Ancylobacter radicis sp. nov., a novel aerobic methylotrophic bacteria associated with plants.

The two novel bacterial strains, designated as VTT and ML, were isolated from roots of cinquefoil (Potentilla sp.) and leaves of meadow-grass (Poa sp.) on the flooded bank of lake, respectively. These isolates were Gram-negative, non-spore-forming, non-motile, rod-shaped cells, utilized methanol, methylamine, and polycarbon compounds as carbon and energy sources. In the whole-cell fatty acid pattern of strains prevailed C18:1ω7c and C19:0cyc. Based on the phylogenetic analysis of 16S rRNA gene sequences, strains VTT and ML were closely related to the representatives of the genus Ancylobacter (98.3-98.5%). The assembled genome of strain VTT has a total length of 4.22 Mbp, and a G + C content is 67.3%. The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values between strain VTT and closely related type strains of genus Ancylobacter were 78.0-80.6%, 73.8-78.3% and 22.1-24.0%, respectively, that clearly lower than proposed thresholds for species. On the basis of the phylogenetic, phenotypic, and chemotaxonomic analysis, isolates VTT and ML represent a novel species of the genus Ancylobacter, for which the name Ancylobacter radicis sp. nov. is proposed. The type strain is VTT (= VKM B-3255T = CCUG 72400T). In addition, novel strains were able to dissolve insoluble phosphates, to produce siderophores and plant hormones (auxin biosynthesis). According to genome analysis genes involved in the biosynthesis of siderophores, polyhydroxybutyrate, exopolysaccharides and phosphorus metabolism, as well as the genes involved in the assimilation of C1-compounds (natural products of plant metabolism) were found in the genome of type strain VTT.

Ancylobacter moscoviensis sp. nov., novel facultatively methylotrophic bacteria from activated sludge and the reclassification of Starkeya novella (Starkey 1934) Kelly et al. 2000 as Ancylobacter novellus comb. nov., Starkeya koreensis Im et al. 2006 as Ancylobacter koreensis comb.nov., Angulomicrobium tetraedrale Vasil'eva et al. 1986 as Ancylobacter tetraedralis comb. nov., Angulomicrobium amanitiforme Fritz et al. 2004 as Ancylobacter amanitiformis comb. nov., and Methylorhabdus multivorans Doronina et al. 1996 as Ancylobacter multivorans comb. nov., and emended description of the genus Ancylobacter.

Three novel facultatively methylotrophic bacteria, strains 3CT, 1A, 8P, were isolated from activated sludges. The isolates were aerobic, Gram-stain-negative, non-motile, non-spore forming rods multiplying by binary fission. The predominant polar lipids were phosphatidylcholine, phosphatidylglycerol, phosphatidylethylethanolamine, phosphatidylmonomethylethanolamine, and diphosphatidylglycerol. The major fatty acids of cells were С18:1ω7c, C19:0ω8c cyclo and C16:0. Levels of 16S rRNA gene similarity indicates that the closely relatives are representatives of the genera Starkeya, Ancylobacter, Angulomicrobium and Methylorhabdus (96.4-99.4%). Genomic comparisons of 3CT and its closest relatives, S. novella DSM 506T and S. koreensis Jip08T, shared 87.3 and 86.8% nucleotide identity and 28.3 and 26.8% digital DNA-DNA hybridization values, respectively. The average amino acid identities between the strain 3CT and representatives of Starkeya, Ancylobacter and Angulomicrobium were in the range of 75.6-84.3%, which combines these strains into a single genus and gives rise to their reclassification. Based on polyphasic analyses, the strains 3CT, 1A, 8P represents a novel species of the genus Ancylobacter, for which the name Ancylobacter moscoviensis sp. nov. is proposed. The type strain is 3CT (= VKM B-3218T = KCTC 62336T). Furthermore, we also suggested the reclassification of Starkeya novella as Ancylobacter novellus comb. nov., Starkeya koreensis as Ancylobacter koreensis comb. nov., Angulomicrobium tetraedrale as Ancylobacter tetraedralis comb. nov., Angulomicrobium amanitiforme as Ancylobacter amanitiformis comb. nov. and Methylorhabdus multivorans as Ancylobacter multivorans comb. nov. with the emended description of the genus Ancylobacter.

Improved cytocompatibility and antibacterial properties of zinc-substituted brushite bone cement based on ß-tricalcium phosphate.

For bone replacement materials, osteoconductive, osteoinductive, and osteogenic properties are desired. The bacterial resistance and the need for new antibacterial strategies stand among the most challenging tasks of the modern medicine. In this work, brushite cements based on powders of Zinc (Zn) (1.4 wt%) substituted tricalcium phosphate (ß-TCP) and non-substituted ß-TCP were prepared and investigated. Their initial and final phase composition, time of setting, morphology, pH evolution, and compressive strength are reported. After soaking for 60 days in physiological solution, the cements transformed into a mixture of brushite and hydroxyapatite. Antibacterial activity of the cements against Enterococcus faecium, Escherichia coli, and Pseudomonas aeruginosa bacteria strains was attested. The absence of cytotoxicity of cements was proved for murine fibroblast NCTC L929 cells. Moreover, the cell viability on the ß-TCP cement containing Zn2+ ions was 10% higher compared to the ß-TCP cement without zinc. The developed cements are perspective for applications in orthopedics and traumatology.

Hansschlegelia quercus sp. nov., a novel methylotrophic bacterium isolated from oak buds.

Novel aerobic, restricted facultatively methylotrophic bacteria were isolated from buds of English oak (Quercus robur L.; strain DubT) and northern red oak (Quercus rubra L.; strain KrD). The isolates were Gram-negative, asporogenous, motile short rods that multiplied by binary fisson. They utilized methanol, methylamine and a few polycarbon compounds as carbon and energy sources. Optimal growth occurred at 25 °C and pH 7.5. The dominant phospholipids were phosphatidylethanolamine, phosphatidylcholine, diphosphatidylglycerol and phoshatidylglycerol. The major cellular fatty acids of cells were C18 : 1 ω7c, 11-methyl C18 : 1 ω7c and C16 : 0. The major ubiquinone was Q-10. Analysis of 16S rRNA gene sequences showed that the strains were closely related to the members of the genus Hansschlegelia: Hansschlegelia zhihuaiae S113T(97.5-98.0 %), Hansschlegelia plantiphila S1T (97.4-97.6 %) and Hansschlegelia beijingensis PG04T(97.0-97.2 %). The 16S rRNA gene sequence similarity between strains DubT and KrD was 99.7 %, and the DNA-DNA hybridization (DDH) result between the strains was 85 %. The ANI and the DDH values between strain DubT and H. zhihuaiae S113T were 80.1 and 21.5  %, respectively. Genome sequencing of the strain DubT revealed a genome size of 3.57 Mbp and a G+C content of 67.0 mol%. Based on the results of the phenotypic, chemotaxonomic and genotypic analyses, it is proposed that the isolates be assigned to the genus Hansschlegelia as Hansschlegelia quercus sp. nov. with the type strain DubT (=VKM B-3284T=CCUG 73648T=JCM 33463T).

Methylopila carotae sp. nov., a facultative methylotroph, isolated from a root of Daucus carota L.

An aerobic facultatively methylotrophic bacterium, designated strain Das4.1T, was isolated from a root of Daucus carota L. The cells of this strain were observed to be Gram-stain negative, asporogenous, non-motile short rods multiplying by binary fission. Strain Das4.1T can utilise methanol, methylamine and a variety of polycarbon compounds as carbon and energy sources. C1-compounds were found to be assimilated via the isocitrate lyase-negative variant of the serine pathway. On medium with 0.5% methanol, growth of strain Das4.1T was observed at pH 5.5-9.0 (optimum, pH 6.0-7.0) and 18-37 °C (optimum, 24-29 °C) and in the presence of 0-2% (w/v) NaCl (optimum, 0.05%). Cells are catalase and oxidase positive and synthesise indole from L-tryptophan. The major fatty acids of methanol-grown cells were identified as C18:1ω7c, C18:0 and 11-methyl-C18:1ω7c. The predominant phospholipids were found to be phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanolamine. The major respiratory quinone was identified as Q-10. The DNA G + C content of strain Das4.1T was determined to be 67.3 mol% (Tm). Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that strain Das4.1T belongs to the genus Methylopila and shows high sequence similarity to Methylopila oligotropha 2395AT (98.4%) and Methylopila capsulata IM1T (98.0%). However, the DNA-DNA relatedness of strain Das4.1T with M. oligotropha 2395AT was only 22 ± 3%. Based on genotypic, chemotaxonomic and physiological characterisation, the isolate can be classified as a novel species of the genus Methylopila, for which the name Methylopila carotae sp. nov. is proposed. The type strain is Das4.1T (= VKM B-3244T = CCUG 72399T).

Ancylobacter sonchi sp. nov., a novel methylotrophic bacterium frоm roots of Sonchus arvensis L.

An aerobic facultatively methylotrophic bacterium was isolated from roots of Sonchus arvensis L. and designated strain OsotT The cells of this strain were Gram-stain-negative, asporogenous, motile short rods multiplying by binary fisson. They utilized methanol, methylamines and a variety of polycarbon compounds as the carbon and energy sources. Methanol was assimilated after sequential oxidation to formaldehyde and CO2 via the ribulose bisphosphate pathway. The organism grew optimally at 22-29 °C and pH 7.5-8.0. The dominant phospholipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and diphosphatidylglycerol (cardiolipin). The major cellular fatty acids of strain OsotT cells grown in R2A medium were C18 : 1ω7c (49.0 %), C19 : 0ω8c cyclo (38.3 %) and C16 : 0 (8.4 %). The major ubiquinone was Q-10. The DNA G+C content of strain OsotT was 66.1 mol% (Tm). On the basis of 16S rRNA gene sequence analysis strain OsotT is phylogenetically related to the members of genus Ancylobacter (97.1-98.8 % sequence similarity). Based on 16S rRNA gene sequence analysis and DNA-DNA relatedness (27-29 %) with type strains of the genus Ancylobacter, the novel isolate is classified as a new species of this genus and named Ancylobacter sonchi sp. nov.; the type strain is OsotT (=VKM B-3145T=JCM 32039T).