Vol 86, No 6 (2017)

The review deals with systematization and generalization of new information concerning the phylogenetic and functional diversity of prokaryotes involved in the methane cycle. Methane is mostly produced by methanogenic archaea, which are responsible for the terminal stage of organic matter decomposition in a number of anoxic ecotopes. Although phylogeny, physiology, and biochemistry of methanogens have been extensively studied, important discoveries were made recently. Thus, members of deep phylogenetic lineages within the Euryarchaeota phylum (Methanomassiliicoccales, “Candidatus Methanofastidiosa,” “Methanonatronarchaeia”) and even outside it (“Ca. Verstraetearchaeota” and “Ca. Bathyarchaeota”) were reported to carry out methyl-reducing methanogenesis. Moreover, evidence was obtained on aerobic methane production by marine heterotrophic bacteria, which demethylate polysaccharide esters of methylphosphonic acid. Methanotrophic microorganisms oxidize methane both aerobically and anaerobically, decreasing significantly the release of this greenhouse gas into the atmosphere. In the presence of oxygen methane is oxidized by methanotrophic members of Alpha- and Gammaproteobacteria, as well as by Verrucomicrobia. Methanotrophic gammaproteobacteria have been recently revealed in hypoxic and even anoxic environments, where they probably oxidize methane either in a trophic consortium with oxygenic phototrophs and/or methylotrophs or using electron acceptors other than oxygen. Anaerobic methane oxidation has been known for a long time. Sulfat- and nitrate-dependent anaerobic methane oxidation carried out by the ANME archaea via reverse methanogenesis are the best studied processes. While metal-dependent anaerobic methane oxidation is considered possible, the mechanisms and agents responsible for this process have not been reliably identified. Intracellular oxygen production during nitrite-dependent anaerobic methane oxidation was shown for bacteria “Ca. Methylomirabilis oxyfera.” These findings stimulate interest in the processes and microorganisms of the methane cycle.

Protective effect of Saccharomyces cerevisiae exometabolites (RF₁ and RF₂) on seven strains of propionic acid bacteria under exposure to bile salts and acid stress was studied. RF₂ had a stronger protective effect than RF₁. Comparison of the analytical profiles of RF₁ and RF₂, separated by RP-HPLC, showed that nearly 80% of RF₁ components were present in RF₂. Separation of S. cerevisiae RF₁ into individual components by means of semi-preparative RP-HPLC was used for structural characterization of the active component of the RF₁ fraction. It was found to contain two pentapeptides (<1 kDa) with identical amino acid sequences (¹Gly-Pro-Tre-Gly-Pro⁵), differing in one hexose residue. The RF₂ separation method based on three-stage liquid chromatography at low and high pressure was developed, which involved analytical high-pressure gel chromatography. The use of the latter method made it possible to separate high-molecular components and perform enrichment of low-molecular peptides. Peptide origin of the RF₂ active substances with molecular masses allegedly below 300 Da was confirmed.

Compaction and biocrystallization of the nucleoid are presently considered as a necessary and important stage in the transformation of the cell ultrastructure during change of microbial cultures strategies from growth to survival. Nucleoid biocrystallization in the stationary phase cells is achieved due to structural regularity of the DNA complexes with the histone-like Dps protein. Our experiments with Escherichia coli mutants, overproducers of the Dps protein, confirmed nucleoid biocrystallization in the late stationary phase cells. Since nucleoid biocrystallization was revealed in E. сoli cells without Dps overproduction at late stages of starvation, it is constitutive in the cycle of development of microbial populations. The present work concentrated on detection of the nucleoid biocrystalline structure in (1) long-starved (21 day in the chemostat mode) bacterial cells (genera Arthrobacter and Pseudomonas), (2) dormant ametabolic (anabiotic) cells of such prokaryotes as archaea and non-spore-forming bacteria, (3) endospores of bacilli, (4) streptomycete exospores, and (5) in the cells surviving in permafrost for (2‒3 Ma). The topics discussed include nucleoid biocrystallization as a necessary stage of maturation of the dormant microbial cells providing for survival and preservation of the species, dynamics of nucleoid biocrystallization during maturation of the dormant cells, and its possible role for the preservation of genetic information in the case of autolysis of most of the cells in a developing culture.

A new obligately methylotrophic bacterium, strain OV^T, was isolated from roots of sedge (Carex sp.). The isolate was represented by aerobic gram-negative motile, non-spore-forming rods, which divided by binary fission. Optimal growth occurred at 22−29°C and pH 7.5−8.5 in the presence of 0.5−2% NaCl; growth was inhibited by 3.5% NaCl. Strain OV^T utilized methanol as the only carbon and energy source. The organism used the KDPG variant of the ribulose monophosphate pathway (RuMP) of С₁ metabolism. Ammonium was assimilated by reductive amination of α-ketoglutarate. The major cellular fatty acids were C_16:0 (45.5%), C_16:1ω7c (40.7%), and C_17cyc (8.0%). The major ubiquinone was Q₈. The dominant phospholipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The DNA G + C content of strain OV^T was 51.4 mol % (T_m). While the 16S rRNA gene sequence of strain OV^T exhibited high similarity to those of Methylobacillus species: M. gramineus Lap^T (99.6%) and M. glycogenes TK 0113^T (98.7%), the DNA-DNA hybridization level between strain OV^T and M. gramineus Lap^T was only 52%. Based on the data obtained, strain OV^T was assigned to the new species Methylobacillus caricis sp. nov. (=VKM B-3158^T = JCM 32031^T).

Two methomyl-degrading bacteria (initially named Disha A and Disha B) were isolated from a pesticide-treated crop field in Baruipur, 24 Parganas (South), West Bengal, India. Both strains could not grow in mineral salt (MS) medium but showed efficient growth in the presence of methomyl. The highest growth was observed in the MS medium containing 0.16% methomyl. When methomyl was supplemented with glucose, no further enhancement of growth was observed, whereas supplementation with yeast extract had a positive effect on growth of both strains, indicating that methomyl could be utilized as the sole source of carbon but not that of nitrogen. In Nutrient Broth and Luria Bertani medium, these strains could tolerate 0.4% methomyl. Optimum pH and temperature for growth of both bacteria in the methomyl-containing MS medium were 7.0 and 30°C, respectively. Protein concentration in the cell-free extracts of bacterial cultures was directly proportional to methomyl concentration in the medium. Disha A was more resistant to the antibiotics amoxicillin and penicillin, as indicated by minimum inhibitory concentration (600 and 500 μg/mL, respectively), which were higher than those obtained for Disha B (350 and 300 μg/mL, respectively). Both Disha A and Disha B were plasmid-bearing, gram-positive, endospore-producing, rod-shaped bacteria. Biochemical studies, 16S rDNA sequencing, and phylogenetic analysis indicated maximum similarity of Disha A to Bacillus cereus ATCC 14579, whereas Disha B showed maximum similarity to Bacillus safensis F0-36b ATCC BAA-1126. The HPLC analysis clearly indicated that B. cereus and B. safensis showed 88.25 and 77.5% of methomyl (Sigma) degradation, respectively within 96 h of growth. This is the first report of Bacillus species that can degrade the carbamate pesticide methomyl and thrive in presence of its high concentrations.

Physicochemical and microbiological characteristics of formation waters low-temperature heavy oil reservoirs (Russia) were investigated. The Chernoozerskoe, Yuzhno-Suncheleevskoe, and Severo-Bogemskoe oilfields, which were exploited without water-flooding, were shown to harbor scant microbial communities, while microbial numbers in the water-flooded strata of the Vostochno-Anzirskoe and Cheremukhovskoe oilfields was as high as 10⁶ cells/mL. The rates of sulfate reduction and methanogenesis were low, not exceeding 1982 ng S^2–/(L day) and 9045 nL СН₄/(L day), respectively, in the samples from water-flooded strata. High-throughput sequencing of microbial 16S rRNA gene fragments in the community of injection water revealed the sequences of the Proteobacteria (74.7%), including Betaproteobacteria (40.2%), Alphaproteobacteria (20.7%), Gammaproteobacteria (10.1%), Deltaproteobacteria (2.0%), and Epsilonproteobacteria (1.6%), as well as Firmicutes (7.9%), Bacteroidetes (4.1%), and Archaea (0.2%). DGGE analysis of microbial mcrA genes in the community of injection water revealed methanogens of the genera Methanothrix, Methanospirillum, Methanobacterium, Methanoregula, Methanosarcina, and Methanoculleus, as well as unidentified Thermoplasmata. Pure cultures of bacteria of the genera Rhodococcus, Pseudomonas, Gordonia, Cellulomonas, etc., capable of biosurfactant production when grown on heavy oil, were isolated. Enrichment cultures of fermentative bacteria producing significant amounts of volatile organic acids (acetic, propionic, and butyric) from sacchariferous substrates were obtained. These acids dissolve the carbonates of oil-bearing rock efficiently. Selection of the efficient microbial technology for enhanced recovery of heavy oil from terrigenous and carbonate strata requires model experiments with microbial isolates and the cores of oil-bearing rocks.

Lactobacillus salivarius belongs to the microbiota of human oral cavity and gastrointestinal tract, as well as of bird and pig intestines. Probiotic activity of various L. salivarius strains has been recently extensively investigated. Production of exopolysaccharides and formation of biofilms as a mechanism providing for resistance to unfavorable factors are also of interest. The goal of this work was to assess the efficiency of microbiological methods for analysis of bacterial concentrations in the cultures of L. salivarius strain NBR2. Samples of lactobacteria grown in liquid medium were collected at equal intervals. The parameters determined were the number of colony-forming units (CFU/mL), share of dead cells by the membrane permeabilization test (LIVE/DEAD) using flow cytometry and fluorescence microscopy, optical density at 595 nm, and pH. After 10 h of cultivation, formation of aggregates commenced, which consisted mainly of living cells and were detected throughout the experiment (30 h). This resulted in underestimation of bacterial abundance determined by plating (CFU/mL). Optical density of the culture and the share of dead cells determined by the LIVE/DEAD method are more reliable criteria of growth of the statically developing L. salivarius culture, which is prone to formation of biofilms and cell aggregates.