Methylovorus mays sp. nov.: A new species of aerobic, obligately methylotrophic bacteria associated with plants

A bacterial strain utilizing methanol as the sole source of carbon and energy was isolated from the maize phyllosphere. Cells are nonpigmented gram-negative motile rods that do not form spores or prosthecae and reproduce by binary fission. The strain does not require vitamins or supplementary growth factors. It is obligately aerobic and urease-, oxidase-, and catalase-positive. The optimum growth temperature is 35–40°C; the optimum pH is 7.0–7.5. The doubling time is 2 h. The bacterium implements the ribulose monophosphate pathway and possesses NAD⁺-dependent 6-phosphogluconate dehydrogenase and enzymes of the glutamate cycle. α-Ketoglutarate dehydrogenase and enzymes of the glyoxylate cycle (isocitrate lyase and malate synthase) are absent. Fatty acids are dominated by palmitic (C_16:0) and palmitoleic (C_16:1) acids. The major phospholipids are phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylcholine. Cardiolipin is present in minor amounts. The dominant ubiquinone is Q₈ The bacterial genome contains genes controlling the synthesis and secretion of cytokinins. The G+C content of DNA is 57.2 mol %, as determined from the DNA thermal denaturation temperature T_m. The bacterium shows low DNA homology (<10%) with restricted facultative methylotrophic bacteria of the genusMethylophilus (M. methylotrophus NCIMB 10515^T andM. leisingerii VKM B-2013¹) and with the obligate methylotrophic bacterium (Methylobacillus glycogenes ATCC 29475^T). DNA homology with the type representative of the genusMethylovorus, M. glucosetrophus VKM B-1745^T, is high (58%). The new isolate was classified as a new species,Methylovorus mays sp. nov.     

<Emphasis Type="Italic">Methylovorus menthalis</Emphasis>, a novel species of aerobic obligate methylobacteria associated with plants

A bacterial strain (MM) utilizing methanol as the only carbon and energy source was isolated from corn mint rhizoplane. The cells of the strain were gram-negative colorless motile rods. Spores and prosthecae were not formed, reproduced by binary fission, and did not require vitamins and growth factors. The organism was strictly aerobic, urease-, oxidase-, and catalase-positive. Used the KDPG variant of the ribulose monophosphate pathway. Possessed NAD⁺ dependent 6-phosphogluconate dehydrogenase activity and enzymes of the glutamate cycle. The activities of α-ketoglutarate dehydrogenase and of the glyoxylate bypass enzymes (isocitrate lyase and malate synthase) were absent. Palmitic (C_16:0) and palmitoleic (C_16:1) acids were predominant in the cell fatty-acid composition. The dominant phospholipids were phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylcholine. The dominant ubiquinone was Q₈. The strain formed indole from tryptophan. The DNA G + C content was 54.5 mol % (T _m). According to the data of the 16S rRNA gene sequencing, strain MM showed high similarity (98–99%) to Methylovorus glucosotrophus VKM B-1745^T and Methylovorus mays VKM B-2221^T, but the level of DNA-DNA homology with these cultures was only 40 and 58%, respectively. The strain was classified as a new species, Methylovorus menthalis sp. nov. (VKM B-2663^T).     

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.).     

Methylophaga murata sp. nov.: a haloalkaliphilic aerobic methylotroph from deteriorating marble

The haloalkaliphilic methylotrophic bacterium (strain Kr3) isolated from material scraped off the deteriorating marble of the Moscow Kremlin masonry has been found to be able to utilize methanol, methylamine, trimethylamine, and fructose as carbon and energy sources. Its cells are gram-negative motile rods multiplying by binary fission. Spores are not produced. The isolate is strictly aerobic and requires vitamin B12 and Na+ ions for growth. It is oxidase- and catalase-positive and reduces nitrates to nitrites. Growth occurs at temperatures between 0 and 42 degrees C (with the optimum temperatures being 20-32 degrees C), pH values between 6 and 11 (with the optimum at 8-9), and NaCl concentrations between 0.05 and 3 M (with the optimum at 0.5-1.5 M). The dominant cellular phospholipids are phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. The major cellular fatty acids are palmitic (C16:0), palmitoleic (C16:1), and octadecenoic (C18:1) acids. The major ubiquinone is Q8. The isolate accumulates ectoine and glutamate, as well as a certain amount of sucrose, to function as osmoprotectants and synthesizes an exopolysaccharide composed of carbohydrate and protein components. It is resistant to heating at 70 degrees C, freezing, and drying; utilizes methanol, with the resulting production of formic acid, which is responsible for the marble-degrading activity of the isolate; and implements the 2-keto-3-deoxy-6-phosphogluconate variant of the ribulose monophosphate pathway. The G+C content of its DNA is 44.6 mol%. Based on 16S rRNA gene sequencing and DNA-DNA homology levels (23-41%) with neutrophilic and alkaliphilic methylobacteria from the genus Methylophaga, the isolate has been identified as a new species, Methylophaga murata (VKM B-2303T = NCIMB 13993T).     

Methylophaga natronica sp. nov., a new alkaliphilic and moderately halophilic, restricted-facultatively methylotrophic bacterium from soda lake of the Southern Transbaikal region

A new, moderately haloalkaliphilic and restricted-facultatively methylotrophic bacterium (strain Bur2T) with the ribulose monophosphate pathway of carbon assimilation is described. The isolate, which utilizes methanol, methylamine and fructose, is an aerobic, Gram-negative, asporogenous, motile short rod multiplying by binary fission. It is auxotrophic for vitamin B12, and requires NaHCO3 or NaCl for growth in alkaline medium. Cellular fatty acids profile consists primarily of straight-chain saturated C16:0, unsaturated C16:1 and C18:1 acids. The major ubiquinone is Q-8. The dominant phospholipids are phosphatidylethanolamine and phosphatidylglycerol. Diphosphatidylglycerol is also present. Optimal growth conditions are 25-29 degrees C, pH 8.5-9.0 and 2-3% (w/v) NaCl. Cells accumulate ectoine and glutamate as the main osmoprotectants. The G + C content of the DNA is 45.0 mol%. Based on 16S rDNA sequence analysis and DNA-DNA relatedness (25-35%) with type strains of marine and soda lake methylobacteria belonging to the genus Methylophaga, the novel isolate was classified as a new species of this genus and named Methylophaga natronica (VKM B-2288T).     

<Emphasis Type="Italic">Methylophaga murata</Emphasis> sp. nov.: a Haloalkaliphilic Aerobic Methylotroph from Deteriorating Marble

The haloalkaliphilic methylotrophic bacterium (strain Kr3) isolated from material scraped off the deteriorating marble of the Moscow Kremlin masonry has been found to be able to utilize methanol, methylamine, trimethylamine, and fructose as carbon and energy sources. Its cells are gram-negative motile rods multiplying by binary fission. Spores are not produced. The isolate is strictly aerobic and requires vitamin B₁₂ and Na⁺ ions for growth. It is oxidase- and catalase-positive and reduces nitrates to nitrites. Growth occurs at temperatures between 0 and 40°C (with the optimum temperatures being 20–32°C), pH values between 6 and 11 (with the optimum at 8–9), and NaCl concentrations between 0.05 and 3 M (with the optimum at 0.5–1.5 M). The dominant cellular phospholipids are phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. The major cellular fatty acids are palmitic (C_16:0), palmitoleic (C_16:1), and octadecenoic (C_18:1) acids. The major ubiquinone is Q₈. It accumulates ectoine and glutamate, as well as a certain amount of sucrose, to function as osmoprotectants and synthesizes an exopolysaccharide composed of carbohydrate and protein components. It is resistant to heating at 70°C, freezing, and drying; utilizes methanol, with the resulting production of formic acid, which is responsible for the marble-degrading activity of the isolate; and implements the 2-keto-3-deoxy-6-phosphogluconate variant of the ribulose monophosphate pathway. The G+C content of its DNA is 44.6 mol %. Based on 16S rRNA gene sequencing and DNA-DNA homology levels (23–41%) with neutrophilic and alkaliphilic methylobacteria from the genus Methylophaga, the isolate has been identified as a new species, Methylophaga murata (VKM B-2303^T = NCIMB 13993^T).__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 511–519.Original Russian Text Copyright © 2005 by Doronina, Lee, Ivanova, Trotsenko.     

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).     

Novel psychrophilic anaerobic spore-forming bacterium from the overcooled water brine in permafrost: description Clostridium algoriphilum sp. nov.

A gram-positive, motile, strict anaerobic spore-forming bacterium was isolated from the over-cooled brine in the permafrost. The optimal temperature for isolate growth was 5-6 degrees C at pH 6.8-7.2. The bacterium was growing on the medium rich in saccharides and disaccharides. Out of polysaccharides tested, only xylan sustained the growth. Fermentation of the hexoses led to the formation of acetate, butyrate, lactate, H2,CO2 and some formate and ethanol. Cell wall peptidoglycan contained meso-diaminopimelic acid. The major fatty acids of the cell wall were C(14:0) and C(16:1c9). The content of G-C pairs in DNA was 31.4 mol%. As phylogenetic analysis has shown, it is closely linked to the members of cluster 1 of Clostridium. It differs from the other species of the genus by the substrates necessary for the growth, products forming as a result of the fermentation and content of the fatty acids in the cell wall. Thus, it was suggested to describe this strain as a new species named Clostridium algoriphilum. Type strain 14D1 was deposited into the Russian Collection of the Microorganisms VKM B-2271T and German Collection of the Microorganisms DSM 16153T .     

The new facultatively chemolithoautotrophic, moderately halophilic, sulfate-reducing bacterium Desulfovermiculus halophilus gen. nov., sp. nov., isolated from an oil field

The new mesophilic, chemolithoautotrophic, moderately halophilic, sulfate-reducing bacterium strain 11-6 could grow at a NaCl concentration in the medium of 30-230 g/l, with an optimum at 80-100 g/l. Cells were vibrios motile at the early stages of growth. Lactate, pyruvate, malate, fumarate, succinate, propionate, butyrate, crotonate, ethanol, alanine, formate, and H2 + CO2 were used in sulfate reduction. Butyrate was degraded completely, without acetate accumulation. In butyrate-grown cells, a high activity of CO dehydrogenase was detected. Additional growth factors were not required. Autotrophic growth occurred, in the presence of sulfate, on H2 + CO2 or formate without other electron donors. Fermentation of pyruvate and fumarate was possible in the absence of sulfate. Apart from sulfate, sulfite, thiosulfate, and elemental sulfur were able to serve as electron acceptors. The optimal growth temperature was 37 degrees C; the optimum pH was 7.2. Desulfoviridin was not detected. Menaquinone MK-7 was present. The DNA G+C content was 55.2 mol %. Phylogenetically, the bacterium represented a separate branch within the cluster formed by representatives of the family Desulfohalobiaceae in the subclass Deltaproteobacteria. The bacterium was assigned to a new genus and species, Desulfovermiculus halophilus gen. nov., sp. nov. The type strain is 11-6T (= VKM B-2364), isolated from the highly mineralized formation water of an oil field.     

A novel plant-associated thermotolerant alkalophilic methylotroph of the genus Paracoccus

Strain GB isolated from the maize rhizosphere is a gram-negative, aerobic, non-spore-forming, nonpigmented, nonmotile, chemolithotrophic, facultatively methylotrophic bacterium. Cells are cocci or short rods. The strain does not require vitamins. Optimum growth in a medium with methanol occurs at 38-42 degrees C at pH 8.0-9.2. The doubling time is 12 h. In addition to methanol, the bacterium can grow on methylamine, dimethylformamide, acetone, thiosulfate + NaHCO3, and in an atmosphere of H2 + CO2 + O2. Methanol and methylamine are oxidized by the respective dehydrogenases to CO2 via formaldehyde and formate, respectively. The CO2 produced is assimilated via the ribulose bisphosphate pathway. Fatty acids are dominated by cyclopropanoic (58-61%), palmitic (24-26%), and octadecanoic (8-9%) acids. The main phospholipids are phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine. The major ubiquinone is Q10. The bacterial genome contains genes controlling the synthesis and secretion of cytokinins. The culture liquid exhibits cytokinin activity. The G + C content of DNA is 62.5 mol %, as determined from the DNA thermal denaturation temperature (Tm). Strain GB shows a moderate degree of DNA-DNA homology (< 40%) with the type representatives of the genus Paracoccus. Based on the data obtained, the bacterium was classified as a new species of this genus, named P. kondratievae.     

Thalassospira permensis sp. nov., a new terrestrial halotolerant bacterium isolated from a naphthalene-utilizing microbial consortium

A halotolerant bacterium, strain SMB34T, was isolated from a naphthalene-utilizing bacterial consortium obtained from primitive technogeneous soil (Vrkhnekamsk salt deposit, Perm region, Russia) by enrichment procedure. The strain itself was unable to degrade naphthalene and grew at NaCl concentrations up to 11% (w/v). The 16S rRNA-based phylogenetic analysis showed that the strain belongs to the genus Thalassospira. The DNA-DNA hybridization values between SMB34T and the type strains of phylogenetically closest species (T. xiamenensis, T. profundimaris and T. tepidiphila) did not exceed 50%. The novel strain could be distinguished from the above species by the cell motility, MALDI/TOF mass spectra of whole cells and a range of physiological and biochemical characteristics. SMB34T also considerably differs from the recently described species T. xianhensis, with the most striking differences in the DNA G + C content (53.7 +/- 1.0 vs. 61.2 +/- 1.0 mol.%) and predominant ubiquinones (Q-10 vs. Q-9). The data obtained suggest strain SMB34T (=VKM B-2527T = NBRC 106175T), designated as the type strain, represents a novel species, named Thalassospira permensis sp. nov.     

Salinicola socius gen. nov., sp. nov., a moderately halophilic bacterium from a naphthalene-utilizing microbial association

A chemoorganotrophic, moderately halophilic bacterium (strain SMB35) has been isolated from a naphthalene-utilizing microbial community obtained from salt mines (Perm region of Russia). Strain SMB35 grows in a wide salinity range, 0.5 to 30% (wt/vol) NaCl. Cells are gram-negative rods motile by means of a single polar flagellum. The predominant fatty acids are 16:1omega7, 16:0, 18:1omega7, and 19 cy. The major lipoquinone is an unsaturated ubiquinone with nine isoprene units (Q-9). The DNA G+C content is 63.0 mol%. The 16S rDNA-based phylogenetic analysis has shown that strain SMB35 formed a separate clade in the cluster of the family Halomonadaceae. The 16S rDNA sequence similarity of the isolate to the members of the family is in the range from 90.6% to 95.1%. The phylogenetic and phenotypic differences from Halomonas elongata (the type species of the genus) and from other members of the family suggest that the isolate represents a novel genus and species, for which the name Salinicola socius gen. nov., sp. nov. is proposed. The type strain is SMB35(T) (=VKM B-2397(T)).     

Molecular cloning of the DNA gyrase genes from Methylovorus sp. strain SS1 and the mechanism of intrinsic quinolone resistance in methylotrophic bacteria

The genes encoding the DNA gyrase A (GyrA) and B subunits (GyrB) of Methylovorus sp. strain SS1 were cloned and sequenced. gyrA and gyrB coded for proteins of 846 and 799 amino acids with calculated molecular weights of 94,328 and 88,714, respectively, and complemented Escherichia coli gyrA and gyrB temperature sensitive (ts) mutants. To analyze the role of type II topoisomerases in the intrinsic quinolone resistance of methylotrophic bacteria, the sequences of the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase and the C subunit (ParC) of topoisomerase IV (Topo IV) of Methylovorus sp. strain SS1, Methylobacterium extorquens AM1 NCIB 9133, Methylobacillus sp, strain SK1 DSM 8269, and Methylophilus methylotrophus NCIB 10515 were determined. The deduced amino acid sequences of the QRDRs of the ParCs in the four methylotrophic bacteria were identical to that of E. coli ParC. The sequences of the QRDR in GyrA were also identical to those in E. coli GyrA except for the amino acids at positions 83, 87, or 95. The Ser83 to Thr substitution in Methylovorus sp. strain SS1, and the Ser83 to Leu and Asp87 to Asn substitutions in the three other methylotrophs, agreed well with the minimal inhibitory concentrations of quinolones in the four bacteria, suggesting that these residues play a role in the intrinsic susceptibility of methylotrophic bacteria to quinolones.     

Description of the erythromycin-producing bacterium Arthrobacter sp. strain NRRL B-3381 as Aeromicrobium erythreum gen. nov., sp. nov

Arthrobacter sp. strain NRRL B-3381T (T = type strain) is a nonmycelial, nonsporulating actinomycete that produces the macrolide antibiotic erythromycin. This bacterium differs in many ways from the type species of the genus Arthrobacter (Arthrobacter globiformis), suggesting that a taxonomic revision is appropriate. The G + C content of strain NRRL B-3381T DNA is 71 to 73 mol%, and the peptidoglycan of this organism contains LL-diaminopimelic acid. Evolutionary distance data obtained from 16S rRNA sequences identified NRRL B-3381T as the deepest branching member of the Nocardioides group of actinomycetes. The principal long-chain fatty acids which we identified that distinguished strain NRRL B-3381T from related G + C-rich bacteria were 10-methyloctadecanoic (tuberculosteric), octadecenoic, and hexadecanoic acids. These characteristics, together with phage typing and biochemical characteristics, form the basis for our recommendation that strain NRRL B-3381 should be the type strain of a new taxon, for which we propose the name Aeromicrobium erythreum.     

Vitamin B12-independent strains of Methylophaga marina isolated from Red Sea algae

Two strains (KM3 and KM5) of halophilic methylobacteria isolated from Red Sea algae do not require vitamin B12 for growth and can use methanol, methylamine, dimethylamine, trimethylamine, dimethyl sulfide, and fructose as sources of carbon and energy. The cells of these strains are gram-negative motile monotrichous (strain KM3) or peritrichous (strain KM5) rods. The strains are strictly aerobic and require Na+ ions but not growth factors for growth. They are oxidase- and catalase-positive and reduce nitrates to nitrites. Both strains can grow in a temperature range of 4 to 37 degrees C (with optimal growth at 29-34 degrees C), at pH between 5.5 and 8.5 (with optimal growth at pH 7.5-8.0), and in a range of salt concentrations between 0.5 and 15% NaCl (with optimal growth at 5-9% NaCl). The phospholipids of these strains are dominated by phosphatidylethanolamine and phosphatidylglycerol and also include phosphatidylcholine, phosphatidylserine, and cardiolipin. The dominant fatty acids are C(16:1omega7c) and C(16:0). The major ubiquinone is Q8. The cells accumulate ectoin, glutamate, and sucrose as intracellular osmoprotectants. The strains implement the 2-keto-3-deoxy-6-phosphogluconate-dependent variant of the ribulose monophosphate pathway. The G+C content of the DNA is 44.4-44.7 mol %. Analysis of the 16S rRNA genes showed that both strains belong to Gammaproteobacteria and have a high degree of homology (99.4%) to Methylophaga marina ATCC 35842T . Based on the data of polyphasic taxonomy, strains KM3 and KM5 are identified as new strains M. marina KM3 (VKM B-2386) and M. marina KM5 (VKM B-2387). The ability of these strains to produce auxins (indole-3-acetic acid) suggests their metabolic association with marine algae.