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.     

Structural-functional organization of R-plasmid pBS222 with a broad range of bacterial hosts

The broad host-range plasmid pBS222 is compatible with broad host-range plasmids of all known incompatibility groups and codes for tetracycline resistance. pBS222 is efficiently mobilized by Inc P-1 plasmid RP4 and is also capable of conjugal transfer with low efficiency to different gramnegative microorganisms. The size of the plasmid (17.2 Kb) has been determined and its physical map has been constructed. The plasmid harbours the unique sites for restriction endonucleases BglII, HindIII, HpaI, KpnI, SmaI and XbaI cleawage. The plasmid derivatives pBS352-pBS355 have been obtained that carry kan- and cam-determinants in addition to tet-gene. Plasmid pBS355 has been used to clone EcoRI-fragments of phage lambda DNA. The plasmid pBS222 regions essential for replication and maintenance have been localized by DNA hybridization analysis of its mini-derivatives pBS356 and 357. pBS222 is a convenient model for investigations of the plasmid replication and maintenance mechanisms in different bacterial hosts as well as for the construction of broad host-range vectors.     

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.     

Plasmids for biphenyl, chlorobiphenyl and metatoluylate degradation from Pseudomonas putida

Pseudomonas putida strain SU83, harbors the pBS311 plasmid coding for the degradation of biphenyl, 2- and 4-chlorbiphenyl, meta- and paratoluylate. The insertional mutants of the plasmid obtained by the transposon Tn5 insertion were isolated. One of the mutants was used for cloning of the biphenyl degradation genes. The plasmid pBS311:: Tn5 DNA was inserted into the BamHI site of the plasmid pBR322 and cloned. 11 recombinants of 354 tested were treated with 0.1% solution of 2,3-dioxybiphenyl. One of them has acquired the yellow colour testifying to conversion of 2,3-dioxyphenyl to "2-hydroxy-6-keto-6-phenylhexa-2,4-diene acid. The recombinant plasmid pBS312 from this clone is 10.5 kb in size, the size of the insert being 6.2 kb. Escherichia coli SU185 cells harbouring pBS312 are able to support metacleavage of 2,3-dioxybiphenyl, 3-methylcatechol and catechol, but not of 4-methylcatechol. The results suggest the cloned fragment to contain a gene for 2,3-dioxybiphenyl-1,2-dioxygenase, the third enzyme for biphenyl catabolism.     

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.     

Streptomycin-3"-phosphotransferases from streptomycin-resistant cells of Escherichia coli strains

Streptomycin-3"-phosphotransferases were isolated and purified from E. coli cells containing plasmids 836, pBS52 or R6K, which determine the microorganisms resistance towards streptomycin and dihydrostreptomycin. Phosphorylation of the 3"-hydroxylic group of dihydrostreptomycin was demonstrated by [13C]-NMR spectrometry. It was shown that streptomycin-3"-phosphotransferase, whose synthesis is determined by plasmid 836 (as well as by plasmid R6K), differs from the analogous enzyme, whose synthesis is operated by plasmid pBS52 in some properties, e. g. dependence of the initial reaction rate on concentrations of antibiotics and ATP, pH-optimum, sensitivity to the buffer ionic strength, stability, etc. Besides, the antiserum against streptomycin-3'-phosphotransferase detected by plasmid pBS52 does not produce cross immunological reactions with the other enzyme.     

Mutants of the plasmid for biodegradation of naphthalene, determining catechol oxidation via the meta-pathway

Most of the known naphthalene biodegradation plasmids determine the process of naphthalene degradation via salicylate and catechol using the meta pathway of catechol degradation. However, Pseudomonas putida strains with plasmids pBS2, pBS216, pBS217 and NPL-1 exert no activity of the enzymes involved in the meta pathway of catechol degradation. When 2-methylnaphthalene was added to the medium as a sole carbon source, mutants growing on this compound were isolated in the strains with the studied plasmids. Plasmid localization of the mutations was established using conjugation transfer as well as by obtaining spontaneous variants that had lost the ability to grow on 2-methylnaphthalene; the respective plasmid mutants were referred to as pBS101, pBS102, pBS103 and pBS105. The strains with the mutant plasmids were tested for the activity of the key enzymes involved in naphthalene catabolism and the activity of catechol-2,3-dioxygenase was found. The data allow one to arrive at the conclusion that plasmids pBS2, pBS216, pBS217 and NPL-1 contain silent genes for the meta pathway of catechol degradation, which are activated by the respective mutations.     

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.     

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.     

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.     

Expression and regulation of a Bacteroides fragilis sucrose utilization system cloned in Escherichia coli.

A Bacteroides fragilis strain isolated from human feces was the source of chromosomal DNA in the construction of plasmid pBS100. The cloned 6-kilobase insert of plasmid pBS100 conferred a sucrose positivity phenotype on transformed cells of Escherichia coli JA221. E. coli JA221(pBS100) cells were able to utilize sucrose as the sole source of carbon because of the presence of sucrase enzyme and sucrose uptake activities. Sucrase activity was inducible in B. fragilis but constitutive in E. coli JA221(pBS100) cells. In sucrose-minimal medium, both B. fragilis and E. coli JA221(pBS100) produced intracellular and extracellular sucrase activities throughout the growth cycle. Osmotic shock experiments performed on E. coli JA221(pBS100) indicated that up to 55% of the sucrase activity was localized in the periplasmic space, 30% was in the cytoplasm, and the remaining 15% was in the cell-free extracellular supernatant fluid. B. fragilis and E. coli JA221(pBS100) actively transported sucrose. Sucrose uptake was induced by sucrose in B. fragilis, whereas the uptake activity in E. coli JA221(pBS100) was constitutive. E. coli JA221(pBS100) appeared to transport sucrose by a phosphotransferase-independent system. B. fragilis transported sucrose only under strictly anaerobic conditions. No uptake activity was detected under aerobic conditions with or without addition of catalase.     

Expression and regulation of a Bacteroides fragilis sucrose utilization system cloned in Escherichia coli

A Bacteroides fragilis strain isolated from human feces was the source of chromosomal DNA in the construction of plasmid pBS100. The cloned 6-kilobase insert of plasmid pBS100 conferred a sucrose positivity phenotype on transformed cells of Escherichia coli JA221. E. coli JA221(pBS100) cells were able to utilize sucrose as the sole source of carbon because of the presence of sucrase enzyme and sucrose uptake activities. Sucrase activity was inducible in B. fragilis but constitutive in E. coli JA221(pBS100) cells. In sucrose-minimal medium, both B. fragilis and E. coli JA221(pBS100) produced intracellular and extracellular sucrase activities throughout the growth cycle. Osmotic shock experiments performed on E. coli JA221(pBS100) indicated that up to 55% of the sucrase activity was localized in the periplasmic space, 30% was in the cytoplasm, and the remaining 15% was in the cell-free extracellular supernatant fluid. B. fragilis and E. coli JA221(pBS100) actively transported sucrose. Sucrose uptake was induced by sucrose in B. fragilis, whereas the uptake activity in E. coli JA221(pBS100) was constitutive. E. coli JA221(pBS100) appeared to transport sucrose by a phosphotransferase-independent system. B. fragilis transported sucrose only under strictly anaerobic conditions. No uptake activity was detected under aerobic conditions with or without addition of catalase.     

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.     

Structural-functional organization of the incompatibility group P plasmid pBS221

Plasmid pBS221 was physically mapped for restriction endonucleases EcoRI, BamHI, BglII, HindIII. The regions essential for the plasmid existence and participating in replication (oriV trfA*) and mobilization (mob) were cloned. The tet determinant and oriV trfA* regions were localized on the physical map of the plasmid. A DNA sequence homologous to genes of Tn501 mer operon was detected in this plasmid. The studies on homology of plasmids RP4 (IncP alpha), R751 (IncP beta) and pBS221 plasmid suggest that the latter belongs to the IncP beta subgroup.     

Plasmid pBS195 with a wide range of hosts, isolated from lactobacilli

The nonconjugative 4.4 kb plasmid pBS195 has been found in Lactobacillus sp. 195 strain resistant to kanamycin and streptomycin. The plasmid pBS195 determining the resistance to kanamycin has a broad host range. It is inherited by the Gram-positive microorganisms (Bacillus subtilis) as well as by Escherichia coli cells, has the cleavage sites for the restriction endonucleases BamHI, EcoRI, HindIII, PstI, KpnI. The restriction map of the plasmid for these enzymes is constructed. The broad host range, efficiently expressed marker, the presence of the unique restriction sites, small size make the plasmid pBS195 promising for the genetic engineering research.     

Nah-genes of Pseudomonas putida: molecular genetic analysis of the plasmid pBS286

The hybridization and restriction analysis of the plasmid pBS286 (73 Kb, the P-9 Inc group) as well as parental plasmids NPL-1, NPL-41 demonstrated that pBS286 plasmid (delta NPL-41::TnA) with the constitutive synthesis of naphthalene dioxygenase carried genes for naphthalene oxidation to salicylate and those participating in degradation of catechol. Restriction map of pBS286 using XhoI restriction endonuclease and that of the nah region using EcoRI, BamHI, SalI and XhoI were established. Structural peculiarities of nah genes from pBS286 are compared with previously described NAH7. Some nah genes were localized. An inverted DNA segment involved in nah gene regulation was shown to be closely linked to a proximal part of the nah1 operon or overlapped. Possible occurrence of a regulatory R locus in this region is suggested.     

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.     

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.