T. P. Tourova

Thioalkalimicrobium aerophilum gen. nov., sp. nov. and Thioalkalimicrobium sibericum sp. nov., and Thioalkalivibrio versutus gen. nov., sp. nov., Thioalkalivibrio nitratis sp. nov. and Thioalkalivibrio denitrificans sp. nov., novel obligately alkaliphilic and obligately chemolithoautotrophic sulfur-oxidizing bacteria from soda lakes

Forty-three strains of obligately chemolithoautotrophic sulfur-oxidizing bacteria were isolated from highly alkaline soda lakes in south-east Siberia (Russia) and in Kenya using a specific enrichment procedure at pH 10. The main difference between the novel isolates and known sulfur bacteria was their potential to grow and oxidize sulfur compounds at pH 10 and higher. The isolates fell into two groups that were substantially different from each other physiologically and genetically. Most of the Siberian isolates belonged to the group with a low DNA G+C content (48.0-51.2 mol%). They were characterized by a high growth rate, a low growth yield, a high cytochrome content, and high rates of oxidation of sulfide and thiosulfate. This group included 18 isolates with a DNA homology of more than 40%, and it is described here as a new genus, Thioalkalimicrobium, with two species Thioalkalimicrobium aerophilum (type species) and Thioalkalimicrobium sibericum. The other isolates, mainly from Kenyan soda lakes, fell into a group with a high DNA G+C content (61.0-65.6 mol%). In general, this group was characterized by a low growth rate, a high molar growth yield and low, but relatively equal, rates of oxidation of thiosulfate, sulfide, elemental sulfur and polythionates. The group included 25 isolates with a DNA homology of more than 30%. It was less compact than Thioalkalimicrobium, containing haloalkalophilic, carotenoid-producing, nitrate-reducing and facultatively anaerobic denitrifying strains. These bacteria are proposed to be assigned to a new genus, Thioalkalivibrio, with three species Thioalkalivibrio versutus (type species), Thioalkalivibrio denitrificans and Thioalkalivibrio nitratis. Phylogenetic analysis revealed that both groups belong to the gamma-Proteobacteria. The Thioalkalimicrobium species were closely affiliated with the neutrophilic chemolithoautotrophic sulfur bacteria of the genus Thiomicrospira, forming a new alkaliphilic lineage in this cluster. In contrast, Thioalkalivibrio was not related to any known chemolithoautotrophic taxa, but was distantly associated with anaerobic purple sulfur bacteria of the genus Ectothiorhodospira.

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.

Phylogenetic diversity and activity of anaerobic microorganisms of high-temperature horizons of the Dagang oil field (P. R. China)

The number of microorganisms of major metabolic groups and the rates of sulfate reduction and methanogenesis processes in the formation waters of the high-temperature horizons of Dagang oil field have been determined. Using cultural methods, it was shown that the microbial community contained aerobic bacteria oxidizing crude oil, anaerobic fermentative bacteria, sulfate-reducing bacteria, and methanogens. Using cultural methods, the possibility of methane production from a mixture of hydrogen and carbon dioxide (H2 + CO2) and from acetate was established, and this result was confirmed by radioisotope methods involving NaH14CO3 and 14CH3COONa. Analysis of enrichment cultures 16S rDNA of methanogens demonstrated that these microorganisms belong to Methanothermobacter sp. (M. thermautotrophicus), which consumes hydrogen and carbon dioxide as basic substrates. The genes of acetate-utilizing bacteria were not revealed. Phylotypes of the representatives of Thermococcus spp. were found among archaeal 16S rDNA. 16S rRNA genes of bacterial clones belong to the orders Thermoanaerobacteriales (Thermoanaerobacter, Thermovenabulum, Thermacetogenium, and Coprothermobacter spp.), Thermotogales, Nitrospirales (Thermodesulfovibrio sp.) and Planctomycetales. 16S rDNA of a bacterium capable of oxidizing acetate in the course of syntrophic growth with H2-utilizing methanogens was found in high-temperature petroleum reservoirs for the first time. These results provide further insight into the composition of microbial communities of high-temperature petroleum reservoirs, indicating that syntrophic processes play an important part in acetate degradation accompanied by methane production.

Microbial Thiocyanate Utilization under Highly Alkaline Conditions

ABSTRACT Three kinds of alkaliphilic bacteria able to utilize thiocyanate (CNS−) at pH 10 were found in highly alkaline soda lake sediments and soda soils. The first group included obligate heterotrophs that utilized thiocyanate as a nitrogen source while growing at pH 10 with acetate as carbon and energy sources. Most of the heterotrophic strains were able to oxidize sulfide and thiosulfate to tetrathionate. The second group included obligately autotrophic sulfur-oxidizing alkaliphiles which utilized thiocyanate nitrogen during growth with thiosulfate as the energy source. Genetic analysis demonstrated that both the heterotrophic and autotrophic alkaliphiles that utilized thiocyanate as a nitrogen source were related to the previously described sulfur-oxidizing alkaliphiles belonging to the gamma subdivision of the division Proteobacteria (theHalomonas group for the heterotrophs and the genusThioalkalivibrio for autotrophs). The third group included obligately autotrophic sulfur-oxidizing alkaliphilic bacteria able to utilize thiocyanate as a sole source of energy. These bacteria could be enriched on mineral medium with thiocyanate at pH 10. Growth with thiocyanate was usually much slower than growth with thiosulfate, although the biomass yield on thiocyanate was higher. Of the four strains isolated, the three vibrio-shaped strains were genetically closely related to the previously described sulfur-oxidizing alkaliphiles belonging to the genus Thioalkalivibrio. The rod-shaped isolate differed from the other isolates by its ability to accumulate large amounts of elemental sulfur inside its cells and by its ability to oxidize carbon disulfide. Despite its low DNA homology with and substantial phenotypic differences from the vibrio-shaped strains, this isolate also belonged to the genusThioalkalivibrio according to a phylogenetic analysis. The heterotrophic and autotrophic alkaliphiles that grew with thiocyanate as an N source possessed a relatively high level of cyanase activity which converted cyanate (CNO−) to ammonia and CO2. On the other hand, cyanase activity either was absent or was present at very low levels in the autotrophic strains grown on thiocyanate as the sole energy and N source. As a result, large amounts of cyanate were found to accumulate in the media during utilization of thiocyanate at pH 10 in batch and thiocyanate-limited continuous cultures. This is a first direct proof of a “cyanate pathway” in pure cultures of thiocyanate-degrading bacteria. Since it is relatively stable under alkaline conditions, cyanate is likely to play a role as an N buffer that keeps the alkaliphilic bacteria safe from inhibition by free ammonia, which otherwise would reach toxic levels during dissimilatory degradation of thiocyanate.

Carboxydocella thermautotrophica gen. nov., sp. nov., a novel anaerobic, CO-utilizing thermophile from a Kamchatkan hot spring.

A novel anaerobic, thermophilic, CO-utilizing bacterium, strain 41(T), was isolated from a terrestrial hot vent on the Kamchatka Peninsula. Strain 41(T) was found to be a Gram-positive bacterium, its cells being short, straight, motile rods. Chains of three to five cells were often observed. The isolate grew only chemolithoautotrophically on CO, producing equimolar quantities of H2 and CO2 (according to the equation CO+H2O --> CO2+H2). Growth was observed in the temperature range 40-68 degrees C, with an optimum at 58 degrees C, and in the pH range 6.5-7.6, with an optimum at pH 7.0. The generation time under optimal conditions for chemolithotrophic growth was 1.1 h. The DNA G+C content was 46 +/- 1 mol%. Growth was completely inhibited by penicillin, ampicillin, streptomycin, kanamycin and neomycin. On the basis of the phenotypic and phylogenetic features, it is proposed that this isolate represents a new genus and species, Carboxydocella thermautotrophica gen. nov., sp. nov. (type strain 41(T) = DSM 12356(T) = VKM B-2282(T)).

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.

Geoalkalibacter ferrihydriticus gen. nov. sp. nov., the first alkaliphilic representative of the family Geobacteraceae, isolated from a soda lake

Investigation of iron reduction in bottom sediments of alkaline soda lakes resulted in the isolation of a new obligately anaerobic iron-reducing bacterium, strain Z-0531, from Lake Khadyn (Tuva, Russia) sediment samples. The cells of strain Z-0531 are short (1.0–1.5 by 0.3–0.5 µm), motile, non-spore-forming, gram-negative rods. The isolate is an obligate alkaliphile, developing in the pH range of 7.8–10.0, with an optimum at pH 8.6. It does not require NaCl but grows at NaCl concentrations of 0–50 g/l. It can oxidize acetate with such electron acceptors as amorphous Fe(III) hydroxide (AFH), EDTA-Fe(III), anthraquinone-2,6-disulfonate (quinone), Mn(IV), and S0. On medium with EDTA-Fe(III), the isolate can oxidize, apart from acetate, ethanol, pyruvate, oxalate, arginine, tartrate, lactate, propionate, and serine. H2 is not utilized. The reduced products formed during growth with AFH are siderite or magnetite, depending on the growth conditions. The isolate is incapable of fermenting sugars, peptides, and amino acids. Yeast extract or vitamins are required as growth factors. The organism is capable of dinitrogen fixation and harbors the nifH gene. The DNA G+C content is 55.3 mol %. 16S rRNA analysis places strain Z-0531 into the family Geobacteraceae. Its closest relative (93% similarity) is Desulfuromonas palmitatis. Based on phenotypic distinctions and phylogenetic position, it is proposed that this strain be assigned to the new genus and species Geoalkalibacter ferrihydriticus gen. nov., sp. nov. (Z-0531T-DSMZ-17813-VKMB-2401).

Carboxydobrachium pacificum gen. nov., sp. nov., a new anaerobic, thermophilic, CO- utilizing marine bacterium from Okinawa Trough

A new anaerobic, thermophilic, CO-utilizing marine bacterium, strain JMT, was isolated from a submarine hot vent in Okinawa Trough. Cells of strain JMT were non-motile thin straight rods, sometimes branching, with a cell wall of the Gram-positive type, surrounded with an S-layer. Chains of three to five cells were often observed. The isolate grew chemolithotrophically on CO, producing equimolar quantities of H2 and CO2 (according to the equation CO+H2O-->CO2+H2) and organotrophically on peptone, yeast extract, starch, cellobiose, glucose, galactose, fructose and pyruvate, producing H2, acetate and CO2. Growth was observed from 50 to 80 degrees C with an optimum at 70 degrees C. The optimum pH was 6.8-7.1. The optimum concentration of sea salts in the medium was 20.5-25.5 g l(-1). The generation time under optimal conditions was 7.1 h. The DNA G+C content was 33 mol %. Growth of isolate JMT was not inhibited by penicillin, but ampicillin, streptomycin, kanamycin and neomycin completely inhibited growth. The results of 16S rDNA sequence analysis revealed that strain JMT belongs to the Thermoanaerobacter phylogenetic group within the Bacillus-Clostridium subphylum of Gram-positive bacteria but represents a separate branch of this group. On the basis of morphological and physiological features and phylogenetic data, this isolate should be assigned to a new genus, for which the name Carboxydobrachium is proposed. The type species is Carboxydobrachium pacificum; the type strain is JMT (= DSM 12653T).

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.

Thioalkalivibrio thiocyanoxidans sp. nov. and Thioalkalivibrio paradoxus sp. nov., novel alkaliphilic, obligately autotrophic, sulfur- oxidizing bacteria capable of growth on thiocyanate, from soda lakes

Nine strains of haloalkaliphilic, obligately autotrophic, sulfur-oxidizing bacteria able to grow with thiocyanate (SCN-) as the sole energy and nitrogen source were isolated from soda lakes in South-East Siberia, Kenya and Egypt after enrichment on sodium carbonate minerals buffered at pH 10 with thiocyanate as the substrate. The isolates fell into two groups that were substantially different in terms of cell morphology, growth parameters and the ability to oxidize carbon disulfide. The bacteria were able to oxidize sulfide, polysulfide, sulfur and tetrathionate, as well as thiocyanate. Two isolates belonged to an extremely halotolerant type growing in the presence of up to 4 M Na+. Cyanate (CNO-) was the main nitrogen-containing intermediate during thiocyanate degradation in both groups. According to DNA-DNA hybridization data and phylogenetic analysis, both groups of isolates belong to a recently described genus of haloalkaliphilic sulfur-oxidizing bacteria, i.e. Thioalkalivibrio, belonging to the gamma-Proteobacteria, in which where they represent two new species. The species name Thioalkalivibrio paradoxus (type strain ARh 1T = DSM 13531T = JCM 11367T) is proposed for the group with barrel-shaped cells, and the species name Thioalkalivibrio thiocyanoxidans (type strain ARh 2T, DSM 13532T = JCM 11368T) is proposed for the group with vibrio-shaped cells. The diagnosis of the genus Thioalkalivibrio is amended according to the new data.