Attractant and repellent induce opposing changes in the four-helix bundle ligand-binding domain of a bacterial chemoreceptor.

Motile bacteria navigate toward favorable conditions and away from unfavorable environments using chemotaxis. Mechanisms of sensing attractants are well understood; however, molecular aspects of how bacteria sense repellents have not been established. Here, we identified malate as a repellent recognized by the MCP2201 chemoreceptor in a bacterium Comamonas testosteroni and showed that it binds to the same site as an attractant citrate. Binding determinants for a repellent and an attractant had only minor differences, and a single amino acid substitution in the binding site inverted the response to malate from a repellent to an attractant. We found that malate and citrate affect the oligomerization state of the ligand-binding domain in opposing way. We also observed opposing effects of repellent and attractant binding on the orientation of an alpha helix connecting the sensory domain to the transmembrane helix. We propose a model to illustrate how positive and negative signals might be generated.

Xiashengella succiniciproducens gen. nov., sp. nov., a succinate-producing bacterium isolated from an anaerobic digestion tank in the family Marinilabiliaceae of the order Bacteroidales.

A strictly anaerobic, motile bacterium, designated as strain Ai-910T, was isolated from the sludge of an anaerobic digestion tank in China. Cells were Gram-stain-negative rods. Optimal growth was observed at 38 °C (growth range 25-42 °C), pH 8.5 (growth range 5.5-10.5), and under a NaCl concentration of 0.06% (w/v) (range 0-2.0%). Major cellular fatty acids were iso-C15 : 0 and anteiso-C15 : 0. The respiratory quinone was MK-7. Using xylose as the growth substrate, succinate was produced as the fermentation product. Phylogenetic analysis based on the 16 S rRNA gene sequences indicated that strain Ai-910T formed a distinct phylogenetic lineage that reflects a new genus in the family Marinilabiliaceae, sharing high similarities to Alkaliflexus imshenetskii Z-7010T (92.78%), Alkalitalea saponilacus SC/BZ-SP2T (92.51%), and Geofilum rubicundum JAM-BA0501T (92.36%). Genomic similarity (average nucleotide identity and digital DNA-DNA hybridization) values between strain Ai-910T and its phylogenetic neighbors were below 65.27 and 16.90%, respectively, indicating that strain Ai-910T represented a novel species. The average amino acid identity between strain Ai-910T and other related members of the family Marinilabiliaceae were below 69.41%, supporting that strain Ai-910T was a member of a new genus within the family Marinilabiliaceae. Phylogenetic, genomic, and phenotypic analysis revealed that strain Ai-910T was distinguished from other phylogenetic relatives within the family Marinilabiliaceae. The genome size was 3.10 Mbp, and the DNA G + C content of the isolate was 42.8 mol%. Collectively, differences of the phenotypic and phylogenetic features of strain Ai-910T from its close relatives suggest that strain Ai-910T represented a novel species in a new genus of the family Marinilabiliaceae, for which the name Xiashengella succiniciproducens gen. nov., sp. nov. was proposed. The type strain of Xiashengella succiniciproducens is Ai-910T (= CGMCC 1.17893T = KCTC 25,304T).

Acidithiobacillus acidisediminis sp. nov., an acidophilic sulphur-oxidizing chemolithotroph isolated from acid mine drainage sediment.

Strain S30A2T, isolated from the acid mine drainage sediment of Mengzi Copper Mine, Yunnan, is proposed to represent a novel species of the sulphur-oxidizing genus Acidithiobacillus. Cells were Gram-stain-negative, non-endospore forming, highly motile with one or two monopolar flagella and rod-shaped. The strain was mesophilic, growing at 30-50 °C (optimum, 38 °C), acidophilic, growing at pH 2.0-4.5 (optimum, pH 2.5), and tolerant of 0-4 % (w/v; 684 mol l-1) NaCl. The 16S rRNA gene-based sequence analysis showed that strain S30A2T belongs to the genus Acidithiobacillus and shows the largest similarity of 96.6 % to the type strain Acidithiobacillus caldus KUT. The genomic DNA G+C content of strain S30A2T was 59.25 mol%. The average nucleotide identity ANIb and ANIm values between strain S30A2T and A. caldus KUT were 70.95 and 89.78 %, respectively and the digital DNA-DNA hybridization value was 24.9 %. Strain S30A2T was strictly aerobic and could utilize elementary sulphur and tetrathionate to support chemolithotrophic growth. The major cellular fatty acid of S30A2T was C19 : 1ω7c. The respiratory quinones were ubiquinone-8 and ubiquinone-7. Based upon its phylogenetic, genetic, phenotypic, physiologic and chemotaxonomic characteristics, strain S30A2T is considered to represent a novel species of the genus Acidithiobacillus, for which the name Acidithiobacillus acidisediminis sp. nov. is proposed. The type strain is S30A2T (=CGMCC 1.17059T=KCTC 72580T).

Chemoreceptors from the commensal gut Roseburia rectibacter bind to mucin and trigger chemotaxis.

Chemotaxis is crucial for bacterial adherence and colonization of the host gastrointestinal tract. Previous studies have demonstrated that chemotaxis affects the virulence of causative pathogens and the infection in the host. However, the chemotactic abilities of non-pathogenic and commensal gut bacteria have rarely been explored. We observed that Roseburia rectibacter NSJ-69 exhibited flagella-dependent motility and chemotaxis to a variety of molecules, including mucin and propionate. A genome-wide analysis revealed that NSJ-69 has 28 putative chemoreceptors, 15 of which have periplasmic ligand-binding domains (LBDs). These LBD-coding genes were chemically synthesized and expressed heterologously in Escherichia coli. Intensive screening of ligands revealed four chemoreceptors bound to mucin and two bound to propionate. When expressed in Comamonas testosteroni or E. coli, these chemoreceptors elicited chemotaxis toward mucin and propionate. Hybrid chemoreceptors were constructed, and results showed that the chemotactic responses to mucin and propionate were dependent on the LBDs of R. rectibacter chemoreceptors. Our study identified and characterized R. rectibacter chemoreceptors. These results will facilitate further investigations on the involvement of microbial chemotaxis in host colonization.

The sulfonamide-resistance dihydropteroate synthase gene is crucial for efficient biodegradation of sulfamethoxazole by Paenarthrobacter species.

Sulfonamide antibiotics (SAs) are serious pollutants to ecosystems and environments. Previous studies showed that microbial degradation of SAs such as sulfamethoxazole (SMX) proceeds via a sad-encoded oxidative pathway, while the sulfonamide-resistant dihydropteroate synthase gene, sul, is responsible for SA resistance. However, the co-occurrence of sad and sul genes, as well as how the sul gene affects SMX degradation, was not explored. In this study, two SMX-degrading bacterial strains, SD-1 and SD-2, were cultivated from an SMX-degrading enrichment. Both strains were Paenarthrobacter species and were phylogenetically identical; however, they showed different SMX degradation activities. Specifically, strain SD-1 utilized SMX as the sole carbon and energy source for growth and was a highly efficient SMX degrader, while SD-2 did could not use SMX as a sole carbon or energy source and showed limited SMX degradation when an additional carbon source was supplied. Genome annotation, growth, enzymatic activity tests, and metabolite detection revealed that strains SD-1 and SD-2 shared a sad-encoded oxidative pathway for SMX degradation and a pathway of protocatechuate degradation. A new sulfonamide-resistant dihydropteroate synthase gene, sul918, was identified in strain SD-1, but not in SD-2. Moreover, the lack of sul918 resulted in low SMX degradation activity in strain SD-2. Genome data mining revealed the co-occurrence of sad and sul genes in efficient SMX-degrading Paenarthrobacter strains. We propose that the co-occurrence of sulfonamide-resistant dihydropteroate synthase and sad genes is crucial for efficient SMX biodegradation. KEY POINTS: • Two sulfamethoxazole-degrading strains with distinct degrading activity, Paenarthrobacter sp. SD-1 and Paenarthrobacter sp. SD-2, were isolated and identified. • Strains SD-1 and SD-2 shared a sad-encoded oxidative pathway for SMX degradation. • A new plasmid-borne SMX resistance gene (sul918) of strain SD-1 plays a crucial role in SMX degradation efficiency.

Alicyclobacillus curvatus sp. nov. and Alicyclobacillus mengziensis sp. nov., two acidophilic bacteria isolated from acid mine drainage.

Two acidophilic strains, designated as ALEF1T and S30H14T, were isolated from acid mine drainage sediment. Cells of both strains were Gram-stain-positive, aerobic, endospore-forming rods. Strains ALEF1T and S30H14T were acidophilic and mesophilic, the former grew at 20-40 °C (optimum, 30 °C) and pH 2.5-4.5 (optimum, pH 3.5), while the latter grew at 20-45 °C (optimum, 30 °C) and pH 2.0-5.5 (optimum, pH 4.5). The 16S rRNA gene-based sequence analysis revealed that strains ALEF1T and S30H14T belonged to the genus Alicyclobacillus, and were phylogenetically close to Alicyclobacillus ferrooxydans TC-34T with 97.1 and 97.4% similarity, respectively. The similarity between the two novel strains was 98.6 %. The average nucleotide identity value between the genome sequences of ALEF1T and S30H14T was 79.5 %, and that between each of the two isolates and A. ferrooxydans TC-34T were 72.0 and 74.3 %. In addition, the digital DNA-DNA hybridization value between ALEF1T and S30H14T was 24.9 %, between strain ALEF1T and A. ferrooxydans TC-34T was 21.7 %, and between S30H14T and A. ferrooxydans TC-34T was 26.3 %, far below the interspecies threshold. Both strains could utilize diverse carbon sources for heterotrophic growth; strain ALEF1T could utilize ferrous iron as the energy source for autotrophic growth. Menaquinone 7 was the only quinone detected in either strain. Both strains contained anteiso-C15 : 0 and anteiso-C17 : 0, while ω-alicyclic fatty acids were not detected. Based on their phylogenetic positions, as well as phenotypic and genomic data, it is considered that strains ALEF1T and S30H14T represent two novel species within the genus Alicyclobacillus, for which the names Alicyclobacillus curvatus sp. nov. (type strain ALEF1T=CGMCC 1.17055T=KCTC 43124T) and Alicyclobacillus mengziensis sp. nov. (S30H14T=CGMCC 1.17050T=KCTC 43125T) are proposed.

Alkaliphilus flagellatus sp. nov., Butyricicoccus intestinisimiae sp. nov., Clostridium mobile sp. nov., Clostridium simiarum sp. nov., Dysosmobacter acutus sp. nov., Paenibacillus brevis sp. nov., Peptoniphilus ovalis sp. nov. and Tissierella simiarum sp. nov., isolated from monkey faeces.

Non-human primates harbour diverse microbiomes in their guts. As a part of the China Microbiome Initiatives, we cultivated and characterized the gut microbiome of cynomolgus monkeys (Macaca fascicularis). In this report, we communicate the characterization and taxonomy of eight bacterial strains that were obtained from faecal samples of captive cynomolgus monkeys. The results revealed that they represented eight novel bacterial species. The proposed names of the eight novel species are Alkaliphilus flagellatus (type strain MSJ-5T=CGMCC 1.45007T=KCTC 15974T), Butyricicoccus intestinisimiae MSJd-7T (MSJd-7T=CGMCC 1.45013T=KCTC 25112T), Clostridium mobile (MSJ-11T=CGMCC 1.45009T=KCTC 25065T), Clostridium simiarum (MSJ-4T=CGMCC 1.45006T=KCTC 15975T), Dysosmobacter acutus (MSJ-2T=CGMCC 1.32896T=KCTC 15976T), Paenibacillus brevis MSJ-6T (MSJ-6T=CGMCC 1.45008T=KCTC 15973T), Peptoniphilus ovalis (MSJ-1T=CGMCC 1.31770T=KCTC 15977T) and Tissierella simiarum (MSJ-40T=CGMCC 1.45012T=KCTC 25071T).

PapA, a peptidoglycan-associated protein, interacts with OmpC and maintains cell envelope integrity.

The bacterial cell envelope is critical to support and maintain cellular life. In Gram-negative bacterial cells, the outer membrane and the peptidoglycan layer are two important parts of the cell envelope and they harbour abundant proteins. Here, we report the identification and characterization of a previously unknown peptidoglycan-associated protein, PapA, from the Gram-negative Comamonas testosteroni. PapA bound peptidoglycan with its C-terminal domain and interacted with the outer-membrane porin OmpC. The PapA-OmpC complex riveted the outer membrane and the peptidoglycan layer, and played a role in maintaining cell envelope integrity. When papA was disrupted, the mutant CNB-1ΔpapA apparently had an outer membrane partly separated from the peptidoglycan layer. Phenotypically, the mutant CNB-1ΔpapA lost chemotactic responses and had longer lag-phase of growth, less flagellation and higher sensitivity to harsh environments. Totally, 1093 functionally unknown PapA homologues were identified from the public NR protein database and they were mainly distributed in Burkholderiales of Betaproteobacteria. Our finding provides a clue that the PapA homologous proteins might function as a rivet to maintain cell envelope integrity in those Gram-negative bacteria.

Bacteria and Metabolic Potential in Karst Caves Revealed by Intensive Bacterial Cultivation and Genome Assembly.

Karst caves are widely distributed subsurface systems, and the microbiomes therein are proposed to be the driving force for cave evolution and biogeochemical cycling. In past years, culture-independent studies on the microbiomes of cave systems have been conducted, yet intensive microbial cultivation is still needed to validate the sequence-derived hypothesis and to disclose the microbial functions in cave ecosystems. In this study, the microbiomes of two karst caves in Guizhou Province in southwest China were examined. A total of 3,562 bacterial strains were cultivated from rock, water, and sediment samples, and 329 species (including 14 newly described species) of 102 genera were found. We created a cave bacterial genome collection of 218 bacterial genomes from a karst cave microbiome through the extraction of 204 database-derived genomes and de novo sequencing of 14 new bacterial genomes. The cultivated genome collection obtained in this study and the metagenome data from previous studies were used to investigate the bacterial metabolism and potential involvement in the carbon, nitrogen, and sulfur biogeochemical cycles in the cave ecosystem. New N2-fixing Azospirillum and alkane-oxidizing Oleomonas species were documented in the karst cave microbiome. Two pcaIJ clusters of the ß-ketoadipate pathway that were abundant in both the cultivated microbiomes and the metagenomic data were identified, and their representatives from the cultivated bacterial genomes were functionally demonstrated. This large-scale cultivation of a cave microbiome represents the most intensive collection of cave bacterial resources to date and provides valuable information and diverse microbial resources for future cave biogeochemical research.IMPORTANCE Karst caves are oligotrophic environments that are dark and humid and have a relatively stable annual temperature. The diversity of bacteria and their metabolisms are crucial for understanding the biogeochemical cycling in cave ecosystems. We integrated large-scale bacterial cultivation with metagenomic data mining to explore the compositions and metabolisms of the microbiomes in two karst cave systems. Our results reveal the presence of a highly diversified cave bacterial community, and 14 new bacterial species were described and their genomes sequenced. In this study, we obtained the most intensive collection of cultivated microbial resources from karst caves to date and predicted the various important routes for the biogeochemical cycling of elements in cave ecosystems.

Candidatus Kaistella beijingensis sp. nov., Isolated from a Municipal Wastewater Treatment Plant, Is Involved in Sludge Foaming.

Biological foaming (or biofoaming) is a frequently occurring problem in wastewater treatment plants (WWTPs) and is attributed to the overwhelming growth of filamentous bulking and foaming bacteria (BFB). Biological foaming has been intensively investigated, with BFB like Microthrix and Skermania having been identified from WWTPs and implicated in foaming. Nevertheless, studies are still needed to improve our understanding of the microbial diversity of WWTP biofoams and how microbial activities contribute to foaming. In this study, sludge foaming at the Qinghe WWTP of China was monitored, and sludge foams were investigated using culture-dependent and culture-independent microbiological methods. The foam microbiomes exhibited high abundances of Skermania, Mycobacterium, Flavobacteriales, and Kaistella. A previously unknown bacterium, Candidatus Kaistella beijingensis, was cultivated from foams, its genome was sequenced, and it was phenotypically characterized. Ca. K. beijingensis exhibits hydrophobic cell surfaces, produces extracellular polymeric substances (EPS), and metabolizes lipids. Ca. K. beijingensis abundances were proportional to EPS levels in foams. Several proteins encoded by the Ca. K. beijingensis genome were identified from EPS that was extracted from sludge foams. Ca. K. beijingensis populations accounted for 4 to 6% of the total bacterial populations in sludge foam samples within the Qinghe WWTP, although their abundances were higher in spring than in other seasons. Cooccurrence analysis indicated that Ca. K. beijingensis was not a core node among the WWTP community network, but its abundances were negatively correlated with those of the well-studied BFB Skermania piniformis among cross-season Qinghe WWTP communities. IMPORTANCE Biological foaming, also known as scumming, is a sludge separation problem that has become the subject of major concern for long-term stable activated sludge operation in decades. Biological foaming was considered induced by foaming bacteria. However, the occurrence and deterioration of foaming in many WWTPs are still not completely understood. Cultivation and characterization of the enriched bacteria in foaming are critical to understand their genetic, physiological, phylogenetic, and ecological traits, as well as to improve the understanding of their relationships with foaming and performance of WWTPs.

Submerged macrophytes recruit unique microbial communities and drive functional zonation in an aquatic system.

Aquatic and wetland systems are widely used for landscapes and water regeneration. Microbiomes and submerged macrophytes (hydrophytes) play essential roles in conversions of organic and inorganic compounds in those ecosystems. The systems were extensively investigated for microbial diversities and compositions. However, little is known about how hydrophytes recruited diverse microbiota and affected functional zonation in aquatic systems. To address this issue, epiphytic leaf and root, sediment, and surrounding water samples were collected from the dragon-shape aquatic system in Beijing Olympic Park. Metagenomic DNAs were extracted and subjected to sequencing. Results showed that epiphytic leaf and root microbiomes and metabolic marker genes were remarkably different from that of surrounding environment. Twenty indicator bacterial genera for epiphytic microbiomes were identified and 50 metabolic marker genes were applied to evaluate the function of epiphytic leaf and root, water, and sediment microbiomes. Co-occurrence analysis revealed highly modularized pattern of metabolic marker genes and indicator bacterial genera related to metabolic functions. These results suggested that hydrophytes shaped microbiomes and drove functional zonation in aquatic systems. KEY POINTS: • Microbiomes of hydrophytes and their surrounding environments were investigated. • Twenty indicator bacterial genera highly specific to epiphytic biofilms were identified. • Epiphytes recruited unique microbiomes and drove functional zonation in aquatic systems.

Tropical and temperate wastewater treatment plants assemble different and diverse microbiomes.

The diversity and assembly of activated sludge microbiomes play a key role in the performances of municipal wastewater treatment plants (WWTPs), which are the most widely applied biotechnological process systems. In this study, we investigated the microbiomes of municipal WWTPs in Bangkok, Wuhan, and Beijing that respectively represent tropical, subtropical, and temperate climate regions, and also explored how microbiomes assembled in these municipal WWTPs. Our results showed that the microbiomes from these municipal WWTPs were significantly different. The assembly of microbiomes in municipal WWTPs followed deterministic and stochastic processes governed by geographical location, temperature, and nutrients. We found that both taxonomic and phylogenetic α-diversities of tropical Bangkok municipal WWTPs were the highest and were rich in yet-to-be-identified microbial taxa. Nitrospirae and ß-Proteobacteria were more abundant in tropical municipal WWTPs, but did not result in better removal efficiencies of ammonium and total nitrogen. Overall, these results suggest that tropical and temperate municipal WWTPs harbored diverse and unique microbial resources, and the municipal WWTP microbiomes were assembled with different processes. Implications of these findings for designing and running tropical municipal WWTPs were discussed. KEY POINTS: • Six WWTPs of tropical Thailand and subtropical and temperate China were investigated. • Tropical Bangkok WWTPs had more diverse and yet-to-be-identified microbial taxa. • Microbiome assembly processes were associated with geographical location.

The ligand-binding domain of a chemoreceptor from Comamonas testosteroni has a previously unknown homotrimeric structure.

Transmembrane chemoreceptors are widely present in Bacteria and Archaea. They play a critical role in sensing various signals outside and transmitting to the cell interior. Here, we report the structure of the periplasmic ligand-binding domain (LBD) of the transmembrane chemoreceptor MCP2201, which governs chemotaxis to citrate and other organic compounds in Comamonas testosteroni. The apo-form LBD crystal revealed a typical four-helix bundle homodimer, similar to previously well-studied chemoreceptors such as Tar and Tsr of Escherichia coli. However, the citrate-bound LBD revealed a four-helix bundle homotrimer that had not been observed in bacterial chemoreceptor LBDs. This homotrimer was further confirmed with size-exclusion chromatography, analytical ultracentrifugation and cross-linking experiments. The physiological importance of the homotrimer for chemotaxis was demonstrated with site-directed mutations of key amino acid residues in C. testosteroni mutants.

Increased number of intratumoral IL-17+ cells, a harbinger of the adverse prognosis of triple-negative breast cancer.

INTRODUCTION: Triple negative breast cancer (TNBC) is an aggressive cancer subtype and lack of effective targeted therapies. It has been recently reported that Interleukin 17 (IL-17), a family of cytokines secreted in tumor microenvironment, affects tumor progression through a variety of molecular pathways. Its role in TNBC is so far still poorly explored. MATERIALS AND METHODS: We employed immunohistochemistry to evaluate the distribution of IL-17+ cells in TNBC with no special type features (TNBC-NST), their association with tumor microangiogenesis, as well as their impact on prognosis of the patients. RESULTS: In comparison to medullary carcinoma with triple-negative molecular features (TNBC-MC), we found a significant increase in IL-17+ cell infiltrates in intratumoral stroma and extratumoral stroma of TNBC-NST. Similarly, stromal cells with co-expression of CD4 and IL-17 were noted in intratumoral and extratumoral stroma in both TNBC-NST and TNBC-MC. In addition, intratumoral IL-17+ cells were positively associated with tumor cell expression of vascular endothelial growth factor A (VEGFA) and with intratumoral tumor microvascular density (MVD). Multivariate analysis identified that intratumoral IL-17+ cells (P = 0.018), MVD (P = 0.039), and TNM stage (P = 0.002) were independent prognostic factors for predicting poor PFS. CONCLUSION: The study indicates that IL-17 is overexpressed in intratumoral stromal cells of TNBC-NST. The overexpression of IL-17 might engage in active tumor microangiogenesis through its signal transduction pathways resulting in increased tumor secretion of VEGFA, and then promote tumor progression. IL-17 might serve as a potential new target for individualized therapy to TNBC-NST patients by development of specific antibodies. Additional study is deemed to further explore the role of IL-17+ stromal cells in breast cancer.

Casimicrobium huifangae gen. nov., sp. nov., a Ubiquitous "Most-Wanted" Core Bacterial Taxon from Municipal Wastewater Treatment Plants.

Microorganisms in wastewater treatment plants (WWTPs) play a key role in the removal of pollutants from municipal and industrial wastewaters. A recent study estimated that activated sludge from global municipal WWTPs harbors 1 × 109 to 2 × 109 microbial species, the majority of which have not yet been cultivated, and 28 core taxa were identified as "most-wanted" ones (L. Wu, D. Ning, B. Zhang, Y. Li, et al., Nat Microbiol 4:1183-1195, 2019, https://doi.org/10.1038/s41564-019-0426-5). Cultivation and characterization of the "most-wanted" core bacteria are critical to understand their genetic, physiological, phylogenetic, and ecological traits, as well as to improve the performance of WWTPs. In this study, we isolated a bacterial strain, designated SJ-1, that represents a novel cluster within Betaproteobacteria and corresponds to OTU_16 within the 28 core taxa in the "most-wanted" list. Strain SJ-1 was identified and nominated as Casimicrobium huifangae gen. nov., sp. nov., of a novel family, Casimicrobiaceae. C. huifangae is ubiquitously distributed and is metabolically versatile. In addition to mineralizing various carbon sources (including carbohydrates, aromatic compounds, and short-chain fatty acids), C. huifangae is capable of nitrate reduction and phosphorus accumulation. The population of C. huifangae accounted for more than 1% of the bacterial population of the activated sludge microbiome from the Qinghe WWTP, which showed seasonal dynamic changes. Cooccurrence analysis suggested that C. huifangae was an important module hub in the bacterial network of Qinghe WWTP.IMPORTANCE The activated sludge process is the most widely applied biotechnology and is one of the best ecosystems to address microbial ecological principles. Yet, the cultivation of core bacteria and the exploration of their physiology and ecology are limited. In this study, the core and novel bacterial taxon C. huifangae was cultivated and characterized. This study revealed that C. huifangae functioned as an important module hub in the activated sludge microbiome, and it potentially plays an important role in municipal wastewater treatment plants.

Crenobacter cavernae sp. nov., isolated from a karst cave, and emended description of the genus Crenobacter.

A Gram-stain-negative, rod-shaped, motile and strictly aerobic novel bacterial isolate, designated strain K1W11S-77T, was obtained from a water sample that was collected from a karst cave in Guizhou province, PR China. The results of a phylogenetic analysis based on 16S rRNA gene sequences indicated that K1W11S-77T represented a member of the genus Crenobacter within the family Neisseriaceae of the phylum Proteobacteria. K1W11S-77T was phylogenetically closely related to Crenobacter luteusYIM 78141T (Their 16S rRNA gene sequence similarity is 95.02 %). Growth of K1W11S-77T occurred at 10-30 °C, at pH 7-9, and in the presence of 0-1 % (w/v) NaCl. The major cellular fatty acids were C12 : 0, C16 : 0, C18:1ω7c and summed feature 3. The major isoprenoid quinone was Q-8. The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol and one unidentified phospholipid. The genome of K1W11S-77T was 3.27 Mb long and encoded 3167 annotated genes. The DNA G+C content of the genomic DNA was 65.3 mol%. On the basis of phylogenetic, phenotypic and chemotaxonomic characteristics, K1W11S-77T is considered to represent a novel species of the genus Crenobacter, for which the name Crenobactercavernae sp. nov. is proposed. The type strain is K1W11S-77T (=CGMCC 1.13527T=NBRC 113452T).

Acidibrevibacterium fodinaquatile gen. nov., sp. nov., isolated from acidic mine drainage.

A heterotrophic and acidophilic bacterial strain, G45-3T, was isolated from acidic mine drainage sampled in Fujian Province, PR China. Cells of strain G45-3T were Gram-stain-negative, non-spore-forming, non-motile and rod-shaped. Catalase and oxidase activities were positive. Strain G45-3T grew aerobically at 20-45 °C (optimum, 37 °C) and at pH 2.5-5.0 (optimum, pH 4.0). Photosynthetic pigments were not produced. Analysis of 16S rRNA gene sequences showed that strain G45-3T was phylogenetically related to different members of the family Acetobacteraceae, and the sequence identities to Acidisphaera rubrifaciens JCM 10600T, Rhodovastum atsumiense G2-11T and Rhodopila globiformis ATCC 35887T were 95.9 , 95.3 and 95.3 %, respectively. Strain G45-3T contained ubiquinone-10 as its respiratory quinone. The major polar lipids were determined to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unidentified aminophospholipid and an unidentified aminolipid. The predominant fatty acids were cyclo-C19 : 0ω8c, C18 : 1ω7c, C16 : 0 and C18 : 0. The genome of G45-3T consists of one chromosome (3 907 406 bp) and three plasmids (68 344, 45 771 and 16 090 bp), with an average G+C content of 65.9 mol%. Based on the results of phenotypic and genomic analyses, it is concluded that strain G45-3T represents a novel species of a new genus, for which the name Acidibrevibacterium fodinaquatile gen. nov., sp. nov. is proposed. A. fodinaquatile is nominated as type species and its type strain is G45-3T (=CGMCC 1.16069T=KCTC 62275T).

Cohnella faecalis sp. nov., isolated from animal faeces in a karst cave.

A Gram-stain-positive, rod-shaped, endospore-forming, motile and aerobic bacterial isolate, designated strain K2E09-144T, was obtained from animal faeces that were collected from a karst cave in Guizhou province, China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain K2E09-144T represents a novel member of the genus Cohnella within the family Paenibacillaceae of the phylum Firmicutes. Strain K2E09-144T was phylogenetically closely related to Cohnella nanjingensis D45T (16S rRNA gene sequence similarity 97.0 %). The major cellular fatty acids were anteiso-C15:0, iso-C16:0 and C16 : 0. The major isoprenoid quinone was menaqinone 7 (MK-7). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified phospholipid, four unidentified aminophospholipids, one glycolipid and one unidentified lipid. The isomer type of diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The genome of strain K2E09-144T comprised 6.43 Mb, and encoded 6029 genes in total. The DNA G+C content of the genomic DNA was 53.3 mol%. Based on its phylogenetic, phenotypic and chemotaxonomic characteristics, strain K2E09-144T is considered to represent a novel species of the genus Cohnella, for which the name Cohnella faecalis sp. nov. is proposed. The type strain is K2E09-144T (=CGMCC 1.13587T=NBRC 113454T).

Vallitalea okinawensis sp. nov., isolated from Okinawa Trough sediment and emended description of the genus Vallitalea.

A polyphasic study was conducted to characterize an obligately anaerobic bacterial strain, S15T, that was isolated from Okinawa Trough sediment. Strain S15T was Gram-stain-negative, non-motile and rod-shaped. Spores were not observed. Strain S15T grew anaerobically at 20-35 °C (optimum at 25-30 °C) and at pH range of 6.0-8.5 (optimum at 7.5). Analysis of 16S rRNA gene sequences showed that strain S15T was phylogenetically related to Vallitalea guaymasensis Ra1766G1T (94.0 %) and Vallitalea pronyensis FatNI3T (93.1 %). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and glycolipids. The predominant fatty acids of strain S15T were iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0 and C16 : 0. The draft genome was 5.86 Mb with a DNA G+C content of 33.9 mol%. A total of 5285 genes were predicted and, of those, 4669 genes were annotated. The genome data supported the result that strain S15T assimilated various carbon sources. On the basis of unique phenotypic, chemotaxonomic and phylogenetic comparisons, strain S15T is proposed to represent a novel species within the genus Vallitalea, and the name Vallitaleaokinawensis sp. nov. is proposed. The type strain is S15T=CGMCC 1.5231T=KCTC 15675T.

Thauera hydrothermalis sp. nov., a thermophilic bacterium isolated from hot spring.

A Gram-stain-negative, non-spore-forming, rod-shaped, motile bacterial strain, designated GD-2T, was isolated from a sediment sample collected from a hot spring in the Tibet Autonomous Region, China. Strain GD-2T grew at a temperature range of 37-55 °C (optimum, 45-50 °C), a pH range of 5.5-11.0 (pH 7.0-7.5) and a NaCl concentration range of 0-4.0 % (0 %). The phylogenetic analysis based on 16S rRNA gene sequencing showed that strain GD-2T represented a member of the genus Thauera within the family Zoogloeaceae. Strain GD-2T was closely related to Thauera linaloolentis 47LolT with the highest 16S rRNA gene sequence similarity of 95.5 %. The whole genomic average nucleotide identity value for GD-2T and 47LolT was 75.3 %. The predominant cellular fatty acids of the strain were C16 : 0, summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), C10 : 0 3-OH and C12 : 0. The main polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, three unidentified phospholipids and two unidentified aminolipids. The major isoprenoid quinone was ubiquinone 8. Genome sequencing revealed that the genome size of GD-2T was 3 059 321 bp with a G+C content of 63.57 mol%. On the basis of phylogenetic, phenotypic and chemotaxonomic characteristics, strain GD-2T is considered to represent a novel species of the genus Thauera, for which the name Thauera hydrothermalis sp. nov. is proposed. The type strain is GD-2T (=NBRC 112472T=CGMCC 1.15527T).