B. B. Kuznetsov

Thermoanaerobacter siderophilus sp. nov., a novel dissimilatory Fe(III)-reducing, anaerobic, thermophilic bacterium

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-anthraquinone-2,6-disulfonic acid, sulfite, thiosulfate, elemental sulfur and MnO2. Strain SR4T did not reduce nitrate or sulfate and was not capable of growth with O2. The fermentation products from glucose were ethanol, lactate, H2 and CO2. The G + C content of DNA was 32 mol%. 16S rDNA sequence analysis placed the organism in the genus Thermoanaerobacter. On the basis of physiological properties and phylogenetic analysis, it is proposed that strain SR4T (= DSM 12299T) should be assigned to a new species, Thermoanaerobacter siderophilus sp. nov.

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).

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.

An Oligonucleotide Primer System for Amplification of the Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Genes of Bacteria of Various Taxonomic Groups

Based on the analysis of GenBank nucleotide sequences of the cbbL and cbbM genes, coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPC), the key enzyme of the Calvin cycle, a primer system was designed that allows fragments of these genes about 800 bp long to be PCR-amplified for various photo- and chemotrophic bacteria. The efficiency of the designed primer system in detection of RuBPC genes was demonstrated in PCR with DNA of taxonomically diverse bacteria possessing RuBPC genes with a known primary structure. Nucleotide sequences of RuBPC gene fragments of bacteria belonging to the genera Acidithiobacillus, Ectothiorhodospira, Magnetospirillum, Methylocapsa, Thioalkalispira, Rhodobacter, and Rhodospirillum were determined to be deposited with GenBank and to be translated into amino acid sequences and subjected to phylogenetic analysis.

Thermanaerovibrio velox sp. nov., a new anaerobic, thermophilic, organotrophic bacterium that reduces elemental sulfur, and emended description of the genus Thermanaerovibrio

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).

Desulfotomaculum alkaliphilum sp. nov., a new alkaliphilic, moderately thermophilic, sulfate-reducing bacterium.

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.

Proposal of Oscillochloridaceae fam. nov. on the basis of a phylogenetic analysis of the filamentous anoxygenic phototrophic bacteria, and emended description of Oscillochloris and Oscillochloris trichoides in comparison with further new isolates.

The nucleotide sequences of the genes of 16S rRNAs were determined for the type strain Oscillochloris trichoides DG-6T and three new strains of Oscillochloris-like mesophilic filamentous green bacteria. Two major clusters have been found within the family Chloroflexaceae by phylogenetic-analysis: one cluster includes thermophilic species of Chloroflexus and the second includes mesophilic strains of Oscillochloris. The degree of relatedness of these clusters was below an intergeneric level, having only 82.5-86.5% of 16S rDNA sequence similarity. These phylogenetic data correlate well with the significant physiological, biochemical and chemotaxonomical differences between members of both groups. Therefore, the Oscillochloris and Chloroflexus clusters should be considered as two separate families. The description of the new family, Oscillochloridaceae fam. nov., and emended descriptions of the genus Oscillochloris and the species Oscillochloris trichoides are presented.

Designing and testing oligonucleotide primers for amplification and sequencing of archaeal 16S rRNA genes

The analysis of 16S rRNA genes is the main tool for investigations in molecular ecology that allows obtaining primary data on biodiversity of prokaryotes in various natural ecosystems. As distinct from the common microbiological techniques, this approach does not require isolation of pure cultures of the members of the natural microbial community, but is based on evaluation of the diversity of 16S rRNA genes (phylotypes) involved in the community. This technique is based on the amplification of a total set of 16S rRNA genes followed with cloning, sequencing, and phylogenetic analysis. It provides for the identification of microorganisms for which pure cultures are still unavailable (noncultivated microorganisms). Moreover, the high sensitivity of this technique allows the detection of very low contents of specific microorganisms, even of single cells [1].

Methylobacterium suomiense sp. nov. and Methylobacterium lusitanum sp. nov., aerobic, pink-pigmented, facultatively methylotrophic bacteria.

Two aerobic, pink-pigmented, facultatively methylotrophic bacteria, strains F20T and RXM(T), are described taxonomically. On the basis of their phenotypic and genotypic properties, the isolates are proposed as novel species of the genus Methylobacterium, Methylobacterium suomiense sp. nov. (type strain F20T = VKM B-2238T = NCIMB 13778T) and Methylobacterium lusitanum sp. nov. (type strain RXMT = VKM B-2239T = NCIMB 13779T).

Thermovenabulum ferriorganovorum gen. nov., sp. nov., a novel thermophilic, anaerobic, endospore-forming bacterium.

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.