A. B. Poltaraus

Application of gyrB and parE sequence similarity analyses for differentiation of species within the genus Geobacillus

The primary structures of the genes encoding the β-subunits of a type II topoisomerase (gyrase, gyrB) and a type IV topoisomerase (parE) were determined for 15 strains of thermophilic bacteria of the genus Geobacillus. The obtained sequences were used for analysis of the phylogenetic similarity between members of this genus. Comparison of the phylogenetic trees of geobacilli constructed on the basis of the 16S rRNA, gyrB, and parE gene sequences demonstrated that the level of genetic distance between the sequences of the genes encoding the β-subunits of type II topoisomerases significantly exceeded the values obtained by comparative analysis of the 16S rRNA gene sequences of Geobacillus strains. It was shown that, unlike the 16S rRNA gene analysis, comparative analysis of the gyrB and parE gene sequences provided a more precise determination of the phylogenetic position of bacteria at the species level. The data obtained suggest the possibility of using the genes encoding the β-subunits of type II topoisomerases as phylogenetic markers for determination of the species structure of geobacilli.

Characterization of the aerobic hydrocarbon-oxidizing enrichments from a high-temperature petroleum reservoir by comparative analysis of DNA- and RNA-derived clone libraries

Enrichment cultures of aerobic hydrocarbon-oxidizing bacteria obtained from the injection and production wells of the Dagang oil field (China) were studied by molecular biological and microbiological methods. This work is the first to report simultaneous isolation of DNA and RNA from enrichment cultures of microorganisms from oil strata with further construction of clone libraries of 16S rRNA genes and 16S crDNA (complementary rDNA). Comparative analysis of the DNA- and RNA-derived clone libraries made it possible to determine the total genomic diversity of microorganisms, as well as to reveal metabolically active microorganisms in these cultures. Phylotypes of bacteria of the genus Geobacillus were found to be dominant in the DNA and RNA clone libraries of the enrichment cultures from the production well. Phylotypes of bacteria belonging to Geobacillus, Pseudomonas, Tepidiphilus, and other genera were detected in the DNA and RNA libraries obtained from the culture from the injection well. Phylotypes of bacteria of the genus Geobacillus were predominant in the RNA library and represented the second-largest group (after pseudomonads) in the DNA library. In the RNA libraries of the alkB genes of both enrichments, three homologs close to alkB-geo1, alkB-geo2, and alkB-geo4 of bacteria of the genus Geobacillus were detected. The occurrence pattern of the alkB transcripts, ribosomal RNA, and the 16S rRNA genes of bacteria of the genus Geobacillus indicates the predominance and functional activity of geobacilli in the enrichment cultures of hydrocarbon-oxidizing bacteria from high-temperature petroleum reservoir.

Chinese hamster protein homologous to human putative protein kinase KIAA0204 is associated with nuclei, microtubules and centrosomes in CHO-K1 cells.

Monoclonal antibody raised against a preparation of loach fish sperm centrosomes was used for screening of cDNA expressing library of Chinese hamster CHO‐K1 cells. Two positive clones appeared to encode 628 amino acid protein fragment that was 72% identical to human KIAA0204 protein, i.e. putative protein kinase. Polyclonal antibodies raised against products of cDNA expression in E. coli recognized 210‐kDa polypeptide in CHO‐K1 cells and immunostained nuclear speckles, centrosomes and microtubules in these cells. The 210‐kDa polypeptide (named MAK‐L) co‐sedimented with exogenous microtubules. Thus, one more protein kinase seems to be associated with the microtubule network in vertebrate cells.

Detection of n-alkane biodegradation genes alkB and ladA in thermophilic hydrocarbon-oxidizing bacteria of the genera Aeribacillus and Geobacillus

Ability to degrade crude oil n-alkanes was revealed in new strains of thermophilic bacilli isolated from petroleum reservoirs and a hot spring: Geobacillus toebii В-1024, Geobacillus sp. 1017, and Aeribacillus pallidus 8m3. The strains utilized С10–С30n-alkanes (В-1024), С10, C11, and С13–С19,22n-alkanes (1017), and C11–C29n-alkanes (8m3). In all three strains, PCR amplification with specific degenerate oligonucleotide primers revealed the alkB gene encoding rubredoxin-dependent alkane monooxygenase. In strains В-1024 and 1017, fragments of the genes homologous to the ladA gene determining flavin-dependent alkane monooxygenase were also amplified. Nucleotide sequences of these genes were practically identical to those of the genes ladAαB23, ladAβB23, and ladBB23, which were revealed previously in Geobacillus thermoleovorans strain B23. For the latter strain, activity of respective enzymes in the oxidation of long-chain n-alkanes has been shown. Thus, simultaneous presence of the alkB and ladA genes coding alkane monooxygenases responsible for oxidation of medium-chain and long-chain n-alkanes in thermophilic bacilli was revealed for the first time.

[Detection and transcription of n-alkane biodegradation genes (alkB) in the genome of a hydrocarbon-oxidizing bacterium Geobacillus subterraneus K].

The diversity and localization of alkB genes in the genome of the hydrocarbon-oxidizing bacterium Geobacillus subterraneus K, as well as the transcription of alkB genes, were studied as functions of culture growth phase and the hydrocarbon substrates utilized. Analysis of 96 clones containing inserted alkB genes revealed six alkB homologs in the strain under study: alkB-geo1, alkB-geo2, alkB-geo3, alkB-geo4, alkB-geo5, and alkB-geo6. In addition, real-time PCR of the total DNA of strain K revealed one more homolog, alkB-geo7. Chromosomal localization of alkB genes was demonstrated in strain K. During exponential growth on n-alkanes of various chain length (n-C16H34 and n-C22H46), formation of mRNA of highly homologous alkB-geo5 and alkB-geo6 genes was observed; in the beginning of the stationary phase, alkB-geo4 mRNA was formed. The functional role and conditions of induction of the enzymes encoded by the rest of the homologs detected in strain K, that is, alkB-geo1, alkB-geo2, and alkB-geo7, remain unknown, which calls for further investigations of alkB genes in thermophilic bacteria under various growth conditions and with the use of various oil hydrocarbons as substrates.

Identification of hydrocarbon-oxidizing Dietzia bacteria from petroleum reservoirs based on phenotypic properties and analysis of the 16S rRNA and gyrB genes

The taxonomic position of hydrocarbon-oxidizing bacterial strains 263 and 32d isolated from formation water of the Daqing petroleum reservoir (PRC) was determined by polyphasic taxonomy techniques, including analysis of the 16S rRNA and the gyrB genes. The major chemotaxonomic characteristics of both strains, including the IV type cell wall, composition of cell wall fatty acids, mycolic acids, and menaquinones, agreed with those typical of Dietzia strains. The DNA G+C content of strains 263 and 32d were 67.8 and 67.6 mol %, respectively. Phylogenetic analysis of the 16S rRNA gene of strain 32d revealed 99.7% similarity to the gene of D. maris, making it possible to identify strain 32d as belonging to this species. The 16S rRNA gene sequence of strain 263 exhibited 99.7 and 99.9% similarity to those of D. natronolimnaea and D. cercidiphylli YIM65002T, respectively. Analysis of the gyrB genes of the subterranean isolates and of a number of Dietzia type strains confirmed classification of strain 32d as a D. maris strain and strain 263 as a D. natronolimnaea strain. A conclusion was made concerning higher resolving power of phylogenetic analysis of the gyrB gene compared to the 16S rRNA gene analysis in the case of determination of the species position of Dietzia isolates.

Draft Genome Sequence of Aeribacillus pallidus Strain 8m3, a Thermophilic Hydrocarbon-Oxidizing Bacterium Isolated from the Dagang Oil Field (China)

ABSTRACT The draft genome sequence of Aeribacillus pallidus strain 8m3, a thermophilic aerobic oil-oxidizing bacterium isolated from production water from the Dagang high-temperature oil field, China, is presented here. The genome is annotated to provide insights into the genomic and phenotypic diversity of the genus Aeribacillus.

Microbiological processes in the Severnyi deep disposal site for liquid radioactive wastes

Local monitoring of physicochemical, radiochemical, and microbiological parameters was performed in the deep horizons of the Severnyi site used for disposal of liquid radioactive waste (LRW). Analysis of the chemical and radiochemical composition of the wastes and formation fluid revealed that the boundary for migration of radionuclides lagged behind that for nonradioactive waste components (sodium nitrate) and tritium. The physicochemical and radiochemical conditions in deep horizons did not prevent microbial growth. The numbers of microorganisms (aerobic organotrophs, denitrifying, fermentative, sulfate-reducing, and methanogenic) were low, as were the rates of sulfate reduction and methanogenesis; they increased in the waste dispersion zone. The microorganisms from deep horizons were able to produce gases (CH4, CO2, N2, and H2S) from possible waste components. Denitrifying bacteria belonged to different Pseudomonas species and reduced nitrate to dinitrogen under the conditions of pH, salinity, temperature, and radioactivity found in the disposal site. These results suggest the need for control of microbiological processes in deep disposal site for liquid RW.

Draft Genome Sequence of Geobacillus subterraneus Strain K, a Hydrocarbon-Oxidizing Thermophilic Bacterium Isolated from a Petroleum Reservoir in Kazakhstan

ABSTRACT The draft genome sequence of Geobacillus subterraneus strain K, a thermophilic aerobic oil-oxidizing bacterium isolated from production water of the Uzen high-temperature oil field in Kazakhstan, is presented here. The genome is annotated for elucidation of the genomic and phenotypic diversity of thermophilic alkane-oxidizing bacteria.

Microbial diversity in formation water and enrichment cultures from the Gangxi bed of the Dagang terrigenous oilfield (PRC)

Microbial diversity and biogeochemical processes of the Gangxi bed with low-mineral water and a temperature gradient from 35 to 54°C were studied. The 16S rRNA gene clone libraries (over 800 clones) were obtained from microbial DNA isolated from formation water and from the primary enrichment cultures for fermenting, sulfate-reducing, methanogenic, and aerobic organotrophic prokaryotes. While both sulfate reduction and methanogenesis were registered in formation water by radioisotope techniques, the genes of sulfate-reducing prokaryotes were not revealed in the 16S rRNA gene clone library from formation water. The 16S rRNA genes of Methanobacterium congolense and Methanococcus vannielii predominated among archaeal sequences retrieved from formation water, while the genes of Methanothermobacter thermoautotrophicus, Methanomethylovorans thermophila, and Methanoculleus sp. predominated in the combined library from enrichment cultures. In the library of Bacteria 16S rRNA genes from formation water, the genes of thermophilic fermentative bacteria of the family Thermoanaerobacteriaceae predominated; the remaining sequences belonged to mesophiles (genera Brevundimonas, Sphingomonas, Oxalicibacterium, and Stenotrophomonas), the phylum Chloroflexi, and unidentified bacteria. The combined library from enrichment cultures, contained, apart from the sequences of the family Thermoanaerobacteriaceae, the genes of fermentative bacteria (genera Anaerobaculum, Coprothermobacter, Thermanaerovibrio, Soehngenia, Bacteroides, and Aminobacterium and the order Thermotogales), of aerobic hydrocarbon-oxidizing bacteria (genera Pannonibacter and Pseudomonas), and of sulfate reducers (genera Desulfomicrobium, Thermodesulfovibrio, and Desulfotomaculum). High coverage was shown for bacterial (97.6%) and archaeal (100%) clone libraries, indicating that a significant portion of the microbial diversity in the studied communities was revealed.