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.     

Degradation of polychloroaromatic insecticides by Pseudomonas aeruginosa containing biodegradation plasmids

The work was aimed at studying the degradation of DDT, its metabolites and analogs by BS 816 and BS 827 strains constructed on the basis of Pseudomonas aeruginosa 640x strains and carrying biodegradation plasmids pBS2 and pBS3, respectively. DDT and kelthane were degraded by the BS 816 strain at a greater rate than by the parent culture. The investigation of enzymes involved in the oxidation of the aromatic cycle has shown that the plasmid-carrying strains possess the activity of metapyrocatechase and salicylate hydroxylase which is absent in P. aeruginosa 640x. The activity of pyrocatechase increased. In contrast to the parent strain where homogentisate induced only homogentisate oxygenase, this compound induces also metapyrocatechase in the constructed strains.     

Plasmid determined degradation of sulfo-aromatic compounds by Pseudomonas sp. BS1304]

Utilized sulfo-aromatic compounds of Pseudomonas sp. BS1304 were isolated. It was shown that the first step of conversion is desulfonation with following meta-cleavage of the substituted aromatic ring. At least two steps are controlled by the plasmid pBS1004 (120 kb), belonging to the IncP-9 incompatibility group. The degradative plasmid marked by Tn5-lac may be mobilized to P. putida, P. aeruginosa, P. mendocina, where a degradative phenotype is expressed.     

Involvement of naphthalene dioxygenase in indole-3-acetic acid biosynthesis by Pseudomonas putida

Two variants of plant growth-promoting strain Pseudomonas putida BS1380 harboring the naphthalene degradative plasmid pBS2 and the recombinant plasmid pNAU64 that contains the genes encoding for naphthalene dioxygenase were constructed by conjugation. The ability of this strain to produce phytohormone indole-3-acetic acid from different carbon sources was studied. Indole-3-acetic acid synthesis by these transconjugants was 15-30 times as much in contrast to a wild-type strain with glucose as the sole carbon source. No difference was observed in other carbon or nitrogen sources. It is suggested that naphthalene dioxygenase is involved in the conversion of indole-3-pyruvic acid to indole-3-acetic acid.     

Cloning of genes degrading 3-chlorobenzoate from Pseudomonas putida strain 87]

The ability of Pseudomonas putida strain 87 to catabolize 3-chlorobenzoate was shown to be mediated by genes of pBS109 plasmid. The plasmid may be transferred by conjugation into P. aeruginosa PAO2175. It seems possible that the pBS109 plasmid codes for pyrocatechase II specific for halogenated catechol, but not catechol. The genes specifying utilization of 3-chlorobenzoate from pBS109 plasmid were cloned in the 5.5 kb BgIII fragment by using broad-host cloning system. The resulting pBS110 plasmid was transferred into P. putida, which results in utilization of 3-chlorobenzoate by transconjugants.     

Hybrid plasmid pBS251 containing genes for n-alkane degradation

The strain of Pseudomonas aeruginosa BS316 utilizing H-alkanes of the C6-C12 series (Alk+) harbours the 96 Md plasmid pBS250. The use of plasmid RP4 to mobilize Alk+ markers in conjugal transfer to Pseudomonas aeruginosa and Pseudomonas putida has resulted in isolation of transconjugants resistant to antibiotics (due to genes coded by plasmid RP4) and capable of growth on H-alkanes. A transconjugant from this series harbours plasmid pBS251, a derivative of plasmid RP4 containing the genes for octane and octanol catabolism. A fragment of DNA inserted into RP4 has a mol mass 3.8 Md, possesses two restriction sites for EcoRI, one site for PstI, is not restricted by SmaI and BamHI restriction endonucleases, and is localized in the region 4.5-5.7 Md on the physical map of plasmid RP4.     

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.     

Comparative study of aromatic ring meta-cleavage enzymes in Pseudomonas strains with plasmid and chromosomal genetic control of the catabolism of biphenyl and m-toluate

It was shown that two different enzymes of aromatic ring oxidative meta-cleavage (2,3-dihydroxybiphenyl-1,2-dioxygenase), DBO and catechol-2,3-dioxygenase, C230) function in Pseudomonas strains with a plasmid and chromosomal genetic control of biphenyl and toluate catabolism. A comparative analysis of DBO's and C230's expressed by the pBS241 biphenyl degradative plasmid in P. putida BS893, pBS311 in P. putida U83, chromosomal genes in P. putida BF and C230 from P. putida PaW160 (pWWO) was carried out. It was found that the DBO's of all strains under study are highly specialized enzymes in respect of 2,3-dihydroxybiphenyl cleavage and are also able to cleave 3-methyl-catechol and catechol (but not 4-methylcatechol) at low rates. In contrast with DBO's, in Pseudomonas strains the substrate specificities of all C230's are variable. The C230's expressed by the D-plasmids pBS241 and pBC311 have a moderate affinity for catechol, 3-methyl- and 4-methylcatechol, but are unable to cleave 2,3-dihydroxybiphenyl. The C230 which is encoded by the chromosomal structure gene from P. putida BF is very similar to C230 which codes for the TOL-plasmid pWWO. These plasmid differ from C230's expressed by biphenyl D-plasmids due to their capability to cleave 2,3-dihydroxybiphenyl in addition to catechol cleavage. All DBO's and C230's under study possess a number of properties that are typical for the enzymes having an oxidative meta-cleaving effect. The different roles of these enzymes in biphenyl and toluate catabolism in Pseudomonas strains are discussed.     

Cloning and localization of the replication and mobilization regions in the D-plasmid of Pseudomonas putida pBS286 (IncP-9) with a broad host range

Rep-mob loci of naphthalene degradative plasmid pBS286 (IncP-9) have been cloned on the Escherichia coli vectors pUC19 and pUBR322. These loci confer to recombinant plasmids pBS952 and pBS953 the ability for effective mobilization by RP4 (IncP-1) and F plasmid, as well as constant maintenance in various gram-negative bacteria. Localization of cloned sequences in the restriction fragments of conservative part of the pBS286 genome was established. The data obtained correlate with the analysis of plasmids pBS950 and pBS951 which are spontaneous mini-derivatives of pBS286 and pBS292 (delta NPL1::Tn1/Tra+ Nah-) plasmids formed during transformation of E. coli HB101 cells. Plasmids pBS952 and pBS953 retain the incompatibility properties of parental IncP-9 replicon. These recombinant derivatives can be used for construction of bhr vectors with required properties and compatible with bhr vectors constructed on the basis of plasmids from the IncP-1 and IncP-4 groups.     

A new pathway for bacterial catabolism of 3-hydroxybenzoic acid

Abstract This paper is the first to describe the transformation of 3-hydroxybenzoate (3-HBA) by Pseudomonas putida BS893 by a new pathway via 2,3-dihydroxybenzoate (2,3-DBA) and catechol. We have compared the intermediates and appropriate enzyme activities in P. putida BS893 (pBS241) and in a cured derivative BS662 (Bph⁻) thereof, for the ascertainment of plasmid or chromosomal genetic control over 3-HBA-catabolism. The results presented show that catabolism of 3-HBA in P. putida BS893 (pBS241) is controlled by chromosomal genes.     

Incompatibility groups of epsilon-caprolactam biodegradation plasmids from bacteria of Pseudomonas genus

Incompatibility of epsilon-caprolactam biodegradation plasmids pBS262, pBS263, pBS264, pBS265, pBS266, pBS267, pBS268, pBS270, pBS276, pBS269 with the tester plasmids of P-1, P-2, P-7, P-9 incompatibility groups in the system of strains of P. putida line BSA, as well as the character of plasmid interaction with the number of P. aeruginosa and P. putida bacteriophages have been studied. The majority of the studied plasmids belongs to IncP-7, IncP-9 or simultaneously to IncP-7 and IncP-9 incompatibility groups. The ability to restrict the growth of some bacteriophages of P. aeruginosa and P. putida has been demonstrated for some plasmids.     

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.     

Oxidation of n-alkanes by Pseudomonas aeruginosa strain carrying the plasmid pBS251

Pseudomonas aeruginosa PAO8 cannot use n-alkanes or their respective alcohols as a sole carbon source. However, it can grow on n-alkanes when plasmid pBS251 is transferred into its cells. The hybrid plasmid pBS251 is a plasmid RP4 containing genes which control the capability to grow on n-alkanes of the C6-C12 series. Studies of n-alkane oxidation by P. aeruginosa PAO8 carrying pBS251 have shown that this plasmid controls the inducible alkane and alcohol oxidizing activities; the subsequent steps of n-alkane oxidation controlled by chromosomal genes are constitutive.     

A strain of Pseudomonas aeruginosa growing on petroleum hydrocarbons

The strain Pseudomonas aeruginosa BS313 was used to isolate mutants that are capable to utilize octane as the sole carbon source. By means of conjugation plasmids of camphor (CAM) and naphthalene (pBS2) biodegradation were inserted into one of the mutant strains P. aeruginosa BS316. The resultant strain P. aeruginosa BS315 shows the capacity to degrade aliphatic, aromatic and cyclic oil hydrocarbons.     

Transformation of low-molecular linear caprolactam oligomers by the caprolactam-degrading bacterium <Emphasis Type="Italic">Pseudomonas putida</Emphasis> BS394(pBS268)

A biosensor based on the most active caprolactam-degrading strain Pseudomonas putida BS394(pBS268) was used in the study of aerobic degradation of linear caprolactam oligomers by bacterial cells. The changes in the respiratory activity of the strain depend quantitatively on caprolactam dimer concentration, making it possible to develop biosensors for detection of caprolactam oligomers in aqueous media. Based on mass spectrometry data, the scheme of transformation of linear caprolactam oligomers by the degrader strain P. putida BS394(pBS268) was proposed for the first time. It was found that oxidative transamination to respective dicarbonic acids may be one of the mechanisms of transformation of linear caprolactam oligomers. According to the scheme proposed, the ability of the caprolactam-degrading strain to transform linear oligomers results from the broad substrate specificities of two enzymes of the caprolactam degradation pathway: 2-oxoglutarate-6-aminohexanoate transaminase and 6-oxohexanoate dehydrogenase. Transformation of linear oligomers is genetically controlled by the CAP biodegradation plasmid pBS268.     

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.     

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).     

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.     

Comparative study of non-conjugative R-plasmids from enterobacteria and Pseudomonas aeruginosa

Nonconjugative R-plasmids pBS76 and pBS94 (Sm Su), pBS95 and pBS96 (Sm Su Ap) isolated from clinical strains of Pseudomonas aeruginosa and plasmids pKMR281-pKMN284 (Sm Su), pKMR285-pKMR286 (Sm Su Tc) isolated from clinical strains of enterobacteria have been studied. Restriction maps of these plasmids are presented in the paper with some of plasmid genes for antibiotic resistance localized on them. The resistance determinants of plasmids pBS95 and pBS96 are shown to be included in transposon Tn3612 analogous to Tn3. Plasmids pBS76, pBS94-96 are of the wide host range and belong to incompatibility group P4 (IncQ). Plasmids pKMR281-pKMR286 are mutually incompatible and share the conspicuous DNA homology. They are inherited only by enterobacteria and are compatible with IncQ plasmids but in contrast to them are mobilized by RP4 plasmid with lower frequency.     

Molecular genetic organization and origin of plasmid pBS52 with a broad range of bacterial hosts

The data are presented on the localization of genetic determinants of resistance to streptomycin, ampicillin and sulfanilamides on the physical map of conjugative R plasmid pBS52 of 38,000 bp which has a broad bacterial host range and belongs to a new incompatibility group. The plasmid has a natural "polylinker" site (less than 200 bp) containing (in the order of arrangement) the recognition sites for restriction enzymes: BamHI-EcoRI-PstI-EcoRV-BglII (PvuII). The comparative analysis shows that pBS52 contains a segment homologous to DNA of plasmid RSE1010 (IncP-4). The evolutionary origin of plasmid pBS52 is discussed. The recA-independent formation of the mini-derivatives of pBS373 and pBS374 types during the transformation of Escherichia coli with pBS52 plasmid DNA has been shown. Plasmids pBS373 and yBS374 are capable of autonomous replication in Pseudomonas putida and P. aeruginosa cells, which is provided by the rep system of IncP-4 replicon.