I. A. Kosheleva

Phenanthrene metabolism by Pseudomonas and Burkholderia strains

Abstract Five pseudomonads and two Burkholderia spp. capable of utilizing naphthalene or phenanthrene as a sole source of carbon and energy were isolated from oil-contaminated soils through enrichment cultures. All the strains studied contained large plasmids. In six strains, they were involved in biodegradation of specific substrates. The major intermediates of phenanthrene metabolism accumulated in the culture liquids were 1-hydroxy-2-naphthoate and 2-hydroxy-1-naphthoate. The strains studied differed in the accumulation pattern of these intermediates. 2-Naphthol, an oxidation product of 2-hydroxy-1-naphthoate, exerted a toxic effect on microorganisms. The phenanthrene-degrading activity of Pseudomonas strains depended on their ability to utilize 1-hydroxy-2-naphthoate. In the Pseudomonas strains under study, phenanthrene metabolism involved the enzymes of naphthalene degradation.

Diversity of IncP-9 plasmids of Pseudomonas

IncP-9 plasmids are important vehicles for degradation and resistance genes that contribute to the adaptability of Pseudomonas species in a variety of natural habitats. The three completely sequenced IncP-9 plasmids, pWW0, pDTG1 and NAH7, show extensive homology in replication, partitioning and transfer loci (an ∼25 kb region) and to a lesser extent in the remaining backbone segments. We used PCR, DNA sequencing, hybridization and phylogenetic analyses to investigate the genetic diversity of 30 IncP-9 plasmids as well as the possibility of recombination between plasmids belonging to this family. Phylogenetic analysis of rep and oriV sequences revealed nine plasmid subgroups with 7–35 % divergence between them. Only one phenotypic character was normally associated with each subgroup, except for the IncP-9β cluster, which included naphthalene- and toluene-degradation plasmids. The PCR and hybridization analysis using pWW0- and pDTG1-specific primers and probes targeting selected backbone loci showed that members of different IncP-9 subgroups have considerable similarity in their overall organization, supporting the existence of a conserved ancestral IncP-9 sequence. The results suggested that some IncP-9 plasmids are the product of recombination between plasmids of different IncP-9 subgroups but demonstrated clearly that insertion of degradative transposons has occurred on multiple occasions, indicating that association of this phenotype with these plasmids is not simply the result of divergent evolution from a single successful ancestral degradative plasmid.

The P-7 incompatibility group plasmids responsible for biodegradation of naphthalene and salicylate in fluorescent pseudomonads

Analysis of seven plasmids (77 to 135 kb in size) of the P-7 incompatibility group that are responsible for the biodegradation of naphthalene and salicylate has shown that the main natural host of IncP-7 plasmids is the species Pseudomonas fluorescens. The IncP-7 plasmids are structurally diverse and do not form groups, as is evident from their cluster analysis. The naphthalene catabolism genes of six of the IncP-7 plasmids are conservative and homologous to the catabolic genes of NAH7 and pDTG1 plasmids. The pAK5 plasmid contains the classical nahA gene, which codes for naphthalene dioxygenase, and the salicylate 5-hydroxylase gene (nagG) sequence, which makes the conversion of salicylate to gentisate possible.

Bacterial Degraders of Polycyclic Aromatic Hydrocarbons Isolated from Salt-Contaminated Soils and Bottom Sediments in Salt Mining Areas

Fifteen bacterial strains capable of utilizing naphthalene, phenanthrene, and biphenyl as the sole sources of carbon and energy were isolated from soils and bottom sediments contaminated with waste products generated by chemical- and salt-producing plants. Based on cultural, morphological, and chemotaxonomic characteristics, ten of these strains were identified as belonging to the genera Rhodococcus, Arthrobacter, Bacillus, and Pseudomonas. All ten strains were found to be halotolerant bacteria capable of growing in nutrient-rich media at NaCl concentrations of 1–1.5 M. With naphthalene as the sole source of carbon and energy, the strains could grow in a mineral medium with 1 M NaCl. Apart from being able to grow on naphthalene, six of the ten strains were able to grow on phenanthrene; three strains, on biphenyl; three strains, on octane; and one strain, on phenol. All of the strains were plasmid-bearing. The plasmids of the Pseudomonas sp. strains SN11, SN101, and G51 are conjugative, contain genes responsible for the degradation of naphthalene and salicylate, and are characterized by the same restriction fragment maps. The transconjugants that gained the plasmid from strain SN11 acquired the ability to grow at elevated NaCl concentrations. Microbial associations isolated from the same samples were able to grow at a NaCl concentration of 2.5 M.Fifteen bacterial strains capable of utilizing naphthalene, phenanthrene, and biphenyl as the sole sources of carbon and energy were isolated from soils and bottom sediments contaminated with waste products generated by chemical- and salt-producing plants. Based on cultural, morphological, and chemotaxonomic characteristics, ten of these strains were identified as belonging to the genera Rhodococcus, Arthrobacter, Bacillus, and Pseudomonas. All ten strains were found to be halotolerant bacteria capable of growing in nutrient-rich media at NaCl concentrations of 1–1.5 M. With naphthalene as the sole source of carbon and energy, the strains could grow in a mineral medium with 1 M NaCl. Apart from being able to grow on naphthalene, six of the ten strains were able to grow on phenanthrene; three strains, on biphenyl; three strains, on octane; and one strain, on phenol. All of the strains were plasmid-bearing. The plasmids of the Pseudomonas sp. strains SN11, SN101, and G51 are conjugative, contain genes responsible for the degradation of naphthalene and salicylate, and are characterized by the same restriction fragment maps. The transconjugants that gained the plasmid from strain SN11 acquired the ability to grow at elevated NaCl concentrations. Microbial associations isolated from the same samples were able to grow at a NaCl concentration of 2.5 M.

The construction and monitoring of genetically tagged, plasmid-containing, naphthalene-degrading strains in soil

A genetically tagged, plasmid-containing, naphthalene-degrading strain, Pseudomonas putida KT2442(pNF142:: TnMod-OTc), has been constructed. The presence of the gfp gene (which codes for green fluorescent protein) and the kanamycin and rifampicin resistance genes in the chromosome of this strain allows the strain’s fate in model soil systems to be monitored, whereas a minitransposon, inserted into naphthalene biodegradation plasmid pNF142 and containing the tetracycline resistance gene, makes it possible to follow the horizontal transfer of this plasmid between various bacteria. Plasmid pNF142::TnMod-OTc is stable in strain P. putida KT2442 under nonselective conditions. The maximal specific growth rate of this strain on naphthalene is found to be higher than that of the natural host of plasmid pNF142. When introduced into a model soil system, the genetically tagged strain is stable and competitive for 40 days. The transfer of labeled plasmid pNF142::TnMod-OTc to natural soil bacteria, predominantly fluorescent pseudomonads, has been detected.

The efficiency of salicylate utilization by Pseudomonas putida strains catabolizing naphthalene via different biochemical pathways.

Abstract Parameters of growth and substrate consumption by plasmid-harbouring Pseudomonas putida strains catabolizing naphthalene via different biochemical pathways under batch cultivation on salicylate were estimated. The values of a specific growth rate, μ , and the yield coefficient, Y , for natural isolates with the functioning genes of the ortho-pathway and silent genes of meta cleavage of catechol oxidation were less than those for isogenous strains with the functioning genes of the meta-pathway. All strains under study with functioning genes of the meta-pathway were shown to possess the activities of the enzymes both of ortho- and meta pathway of catechol oxidation: catechol-1,2-dioxygenase and catechol-2,3-dioxygenase. The comparison of the functioning of the genes of the ortho- and meta-pathways was carried out in an isogenous host background. An approach to estimate the efficiency of the biodegradation process using the kinetics of microbial growth and substrate consumption under batch cultivation was proposed.

Growth and survival of Pseudomonas putida strains degrading naphthalene in soil model systems with different moisture levels

The effect of different moisture levels (from 20 to 70%) on the growth and survival of Pseudomonas putida strains G7 and BS3701 degrading naphthalene was studied in soil model systems. P.putida G7 contains plasmid NAH7 and P.putida BS3701 harbours plasmids pBS1141 and pBS1142. A mathematical model is proposed to describe the observed dynamics of the number of viable bacterial cells. Naphthalene and soil organic matter were considered as substrates available to bacteria. Data fitting allowed the estimation of model parameters characterizing microbial growth rate, utilization rate of substrates, specific maintenance rate and yield coefficient. Both the maximum bacterial concentration and the highest yield coefficient were observed at a soil moisture level of 40%. This optimal moisture level is close to but less than the water capacity (48%) of the soil used.

Pseudomonas putida as a receptor element of microbial sensor for naphthalene detection

Abstract Pseudomonas putida BS238 carrying the naphthalene degradative plasmid pBS2 was immobilized by adsorption on chromatographic paper and fixed on the surface of a Clark electrode. Naphthalene activated the respiratory activity of the cells, which was the basis for determining its concentration in one-component aqueous media. The lower limit of naphthalene detection was in the region of 0·9 μ m . The maximal responses of the sensor were observed at pH 6·0–6·2, 20–25°C and a salt concentration 2·9–3·2 g ion/litre. The sensor responded weakly to the presence of biphenyl, monocyclic aromatic compounds, ethanol, carbohydrates and organic acids in the medium. A sensor based on an isogenic strain without plasmid pBS2 had no response to naphthalene. The possibility of improving the selectivity of naphthalene detection using a pair of sensors based on the original and isogenic strains is discussed.

Degradation of Phenanthrene by Mutant Naphthalene-Degrading Pseudomonas putida Strains

Five naphthalene- and salicylate-utilizing Pseudomonas putida strains cultivated for a long time on phenanthrene produced mutants capable of growing on this substrate and 1-hydroxy-2-naphthoate as the sole sources of carbon and energy. The mutants catabolize phenanthrene with the formation of 1-hydroxy-2-naphthoate, 2-hydroxy-1-naphthoate, salicylate, and catechol. The latter products are further metabolized by the meta- and ortho-cleavage pathways. In all five mutants, naphthalene and phenanthrene are utilized with the involvement of plasmid-born genes. The acquired ability of naphthalene-degrading strains to grow on phenanthrene is explained by the fact that the inducible character of the synthesis of naphthalene dioxygenase, the key enzyme of naphthalene and phenanthrene degradation, becomes constitutive.

Salicylate degradation by Pseudomonas putida strains not involving the “Classical” nah2 operon

Genetic systems for salicylate catabolism were analyzed in 12 strains of Pseudomonas putida, isolated from polluted soil samples collected in the Murmansk and Tula oblasts. All of the studied P. putida strains utilize salicylate in the ortho-pathway of catechol cleavage without employing the enzymes of the “classical” nah2 operon. The data demonstrates that salicylate degradation in the studied strains is performed with the involvement of the salicylate hydroxylase gene analogous to the nahU gene of strain P. putida ND6. New variants of salicylate hydroxylase genes nahG1 and nahU were found.