S. A. Bykova

A Study of Nucleotide Sequences of nifH Genes of Some Methanotrophic Bacteria

Using a previously developed primer system, nifH gene fragments 450 nucleotides long were amplified, cloned, and sequenced for representatives of nitrogen-fixing methanotrophic bacteria of the genera Methylococcus, Methylocystis, and Methylosinus. Fragments of nifH genes were also detected and sequenced in representatives of the genera Methylomonas and Methylobacter, which were not considered diazotrophs until recently. Phylogenetic analysis revealed the remoteness of nifH gene sequences of methanotroph types I and II. At the same time, a close relationship was found between nifH of type I methanotrophs and representatives of γ-proteobacteria and between nifH genes of type II methanotrophs and representatives of α-proteobacteria. The results obtained in this study are in good accordance with the data of phylogenetic analysis based on 16S rRNA sequence comparison with the only exception being Methylococcus capsulatus strains, whose nifH genes proved to be closely related to nifH genes of Methylocystis and Methylosinus representatives. Our findings extend the database of primary sequences of nifH genes and allow the contribution of methanotrophs to the process of nitrogen fixation to be estimated.

[Phenotypic features of Ferroplasma acidiphilum strains Yt and Y-2].

Earlier, we described a new family of mesophilic, strictly autotrophic Fe2+-oxidizing archaebacteria, Ferroplasmaceae, which belongs to the order Thermoplasmales and includes the genus Ferroplasma and the species F. acidiphilum (strain YT) [1]. The present work is concerned with a comparative study of phenotypic characteristics of the type strain YТ and a new strain, F. acidiphilum Y-2, isolated from dense pulps during oxidation of gold-containing arsenopyrite/pyrite concentrates from the Bakyrchikskoe (Kazakhstan) and Olimpiadinskoe (Krasnoyarsk krai) ore deposits, respectively. The G+C content of DNA from strains YT and Y-2 comprised 35.1 and 35.2 mol %, respectively; the level of DNA–DNA homology between the strains was 84%. Restriction profiles of chromosomal DNA from both strains exhibited a similarity coefficient of 0.87. Genotypic characteristics of these strains indicate their affiliation to the same species. The cells of both strains are polymorphic and lack cell walls. Strains of F. acidiphilum oxidized ferrous iron and pyrite as the sole source of energy and fixed carbon dioxide as the sole carbon source. The strains required yeast extract as a growth factor. Optimum pH for cell growth ranged from 1.7 to 1.8; the temperature optima for the growth of strains YT and Y-2 were 34–36 and 40–42°С, respectively. Comparative analysis of the total lipids revealed their close similarity in the strains; two glycophospholipids comprised 90% of the total lipids: lipid I, β-D-glucopyranosylcaldarchaetidylglycerol (about 55%), and lipid II, trihexosylcaldarchaetidylglycerol (26%), whose isopranyl chains contained no cyclopentane rings. The carbohydrate fraction of lipid I hydrolysate contained only D-glucose, whereas hydrolysate of lipid II contained both D-glucose and D-galactose in a molar ratio of 2 : 1. Thus, it was established that the intraspecies phylogenetic divergence within F. acidiphilum is manifested in the two strains by different temperature optima against a background of similarity in other phenotypic properties.

Molecular analysis of high-affinity methane-oxidizing enrichment cultures isolated from a forest biocenosis and agrocenoses

Methane oxidation by microorganisms inhabiting aerobic soils is a key process involved in the regulation of the concentration of this significant greenhouse gas in the atmosphere; however, the microorganisms responsible for this process remain unknown. Three stable methane-oxidizing cultures were isolated from samples of forest soils (FS) and agricultural soils (AS) of Moscow oblast, as well as from soil samples collected from a Belgian agrocenosis (BS). The obtained enrichment cultures exhibit a high affinity for methane; their km values range from 54.2 to 176.8 nM CH4 and are comparable to those of aerobic soils. Analysis of the fragments of the ribosomal (16S rRNA) and functional (pmoA) genes of methanotrophs by PCR— DGGE and cloning demonstrated the presence of bacteria belonging to the genera Methylocystis in FS, Methylosinus in AS and BS, and Methylocella in BS. It was established that Methylocystis and Methylosinus detected in the enrichment cultures contain the genes encoding the synthesis of the active center of two membrane-bound particulate methane monooxygenases; it is likely that one of these genes (pmoA2) is responsible for the capacity of these microorganisms for oxidation of atmospheric methane.

Seasonal dynamics of atmospheric methane oxidation in gray forest soils

Seasonal fluctuations in the methane fluxes in the soil–atmosphere system were determined for gray forest soils of Central Russia. Consumption of atmospheric methane was found to exceed methane emission in gray forest soils under forest and in the agrocenosis. The average annual rates of atmospheric methane consumption by the soil under forest and in the agrocenosis were 0.026 and 0.008 mg C-CH4/(m2 h), respectively. The annual rate of atmospheric methane oxidation in the gray forest soils of Moscow oblast was estimated to be 0.68 kton. Seasonal fluctuations in the methane oxidation activity were due to changes in the hydrothermal conditions and in the reserves of readily decomposable organic matter and mineral nitrogen, as well as to changes in the activity of methane oxidizers.

Physicochemical and Biological Factors Affecting Atmospheric Methane Oxidation in Gray Forest Soils

The decline of methane oxidizing activities in gray forest soil upon its conversion into arable land was shown to be caused by major changes in biotic and physicochemical properties of soil. Using the method of immune serums, methane-oxidizing bacteria were detected in both forest and agricultural soils, but their populations differed significantly in both abundance and composition. In the forest soil, the number of methanotrophs was an order of magnitude higher than in arable soil, amounting to 3.5 × 108 and 0.24 × 108 cells/g soil, respectively. All methane-oxidizing bacteria identified in the forest soil belonged to the genus Methylocystis, and 94% of these were represented by a single species, M. parvus. The arable soil was dominated by type I methanotrophs (Methylobacter and Methylomonas, 67.6%), occurring along with bacteria of the genus Methylocystis. In addition, arable soil is characterized by a low content of microbial biomass, lower porosity and water resistance of soil aggregates, and the predominance of nitrogen mineralization processes over those of nitrogen immobilization. These factors can also contribute to lower rates of methane oxidation in arable soil as compared to forest soil.

[Heliobacterium sulfidophilum sp. Nov. and Heliobacterium undosum sp. Nov.: sulfide-oxidizing Heliobacteria from thermal sulfidic springs].

Two new species of heliobacteria isolated from cyanobacterial mats of two alkaline sulfidic hot springs are formally described. Strains BR4 and BG29 are assigned to anoxygenic phototrophic bacteria of the familyHeliobacteriaceae, since they possess the unique properties of this taxon: strict anaerobiosis, formation of bacteriochlorophyllg, the lack of extensive intracytoplasmic membranes and chlorosomes, an unusual cell wall structure, and phylogenetic relatedness to the low G+C gram-positive eubacteria. Based on the 16S rDNA sequence similarity, strains BR4 and BG29 are assigned to the genusHeliobacterium and described as two new species of this genus:Heliobacterium sulfidophilum sp. nov. andHeliobacterium undosum sp. nov. The G+C content of the DNA is 51.3 mol % inHbt. sulfidophilum and 57.2-57.7 mol % inHbt. undosum. The cells ofHbt. sulfidophilum are rods, and the cells ofHbt. undosum are slightly twisted spirilla or short rods. Both new bacteria are motile by peritrichous flagella.Hbt. sulfidophilum produces endospores. The new bacteria are strict anaerobes growing photoheterotrophically on a limited range of organic compounds. In the dark, they can switch from photosynthesis to the slow fermentation of pyruvate. Biotin is required as a growth factor. Both species are highly tolerant to sulfide (up to 2 mM at pH 7.5) and oxidize it photoheterotrophically to elemental sulfur; photoautotrophic growth was not observed. The temperature optimal for growth ofHbt. sulfidophilum andHbt undosum is 30–35‡C, and the optimal pH is 7–8.

[Clusterization of halophilic and halotolerant eubacteria using whole-cell protein electrophoresis data].

Total cell proteins of the nineteen halophilic and halotolerant eubacteria isolated from marine sediments and highly mineralized formation waters of oil fields were investigated by SDS gel electrophoresis. The microorganisms studied, phenotypically identified as belonging to the generaDietzia, Rhodococcus, Staphylococcus, Cytophaga, Brevibacterium, andArchangium, were found to form clearly distinguishable clusters (20–33% similarity at the generic level) on the dendrogram derived from electrophoretic protein patterns. Protein similarity data confirmed the heterogeneity ofRhodococcus maris and its relatedness to the genusDietza.