Rhizosphere strain of Pseudomonas chlororaphis capable of degrading naphthalene in the presence of cobalt/nickel

Combination of genetic systems of degradation of polyaromatic hydrocarbons, resistance to heavy metals, and promotion of plant growth/protection is one of the approaches to the creation of polyfunctional strains for phytoremediation of soils after combined contamination with organic pollutants and heavy metals. A plant-growth-promoting rhizosphere strain Pseudomonas chlororaphis PCL1391 (pBS216*, pBS501) has been obtained, in which the nah operon of plasmid pBS216 provides naphthalene biodegradation and the cnr-like operon of plasmid pBS501 provides resistance to cobalt and nickel due to the withdrawal of heavy metal cations from the cells. In the presence of 100 microM of nickel, the viability, growth rate, and naphthalene biodegradation efficiency of the resistant strain PCL1391 (pBS216*, pBS501) were much higher as compared with the sensitive PCL1391 (pBS216). During the growth of the resistant strain, in contrast to the sensitive strain, nickel (100 microM) had no inhibiting effect on the activity of the key enzymes of naphthalene biodegradation.     

Rhizosphere strain of <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> capable of degrading naphthalene in the presence of cobalt/nickel

Combination of genetic systems of degradation of polyaromatic hydrocarbons, resistance to heavy metals, and promotion of plant growth/protection is one of the approaches to the creation of polyfunctional strains for phytoremediation of soils after co-contamination with organic pollutants and heavy metals. A plant-growth-promoting rhizosphere strain Pseudomonas chlororaphis PCL1391 (pBS216^*, pBS501) has been obtained, in which the nah operon of plasmid pBS216 provides naphthalene biodegradation and the cnr-like operon of plasmid pBS501 provides resistance to cobalt and nickel due to the extrusion of heavy metal cations from the cells. In the presence of 100 μM of nickel, the viability, growth rate, and naphthalene biodegradation efficiency of the resistant strain PCL1391 (pBS216^*, pBS501) were much higher as compared with the sensitive PCL1391 (pBS216). During the growth of the resistant strain, in contrast to the sensitive strain, nickel (100 μM) had no inhibiting effect on the activity of the key enzymes of naphthalene biodegradation. Original Russian Text ? T.V. Siunova, T.O. Anokhina, A.V. Mashukova, V.V. Kochetkov, A.M. Boronin, 2007, published in Mikrobiologiya, 2007, Vol. 76, No. 2, pp. 212–218.     

Effect of sodium salicylate on the population dynamics of a rhizospheric strain of Pseudomonas aureofaciens in soil and on wheat roots

The effect of sodium salicylate on the population dynamics of the rhizobacterium Pseudomonas aureofaciens BS1393 and its variant bearing the naphthalene biodegradation plasmid pBS216 was studied in the wheat rhizoplane and adjacent soil. Optimum salicylate concentration for the maintenance of the plasmid-bearing strain and for the normal growth of wheat was found to be 250 micrograms/g soil. When the soil was supplemented with salicylate, the population of P. aureofaciens BS1393(pBS216) in the wheat rhizoplane and adjacent soil was, respectively, 4- and 20-fold higher than that of the parent strain lacking the plasmid.     

Effect of naphthalene biodegradation plasmids on physiological characteristics of rhizospheric bacteria of the genus Pseudomonas

Specific growth rate, duration of the lag phase, stability of plasmids, and activities of the key enzymes involved in naphthalene biodegradation were studied in rhizospheric pseudomonades carrying structurally similar plasmids pOV17 and pBS216. It was demonstrated that these plasmids determined various levels of catechol 2,3-dioxygenase activities. The structural rearrangements in the plasmid pBS216 could "switch off" the genes of catechol oxidation meta-pathway. It was shown that certain combinations of biodegradation plasmids and bacterial hosts, such as Pseudomonas chlororaphis PCL1391(pBS216), P. chlororaphis PCL1391(pOV17), and P. putida 53a(pOV17), were considerably more efficient than natural variants in their growth characteristics and stability of the biodegradation activity, having a potential for bioremediation of soils polluted with polycyclic aromatic hydrocarbons (PAHs).     

Effect of Sodium Salicylate on the Population Dynamics of the Rhizobacterium Pseudomonas aureofaciens in the Wheat Rhizoplane and Adjacent Soil

The effect of sodium salicylate on the population dynamics of the rhizobacterium Pseudomonas aureofaciens BS1393 and its variant bearing the naphthalene biodegradation plasmid pBS216 was studied in the wheat rhizoplane and adjacent soil. Optimum salicylate concentration for the maintenance of the plasmid-bearing strain and for the normal growth of wheat was found to be 250 g/g soil. When the soil was supplemented with salicylate, the population of P. aureofaciens BS1393(pBS216) in the wheat rhizoplane and adjacent soil was, respectively, 4- and 20-fold higher than that of the parent strain lacking the plasmid.     

Effects of Naphthalene Degradative Plasmids on the Physiological Characteristics of Rhizosphere Bacteria of the Genus <Emphasis Type="Italic">Pseudomonas</Emphasis>

We studied the specific growth rate, duration of the lag phase, stability of plasmids, and activities of the key enzymes involved in naphthalene biodegradation in rhizosphere pseudomonades carrying the structurally similar plasmids pOV17 and pBS216. It was demonstrated that these plasmids determined various levels of catechol-2,3-dioxygenase activities. The structural rearrangements in the plasmid pBS216 could “switch off” the genes of the catechol oxidation meta-pathway. It was shown that certain combinations of degradative plasmids and bacterial hosts, such as Pseudomonas chlororaphis PCL1391(pBS216), P. chlororaphis PCL1391(pOV17), and P. putida 53a(pOV17), were considerably more efficient than natural variants in their growth characteristics and the stability of the biodegradation activity, having a potential for bioremediation of soils polluted with polycyclic aromatic hydrocarbons (PAHs).     

Plant growth-promoting Pseudomonas bearing catabolic plasmids: Naphthalene degradation and effect on plants

Two IncP-9 naphthalene degradative plasmids pOV17 and pBS216 were transferred into plant growth-promoting Pseudomonas which were represented by species P. aureofaciens, P. chlororaphis, P. fluorescens, and P. putida. The strains with the same plasmid differed significantly by their growth parameters, stability of the plasmid and plant protective effect from naphthalene action. Strains P. putida 53a(pOV17) and P. chlororaphis PCL1391(pOV17) demonstrated higher population number in the rhizosphere. Moreover, they protected the mustard plants from naphthalene toxic influence more effectively than the wild type strain P. aureofaciens OV17(pOV17). The activity of catechol-2,3-dioxygenase in the strains with the plasmid pOV17 was higher than that in strains with the plasmid pBS216. The strain P. putida 53a(pOV17) with high catechol-2,3-dioxygenase activity has been demonstrated to have the best protective effect. The strain P. putida 53a(pBS216) without catechol dioxygenases activities did not have protective effect but suppressed the plant germination.     

Effect of catabolic plasmids on physiological parameters and efficiency of oil destruction by Pseudomonas bacteria

The ability of microbial degraders of polycyclic aromatic hydrocarbons to grow at 24°C in liquid mineral medium supplemented with oil as the sole source of carbon and energy was studied. Growth characteristics (CFU) and the level of oil destruction by plasmid-bearing and plasmid-free strains were determined after seven days of cultivation. The presence of catabolic plasmids in the degrader strains, including rhizosphere pseudomonads, was shown to increase cell growth and enhance the level of oil degradation. Strain Pseudomonas chlororaphis BS1391 bearing plasmid pBS216 was found to be the most effective oil degrader.     

Effect of catabolic plasmids on physiological parameters and efficiency of oil destruction by bacteria of the genus Pseudomonas

The ability of microbial degraders of polycyclic aromatic hydrocarbons to grow at 24 degrees C in liquid mineral medium supplemented with oil as the sole source of carbon and energy was studied. Growth characteristics (CFU) and the level of oil destruction by plasmid-bearing and plasmid-free strains were determined after seven days of cultivation. The presence of catabolic plasmids in the degrader strains, including rhizosphere pseudomonads, was shown to increase cell growth and enhance the level of oil degradation. Strain Pseudomonas chlororaphis BS 1391 bearing plasmid pBS216 was found to be the most effective oil degrader.     

Phenanthrene degradation by bacteria of the genera Pseudomonas and Burkholderia in model soil systems

Degradation of phenanthrene by strains Pseudomonas putida BS3701 (pBS1141, pBS1142), Pseudomonas putida BS3745 (pBS216), and Burkholderia sp. BS3702 (pBS1143) was studied in model soil systems. The differences in accumulation and uptake rate of phenanthrene intermediates between the strains under study have been shown, Accumulation of 1-hydroxy-2-naphthoic acid in soil in the course of phenanthrene degradation by strain BS3702 (pBS143) in a model system has been revealed. The efficiency of phenanthrene biodegradation was assessed using the mathematical model proposed previously for assessment of naphthalene degradation efficiency. The efficiency of degradation of both phenanthrene and the intermediate products of its degradation in phenanthrene-contaminated soil is expected to increase with the joint use of strains P. putida BS3701 (pBS1141, pBS1142) and Burkholderia sp. BS3702 (pBS1143).     

Molecular classification of IncP-9 naphthalene degradation plasmids

A large collection of naphthalene-degrading fluorescent Pseudomonas strains isolated from sites contaminated with coal tar and crude oil was screened for the presence of IncP-9 plasmids. Seventeen strains were found to carry naphthalene catabolic plasmids ranging in size from 83 to 120 kb and were selected for further study. Results of molecular genotyping revealed that 15 strains were closely related to P. putida, one to P. fluorescens, and one to P. aeruginosa. All catabolic plasmids found in these strains, with the exception of pBS216, pSN11, and p8909N-1, turned out to belong to IncP-9 beta-subgroup. Plasmids pBS216, pSN11, and p8909N-1 were identified as members of IncP-9 delta-subgroup. One plasmid, pBS2, contains fused replicons of IncP-9beta and IncP-7 groups. RFLP analyses of the naphthalene catabolic plasmids revealed that organisation of the replicon correlates well with the overall plasmid structure. Comparative PCR studies with conserved oligonucleotide primers indicated that genes for key enzymes of naphthalene catabolism are highly conserved among all studied plasmids. Three bacterial strains, P. putida BS202, P. putida BS3701, and P. putida BS3790, were found to have two different salicylate hydroxylase genes one of which has no similarity to the "classic" enzyme encoded by nahG gene. Discovery of a large group of plasmid with unique nahR suggested that the regulatory loop may also represent a variable part of the pathway for catabolism of naphthalene in fluorescent Pseudomonas spp.     

The production of phenazine antibiotics by the Pseudomonas aureofaciens strain with plasmid-controlled resistance to cobalt and nickel

Plasmid pBS501 responsible for the resistance of the wild-type Pseudomonas sp. BS501 (pBS501) to cobalt and nickel ions was conjugatively transferred to the rhizosphere Pseudomonas aureofaciens strain BS1393, which is able to synthesize phenazine antibiotics and to suppress a wide range of phytopathogenic microorganisms. The transconjugant P. aureofaciens BS1393 (pBS501) turned out to be resistant to cobalt and nickel with an MIC of 8 mM. When grown in a synthetic medium with 0.25 mM cobalt, the transconjugant accumulated 6 times more cobalt than the wild-type strain BS501 (pBS501) (1.2 and 0.2 microgram Co/mg protein). Electron microscopic studies showed that cobalt accumulates on the surface of transconjugant cells in the form of electron-opaque granules. In a culture medium with 2 mM cobalt or nickel, strain BS1393 produced phenazine-1-carboxylic acid in trace amounts. The transconjugant P. aureofaciens BS1393 (pBS501) produced this antibiotic in still smaller amounts. Unlike the parent strain BS1393, the transconjugant P. aureofaciens BS1393 (pBS501) was able to suppress in vitro the growth of the phytopathogenic fungus Gaeumannomyces graminis var. tritici 1818 in a medium containing 0.5 mM cobalt or nickel.     

The Production of Phenazine Antibiotics by the Pseudomonas aureofaciens Strain with Plasmid-Controlled Resistance to Cobalt and Nickel

Plasmid pBS501, responsible for the resistance of the wild-type Pseudomonas sp. BS501(pBS501) to cobalt and nickel ions, was conjugatively transferred to the rhizosphere Pseudomonas aureofaciens strain BS1393, which is able to synthesize phenazine antibiotics and to suppress a wide range of phytopathogenic microorganisms. The transconjugant P. aureofaciens BS1393(pBS501) turned out to be resistant to cobalt and nickel with an MIC of 8 mM. When grown in a synthetic medium with 0.25 mM cobalt, the transconjugant accumulated 6 times more cobalt than the wild-type strain BS501(pBS501) (1.2 versus 0.2 g Co/mg protein). Electron microscopic studies showed that cobalt accumulates on the surface of transconjugant cells in the form of electron-opaque granules. In a culture medium with 2 mM cobalt or nickel, strain BS1393 produced phenazine-1-carboxylic acid in trace amounts. The transconjugant P. aureofaciens BS1393(pBS501) produced this antibiotic in still smaller amounts. Unlike the parent strain BS1393, the transconjugant P. aureofaciens BS1393(pBS501) was able to suppress in vitro the growth of the phytopathogenic fungus Gaeumannomyces graminis var. tritici1818 in a medium containing 0.5 mM cobalt or nickel.     

Phenanthrene degradation by bacteria of the genera <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Burkholderia</Emphasis> in model soil systems

Degradation of phenanthrene by strains Pseudomona, Moscow, KMK, 2004simova, I.A. and Chernov, I.s putida BS3701 (pBS1141, pBS1142), Pseudomonas putida BS3745 (pBS216), and Burkholderia sp. BS3702 (pBS1143) were studied in model soil systems. The differences in accumulation and uptake rate of phenanthrene intermediates between the strains under study have been shown. Accumulation of 1-hydroxy-2-naphthoic acid in soil in the course of phenanthrene degradation by strain BS3702 (pBS1143) in a model system has been revealed. The efficiency of phenanthrene biodegradation was assessed using the mathematical model proposed previously for assessment of naphthalene degradation efficiency. The efficiency of degradation of both phenanthrene and the intermediate products of its degradation in phenanthrene-contaminated soil is expected to increase with the joint use of strains P. Putida BS3701 (pBS1141, pBS1142) and Burkholderia sp. BS3702 (pBS1143).     

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.     

Growth and plasmid-encoded naphthalene catabolism of Pseudomonas putida in batch culture

Abstract The growth characteristics of Pseudomonas putida plasmid-harbouring strains which catabolize naphthalene via various pathways in batch culture with naphthalene as the sole source of carbon and energy have been investigated. The strains under study were constructed using the host strain P. putida BS394 which contained various naphthalene degradation plasmids. The highest specific growth rate was ensured by the plasmids that control naphthalene catabolism through the meta-pathway of catechol oxidation. The strains metabolizing catechol via the ortho -pathway grew at a lower rate. The lowest growth rate was observed with strain BS291 harbouring plasmid pBS4 which controls naphthalene catabolism via the gentisic acid pathway. Various pathways of naphthalene catabolism appear to allow these strains to grow at various rates which should be taken into account when constructing efficient degraders of polycyclic aromatic compounds.     

Effect of arsenite on the physiological and cytological properties of Pseudomonas putida strains capable of degrading polycyclic aromatic hydrocarbons]

Some physiological and cytological properties of Pseudomonas putida strains resistant to arsenite and capable of degrading polycyclic aromatic hydrocarbons were studied. The resistance of P. putida BS202 (NPL-1) to arsenite proved to be determined by chromosomal genes, while the arsenite resistance of P. putida BS238 (pBS2; pBS3031) was by plasmid-borne genes. Arsenite affected the pattern and rate of growth of strain BS202 (NPL-1) in media with naphthalene or salicylate as carbon sources; particularly, it lengthened the lag phase. Electron-microscope analysis of the strains studied did not reveal any arsenite-induced destructive changes in the cell envelope. At the same time, arsenite in the growth medium induced some alterations in the structure of the outer membrane of strain BS202 (NPL-1) and the cytoplasmic membrane of strain BS238 (pBS2; pBS3031) and, in both strains, led to an increase in the density of intramembrane particles on the EF face of the freeze-fractured cytoplasmic membrane. Arsenite resistance probably evidently protects cells of both strains from greater damage. Physiological and cytological data suggest that the mechanisms of arsenite resistance in the strains studied are different.     

Kelthane degradation by genetically engineered Pseudomonas aeruginosa BS827 in a soil ecosystem

The aim of this study was to study the degradation of kelthane by Pseudomonas aeruginosa BS827, which carried the plasmid pBS3. This plasmid encodes naphthalene oxidation. The strain was able to survive in the presence of kelthane and to retain its degradative ability. Kelthane also stabilized the biodegradative plasmid that was preserved by 70 to 100% of the cell population. Cells deficient in Nah or Sal characters were less effective in degrading kelthane, whereas plasmid-free cells lost this ability completely. Evidently, the degradative activity of P. aeruginosa BS827 was conditioned by plasmid determinants coupled with genes of the plasmid pBS3 Nah region.     

Catabolism of biphenyl by Pseudomonas putida BS 893 strain containing the biodegradation plasmid pBS241

The isolation and identification of biphenyl catabolism products in Pseudomonas putida BS 893 (pBS241) showed the presence of benzoic, m-hydroxybenzoic and cinnamic acids. The two latter compounds were not found in biphenyl degradation by other bacterial strains. P. putida BS 893 (pBS241) differed from other biphenyl-positive Pseudomonas strains in the enzyme activity. These differences may stem from peculiarities in the pathway of biphenyl catabolism controlled by plasmid pBS241.     

Kelthane degradation by genetically engineered Pseudomonas aeruginosa BS827 in a soil ecosystem.

The aim of this study was to study the degradation of kelthane by Pseudomonas aeruginosa BS827, which carried the plasmid pBS3. This plasmid encodes naphthalene oxidation. The strain was able to survive in the presence of kelthane and to retain its degradative ability. Kelthane also stabilized the biodegradative plasmid that was preserved by 70 to 100% of the cell population. Cells deficient in Nah or Sal characters were less effective in degrading kelthane, whereas plasmid-free cells lost this ability completely. Evidently, the degradative activity of P. aeruginosa BS827 was conditioned by plasmid determinants coupled with genes of the plasmid pBS3 Nah region.