[Thermotolerant oil-degrading bacteria isolated from soil and water of geographically distant regions].

Oil-degrading bacteria were isolated from soil and water samples taken in Russia, Kazakhstan, and the Antarctic; 13 of 86 strains proved to be thermotolerant. These bacteria utilized crude oil at 45­50°C; their growth optimum (35­37°C) and range (20­53°C) differ from those of mesophilic bacteria. Thermotolerant strains were identified as representatives of the genera Rhodococcus and Gordonia. It was shown that their ability to degrade petroleum products does not differ at 24 and 45°C. The strains Rhodococcus sp. Par7 and Gordonia sp. 1D utilized 14 and 20% of the oil, respectively, in 14 days at 45°C. All of the isolated thermotolerant bacteria grew in a medium containing 3% NaCl; the medium for the strains Gordonia amicalis 1B and Gordonia sp. 1D contained up to 10% NaCl. The bacteria G. amicalis and Rhodococcus erythropolis were able to utilize crude oil and individual hydrocarbons at higher (up to 50°C) temperatures.

[Oil biodegradation by microbial-plant associations].

The degradation of petroleum hydrocarbons by plant-microbial associations, as well as the peculiarities of the interaction between microorganisms in consortium and the associated plants, have been studied. It was shown that degrader microorganisms that are part of the consortium Rhodococcus erythropolis S26, Acinetobacter baumannii 1 B, Acinetobacter baumannii 7, and Pseudomonas putida F701 were effective in the degradation of oil and were good colonizers of plant roots (barley). The efficiency of oil degradation increases when microorganisms and plants are used together.

[Comparative characteristics of free-living ultramicroscopical bacteria obtained from extremal biotopes].

We isolated 50 strains of free-living ultrasmall bacteria with a cell volume that varies from 0.02 to 1.3 microm3 from a range of extremal natural biotopes, namely permafrost soils, oil slime, soils, lake silt, thermal swamp moss, and the skin integuments of the clawed frog, Xenopus laevis. Of them, 15 isolates, characterized by a cell size of less than 0.1 microm3 and a genome size from 1.5 to 2.4 Mb, were subsumed to ultramicrobacteria belonging to different philogenetic groups (Alphaproteobacteria, Bacteroidetes, Actinobacteria) and genera (Kaistia, Chryseobacterium, Microbacterium, Leucobacter, Leifsonia, and Agrococcus) of the Bacteria domain. They are free-living mesophilic heterotrophic aerobic bacteria. The representatives of Kaistia and Chryseobacterium genera were capable of facultative parasitism on other species of chemo-organotrophic bacteria and cyanobacteria. The ultramicrobacteria differed in their morpholgy, cell ultrastructural organization, and physiological and biochemical features. According to the fine structure of their cell walls, the isolates were subdivided into two groups, namely Gram-positive and Gram-negative forms.

[Bioremediation of oil-polluted soils: using the [13C]/[12C] ratio to characterize microbial products of oil hydrocarbon biodegradation].

We compared data on the extent of bioremediation in soils polluted with oil. The data were obtained using conventional methods of hydrocarbon determination: extraction gas chromatography-mass spectrometry, extraction IR spectroscopy, and extraction gravimetry. Due to differences in the relative abundances of the stable carbon isotopes (13C/12C) in oil and in soil organic matter, these ratios could be used as natural isotopic labels of either substance. Extraction gravimetry in combination with characteristics of the carbon isotope composition of organic products in the soil before and after bioremediation was shown to be the most informative approach to an evaluation of soil bioremediation. At present, it is the only method enabling quantification of the total petroleum hydrocarbons in oil-polluted soil, as well as of the amounts of hydrocarbons remaining after bioremediation and those microbially transformed into organic products and biomass.

[IncP-7 plasmids' classification based on structural diversity of their basic replicons].

The structural diversity of basic replicons and repB gene of the IncP-7 plasmids' collection was firstly assessed on the basis of PCR, restriction analysis and partial sequencing. It has been revealed that DNA fragment containing gene for UvrD-like helicase RepB is a part of all known P-7 replicons, but often serves as hot place for diverse IS-elements invasion. The first system of P-7 plasmids' classification has been worked out on the basis of determined repA-oriV-par WABC nucleotide divergency. Most degradation plasmids established to be belonging to large beta-subgroup, streptomycin resistance plasmid Rms148 (IncP-7 archetype)--to alpha-subgroup, carbazole degradation plasmid pCAR1 and NAH/SAL-plasmids from pY-line (Yamal oil deposits)--to gamma-subgroup and CAP-plasmid pBS270 with potentially reduced P-7 replicon--to delta-subgroup. It has been observed that the type of IncP-7 basic replicon molecular organization does not correlate with fixed phenotypic character in most cases, that is plasmids encoding different phenotypic markers could be members of the same P-7 subgroup.

[The new gene encoding TrfA-type replication initiator has been found on caprolactam/salicylate degradation plasmid pBS270].

The mini-replicon of pseudomonads' caprolactam/salicylate degradation plasmid pBS270 (105 kb, contains incompatibility determinants of P-7 group) has been obtained and its nucleotide sequence has been determined. The new gene encoding TrfA-like replication initiator has been found on this replicon. Poor homology of this replication initiator with known proteins of TrfA-family allows us to classify obtained replicon as IncP-1-like. The pBS270mini reveals chimeric nature.

[scpA the new salicylate hydroxylase gene localized on salicylate/caprolactam degradation plasmids].

Both caprolactam and salicylate biodegradation by Pseudomonas salicylate/caprolactam degraders is controlled by large conjugative plasmids (SAL/CAP). Some of these plasmids determined to be the members of IncP-7 group. The new salicylate 1-hydroxylase gene (scpA) on SAL/CAP-plasmids has been detected and partially sequenced. Gene scpA was equally related to closest homologs nahG (NAH7), salA (P. reinekei MT1) and nahU (pND6-1), but identity of scpA to these genes did not exceed 72-74%. Synthesis of salicylate 1-hydroxylase ScpA was not induced by salicylate. This enzyme had wide substrate specificity and exhibited highest specific activity with 4-methylsalicylate and nonsubstituted salicylate. Besides pseudomonad's salicylate degradative conjugative plasmids without "classical" nah2-operon and harboring only salicylate 1-hydroxylase gene nahU have been firstly described.

[Organization and maintenance features of IncP-7 naphthalene degradation plasmid pFME5 basic replicon].

A basic replicon of the naphthalene degradation plasmid pFME5 (80 kb, IncP-7) has been constructed and sequenced. The nucleotide sequence of pFME5mini is almost identical to replicons of the pND6-1 subgroup, which was separated based on the reA-oriV homology in our previous work. The basic replicon of pFME5 is capable of replication and stable maintenance exclusively in Pseudomonas species. An analysis of the deletion mutation indicated that, in contrast to the parWAB region, the parC gene is not essential for the stability of pFME5mini and can be a common feature of IncP-7 replicons. We revealed that par-defective mutants of pFME5mini were slowly eliminated from the bacterial population in a nonselective medium compared to their pCAR1-based counterparts. Designed primers specific to the repA and parC genes can be used to detect IncP-7 plasmids, while primers specific to two variants of parA can be used for intragroup classification.

[Phylogenetic analysis of the genes for naphthalene and phenanthrene degradation in Burkholderia sp. strains].

The genetic systems responsible for naphthalene and phenanthrene catabolism have been analyzed in the five strains of Burkholderia sp. isolated from soil samples (West Siberia) contaminated by heavy residual fuel oil and in the strain Burkholderia sp. BS3702 from the laboratory collection isolated from soil samples of the coke gas works (Vidnoe, Moscow oblast). The results of this work demonstrate that naphthalene and phenanthrene degradation in the above strains is encoded by the sequences not homologous to the classical nah genes of pseudomonades. In the Burkholderia sp. BS3702 strain, the initial stages of phenanthrene degradation and the subsequent stages of salicylate degradation are controlled by the sequences of different evolutionary origins (phn and nag genes).

[The structure of replication initiation region of Pseudomonas IncP-7 streptomycin resistance plasmid Rms148].

Pseudomonads' IncP-7 plasmids make significant contribution to the environmental biodegradative potential and sometimes harbour antibiotic resistance genes. More than 30 years plasmid Rms148 is used as archetypal P-7 plasmid in microbiological incompatibility tests. However, the structure of its basic replicon was not described up to now, as well as phylogenetic relationships between all known plasmids within the IncP-7 group were not studied. In the frames of this work we have constructed two primer pairs to amplify main components of P-7 replication initiation region, and subsequent screening of repA intragenic polymorphism was made using laboratory collection of IncP-7 plasmids. Minimal replicon of Rms148 was constructed and its nucleotide sequence was determined to be identical to repA-oriVof known P-7 plasmids on 81-83% and forming separate branch on appropriate phylogenetic tree. Additionally, repA seems to be more conservative between group members compared with putative oriV region. Deduced amino acid sequence and predicted secondary and tertiary structures of Rms148 RepA protein allow us to make assumption about similar to unclassified cryptic plasmid pPS10 model of replication initiation for IncP-7 group members.

[Ratio [13C]/[12C] as an index for express estimation of hydrocarbon-oxidizing potential of microbiota in soil polluted with crude oil].

The hydrocarbon-oxidizing potential of soil microbiota and hydrocarbon-oxidizing microorganisms introduced into soil was studied based on the quantitative and isotopic characteristics of carbon in products formed in microbial degradation of oil hydrocarbons. Comparison of CO2 production rates in native soil and that polluted with crude oil showed the intensity of microbial mineralization of soil organic matter (SOM) in the presence of oil hydrocarbons to be higher as compared with non-polluted soil, that is, revealed a priming effect ofoil. The amount of carbon of newly synthesized organic products (cell biomass and exometabolites) due to consumed petroleum was shown to significantly exceed that of SOM consumed for production of CO2. The result of microbial processes in oil-polluted soil was found to be a potent release of carbon dioxide to the atmosphere.

[Rhizosphere bacteria Pseudomonas aureofaciens and Pseudomonas chlororaphis oxidizing naphthalene in the presence of arsenic].

Rhizosphere strains of P. aureofaciens BS1393(pBS216, pKS1) and P. chlororaphis PCL1391(pBS216, pKS1), exhibiting the ability to stimulate the growth of plants and protect them from phytopathogens, have been obtained. In these strains, plasmid pBS216 ensures naphthalene degradation and plasmid pKS1 confers resistance to arsenic. In the presence of arsenic and naphthalene, the number of living cells and the growth rate of the arsenic-resistant strains were higher than those of the arsenic-sensitive strains BS1393(pBS216) and PCL1391(pBS216). During the cultivation of the resistant strains, arsenic had no inhibitory effect on the activity of the key enzymes of naphthalene biodegradation, except for catechol-2,3-dioxygenase. In a model system containing plant-microbial associations, strains BS1393(pBS216, pKS1) and PCL1391(pBS216, pKS1) degraded as much as 97% of added naphthalene in the presence of arsenic.

[The cnr-like operon in strain Comamonas sp. encoding resistance to cobalt and nickel].

Plasmid pBS501 was detected in the strain Comamonas sp. BS501. This plasmid specifies high level of induced resistance (5 mM) to cobalt/nickel both in the host strain and in related strains C. testosteroni B-1241 and C. acidovorans B-1251. Hybridization analysis revealed a homology of pBS501 restriction fragments with the only well-characterized operon cnrXYHCBAT that resides in plasmid pMOL28 from Cupriavidus metallidurans CH34. Essential differences in the structural organization of the cobalt/nickel resistance determinant were found between plasmid pBS501 and the cnr-operon.

[Obtaining of the mutant Yarrowia lipolytica strains producing citric acid from glucose].

The possibility of obtaining mutant yeasts Yarrowia lipolytica VKM Y-2373 with increased ability to synthesize citric acid from glucose by using UV irradiation and N-methyl-NT-nitro-N-nitrosoguanidine was studied. Of 1500 colonies of the Y. lipolytica treated with either UV or N-methyl-NT-nitro-N-nitrosoguanidine, three mutants were selected that displayed higher (by 23%) biosynthetic ability as compared with the initial strain. Additionally, three mutants were selected from 1000 colonies of the Y. lipolytica exposed to a combined action of UV and N-methyl-NT-nitro-N-nitrosoguanidine; their biosynthetic activity exceeded that of the initial strain by 43.9%. The selective media with citrate and acetate were developed for a rapid selection of mutants as well as the express methods for the detection of active citric acid producers on the solid media with chalk and bromocresol containing a limiting concentration of amine nitrogen and an excess of glucose.

Epiphytic microorganisms degrading aromatic hydrocarbons from the phyllosphere of urban woody plants.

From the leaves of three urban trees (Tilia sp., Acer sp., and Fraxinus sp.), 180 strains degrading phenanthrene, naphthalene, and salicylate were isolated by direct plating and enrichment cultures. The leaves of each tree species were characterized by a specific profile of aromatic hydrocarbon-degrading microflora. Members of the type Actinobacteria were predominant in the case of direct plating on media with phenanthrene and naphthalene. Enrichment cultures with phenanthrene and salicylate were shown to yield microbial consortia, the composition of which changed with time. Members of the type Proteobacteria were predominant in these consortia. No plasmids of polycyclic aromatic hydrocarbon degradation of the P-7 and P-9 incompatibility groups were revealed in the studied strains.

Sporulation of Bacillus subtilis in Binary Cultures with Ultramicrobacteria.

The effect of ultramicrobacterial epibionts of the genera Kaistia (strain NF1), Chryseobacterium (strain NF4), and Stenotrophomonas (strain FM3) on the process of sporulation of Bacillus subtilis ATCC 6633 was studied. The investigated strains of ultramicrobacteria (UMB) were found to inhibit the sporulation process of B. subtilis ATCC 6633 in binary mixed cultures, exhibiting a 3-day delay of the onset of sporulation compared to the control one, an extended period of the prospore maturation, formation of the fraction of immature spores, and development of ultrastructural defects in many endospores. Thus, investigation of binary mixed cultures of B. subtilis and UMB revealed that, apart from suppression of reproduction and lysis of host vegetative cells, inhibition of spore formation and destruction of endospores was yet another feature of intermicrobial parasitism. The UMB parasites of the studied genera are assumed to participate in the regulation of development and reproduction of B. subtilis in natural habitats of this spore-forming bacterium.

[Change in the composition of a bacterial association degrading aromatic compounds during oil sludge detoxification in a continuous-flow microbial reactor].

Analysis of oil sludge by direct plating and enrichment cultivation revealed 16 strains degrading aromatic compounds. After 30 days of cultivation in a continuous-flow microbial reactor, 17 more degrader strains were isolated. Genotyping of these strains showed that they were taxonomically diverse, and the range of strains degrading naphthalene, benzene, toluene, ethylbenzene, and xylenes depended on isolation methods. Direct plating yielded more aromatic degraders than enrichment cultivation. A microbial association different from that existing before the enrichment cultivation was obtained in the laboratory continuous-flow reactor.

[Modeling of lactic acid fermentation of leguminous plant juices].

Lactic acid fermentation of leguminous plant juices was modeled to provide a comparative efficiency assessment of the previously selected strains of lactic acid bacteria as potential components of starter cultures. Juices of the legumes fodder galega, red clover, and alfalfa were subjected to lactic acid fermentation in 27 variants of experiment. Local strains (Lactobacillus sp. RS 2, Lactobacillus sp. RS 3, and Lactobacillus sp. RS 4) and the collection strain Lactobacillus plantarum BS 933 appeared the most efficient (with reference to the rate and degree of acidogenesis, ratio of lactic and acetic acids, and dynamics of microflora) in fermenting fodder galega juice; Lactobacillus sp. RS 1, Lactobacillus sp. RS 2, Lactobacillus sp. RS 3, Lactobacillus sp. RS 4, and L. plantarum BS 933 were the most efficient for red clover juice. Correction of alfalfa juice fermentation using the tested lactic acid bacterial strains appeared inefficient, which is explainable by its increased protein content and a low level of the acids produced during fermentation.

[Search for active psychrotrophic microbial oil degraders and their characterization].

The ability of 96 microbial strains degrading oil and 32 strains degrading polycyclic aromatic hydrocarbons (PAHs) to consume diesel fuel and oil at 4-6 degrees C and 24 degrees C and at elevated NaCl concentrations was studied. The temperature range, salt tolerance, ability to produce bioemulsifiers, range of substrates, and antibiotic resistance were determined. The eleven most active oil-degrading and PAH-degrading strains were genotyped by a polymerase chain reaction with BoxA1R primers and a restriction analysis of ribosomal DNA amplicons. For six strains, the degree of oil degradation at 4-6 degrees C was higher than at 24 degrees C. For the most active strains, the degree of oil degradation in liquid mineral medium ranged from 15 to 26% at 24 degrees C and from 28 to 47% at 4-6 degrees C. An artificial association of six of the strains degraded the oil by 46% at 24 degrees C.