Physical map of the aromatic amine and m-toluate catabolic plasmid pTDN1 in Pseudomonas putida: location of a unique meta-cleavage pathway

A restriction endonuclease map was derived for the aromatic amine and m-toluate catabolic plasmid pTDN1 present in Pseudomonas putida UCC22, a derivative of P. putida mt-2. The plasmid is 79 +/- 1 kbp in size and can be divided into a restriction-site-deficient region of 51 +/- 1 kbp and a restriction-site-profuse region of 28 kbp which begins and ends with directly repeating sequences of at least 2 kbp in length. A mutant plasmid isolated after growth of the host on benzoate had lost the restriction-profuse region by a straightforward recombinational loss retaining one copy of the direct repeat. Analysis of clones, deletion and Tn5 insertion mutants strongly suggested that the meta-cleavage pathway of pTDN1 was situated in the region readily deleted. The catechol 2,3-dioxygenase (C23O) gene of pTDN1 showed no hybridization or restriction homology to previously described C23O genes of TOL plasmids pWW0 and pWW15. In addition, there was little homology between intact pTDN1, pWW0 and pWW15, suggesting the presence of a unique meta-cleavage pathway. We also demonstrated that pTDN1 did not originate from P. putida mt-2 chromosome.     

Physical and functional mapping of two cointegrate plasmids derived from RP4 and TOL plasmid pDK1

Cointegrate plasmids were formed in vivo between the broad-host-range R-plasmid RP4 and two catabolic plasmids derived from Pseudomonas putida HS1. One of these was the wild-type plasmid pDK1 encoding the complete inducible toluene/xylene (TOL) catabolic pathway and one was pDKT1, a deletion derivative of pDK1 selected after growth of HS1 on benzoate and supporting growth on only toluene. The two plasmids formed, pDK2 and pDKT2 respectively, each consisted of a complete RP4 replicon in which was an insert of the parent plasmid DNA respectively 40 and 20 kbp in size. The detailed restriction maps of the two plasmids were determined and many of the catabolic genes were located by subcloning and enzyme assay of recombinant plasmids in Escherichia coli and Pseudomonas hosts. The insert in pDK2 contained both operons of the catabolic pathway, the 'upper pathway' operon (xylCAB) and the meta pathway operon (xylDLEGF(I,J,K)H), and a region identified as having the function of the regulator gene xylS. The insert in pDKT2 contained only the upper pathway operon and the regulatory region. Within each of the three coding regions there was great similarity with the same regions on TOL plasmids pWW0 and pWW53-4 apparent (a) by the same order of the genes, (b) by a similar pattern of restriction sites and (c) by hybridization studies. However, the order and orientations of the three coding regions differed from those previously described for both pWW0 and pWW53-4. The significance of these findings to the evolution of TOL plasmids is discussed.     

Loss of Tdn catabolic genes by deletion from and curing of plasmid pTDN1 in Pseudomonas putida: rate and mode of loss are substrate and pH dependent

The ability to degrade aromatic amines and m-toluate (Tdn+ phenotype), encoded by plasmid pTDN1, was lost from Pseudomonas putida hosts after subculture in benzoate, succinate, acetate and glucose minimal medium, the fastest rate of loss occurring where benzoate was the substrate. Tdn- cells had either lost the entire pTDN1 plasmid or suffered a recombinational deletion of a specific 26 kbp region. Proportional increase of Tdn- cells resulted from their growth-rate advantage, and additionally, where benzoate was the substrate, from its metabolism via the chromosomal ortho-cleavage pathway incorporating a short lag phase. The ratio of whole plasmid loss to deletion was substrate and pH dependent. Deletion of catabolic genes was not required for loss of pTDN1 but by comparison was a prerequisite for loss of TOL plasmid pWW0. It appeared that m-toluate and benzoate were channelled via chromosomally encoded benzoate oxygenase and dihydroxycyclohexadiene carboxylate dehydrogenase prior to pTDN1 encoded meta-cleavage.     

Evolutionary conservation of genes coding for meta pathway enzymes within TOL plasmids pWW0 and pWW53.

Pseudomonas putida MT53 contains a TOL plasmid, pWW53, that encodes toluene-xylene catabolism. pWW53 is nonconjugative, is about 105 to 110 kilobase pairs (kbp) in size, and differs significantly in its restriction endonuclease digestion pattern and incompatibility group from the archetypal TOL plasmid pWW0. An RP4::pWW53 cointegrate plasmid, pWW53-4, containing about 35 kbp of pWW53 DNA, including the entire catabolic pathway genes, was formed, and a restriction map for KpnI, HindIII, and BamHI was derived. The entire regulated meta pathway genes for the catabolism of m-toluate were cloned into pKT230 from pWW53 on a 17.5-kbp HindIII fragment. The recombinant plasmid supported growth on m-toluate when mobilized into plasmid-free P. putida PaW130. A restriction map of the insert for 10 restriction enzymes was derived, and the locations of xylD, xylL, xylE, xylG, and xylF were determined by subcloning and assaying for their gene products in both Escherichia coli and P. putida hosts. Good induction of the enzymes by m-toluate and m-methylbenzyl alcohol but not by m-xylene was measured in P. putida, but little or no regulation was found in E. coli. The restriction map and the gene order showed strong similarities with published maps of the DNA encoding both the entire meta pathway operon (xylDLEGFJIH) and the regulatory genes xylS and xylR on the archetype TOL plasmid pWW0, suggesting a high degree of conservation in DNA structure for the catabolic operon on the two different plasmids.     

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.     

Adaptation of Pseudomonas putida mt-2 to growth on aromatic amines

Pseudomonas putida mt-2 (ATCC 33015) carrying the TOL plasmid pWW0 could adapt to growth on the aromatic amines aniline and m- and p-toluidine. In strain UCC2, a derivative adapted to rapid growth on these compounds, they were oxidatively deaminated to catechol or 4-methylcatechol, which in turn were dissimilated by a meta-cleavage pathway. The aniline/toluidine oxygenase and the meta-cleavage pathway enzymes were inducible by aromatic amines. Evidence is presented that in strain UCC2, plasmid pWW0 has undergone deletion of its catabolic genes, and that it is a novel plasmid, pTDN1, which is involved in the catabolism of aniline and m- and p-toluidine. The meta-cleavage pathway genes which are carried by pTDN1 were shown not to have originated in pWW0.     

Comparison of the meta pathway operons on NAH plasmid pWW60-22 and TOL plasmid pWW53-4 and its evolutionary significance

The regulated meta pathway operon for the catabolism of salicylate on the naphthalene plasmid pWW60-22 was cloned into the broad-host-range vector pKT230 on a 17.5 kbp BamHI fragment. The recombinant plasmid conferred the ability to grow on salicylate when mobilized into plasmid-free Pseudomonas putida PaW130. A detailed restriction map of the insert was derived and the locations of some of the genes were determined by subcloning and assaying for their gene products in Escherichia coli and P. putida hosts. The existence of a regulatory gene was demonstrated by the induction of enzyme activities in the presence of salicylate. DNA-DNA hybridization indicated a high degree of structural homology between the pWW60-22 operon and the analogous meta pathway operon on TOL plasmid pWW53-4. The data are consistent with the structural genes being arranged in an identical linear array and suggest an evolutionary link between the two catabolic systems.     

Isolation and characterization of hydrogen peroxide producing Aerococcus sp. from soil samples

Genes involved in 4-methyl-o-phthalate and 4-hydroxy-iso-phthalate catabolism reside on a 226-232 kbp catabolic plasmid termed MOP. This was confirmed by transformation and conjugation into an isogenic heat-cured (MOP-) derivative of the wild-type isolate, identified and termed Pseudomonas cepacia Pc701. Transformation confirmed the presence of Tn1 in MOP derived from Pc704, a mutant deficient in 4-methyl-o-phthalate catabolism. pCS1, a recombinant plasmid bearing MOP DNA, complemented MOP::Tn1 restoring the ability of Pc704 to grow on 4-methyl-o-phthalate. DNA-DNA hybridization using pCS1 as probe confirmed that loss of 4-methyl-o-phthalate catabolism by Pc704 was the result of Tn1 insertion into a 2.1 kbp HindIII fragment of MOP.     

pWW174: A large plasmid from Acinetobacter calcoaceticus encoding benzene catabolism by the β-ketoadipate pathway

Acinetobacter calcoaceticus RJE74 contains a large transmissible catabolic plasmid, pWW174, of about 200 kb, which encodes its ability to grow on benzene (Bzn+). pWW174 was unstable in Acinetobacter hosts and was lost at high frequency in the absence of selection for Bzn+. The catabolic pathway appeared to be via benzene cis-glycol, catechol and the beta-ketoadipate (ortho) pathway. pWW174 encodes a catechol 1,2-oxygenase which is significantly more thermolabile than the chromosomally determined enzyme. pWW174 was able to complement all cat mutants (catechol to central metabolites) of A. calcoaceticus ADP1 (BD413) tested. Two regions of the plasmid were cloned, one carrying catA, the gene for catechol 1,2-oxygenase, and another carrying catBCDE, the subsequent four enzymes of the beta-ketoadipate pathway: these two regions appeared to be separated by at least 10 kbp. Hybridization indicated homology between the plasmid cat genes and the corresponding chromosomal genes of ADP1.     

Genetic analysis of chromosomal operons involved in degradation of aromatic hydrocarbons in Pseudomonas putida TMB.

The catabolic pathway for the degradation of aromatic hydrocarbons encoded by Pseudomonas putida TMB differs from the TOL plasmid-encoded pathway as far as regulation of the upper pathway is concerned. We found, by analyzing Tn5-induced mutants and by Southern blot hybridization with appropriate probes derived from the TOL plasmid pWW0, that the catabolic genes of strain TMB were located on the bacterial chromosome and not on the 84-kb plasmid harbored by this strain. The catabolic genes of TMB and pWW0 had sequence homology, as shown by Southern blot hybridization, but differed significantly in their restriction patterns. The analysis of the mutants suggests that a regulatory mechanism similar to that present in pWW0 coexists in TMB with a second mode of regulation which is epistatic on the former and that the chromosomal region carrying the catabolic genes is prone to rearrangements and deletions.     

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.     

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.     

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.     

The presence of two complete homologous meta pathway operons on TOL plasmid pWW53

pWW53 is a 110 kbp catabolic plasmid which encodes the complete pathway for the utilization of toluene and the xylenes. The upper pathway operon xylCAB is located between two homologous but distinct meta pathway operons, xylDLEGF(I,J,K)H, which are in direct repeat. These have each been cloned on large HindIII restriction fragments HA (17.5 kbp) and HB (15.6 kbp), the restriction sites of which have been mapped. During growth of MT53 on benzoate, mutants which have lost the ability to grow on hydrocarbons such as m-xylene (Mxy-) but which retain the ability to grow on their carboxylic acid metabolites such as m-toluate (Mtol+) take over the culture before ultimately being displaced by plasmid-free strains which are Mxy- Mtol-. The plasmids in the Mxy- Mtol+ mutants are formed by a large deletion between homologous regions of the two duplicate meta pathway operons. This causes the loss of the intervening xylCAB operon and the formation of a hybrid xylDLEGF(I, J, K)H operon, starting with the genes originally on HA and terminating with the genes originally on HB.     

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.     

Genomic and functional analysis of the IncP-9 naphthalene-catabolic plasmid NAH7 and its transposon Tn4655 suggests catabolic gene spread by a tyrosine recombinase

The naphthalene-catabolic (nah) genes on the incompatibility group P-9 (IncP-9) self-transmissible plasmid NAH7 from Pseudomonas putida G7 are some of the most extensively characterized genetic determinants for bacterial aerobic catabolism of aromatic hydrocarbons. In contrast to the detailed studies of its catabolic cascade and enzymatic functions, the biological characteristics of plasmid NAH7 have remained unclear. Our sequence determination in this study together with the previously deposited sequences revealed the entire structure of NAH7 (82,232 bp). Comparison of NAH7 with two other completely sequenced IncP-9 catabolic plasmids, pDTG1 and pWW0, revealed that the three plasmids share very high nucleotide similarities in a 39-kb region encoding the basic plasmid functions (the IncP-9 backbone). The backbone of NAH7 is phylogenetically more related to that of pDTG1 than that of pWW0. These three plasmids carry their catabolic gene clusters at different positions on the IncP-9 backbone. All of the NAH7-specified nah genes are located on a class II transposon, Tn4655. Our analysis of the Tn4655-encoded site-specific recombination system revealed that (i) a novel tyrosine recombinase, TnpI, catalyzed both the intra- and intermolecular recombination between two copies of the attI site, (ii) the functional attI site was located within a 119-bp segment, and (iii) the site-specific strand exchange occurred within a 30-bp segment in the 41-bp CORE site. Our results and the sequence data of other naphthalene-catabolic plasmids, pDTG1 and pND6-1, suggest a potential role of the TnpI-attI recombination system in the establishment of these catabolic plasmids.     

TOL plasmid pWW0 in constructed halobenzoate-degrading Pseudomonas strains: enzyme regulation and DNA structure.

WR211 and WR216 are derivatives of halobenzoate-degrading Pseudomonas sp. strain B13 into which the 117-kilobase TOL degradative plasmid pWW0 has been transferred from Pseudomonas putida mt-2. WR211 has lost the ability to grow on the TOL-specific substrate m-xylene but retains the ability to grow on its metabolite, m-toluate. An analysis of the induction of enzymes was consistent with WR211 carrying a nonfunctional regulatory gene, xy1R, WR216 is a spontaneous derivative of WR211 which grows on one of the TOL substrates and yet expresses the nonspecific toluate oxidase, which enables it to grow on the novel substrate 4-chlorobenzoate. In addition to the xy1R lesion inherited from WR211, WR216 appears to carry a mutation in the structural gene for catechol 2,3-oxygenase, xy1E. The plasmids in both strains were analyzed by restriction endonuclease digestion. pWW0-1211 in WR211 has a large deletion (39 kilobases) compared with pWW0 and appears to be identical to a previously described plasmid (pWW0-8) which encodes none of the TOL degradative functions. pWW0-1216 in WR216 has undergone a major structural reorganization relative to its parent, pWW0-1211. This plasmid has a smaller deletion (19 kilobases), which is staggered relative to the deletion in pWW0-1211, and in addition it has two 3-kilobase insertions of unknown origin, one of which appears to cause the xylE mutation.     

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.     

Mathematical analysis of catabolic function loss in a population of Pseudomonas putida mt-2 during non-limited growth on benzoate

Pseudomonas putida mt-2, harbouring the TOL plasmid PWW0, was grown continuously on benzoate in a phauxostat at a non-limited rate. The gradual decrease in the population carrying the complete TOL plasmid was caused predominantly by a growth-rate advantage of spontaneous mutants carrying a partially deleted plasmid (TOL- cells). The growth-rate difference (v) was quantified both by measuring the increase in the dilution rate (from 0.68 to 0.79 h-1; v = 0.11 h-1) and by mathematical analysis of the ingrowth of TOL- cells (v = 0.12 h-1). The latter procedure also established that the segregation rate was of the order of magnitude 10(-5) h-1. Similar values for the growth-rate advantage and the segregation rate were found when both benzoate and succinate were present in non-limiting concentrations. It is suggested that the growth-rate disadvantage of the wild-type strain is caused by inhibitory effects of an intermediate in the degradation of benzoate via the plasmid-encoded meta-pathway.