A survey of Methylobacterium species and strains reveals widespread production and varying profiles of cytokinin phytohormones.

BACKGROUND: Symbiotic Methylobacterium strains comprise a significant part of plant microbiomes. Their presence enhances plant productivity and stress resistance, prompting classification of these strains as plant growth-promoting bacteria (PGPB). Methylobacteria can synthesize unusually high levels of plant hormones, called cytokinins (CKs), including the most active form, trans-Zeatin (tZ). RESULTS: This study provides a comprehensive inventory of 46 representatives of Methylobacterium genus with respect to phytohormone production in vitro, including 16 CK forms, abscisic acid (ABA) and indole-3-acetic acid (IAA). High performance-liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analyses revealed varying abilities of Methylobacterium strains to secrete phytohormones that ranged from 5.09 to 191.47 pmol mL-1 for total CKs, and 0.46 to 82.16 pmol mL-1 for tZ. Results indicate that reduced methanol availability, the sole carbon source for bacteria in the medium, stimulates CK secretion by Methylobacterium. Additionally, select strains were able to transform L-tryptophan into IAA while no ABA production was detected. CONCLUSIONS: To better understand features of CKs in plants, this study uncovers CK profiles of Methylobacterium that are instrumental in microbe selection for effective biofertilizer formulations.

Methylobacterium radiodurans sp. nov., a novel radiation-resistant Methylobacterium.

A Gram-negative, aerobic, flagellated, rod-shaped, and pink-pigmented bacterium, strain 17Sr1-43 T, was isolated from a soil sample collected in Nowongu, Seoul, Korea. The isolate could grow at 18-37 °C (optimum, 28-30 °C), pH 6.0-8.0 (optimum, pH 7.0) and in the presence of 0-1.0% (w/v) NaCl (optimum, 0%) with aeration. The major cellular fatty acids were summed feature 8 (C18:1 ω7c and/or C18:1 ω6c) and summed feature 2 (iso-C16:1 I and/or C14:0 3-OH). The predominant respiratory quinone was Q-10 and the major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phospholipid, and diphosphatidylglycerol. The G + C content of genomic DNA was 69.1 mol%. Strain 17Sr1-43 T was closely related to Methylobacterium gregans KACC 14808 T (98.4% 16S rRNA gene sequence similarity), Methylobacterium hispanicum KACC 11432 T (97.9%), and Methylobacterium phyllosphaerae CBMB27T (96.1%). The complete genome of strain 17Sr1-43 T contains essential genes related to DNA repair processes including bacterial RecBCD dependent pathway and UmuCD system. Based on the phenotypic, genotypic, and chemotaxonomic characteristics, strain 17Sr1-43 T represents a novel species in the genus Methylobacterium, for which the name Methylobacterium radiodurans sp. nov. is proposed. The type strain is strain 17Sr1-43 T (= KCTC 52906 T = NBRC 112875 T).

Natural Bacterial Assemblages in Arabidopsis thaliana Tissues Become More Distinguishable and Diverse during Host Development.

To study the spatial and temporal dynamics of bacterial colonization under field conditions, we planted and sampled Arabidopsis thaliana during 2 years at two Michigan sites and surveyed colonists by sequencing 16S rRNA gene amplicons. Mosaic and dynamic assemblages revealed the plant as a patchwork of tissue habitats that differentiated with age. Although assemblages primarily varied between roots and shoots, amplicon sequence variants (ASVs) also differentiated phyllosphere tissues. Increasing assemblage diversity indicated that variants dispersed more widely over time, decreasing the importance of stochastic variation in early colonization relative to tissue differences. As tissues underwent developmental transitions, the root and phyllosphere assemblages became more distinct. This pattern was driven by common variants rather than those restricted to a particular tissue or transiently present at one developmental stage. Patterns also depended critically on fine phylogenetic resolution: when ASVs were grouped at coarse taxonomic levels, their associations with host tissue and age weakened. Thus, the observed spatial and temporal variation in colonization depended upon bacterial traits that were not broadly shared at the family level. Some colonists were consistently more successful at entering specific tissues, as evidenced by their repeatable spatial prevalence distributions across sites and years. However, these variants did not overtake plant assemblages, which instead became more even over time. Together, these results suggested that the increasing effect of tissue type was related to colonization bottlenecks for specific ASVs rather than to their ability to dominate other colonists once established.IMPORTANCE Developing synthetic microbial communities that can increase plant yield or deter pathogens requires basic research on several fronts, including the efficiency with which microbes colonize plant tissues, how plant genes shape the microbiome, and the microbe-microbe interactions involved in community assembly. Findings on each of these fronts depend upon the spatial and temporal scales at which plant microbiomes are surveyed. In our study, phyllosphere tissues housed increasingly distinct microbial assemblages as plants aged, indicating that plants can be considered collections of tissue habitats in which microbial colonists-natural or synthetic-are established with differing success. Relationships between host genes and community diversity might vary depending on when samples are collected, given that assemblages grew more diverse as plants aged. Both spatial and temporal trends weakened when colonists were grouped by family, suggesting that functional rather than taxonomic profiling will be necessary to understand the basis for differences in colonization success.

Methylobacterium planium sp. nov., isolated from a lichen sample.

A novel bacterial strain, designated YIM 132548 T, was isolated from Lepraria sp. lichen collected from Yunnan province, south-west PR China. The organism was Gram-stain negative, aerobic and methylotrophic. The cell was catalase positive and oxidase negative, asporogenous, rod-shaped and motile with three polar flagella. The strain could grow at 15-30 °C (optimum, 20 °C), at pH 6.0-9.0 (optimum, pH 7.0) and does not grow in the presence of NaCl. According to the 16S rRNA gene sequence analysis, strain YIM 132548 T showed high levels of 16S rRNA gene sequence similarity with Methylobacterium soli YIM 48816 T (97.6%) and Methylobacterium durans NBRC 112876 T (97.3%), less than 97.0% with other validly named type strains of the genus Methylobacterium. Ubiquinone Q-10 was the predominant respiratory ubiquinone. The predominant cellular fatty acid was identified as summed feature 8 (C18:1ω7c). The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine. The DNA G + C content of the draft genome sequence is 70.2 mol%. The average nucleotide identity and digital DNA-DNA hybridizations values of strain YIM 132548 T with M. soli YIM 48816 T and M. durans NBRC 112876 T were 87.0% and 82.0%, 40.6% and 27.2% based on draft genome sequences, respectively. On the basis of phylogenetic, chemotaxonomic, phenotypic and genomic data, strain YIM 132548 T is concluded to represent a novel species of the genus Methylobacterium, for which the name Methylobacterium planium sp. nov. is proposed. The type strain is YIM 132548 T (= CGMCC 1.17323 T = NBRC 114056 T).

Impact of plants on the diversity and activity of methylotrophs in soil.

BACKGROUND: Methanol is the second most abundant volatile organic compound in the atmosphere, with the majority produced as a metabolic by-product during plant growth. There is a large disparity between the estimated amount of methanol produced by plants and the amount which escapes to the atmosphere. This may be due to utilisation of methanol by plant-associated methanol-consuming bacteria (methylotrophs). The use of molecular probes has previously been effective in characterising the diversity of methylotrophs within the environment. Here, we developed and applied molecular probes in combination with stable isotope probing to identify the diversity, abundance and activity of methylotrophs in bulk and in plant-associated soils. RESULTS: Application of probes for methanol dehydrogenase genes (mxaF, xoxF, mdh2) in bulk and plant-associated soils revealed high levels of diversity of methylotrophic bacteria within the bulk soil, including Hyphomicrobium, Methylobacterium and members of the Comamonadaceae. The community of methylotrophic bacteria captured by this sequencing approach changed following plant growth. This shift in methylotrophic diversity was corroborated by identification of the active methylotrophs present in the soils by DNA stable isotope probing using 13C-labelled methanol. Sequencing of the 16S rRNA genes and construction of metagenomes from the 13C-labelled DNA revealed members of the Methylophilaceae as highly abundant and active in all soils examined. There was greater diversity of active members of the Methylophilaceae and Comamonadaceae and of the genus Methylobacterium in plant-associated soils compared to the bulk soil. Incubating growing pea plants in a 13CO2 atmosphere revealed that several genera of methylotrophs, as well as heterotrophic genera within the Actinomycetales, assimilated plant exudates in the pea rhizosphere. CONCLUSION: In this study, we show that plant growth has a major impact on both the diversity and the activity of methanol-utilising methylotrophs in the soil environment, and thus, the study contributes significantly to efforts to balance the terrestrial methanol and carbon cycle. Video abstract.

Methylobacterium nonmethylotrophicum sp. nov., isolated from tungsten mine tailing.

A novel pink-pigmented strain, designated 6HR-1T, was isolated from tungsten mine tailings in Jiangxi Province, PR China. Cells were Gram-stain-negative, aerobic, non-spore-forming, rod-shaped and motile with a polar flagellum (monotrichous). It could not utilize methanol, methylamine, formaldehyde or formate as a sole carbon source. The methanol dehydrogenase mxaF gene was absent but the xoxF gene was present. Phylogenomic and 16S rRNA gene phylogenetic analyses clearly showed that strain 6HR-1T was affiliated to the genus Methylobacterium and closely related to 'Methylobacterium terrae' 17Sr1-28T (98.6 %), Methylobacterium platani JCM 14648T (97.7 %), Methylobacterium variabile DSM 16961T (97.7 %) and Methylobacterium currus KACC 19662T (97.4 %). The average nucleotide identity and digital DNA-DNA hybridization values between strain 6HR-1T and its closely related type species were 87.4-88.7 and 33.2-36.3 %, respectively. It had summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) as the major fatty acid and ubiquinone 10 as the predominant respiratory quinone. Polyphasic characterization supported that strain 6HR-1T represents a novel species of the genus Methylobacterium, for which the name Methylobacterium nonmethylotrophicum sp. nov. is proposed with the type strain 6HR-1T (=GDMCC 1.662T=KCTC 42760T).

Phenotypic diversity of Methylobacterium associated with rice landraces in North-East India.

The ecology and distribution of many bacteria is strongly associated with specific eukaryotic hosts. However, the impact of such host association on bacterial ecology and evolution is not well understood. Bacteria from the genus Methylobacterium consume plant-derived methanol, and are some of the most abundant and widespread plant-associated bacteria. In addition, many of these species impact plant fitness. To determine the ecology and distribution of Methylobacterium in nature, we sampled bacteria from 36 distinct rice landraces, traditionally grown in geographically isolated locations in North-East (NE) India. These landraces have been selected for diverse phenotypic traits by local communities, and we expected that the divergent selection on hosts may have also generated divergence in associated Methylobacterium strains. We determined the ability of 91 distinct rice-associated Methylobacterium isolates to use a panel of carbon sources, finding substantial variability in carbon use profiles. Consistent with our expectation, across spatial scales this phenotypic variation was largely explained by host landrace identity rather than geographical factors or bacterial taxonomy. However, variation in carbon utilisation was not correlated with sugar exudates on leaf surfaces, suggesting that bacterial carbon use profiles do not directly determine bacterial colonization across landraces. Finally, experiments showed that at least some rice landraces gain an early growth advantage from their specific phyllosphere-colonizing Methylobacterium strains. Together, our results suggest that landrace-specific host-microbial relationships may contribute to spatial structure in rice-associated Methylobacterium in a natural ecosystem. In turn, association with specific bacteria may provide new ways to preserve and understand diversity in one of the most important food crops of the world.

Methylobacterium terricola sp. nov., a gamma radiation-resistant bacterium isolated from gamma ray-irradiated soil.

A gamma radiation-resistant and pink-pigmented bacterial strain, designated as 17Sr1-39T, was isolated from a gamma ray-irradiated soil sample collected in the Republic of Korea. Cells were Gram-stain-negative, strictly aerobic, flagellated, asporogenous, rod-shaped and methylotrophic. Results of 16S rRNA gene sequence analysis showed that strain 17Sr1-39T was phylogenetically related to Methylobacterium currus PR1016AT (97.3 %), Methylobacterium aquaticum DSM 16371T (97.2 %), Methylobacterium platani PMB02T (97.0 %), Methylobacterium frigidaeris IER25-16T (96.6 %), Methylobacterium terrae 17Sr1-28T (96.6 %) and Methylobacterium organophilum JCM 2833T (93.4 %). The G+C content calculated based on the genome sequence was 70.4 mol%. The average nucleotide identity and in silico DNA-DNA hybridization values between strain 17Sr1-39T and M. currus, M. aquaticum, M. platani, M. frigidaeris, M. terrae and M. organophilum were 77.3-89.9 and 22-38.2 %, respectively. The predominant fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The predominant quinone was ubiquinone 10 and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Based on the data from phenotypic tests and genotypic differences between strain 17Sr1-39T and its close phylogenetic relatives, strain 17Sr1-39T represented a new species belonging to the genus Methylobacterium, for which the name Methylobacterium terricola sp. nov. (=KACC 52905T=NBRC 112874T) is proposed.

Diversity of Methylobacterium spp. in the Rice of the Vietnamese Mekong Delta.

The Vietnamese Mekong delta is one of the largest rice-producing areas globally. Methylobacterium spp. are persistent colonizers of the rice plant and exert beneficial effects on plant growth and health. Sixty-one Methylobacterium strains belonging to seven species were predominantly isolated from the phyllosphere of rice cultivated in six Mekong delta provinces. Inoculation tests revealed that some strains exhibited plant growth-promoting activity. Moreover, three strains possessed the novel characteristics of inducing leaf bleaching and killing rice seedlings. These results revealed the complex diversity of Methylobacterium in Mekong delta rice and that healthy and productive rice cultivation requires a proper balance of Methylobacterium.

Methylobacterium crusticola sp. nov., isolated from biological soil crusts.

A pink-pigmented, Gram-negative, rod-shaped, obligate aerobic bacterial strain, MIMD6T, was isolated from biological soil crusts in PR China. Cells grew at 20-37 °C (optimum, 30 °C), at pH 6-8 (optimum, pH 7) and with 0-1 % (w/v) NaCl (optimum, 0 %). Strain MIMD6T could use methanol or formate as a sole carbon source to grow, and carried methanol dehydrogenase genes mxaF and xoxF, supporting its methylotrophic metabolism. The respiratory quinone was ubiquinone Q-10, the major fatty acids were C18 : 1ω7c (87.3 %), and the major polar lipids were diphosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, one unknown aminolipid and one unidentified glycolipid. The results of phylogenetic analyses based on the sequences of the 16S rRNA gene, seven housekeeping genes (dnaK, recA, rimO, rpIK, rpmG, rpsR and rpoB) and methanol dehydrogenase genes indicated that strain MIMD6T formed a phylogenetic linage with members of the genus Methylobacterium. Strain MIMD6T was most closely related to Methylobacterium isbiliense DSM 17168T and Methylobacterium nodulans LMG 21967T with 16S rRNA gene sequence similarities of 95.7 and 95.2 %, respectively. The genomic DNA G+C content calculated via draft genome sequencing was 73.0 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain MIMD6T and the type strains of other Methylobacterium species were 70.7-82.0 and 24.6-30.0 %, respectively. Based on phenotypic, chemotaxonomic and phylogenetic characteristics, strain MIMD6T represents a novel species of the genus Methylobacterium, for which the name Methylobacterium crusticola sp. nov. is proposed. The type strain is MIMD6T (=KCTC 52305T=MCCC 1K01311T).

Methylobacterium segetis sp. nov., a novel member of the family Methylobacteriaceae isolated from soil on Jeju Island.

A bacterial strain, 17J42-1T, was isolated from a soil sample collected on Jeju Island, Republic of Korea. Colonies grown on R2A agar were pink in color, and cells were Gram-stain negative, short and rod-shaped. Analysis of 16S rRNA gene sequences identified this strain as a member of the genus Methylobacterium in the family Methylobacteriaceae, with high levels of 16S rRNA sequence similarity shared with Methylobacterium oxalidis 35aT (98.6%), Methylobacterium jeotgali S2R03-9T (97.5%), and Methylobacterium soli YIM 48816T (97.3%). Cells grew at 15-35 °C, pH 5-9, and in the presence of 0-1.0% NaCl. The genomic G + C content was 70.2 mol% based on the whole genome analysis. The predominant respiratory quinone was ubiquinone Q-10, the major fatty acid was C18:1ω7c (85.3%), and the major polar lipids were phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The phenotypic and chemotaxonomic data support the affiliation of strain 17J42-1T with the genus Methylobacterium. However, the DNA-DNA relatedness between the isolate and its closest phylogenetic neighbors was lower than 38%. The OrthoANI and dDDH values between strain 17J42-1T and the closest type strain Methylobacterium oxalidis NBRC 107715T were calculated to be 85.9% and 30.6%, respectively. The results of 16S rRNA gene sequence similarity analysis, DNA-DNA hybridization analysis, and the observed differentiating phenotypic properties from other closely related taxa clearly indicate that strain 17J42-1T represents a novel species in the genus Methylobacterium, for which the name Methylobacterium segetis sp. nov. is proposed. The type strain is 17J42-1T (= KCTC 62267T = JCM 33059T).

Genomics of Aerobic Photoheterotrophs in Wheat Phyllosphere Reveals Divergent Evolutionary Patterns of Photosynthetic Genes in Methylobacterium spp.

Phyllosphere is a habitat to a variety of viruses, bacteria, fungi, and other microorganisms, which play a fundamental role in maintaining the health of plants and mediating the interaction between plants and ambient environments. A recent addition to this catalogue of microbial diversity was the aerobic anoxygenic phototrophs (AAPs), a group of widespread bacteria that absorb light through bacteriochlorophyll α (BChl a) to produce energy without fixing carbon or producing molecular oxygen. However, culture representatives of AAPs from phyllosphere and their genome information are lacking, limiting our capability to assess their potential ecological roles in this unique niche. In this study, we investigated the presence of AAPs in the phyllosphere of a winter wheat (Triticum aestivum L.) in Denmark by employing bacterial colony based infrared imaging and MALDI-TOF mass spectrometry (MS) techniques. A total of ∼4,480 colonies were screened for the presence of cellular BChl a, resulting in 129 AAP isolates that were further clustered into 21 groups based on MALDI-TOF MS profiling, representatives of which were sequenced using the Illumina NextSeq and Oxford Nanopore MinION platforms. Seventeen draft and four complete genomes of AAPs were assembled belonging in Methylobacterium, Rhizobium, Roseomonas, and a novel Alsobacter. We observed a diverging pattern in the evolutionary rates of photosynthesis genes among the highly homogenous AAP strains of Methylobacterium (Alphaproteobacteria), highlighting an ongoing genomic innovation at the gene cluster level.

Methylobacterium oryzihabitans sp. nov., isolated from water sampled from a rice paddy field.

Strain TER-1T was isolated from water sampled from a rice paddy field in Taiwan. Cells were Gram-negative, aerobic, motile, rod-shaped and formed pink-coloured colonies. Optimal growth occurred at 25-30 °C, pH 6-7 and in the presence of 0.5 % NaCl. Strain TER-1T could grow on C1 compounds such as methanol, formic acid, methylamine and dimethylamine as sole carbon source, and carry methanol dehydrogenase gene, which supports its methylotrophic metabolism. The results of phylogenetic analyses based on the 16S rRNA gene sequence, methanol dehydrogenase gene sequence and coding sequences of 92 protein clusters indicated that strain TER-1T formed a phylogenetic lineage in the genus Methylobacterium. Strain TER-1T was most closely related to Methylobacterium isbiliense AR24T with 96.8 % 16S rRNA gene sequence similarity. Strain TER-1T showed 77.1-82.8 % average nucleotide identity and 16.4-20.2 % digital DNA-DNA hybridization identity with the strains of other Methylobacterium species. The major fatty acid of strain TER-1T was C18 : 1ω7c. The predominant hydroxy fatty acid was C18 : 0 3-OH. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and two uncharacterized lipids. The only isoprenoid quinone was Q-10. The genomic DNA G+C content of strain TER-1T was 71.9 mol%. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain TER-1T should be classified in a novel species of the genus Methylobacterium, for which the name Methylobacterium oryzihabitans sp. nov. is proposed. The type strain is TER-1T (=BCRC 81157T=LMG 30931T=KCTC 62864T).

Methylobacterium terrae sp. nov., a radiation-resistant bacterium isolated from gamma ray-irradiated soil.

A Gram-stain-negative, asporogenous, aerobic rods, motile by means of a single polar flagellum, catalase- and oxidase-positive, methylotrophic bacterium, designated 17Sr1-28T, was isolated from gamma ray-irradiated soil. The 16S rRNA gene sequence analysis showed that strain 17Sr1-28T was phylogenetically related to Methylobacterium currus PR1016AT (96.8%), Methylobacterium platani PMB02T (96.2%), Methylobacterium aquaticum DSM 16371T (96.3%), Methylobacterium tarhaniae N4211T (96.4%), Methylobacterium frigidaeris IER25-16T (95.8%), and Methylobacterium organophilum JCM 2833T (92.7%). The G+C content calculated based on genome sequence was 71.6%. The average nucleotide identity and in silico DNA-DNA hybridization values between strain 17Sr1- 28T and M. currus, M. platani, M. aquaticum, M. tarhaniae, M. frigidaeris, and M. organophilum were 77.7-90.4% and 22-39.6%, respectively. The major fatty acids of strain 17Sr1-28T were summed feature 8 (C18:1ω7c and/or C18:1ω6c), and summed feature 3 (C16:1ω7c and/or C16:1ω6c). The predominant quinone was ubiquinone 10 and the major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol. On the basis of the data from phenotypic tests and genotypic differences between strain 17Sr1-28T and its close phylogenetic relatives, strain 17Sr1-28T represents a new species belonging to the genus Methylobacterium, for which the name Methylobacterium terrae sp. nov. (= KCTC 52904T = NBRC 112873T) is proposed.

Methylotrophs and Methylotroph Populations for Chloromethane Degradation.

Chloromethane is a halogenated volatile organic compound, produced in large quantities by terrestrial vegetation. After its release to the troposphere and transport to the stratosphere, its photolysis contributes to the degradation of stratospheric ozone. A better knowledge of chloromethane sources (production) and sinks (degradation) is a prerequisite to estimate its atmospheric budget in the context of global warming. The degradation of chloromethane by methylotrophic communities in terrestrial environments is a major underestimated chloromethane sink. Methylotrophs isolated from soils, marine environments and more recently from the phyllosphere have been grown under laboratory conditions using chloromethane as the sole carbon source. In addition to anaerobes that degrade chloromethane, the majority of cultivated strains were isolated in aerobiosis for their ability to use chloromethane as sole carbon and energy source. Among those, the Proteobacterium Methylobacterium (recently reclassified as Methylorubrum) harbours the only characterisized 'chloromethane utilization' (cmu) pathway, so far. This pathway is not representative of chloromethane-utilizing populations in the environment as cmu genes are rare in metagenomes. Recently, combined 'omics' biological approaches with chloromethane carbon and hydrogen stable isotope fractionation measurements in microcosms, indicated that microorganisms in soils and the phyllosphere (plant aerial parts) represent major sinks of chloromethane in contrast to more recently recognized microbe-inhabited environments, such as clouds. Cultivated chloromethane-degraders lacking the cmu genes display a singular isotope fractionation signature of chloromethane. Moreover, 13CH3Cl labelling of active methylotrophic communities by stable isotope probing in soils identify taxa that differ from the taxa known for chloromethane degradation. These observations suggest that new biomarkers for detecting active microbial chloromethane-utilizers in the environment are needed to assess the contribution of microorganisms to the global chloromethane cycle.

Methylobacterium currus sp. nov., isolated from a car air conditioning system.

A novel bacterial strain, designated PR1016AT, was isolated from a car air conditioning system. This rod-shaped strain showed catalase and oxidase activities, was aerobic and methylotrophic, and had a reddish pink colour. The genomic DNA G+C content of strain PR1016AT was 70.2 mol%, as determined by genome sequencing. Phylogenetic analysis based on 16S rRNA gene sequence similarity showed that strain PR1016AT was most closely related to Methylobacterium aquaticum GR16T (98.86 %), M. variabile GR3T (98.43 %), M. platani PMB 02T (98.36 %) and M. tarhaniae N4211T (98.14 %). The average nucleotide identity and digital DNA-DNA hybridization values between strain PR1016AT and M. aquaticum GR16T, M. platani PMB02T and M. variabile GR3T were 88.61, 88.14 and 87.88 %, and 36.4, 35.8 and 34.7 %, respectively. Numerous insertion sequences are present in the genome of strain PR1016AT, which has a larger genome than the four Methylobacterium species described above. Cells grew at 18-42 °C (optimum, 30 °C), at pH 4.0-9.0 (optimum, pH 7.0) and in the presence of 0-1.0 % (w/v) NaCl (optimum, 0 %). The major respiratory quinone was Q10. Fatty acid methyl ester analysis revealed that summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) was the predominant cellular fatty acid in strain PR1016AT. Two-dimensional TLC indicated that the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. The genotypic and phenotypic characteristics indicate that strain PR1016AT represents a novel species of the genus Methylobacterium, for which the name Methylobacterium currus sp. nov. is proposed. The type strain is PR1016AT (=KACC 19662T=JCM 32670T).

Review of the genus Methylobacterium and closely related organisms: a proposal that some Methylobacterium species be reclassified into a new genus, Methylorubrum gen. nov.

The genus Methylobacterium, when first proposed by Patt et al. in 1976, was a monospecific genus created to accommodate a single pink pigmented facultatively methylotrophic bacterium. The genus now has over 50 validly published species, however, the percentage 16S rRNA sequence divergence within Methylobacterium questions whether or not they can still be accommodated within one genus. Additionally, several strains are described as belonging to Methylobacterium, but nodulate legumes and in some cases are unable to utilize methanol as a sole carbon source. This study reviews and discusses the current taxonomic status of Methylobacterium. Based on 16S rRNA gene, multi-locus sequence analysis, genomic and phenotypic data, the 52 Methylobacterium species can no longer be retained in one genus. Consequently, a new genus, Methylorubrum gen. nov., is proposed to accommodate 11 species previously held in Methylobacterium. The reclassified species names are proposed as: Methylorubrum aminovorans comb. nov. (type strain TH-15T=NCIMB 13343T=DSM 8832T), Methylorubrum extorquens comb. nov. (type strain NCIMB 9399T=DSM 1337T), Methylorubrum podarium comb. nov. (type strain FM4T=NCIMB 14856T=DSM 15083T), Methylorubrum populi comb. nov. (type strain BJ001T=NCIMB 13946T=ATCC BAA-705T), Methylorubrum pseudosasae comb. nov. (type strain BL44T=ICMP 17622T=NBRC 105205T), Methylorubrum rhodesianum comb. nov. (type strain NCIMB 12249T=DSM 5687T), Methylorubrum rhodinum comb. nov. (type strain NCIMB 9421T=DSM 2163T), Methylorubrum salsuginis comb. nov. (type strain MRT=NCIMB 14847T=NCCB 100140T), Methylorubrum suomiense comb. nov. (type strain F20T=NCIMB 13778T=DSM 14458T), Methylorubrum thiocyanatum comb. nov. (type strain ALL/SCN-PT=NCIMB 13651T=DSM 11490T) and Methylorubrum zatmanii comb. nov. (type strain NCIMB 12243T=DSM 5688T). The taxonomic position of several remaining species is also discussed.

Distribution of 1-aminocyclopropane-1-carboxylate deaminase and D-cysteine desulfhydrase genes among type species of the genus Methylobacterium.

The presence of 1-aminocyclopropane-1-carboxylate (ACC) deaminase determines the ability of bacteria to increase the resistance of plants to various types of stress. The genes of ACC deaminase (acdS) and the closely related enzyme D-cysteine desulfhydrase (dcyD) were searched in type strains of various representatives of the genus Methylobacterium. Using PCR screening and in silico searching in the available complete genome sequences of type strains, the genes were found in 28 of 48 species of the genus. Phylogenetic analysis of amino acid sequences of proteins revealed two large groups of sequences of the AcdS protein and one of the DcyD protein. The distribution of these groups correlates well with the phylogenetic tree based on the sequences of the 16S rRNA genes, which apparently indicates a different evolutionary adaptation to association with plants in the representatives of these groups. For the first time for aerobic methylotrophs it was demonstrated that the gene dcyD encodes D-cysteine desulfhydrase by cloning and recombinant protein characterization.

Methylobacterium frigidaeris sp. nov., isolated from an air conditioning system.

A reddish pink-pigmented, Gram-stain-negative, aerobic and methylotrophic bacterial strain, designated strain IER25-16T, was isolated from a laboratory air conditioning system in the Republic of Korea. Cells were motile rods showing catalase- and oxidase-positive reactions. Strain IER25-16T grew at 10-40 °C (optimum, 30 °C), at pH 4.0-7.0 (optimum, pH 5.0-7.0) and in the presence of 0-1.0 % (w/v) NaCl (optimum, 0 %). The major respiratory quinone was ubiquinone-10 and ubiquinone-9 was also detected as the minor respiratory quinone. Summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c) was detected as the predominant fatty acids. The genomic DNA G+C content of strain IER25-16T was 70.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that strain IER25-16T belonged to the genus Methylobacterium of the class Alphaproteobacteria. Strain IER25-16T was most closely related to Methylobacterium platani PMB02T (97.9 %), Methylobacterium aquaticum GR16T (97.9 %) and Methylobacterium tarhaniae N4211T (97.5 %). The average nucleotide identity and in silico DNA-DNA hybridization values between strain IER25-16T and M. platani, M. aquaticum and M. tarhaniae were 88.3, 88.8 and 89.6 % and 36.2, 37.3 and 39.3 %, respectively. The phenotypic and chemotaxonomic features and the phylogenetic inference clearly suggested that strain IER25-16T represents a novel species of the genus Methylobacterium, for which the name Methylobacteriumfrigidaeris sp. nov. is proposed. The type strain is strain IER25-16T (=KACC 19280T=JCM 32048T).

Biodiversity of aerobic methylobacteria associated with the phyllosphere of the southern Moscow Oblast.

During the summer period (15­25°C), 34 strains of methylotrophic bacteria associated with different species of herbs, shrub, and trees in Pushchino (Moscow oblast, Russia) were isolated on the medium with methanol. Predominance of pink-colored Methylobacterium strains in the phyllosphere of many plants was confirmed by microscopy, enumeration of the colonies from grass leaves, and sequencing of the 16S rRNA genes. Colorless and yellow-pigmented methylotrophs belonged to the genera Methylophilus, Methylobacillus, Hansschlegelia, Methylopila, Xanthobacter, and Paracoccus. All isolates were able to synthesize plant hormones auxins from L-tryptophan (5−50 µg/mL) and are probably plant symbionts.