Excision and integration of degradative pathway genes from TOL plasmid pWW0.

WR211 is a transconjugant resulting from transfer of the 117-kilobase (kb) TOL degradative plasmid pWW0 into Pseudomonas sp. strain B13. The plasmid of this strain, pWW01211, is 78 kb long, having suffered a deletion of 39 kb. We show that WR211 contains the 39 kb that is missing from its plasmid, together with at least an additional 17 kb of pWW0 DNA integrated in another part of the genome, probably the chromosome. The ability of WR211 to grow on the TOL-specific substrate m-toluate is the result of expression of the TOL genes in this alternative location, whereas its inability to grow on m-xylene is caused by insertional mutagenesis by 3 kb of DNA of unknown origin in the xylR gene of this DNA. The resident plasmid pWW01211 plays no part in the degradative phenotype of WR211 since it can be expelled by mating in incompatible IncP9 resistance plasmid R2 or pMG18 without loss of the phenotype. This alternatively located DNA can be rescued back into the R2 and pMG18 plasmids as R2::TOL and pMG18::TOL recombinants by mating out into plasmid-free recipients and selecting for Mtol+ transconjugants. In all cases examined, these plasmids contained the entire R plasmid into which is inserted 59 kb of DNA, made up of 56 kb of pWW0 DNA and the 3-kb xylR insertion. Selection for faster growth on benzoate can lead to precise excision of the 39 kb from the TOL region of an R2::TOL recombinant, leaving a residual and apparently cryptic 17-kb segment of pWW0 DNA in the R plasmid.     

A comparative study of the NAH and TOL catabolic plasmids in Pseudomonas putida

A comparative study of the NAH and TOL catabolic plasmids was carried out to provide information for future genetic manipulation experiments involving these two plasmids. The plasmids were studied in a strain of P. putida and its mutant derivatives. The NAH and TOL plasmids were found to be incompatible. Under the conditions used in these experiments the TOL plasmid transferred into some strains into which NAH was unable to transfer. The use of mutants to remove certain catabolic activities encoded by the bacterial host cell facilitated the allocation of growth genotypes to the NAH and TOL plasmids. TOL encoded the degradation of benzoate, m-toluate and p-toluate, whereas NAH encoded the degradation of naphthalene and salicylate. The other plasmid-associated growth phenotypes were partly plasmid-specified and partly specified by the host cell. The pH optimum of the catechol 2,3-dioxygenase specified by the TOL plasmid was approximately 6.7, whereas that of the NAH-encoded enzyme was approximately 8.3.     

Recombination of the bph (Biphenyl) Catabolic Genes from Plasmid pWW100 and Their Deletion during Growth on Benzoate

Pseudomonas sp. strain CB406 was isolated from polychlorinated biphenyl-contaminated soil and harbors a nontransmissible plasmid, pWW100, of approximately 200 kb which carries the genes required for biphenyl and 4-chlorobiphenyl catabolism. The catabolic phenotype was mobilized following the construction in vivo of a cointegrate plasmid containing functional upper and lower biphenyl operons inserted into the broad-host-range R plasmid RP4. The Bph⁺ phenotype carried by pWW100 was stable in nonselective media but was unstable during growth on benzoate, where the sequential selection of two species of bph deletion derivatives occurs at high frequency. This mirrors observations made with TOL plasmids (encoding toluene and xylene catabolism) grown under similar conditions. Subcloning of dioxygenase genes involved in biphenyl catabolism confirmed the localization of the bph genes on the wild-type plasmid and the RP4 cointegrate plasmid.     

Genetic rearrangements in plasmids specifying total degradation of chlorinated benzoic acids

Summary Growth in a chemostat of the 3-chlorobenzoatepositive Pseudomonas putida cells harboring the plasmid pAC25, in presence of cells harboring the TOL plasmid, allows emergence of cells that can also utilize 4-chlorobenzoate (4Cba). Isolation of plasmid DNA from such cells demonstrate the deletion of a 11kb (Kilobase pair) EcoR1 fragment from the pAC25 plasmid; a portion of the TOL plasmid (41.5 kb TOL^*) is also found to be transposed onto the chromosome of such cells. Further enrichment of the 4-chlorobenzoate-positive cells with 3,5-dichlorobenzoate (3,5-Dcb) as a sole carbon source has produced cells that can also slowly utilize 3,5-dichlorobenzoate. Isolation of plasmid DNA from such cells demonstrates the appearance of a second plasmid (pAC29). Restriction hybridization of pAC29 EcoRI fragments with pAC25 and TOL demonstrates that pAC29 is derived primarily by duplication of a segment of the pAC27 plasmid and a fragment from TOL, with further mutational divergence. Southern hybridization of the EcoRI-digested chromosomal DNA with ³²P-labeled TOL, pTS11 and pTS71 plasmid DNAs demonstrates the presence of the TOL^* transposon containing xylD, G, E and F genes in both 4Cba⁺ (pAC27⁺) and 3,5-DCb⁺ (pAC27⁺, pAC29⁺) cells. Isolation of plasmid DNA from 3,5-Dcb⁺ faster growing variants, obtained from slow-growing pAC27⁺ pAC29⁺ cells, demonstrates the presence of a single type of plasmid, with identical size and EcoRI digestion profile as pAC27. The implications of gene duplications and subsequent homologous recombination with regard to the biochemical pathway of 3,5-dichlorobenzoate degradation have been discussed.     

Intermolecular recE4-independent recombination in Bacillus subtilis: formation of plasmid pKBT1

Summary The plasmid pKBT1 was derived by in vivo recE4-independent recombinational event(s) yielding a structure containing regions of plasmid and chromosomal origin. BamHI digests of plasmid pUB110 (Kan^r/Neo^r) and Bg/II digests of pTL12 (Tmp^r, leuA) were mixed, ligated and used to transform competent cells of a recE4 strain of Bacillus subtilis. Kanamycin-resistant transformants were electrophoretically screened for hybrid plasmids. Plasmid pKBT1 (8.0 kb) was smaller than pTL12 (10.4 kb) but larger than monomeric pUB110 (4.5 kb). Plasmid PKBT1 was stably maintained in recE4 strains of B. subtilis and conferred kanamycin resistance but did not specify trimethoprim resistance or leucine prototrophy. At least 86% of the pUB110 monomer length was present in pKBT1 and was completely contained within a single 5.58 kb HindIII fragment. The other segment of pKBT1 was of chromosomal origin as evidenced by lack of homology to pTL12 and strong hybridization to B. subtilis chromosomal DNA. At least one of the in vivo recE4-independent event(s) which produced pKBT1 must have involved intermolecular recombination between transforming and chromosomal DNA. This finding differs from previous reports in which recE4-independent recombination involving pUB110 sequences was a strictly intramolecular event.     

Construction of a partial diploid for the degradative pathway encoded by the TOL plasmid (pWW0) from Pseudomonas putida mt-2: Evidence for the positive nature of the regulation by the xylR gene

Summary The hypothesis that the early enzymes of the degradative pathway determined by the TOL plasmid pWW0 are positively regulated by the product of the xylR gene has been tested by constructing a strain which is a partial diploid for the TOL genes. Two parental plasmids were first constructed by in vivo methods, neither of which could determine the ability to grow on m-xylene, one of the primary substrates of the plasmid degradative pathway, because of mutations. One of these, pWW0-216, was a derivative of pWW0 but carried a xylR ^- allele and a copy of the Tn401 transposon, encoding carbenicillin resistance. The other plasmid, pWW0-152, was a derivative of the promiscuous R plasmid RP4 into which had been translocated part of a pWW0 plasmid carrying a wild type xylR ⁺ allele but with a defective xylA, the structural gene for xylene oxidase. When these two plasmids were mated into the same strain, all the transconjugants examined grew on m-xylene and one representative of these, PaW 219, was shown to contain induced levels of xylene oxidase when grown under inducing conditions. The possibility that ability to utilise m-xylene was due to recombination between or reversion of the coexisting plasmids was eliminated by demonstrating that the two parental plasmids segregated on mating out from PaW 219. It is concluded therefore that xylR ⁺ is transdominant to xylR ^-, and that its gene product is a positive regulator.     

Evolutionary relationships between catabolic pathways for aromatics: Conservation of gene order and nucleotide sequences of catechol oxidation genes of pWW0 and NAH7 plasmids

Summary TOL plasmid pWW0 and plasmid NAH7 encode catabolic enzymes required for oxidative degradation of toluene and naphthalene, respectively. The gene order of the catabolic operon of NAH7 for salicylate oxidation was determined to be: promoter-nahG (the structural gene for salicylate hydroxylase)-nahH (catechol 2,3-dioxygenase)-nahI (hydroxymuconic semialdehyde dehydrogenase)-nahN (hydroxymuconic semialdehyde hydrolase)-nahL (2-oxopent-4-enoate hydratase). This order is identical to that of the isofunctional genes of TOL plasmid pWW0. The complete nucleotide sequence of nahH was determined and compared with that of xylE, the isofunctional gene of TOL plasmid pWW0. There were 20% and 16% differences in their nucleotide and amino acid sequences, respectively. The homology between the NAH7 and TOL pWW0 plasmids ends upstream of the Shine-Dalgarno sequences of nahH and xylE, but the homology continues downstream of these genes. This observation suggested that genes for the catechol oxidative enzymes of NAH7 and TOL pWW0 were derived from a common ancestral sequence which was transferred as a discrete segment of DNA between plasmids.     

Complete sequence determination combined with analysis of transposition/site-specific recombination events to explain genetic organization of IncP-7 TOL plasmid pWW53 and related mobile genetic elements

Recent studies have indicated that the evolutionarily common catabolic gene clusters are loaded on structurally diverse toluene-catabolic (TOL) plasmids and their residing transposons. To elucidate the mechanisms supporting the diversification of catabolic plasmids and transposons, we determined here the complete 107,929 bp sequence of pWW53, a TOL plasmid from Pseudomonas putida MT53. pWW53 was found to belong to the IncP-7 incompatibility group that play important roles in the catabolism of several xenobiotics. pWW53 carried two distinct transposase-resolvase gene clusters (tnpAR modules), five short terminal inverted repeats (IRs), and three site-specific resolution (res) sites that are all typical of class II transposons. This organization of pWW53 suggested the four possible transposable regions, Tn4657 to Tn4660. The largest 86 kb region (Tn4657) spanned the three other regions, and Tn4657 and Tn4660 (62 kb) covered all of the 36 xyl genes for toluene catabolism. Our subsequent transposition experiments clarified that the three transposons, Tn4657 to Tn4659, indeed exhibit their transposability, and that pWW53 also generated another 37 kb toluene-catabolic transposon, Tn4656, which carried the two separated and inversely oriented segments of pWW53: the tnpRA-IR module of Tn4658 and a part of xyl gene clusters on Tn4657. The Tn4658 transposase was able to mediate the transposition of Tn4658, Tn4657, and Tn4656, while the Tn4659 transposase catalyzed only the transposition of Tn4659. Tn4656 was formed by the Tn4658 resolvase-mediated site-specific inversion between the two inversely oriented res sites on pWW53. These findings and comparison with other catabolic plasmids clearly indicate multiple copies of transposition-related genes and sites on one plasmid and their recombination activities contribute greatly to the diversification of plasmid structures as well as wide dissemination of the evolutionary common gene clusters in various plasmids.     

Spontaneous deletions in the TOL plasmid pWW20 which give rise to the B3 regulatory mutants of Pseudomonas putida MT20

The size of the TOL plasmid pWW20 from Pseudomonas putida MT20, as measured by analysis of agarose electrophoresis gels after restriction endonuclease hydrolysis, was 270-280 kilobase pairs (kb). During growth on benzoate, MT20 segregates strains carrying mutations in the plasmid regulatory gene xylS; these so-called B3 strains retain the ability to grow on m-xylene (Mxy+) but do not grow on its metabolite m-toluate (Mtol-) and have also lost the ability to transfer the plasmid (Tra-). Analysis of restriction digests of plasmid DNA from seven such segregants, independently isolated, showed that pWW20 had undergone extensive deletions of 90-100 kb. All the deleted plasmids had lost a common core of DNA, of about 72-80 kb, but in class A mutants the deletion extended at one end of this core and in class B mutants at the other end. Class A and B mutants also differed in their rate of growth on m-xylene as a result of differences in the level of expression of their plasmid-coded catabolic enzymes. This suggests that an additional gene, involved in regulating levels of gene expression, is located in the region uniquely deleted in the class B mutants.     

Localization of camphor degradative plasmids on the chromosome of Pseudomonas putida strains PaW]

Camphor degradative plasmids (CAM, pRK1) are preferentially situated on chromosomes of Pseudomonas putida strains PaW. After having been transferred into Cam+ strains, the TOL plasmid pWWO dissociates into the cryptic plasmid pWWO-8 and chromosome-borne transposon Tn4651. The opposite situation, i.e. reconstruction of the TOL plasmid pWWO from the cryptic plasmid pWWO-8 and chromosome-borne catabolic operons of the pWWO plasmid has been described. Cam- derivatives of the CAM plasmid were obtained in vivo which contain the TOL plasmid transposons Tn4651 or Tn4652 as obligatory structural elements. These plasmids as well as pWWO-8 determine conjugational mobilization of chromosome-located cam operons followed by their integration into the chromosome of recipient.     

Restriction maps and homologies of the three plasmids of Agrobacterium rhizogenes strain A4

Agrobacterium rhizogenes strain A4 is a virulent agropine-type strain possessing three plasmids: plasmid a (pArA4a, 180 kb) is not necessary for plant transformation, plasmid b (250 kb) is the root-inducing plasmid (pRiA4), and plasmid c (pArA4c) is a cointegrate of pArA4a and pRiA4. The total plasmid DNA (pArA4) of strain A4 was cloned in the cosmid pHSG262 and the library obtained was used to establish BamHI maps of the three plasmids. The plasmids a and Ri have an apparently identical region and a partly homologous region, and are different in the remaining regions including their origins of replication. Another agropine-type A. rhizogenes strain, HRI, bears only one plasmid, which is the Ri plasmid (pRiHRI). pRiHRI and pRiA4 present the same restriction maps for a great part, but are different in a region of 48 kb; however, this region of pRiHRI is found unmodified in pArA4a and may have a role in the virulence of the bacteria. The comparison between the restriction maps of the plasmids of strain A4 leads us to propose that the recombination event leading to pArA4c formation occurs within the identical regions of pArA4a and pRiA4. In addition, the comparison with the already established map of pRiHRI suggests that strain HRI could have been derived from a recombination event between the two homologous regions of pArA4c with subsequent loss of the smaller plasmid.     

Retrotransfer of DNA in the rhizosphere

Retrotransfer of DNA refers to the phenomenon by which a plasmid travels from a host strain to a recipient one and returns to the original host, bringing with it DNA from the recipient. The resultant host strain with DNA from the recipient is called a retrotransconjugant. The retrotransfer phenomenon mediated by the TOL plasmid pWW0 and other plasmids has been documented on plates under optimal laboratory culture conditions, but never under natural conditions. In this work, we show that retrotransfer mediated by the IncP9 TOL pWW0 plasmid occurs in the rhizosphere, a niche in which the continuous supply of nutrients via root exudates allows cells to reach a high density. This suggests that this unusual sexual fertilization may be of great importance in lateral gene transfer. We also show that retrotransfer of DNA seems to require co-integration of the plasmid and the host chromosome and subsequent resolution, because a TOL plasmid with a mutation in the tnpR gene, encoding the resolvase of the Tn 4653 of the TOL plasmid, was self-transferred between Pseudomonas strains, but unable to mobilize chromosome.     

A host-vector system for gene cloning in the cyanobacterium Synechocystis PCC 6803

Summary Synechocystis 6803 contains at least four cryptic plasmids of 2.27 kb (pUS1, pUS2 and pUS3) and 5.20 kb (pUS4). The 1.70 kb HpaI fragments of the related plasmids pUS2 and pUS3 were cloned into the Ap^r gene of the E. coli plasmid pACYC177, yielding the Km^r hybrid plasmids pUF12 and pUF3 respectively. pUF3 recombines in Synechocystis 6803 with a 2.27 kb plasmid giving the Km^r shuttle vector pUF311. The 1.35 kb HaeII fragment containing the Cm² gene of the E. coli plasmid pACYC184 was cloned in pUF311 generating the Cm^r Km^r shuttle vector pFCLV7. Wild-type cells of Synechocystis 6803 are transformed, albeit poorly, by the plasmids pUF3, pUF12 and pFCLV7. pFCLV7 very efficiently transforms the SUF311 strain of Synechocystis 6803 containing pUF311 as a resident plasmid. This is due to recombination between the homologous parts of pFCLV7 and pUF311. For the same reason the strain SUF311 is also efficiently transformable by E. coli plasmids, as shown for pLF8, provided that they have some homology with the E. coli part of pUF311.The combined use of Synechocystis 6803 strain SUF311 and of plasmids pFCLV7 and pLF8 generates an efficient host-vector system for gene cloning in this facultatively heterotrophic cyanobacterium.     

Complete sequence of the IncP-9 TOL plasmid pWW0 from Pseudomonas putida

The TOL plasmid pWW0 (117 kb) is the best studied catabolic plasmid and the archetype of the IncP-9 plasmid incompatibility group from Pseudomonas. It carries the degradative (xyl) genes for toluenes and xylenes within catabolic transposons Tn4651 and Tn4653. Analysis of the complete pWW0 nucleotide sequence revealed 148 putative open reading frames. Of these, 77 showed similarity to published sequences in the available databases predicting functions for: plasmid replication, stable maintenance and transfer; phenotypic determinants; gene regulation and expression; and transposition. All identifiable transposition functions lay within the boundaries of the 70 kb transposon Tn4653, leaving a 46 kb sector containing all the IncP-9 core functions. The replicon and stable inheritance region was very similar to the mini-replicon from IncP-9 antibiotic resistance plasmid pM3, with their Rep proteins forming a novel group of initiation proteins. pWW0 transfer functions exist as two blocks encoding putative DNA processing and mating pair formation genes, with organizational and sequence similarity to IncW plasmids. In addition to the known Tn4651 and IS1246 elements, two additional transposable elements were identified as well as several putative transposition functions, which are probably genetic remnants from previous transposition events. Genes likely to be responsible for known resistance to ultraviolet light and free radicals were identified. Other putative phenotypic functions identified included resistance to mercury and other metal ions, as well as to quaternary ammonium compounds. The complexity and size of pWW0 is largely the result of the mosaic organization of the transposable elements that it carries, rather than the backbone functions of IncP-9 plasmids.     

Establishment of New Genetic Traits in a Microbial Biofilm Community

Conjugational transfer of the TOL plasmid (pWWO) was analyzed in a flow chamber biofilm community engaged in benzyl alcohol degradation. The community consisted of three species, Pseudomonas putida RI, Acinetobacter sp. strain C6, and an unidentified isolate, D8. Only P. putida RI could act as a recipient for the TOL plasmid. Cells carrying a chromosomally integrated lacI^q gene and a lacp-gfp-tagged version of the TOL plasmid were introduced as donor strains in the biofilm community after its formation. The occurrence of plasmid-carrying cells was analyzed by viable-count-based enumeration of donors and transconjugants. Upon transfer of the plasmids to the recipient cells, expression of green fluorescence was activated as a result of zygotic induction of the gfp gene. This allowed a direct in situ identification of cells receiving the gfp-tagged version of the TOL plasmid. Our data suggest that the frequency of horizontal plasmid transfer was low, and growth (vertical transfer) of the recipient strain was the major cause of plasmid establishment in the biofilm community. Employment of scanning confocal laser microscopy on fixed biofilms, combined with simultaneous identification of P. putida cells and transconjugants by 16S rRNA hybridization and expression of green fluorescence, showed that transconjugants were always associated with noninfected P. putida RI recipient microcolonies. Pure colonies of transconjugants were never observed, indicating that proliferation of transconjugant cells preferentially took place on preexisting P. putida RI microcolonies in the biofilm.     

The effect of lipophilic weak acids on the segregational stability of TOL plasmids in Pseudomonas putida

The effect of various lipophilic weak acids on the stability of certain TOL plasmids was investigated. Benzoate induced deletion of TOL plasmid DNA in Pseudomonas putida MT15, followed by loss of the plasmid; this effect was pH- and concentration-dependent, suggesting that undissociated benzoic acid was a more effective curing agent than the benzoate anion. Plasmid loss always approached a frequency of 100% after a lag and apparently depended on the prior occurrence of deletions, although deleted plasmid was stably maintained in the absence of the acid. m-Toluate, acetate and butyrate also induced deletions and plasmid loss at high frequencies, although these acids were less effective than benzoate. Benzoate inhibited the growth of plasmid-containing cells rather than permitting faster growth of cured cells on benzoate. Similar results were obtained with P. putida strains MT20 and MT84, which contain different TOL plasmids. We suggest that lipophilic weak acids induced deletions, possibly by excision of a transposon-like region, and disrupted the segregation of deleted plasmid.     

Site specific recombination involved in the generation of small plasmids

Summary The physical structures of seven small plasmids, Rsc10, Rsc11, Rsc12, Rsc13, Rsc15, Rsc10-1 and pEM1 were analyzed. Molecular lengths of these plasmids were determined to range from 7.65 to 19.8 kilobases or kb. Electron microscope heteroduplex analysis of these plasmids show that the plasmids were all derived from pKN102 (86.3kb) in a complicated process that takes place by a series of deletion and, in some cases, transposition events. Rsc10 and Rsc11 were each formed by a simple deletion event from the parental plasmid. The physical structures of Rsc13 and pEM1 suggest that these plasmids must have been derived by a single and two successive deletion events from Rsc11. In the formation of these plasmids, all the deletions occured at the ends of the transposon, Tn3, which confers ampicillin resistance (amp) to the plasmid, or at the ends of the insertion sequence, IS1. Rsc15 was assumed to be formed in a two step process. The first step was a deletion event to form Rsc10-1 which occurs at one end of the IS1 present in pKN102. At first, the deletion event leaves out the ampicillin gene but in the second step Tn3 is transposed to the newly formed plasmid, Rsc10-1. Rsc12 is believed to have been formed in a similar fashion; first, a series of deletions and second, the transposition of Tn3.Studies on these small plasmids enabled us to also map the regions of the replication genes and ampicillin resistance on pKN102.     

Plasmid content of salt stress-tolerant <Emphasis Type="Italic">Rhizobium</Emphasis> strains from Egyptian soils nodulating common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Plasmid profile analysis is useful to characterize Rhizobium strains within the same species. Among the 16 Rhizobium strains examined, 14 had distinct plasmid profiles. The size of plasmids ranged from 40 to 650 kb, and three plasmids of 650, 510 and 390 kb were common to several strains. Plasmid analysis revealed that Rhizobium etli contained a mega-plasmid, similar in size to Rhizobium tropici. All the salt-tolerant strains examined had a plasmid of 250 kb, except for strain EBRI 29. This suggests that this plasmid may play an important adaptive role under salt stress conditions.     

Characterization of new plasmids from methylotrophic bacteria

Several tens of methanol-utilizing bacterial strains isolated from soil were screened for the presence of plasmids. From the obligate methylotrophMethylomonas sp. strain R103a plasmid pIH36 (36 kb) was isolated and its restriction map was constructed. In pink-pigmented facultative methylotrophs (PPFM), belonging to the genusMethylobacterium four plasmids were detected: plasmids pIB200 (200 kb) and pIB14 (14 kb) in the strain R15d and plasmids pWU14 (14 kb) and pWU7 (7.8 kb) in the strain M17. Because of the small size and the presence of several unique REN sites (HindIII, EcoRI, NcoI), plasmid pWU7 was chosen for the construction of a vector for cloning in methylotrophs. Cointegrates pKWU7A and pKWU7B were formed between pWU7 and theE. coli plasmid pK19 Km^r, which were checked for conjugative transfer fromE. coli into the methylotrophic host.