N. V. Doronina

Methyloferula stellata gen. nov., sp. nov., an acidophilic, obligately methanotrophic bacterium that possesses only a soluble methane monooxygenase.

Two strains of aerobic methanotrophic bacteria, AR4(T) and SOP9, were isolated from acidic (pH 3.8-4.0) Sphagnum peat bogs in Russia. Another phenotypically similar isolate, strain LAY, was obtained from an acidic (pH 4.0) forest soil in Germany. Cells of these strains were Gram-negative, non-pigmented, non-motile, thin rods that multiplied by irregular cell division and formed rosettes or amorphous cell conglomerates. Similar to Methylocella species, strains AR4(T), SOP9 and LAY possessed only a soluble form of methane monooxygenase (sMMO) and lacked intracytoplasmic membranes. Growth occurred only on methane and methanol; the latter was the preferred growth substrate. mRNA transcripts of sMMO were detectable in cells when either methane or both methane and methanol were available. Carbon was assimilated via the serine and ribulose-bisphosphate (RuBP) pathways; nitrogen was fixed via an oxygen-sensitive nitrogenase. Strains AR4(T), SOP9 and LAY were moderately acidophilic, mesophilic organisms capable of growth between pH 3.5 and 7.2 (optimum pH 4.8-5.2) and at 4-33 °C (optimum 20-23 °C). The major cellular fatty acid was 18 : 1ω7c and the quinone was Q-10. The DNA G+C content was 55.6-57.5 mol%. The isolates belonged to the family Beijerinckiaceae of the class Alphaproteobacteria and were most closely related to the sMMO-possessing methanotrophs of the genus Methylocella (96.4-97.0 % 16S rRNA gene sequence similarity), particulate MMO (pMMO)-possessing methanotrophs of the genus Methylocapsa (96.1-97.0 %), facultative methylotrophs of the genus Methylovirgula (96.1-96.3 %) and non-methanotrophic organotrophs of the genus Beijerinckia (96.5-97.0 %). Phenotypically, strains AR4(T), SOP9 and LAY were most similar to Methylocella species, but differed from members of this genus by cell morphology, greater tolerance of low pH, detectable activities of RuBP pathway enzymes and inability to grow on multicarbon compounds. Therefore, we propose a novel genus and species, Methyloferula stellata gen. nov., sp. nov., to accommodate strains AR4(T), SOP9 and LAY. Strain AR4(T) ( = DSM 22108(T)  = LMG 25277(T)  = VKM B-2543(T)) is the type strain of Methyloferula stellata.

Aerobic Methylobacteria Are Capable of Synthesizing Auxins

Obligately and facultatively methylotrophic bacteria with different pathways of C1 metabolism were found to be able to produce auxins, particularly indole-3-acetic acid (IAA), in amounts of 3–100 μg/ml. Indole-3-pyruvic acid and indole-3-acetamide were detected only in methylobacteria with the serine pathway of C1 metabolism (Methylobacterium mesophilicumand Aminobacter aminovorans).The production of auxins by methylobacteria was stimulated by the addition of L-tryptophan to the growth medium and was inhibited by ammonium ions. The methylobacteria under study lacked tryptophan decarboxylase and tryptophan side-chain oxidase. At the same time, they were found to contain several aminotransferases. IAA is presumably synthesized by methylobacteria through indole-3-pyruvic acid.

Aerobic methylotrophic bacteria as phytosymbionts

This paper deals with the physiological, biochemical, and molecular genetic aspects of the interaction of aerobic methylotrophic bacteria with plants by means of phytohormones (such as cytokinins and auxins) and other physiologically active substances (vitamins, exopolysaccharides, bioprotectants, and others). An overview of the field and the prospects of research in the field of bacteria–plant interactions and the application of aerobic methylotrophs in plant biotechnology is discussed.

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).

Hyphomicrobium chloromethanicum sp. nov. and Methylobacterium chloromethanicum sp. nov., chloromethane-utilizing bacteria isolated from a polluted environment.

Two chloromethane-utilizing facultatively methylotrophic bacteria, strains CM2T and CM4T, were isolated from soil at a petrochemical factory. On the basis of their morphological, physiological and genotypical properties, strain CM2T (= VKM B-2176T = NCIMB 13687T) is proposed as a new species of the genus Hyphomicrobium, Hyphomicrobium chloromethanicum, and strain CM4T (= VKM B-2223T = NCIMB 13688T) as a new species of the genus Methylobacterium, Methylobacterium chloromethanicum.

Facultative and obligate aerobic methylobacteria synthesize cytokinins

The presence and expression of genes controlling the synthesis and secretion of cytokinins by the pink-pigmented facultative methylotroph Methylobacterium mesophilicum VKM B-2143 with the serine pathway and nonpigmented obligate methylotroph Methylovorus mays VKM B-2221 with the ribulose monophosphate pathway of C1 metabolism were shown using the polymerase chain reaction (PCR) and reverse transcription–PCR methods. The presence of the corresponding mRNA in M. mesophilicum cells grown on methanol or succinate suggests that the expression of these genes is constitutive. The cytokinin activity of culture liquid and its fractions was determined by a biotest with Amarantus caudatus L. seedlings. Using enzyme-linked immunosorbent analysis, we detected zeatin (riboside) in the culture liquid of both bacteria studied. The data obtained show that the aerobic methylobacteria are phytosymbionts that are able to utilize the single- and polycarbon compounds secreted by symbiotic plants and to synthesize cytokinins.

Methylarcula marina gen. nov., sp. nov. and Methylarcula terricola sp. nov.: novel aerobic, moderately halophilic, facultatively methylotrophic bacteria from coastal saline environments.

A new genus, Methylarcula, with two new species, Methylarcula marina and Methylarcula terricola, are proposed for strains h1T and h37T of moderately halophilic facultatively methylotrophic bacteria isolated from the coastal saline habitats. These methylobacteria are aerobic, Gram-negative, asporogenous, non-motile, colourless rods that multiply by binary fission. Their cellular fatty acids profiles consist primarily of straight-chain unsaturated (C18:1; 70-80%), saturated (C18:0; 14-16%) and cyclopropane (C19:0; 5-6%) acids. The major ubiquinone is Q-10. The dominant phospholipids are phosphatidylethanolamine and phosphatidylcholine. Both strains could use methylamine, some sugars and organic acids as carbon and energy sources. They grew well under optimal conditions (29-35 degrees C, pH 7.5-8.5, 0.5-1.0 M NaCl) and accumulated intracellularly poly-beta-hydroxybutyrate and the compatible solute ectoine. The ectoine pool was found to increase upon increasing the external NaCl concentration and accounted for 18% of the dry cellular weight. Both strains oxidized methylamine by the N-methylglutamate (N-MG) pathway enzymes (gamma-glutamylmethylamide synthetase/lyase and N-MG synthetase/lyase) to formaldehyde and assimilated it via the icl- serine pathway. The DNA G+C content was 60-4 mol% for Methylarcula marina h1T and 57.1 mol% for Methylarcula terricola h37T. The DNA-DNA hybridization value between strains hl and h37 was 25-30%, although they had a low level of DNA relatedness (5-7%) with the type strains of the serine pathway methylobacteria belonging to the genera Methylobacterium, Aminobacter, Methylorhabdus and Methylopila. A comparative 16S rDNA sequence-based phylogenetic analysis placed the two species of Methylarcula into a separate branch of the alpha-3 subclass of the Proteobacteria. The type strains of the new species are Methylarcula marina h1T (= VKM B-2159T) and Methylarcula terricola h37T (= VKM B-2160T).

Isolation and Characterization of a New Facultatively Methylotrophic Bacterium: Description of Methylorhabdus multivorans, gen. nov., sp. nov.

Summary A new Gram-negative aerobic, asporogenous, nonmotile, colourless, facultatively methylotrophic bacterium with the serine pathway of C1 assimilation is described. The organism (strain DM13) is a short rod multiplying by fission with the formation of a constriction connecting freshly divided cells. The G+C content of DNA is 66,2 mol %. The major cellular fatty acids are cis-vaccenic, lactobacillic and palmitic acid. The major ubiquinone is Q-10. The bacterium utilizes dichloromethane, methanol and methylamine as C1 sources of carbon and energy as well as a wide range of polycarbon compounds. It shows a low degree of DNA-DNA similarity ( Pseudomonas, Methylobacterium, Methylomicrobium, Hyphomicrobium , and Aminobacter . Based on morphological, pheno- and genotypic distinctions, the isolate is proposed to represent a new genus — Methylorhabdus with the type strain Methylorhabdus multivorans gen. nov., sp. nov. The characteristics of the new genus and species are presented.

Methylopila capsulata gen. nov., sp. nov., a novel non-pigmented aerobic facultatively methylotrophic bacterium

A new genus, Methylopila, and one new species are described for a group of seven strains of facultatively methylotrophic bacteria with the serine pathway of C1 assimilation. These bacteria are aerobic, Gram-negative, non-spore--forming, motile, colourless rods that multiply by binary fission. Their DNA base content ranges from 66 to 70 mol % G + C. Their cellular fatty acid profile consists primarily of C18:1 omega 7 cis-vaccenic and C19:0 cyclopropane acids. The major hydroxy acid is 3-OH C14:0. The main ubiquinone is Q-10. The dominant cellular phospholipids are phosphatidylethanolamine and phosphatidylcholine. The new isolates have a low level of DNA-DNA homology (5-10%) with the type strains of the serine pathway methylobacteria belonging to the genera Methylobacterium, Aminobacter, Hyphomicrobium and Methylorhabdus. Another approach, involving 16S rRNA gene sequence analysis of strain IM1T, has shown that the new isolates represent a separate branch within the alpha-2 subclass of the Proteobacteria. The type species of the new genus is Methylopila capsulata sp. nov., with the type strain IM1T (= VKM B-1606T).

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).