealed that 4 of these cellulases and 2 of these ß-glucosidases functioned during cellulose hydrolysis. Thin-layer chromatography analysis revealed extracellular ß-glucosidases play a major role in carboxyl methyl cellulose (CMC) degradation of products in culture supernatants. In this study, 2 of the identified secreted ß-glucosidases, AaBGL1 and AaBGL2, were functionally expressed in Escherichia coli and found to have ß-glucosidase activity with wide substrate specificities, including for p-nitrophenyl ß-D-glucopyranoside (pNPG), p-nitrophenyl-beta-D-cellobioside (pNPC), and cellobiose. Moreover, AaBGL1 and AaBGL2 had high tolerances for glucose. After adding these ß-glucosidases to commercial cellulases, the degradation rates of CMC, Avicel, birch sawdust, and corncob powder increased by 37, 42, 33, and 9%, respectively. Overall, this work identifies an alternative potential source of ß-glucosidases with potential applications in commercial cellulose utilization and the bioenergy industry.

The Hybrid Strategy of Thermoactinospora rubra YIM 77501T for Utilizing Cellulose as a Carbon Source at Different Temperatures.

Thermoactinospora rubra YIM 77501T is an aerobic, Gram-positive, spore-forming and cellulose degrading thermophilic actinomycete isolated from a sandy soil sample of a volcano. Its growth temperature range is 28-60°C. The genomic sequence of this strain revealed that there are 27 cellulase genes belonging to six glycoside hydrolase families. To understand the strategy that this strain uses to utilize carbon sources such as cellulose at different temperatures, comparative transcriptomics analysis of T. rubra YIM 77501T was performed by growing it with cellulose (CMC) and without cellulose (replaced with glucose) at 30, 40, and 50°C, respectively. Transcriptomic analyses showed four cellulase genes (TrBG2, TrBG3, TrBG4, and ThrCel6B) were up-regulated at 30, 40, and 50°C. The rate of gene expression of TrBG2, TrBG3, TrBG4, and ThrCel6B were 50°C > 30°C > 40°C. One cellulase gene (TrBG1) and two cellulase genes (TrBG5 and ThrCel6A) were up-regulated only at 30 and 50°C, respectively. These up-regulated cellulase genes were cloned and expressed in Escherichia coli. The enzymatic properties of up-regulated cellulases showed a variety of responses to temperature. Special up-regulated cellulases TrBG1 and ThrCel6A displayed temperature acclimation for each growth condition. These expression patterns revealed that a hybrid strategy was used by T. rubra to utilize carbon sources at different temperatures. This study provides genomic, transcriptomics, and experimental data useful for understanding how microorganisms respond to environmental changes and their application in enhancing cellulose hydrolysis for animal feed and bioenergy production.

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.

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.

High-quality draft genome sequence of the Thermus amyloliquefaciens type strain YIM 77409(T) with an incomplete denitrification pathway.

Thermus amyloliquefaciens type strain YIM 77409(T) is a thermophilic, Gram-negative, non-motile and rod-shaped bacterium isolated from Niujie Hot Spring in Eryuan County, Yunnan Province, southwest China. In the present study we describe the features of strain YIM 77409(T) together with its genome sequence and annotation. The genome is 2,160,855 bp long and consists of 6 scaffolds with 67.4 % average GC content. A total of 2,313 genes were predicted, comprising 2,257 protein-coding and 56 RNA genes. The genome is predicted to encode a complete glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle. Additionally, a large number of transporters and enzymes for heterotrophy highlight the broad heterotrophic lifestyle of this organism. A denitrification gene cluster included genes predicted to encode enzymes for the sequential reduction of nitrate to nitrous oxide, consistent with the incomplete denitrification phenotype of this strain.

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).