A Sb(III)-specific efflux transporter from Ensifer adhaerens E-60.

Antimony is pervasive environmental toxic substance, and numerous genes encoding mechanisms to resist, transform and extrude the toxic metalloid antimony have been discovered in various microorganisms. Here we identified a major facilitator superfamily (MFS) transporter, AntB, on the chromosome of the arsenite-oxidizing bacterium Ensifer adhaerens E-60 that confers resistance to Sb(III) and Sb(V). The antB gene is adjacent to gene encoding a LysR family transcriptional regulator termed LysRars, which is an As(III)/Sb(III)-responsive transcriptional repressor that is predicted to control expression of antB. Similar antB and lysRars genes are found in related arsenic-resistant bacteria, especially strains of Ensifer adhaerens, and the lysRars gene adjacent to antB encodes a member of a divergent subgroup of putative LysR-type regulators. Closely related AntB and LysRars orthologs contain three conserved cysteine residues, which are Cys17, Cys99, and Cys350 in AntB and Cys81, Cys289 and Cys294 in LysRars, respectively. Expression of antB is induced by As(III), Sb(III), Sb(V) and Rox(III) (4-hydroxy-3-nitrophenyl arsenite). Heterologous expression of antB in E. coli AW3110 (Δars) conferred resistance to Sb(III) and Sb(V) and reduced the intracellular concentration of Sb(III). The discovery of the Sb(III) efflux transporter AntB enriches our knowledge of the role of the efflux transporter in the antimony biogeochemical cycle.

Keguizhuia sedimenti gen. nov., sp. nov., isolated from river sediment represents a novel genus of the family Oxalobacteraceae.

A Gram-stain-negative bacterium, designated as R-40T, was isolated from sediment of the Mulong river in Mianyang city, Sichuan province, PR China. The cells of strain R-40T were aerobic non-motile and formed translucent white colonies on R2A agar. Growth occurred at 15-37 °C (optimum 30 °C), pH 5.0-9.0 (optimum 7.0) and salinities of 0-3.0 % (w/v, optimum 0 %). R-40T showed 95.2-96.6 % 16S rRNA gene sequence similarities with the type strains of species of the genera Oxalicibacterium, Herminiimonas, Lacisediminimonas, Paucimonas, Herbaspirillum and Noviherbaspirillum in the family Oxalobacteraceae. The results of phylogenetic analysis based on genome sequences indicated that the strain was clustered with type strains of species of the genera Oxalicibacterium and Herminiimonas in the family Oxalobacteraceae but formed a distinct lineage. The average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH) and average amino acid identity (AAI) values between R-40T and type strains of species of the genera Oxalicibacterium, Herminiimonas, Lacisediminimonas, Paucimonas, Herbaspirillum and Noviherbaspirillum ranged from 69.3 to 74.1 %, from 18.2 to 21.4 % and from 60.1 to 67.4 %, respectively. The major cellular fatty acids were C16 : 0, C17 : 0 cyclo and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The major quinone was ubiquinone-8 (Q-8). The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phospholipid and small amounts of glycophospholipids. The genome size of R-40T was 5.1 Mbp with 54.0 % DNA G+C content. On the basis of the evidence presented in this study, strain R-40T represents a novel species of a novel genus in the family Oxalobacteraceae, for which the name Keguizhuia sedimenti gen. nov., sp. nov. (type strain R-40T=MCCC 1K08818T=KCTC 8137T) is proposed.

Functional characterization of a novel violacein biosynthesis operon from Janthinobacterium sp. B9-8.

Violacein is a secondary metabolite mainly produced by Gram-negative bacteria that is formed from tryptophan by five enzymes encoded by a single operon. It is a broad-spectrum antibacterial pigment with various important biological activities such as anti-tumor, antiviral, and antioxidative effects. The newly discovered violacein operon vioABCDE was identified in the genome of the extremophile Janthinobacterium sp. B9-8. The key enzyme-encoding genes were cloned to construct the multigene coexpression plasmids pET-vioAB and pRSF-vioCDE. The violacein biosynthesis pathway was heterologously introduced into engineered Escherichia coli VioABCDE and VioABCDE-SD. The factors affecting violacein production, including temperature, pH, inoculum size, carbon and nitrogen source, precursor, and inducers were investigated. The violacein titer of VioABCDE-SD reached 107 mg/L in a two-stage fermentation process, representing a 454.4% increase over the original strain. The violacein operon from B9-8 provides a new microbial gene source for the analysis of the violacein synthesis mechanism, and the constructed engineering E. coli strains lay a foundation for the efficient and rapid synthesis of other natural products.Key points• The newly discovered violacein operon vioABCDE was identified in the genome of the extremophile Janthinobacterium sp. B9-8.• The violacein synthesis pathway was reconstructed in E. coli using two compatible plasmids.• A two-stage fermentation process was optimized for improved violacein accumulation.

Functional characterization of the methylarsenite-inducible arsRM operon from Noviherbaspirillum denitrificans HC18.

Microbial arsenic methylation by arsenite (As(III)) S-adenosylmethionine methyltransferases (ArsMs) can produce the intermediate methylarsenite (MAs(III)), which is highly toxic and is used by some microbes as an antibiotic. Other microbes have evolved mechanisms to detoxify MAs(III). In this study, an arsRM operon was identified in the genome of an MAs(III)-methylation strain Noviherbaspirillum denitrificans HC18. The arsM gene (NdarsM) is located downstream of an open reading frame encoding an MAs(III)-responsive transcriptional regulator (NdArsR). The N. denitrificans arsRM genes are co-transcribed whose expression is significantly induced by MAs(III), likely by alleviating the repressive effect of ArsR on arsRM transcription. Both in vivo and in vitro assays showed that NdArsM methylates MAs(III) to dimethyl- and trimethyl-arsenicals but does not methylate As(III). Heterologous expression of NdarsM in arsenic-sensitive Escherichia coli AW3110 conferred resistance to MAs(III) but not As(III). NdArsM has the four conserved cysteine residues present in most ArsMs, but only two of them are essential for MAs(III) methylation. The ability to methylate MAs(III) by enzymes such as NdArsM may be an evolutionary step originated from enzymes capable of methylating As(III). This finding reveals a mechanism employed by microbes such as N. denitrificans HC18 to detoxify MAs(III) by further methylation.

Molecular signatures of Janthinobacterium lividum from Trinidad support high potential for crude oil metabolism.

BACKGROUND: Janthinobacterium lividum is considered to be a psychrotrophic bacterial species. For the first time in the literature, J. lividum strains were isolated from Trinidad presenting with atypical features - hydrocarbonoclastic and able to survive in a tropical environment. METHODS: Identification of the Trinidad strains was carried out through 16S rRNA phylogenetic analysis. Gene-specific primers were designed to target the VioA which encodes violacein pigment and the EstA/B gene which encodes secreted extracellular lipase. Bioinformatics analyses were carried out on the nucleotide and amino acid sequences of VioA and EstA/B genes of the Trinidad Janthinobacterium strains to assess functionality and phylogenetic relatedness to other Janthinobacterium sequences specifically and more broadly, to other members of the Oxalobacteraceae family of betaproteobacteria. RESULTS: 16S rRNA confirmed the identity of the Trinidad strains as J. lividum and resolved three of the Trinidad strains at the intra-specific level. Typical motility patterns of this species were recorded. VioAp sequences were highly conserved, however, synonymous substitutions located outside of the critical sites for enzyme function were detected for the Trinidad strains. Comparisons with PDB 6g2p model from aa231 to aa406 further indicated no functional disruption of the VioA gene of the Trinidad strains. Phylogeny of the VioA protein sequences inferred placement of all J. lividum taxa into a highly supported species-specific clade (bs = 98%). EstA/Bp sequences were highly conserved, however, synonymous substitutions were detected that were unique to the Trinidad strains. Phylogenetic inference positioned the Trinidad consensus VioA and EstA protein sequences in a clearly distinct branch. CONCLUSIONS: The findings showed that the primary sequence of VioAp and EstA/Bp were unique to the Trinidad strains and these molecular signatures were reflected in phylogenetic inference. Our results supported chemotaxis, possible elective inactivation of VioA gene expression and secreted lipase activity as survival mechanisms of the Trinidad strains in petrogenic conditions.

Duganella aceris sp. nov., isolated from tree sap and proposal to transfer of Rugamonas aquatica and Rugamonas rivuli to the genus Duganella as Duganella aquatica comb. nov., with the emended description of the genus Rugamonas.

A Gram-reaction-negative, strictly aerobic, betaproteobacterial strain, designated SAP-35T, was isolated from sap extracted from Acer pictum in Mt. Halla in Jeju, Republic of Korea, and its taxonomic status was examined by a polyphasic approach. Cells of the organism were non-sporulating, motile rods and grew at 4-30 °C, pH 6-7 and in the absence of NaCl. 16S rRNA gene- and whole genome-based phylogenetic analyses showed that strain SAP-35T belonged to the family Oxalobacteraceae and was closely related to Rugamonas rivuli (98.9% 16S rRNA gene sequence similarity) and Rugamonas aquatica (98.4%). The phylogenomic clustering and average amino acid identity values supported that strain SAP-35T belonged to the genus Duganella and two Rugamonas species should be transferred to the genus Duganella. The major isoprenoid quinone of the isolate was Q-8. The major polar lipids were phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and an unidentified aminophospholipid. The predominant fatty acids were summed feature 3, C16:0 and C17:0 cyclo. The G + C content of genome was 64.9%. The average nucleotide identity and dDDH values between strain SAP-35T and the members of the genera Rugamonas and Duganella were < 85.1% and < 49%, respectively. Based on the combined data presented here, strain SAP-35T (= KCTC 72227T = NBRC 113903T) represents a novel species of the genus Duganella, for which the name Duganella aceris sp. nov. is proposed. Also, Rugamonas aquatica Lu et al. (Int J Syst Evol Microbiol 70: 3328-3334, 2020) and Rugamonas aquatica Lu et al. 2020 are reclassified as Duganella aquatica comb. nov., with the emended description of the genus Rugamonas.

Noviherbaspirillum pedocola sp. nov., isolated from oil-contaminated experimental soil.

An orange-coloured, rod-shaped, and aerobic bacterial strain DKR-6 T was isolated from oil-contaminated experimental soil. The strain was Gram-stain-negative, catalase and oxidase positive, and grew at temperature 10-42 °C, at pH 5.5-9.5, and at 0-3.0% (w/v) NaCl concentration. The phylogenetic analysis and 16S rRNA gene sequence analysis suggested that the strain DKR-6 T was affiliated to the genus Noviherbaspirillum, with the closest species being Noviherbaspirillum massiliense JC206T (96.3% sequence similarity). The chemotaxonomic profiles revealed the presence of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylcholine as the principal polar lipids; C16:0, C17:0 cyclo, summed feature 3 (C16:1ω7c and/or C16: 1ω6c), and summed feature 8 (C18:1ω7c/or C18:1ω6c) as the main fatty acids; and Q-8 as a sole ubiquinone. The DNA G + C content was 61.6%. The polyphasic taxonomic features illustrated in this study clearly implied that strain DKR-6 T represents a novel species in the genus Noviherbaspirillum, for which the name Noviherbaspirillum pedocola sp. nov. is proposed with the type strain DKR-6 T (= KACC 22074 T = NBRC 114727 T).

Genome insight and description of antibiotic producing Massilia antibiotica sp. nov., isolated from oil-contaminated soil.

An ivory-coloured, motile, Gram-stain-negative bacterium, designated TW-1T was isolated from oil-contaminated experimental soil in Kyonggi University. The phylogenetic analysis based on 16S rRNA gene sequence revealed, strain TW-1T formed a lineage within the family Oxalobacteraceae and clustered as members of the genus Massilia. The closest members were M. pinisoli T33T (98.8% sequence similarity), M. putida 6NM-7T (98.6%), M. arvi THG-RS2OT (98.5%), M. phosphatilytica 12-OD1T (98.3%) and M. niastensis 5516S-1T (98.2%). The sole respiratory quinone is ubiquinone-8. The major cellular fatty acids are hexadeconic acid, cis-9, methylenehexadeconic acid, summed feature 3 and summed feature 8. The major polar lipids are phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. The DNA G + C content of the type strain is 66.3%. The average nucleotide identity (ANI) and in silico DNA-DNA hybridization (dDDH) relatedness values between strain TW-1T and closest members were below the threshold value for species demarcation. The genome size is 7,051,197 bp along with 46 contigs and 5,977 protein-coding genes. The genome showed 5 putative biosynthetic gene clusters (BGCs) that are responsible for different secondary metabolites. Cluster 2 showed thiopeptide BGC with no known cluster blast, indicating TW-1T might produce novel antimicrobial agent. The antimicrobial assessment also showed that strain TW-1T possessed inhibitory activity against Gram-negative pathogens (Escherichia coli and Pseudomonas aeruginosa). This is the first report of the species in the genus Massilia which produces antimicrobial compounds. Based on the polyphasic study, strain TW-1T represents novel species in the genus Massilia, for which the name Massilia antibiotica sp. nov. is proposed. The type strain is TW-1T (= KACC 21627T = NBRC 114363T).

Description of Massilia rubra sp. nov., Massilia aquatica sp. nov., Massilia mucilaginosa sp. nov., Massilia frigida sp. nov., and one Massilia genomospecies isolated from Antarctic streams, lakes and regoliths.

Bacteria of the genus Massilia often colonize extreme ecosystems, however, a detailed study of the massilias from the Antarctic environment has not yet been performed. Here, sixty-four Gram-stain-negative, aerobic, motile rods isolated from different environmental samples on James Ross Island (Antarctica) were subjected to a polyphasic taxonomic study. The psychrophilic isolates exhibited slowly growing, moderately slimy colonies revealing bold pink-red pigmentation on R2A agar. The set of strains exhibited the highest 16S rRNA gene sequence similarities (99.5-99.9%) to Massilia violaceinigra B2T and Massilia atriviolacea SODT and formed several phylogenetic groups based on the analysis of gyrB and lepA genes. Phenotypic characteristics allowed four of them to be distinguished from each other and from their closest relatives. Compared to the nearest phylogenetic neighbours the set of six genome-sequenced representatives exhibited considerable phylogenetic distance at the whole-genome level. Bioinformatic analysis of the genomic sequences revealed a high number of putative genes involved in oxidative stress response, heavy-metal resistance, bacteriocin production, the presence of putative genes involved in nitrogen metabolism and auxin biosynthesis. The identification of putative genes encoding aromatic dioxygenases suggests the biotechnology potential of the strains. Based on these results four novel species and one genomospecies of the genus Massilia are described and named Massilia rubra sp. nov. (P3094T=CCM 8692T=LMG 31213T), Massilia aquatica sp. nov. (P3165T=CCM 8693T=LMG 31211T), Massilia mucilaginosa sp. nov. (P5902T=CCM 8733T=LMG 31210T), and Massilia frigida sp. nov. (P5534T=CCM 8695T=LMG 31212T).

First Complete Genome Sequences of Janthinobacterium lividum EIF1 and EIF2 and Their Comparative Genome Analysis.

We present the first two complete genomes of the Janthinobacterium lividum species, namely strains EIF1 and EIF2, which both possess the ability to synthesize violacein. The violet pigment violacein is a secondary metabolite with antibacterial, antifungal, antiviral, and antitumoral properties. Both strains were isolated from environmental oligotrophic water ponds in Göttingen. The strains were phylogenetically classified by average nucleotide identity (ANI) analysis and showed a species assignment to J. lividum with 97.72% (EIF1) and 97.66% (EIF2) identity. These are the first complete genome sequences of strains belonging to the species J. lividum. The genome of strain EIF1 consists of one circular chromosome (6,373,589 bp) with a GC-content of 61.98%. The genome contains 5,551 coding sequences, 122 rRNAs, 93 tRNAs, and 1 tm-RNA. The genome of EIF2 comprises one circular chromosome (6,399,352 bp) with a GC-content of 61.63% and a circular plasmid p356839 (356,839 bp) with a GC-content of 57.21%. The chromosome encodes 5,691 coding sequences, 122 rRNAs, 93 tRNAs, and 1 tm-RNA and the plasmid harbors 245 coding sequences. In addition to the highly conserved chromosomally encoded violacein operon, the plasmid comprises a nonribosomal peptide synthetase cluster with similarity to xenoamicin, which is a bioactive compound effective against protozoan parasites.

Glaciimonas soli sp. nov., a soil bacterium isolated from the forest of a high elevation mountain.

A Gram-negative, psychrophilic bacterium, designated strain GS1T, was isolated from a forest soil sample collected from the West Peak of Mt. Yushan, Yushan National Park, Taiwan. Cells grown in broth cultures were mostly non-motile and non-flagellated, whereas motile cells with monotrichous, subpolar flagella were also observed. The novel strain grew over a temperature range of 4-25 °C with optimum growth at 10-15 °C. It grew aerobically and was not capable of anaerobic growth by fermentation of D-glucose or other carbohydrates. Ubiquinone 8 was the predominant isoprenoid quinone. The major polar lipids comprised phosphatidylethanolamine, diphosphatidylglycerol and dimethylaminoethanol. Cellular fatty acids were dominated by C16:1ω7c (35.2%), C16:0 (19.5%), C18:1ω7c (18.8%) and C17:0ω7c cyclo (15.5%). The DNA G + C content was 49.2 mol% evaluated according to the genomic sequencing data. Strain GS1T shared more than 96.5% 16S rRNA gene sequence similarities with type strains of four Collimonas species (97.2-97.5%), three Glaciimonas species (97.3% for each of the three) and Oxalicibacterium solurbis (96.5%). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain GS1T formed a stable genus-level clade with type strains of species in the genus Glaciimonas in the family Oxalobacteraceae and GS1T was an outgroup with respect to these Glaciimonas species. Characteristically, strain GS1T could be easily distinguished from the recognised Glaciimonas species by exhibition of swimming motility with monotrichous, subpolar flagellum in some of the cells, ability to grow in NaCl at 2% but not at 3% and the distinguishable fatty acid profiles. On the basis of the polyphasic taxonomic data from this study, strain GS1T is considered to represent a novel species of the genus Glaciimonas, for which the name Glaciimonas soli sp. nov. is proposed. The type strain is GS1T (= JCM 33275T = BCRC 81091T).

Collimonas antrihumi sp. nov., isolated from a natural cave and emended description of the genus Collimonas.

A novel bacterium, designated strain C3-17T, was isolated from a natural cave in Jeju, Republic of Korea. Cells of the organism were Gram-stain-negative, strictly aerobic, non-sporulating, non-motile rods. The polar lipids present were phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminophospholipids, an unidentified aminolipid and an unidentified lipid. The sole isoprenoid quinone was Q-8. The predominant fatty acids were C16 : 0 and summed feature 3, and the DNA G+C content was 54.5 mol%. A phylogenetic tree based on 16S rRNA gene sequences showed that strain C3-17T belonged to the family Oxalobacteraceae and was most closely related to the type strains of the genus Collimonas. 16S rRNA gene sequence similarities between the novel isolate and the closest neighbours, Collimonas pratensis Ter91T, Collimonas fungivorans Ter6T and Collimonas arenae NCCB 100031T were 98.7, 98.5 and 98.1 %, respectively. On the basis of data obtained by polyphasic analyses and DNA-DNA hybridization, strain C3-17T (=KACC 19055T=DSM 104040T) represents a novel species of the genus Collimonas, for which the name Collimonasantrihumi sp. nov. is proposed.

Massilia armeniaca sp. nov., isolated from desert soil.

A Gram-stain-negative, aerobic, motile and rod-shaped bacterium, strain ZMN-3T, was isolated from desert soil sample collected from Ongniod Qi, Inner Mongolia, China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZMN-3T was affiliated with the genus Massilia and showed the highest similarity to Massilia humi THG S6-8T (98.9 %) and Massilia buxea A9T (98.2 %). In partial gyrB and lepA sequences, the highest similarity of strain ZMN-3T and M. humi THG S6-8T were 95.9 and 96.8 %, respectively. The DNA-DNA hybridization value between strain ZMN-3T and its closely related type strains were all below 70 %. The major respiratory quinone of strain ZMN-3T was Q-8 and the major cellular fatty acids consisted of summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. The predominant polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified phospholipid. The DNA G+C content of strain ZMN-3T was 66.3 mol%. On the basis of this polyphasic taxonomic study, strain ZMN-3T is considered to represent a novel species of the genus Massilia, for which the name Massilia armeniaca sp. nov. is proposed. The type strain is ZMN-3T (=CGMCC 1.16209T=DSM 104676T).

Physiological and genomic insights into the lifestyle of arsenite-oxidizing Herminiimonas arsenitoxidans.

Arsenic, a representative toxic metalloid, is responsible for serious global health problems. Most organisms possess arsenic resistance strategies to mitigate this toxicity. Here, we reported a microorganism, strain AS8, from heavy metal/metalloid-contaminated soil that is able to oxidize arsenite, and investigated its physiological and genomic traits. Its cells were rod-shaped and Gram-negative, and formed small beige-pigmented colonies. 16S rRNA-based phylogenetic analysis indicated that the strain belongs to the genus Herminiimonas and is closely related to Herminiimonas glaciei UMB49T (98.7% of 16S rRNA gene sequence similarity), Herminiimonas arsenicoxydans ULPAs1T (98.4%), and Herminiimonas saxobsidens NS11T (98.4%). Under chemolithoheterotrophic conditions, the strain utilized some organic acids and amino acids as carbon and/or nitrogen sources but not electron sources. Further, the strain grew as a sulfur oxidizer in a complex medium (trypticase soy agar). Unexpectedly, most carbohydrates failed to support its growth as sole carbon sources. Genome sequencing supported these observations, and very few ABC transporters capable of oligo/monosaccharide uptake were identified in the AS8 genome. The genome harbored genes required for the colonization, flagella biosynthesis, urea degradation, and heavy metal and antibiotic resistance. Based on these polyphasic and genomic analyses, we propose that the strain AS8 be named Herminiimonas arsenitoxidans.

Undibacterium amnicola sp. nov., isolated from a freshwater stream.

Strain KYPY9T, isolated from the Funglin Stream in Taiwan, was characterized using a polyphasic taxonomy approach. Cells of KYPY9T were Gram-staining-negative, strictly aerobic, motile by means of a single polar flagellum, rod-shaped and formed light yellow colonies. Optimal growth occurred at 20-25 °C, at pH 7 and with 0 % NaCl. The results of phylogenetic analyses based on 16S rRNA gene sequences indicated that KYPY9T represented a member of the genus Undibacterium and the sequence similarity between the 16S rRNA genes of KYPY9T and of the type strains of other species of the genus Undibacterium ranged from 94.1 to 98.0 %. The closest relatives of KYPY9T were Undibacterium seohonense SHS5-24T (98.0 %) and Undibacterium macrobrachii CMJ-9T (97.0 %). KYPY9T had summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0 as the predominant fatty acids. The major cellular hydroxy fatty acid was C10 : 0 3-OH. KYPY9T had phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol as the predominant polar lipids. The polyamine profile was composed of the major compound 2-hydroxyputrescine and moderate amounts of putrescine. The major respiratory quinone was Q-8 and the DNA G+C content was 47.4 mol%. The DNA-DNA relatedness of KYPY9T with respect to Undibacterium seohonense SHS5-24T and Undibacterium macrobrachii CMJ-9T was less than 35 %. On the basis of the phylogenetic inference and phenotypic data, KYPY9T was recognized as a representative of a novel species within the genus Undibacterium. The name Undibacterium amnicola sp. nov. is proposed, with KYPY9T (=BCRC 81009T=LMG 29730T=KCTC 52442T) as the type strain.

Massilia agri sp. nov., isolated from reclaimed grassland soil.

A light yellow-coloured, Gram-stain-negative, motile and rod-shaped bacterium, designated strain K-3-1T, was isolated from reclaimed grassland soils of Belbari, Morang, Nepal. It was able to grow at 4-45 °C, at pH 5.0-10.0, and at 0-2 % (w/v) NaCl concentrations. This strain was taxonomically characterized by a polyphasic approach. Based on the 16S rRNA gene sequence analysis, strain K-3-1T belongs to the genus Massilia and is closely related to Massilia consociata CCUG 58010T (98.3 % sequence similarity), Massilia tieshanensis TS3T (98.1 % sequence similarity), Massilia kyonggiensis TSA1T (98.1 % sequence similarity), Massilia yuzhufengensisY1243-1T (98.1 % sequence similarity), Massilia haematophila CCUG 38318T (98.0 % sequence similarity), Massilia varians CCUG 35299T (97.9 % sequence similarity), Massilia niastensis 5516 S-1T (97.6 % sequence similarity) and Massilia alkalitolerans YIM 31775T (97.5 % sequence similarity). The predominant respiratory quinone was ubiquinone-8. The polar lipid profile revealed the presence of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The predominant fatty acids of strain K-3-1T were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0, C12 : 0, C10 : 0 3-OH and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The genomic DNA G+C content of this novel strain was 66.8 mol%. The DNA-DNA relatedness between strain K-3-1T and its closest reference strains were significantly lower than the threshold value of 70 %. The morphological, physiological, chemotaxonomic and phylogenetic analyses clearly distinguished this strain from its closest phylogenetic neighbours. Thus, strain K-3-1T represents a novel species of the genus Massilia, for which the name Massilia agri sp. nov. is proposed. The type strain is K-3-1T (=KEMB 9005-446T=KACC 19000T=JCM 31661T).

Massilia solisilvae sp. nov., Massilia terrae sp. nov. and Massilia agilis sp. nov., isolated from forest soil in South Korea by using a newly developed culture method.

Using a newly developed culture method for not yet cultured soil bacteria, three Gram-stain-negative, aerobic, non-spore-forming, motile, and rod-shaped bacteria (strain designated J18T, J11T and J9T) were isolated from forest soil at Kyonggi University, South Korea. Isolates were subjected to a taxonomic study by using a polyphasic approach. According to a phylogenetic tree based on 16S rRNA gene sequences, strains J18T, J11T and J9T belonged to the genus Massilia and clustered with Massilia haematophila CCUG 38318T (similarity range: 97.6~98.0 %). The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol, and the genomic DNA G+C contents of strains J18T, J11T and J9T were 63.4, 68.7 and 64.5 mol%, respectively. The major polyamines were putrescine and 2-hydroxyputescine, which were detected in all three strains. DNA-DNA between the three tested strains and the reference strains much lower than 70 %, the recommended threshold value for the delineation of genomic species. The predominant respiratory quinine was ubiquinone-8 (Q-8) and the major cellular fatty acids were Summed feature 3 (C16 : 1ω6c/C16 : 1ω7c) and C16 : 0. On the basis of phenotypic and genotypic data and DNA-DNA hybridization results, the three isolates are considered to represent three novel species of the genus Massilia, for which the names Massilia solisilvae sp. nov. for type strain J18T (=KEMB 9005-366T=JCM 31607T), Massilia terrae sp. nov. for type strain J11T (=KEMB 9005-360T=JCM 31606T) and Massilia agilis sp. nov. for type strain J9T (=KEMB 9005-359T=JCM 31605T) are proposed.

LRLSHMDA: Laplacian Regularized Least Squares for Human Microbe-Disease Association prediction.

An increasing number of evidences indicate microbes are implicated in human physiological mechanisms, including complicated disease pathology. Some microbes have been demonstrated to be associated with diverse important human diseases or disorders. Through investigating these disease-related microbes, we can obtain a better understanding of human disease mechanisms for advancing medical scientific progress in terms of disease diagnosis, treatment, prevention, prognosis and drug discovery. Based on the known microbe-disease association network, we developed a semi-supervised computational model of Laplacian Regularized Least Squares for Human Microbe-Disease Association (LRLSHMDA) by introducing Gaussian interaction profile kernel similarity calculation and Laplacian regularized least squares classifier. LRLSHMDA reached the reliable AUCs of 0.8909 and 0.7657 based on the global and local leave-one-out cross validations, respectively. In the framework of 5-fold cross validation, average AUC value of 0.8794 +/-0.0029 further demonstrated its promising prediction ability. In case studies, 9, 9 and 8 of top-10 predicted microbes have been manually certified to be associated with asthma, colorectal carcinoma and chronic obstructive pulmonary disease by published literature evidence. Our proposed model achieves better prediction performance relative to the previous model. We expect that LRLSHMDA could offer insights into identifying more promising human microbe-disease associations in the future.

Noviherbaspirillum agri sp. nov., isolated from reclaimed grassland soil, and reclassification of Herbaspirillum massiliense (Lagier et al., 2014) as Noviherbaspirillum massiliense comb. nov.

A straw-coloured, Gram-staining-negative, aerobic, motile and rod-shaped bacterium, designated strain K-1-15T, was isolated from reclaimed grassland soil from Biratnagar, Morang, Nepal. This strain was non-spore-forming, catalase-negative and oxidase-positive. It was able to grow at 10-45 °C, pH 6.5-9.5 and 0-1.5 % (w/v) NaCl concentration. This strain was taxonomically characterized by a polyphasic approach. Based on the results of 16S rRNA gene sequence analysis, K-1-15T formed a distinct lineage within the family Oxalobacteraceae and was most closely related to members of the genera Herbaspirillum(96.99-95.34 % sequence similarity), Noviherbaspirillum(96.72-95.45 % sequence similarity) and Paraherbaspirillum (95.85 % sequence similarity). The only respiratory quinone was ubiquinone-8. The polar lipid profile revealed the presence of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. The major fatty acids of K-1-15T were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16  :   0, summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), C10 : 0 3-OH, and iso-C16 : 0. The genomic DNA G+C content of this novel strain was 65.2 mol %. The DNA-DNA relatedness between K-1-15T and Herbaspirillum massiliense DSM 25712T and Noviherbaspirillum soli LMG 26149T were 18.3 and 13.7 % repectively. On the basis of the results of morphological, physiological, chemotaxonomic and phylogenetic analyses, K-1-15T represents a novel species of the genus Noviherbaspirillum in the family Oxalobacteraceae, for which the name Noviherbaspirillum agri sp. nov. is proposed. The type strain is K-1-15T (=KEMB 9005-422T=KACC 18909T=JCM 31463T). Based on new data obtained in this study, we also propose the reclassification of Herbaspirillum massiliense as Noviherbaspirillum massiliense comb. nov. (type strain JC206T=CSUR P159T=DSM 25712T).

Noviherbaspirillum humi sp. nov., isolated from soil.

Two novel Gram-stain negative, motile, non-spore forming, facultative aerobic and short rod shaped bacterial strains, designated U15(T) and U32, were isolated from soil obtained from Ukraine. The sequence similarity of the 16S rRNA gene between strains U15(T) and U32 was found to be 99.5 %. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that new bacteria belong to the genus Noviherbaspirillum. The closest member of the genus was found to be Noviherbaspirillum malthae (97.0 %) followed by Noviherbaspirillum suwonensis (96.3 %). The novel isolates was observed to grow optimally at 30 °C and pH 7.0. The major fatty acids present in the two strains were identified as summed feature 3 (C16:1 ω7c/C16:1 ω6c), C16:0, and summed feature 8 (C18:1 ω7c/C18:1 ω6c). Ubiquinone 8 was identified as the respiratory quinone component for both the strains. The polar lipid (L) profile contained phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, aminophospholipid, unidentified aminolipid and unidentified Ls, and putrescine and 2-hydroxyputrescine as major polyamines. The G+C content of the DNA for the strain U15(T) was found to be 61.2 mol%. The DNA-DNA relatedness between U15(T) and U32 and closely related species was less than 40 %. Based on the polyphasic taxonomic analysis, a new species, Noviherbaspirillum humi sp. nov., is proposed. The type strain is strain U15(T) = JCM 19873(T) = KEMB 7305-102(T).