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Том 87, № 3 (2018)

Experimental Articles

Diversity of the alkB Genes of n-Alkane Biodegradation in Thermophilic Hydrocarbon-Oxidizing Bacteria of the Genera Geobacillus, Parageobacillus, and Aeribacillus

Tourova T., Sokolova D., Semenova E., Poltaraus A., Nazina T.

Аннотация

Analysis of complete genomes of thermophilic bacteria of the genus Geobacillus, oxidizing n-alkanes and crude oil revealed the ladA gene and its homologues. In the genomes of some strains, the ladA gene was not detected, although they were capable of growth on n-alkanes. Cloning with degenerate primers has previously revealed eight homologues of the alkB gene (alkB-geo1–alkB-geo8) encoding alkane hydroxylases in Geobacillus. In the present work, investigation of the alkB genes of several new strains of thermophilic, hydrocarbon-oxidizing bacilli was carried out. In the clone libraries obtained using degenerate primers for the alkB genes from five strains of the genera Geobacillus, Parageobacillus, and Aeribacillus, mostly the universal homologous genes alkB-geo1 and alkB-geo4 were revealed. Additional PCR amplification with specific primers for each of the eight known alkB homologues revealed the universal homologous genes only in some of the studied strains. A correlation was found between the set of the alkB-geo3alkB-geo6 homologus genes from Geobacillus subterraneus strain K amplified with specific primers and the previously revealed a set of these homologous genes transcripted at different cultivation conditions. This correlation may be due to accumulation of the copies of individual homologues under different cultivation conditions, which results in higher sensitivity of specific primers. The least common homologue, alkB-geo7, which was not detected in the relevant clone libraries, was found in two strains, indicating the possibility of development of more specific primers for amplification of this homologue in order to reveal hydrocarbon-oxidizing bacteria of related genera Geobacillus–Parageobacillus in molecular ecological research.

Microbiology. 2018;87(3):301-307
pages 301-307 views

Laccase Production and Humic Acids Decomposition by Microscopic Soil Fungi

Zavarzina A., Semenova T., Belova O., Lisov A., Leontievskii A., Ivanova A.

Аннотация

Laccase (para-diphenol:oxygen oxidoreductase, EC 1.10.3.2) is a phenol oxidase widespread in fungi and bacteria. In basidiomycetes, this enzyme is involved in the transformation of lignin and humic substances (HS) in soil. The role of laccases of soil ascomycetes and deuteromycetes in HS degradation is not established, and conditions of the enzyme production have been poorly studied. In the present work soil micromycetes, potential laccase producers, were isolated from typical soils of the forest, steppe, and foreststeppe zones of European Russia by plating on agar media with ABTS (2,2'-azino-bis(3-ethylbenzothiazoline- 6-sulphonic acid, sodium salt)) as the substrate. Their abundance, species composition, conditions of laccase production, and its relation to humic acids (HA) degradation in liquid and solid media were studied. Out of 68 strains isolated, 20 exhibited ABTS oxidation at initial plating on agar media. In pure cultures on agar media, oxidation was less pronounced, but in the presence of HA laccase production by some strains was higher than without HA. Significant and weak extracellular laccase production in liquid medium was observed for Acremonium murorum (Corda) W. Gams Z1710 and Botritis cinerea Pers. ex Fries Z1711, respectively. The level of laccase production by A. murorum was the same without inducers and in the presence of HA, while B. cinerea produced laccase only without inducers. No direct correlation was found between the presence of laccase and/or its activity and ability of the fungi to decolorize (degrade) HA. In liquid media active laccase producer A. murorum caused lower HA decolorization (43%) than B. cinerea (62%) and the fungi lacking extracellular laccase (54–81%). The role of micromycete oxidative systems in HA degradation requires further investigation.

Microbiology. 2018;87(3):308-316
pages 308-316 views

Anaerobic Methane Oxidation in Enrichment Cultures from Deep Sediments of a Mud Volcano Peschanka (South Baikal)

Lomakina A., Mamaeva E., Pogodaeva T., Kalmychkov G., Khal’zov I., Zemskaya T.

Аннотация

Under laboratory conditions, the microbial communities of bottom sediments of a mud volcano Peschanka (Lake Baikal) were found to carry out anaerobic methane oxidation (AOM). After 16 days of anaerobic cultivation of the enrichment cultures, methane content in the gas phase decreased, and microbial consortia were established. The content of carbon, nitrogen, and oxygen determined by energy dispersive X-ray spectroscopy (EDS) was higher than in the nearby sediment particles. The presence of bacteria of the NC10 phylum and archaea of the ANME-2d cluster was established by fluorescent in situ hybridization (FISH).

Microbiology. 2018;87(3):317-325
pages 317-325 views

Physiological and Morphological Characteristics of Acidophilic Bacteria Leptospirillum ferriphilum and Acidithiobacillus thiooxidans, Members of a Chemolithotrophic Microbial Consortium

Panyushkina A., Tsaplina I., Kondrat’eva T., Belyi A., Bulaev A.

Аннотация

A thermoacidophilic consortium of chemolithotrophic microorganisms oxidizing the concentrate of high-pyrrhotite pyrite−arsenopyrite ore at 38–40°C was isolated. The most active members of the consortium were identified as Leptospirillum ferriphilum, Acidithiobacillus thiooxidans, Ferroplasma acidiphilum, and Sulfobacillus thermotolerans. Leptospirillum and Thiobacillus species were the most numerous members of the consortium and had the highest activity of ferrous iron and sulfur oxidation, respectively. The optimal temperature values for the growth of both isolates were within 35–38°C. The optimal ranges of initial pH were 1.0–1.2 and 1.75–1.85 for leptospirilla and 1.7–3.3 for thiobacilli with the pH optimum of 1.9. Significant polymorphism and specific cyclic growth with formation of vibrios, spirilla, rods with different end shape, spiral filaments, numerous “pseudococci,” and densely packed spiral filaments surrounded by a sheath were revealed for the Leptospirillum isolate. Two latter morphoforms of L. ferriphilum were not previously described. Differences in ability of the morphoforms to oxidize Fe2+ were revealed. For the first time, the possibility of growth in the presence of organic substances was demonstrated for A. thiooxidans. The rates of growth and substrate oxidation, cell size, and the maximal cell yield decreased insignificantly in comparison with the lithoautotrophic strain.

Microbiology. 2018;87(3):326-338
pages 326-338 views

Cytophysiological Characteristics of the Vegetative and Dormant Cells of Stenotrophomonas sp. Strain FM3, a Bacterium Isolated from the Skin of a Xenopus laevis Frog

Suzina N., Ross D., Shorokhova A., Abashina T., Polivtseva V., Esikova T., Machulin A., Mulyukin A., Duda V.

Аннотация

A bacterial strain (FM3) that is closely related to Stenotrophomonas acidaminiphila and S. maltophilia, was isolated from the skin surface of the frog Xenopus laevis. Cytophysiological studies on vegetative cells and cyst-like cells (CLCs) that were obtained in model experiments addressed the dynamics of transition of vegetative cells to the dormant state and their reversion to vegetative growth. The ultrastructural organization of the vegetative and dormant cells of strain FM3 possesses unique properties. Cultures that developed after inoculating vegetative cells were characterized by: (1) resistance to physical factors and sterilization procedures; (2) high antimicrobial activity with respect to some gram-positive and gram-negative bacteria; (3) resistance to polypeptide antibiotics; (4) the presence of an easily detaching S-layer on the cell surface; (5) the ability to secret outer membrane vesicles into the intercellular space; and (6) formation of S-layerderived tubular structures associated with outer membrane vesicles that are regularly arranged within the tubes. Dormant cells were characterized by: (1) resistance to dehydration; (2) resistance to high temperatures; and (3) the preservation of the S-layer on the surface of cystlike cells (CLCs). Depending on experimental conditions, strain FM3 formed three CLC morphotypes, which differed in their abundance and ultrastructural organization. The experimental conditions used for CLC formation approximated those under which bacteria survive in hospitals. A model of intermicrobial parasitism is suggested that applies to motile FM3 cells during the development of their populations (cultures).

Microbiology. 2018;87(3):339-349
pages 339-349 views

Probiotic Evaluation of Antimicrobial Lactobacillus plantarum VJC38 Isolated from the Crop of Broiler Chicken

Nallala V., Jeevaratnam K.

Аннотация

The aim of this study was to evaluate probiotic properties of antimicrobial Lactobacillus plantarum VJC38 in vitro. L. plantarum VJC38 was isolated from the crop of broiler chicken and characterized using dnaK gene sequence. The inhibitory activities of L. plantarum VJC38 against bacterial and fungal pathogens were evaluated. Antifungal compounds secreted by the strain VJC38 were identified using Gas Chromatography and Mass Spectrometry (GC-MS). The strain was evaluated for its tolerance to low pH, resistance to bile salts, auto-aggregation, co-aggregation with pathogenic Escherichia coli, cell surface hydrophobicity, cholesterol lowering activity, β-galactosidase production, adhesion ability to Caco-2 cells, mucin degradation, hemolytic activity and biogenic amine production. Phylogenetic analysis of dnaK gene of bacterial strain VJC38 showed 99% sequence similarity to Lactobacillus plantarum var. plantarum. It showed effective inhibition against food spoiling and pathogenic organisms like Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Aspergillus niger, Penicillium expansum and Eurotium species. The antifungal compound phenol- 2,4-bis(1,1-dimethylethyl) (PD) was identified in the culture filtrate of L. plantarum VJC38 and reported to have inhibition against Aspergillus species. L. plantarum VJC38 exhibited tolerance to low pH, resistance to bile salts, bile salt hydrolase activity, auto-aggregation (87.5%), co-aggregation with Escherichia coli (55.7%), cholesterol lowering activity (64%), β-galactosidase production (1206 MU), adherence to Caco-2 cells (11%), negative for mucin degradation, hemolytic activity and biogenic amine production. L. plantarum VJC38 could be a good candidate for further investigation in vivo to elucidate its health benefits and to evaluate its technological properties as a bio-protective strain.

Microbiology. 2018;87(3):350-362
pages 350-362 views

Isolation of Two Radiation Resistant and Desiccation Tolerant Bacteria, Modestobacter sp. A2 and Maritalea sp. B9, from Gandom Beryan Hill in the Lut Desert of Iran

Shirsalimian M., Akhavan Sepahy A., Amoozegar M., Kalantar S., Dabbagh R.

Аннотация

Although dryland ecosystems are the most abundant terrestrial biomes on the Earth, relatively little is known about their microbial diversity and potential metabolic activities. Therefore, the bacterial diversity of the Lut Desert in Iran has been remained largely obscure. In this study, ionizing radiation resistant bacteria from arid Gandom Beryan region was investigated by a culture-dependent method. After exposing the soil and surface sand samples to different periods of dehydration in a desiccator containing silica gel, two nonendospore- forming bacterial isolates were recovered by plating on R2A and TSA agar media and then subjected to a desiccation and ionizing radiation resistance assay. The isolates A2 and B9 were still recovered after 8 weeks in a desiccator containing silica gel and were moderately resistant to gamma radiation with a D10 value between 2 and 4 kGy. Strains A2 and B9 were affiliated with Modestobacter muralis MDVD1T (99.7% similarity) and Maritalea mobilis E6T (97.3% similarity) respectively, using 16S rRNA gene sequence analysis. This is the first report of radiation resistant bacteria which belongs to the genus Maritalea.

Microbiology. 2018;87(3):363-371
pages 363-371 views

Phylogenetic Diversity of the Sulfur Cycle Bacteria in the Bottom Sediments of the Chersonesus Bay

Bryukhanov A., Vlasova M., Malakhova T., Perevalova A., Pimenov N.

Аннотация

The Black Sea is the largest meromictic basin, in the bottom sediments of which a powerful biogenic process of sulfide production occurs. The goal of the present work was to obtain data on phylogenetic diversity of the sulfur cycle microorganisms (sulfate-reducing and sulfur-oxidizing bacteria) in the Black Sea coastal gas-saturated bottom sediments. The samples were collected in the Chersonesus (Blue) Bay near Sevastopol from whitish bacterial mats of sulfurettes, and from the upper layer of the nearby seabed. Using DNA isolated from the native samples and obtained enrichment cultures, PCR analysis was performed with oligonucleotide primers specific to the fragments of the 16S rRNA genes of the main subgroups of sulfatereducing bacteria (SRB) and to the fragments of the dsrB gene (both reductive and oxidative types), encoding the β-subunit of dissimilatory (bi)sulfite reductase, the key enzyme in the sulfur cycle, inherent in both sulfate- reducing and sulfur-oxidizing microorganisms. The presence of 16S rRNA gene fragments specific to the genera Desulfobacterium, Desulfobacter, Desulfococcus–Desulfonema–Desulfosarcina, and Desulfovibrio–Desulfomicrobium was detected in the DNA samples isolated from coastal bottom bacterial mats. Usage of denaturing gradient gel electrophoresis (DGGE) with subsequent sequencing of reamplified dsrB gene fragments revealed that according to deduced amino acid sequences encoded by the dsrB gene (reductive type), SRB from the coastal gas-saturated bottom sediments of the Black Sea had the highest homology (92−99%) with the dsrB gene of cultured SRB belonging to the genera Desulfovibrio, Desulfatitalea, Desulfobacter, and Desulfobacterium, as well as with uncultured SRB strains from various marine habitats, such as bottom sediments of the Northern and Japanese seas. Deduced amino acid sequences encoded by the oxidative dsrB gene had the highest homology (90−99%) with the relevant sequences of the genera Thiocapsa, Thiobaca, Thioflavicoccus, and Thiorhodococcus.

Microbiology. 2018;87(3):372-381
pages 372-381 views

Composition of Microbial Communities in Sediments from Southern Baikal Containing Fe/Mn Concretions

Zemskaya T., Lomakina A., Mamaeva E., Zakharenko A., Likhoshvai A., Galach’yants Y., Müller B.

Аннотация

Massive parallel sequencing of the 16S rRNA gene fragments was used to investigate the composition and diversity of microbial communities in sediments from Southern Baikal to a depth of 9 cm with 1-cm step. In the layers from the sediment surface to the lower border of oxygen penetration (2 cm), organotrophic bacteria with high similarity to the heterotrophic species Luteolibacter luojiensis constituted the largest fraction of the community. In the formation zone of Fe/Mn crusts (3–5 cm), Proteobacteria and Actinobacteria predominated in the community, while the share of Cyanobacteria was considerable. The lower reduced layers showed an increased contribution of the Bacteroidetes, while the shares of the taxa predominant in the higher layers remained significant. Analysis of archaeal 16S rRNA gene amplicons revealed predominance of the soil and aquatic Thaumarchaeota (Marine Group I lineage), which are involved in anaerobic ammonium oxidation, practically in all sediment layers. The buried oxidized layer (6–7 cm), where members of the uncultured Marine Benthic Group D lineage of the order Thermoplasmatales (Euryarchaeota) predominated, was an exception in this regard. Small numbers of archaea of the Baikal-1 lineages (below 1%) were observed in the communities from the 6–7 and 7–8 cm layers, while the archaea involved in anaerobic methane oxidation (including the ANME-2d group) were not detected.

Microbiology. 2018;87(3):382-392
pages 382-392 views

Taxonomic Attribution of “Oscillatoriales” Strains within the Bacteriological System of Cyanobacteria: Identification Algorithm for Operational Genera

Velichko N., Emelijanova M., Averina S., Pinevich A., Pinevich A.

Аннотация

In “Oscillatoriales” cyanobacteria (Cyanophyceae), relatively simple and uniform morphology superimposes on high genetic diversity that impedes reliable identification. The system of Cyanobacteria set forth in Bergey’s Manual of Systematic Bacteriology-2001/Systematics of Archaea and Bacteria-2015 deals with operational taxa—form-genera (“larger” genera represented by strains) unlike true cyanophycean genera represented by species. Form-genera were established on morphological criteria shared with Cyanophyceae, although they were typified by Pasteur Culture Collection (PCC) strains. Despite being important in determinative cyanobacteriology, old diagnoses of form-genera should be reappraised because, in them: (i) vague and/or ephemeral morphological characters are considered taxonomically significant; (ii) phylogenetic character, such as 16S rRNA gene sequence (16S) is missing. We identified 32 “Oscillatoriales” strains from CALU collection (St. Petersburg University, Russia) basing on core morphology traits, 16S of PCC type strains, and 16S from GenBank database. We proposed that, in experimentally oriented and ecology oriented studies, unequivocal identification can be attained via triple match: streamlined form-genus diagnosis— 16S of PCC reference strain—GenBank most similar 16S. Additionally, we traced the phylogeny of “Oscillatoriales” form-genera via 16S clustering and HIP1 fingerprinting, and suggested that these operational taxa should be replaced with monophyletic assemblages. Nucleotide sequence data reported are available in the GenBank database under the accession numbers KX263921−KX263950.

Microbiology. 2018;87(3):393-406
pages 393-406 views

Yeast Communities of the Moscow City Soils

Tepeeva A., Glushakova A., Kachalkin A.

Аннотация

Yeast abundance and diversity were studied in the soils (topsoil) of Moscow city: urban soils under lawn vegetation and close to the areas of household waste disposal, as well as in zonal soddy-podzolic soils (retisols) in parks (Losiny Ostrov and Izmailovo). The numbers of soil yeasts were similar in all studied urban biocenoses (on average ~3.5 × 103 CFU/g). From all studied soils, 54 yeast species were isolated. The highest yeast diversity was found in the soils adjacent to the areas of household waste storage. Soils from different urban sites were found to have different ratios of ascomycetous and basidiomycetous yeasts: basidiomycetes predominated in urban soils under lawn vegetation, while in the areas close to the waste disposal sites their share was considerably lower. The differences between the studied urban soils were also found in the structure of soil yeast complexes. In urban soils with high anthropogenic impact, the isolation frequency of clinically important yeast species (Candida parapsilosis, C. tropicalis, Diutina catenulata, and Pichia kudriavzevii) was as high as 35% of all studied samples, while its share in the community was 17%. The factors responsible for development of specific features of yeast communities in various urban soils are discussed in the paper.

Microbiology. 2018;87(3):407-415
pages 407-415 views

Phylogenetic Diversity of Microorganisms from the Sludge of a Biogas Reactor Processing Oil-Containing and Municipal Waste

Nazina T., Sokolova D., Babich T., Semenova E., Borzenkov I., Bidzhieva S., Merkel A., Khisametdinov M., Tourova T.

Аннотация

High-throughput sequencing of the 16S rRNA gene fragments was used to determine the phylogenetic diversity of prokaryotes, including human pathogens, in the liquid phase of the sludge of a biogas reactor processing oil-containing and municipal waste. A unique microbial community was found to develop in the sludge, which comprised the microorganisms of municipal wastewater (bacteria of human feces) and specific groups of aerobic and anaerobic microorganisms, which possibly arrived with oil-containing water. In the 16S rRNA gene library, the sequences of representatives of Firmicutes prevailed (54.9%), which belonged to anaerobic bacteria of the genera Gelria (26.6%), Syntrophomonas (6.0%), Lutispora (2.0%), and uncultured Clostridia (group MBA03, 11.1%). The Proteobacteria sequences (20.7%) belonged mostly to the metabolically diverse members of the genus Pseudomonas (13.8%). The phylum Bacteroidetes (7%) was represented by uncultured bacteria (VadinBC27 wastewater-sludge group), while members of the phylum Cloacimonetes were mainly syntrophic bacteria Candidatus Cloacamonas (7.5%). The sequences of bacteria commonly occurring in oilfields (Clostridia, Anaerolinea, Bacteroidetes, sulfate-reducing Deltaproteobacteria, members of the family Syntrophaceae, and of the genera Thauera, Pseudomonas, Dechloromonas, and Petrimonas) were revealed. No sequences of bacteria known to be pathogenic to humans were found. The cultured microorganisms were aerobic organotrophic and anaerobic fermenting, denitrifying, and methanogenic prokaryotes. Fermenting and methanogenic enrichments grew on a broad range of organic substrates (sucrose, glycerol, starch), producing volatile fatty acids (acetate, n-butyrate, and propionate), gases (Н2, СО2, and CH4), and decreasing pH of the medium from 7.0 to 4.5–5.0. The possible application of the biogas reactor sludge as a source of fermenting and methanogenic anaerobic prokaryotes, as well as of aerobic hydrocarbonoxidizing bacteria for oilfield introduction and for production of new preparations for enhanced oil recovery and for bioremediation of oil contamination is discussed.

Microbiology. 2018;87(3):416-424
pages 416-424 views

Community Structure of Rhizomicrobiomes in Four Medicinal Herbs and Its Implication on Growth Management

Shi S., Tian L., Ma L., Tian C.

Аннотация

Medicinal plants are the basic materials of traditional Chinese medicine. Soil characteristics and microbial contribution play important roles in the growth and product quality of medicinal plants, but the link between them in the rhizosphere of medicinal plants has been overlooked. Accordingly, Mentha haplocalyx, Perilla frutescens, Glycyrrhiza uralensis, and Astragalus membranaceus, four plants used in traditional Chinese medicines, were investigated in this study in order to elucidate bacterial and arbuscular mycorrhizal fungal (AMF) diversity in the rhizosphere and its possible association with soil quality. DGGE-based 16S rRNA and 18S rRNA gene sequencing results indicated that the diversity of both bacteria and AMF in Glycyrrhiza uralensis and Astragalus membranaceus was significantly higher than those in Mentha haplocalyx and Perilla frutescens, suggesting that medicinal plants have different preferences even under the same conditions. In addition, enzymatic activities and nutrition were enhanced in the rhizospheric soil of Mentha haplocalyx and Perilla frutescens, and the correlation among AMF diversity, soil enzymatic activities and nutrition was confirmed using RDA analysis. These results suggest the potential to grow medicinal plants with a reasonable rotation or intercrop in order to maintain long-term continuous soil development.

Microbiology. 2018;87(3):425-436
pages 425-436 views

Short Communications

Stimulation of Violacein Biosynthesis in Chromobacterium violaceum Biofilms in the Presence of Dimethyl Sulfoxide

Mart’yanov S., Letarov A., Ivanov P., Plakunov V.
Microbiology. 2018;87(3):437-440
pages 437-440 views

Erratum

Erratum to: “Bacterial Complexes of Khibiny Mountains Lichens Revealed in Cladonia uncialis, C. portentosa, Alectoria ochroleuca, and Nephroma arcticum” [Microbiology 87, 79 (2018)]

Pankratov T.

Аннотация

In section ACKNOWLEDGMENTS, project number should read 16-04-00966а.

Microbiology. 2018;87(3):441-441
pages 441-441 views

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