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.

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.

[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.

[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.

[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.

[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.

[Occurrence of the SAL+ phenotype in soil pseudomonads].

Genetic systems of salicylate catabolism were studied in 75 strains of fluorescent pseudomoriads and in 30 exogenously isolated SAL plasmids. All exogenously isolated SAL plasmids were found to contain the classical nahG gene in combination with the genes of the meta-pathway of catechol cleavage. In most studied strains, salicylate catabolism was controlled by the chromosomal genes, the nah Ugene being the key gene ofsalicylate utilization and subsequent catechol cleavage occurring via the ortho-pathway. It is suggested that the nah U-like sequences play a key role in occurrence of the Sal+ phenotype in strains degrading salicylate, but not naphthalene.

[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.

[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.

[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.