B. Kuznetsov

Three stable methane-oxidizing enrichment cultures, SB26, SB31, and SB31A were analyzed by transmission electron microscopy and by serological and molecular techniques. Electron microscopy revealed the presence of both type I and type II methanotrophs in SB31 and SB31A enrichments; only type II methanotrophs were found in SB26 enrichment. Methylosinus trichosporium was detected in all three enrichments by the application of species-specific antibodies. Additionally, Methylocystis echinoides was found in SB26 culture; Methylococcus capsulatus, in SB31 and SB31A; and Methylomonas methanica, in SB31. The analysis with pmoA and nifH gene sequences as phylogenetic markers revealed the presence of Methylosinus/Methylocystis group in all communities. Moreover, the analysis of pmoA sequences revealed the presence of Methylomonas in SB31. Methylocella was detected in SB31 and SB31A enrichments only by nifH analysis. It was concluded that the simultaneous application of different approaches r...

A thermophilic, anaerobic, spore-forming, dissimilatory Fe(III)-reducing bacterium, designated strain SR4T, was isolated from sediment of newly formed hydrothermal vents in the area of the eruption of Karymsky volcano on the Kamchatka peninsula. Cells of strain SR4T were straight-to-curved, peritrichous rods, 0.4-0.6 micron in diameter and 3.5-9.0 microns in length, and exhibited a slight tumbling motility. Strain SR4T formed round, refractile, heat-resistant endospores in terminally swollen sporangia. The temperature range for growth was 39-78 degrees C, with an optimum at 69-71 degrees C. The pH range for growth was 4.8-8.2, with an optimum at 6.3-6.5. Strain SR4T grew anaerobically with peptone as carbon source. Amorphous iron(III) oxide present in the medium stimulated the growth of strain SR4T; cell numbers increased with the concomitant accumulation of Fe(II). In the presence of Fe(III), strain SR4T grew on H2/CO2 and utilized molecular hydrogen. Strain SR4T reduced 9,10-anthr...

A new genus and species of bacteria capable of ammonium oxidation under anaerobic conditions in the presence of nitrite is described. The enrichment culture was obtained from the Moscow River silt by sequential cultivation in reactors with selective conditions for anaerobic ammonium oxidation. Bacterial cells were coccoid, -0.4 x 0.7 mm, with the intracellular membrane structures typical of bacteria capable of anaerobic ammonium oxidation (anammoxosome and paryphoplasm). The cells formed aggregates 5-25 μm in diameter (10 μm on average). They were readily adhered to solid surfaces. The cells were morphologically labile, they easily lost their content and changed their morphology during fixation for electron microscopy. The organism was capable of ammonium oxidation with nitrite. The semisaturation constants Ks for nitrite and ammonium were 0.38 mg N-NO2/L and 0.41 mg N-NH4/L, respectively. The maximal nitrite concentrations for growth were 90 and 75 mg N-NO2/L for single and continuous application, respectively. The doubling time was 32 days, μ(max) = 0.022 day(-1), the optimal temperature and pH were 20 degrees C and 7.8-8.3, respectively. According to the 16S rRNA gene sequencing, the bacterium was assigned to a new genus and species within the phylum Planctomycetes. The proposed name for the new bacterium is Candidatus Anammoximicrobium moscowii gen. nov., sp. nov. (a microorganisms carrying out anaerobia ammonium oxidation, isolated in the Moscow region).

A method for indirect DNA extraction from various soils significantly differing in their physicochemical properties has been developed. The proposed method is based on cell desorption from soil particles using a Tris-EDTA (TE) buffer supplemented with polyvinylpolypyrrolydone (PVPP) and sodium dodecylsulfate (SDS). Subsequent cell lysis and purification of DNA preparations methods based on alkaline lysis followed by chromatography on ion-exchange resins were described by us earlier. The purity of the DNA preparations obtained did not depend on the type of soil. It was shown that the DNA preparations can be used for the amplification of rather large fragments, e.g., sequences spanning the complete 16S rRNA gene.

Phylogeny of anoxygenic filamentous phototrophic bacteria (AFPB) of the family Oscillochloridaceae (Oscillochloris trichoides DG6T and the recently isolated strains Oscillochloris sp. R and C6) was studied based on comparative analyses of the genes coding for 16S rRNA (rrs), ribulose- 1,5-bisphosphate carboxylase/oxygenase (cbbL), and nitrogenase (nifH). The sequences of the genes studied proved to be identical in the three strains, which is in agreement with data obtained earlier that showed lack of differentiating phenotypic distinctions between these strains; therefore, it is proposed that the new strains should be identified as representatives of the species O. trichoides. Using an earlier designed system of oligonucleotide primers and a specially designed additional primer, fragments of the cbbL genes of the "red-like" form I RuBPC were amplified and sequenced for all of the O. trichoides strains. Analysis of the cbbL genes suggested a separate position of the bacteria studied in the phylogenetic tree, where O. trichoides strains formed an independent branch, which, apart from this species, also included the only studied species of gram-positive facultatively chemoautotrophic bacteria, Sulfobacillus acidophilus. In the phylogenetic tree inferred from the analysis of nifH genes, the bacteria under study also formed a new separate branch, deviating near the root, which indicated lack of relatedness between them and other phototrophic bacteria. The data obtained support the conclusion that AFPB has an ancient origin and their identification as one of the main evolutionary lineages of eubacteria, which was made based on the analysis of ribosomal genes.

The interspecies relationships between Bacillus thuringiensis strains producing different types of δ-endotoxins were studied using a range of molecular-biological methods. Analysis of the 16S rRNA nucleotide sequence, the 16S to 23S rRNA intergenic spacer sequence, and the 5′-terminal region of 23S rRNA allowed the studied strains to be subdivided into three groups based on the pattern of nucleotide substitutions. In

ABSTRACT A fraction of magnetotactic bacteria was isolated by magnetic separation from the water and silt samples collected from the Ol’khovka River (Kislovodsk, Russia). A 16S rRNA clone library was obtained from the total DNA of the fraction by PCR amplification and molecular cloning. Phylogenetic analysis of 67 16S rRNA gene sequences of randomly selected clones demonstrated that two phylotypes of magnetotactic bacteria were present in the library: the first phylotype consisted of 42 sequences and the second one included only one sequence. The remaining 24 sequences belonged to non-magnetotactic bacteria. According to the results of phylogenetic analysis, both phylotypes were magnetotactic cocci; the predominant sequences were almost identical to the 16S rRNA sequence of the freshwater coccus TB24 (X81185.1) identified earlier among the magnetotactic bacteria isolated from Lake Chiemsee (Bavaria). The phylotype represented by a single sequence formed a separate branch in the dendrogram, with 97% similarity between its sequence and that of TB24. The discovered phylotypes formed with the sequences of uncultured freshwater magnetotactic cocci a separate branch within the class Alphaproteobacteria and presumably belonged to a separate family within the recently described order Magnetococcales. Despite the fact that phylogenetic analysis of the 16S rRNA clone library did not reveal any phylotypes of magnetotactic spirilla, after the secondary enrichment of the fraction of magnetotactic bacteria using the “race track” technique, a new strain of magnetotactic spirilla, Magnetospirillum SO-1, was isolated. The closest relative of strain SO-1 was the previously described magnetotactic spirillum Magnetospirillum magneticum AMB-1.

ABSTRACT A recently developed PCR-fingerprinting method, the so-called DIR (diverged inverted repeats)-PCR, was used for quick search for molecular markers of Bacillus thuringiensis subspecies carrying the cry1 genes. The analysis of the fingerprints obtained with this method made it possible to reveal PCR fragments characteristic of the subspecies that produce proteins toxic for insects of the order Lepidoptera. Cloning and sequencing of these fragments allowed systems of SCAR (sequence characterized amplified region) primers to be designed, which are specific to the above group of B. thuringiensis strains. Comparison of the specific fragments with sequences available in the GenBank database revealed their homology with the rpoC gene family and the adjacent spacer region, suggesting chromosomal localization of these markers. This increases the reliability of the designed system of SCAR primers, because plasmids may be lost or transferred by transformation between closely related strains. It was demonstrated that the DIR-PCR method allows markers to be elaborated that are linked to diagnostic genotypic and phenotypic characteristics of bacteria. Key wordsbacilli–genomic fingerprints–PCR–SCAR primers

A thermophilic, anaerobic, spore-forming bacterium (strain Z-9801T) was isolated from a terrestrial hydrothermal source in the Uzon caldera on the Kamchatka peninsula. Cells of strain Z-9801T were straight, sometimes branched rods, 0.5-0.6 microm in diameter and 1.5-7.0 microm in length, with peritrichous flagella. The temperature range for growth was 45-76 degrees C, with an optimum at 63-65 degrees C. The pH range for growth was 4.8-8.2, with an optimum at 6.7-6.9. The substrates utilized by strain Z-9801T included peptone, yeast extract, beef extract, Casamino acids, starch, pyruvate, melibiose, sucrose, fructose, maltose, xylose and ribose. The fermentation products from melibiose were ethanol, acetate, H2 and CO2. Strain Z-9801T used H2 in the presence of Fe(III) and an organic electron donor. Strain Z-9801T reduced Fe(III), Mn(IV), nitrate, fumarate, sulfite, thiosulfate, elemental sulfur and 9,10-anthraquinone 2,6-disulfonate. The G+C content of strain Z-9801T DNA was 36 mol%. 16S rDNA sequence analysis revealed that the isolated organism forms a separate branch within the Bacillus/Clostridium group. On the basis of physiological properties and phylogenetic analysis, it is proposed that strain Z-9801T (= DSM 14006T = UNIQEM 210T) should be assigned to a novel species of a new genus, Thermovenabulum ferriorganovorum gen. nov., sp. nov.

A moderately thermophilic, organotrophic bacterium with vibrioid cells was isolated from a sample of a cyanobacterial mat from caldera Uzon, Kamchatka, Russia, and designated strain Z-9701T. Cells of strain Z-9701T were curved, Gram-negative rods, 0.5-0.7 x 2.5-5.0 microm in size, with tapering ends and with fast, wavy movement by means of lateral flagella located on the concave side of the cell. Colonies were small, white, irregular or round, 0.2 mm in diameter, and with even edges. Strain Z-9701T was an obligate anaerobe with a temperature optimum at 60-65 degrees C and a pH optimum at 7.3. It fermented glucose, fructose, mannose, N-acetyl-D-glucosamine, adonite, arginine, serine, peptone, yeast extract and Casamino acids. The fermentation products formed during growth on glucose were acetate, lactate, H2, CO2 and ethanol. Strain Z-9701T reduced elemental sulfur to H2S during organotrophic growth with glucose or peptides as energy and carbon sources. In the presence of S0, strain Z-9701T was capable of lithotrophic growth with molecular hydrogen as energy substrate and 0.1 g yeast extract l(-1) as carbon source. Sulfate, thiosulfate, nitrate, Fe(III) and sulfite were not reduced and did not stimulate growth. The G+C content of strain Z-9701T DNA was 54.6 mol%. The results of 16S rDNA sequence analyses revealed that strain Z-9701T belongs to the cluster within the Clostridium group formed by Thermanaerovibrio acidaminovorans, Dethiosulfovibrio peptidovorans, Anaerobaculum thermoterrenum and Aminobacterium colombiense, but the level of sequence similarity with the members of this cluster was not very high (87.6-92.2%). Among these organisms, Thermanaerovibrio acidaminovorans is phenotypically close to strain Z-9701T. However, the two organisms showed a relatively low level of similarity of their 16S rRNA sequences (92.2%) and of DNA-DNA hybridization (15 +/- 1%). Nevertheless, on the basis of the similar morphology and physiology of the new isolate and Thermanaerovibrio acidaminovorans, strain Z-9701T was placed in the genus Thermanaerovibrio and a new species, Thermanaerovibrio velox, proposed for it. The type strain is Z-9701T (= DSM 12556T).

An obligately anaerobic, hyperthermophilic, organoheterotrophic archaeon, strain Z-1312(T), was isolated from a freshwater hot spring of the Uzon caldera (Kamchatka Peninsula, Russia). The cells were regular cocci, 1-4 microm in diameter, with one long flagellum. The cell envelope was composed of a globular layer attached to the cytoplasmic membrane. The temperature range for growth was 63-89 degrees C, with an optimum between 80 and 82 degrees C. The pH range for growth at 80 degrees C was 4.8-6.8, with an optimum at pH 6.0. Strain Z-1312(T) grew by hydrolysis and/or fermentation of a wide range of polymeric and monomeric substrates, including agarose, amygdalin, arabinose, arbutin, casein hydrolysate, cellulose (filter paper, microcrystalline cellulose, carboxymethyl cellulose), dextran, dulcitol, fructose, lactose, laminarin, lichenan, maltose, pectin, peptone, ribose, starch and sucrose. No growth was detected on glucose, xylose, mannitol or sorbitol. Growth products when sucrose or starch were used as the substrate were acetate, H(2) and CO(2). Elemental sulfur, thiosulfate and nitrate added as potential electron acceptors for anaerobic respiration did not stimulate growth when tested with starch as the substrate. H(2) at 100 % in the gas phase did not inhibit growth on starch or peptone. The G+C content of the DNA was 42.5 mol%. 16S rRNA gene sequence analysis placed the isolated strain Z-1312(T) as a member of the genus Desulfurococcus, where it represented a novel species, for which the name Desulfurococcus fermentans sp. nov. (type strain Z-1312(T) = DSM 16532 (T) = VKM V-2316(T)) is proposed.

A rod-shaped heliobacterium motile by peritrichous flagella, designated strain OS-H1, was isolated from a sample of shoreline soil of the soda lake Ostozhe (pH 9.2, total salt content 0.22%) located in the steppe of south-east Siberia. In the first few transfers, the isolate produced heat-resistant endospores. Like other heliobacteria, strain OS-H1 contained bacteriochlorophyll g and lacked intracytoplasmic membranes. The new isolate was a strict anaerobe and photoheterotroph. In the light and in the presence of organic compounds, strain OS-H1 oxidized sulfide to elemental sulfur and polysulfides, but was not capable of photoautotrophic growth. The isolate was an obligate alkaliphile able to grow at pH 8-10.2. The best growth was observed at pH 8.5-9.5, a temperature of 30 degrees C and at 5-10 g sodium carbonate l(-1). Biotin was required as a growth factor. The G+C content of strain OS-H1 was 45.0 mol%. Comparison of the 16S rRNA gene sequence to that of phototrophic bacteria showed strain OS-H1 to group within gram-positive bacteria of the family Heliobacteriaceae with the closest relationship to Heliorestis daurensis (95.6% similarity). Based on physiological, genetic and chemotaxonomic characteristics, the new heliobacterium is described as a new species of the genus Heliorestis, Heliorestis baculata.

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 remoteness of nifH genes sequences of methanotroph types I and II. At the same time, close relationship was found between nifH of type I methanotrophs and representatives of gamma-proteobacteria and between nifH genes of type II methanotrophs and representatives of alpha-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 of Methylococcus capsulatus strains, whose nifH genes proved to be closely related to nifH genes...

The phylogenetic relationships of 12 aerobic dichloromethane-degrading bacteria that implement different C1-assimilation pathways was determined based on 16S ribosomal RNA sequences and DNA–DNA hybridization data. The restricted facultative methylotroph “Methylophilus leisingerii” DM11 with the ribulose monophosphate pathway was found to belong to the genus Methylophilus cluster of the beta subclass of Proteobacteria. The facultative methylotroph Methylorhabdus multivorans DM13 was assigned

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 family Heliobacteriaceae, since they possess the unique properties of this taxon: strict anaerobiosis, formation of bacteriochlorophyll g, 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 genus Heliobacterium and described as two new species of this genus: Heliobacterium sulfidophilum sp. nov. and Heliobacterium undosum sp. nov. The G + C content of the DNA is 51.3 mol % in Hbt. sulfidophilum and 57.2-57.7 mol % in Hbt. undosum. The cells of Hbt. sulfidophilum are rods, and the cells of Hbt. 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 of Hbt. sulfidophilum and Hbt. undosum is 30-35 degrees C, and the optimal pH is 7-8.

The community of anoxygenic phototrophic bacteria (APB) in the water column of the Kislo-Sladkoe stratified lake recently isolated from the sea (White Sea, Kandalaksha Bay) was investigated in September 2010. The water of the sulfide-rich zone was greenish-brown due to intense development of green sulfur bacteria (GSB). Nine APB strains were isolated from the water samples: three belonging to GSB, five, to purple sulfur bacteria (PSB), and one, to purple nonsulfur bacteria (PNB). GSB predominated in the phototrophic community of the chemocline. Unexpectedly, two morphologically different green-colored GSB strains were found to be phylogenetically identical and related to the brown-colored @Chlorobium phaeovibrioides (99% similarity according to the 16S rRNA gene sequencing). Homology to the closest green-colored species (Chlorobium luteolum) was 98%. Two morphologically and physiologically similar PSB strains (TcrPS10 and AmPS10) had rounded cells containing okenonokenonee and gas vesicles. According to the 16S rRNA gene sequencing, these strains were most closely related (99%) to two different Thiocapsa species: Tca. marina (containing okenonokenonee and no gas vesicles) and Tca. rosea (containing spirilloxanthin and gas vesicles). The remaining isolates of purple bacteria were similar to the already described APB species.

... We are grateful to Dr Dhugal J. Lindsay for useful suggestions and critical reading of this manuscript. ... JAMSTEC J Deep-Sea Res 9:271–282 (in Japanese with English abstract) Colodner D, Lin J, Von Damm K, Buttermore L, Kozlowski R, Charlou JL, Donval JP, Wilson C, The ...

A novel genus, Albibacter, with one species, Albibacter methylovorans sp. nov., is proposed for a facultatively chemolithotrophic and methylotrophic bacterium (strain DM10T) with the ribulose bisphosphate (RuBP) pathway of C1 assimilation. The bacterium is a Gram-negative, aerobic, asporogenous, nonmotile, colourless rod that multiplies by binary fission. The organism utilizes dichloromethane, methanol, methylamine, formate and CO2/H2, as well as a variety of polycarbon compounds, as carbon and energy sources. It is neutrophilic and mesophilic. The major cellular fatty acids are straight-chain unsaturated C18:1, saturated C16:0 and cyclopropane C19:0 acids. The main ubiquinone is Q-10. The dominant phospholipids are phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl choline and cardiolipin. The DNA G+C content is 66.7 mol%. Strain DM10T has a very low degree of DNA-DNA hybridization (4-7%) with the type species of the genera Paracoccus, Xanthobacter, Blastobacter, Angulomicrobium, Ancylobacter and Ralstonia of RuBP pathway methylobacteria. Another approach, involving comparative 16S rDNA analysis, has shown that the novel isolate represents a separate branch within the alpha-2 subgroup of the Proteobacteria. The type species of the new genus is Albibacter methylovorans sp. nov.; the type strain is DM10T (= VKM B-2236T = DSM 13819T).

ABSTRACT A new moderately thermophilic, alkaliphilic, sulfate-reducing, chemolithoheterotrophic bacterium, strain S1T, was isolated from a mixed cow/pig manure with neutral pH. The bacterium is an obligately anaerobic, non-motile, Gram-positive, spore-forming curved rod growing within a pH range of 8.0-9.15 (optimal growth at pH 8.6-8.7) and temperature range of 30-58 degrees C (optimal growth at 50-55 degrees C). The optimum NaCl concentration for growth is 0.1%. Strain S1T is an obligately carbonate-dependent alkaliphile. The G+C content of the DNA is 40.9 mol%. A limited number of compounds are utilized as electron donors, including H2+acetate, formate, ethanol, lactate and pyruvate. Sulfate, sulfite and thiosulfate, but not sulfur or nitrate, can be used as electron acceptors. Strain S1T is able to utilize acetate or yeast extract as sources of carbon. Analysis of the 16S rDNA sequence allowed strain S1T (= DSM 12257T) to be classified as a representative of a new species of the genus Desulfotomaculum, Desulfotomaculum alkaliphilum sp. nov.

... DNA was extracted from bacteria using a combination of a modified method of Birn-boim–Doly [18] and the Wizard-technology of the Promega ... The mixture was sonicated using a UZDN-2T (Russia) ultrasound disintegrator at a maximum power and 22 kHz for 2 min at 4°C. Then ...

Genetic constructs coding for the fusion of the Mam12 membrane protein from the magnetotactic bacteria Magnetospirilium magnetotacticum with one B-domain (mb) or two B or Z domains (mbb and mzz, respectively) of Staphylococcus aureus protein A have been synthesized. Expression, fractionation, and purification of fusion proteins have been performed, and the activity of the modified proteins has been characterized. ELISA results showed that all modified proteins displayed immunoglobulin-binding activity.

We studied the compositions of microbial associations isolated from soils where nontransgenic and transgenic late blight-resistant lines of potato varieties Lugovskoi, Charodei, and Golubizna had been grown. The analysis was based on denaturing gradient gel electrophoresis of total amplificates of 16S rRNA gene fragments and analysis of libraries of nifH gene fragments. Neither method revealed significant differences in the structure of the microbial associations isolated from soils with control or transgenic plants. The minor differences detected in the microflora ranges were no greater than those in the rhizospheres of different nontransgenic potato varieties.