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.

AcdR protein is an activator of transcription of 1-aminocyclopropane-1-carboxylate deaminase in Methylobacterium radiotolerans JCM 2831.

It has been previously shown that a number of plant associated methylotrophic bacteria contain an enzyme aminocyclopropane carboxylate (ACC) deaminase (AcdS) hydrolyzing ACC, the immediate precursor of ethylene in plants. The genome of the epiphytic methylotroph Methylobacterium radiotolerans JCM2831 contains an open reading frame encoding a protein homologous to transcriptional regulatory protein AcdR of the Lrp (leucine-responsive regulatory protein) family. The acdR gene of M. radiotolerans was heterologously expressed in Escherichia coli and purified. The results of gel retardation experiments have shown that AcdR specifically binds the DNA fragment containing the promoter-operator region of the acdS gene. ACC decreased electrophoretic mobility of the AcdR-DNA complex whereas leucine had no effect on the complex mobility. The mutant strains of M. radiotolerans obtained by insertion of a tetracycline cassette in the acdS or acdR gene lost the ACC-deaminase activity but the strains with complementation of the mutation recovered this function. The acdS- mutant but not acdR- strain expressed the xylE reporter gene under the control of acdS promoter region thus resulting in a catechol 2,3-dioxygenase activity. This suggested that AcdR in vivo functions as activator of transcription of the acdS gene. The results obtained in this study showed that in phytosymbiotic methylotroph Methylobacterium radiotolerans AcdR mediates activation of the acdS gene transcription in the presence of an inducer ACC or 2-aminoisobutyrate and the excess of the regulatory protein assists in transcription initiation even in the absence of the inducer. The model of regulation of acdS transcription in M. radiotolerans was proposed.

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).

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.

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).

Methylobacillus methanolivorans sp. nov., a novel non-pigmented obligately methylotrophic bacterium.

Three strains of obligately methylotrophic Betaproteobacteria (ZT, SP and M3) with the ribulose monophosphate pathway of C1 assimilation are described. The isolates were strictly aerobic, Gram-stain-negative, asporogenous, motile (strains ZT and M3) or non-motile (strain SP) rods that multiplied by binary fisson, and were mesophilic and neutrophilic. All three strains utilized methanol but only strains SP and M3 utilized methylamine as carbon and energy sources. The prevailing cellular fatty acids were straight-chain saturated C16 : 0 and unsaturated C16 : 1ω7c acids. The major ubiquinone was Q-8. The predominant phospholipids were phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Ammonia was assimilated by glutamate dehydrogenase. The DNA G+C contents of strains ZT, SP and M3 were 51.0, 52.0 and 52.0 mol% (Tm), respectively. Levels of 16S rRNA gene sequence similarity between the three strains were very high (99.9-100 %), and they shared high levels of DNA-DNA relatedness (88-98 %). Based on 16S rRNA gene sequence analysis and DNA-DNA relatedness (19-30 %) with the type strains of the genus Methylobacillus, the novel isolates ZT, SP and M3 are classified as representing a novel species of this genus, for which the name Methylobacillus methanolivorans sp. nov. is proposed. The type strain is ZT (=VKM B-3037T=JCM 31401T=CCUG 68999T).

Draft Genome Sequence of Methylophaga muralis Bur 1, a Haloalkaliphilic (Non-Methane-Utilizing) Methylotroph Isolated from a Soda Lake.

The draft genome sequence of Methylophaga muralis strain Bur 1 (VKM B-3046T), a non-methane-utilizing methylotroph isolated from a soda lake, is reported here. Strain Bur 1 possesses genes for methanol and methylamine (methylamine dehydrogenase and N-methylglutamate pathway) oxidation. Genes for the biosynthesis of ectoine were also found.

Draft Genome Sequence of Methyloligella halotolerans С2T, a New Halotolerant Methylotroph, Accumulating Poly-3-Hydroxybutyrate and Ectoine.

Methyloligella halotolerans С2T is a moderate halophilic obligate methylotroph, accumulating ultra-high-molecular-weight poly-3-hydroxybutyrate (up to 8 to 10 MDa) from methanol. Here we report a draft genome and annotation of Methyloligella halotolerans C2T (VKM B-2706T = CCUG 61687T = DSM 25045T).

Functional and genomic diversity of methylotrophic Rhodocyclaceae: description of Methyloversatilis discipulorum sp. nov.

Three strains of methylotrophic Rhodocyclaceae (FAM1(T), RZ18-153 and RZ94) isolated from Lake Washington sediment samples were characterized. Based on phylogenetic analysis of 16S rRNA gene sequences the strains should be assigned to the genus Methyloversatilis. Similarly to other members of the family, the strains show broad metabolic capabilities and are able to utilize a number of organic acids, alcohols and aromatic compounds in addition to methanol and methylamine. The main fatty acids were 16:1ω7c (49-59%) and 16:0 (32-29%). Genomes of all isolates were sequenced, assembled and annotated in collaboration with the DOE Joint Genome Institute (JGI). Genome comparison revealed that the strains FAM1T, RZ18-153 and RZ94 are closely related to each other and almost equally distant from two previously described species of the genus Methyloversatilis, Methyloversatilis universalis and Methyloversatilis thermotolerans. Like other methylotrophic species of the genus Methyloversatilis, all three strains possess one-subunit PQQ-dependent ethanol/methanol dehydrogenase (Mdh-2), the N-methylglutamate pathway and the serine cycle (isocitrate lyase/malate synthase, Icl/ms(+) variant). Like M. universalis, strains FAM1(T), RZ18-153 and RZ94 have a quinohemoprotein amine dehydrogenase, a tungsten-containing formaldehyde ferredoxin oxidoreductase, phenol hydroxylase, and the complete Calvin cycle. Similarly to M. thermotolerans, the three strains possess two-subunit methanol dehydrogenase (MxaFI), monoamine oxidase (MAO) and nitrogenase. Based on the phenotypic and genomic data, the strains FAM1(T), RZ18-153 and RZ94 represent a novel species of the genus Methyloversatilis, for which the name Methyloversatilis discipulorum sp. nov. is proposed. The type strain is FAM1(T) ( = JCM 30542(T) = VKM = B-2888(T)).

Methylobrevis pamukkalensis gen. nov., sp. nov., a halotolerant restricted facultative methylotroph isolated from saline water.

An aerobic halotolerant restricted facultatively methylotrophic bacterium was isolated from a saline hot spring in Pamukkale, Turkey, and designated strain PK2(T). The cells of this strain were Gram-stain-negative, asporogenous, motile short rods multiplying by binary fission. They utilized methanol, methylamine and mannitol as carbon and energy sources. The organism grew optimally at 30 °C in media containing 85 mM NaCl and at pH 7.5-8.0. C1 compounds were assimilated via the isocitrate-lyase-positive variant of the serine pathway. Poly-ß-hydroxybutyrate and the compatible solute ectoine were found in the cells. The dominant phospholipids were phosphatidylethanolamine and phosphatidylmonomethylethanolamine. The major cellular fatty acids of methanol-grown cells were C(18 : 1)ω7 and C(16 : 1)ω7c. The main ubiquinone was Q-10. The DNA G+C content was 67.9 mol% (T(m)). The 16S rRNA gene sequence suggests that strain PK2(T) is affiliated with the order Rhizobiales within the class Alphaproteobacteria , being most closely related to Mesorhizobium gobiense CCBAU 83330(T) (94% similarity). A novel genus and species, Methylobrevis pamukkalensis gen. nov., sp. nov., is proposed on the basis of phenotypic and genotypic data, with PK2(T) (VKM B-2849(T) = JCM 30229(T)) as the type strain.

Methyloversatilis thermotolerans sp. nov., a novel thermotolerant facultative methylotroph isolated from a hot spring.

A newly isolated facultatively methylotrophic bacterium (strain 3t(T)) was investigated. Cells of the isolate were Gram-stain-negative, asporogenous, non-motile rods that multiplied by binary fission. The strain utilized methanol, methylamine and a variety of multicarbon compounds as carbon and energy sources. Growth occurred at pH 6.5-8.5 (optimally at 7.0-7.5) and at 10-45 °C (optimally at 30-37 °C). The major fatty acids of methanol-grown cells were C16 : 1ω7c and C16 : 0. The predominant phospholipids were phosphatidylethanolamine and phosphatidylglycerol. The major ubiquinone was Q-8. Strain 3t(T) possessed pyrroloquinoline quinone (PQQ)-linked methanol dehydrogenase and assimilated C1 units at the level of formaldehyde and CO2 via the serine cycle. The DNA G+C content of the strain was 63.6 mol% (Tm). On the basis of 16S rRNA gene sequence similarity (98.1 %) and rather low DNA-DNA relatedness (30 %) with the type strain of the type species of the genus Methyloversatilis (Methyloversatilis universalis FAM5(T)), and physiological and biochemical characteristics, the isolate was classified as a representative of a new species of the genus and named Methyloversatilis thermotolerans 3t(T) ( = VKM B-2692(T) = CCUG 61694(T) = DSM 25156(T)).

1-Aminocyclopropane-1-carboxylate (ACC) deaminases from Methylobacterium radiotolerans and Methylobacterium nodulans with higher specificity for ACC.

The 1-aminocyclopropane-1-carboxylate (ACC) deaminases (EC 3.4.99.7), the key enzymes of degradation of the precursor of the phytohormone ethylene, have not been well studied despite their great importance for plant-bacterial interactions. Using blast, the open reading frames encoding ACC deaminases were found in the genomes of epiphytic methylotroph Methylobacterium radiotolerans JCM2831 and nodule-forming endosymbiont Methylobacterium nodulans ORS2060. These genes were named acdS and cloned; recombinant proteins were expressed and purified from Escherichia coli. The enzyme from M. nodulans displayed the highest substrate specificity among all of the characterized ACC deaminases (Km 0.80 ± 0.04 mM), whereas the enzyme from M. radiotolerans had Km 1.8 ± 0.3 mM. The kcat values were 111.8 ± 0.2 and 65.8 ± 2.8 min(-1) for the enzymes of M. nodulans and M. radiotolerans, respectively. Both enzymes are homotetramers with a molecular mass of 144 kDa, as was demonstrated by size exclusion chromatography and native PAGE. The purified enzymes displayed the maximum activity at 45-50 °C and pH 8.0. Thus, the priority data have been obtained, extending the knowledge of biochemical properties of bacterial ACC deaminases.

Methyloligella halotolerans gen. nov., sp. nov. and Methyloligella solikamskensis sp. nov., two non-pigmented halotolerant obligately methylotrophic bacteria isolated from the Ural saline environments.

Two newly isolated halotolerant obligately methylotrophic bacteria (strains C2(T) and SK12(T)) with the serine pathway of C1 assimilation are described. The isolates are strictly aerobic, Gram negative, asporogenous, non-motile rods, forming rosettes, multiplying by binary fission. Mesophilic and neutrophilic, accumulate intracellularly compatible solute ectoine and poly-ß-hydroxybutyrate. The novel strains are able to grow at 0 up to 16% NaCl (w/v), optimally at 3-5% NaCl. The major cellular fatty acids are C18:1ω7c and C19:0cyc and the prevailing quinone is Q-10. The predominant phospholipids are phosphatidylcholine, phosphatidylglycerol, phosphatidyldimethylethanolamine and phosphatidylethanolamine. Assimilate NH4(+) by glutamate dehydrogenase and via the glutamate cycle (glutamine synthetase and glutamate synthase). The DNA G+C contents of strains C2(T) and SK12(T) are 60.9 and 60.5 mol% (Tm), respectively. 16S rRNA gene sequence similarity between the two new isolates are 99% but below 94% with other members of the Alphaproteobacteria thus indicating that they can be assigned to a novel genus Methyloligella. Rather low level of DNA-DNA relatedness (53%) between the strains C2(T) and SK12(T) indicates that they represent two separate species of the new genus, for which the names Methyloligella halotolerans gen. nov., sp. nov. and Methyloligella solikamskensis sp. nov. are proposed. The type strain of Methyloligella halotolerans is C2(T) (=VKM B-2706(T)=CCUG 61687(T)=DSM 25045(T)) and the type strain of Methyloligella solikamskensis is SK12(T) (=VKM B-2707(T)=CCUG 61697(T)=DSM 25212(T)).

Methylopila musalis sp. nov., an aerobic, facultatively methylotrophic bacterium isolated from banana fruit.

A newly isolated, facultatively methylotrophic bacterium (strain MUSA(T)) was investigated. The isolate was strictly aerobic, Gram-stain-negative, asporogenous, motile, rod-shaped and multiplied by binary fission. The strain utilized methanol, methylamine and an apparently narrow range of multi-carbon compounds, but not methane, dichloromethane or CO2/H2, as the carbon and energy sources. Growth occurred at pH 5.5-9.5 (optimum, pH 7.0) and 16-40 °C (optimum, 28-30 °C). The major fatty acids of methanol-grown cells were C18 : 1ω7c, C18 : 0 and 11-methyl-C18 : 1ω7c . The predominant phospholipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and phosphatidylmonomethylethanolamine. The major ubiquinone was Q-10. The strain had methanol and methylamine dehydrogenases as well as the enzymes of the N-methylglutamate pathway (lyases of γ-glutamylmethylamide and N-methylglutamate). C1 assimilation occurs via the isocitrate lyase-negative serine pathway. Ammonium was assimilated by glutamate dehydrogenase and the glutamate cycle (glutamate synthase/glutamine synthetase). The DNA G+C content of the strain was 64.5 mol% (determined from the melting temperature). Based on 16S rRNA gene sequence similarity (97.0-98.9 %) and DNA-DNA relatedness (36-38 %) with representatives of the genus Methylopila (Methylopila capsulata IM1(T) and Methylopila jiangsuensis JZL-4(T)) the isolate was classified as a novel species of the genus Methylopila, for which the name Methylopila musalis sp. nov. is proposed. The type strain is MUSA(T) ( = VKM B-2646(T) = DSM 24986(T) = CCUG 61696(T)).

Methylorosula polaris gen. nov., sp. nov., an aerobic, facultatively methylotrophic psychrotolerant bacterium from tundra wetland soil.

Three strains of Gram-negative, aerobic, motile bacteria with bipolar flagella were isolated from acidic tundra wetland soils near the city of Vorkuta and from the Chukotka and Yugorsky Peninsulas and designated strains V-022(T), Ch-022 and Ju-022. The cells were rod-shaped, 0.5-0.6 µm in width and 1.3-4.5 µm in length and reproduced by irregular fission. These bacteria were facultative methylotrophs that used methanol, methylamines and a wide range of other sources of carbon and energy such as sugars and polysaccharides, ethanol and amino acids. The isolates used the Calvin-Benson pathway for the assimilation of one-carbon compounds and were unable to fix nitrogen. The new strains were moderately acidophilic and psychrotolerant, capable of growth over a pH range of 4.0 to 7.8, with optimum growth at pH 5.5-6.0. Growth occurred between 4 and 30 °C (optimum 20-25 °C). The principal phospholipid fatty acid was C(18:1)ω7c. The DNA G+C content of strain V-022(T) was 65.2 mol%. Analysis of the 16S rRNA gene sequences revealed that all three isolates V-022(T), Ch-022 and Yu-022 exhibited almost identical 16S rRNA gene sequences (99.9% gene sequence similarity) and formed a new lineage within the class Alphaproteobacteria. The name Methylorosula polaris is suggested to accommodate this new genus and novel species with strain V-022(T) (=DSM 22001(T)=VKM V-2485(T)) as the type strain of the type species.

Methylophilus glucosoxydans sp. nov., a restricted facultative methylotroph from rice rhizosphere.

Two restricted facultatively methylotrophic strains, designed B(T) and P, were isolated from rice roots. The isolates were strictly aerobic, Gram-negative, asporogenous, mesophilic, neutrophilic, motile rods that multiplied by binary fission and were able to synthesize indole-3-acetate. The cellular fatty acid profiles of the two strains were dominated by C(16:0), C(16:1)ω7c and C(16:0) 2-OH. The major ubiquinone was Q-8. The predominant phospholipids were phosphatidylethanolamine and phosphatidylglycerol. Cardiolipin (diphosphatidylglycerol) was absent. The two strains assimilated methanol carbon at the level of formaldehyde via the ribulose monophosphate cycle (2-keto-3-deoxy-6-phosphogluconate variant). They lacked α-ketoglutarate dehydrogenase and glutamate dehydrogenase. They assimilated ammonium via the glutamate cycle enzymes glutamine synthetase and glutamate synthase. The DNA G+C contents of strains B(T) and P were 52.5 and 51.5 mol% (T(m)), respectively. The level of DNA-DNA reassociation between these strains was 78%, indicating that they belong to one species. Phylogenetic analysis of strain B(T) based on 16S rRNA and methanol dehydrogenase (mxaF) gene sequences showed a high level of similarity to members of the genus Methylophilus. As the two isolates were clearly distinct from all recognized members of the genus Methylophilus based on phenotypic data and levels of DNA-DNA relatedness (30-46%), they are considered to represent a novel species, for which the name Methylophilus glucosoxydans sp. nov. is proposed; the type strain is B(T) (=VKM B-1607(T)=CCUG 59685(T)=DSM 5898(T)).

Methylophaga lonarensis sp. nov., a moderately haloalkaliphilic methylotroph isolated from the soda lake sediments of a meteorite impact crater.

A moderately haloalkaliphilic methylotrophic bacterium possessing the ribulose monophosphate pathway for carbon assimilation, designated MPL(T), was isolated from Lonar Lake sediment microcosms that were oxidizing methane for two weeks. The isolate utilized methanol and was an aerobic, Gram-negative, asporogenous, motile, short rod that multiplied by binary fission. The isolate required NaHCO(3) or NaCl for growth and, although not auxotrophic for vitamin B(12), had enhanced growth with vitamin B(12). Optimal growth occurred with 0.5-2% (w/v) NaCl, at 28-30 °C and at pH 9.0-10.0. The cellular fatty acid profile consisted primarily of straight-chain saturated C(16:0) and unsaturated C(16:1)ω7c and C(18:1)ω7c. The major ubiquinone was Q-8. The dominant phospholipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Cells accumulated ectoine as the main compatible solute. The DNA G+C content was 50.0 mol%. The isolate exhibited 94.0-95.4% 16S rRNA gene sequence similarity with the type strains of methylotrophs belonging to the genus Methylophaga and 31% DNA-DNA relatedness with the reference strain, Methylophaga alcalica VKM B-2251(T). It is proposed that strain MPL(T) represents a novel species, Methylophaga lonarensis sp. nov. (type strain MPL(T)=VKM B-2684(T)=MCC 1002(T)).

Methylobacillus arboreus sp. nov., and Methylobacillus gramineus sp. nov., novel non-pigmented obligately methylotrophic bacteria associated with plants.

Two newly isolated obligate methanol-utilizing bacteria (strains Iva(T) and Lap(T)) with the ribulose monophosphate pathway of C(1) assimilation are described. The isolates are strictly aerobic, Gram negative, asporogenous, motile rods multiplying by binary fission, mesophilic and neutrophilic, synthesize indole-3-acetate. The prevailing cellular fatty acids are straight-chain saturated C(16:0) and unsaturated C(16:1) acids. The major ubiquinone is Q-8. The predominant phospholipids are phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Ammonia is assimilated by glutamate dehydrogenase. The DNA G+C contents of strains Iva(T) and Lap(T) are 54.0 and 50.5mol% (T(m)), respectively. Based on 16S rRNA gene sequence analysis and DNA-DNA relatedness (38-45%) with type strains of the genus Methylobacillus, the novel isolates are classified as the new species of this genus and named Methylobacillus arboreus Iva(T) (VKM B-2590(T), CCUG 59684(T), DSM 23628(T)) and Methylobacillus gramineus Lap(T) (VKM B-2591(T), CCUG 59687(T), DSM 23629(T)). The GenBank accession numbers for the 16S rRNA gene and mxaF gene sequences of the strains Iva(T) and Lap(T) are GU937479, GU937478 and HM030736, HM030735, respectively.