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.     

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.     

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.     

Analysis of the conjugation system of plasmid pBS1001 with a broad range of hosts]

Tn5-induced tra mutations were localized on the physical map of a broad host range plasmid pBS1001. Mutations were united into three clusters covering 25% of plasmid DNA. They were distributed in 7 groups by complementation analysis. It was shown that coexistence of tra mutants of pBS1001 and RP4 within the same cell did not restore conjugation properties of both plasmids. High frequency mobilization of some known vectors by pBS1001 was demonstrated.     

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.     

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.     

Cloning of Pseudomonas putida genes responsible for the primary stages of oxidation of naphthalene in Escherichia coli cells

Data on cloning Pseudomonas putida D-plasmid pBS286 (IncP-9) genes which are responsible for primary stages of naphthalene oxidation as well as data on the expression of these genes in Escherichia coli cells are presented. Recombinant plasmid pBS959 containing the whole constitutive nahA locus encoding naphthalene dioxygenase, a key enzyme of the naphthalene oxidation pathway, has been constructed on the basis of the pUC19 vector. An evidence has been obtained that at least a portion of the sequence of structural nahB gene is cloned in the recombinant pBS959 plasmid. Constitutive expression of the nahA gene is controlled by its own promoter and leads to conversion of indole to indigo in E. coli cells, strain HB101. Plasmid pBS959 is characterized by high structural stability; some instability observed is due to segregation.     

Identification and nucleotide sequence of the minimal replicon of the low-copy-number plasmid pBS2

The plasmid pBS2 has a low copy number and is endogenous to Bacillus subtilis. The replication of this plasmid depends on the function of most of the host's dna genes including dnaB, which is unique to B. subtilis and is required for both the initiation of chromosome replication and the DNA-membrane association. We have identified the region that is essential for the replication of pBS2 and determined the complete 2279-bp nucleotide sequence of this region. In this region, there are two stretches of sequence homologous to the 18-bp consensus sequence which commonly appears at the origin of replication of plasmids pUB110 and pC194. The entire region contains six sizable open reading frames. Two of them are probably translated. One open reading frame, designated ORF A, coding for 269 amino acids, has significant homology, in terms of amino acid sequence, with the open reading frame of the gene for the Rep U protein of plasmid pUB110. The similarities between pBS2 and other plasmids suggest that the pBS2 may also replicate as a rolling circle, which appears to be the salient feature of a mechanism of replication that is common to small plasmids in gram-positive bacteria.     

Genetic and physical studies of recombinant plasmids formed between an R plasmid of compatibility group FI and sex factor F of HfrH.

Recombinant plasmids between an R plasmid of the FI group (R162/3) and the sex factor F or HfrH were produced after the conjugal transfer of this R plasmid into HfrH. Three types of recombinant plasmids were identified after the mating of HfrH (R162/3) with recA and rec+ recipients. One specimen of each type (pIP218, pIP222, pIP226) was studied in this report. All three recombinant plasmids carry the same genetic information for resistance to antibiotics (CSSuT) retained from R162/3. pIP218 retained all the other properties from F of HfrH: derepression for pilus synthesis, mobilization of the chromosome for the proximally transferred HfrH genes (thr, leu, proA), interference with T7 propagation, and ability to be cured by acridine orange. pIP222 retained from F of HfrH the derepression for pilus synthesis and the same polarity of chromosome transfer (thr, leu, proA), while pIP226 retained the interference with T7 propagation and acridine orange curing. Physical studies revealed that replication control and/or recovery of F and pIP218 as covalent circles of deoxyribonucleic acid are similar, and are different from R162/3. The new plasmids are more likely the result of a substitutive recombination event than a fusion. We propose genetic maps of these recombinant plasmids, showing the unequal participation of the parental plasmids in their formation.     

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.     

Localization in Escherichia coli of transcribed regions of the broad host range plasmid pBS222

The set of insertions of the CmR-gene region devoid of the promoter into the broad host-range plasmid pBS222 regions transcribed in Escherichia coli cells has been obtained and characterized. Three transcribed regions of the plasmid that are dispensable for life functions of the plasmid have been identified by the insertions technique. The deleted plasmid derivatives have been isolated suitable for using as the broad host-range vectors.     

Use of the plasmid R89S replicon for constructing integration vectors

It has been shown that the plasmid R89S derivatives can be used as integrative vectors for bacteria in which the plasmid is unable to replicate autonomously. The chromosomal and plasmid fragments of phototrophic bacterium Rhodobacter sphaeroides have been cloned in plasmid pVZ365, a SmRKmR-derivative of R89S. The obtained recombinant plasmids were mobilized into R. sphaeroides cells by the I pcP-group conjugative plasmid R751. The frequencies of the SmR-transconjugants formation are 3.7.10(-5) to 5.6.10(-3) per recipient cell. The formation of the SmR-transconjugants has not been revealed in case of the plasmid pVZ365 mobilization. The recombinant molecules containing R. sphaeroides plasmid fragments have been shown to integrate into endogenous plasmids and form cointegrates with them.     

Genetic characteristics and physical organization of the R-plasmid pBS52 with a broad range of bacterial hosts

The genetic and physical data on Pseudomonas aeruginosa plasmid pBS52 coding for the resistances to ampicillin, streptomycin and sulfonamids have been obtained. This conjugative plasmid is transferable to a broad range of gram-negative bacterial hosts and compatible with the broad host-range plasmids from all known incompatibility groups. The plasmid size has been determined (38 Kb) and a physical map has been constructed using restriction endonucleases EcoRI, EcoRV, BamHI, BglII, PstI, PvuII, SalI, SlaI. The presence of a fragment, approximately 200 bp in size, which contains the sites for many of widely used restriction endonucleases is a characteristic feature of the plasmid pBS52.     

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).     

Mutations of plasmid pBS286 blocking the initial stages of naphthalene oxidation induced by Tn5

A collection of Tn5 transposon Nah- mutants of the plasmid pBS286 was obtained. The insertion sites were localized and orientation of Tn5 determined. The mutants obtained were biochemically analyzed, the nah-region map of the plasmid being elaborated. Structural genes of the nah operon were shown to be organized similarly to those of the nah1 operon of the NAH7 plasmid discussed in the literature. The data obtained are in favour of the previously published information on the presence of elements operating the pBS286 plasmid. The results are given indicating a possibility of regulating the expression of catechol splitting meta-pathway genes with participation of products on early stages of naphthalene oxidation.     

Restriction mapping of recombinant plasmids carrying the genes for arginine biosynthesis in Escherichia coli K-12

ArgA and argECBH genes of Escherichia coli K-12 were cloned on the pBR322 vector. Restriction maps of the recombinant plasmids were constructed. Deletion mutants of these recombinant plasmids, retaining the functional argA and argE genes, were obtained using different restriction enzymes. All of the recombinant derivatives have the replication properties of the pBR322 vector.     

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.     

Identification and characterization of a native Dichelobacter nodosus plasmid, pDN1

The gram-negative anaerobe Dichelobacter nodosus is the primary causative agent of ovine footrot, a mixed bacterial infection of the hoof. We report here the characterization of a novel native plasmid, pDN1, from D. nodosus. Sequence analysis has revealed that pDN1 has a high degree of similarity to broad-host-range plasmids belonging, or related, to Escherichia coli incompatibility group Q. However, in contrast to these plasmids, pDN1 encodes no antibiotic resistance determinants, lacks genes E and F, and hence is smaller than all previously reported IncQ plasmids. In addition, pDN1 belongs to a different incompatibility group than the IncQ plasmids to which it is related. However, pDN1 does contain the replication and mobilization genes that are responsible for the extremely broad host range characteristic of IncQ plasmids, and derivatives of pDN1 replicate in E. coli. In addition, the mobilization determinants of pDN1 are functional, since derivatives of pDN1 are mobilized by the IncPalpha plasmid RP4 in E. coli.