Five isolates, designated TA2, TA4, TA25^T, KOx^T and NS15^T were isolated in previous studies by enrichment in mineral medium with potassium oxalate as the sole carbon source and were characterized using a polyphasic approach. The isolates were Gram-reaction-negative, aerobic, non-spore-forming rods. Phylogenetic analyses based on 16S rRNA and DNA gyrase B subunit (gyrB) gene sequences confirmed that the isolates belonged to the genus Pandoraea and were most closely related to Pandoraea sputorum and Pandoraea pnomenusa (97.2–99.7 % 16S rRNA gene sequence similarity). The isolates could be differentiated from their closest relatives on the basis of several phenotypic characteristics. The major cellular fatty acid profiles of the isolates comprised C_16 : 0, C_18 : 1ω7c, C_17 : 0 cyclo and summed feature 3 (C_16 : 1ω7c and/or iso-C_15 : 0 2-OH). On the basis of DNA–DNA hybridization studies and phylogenetic analyses, the isolates represent three novel species within the genus Pandoraea, for which the names Pandoraea oxalativorans sp. nov. (TA25^T = NBRC 106091^T = CCM 7677^T = DSM 23570^T), Pandoraea faecigallinarum sp. nov. (KOx^T = NBRC 106092^T = CCM 2766^T = DSM 23572^T) and Pandoraea vervacti sp. nov. (NS15^T = NBRC 106088^T = CCM 7667^T = DSM 23571^T) are proposed.

Methylobacterium species are ubiquitous α-proteobacteria that reside in the phyllosphere and are fed by methanol that is emitted from plants. In this study, we applied whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (WC-MS) to evaluate the diversity of Methylobacterium species collected from a variety of plants. The WC-MS spectrum was reproducible through two weeks of cultivation on different media. WC-MS spectrum peaks of M. extorquens strain AM1 cells were attributed to ribosomal proteins, but those were not were also found. We developed a simple method for rapid identification based on spectra similarity. Using all available type strains of Methylobacterium species, the method provided a certain threshold similarity value for species-level discrimination, although the genus contains some type strains that could not be easily discriminated solely by 16S rRNA gene sequence similarity. Next, we evaluated the WC-MS data of approximately 200 methylotrophs isolated from various plants with MALDI Biotyper software (Bruker Daltonics). Isolates representing each cluster were further identified by 16S rRNA gene sequencing. In most cases, the identification by WC-MS matched that by sequencing, and isolates with unique spectra represented possible novel species. The strains belonging to M. extorquens, M. adhaesivum, M. marchantiae, M. komagatae, M. brachiatum, M. radiotolerans, and novel lineages close to M. adhaesivum, many of which were isolated from bryophytes, were found to be the most frequent phyllospheric colonizers. The WC-MS technique provides emerging high-throughputness in the identification of known/novel species of bacteria, enabling the selection of novel species in a library and identification without 16S rRNA gene sequencing.

Two strains of aerobic, non-spore-forming, Gram-negative, rod-shaped bacteria (ND5 and MY14T), previously isolated from urban soil using the membrane

A pink-pigmented, facultatively methylotrophic bacterium, strain 35a^T, was isolated from the leaves ofOxalis corniculata. Cells of strain 35a^Twere Gram-reaction-negative, motile, non-spore-forming rods. The highest 16S rRNA gene pairwise sequence similarities for strain 35a^Twere found with the strains of Methylobacterium iners 5317S-33^T(96.7 %), ‘Methylobacterium soli’ YIM 48816 (96.6 %) and Methylobacterium jeotgali S2R03-9^T(96.3 %). 16S rRNA gene sequence similarities with the type strains of all other recognized species of the genus Methylobacterium were below 96 %. Major cellular fatty acids were C_18 : 1ω7c, C_18 : 0and C_16 : 0. The results of DNA–DNA hybridization experiments, analysis ofcpn60gene sequences, fatty acid profiles, whole-cell MALDI-TOF/MS spectral pattern analysis, and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain 35a^Tfrom its nearest phylogenetic neighbours. Strain 35a^Tis therefore considered to represent a novel species within the genus Methylobacterium , for which the nameMethylobacterium oxalidissp. nov. is proposed. The type strain is 35a^T( = DSM 24028^T = NBRC 107715^T).