Methylobacterium bullatum sp. nov., a methylotrophic bacterium isolated from Funaria hygrometrica

A novel, pink-pigmented aerobic, facultatively methylotrophic bacterial strain (F3.2^T) isolated from the phyllosphere of Funaria hygrometrica, was analyzed using a polyphasic approach. Cells were Gram-negative, motile rods, strictly aerobic and non-spore-forming and exhibited surface structures varying in quantity, distribution and morphology. The isolate grew at 10–33 °C over a pH range of 5.5–8.0 and in the presence of less than 1.0% NaCl. Strain F3.2^T shared less than 70% DNA–DNA binding to the next type strain of the genus Methylobacterium (M. adhaesivum DSM 17169^T). In addition to the major cellular fatty acid C_18:1ω7c (81.7%), present in all Methylobacterium species (and also members of the genus Alphaproteobacteria), a high value (11.7%) of the fatty acids (summed feature) C_16:1ω7c and/or iso-C_15:02OH was determined. Phylogenetic analyses based on 16S rDNA and methanol dehydrogenase gene sequences, DNA–DNA hybridization values, chemotaxonomic and phenotypic characteristics indicate that the strain F3.2^T represents a novel species within the genus Methylobacterium. We propose the name Methylobacterium bullatum sp. nov. for this species. The type strain is the strain F3.2^T (DSM 21893^T = LMG 24788^T).     

Methylobacterium pseudosasae sp. nov., a pink-pigmented, facultatively methylotrophic bacterium isolated from the bamboo phyllosphere

A pink-pigmented, Gram negative, aerobic, facultatively methylotrophic bacterium, strain BL44^T, was isolated from bamboo leaves and identified as a member of the genus Methylobacterium. Phylogenetic analysis based on 16S rRNA gene sequences showed similarity values of 98.7–97.0 % with closely related type strains and showed highest similarity to Methylobacterium zatmanii DSM 5688^T (98.7 %) and Methylobacterium thiocyanatum DSM 11490^T (98.7 %). Methylotrophic metabolism in this strain was confirmed by PCR amplification and sequencing of the mxaF gene coding for the α-subunit of methanol dehydrogenase. Strain BL44^T produced three known quorum sensing signal molecules with similar retention time to C₈, C₁₀ and C₁₂-HSLs when characterized by GC–MS. The fatty acid profiles contained major amounts of C_18:1 ω7c, iso-3OH C_17:0 and summed feature 3 (C_16:1 ω7c and/or iso-C_15:0 2-OH), which supported the grouping of the isolate in the genus Methylobacterium. The DNA G+C content was 66.9 mol%. DNA relatedness of the strain BL44^T to its most closely related strains ranged from 12–43.3 %. On the basis of the phenotypic, phylogenetic and DNA–DNA hybridization data, strain BL44^T is assigned to a novel species of the genus Methylobacterium for which the name Methylobacterium pseudosasae sp. nov. is proposed (type strain BL44^T = NBRC 105205^T = ICMP 17622^T).     

Prokaryotic triterpenoids. New bacteriohopanetetrol cyclitol ethers from the methylotrophic bacterium Methylobacterium organophilum

Together with bacteriohopanetetrol, which is identical to bacteriohopanetetrol isolated from Bacillus acidocaldarius, three other bacteriohopane derivatives were isolated from the methylotrophic bacterium Methylobacterium organophilum. Three different kinds of polar moieties were found linked to the C-35 hydroxyl group of bacteriohopanetetrol: glucosamine linked through a glycosidic bond and two new polyhydroxylated methylcyclopentanoids differing one from each other by the presence of an amino or a guanidino group and linked through an ether bond. The significance of the presence of these compounds in terms of membrane reinforcement and DNA/triterpenoid interactions is discussed.     

Methylophilus quaylei sp. nov., a new aerobic obligately methylotrophic bacterium

A new obligately methylotrophic bacterium (strain MTT) with the ribulose monophosphate pathway of carbon assimilation is described. The isolate, utilizing only methanol, is an aerobic, Gram-negative, asporogenous, non-motile short rod multiplying by binary fission. Its cellular fatty acids profile consists primarily of straight-chain saturated C16:0 and unsaturated C16:l acids. The major ubiquinone is Q-8. The dominant phospholipids are phosphatidylethanolamine and phosphatidylglycerol. Diphosphatidylglycerol (cardiolipin) is absent. Optimal growth conditions are 25-29 degree C, pH 6.5 - 7.5, 0.5% CH3OH and 0.05% NaCl. Strain MTT lacks alpha-ketoglutarate dehydrogenase, the glyoxylate shunt enzymes, and glutamate dehydrogenase. Ammonium is assimilated by the operation of the glutamate cycle enzymes: glutamine synthetase and glutamate synthase. An exopolysaccharide consisting of rhamnose, glucose and galactose is formed under nitrogen limitation. The G + C content of the DNA is 54.0 mol%. Based on 16S rDNA sequence analysis and DNA-DNA relatedness (29-34%) with type strains of the genus Methylophilus, the novel isolate was classified as a new species of this genus and named Methylophilus quaylei MTT (VKM B-2338T, DSMZ, etc.).     

Preliminary crystallographic study of a pseudoazurin from methylotrophic bacterium, Methylobacterium extorquens AM1

Single crystals of pseudoazurin, one of the blue copper proteins produced by methylotrophic bacterium Methylobacterium extorquens AM1, have been obtained by the method of vapor diffusion with ammonium sulfate as a precipitant at pH 8.0. Crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit cell dimensions of a = 52.619(4) A, b = 63.280(6) A, c = 35.133(4) A. The asymmetric unit includes one molecule of pseudoazurin (Vm = 2.18 A3/dalton). The crystals are so stable against X-ray irradiation that diffraction intensities of the native crystal up to 1.68 A resolution could be collected from only one crystal. Among the many heavy-metal reagents examined, uranyl acetate gave an effective isomorphous derivative.     

Methylobacterium soli sp. nov. a methanol-utilizing bacterium isolated from the forest soil

A Gram-negative, pink-pigmented, non-spore-forming rod shaped, methanol-utilizing bacterium, strain YIM 48816(T), was isolated from forest soil collected from Sichuan province, China. Strain YIM 48816(T) can grow at 4-37 °C, pH 5.0-7.0 and 0% NaCl (w/v). Based on 16S rRNA gene sequence similarity studies, it belonged to the genus Methylobacterium, and formed a phyletic line. The 16S rRNA gene sequence similarities were 96.2% to Methylobacterium mesophilicum DSM 1708(T) and 96.0% to Methylobacterium brachiatum DSM 19569(T), and the phylogenetic similarities to all other Methylobacterium species with validly published names were less than 96.0%. The major menaquinones detected were Q-10 (97.14%) and Q-9 (2.86%). The major fatty acids were C18:1 ω7c (80.84%). The DNA G + C content was 66.2 mol%. It is apparent from the genotypic and phenotypic data that strain YIM 48816(T) belongs to a novel species of the genus Methylobacterium, for which the name Methylobacterium soli sp. nov. is proposed. The type strain is YIM 48816(T) (CCTCC AA 208027(T) = KCTC 22810(T)).     

Statistical media optimization for growth and PHB production from methanol by a methylotrophic bacterium

Media components were optimized by statistical design for cell growth and PHB production of Methylobacterium extorquens DSMZ 1340. Four important components of growth media were optimized by central composite design. The growth increased from an OD = 1.35 for Choi medium as control to an OD = 2.15 for optimal medium. Then media components for PHB production were optimized. Optimization of five important factors was conducted by response surface method. The optimal composition of PHB production medium was found to be at 7.8 (g/L) Na₂HPO₄ · 12H₂O, and surprisingly at zero concentration of (NH₄)₂SO₄, KH₂PO₄, MgSO₄ and MnSO₄. The PHB production was found to be 2.95 (g/L) at this medium. RSM results indicated that a deficiency of nitrogen and magnesium is crucial for PHB accumulation in this microorganism. Also, PHB production was carried out in a 5 L fermentor at the optimum condition which resulted in 9.5 g/L PHB and 15.4 g/L cell dry weight with 62.3% polymer content.     

Alcohol dehydrogenases from a facultative methylotrophic bacterium.

Alcohol-oxidizing enzymes of the facultative methylotroph PAR were investigated after growth of the bacteria on methanol and ethanol. During methanol growth only a phenazine methosulfate-linked alcohol dehydrogenase was detected. This enzyme had broad specificity for primary alcohols and was also capable of oxidation of secondary alcohols. It had a molecular weight of 112,000, was composed of two subunits of equal molecular weight, and showed an absolute requirement for ammonium ion for activation. During ethanol growth this enzyme was absent and was replaced by a typical nicotinamide adenine dinucleotide-linked alcohol dehydrogenase of molecular weight 150,000. The latter enzyme also had broad specificity but could not oxidize methanol. This enzyme was not found during methanol growth. These data show that the organism has two distinctly separate mechanisms for oxidation of alcohols.     

The fractionation of chlorine isotopes by the aerobic methylotrophic bacterium Methylobacterium dichloromethanicum grown on dichloromethane

Methylobacterium dichloromethanicum was found to be able to utilize dichloromethane (DCM) as the source of carbon and energy with the production of biomass, CO2, and HCl. A comparative analysis of abundances of the major DCM isotopomers 35Cl(2)12C1H2, 35Cl37Cl12C1H2 and 37Cl(2)12CH2 made it possible to estimate the fractionation of chlorine isotopes during the bacterial metabolism of DCM. The kinetic chlorine isotope effects for 35Cl37Cl12C1H2 (m/z 86) and 37Cl(2)12C1H2 (m/z 88) relative to 35Cl(2)12C1H2 (m/z 84) turned out to be alpha 86/84 = 1.006 +/- 0.002 and alpha 88/84 = 1.023 +/- 0.003, respectively. The inference is made that the growth of M. dichloromethanicum on DCM is accompanied by the mass-independent fractionation of the DCM isotopomers.     

Acetate utilization by a methylotrophic bacterium

The mode of acetate ultilization in the Gram-negative diplococcoid bacterium PAR was investigated. This organism uses the isocitrate lyase-negative serine pathway during growth on C₁-compounds and was found to lack isocitrate lyase on acetate growth also. Enzyme assays revealed the absence of the glycerate and hydroxyaspartate pathways for the metabolism of C₂-compounds. Pulse-labeling experiments indicated the rapid formation of glycine, glutamate, and malate. The rapid formation of malate and the presence of malate synthase suggest that glyoxylate is an intermediate formed from acetate by a means other than the conventional isocitrate cleavage reaction.     

Isolation and characterization ofSporomusa acidovorans sp. nov., a methylotrophic homoacetogenic bacterium

Exopolysaccharides (EPS) of nodulating strains of Rhizobium trifolii and Rhizobium leguminosarum added to red clover seedlings before inoculation reduced the number of nodules. The inhibition of the nodulation was correlated with the amount of EPS. The preparations of EPS from mutants defective in early stages of nodulation (Roa^- or Hac^-) did not affect the nodulation, whereas EPS from mutants deficient in late stages (post Hac^-) exerted an inhibitory effect.Inactive preparation of EPS contained less O-acetyl groups and pyruvic acid residues. Deacetylation and depyruvylation of EPS from R. trifolii Nod⁺ abolished it inhibitory effect. It was concluded that noncarbohydrate substitutions (acetate, pyruvate) are involved in EPS effect.Abbreviations CPS capsular polysaccharide - EPS exopolysaccharide - LPS lipopolysaccharide - Nod nodulation - Fix nitrogen fixation - Hac root hairs curling - Roa root adhesion     

Production of poly-3-hydroxybutyrate from lactose and whey by Methylobacterium sp. ZP24

Methylobacterium sp. ZP24, isolated from a local pond, is able to grow in a medium containing 12 g l⁻¹ lactose as a sole source of carbon, giving 5·25 g l⁻¹ biomass yield and poly-3-hydroxybutyrate (PHB) up to 59% of its dry weight in 40 h. The isolate was also able to utilize cheese whey and produce 1·1 g l⁻¹ PHB. Addition of ammonium sulphate increased the production of PHB from whey 2·5-fold. The potential of Methylobacterium sp. ZP24 in PHB production from cheese whey is discussed.     

Methylobacillus rhizosphaerae sp. nov., a novel plant-associated methylotrophic bacterium isolated from rhizosphere of red pepper

A novel plant-associated obligate methylotrophic bacterium, designated strain Ca-68^T, was isolated from the rhizosphere soil of field-grown red pepper from India. The isolates are strictly aerobic, Gram negative, motile rods multiplying by binary fission and formaldehyde is assimilated via the ribulose monophosphate pathway. A comparative 16S rRNA gene sequence-based phylogenetic analysis placed the strain in a clade with the species Methylobacillus flagellatus, Methylobacillus glycogens and Methylobacillus pratensis, with which it showed pairwise similarity of 97.8, 97.4 and 96.2 %, respectively. The major fatty acids are C_16:0, C_10:0 3OH and C_16:1 ω7c. The G+C content of the genomic DNA is 59.7 mol%. The major ubiquinone is Q-8. Dominant phospholipids are phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Based on 16S rRNA gene sequence analysis and DNA–DNA relatedness (14–19 %) with type strains of the genus Methylobacillus, the novel isolate was classified as a new species of this genus and named Methylobacillus rhizosphaerae Ca-68^T (=KCTC 22383^T = NCIMB 14472^T).     

Hansschlegelia plantiphila gen. nov. sp. nov., a new aerobic restricted facultative methylotrophic bacterium associated with plants

A new genus, Hansschlegelia, and a new species, Hansschlegelia plantiphila, are proposed for three strains of methanol-utilizing bacteria isolated from lilac buds (strain S(1)(T)), linden buds (strain S(2)) and blue spruce needles (strain S(4)), which were selected in winter at -17 degrees C. These bacteria are aerobic, Gram-negative, colorless, non-motile short rods that multiply by binary fission and employ the ribulose bisphosphate (RuBP) and the serine pathways for C(1) assimilation. The strains have a limited number of growth substrates and use methanol, methylamine, formate, CO(2)/H(2) and glycerol as carbon and energy sources. Only strain S(1)(T) grows with ethanol and inulin. The strains are neutrophilic and mesophilic, and synthesize phytohormones (auxins and cytokinins) and vitamin B(12). Their major cellular fatty acids are saturated C(16:0), straight-chain, unsaturated C(18:1)(omega)(7) and cyclopropane C(19 cyc) acids. The main ubiquinone is ubiquinone-10 (Q-10). The dominant phospholipids are phosphatidylethanolamine, phosphatidylcholine and diphosphatidylglycerol (cardiolipin). The DNA G+C content is 68.5+/-0.2 mol%. The strains share almost identical 16S rRNA gene sequences, a high DNA-DNA hybridization value (72-86%) and represent a novel lineage of autotrophic methanol-utilizing bacteria within the Alphaproteobacteria. Collectively, these strains comprise a new genus and species H. plantiphila gen. nov., sp. nov., with strain S(1)(T) (VKM B-2347(T), NCIMB 14035(T)) as the type strain.     

Methylopila helvetica sp. nov. and Methylobacterium dichloromethanicum sp. nov.--novel aerobic facultatively methylotrophic bacteria utilizing dichloromethane

Eight strains of Gram-negative, aerobic, asporogenous, neutrophilic, mesophilic, facultatively methylotrophic bacteria are taxonomically described. These icl- serine pathway methylobacteria utilize dichloromethane, methanol and methylamine as well as a variety of polycarbon compounds as the carbon and energy source. The major cellular fatty acids of the non-pigmented strains DM1, DM3, and DM5 to DM9 are C18:1, C16:0, C18:0, Ccy19:0 and that of the pink-pigmented strain DM4 is C18:1. The main quinone of all the strains is Q-10. The non-pigmented strains have similar phenotypic properties and a high level of DNA-DNA relatedness (81-98%) as determined by hybridization. All strains belong to the alpha-subgroup of the alpha-Proteobacteria. 16S rDNA sequence analysis led to the classification of these dichloromethane-utilizers in the genus Methylopila as a new species - Methylopila helvetica sp.nov. with the type strain DM9 (=VKM B-2189). The pink-pigmented strain DM4 belongs to the genus Methylobacterium but differs from the known members of this genus by some phenotypic properties, DNA-DNA relatedness (14-57%) and 16S rDNA sequence. Strain DM4 is named Methylobacterium dichloromethanicum sp. nov. (VKM B-2191 = DSMZ 6343).     

Genome sequence of Herbaspirillum sp. strain GW103, a plant growth-promoting bacterium

Herbaspirillum sp. strain GW103 was isolated from rhizosphere soil of the reed Phragmites australis on reclaimed land. Here we report the 5.05-Mb draft genome sequence of the strain, providing bioinformation about the agronomic benefits of this strain, such as multiple traits relevant to plant root colonization and plant growth promotion.     

Production of feather protein hydrolysate by keratinolytic bacterium Vibrio sp. kr2

A feather protein hydrolysate was produced using the keratinolytic bacterium Vibrio sp. strain kr2. Complete feather degradation was observed in medium containing up to 60 g L(-1) raw feathers. Cultivation on 40, 60 or 80 g L(-1) feathers for five days resulted in similar amounts of soluble protein, reaching maximum values around 2.5 g L(-1). Maximum yields of soluble protein were achieved at 30 degrees C and initial pH ranging from 6.0 to 8.0. Strain kr2 was effective in producing keratin hydrolysate from chicken feathers. Bacterial feather hydrolysate has the potential for utilization as an ingredient in animal feed or as organic fertilizer, thereby reducing the environmental impact of feather waste from the poultry industry.     

Production of RNA by a marine photosynthetic bacterium, Rhodovulum sp.

The marine photosynthetic bacterium, Rhodovulum sp. PS88, produces RNA not only in cells but also as an extracellular polymeric substance during aerobic continuous cultivation in the dark. At a dilution rate of 0.32–0.5 h^–1, the maximum RNA production was 460 mg RNA l^–1 broth (200 mg RNA g^–1 suspended solids) which is a value about 2–3 times more than that of yeast cells.