Genome-based reclassification of Bacillus plakortidis Borchert et al. 2007 and Bacillus lehensis Ghosh et al. 2007 as a later heterotypic synonym of Bacillus oshimensis Yumoto et al. 2005; Bacillus rhizosphaerae Madhaiyan et al. 2011 as a later heterotypic synonym of Bacillus clausii Nielsen et al. 1995 Abstract In the present study, phylogenetic and genome-based comparison was carried out to clarify the taxonomic positions of alkaliphilic Bacillus species, Bacillus plakortidis, Bacillus lehensis, Bacillus oshimensis, Bacillus rhizosphaerae and Bacillus clausii. Phylogenetic trees based on 16S rRNA gene sequences and concatenated protein marker genes were constructed. Average nucleotide identity (ANI) values were calculated to compare genetic relatedness. In phylogenetic trees, B. plakortidis DSM 19153T, B. lehensis DSM 19099T, and B. oshimensis DSM 18940T; B. rhizosphaerae DSM 21911T and B. clausii DSM 8716T clade together. The average nucleotide identity (ANI) values between B. oshimensis DSM 18940T, B. plakortidis DSM 19153T and B. lehensis DSM 19099T ranged from 98.7–98.8%, while the ANI values between B. rhizosphaerae DSM 21911T and B. clausii DSM 8716T were 95.2–95.5%. The ANI values were higher than the recognized threshold value for bacterial species delineation. Based on phylogenetic and genome comparison we propose reclassification of B. plakortidis and B. lehensis as a later heterotypic synonym of B. oshimensis; B. rhizosphaerae as a later heterotypic synonym of B. clausii.

Isolation, functional characterization and efficacy of biofilm-forming rhizobacteria under abiotic stress conditions Abstract Abiotic stresses such as salinity, drought and excessive heat are associated with significant loss of crop productivity globally, and require effective strategies for their reduction or tolerance. Biofilm-forming rhizobacteria, which harbor multifarious plant growth promoting traits and tolerance to abiotic stress, are believed to benefit plant health and production even under environmental stresses. The primary objective of this study was to investigate indigenous biofilm-forming rhizobacteria (Pseudomonas spp., Bacillus sp., Pantoea sp., Brevibacterium sp. and Acinetobacter sp.) for their functional diversity relevant to plant growth promoting activities, biofilm development and tolerance to abiotic stress conditions. The most promising isolates among FAP1, FAP2, FAP3, FAP4, FAP5, FAP10, FAB1, FAB3 and FAA1 were selected. Rhizobacteria exhibited high tolerance to salinity (1.5 M NaCl) and drought stress (up to 55% PEG-6000) conditions in vitro. The isolates demonstrated varying levels of PGP activities (IAA production and phosphate solubilization), biofilm development, and production of alginate and exopolysaccharides in the presence of salinity, drought stress and elevated temperature. Further efficacy of the isolates was demonstrated by inoculating to wheat (Triticum aestivum L.) plants in greenhouse conditions under both normal and drought stress for up to 30 days inoculation. The plant growth potential of the isolates was in the order of FAP3 > FAB3 > FAB1 > FAP10 > FAP5 > FAP4 > FAA1 > FAP2 > FAP1. The present study resulted in successful selection of promising PGPR as identified by 16S rRNA gene sequence analysis. Field study is needed to evaluate their relative performance in both ‘normal’ and stressed environments in order to be exploited for plant stress management.

Halorubrum amylolyticum sp. nov., a novel halophilic archaeon isolated from a salt mine Abstract A pleomorphic and non-motile halophilic archaeon forming light-red pigmented colonies, strain ZC67T, was isolated from the Yuanyongjing Salt Mine, Yunnan, China. Based on similarity search and phylogenetic analysis of the 16S rRNA gene sequence, strain ZC67T belongs to the genus Halorubrum and is closely related to the species of Halorubrum (Hrr.) saccharovorum JCM 8865T, Hrr. persicum C49T, Hrr. halophilum B8T, Hrr. lipolyticum 9-3T, Hrr. salsamenti Y69T and Hrr. depositum Y78T with 16S rRNA gene sequence similarities of 99.0%, 98.7%, 98.5%, 98.4%, 98.1% and 97.7%, respectively. The values of average nucleotide identity (ANI) and average amino-acid identity (AAI) between strain ZC67T and its close relatives were less than 90.5% and 89.3%, respectively. In silico DNA-DNA hybridization (DDH) analysis showed that DNA-DNA relatedness between strain ZC67T and its relatives is less than 45%. Values of ANI, AAI and in silico DDH were clearly below the thresholds used for the delineation of a new species. The major polar lipids of strain ZC67T were similar to other neutrophilic members in the genus Halorubrum containing phosphatidylglycerol, phosphatidylglycerolphosphate methyl ester, phosphatidylglycerol sulfate and sulfated mannosyl-glucosyl-glycerol diether-1. The DNA G+C content was determined to be 66.3 mol% (based on the draft genome). Combined with other diagnostic characteristics, e.g. phenotypic and chemotaxonomic differences, strain ZC67T is concluded to represent a novel species in the genus Halorubrum, for which the name Halorubrum amylolyticum sp. nov. is proposed. The type strain is ZC67T (=CGMCC 1.15718T = JCM 31850T).

Chachezhania antarctica gen. nov., sp. nov., a novel member of the family ‘ Rhodobacteraceae ’ isolated from Antarctic seawater Abstract A Gram-stain-negative, aerobic, non-flagellated, rod-shaped bacterium, designated strain SM1703T, was isolated from Antarctic seawater collected near the Chinese Antarctic Great Wall Station, King George Island, West Antarctica. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SM1703T formed a distinct phylogenetic lineage within the family ‘Rhodobacteraceae’, sharing high 16S rRNA gene sequence similarity with Marivita litorea (95.5%). The strain grew at 10–37 °C (optimum, 25 °C) and with 0.5–13% (w/v) NaCl (optimum, 3–5%). The major cellular fatty acids were C19:0 cyclo ω8c, C18:1ω7c, C18:1 2-OH and C16:0 2-OH. The major polar lipids were phosphatidylglycerol, phosphatidylcholine, an unidentified aminolipid and an unidentified lipid. The genomic DNA G+C content of strain SM1703T was 64.6 mol%. Based on the results of the polyphasic characterization for strain SM1703T, it is classified as the representative of a novel species in a new genus of the family ‘Rhodobacteraceae’, for which the name Chachezhania antarctica gen. nov., sp. nov. is proposed. The type strain of Chachezhania antarctica is SM1703T (= MCCC 1K03470T = KCTC 62794T = CCTCC AB 2018351T).

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

Rubellimicrobium rubrum sp. nov., a novel bright reddish bacterium isolated from a lichen sample Abstract A novel strain, YIM 131921T, was isolated from a Physcia sp. lichen collected from the South Bank Forest of the Baltic Sea. The strain is Gram-negative, catalase positive and oxidase negative, strictly aerobic, asporogenous, non-motile and reddish brown in colour. The temperature and pH for growth were found to be 20–30 °C (optimum 28 °C) and pH 6.5–12.0 (optimum pH 7.0 ± 0.5). No growth was observed in the presence of NaCl. Based on 16S rRNA gene sequence similarity, strain YIM 131921T shares high similarities with Rubellimicrobium roseum YIM 48858T (98.3%), followed by Rubellimicrobium mesophilum MSL-20T (96.8%), Rubellimicrobium aerolatum 5715S-9T (96.1%) and Rubellimicrobium thermophilum DSM 16684T (96.0%). Phylogenetic trees showed YIM 131921T forms a cluster with type strains of the genus Rubellimicrobium. The predominant cellular fatty acids (> 20%) were identified as summed feature 8 (C18:1ω7c) and C16:0. Q-10 was found to be the predominant respiratory ubiquinone. The polar lipids were identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, glycolipid, phospholipids and an unidentified aminolipid. The DNA G + C content of the draft genome sequence is 66.6 mol%. Strain YIM 131921T showed an average nucleotide identity value of 80.3% and a digital DNA–DNA hybridizations value of 26.1% with the reference strain R. roseum YIM 48858T based on draft genome sequences. Based on comparative analyses of phenotypic, molecular, chemotaxonomic data and genomic comparisons, strain YIM 131921T is concluded to represent a novel species of the genus Rubellimicrobium, for which the name Rubellimicrobium rubrum sp. nov. is proposed. The type strain is YIM 131921T (= CGMCC 1.13958T = NBRC 114054T = KCTC 72461T).

Genetic marker-based multi-locus sequence analysis for classification, genotyping, and phylogenetics of the family Bifidobacteriaceae as an alternative approach to phylogenomics Abstract Bifidobacteria are widely known for their probiotic potential; however, little is known regarding the ecological significance and potential probiotic effects of the phylogenetically related ‘scardovial’ genera (Aeriscardovia, Alloscardovia, Bombiscardovia, Galliscardovia, Neoscardovia, Parascardovia, Pseudoscardovia and Scardovia) and Gardnerella classified with bifidobacteria within the Bifidobacteriaceae family. Accurate classification and genotyping of bacteria using certain housekeeping genes is possible, whilst current phylogenomic analyses allow for extremely precise classification. Studies of applicable genetic markers may provide results comparable to those obtained from phylogenomic analyses of the family Bifidobacteriaceae. Segments of the glyS (624 nucleotides), pheS (555 nucleotides), rpsA (630 nucleotides), and rpsB (432 nucleotides) genes and their concatenated sequence were explored. The mean glyS, pheS, rpsB and rpsA gene sequence similarities calculated for Bifidobacterium taxa were 84.8, 85.2, 90.2 and 86.8%, respectively. Interestingly, the average value of the Average Nucleotide Identity among 67 type strains of the family Bifidobacteriaceae (84.70%) calculated based on values published recently was in agreement with the average pairwise similarity (84.6%) among 75 type strains of Bifidobacteriaceae family computed in this study using the concatenated sequences of four gene fragments. Similar to phylogenomic analyses, several gene sequence and phylogenetic analyses revealed that concatenated gene regions allow for classification of Bifidobacteriaceae strains into particular phylogenetic clusters and groups. Phylogeny reconstructed from the concatenated sequences assisted in defining two novel phylogenetic groups, the Bifidobacterium psychraerophilum group consisting of B. psychraerophilum, Bifidobacterium crudilactis and Bifidobacterium aquikefiri species and the Bifidobacterium bombi group consisting of B. bombi, Bifidobacterium bohemicum and Bifidobacterium commune.

New genus-specific primers for PCR identification of Rubrobacter strains Abstract A set of oligonucleotide primers, Rubro223f and Rubro454r, were found to amplify a 267 nucleotide sequence of 16S rRNA genes of Rubrobacter type strains. The primers distinguished members of this genus from other deeply-rooted actinobacterial lineages corresponding to the genera Conexibacter, Gaiella, Parviterribacter, Patulibacter, Solirubrobacter and Thermoleophilum of the class Thermoleophilia. Amplification of DNA bands of about 267 nucleotides were generated from environmental DNA extracted from soil samples taken from two locations in the Atacama Desert. Sequencing of a DNA library prepared from the bands showed that all of the clones fell within the evolutionary radiation occupied by the genus Rubrobacter. Most of the clones were assigned to two lineages that were well separated from phyletic lines composed of Rubrobacter type strains. It can be concluded that primers Rubro223f and Rubro454r are specific for the genus Rubrobacter and can be used to detect the presence and abundance of members of this genus in the Atacama Desert and other biomes.

Amnibacterium setariae sp. nov., an endophytic actinobacterium isolated from dried foxtail Abstract A Gram-stain positive, short rod-shaped, aerobic, motile by means of gliding, yellow-pigmented actinobacterium, designated strain DD4aT, was isolated from dry yellow foxtail. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DD4aT is closely related to Amnibacterium soli MB78T (98.4% similarity), Amnibacterium kyonggiense KSL51201-037T (98.2%) and Amnibacterium endophyticum 1T4Z-3T (97.43%). Strain DD4aT forms yellow colonies on R2A agar medium. The peptidoglycan was found to contains diaminopimelic acid (which is a diagnostic cell wall diamino acid), alanine, glutamic acid and lysine. The polar lipids diphosphatidylglycerol, phosphatidylglycerol, six unidentified glycolipids and an unidentified polar lipid were found to be present in strain DD4aT. The major cellular fatty acids anteiso-C15:0 (42.9%) and iso-C16:0 (34.6%) were found in strain DD4aT. The predominant respiratory quinones were found to be MK-11 and MK-12. The DNA G+C content of strain DD4aT is 73.9 mol%. DNA–DNA relatedness of strain DD4aT with A. soli MB78T, A. kyonggiense KSL51201-037T, and A. endophyticum 1T4Z-3T were 53.3% (± 1.1%), 47.0% (± 0.5%), and 47.9% (± 0.9%), respectively. The digital DNA–DNA hybridisation and average nucleotide identity values between strain DD4aT and A. kyonggiense KSL51201-037T were determined to be 26.1% and 82.7%. On the basis of phenotypic, genotypic, chemotaxonomic and phylogenetic analysis, DD4aT represents a novel member of the genus Amnibacterium, for which the name Amnibacterium setariae sp. nov., is proposed. The type strain of Amnibacterium setariae is DD4aT (= KACC 19817T = JCM 32878T).

Pedobacter changchengzhani sp. nov., isolated from soil of Antarctica Abstract A Gram-negative, aerobic, non-motile, pink and rod-shaped bacterium, designated E01020T, was isolated from soil collected from the Chinese Great Wall Station, Antarctica. Comparative analysis of 16S rRNA gene sequences revealed that strain E01020T is a member of the genus Pedobacter, related to Pedobacter alluvionis DSM 19624T (96.8% similarity), Pedobacter agri JCM 15120T (96.5% similarity) and Pedobacter chinensis JDX94T (96.3% similarity). The dDDH values and ANI values of strain E01020T with closely related strains indicate that it can be distinguished from them as a novel species. The DNA G+C content was determined to be 35.2 mol%. The growth of strain E01020T was observed at 4–25 °C (optimal 20 °C), in the presence of 0–1% NaCl (w/v, optimal 0%) and at pH 6.0–8.0 (optimal pH 7.0). Strain E01020T was found to contained menaquinon-7 (MK-7) as only respiratory quinone, iso-C15:0, iso-C17:0 3-OH and Summed feature 3 (C16:1ω6c and/or C16:1ω7c) as major fatty acids. The major polar lipids were phosphatidylethanolamine, one unidentified lipid and two unidentified aminolipids. On the basis of the results of the phylogenetic and phenotypic analyses, it was suggested that strain E01020T represents a novel species of the genus Pedobacter, for which the name Pedobacter changchengzhani sp. nov. is proposed. The type strain is E01020T (= KCTC 62990T = MCCC 1H00357T).