Obligate methylotroph <Emphasis Type="Italic">Methylobacillus arboreus</Emphasis> Iva<Superscript>T</Superscript> synthesizes a plant hormone,gibberellic acid GA<Subscript>3</Subscript>

An obligate methylotroph Methylobacillus arboreus Iva^Т (VKM B-2590^Т, CCUG 59684^T, DSM 23628T) is the first known aerobic methylotrophic bacterium capable of synthesis of the bioactive gibberellic acid GA₃. Primary separation and identification of gibberellic acid from the culture liquid of methanol-grown culture were carried out using thin-layer chromatography and high-performance liquid chromatography. The concentration and structure of the gibberellic acid GA₃ were determined by liquid chromatography?mass spectrometry (LC/MS). Biological activity of the isolated compound was confirmed by tests on sprouts of lettuce (Laсtuca sativa L.).     

Methylovorus mays--novel species of aerobic, obligatory 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 degrees 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. alpha-Ketoglutarate dehydrogenase and enzymes of the glyoxylate cycle (isocitrate lyase and malate synthase) are absent. Fatty acids are dominated by palmitic (C16:0) and palmitoleic (C16:1) 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 (Tm). The bacterium shows low DNA homology (< 10%) with restricted facultative methylotrophic bacteria of the genus Methylophilus (M. methylotrophus NCIMB 10515T and M. leisingerii VKM B-2013T) and with the obligate methylotrophic bacterium (Methylobacillus glycogenes ATCC 29475T). DNA homology with the type representative of the genus Methylovorus, M. glucosetrophus VKM B-1745T, is high (58%). The new isolate was classified as a new species, Methylovorus mays sp. now.     

Ancylobacter dichloromethanicus sp. nov. – a new aerobic facultatively methylotrophic bacterium utilizing dichloromethane

A novel aerobic facultative methylotroph was isolated from contaminated soil. The organism (strain DM16) is a Gram-negative asporogenous non-motile curved rod multiplying by binary fission. Cells are neutrophilic and mesophilic. This strain utilized dichloromethane, methanol, formate and formaldehyde along with a variety of polycarbon compounds. Strain DM16 employs the ribulosebisphosphate pathway for C₁ assimilation. The DNA G+C content is 64.5 mol%. The major ubiquinone is Q-10. The dominant cellular fatty acids are 18:1ω7c (58.6%), cyclo-19:0ω8c (34.8%) and 16:0 (3.2%). Sequencing of the 16S rRNA gene and DNA–DNA hybridization experiments clearly indicated that this methylotroph should be classified as a new species within genus Ancylobacter – Ancylobacter dichloromethanicus sp. nov. with the type strain DM16^T (DSM 21507^T=VKM B-2484^T).     

Analysis of the anaerobic microbial community capable of degrading p-toluene sulphonate

Three strains of Clostridium sp., 14 (VKM B-2201), 42 (VKM B-2202), and 21 (VKM B-2279), two methanogens, Methanobacterium formicicum MH (VKM B-2198) and Methanosarcina mazei MM (VKM B-2199), and one sulfate-reducing bacterium, Desulfovibrio sp. SR1 (VKM B-2200), were isolated in pure cultures from an anaerobic microbial community capable of degrading p-toluene sulfonate. Strain 14 was able to degrade p-toluene sulfonate in the presence of yeast extract and bactotryptone and, like strain 42, to utilize p-toluene sulfonate as the sole sulfur source with the production of toluene. p-Toluene sulfonate stimulated the growth of Ms. mazei MM on acetate. The sulfate-reducing strain Desulfovibrio sp. SR1 utilized p-toluene sulfonate as an electron acceptor. The putative scheme of p-toluene sulfonate degradation by the anaerobic microbial community is discussed.     

Role of surfactants in optimizing fluorene assimilation and intermediate formation by Rhodococcus rhodochrous VKM B-2469

Biodegradation of fluorene by Rhodococcus rhodochrous VKM B-2469 was investigated and optimized by adding non-ionic surfactants to the liquid media. The utilization of 1-1.5% Tween 60 or 1% Triton X100 allowed to solubilize 1 mM fluorene over 150 times more than in water medium (from 9-11 microM to above 1.5 mM at 28 degrees C). We observed that Tween 60 was useful to enhance the fluorene biodegradation rates further supporting R. rhodochrous VKM B-2469 growth as an additional carbon source and to decrease fluorene toxicity for bacterial cells whereas Triton X100 resulted to be toxic for this strain. An additional enzyme induction step before starting the bioconversion process and the increase of incubation temperature during fluorene bioconversion led to further improvements in rates of fluorene utilization and formation of its intermediates. In the optimized conditions 1 mM fluorene was degraded completely within 24h of incubation. Some intermediates in fluorene degradation built up during the process reaching maxima of 31% for 9-hydroxyfluorene, 2.1% for 9-fluorenone and 1.9% for 2-hydroxy-9-fluorenone (starting from 1 mM substrate). In the presence of Tween 60 the appearance and following conversion of 2-hydroxy-9-fluorenone was observed for R. rhodochrous VKM B-2469 revealing the existence of a new pathway of 9-fluorenone bioconversion.     

Cell wall teichoic acids of Bacillus licheniformis VKM B-511T, Bacillus pumilus VKM B-508T, and other strains previously assigned to Bacillus pumilus

The teichoic acids (TAs) of type strains, viz. Bacillus licheniformis VKM B-511^T and Bacillus pumilus VKM B-508^T, as well as phylogenetically close bacteria VKM B-424, VKM B-1554, and VKM B-711 previously assigned to Bacillus pumilus on the basis of morphological, physiological, and biochemical properties, were investigated. Three polymers were found in the cell wall of each of the 5 strains under study. Strains VKM B-508^T, VKM B-424, and VKM B-1554 contained polymers of the same core: unsubstituted 1,3-poly(glycerol phosphate) (TA I) and 1,3-poly(glycerol phosphate) with O-D-Ala and N-acetyl-??-D-glucosamine substituents (TA II and TA III??, respectively). The cell walls of two remaining strains contained TA I, TA II, and a poly(glycosylpolyol phosphate) with the following structure of repeating units: -6)-??-D-GlcpNAc(1??1)-snGro-(3-P-(TA III?) in ??Bacillus pumilus?? VKM B-711 (100% 16S rRNA gene similarity with the type strain of Bacillus safensis) and -6)-??-D-Galp-(1??2)-snGro-(3-P-(TA III?) in Bacillus licheniformis VKM B-511^T. The simultaneous presence of three different TAs in the cell walls was confirmed by the NMR spectroscopic DOSY methods. The structure of the polymers and localization of O-D-Ala residues were investigated by the chemical and NMR spectroscopic methods.     

Disaccharide 1-phosphate polymers of some representatives of the Bacillus subtilis group

Disaccharide 1-phosphate polymers as well as teichoic acids of various structures have been found in the cell walls of the representatives of the Bacillus subtilis group, namely Bacillus subtilis subsp. spizizenii VKM B-720 and VKM B-916, B. subtilis VKM B-517, and Bacillus vallismortis VKM B-2653^T. Disaccharide 1-phosphate polymers are composed of repeating units of the following structure: -P-4)-β-D-GlcpNAc-(1→6)-α-D-Galp-(1-, the N-acetylglucosamine residues are partially acetylated at positions O3 and O6 (VKM B-720 and VKM B-916); -P-4)-β-D-Glcp-(1→6)-α-D-GlcpNAc-(1-, the glucopyranose residues are partially acetylated at positions O2 or O3 (VKM B-517); -P-6)-α-D-GlcpNH ₃ ⁺ /α-D-GlcpNAc-(1→2)-α-D-Glcp-(1-, the N-acetylglucosamine residues are partially deacetylated (VKM B-2653^T). The structures of the two last disaccharide 1-phosphate polymers have not been reported so far for Gram-positive bacteria. The teichoic acids in the studied strains are O-D-alanyl-1,5-poly(ribitol phosphates) substituted with β-D-glucopyranose (VKM B-517, VKM B-720, VKM B-916) or 2-acetamido-2-deoxy-β-D-glucopyranose (VKM B-2653^T). The structures of the phosphate-containing polymers have been studied by chemical methods and by NMR spectroscopy.     

Analysis of the Anaerobic Microbial Community Capable of Degrading p-Toluene Sulfonate

Three strains of Clostridium sp., 14 (VKM B-2201), 42 (VKM B-2202), and 21 (VKM B-2279), two methanogens, Methanobacterium formicicum MH (VKM B-2198) and Methanosarcina mazei MM (VKM B-2199), and one sulfate-reducing bacterium, Desulfovibrio sp. SR1 (VKM B-2200), were isolated in pure cultures from an anaerobic microbial community capable of degrading p-toluene sulfonate. Strain 14 was able to degrade p-toluene sulfonate in the presence of yeast extract and bactotryptone and, like strain 42, to utilize p-toluene sulfonate as the sole sulfur source with the production of toluene. p-Toluene sulfonate stimulated the growth of Ms. mazei MM on acetate. The sulfate-reducing strain Desulfovibrio sp. SR1 utilized p-toluene sulfonate as an electron acceptor. The putative scheme of p-toluene sulfonate degradation by the anaerobic microbial community is discussed.     

Carbohydrate-containing cell wall polymers of some strains of the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> group

A comparative study of the structures of carbohydrate-containing cell wall polymers isolated from the strains of the Bacillus subtilis group was performed by means of chemical and NMR spectroscopic meth ods. Polymers of different structure were revealed, namely, 1,3-poly(glycerol phosphates) with β-glucopyranose in Bacillus subtilis strains VKM B-520, VKM B-723, and VKM B-763 (= VKM B-911); 1,5-poly(ribitol phosphate) with α-glucopyranose in B. subtilis strains VKM B-722 and VKM B-922 (the structure is reported for the first time); and simultaneously two polymers in B. subtilis VKM B-761, 1,5-poly(ribitol phosphate) with β-glucopyranose and the disaccharide 1-phosphate polymer with the following repeating unit: -6)-α-D-Galp-(1-P-4)-gB-D-GlcpNAc-(1-, in which the hydroxyls at C3 and C6 of glucosamine residues are partially O-acetylated (the structure is reported for the first time). Heterogeneity of the B. subtilis group is con firmed by variations in the structure and composition of the cell wall polymers. The cell surface polymers are useful for discrimination of closely related bacilli strains and are cell wall marker components that may be an indispensable element of the Bacillus subtilis group taxonomy along with the genomosystematic methods.     

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.     

Teichoic acids of three type strains of the Bacillus subtilis group, Bacillus mojavensis VKM B-2650, Bacillus amyloliquefaciens subsp. amyloliquefaciens VKM B-2582, and Bacillus sonorensis VKM B-2652

Cell walls of three type strains of the Bacillus subtilis group, Bacillus mojavensis VKM B-2650, Bacillus amyloliquefaciens subsp. amyloliquefaciens VKM B-2582, and Bacillus sonorensis VKM B-2652, are characterized by the individual set of teichoic acids. All strains contained 1,3-poly(glycerol phosphates), unsubstituted, acylated with D-alanine, and glycosylated. The latter differ in the nature of the monosaccharide residue. Teichoic acids of B. mojavensis VKM B-2650^T and B. amyloliquefaciens subsp. amyloliquefaciens VKM B-2582^T contained α-glucopyranose, while those of B. sonorensis VKM B-2652^T contained β-glucopyranose and N-acetyl-α-D-glucosamine. Moreover, cell walls of B. mojavensis VKM B-2650^T contained a teichoic acid of poly(glycosylglycerol phosphate) nature with the following structure of the repeating unit: -4)-α-D-α-D-GlcpNAc-(1 → 3)]-Glcp-(1 → 2)-sn-Gro-(3-P-. The type strains have been characterized according to the composition of cell wall sugars and polyols. Application of teichoic acids (set and structure) as chemotaxonomic characteristics is discussed for six type strains of the Bacillus subtilis group. Polymer structures were determined by chemical and NMR spectroscopic techniques.     

Characterization of Rhodococcus wratislaviensis,a New Gram-Positive Facultative Methylotroph,and Properties of Its C1 Metabolism

Microbiology - A facultative methylotroph, strain 2AzMo (VKM Ac-2782), was isolated from the coastal zone of the Sea of Azov. The cells of the isolate are aerobic gram-positive nonmotile rods....     

Biogeochemistry of methane and methanogenic archaea in permafrost

This study summarizes the findings of our research on the genesis of methane, its content and distribution in permafrost horizons of different age and origin. Supported by reliable data from a broad geographical sweep, these findings confirm the presence of methane in permanently frozen fine-grained sediments. In contrast to the omnipresence of carbon dioxide in permafrost, methane-containing horizons (up to 40.0 mL kg(-1)) alternate with strata free of methane. Discrete methane-containing horizons representing over tens of thousands of years are indicative of the absence of methane diffusion through the frozen layers. Along with the isotopic composition of CH(4) carbon (delta(13)C -64 per thousand to -99 per thousand), this confirms its biological origin and points to in situ formation of this biogenic gas. Using (14)C-labeled substrates, the possibility of methane formation within permafrost was experimentally shown, as confirmed by delta(13)C values. Extremely low values (near -99 per thousand) indicate that the process of CH(4) formation is accompanied by the substantial fractionation of carbon isotopes. For the first time, cultures of methane-forming archaea, Methanosarcina mazei strain JL01 VKM B-2370, Methanobacterium sp. strain M2 VKM B-2371 and Methanobacterium sp. strain MK4 VKM B-2440 from permafrost, were isolated and described.     

Novel laccase—producing ascomycetes

Screening of ascomycetes producing laccases during growth on agar medium or submerged cultivation in the presence of various natural sources of carbon and energy (grain crops and potato) was carried out. The conditions of submerged cultivation of the most active strains (Myrothecium roridum VKM F-3565, Stachybotrys cylindrospora VKM F-3049, and Ulocladium atrum VKM F-4302) were optimized for the purpose of increasing laccase activity. The pH-optima and substrate selectivity of laccases in the culture liquid of the strains in relation to ABTS and phenolic compounds (2,6-dimethoxyphenol, syringaldazine, ferulic acid, p-coumaryl alcohol, and coniferyl alcohol) were investigated. High laccase activity at neutral pH was shown for the culture liquids of M. roridum VKM F-3565 and S. cylindrospora VKM F-3049 strains that provides prospects for using laccases of these strains in various cell biotechnologies.     

Production of mycophenolic acid by fungi of the genus Penicillium link

Out of 36 strains of fungi of the genus Penicillium, some of which were isolated from ancient permafrost soils, 14 strains synthesized mycophenolic acid (MPA). Maximal (over 500 mg/l) accumulation of MPA in culture liquid was observed in P. brevicompactum strains (VKM F-457, VKM F-477, and VKM F-1150). This was the first study to detect MPA in representatives of the species P. rugulosum; in three strains of this species (VKM FW-665, VKM FW-717, and VKM FW-733), the level of MPA accumulation exceeded 300 mg/l. The time course of the synthesis of MPA by the P. rugulosum strain VKM FW-733 was studied. It was shown that the synthesis of this metabolite was dramatically intensified at the stationary growth phase (ten days).     

Down-regulation of <Emphasis Type="Italic">S</Emphasis>-adenosyl-<Emphasis Type="SmallCaps">l</Emphasis>-homocysteine hydrolase reveals a role of cytokinin in promoting transmethylation reactions

S-adenosyl-L: -homocysteine hydrolase (SAHH) is a key enzyme for maintenance of cellular transmethylation potential. Although a cytokinin-binding activity had been hypothesized for SAHH, the relation between cytokinin and transmethylation reactions has not been elucidated. Here we show that, of the two Arabidopsis thaliana SAHH genes, AtSAHH1 has a much higher expression level than AtSAHH2. A T-DNA insertion mutant of AtSAHH1 (sahh1-1) and the RNA interference (RNAi) plants (dsAtSAHH2) accumulated a higher level of cytokinins, exhibited phenotypic changes similar to those of cytokinin-overproducers, and their global DNA methylation status was reduced. On the other hand, cytokinins positively regulate the transmethylation pathway genes, including AtSAHH1, AtADK1 (for adenosine kinase), and this regulation involves the cytokinin activity. Furthermore, expression of three cytosine DNA methyltransferase genes examined was inducible by cytokinin treatment. Unlike adenine and adenosine which are SAHH inhibitors, the adenine-type cytokinins have no effect on SAHH activity at protein level. Changing of endogenous cytokinin levels by transgene expression resulted in alterations of DNA methylation status in the sahh1-1 background, suggesting that cytokinins promote DNA methylation, at least under transmethylation stringent conditions. These data demonstrate that the phytohormone cytokinin plays a role in promoting transmethylation reactions, including DNA methylation.