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.     

Gene organization of the first catabolic operon of TOL plasmid pWW53: production of indigo by the xylA gene product.

The entire operon coding for the enzymes responsible for conversion of toluenes to benzoates has been cloned from TOL plasmid pWW53 and the position of the genes accurately located. The coding region was 7.4 kilobase pairs (kbp) long, and the gene order was operator-promoter region (OP1)-a small open reading frame-xylC (1.6 kbp)-xylA (2.9 kbp)-xylB (1.8 kbp). Within the coding region there was considerable homology with the isofunctional region of the archetypal TOL plasmid pWW0. A central region of 2.9 kbp complemented an xylA (for xylene oxygenase) mutant of Pseudomonas putida mt-2 and was also capable of conferring the ability to convert indole to indigo on strains of Escherichia coli and P. putida. This reaction has been reported previously only for dioxygenases involved in aromatic catabolism but not for monooxygenases. It is proposed that the region encodes xylene oxygenase activity capable of direct monohydroxylation of indole to 3-hydroxyindole (oxindole), which then spontaneously dimerizes to form indigo.     

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.     

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.     

Naturally occurring TOL plasmids in Pseudomonas strains carry either two homologous or two nonhomologous catechol 2,3-oxygenase genes

Structural genes for catechol 2,3-oxygenase (C23O) were cloned from the TOL plasmids pWW5, pWW14, pWW74, pWW84, and pWW88 isolated from Pseudomonas strains of diverse geographical origins. Each pKT230-based C23O+ recombinant plasmid carried a 2.05-kilobase XhoI insert which showed strong homology in Southern hybridizations with the xylE gene from the archetype TOL plasmid pWW0. Fragments were mapped for restriction endonuclease sites and were classified into two closely related groups on the basis of restriction maps. C23O structural genes were located on cloned fragments by a combination of subcloning and site-specific mutagenesis. All five TOL plasmids examined yielded clones whose maps differed from that of xylE of pWW0 by only a single XbaI site, but in addition plasmids pWW5, pWW74, and pWW88 carried a second, homologous C23O gene with seven further restriction site differences. The remaining plasmids, pWW14 and pWW84, carried a second nonhomologous C23O gene related to the second C23O gene (C23OII) of TOL plasmid pWW15 described previously (H. Keil, M. R. Lebens, and P. A. Williams, J. Bacteriol. 163:248-255, 1985). Thus, each naturally occurring TOL plasmid in this study appears to carry genes for two meta cleavage dioxygenases.     

Loss of the toluene-xylene catabolic genes of TOL plasmid pWW0 during growth of Pseudomonas putida on benzoate is due to a selective growth advantage of 'cured' segregants

During growth on benzoate-minimal medium Pseudomonas putida mt-2 (PaW1) segregates derivative ('cured') strains which have lost the ability to use the pathway encoded by its resident catabolic plasmid pWW0. Experiments with two plasmids identical to pWW0 but each with an insert of Tn401, which confers resistance to carbenicillin, suggested that the 'benzoate curing' occurs far more frequently by the specific deletion of the 39 kbp region carrying the catabolic genes than by total plasmid loss. This effect was not pH-dependent, and was not produced during growth on other weak organic acids, such as succinate or propionate, or when benzoate was present in the medium with an alternative, preferentially used carbon source such as succinate. Growth on benzoate did not cause loss from strain PaW174 of the plasmid pWW0174, a derivative of pWW0 which has deleted the 39 kbp region but carries Tn401. Similarly the naphthalene-catabolic plasmid pWW60-1, of the same incompatibility group as pWW0, was not lost from PaW701 during growth on benzoate. Competition between wild-type PaW1 and PaW174, which has the 'cured' phenotype, showed that the latter has a distinct growth advantage on benzoate over the wild-type even when initially present as only 1% of the population: when PaW174 was seeded at lower cell ratios, spontaneously 'cured' derivatives of PaW1 took over the culture after 60-80 generations, indicating that they are present in PaW1 cultures at frequencies between 10(-2) and 10(-3). We conclude that the progressive takeover of populations of PaW1 only occurs when benzoate is present as the sole growth source and that neither benzoate, nor other weak acids, affect plasmid segregation or deletion events: a sufficient explanation is that the 'cured' segregants grow faster than the wild-type using the chromosomally determined beta-ketoadipate pathway.     

Molecular analysis of regulatory and structural xyl genes of the TOL plasmid pWW53-4

pWW53-4 is a cointegrate between RP4 and the catabolic plasmid pWW53 from Pseudomonas putida MT53, which contains 36 kbp of pWW53 DNA inserted close to the oriV gene of RP4; it encodes the ability to grow on toluene and the xylenes, characteristic of pWW53, as well as resistance to tetracycline, kanamycin and carbenicillin, characteristic of RP4. A physical map of the 36 kbp insert of pWW53 DNA for 11 restriction enzymes is presented, showing that the relative positions of the two xyl operons are different from those on the archetypal TOL plasmid pWW0. The location of the genes for 4-oxalocrotonate decarboxylase (xylI) and 4-oxalocrotonate tautomerase (xylH) were shown by subcloning and enzyme assay to lie at the distal end of the meta pathway operon. Although 2-oxopent-4-enoate hydratase (xylJ) and 4-hydroxy-2-oxovalerate aldolase (xylK) could be detected on a large cloned HindIII fragment, they could not be accurately located on smaller subcloned DNA, but the only credible position for them is between xylF and xylI. The gene order in the meta pathway operon is therefore xylDLEGF(J,K)IH. The regulatory genes xylS and xylR were located close to and downstream of the meta pathway operon, and the restriction map of the DNA in this region, as has previously been shown for the two operons carrying the structural genes, shows similarities with the corresponding region on pWW0. Evidence is also presented for the existence of two promoters, termed P3 and P4, internal to the meta pathway operon which support low constitutive expression of the structural genes downstream in Pseudomonas hosts but not in E. coli.     

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.     

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.     

Survival in soils of an herbicide-resistant Pseudomonas putida strain bearing a recombinant TOL plasmid

Pseudomonas putida EEZ15(pWW0-EB62) is a phosphinothricin (PPT)-resistant strain with a recombinant TOL plasmid which allows the strain to grow on p-ethylbenzoate. The survival of this strain in sterile agricultural soils depends on the physicochemical properties of the soil. The recombinant pWW0-EB62 plasmid and its catabolic functions were stable for periods of up to 1 month in bacteria introduced in unamended soils and only conferred selective advantage to the host bacteria without the plasmid or with the natural pWW0 plasmid when the soils were amended with low amounts of p-ethylbenzoate. The addition to soils of aromatics that are cometabolized by P. putida EEZ15(pWW0-EB62) had a detrimental effect on the survival of the bacteria, whereas low amounts of aromatics that are not metabolized by this bacterium had no effect on their survival. Survival of P. putida EEZ15(pWW0-EB62) was better at 4 and 25 degrees C than at 37 degrees C. The host bacterium carrying the recombinant pWW0-EB62 plasmid was established in unsterile soils.     

Survival in soils of an herbicide-resistant Pseudomonas putida strain bearing a recombinant TOL plasmid.

Pseudomonas putida EEZ15(pWW0-EB62) is a phosphinothricin (PPT)-resistant strain with a recombinant TOL plasmid which allows the strain to grow on p-ethylbenzoate. The survival of this strain in sterile agricultural soils depends on the physicochemical properties of the soil. The recombinant pWW0-EB62 plasmid and its catabolic functions were stable for periods of up to 1 month in bacteria introduced in unamended soils and only conferred selective advantage to the host bacteria without the plasmid or with the natural pWW0 plasmid when the soils were amended with low amounts of p-ethylbenzoate. The addition to soils of aromatics that are cometabolized by P. putida EEZ15(pWW0-EB62) had a detrimental effect on the survival of the bacteria, whereas low amounts of aromatics that are not metabolized by this bacterium had no effect on their survival. Survival of P. putida EEZ15(pWW0-EB62) was better at 4 and 25 degrees C than at 37 degrees C. The host bacterium carrying the recombinant pWW0-EB62 plasmid was established in unsterile soils.     

TOL plasmid pWW0 in constructed halobenzoate-degrading Pseudomonas strains: prevention of meta pathway.

The hybrid pathway for chlorobenzoate metabolism was studied in WR211 and WR216, which were derived from Pseudomonas sp. B13 by acquisition of TOL plasmid pWW0 from Pseudomonas putida mt-2. Chlorobenzoates are utilized readily by these strains when meta cleavage of chlorocatechols is suppressed. When WR211 utilizes 3-chlorobenzoate (3CB), the expression of catechol 2,3-dioxygenase (C23O) and the catabolic activities for chloroaromatics via the ortho pathway coexist as a consequence of inactivation of the meta cleavage activity by 3-chlorocatechol. Utilization of 4-chlorobenzoate (4CB) by WR216 presupposes the suppression of C23O by a spontaneous mutation in the structural gene, so that 4-chlorocatechol is not misrouted into the meta pathway. Such C23O- mutants were also selected when WR211 was grown continuously on 3CB. Our data explain why the phenotypic characters 3CB+ and Mtol+ (m-toluate) are compatible, whereas 4CB+ and Mtol+ are incompatible.     

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.     

Characterization of a novel TOL-like plasmid from Pseudomonas putida involved in 1,2,4-trimethylbenzene degradation.

A strain of Pseudomonas putida (TMB) was found to resemble P. putida mt-2 (PaW1) in its ability to degrade 1,2,4-trimethylbenzene, toluene, m-xylene, and p-xylene via oxidation of a methyl substituent and reaction of the meta fission pathway, but a different regulatory model is suggested. The ability of P. putida TMB to degrade these substrates was encoded by plasmid pGB (85 kilobase pairs), which showed considerable differences in size, restriction patterns, and DNA sequence from those of plasmid pWWO of strain PaW1.     

4-Oxalocrotonate tautomerase, an enzyme composed of 62 amino acid residues per monomer.

The xylH gene encoding 4-oxalocrotonate tautomerase (4-OT) has been located on a subclone of the Pseudomonas putida mt-2 TOL plasmid pWW0 and inserted into an Escherichia coli expression vector. Several of the genes of the metafission pathway encoded by pWW0 have been cloned in E. coli, but the overexpression of their gene products has met with limited success. By utilizing the E. coli alkaline phosphatase promoter (phoA) coupled with the proper positioning of a ribosome-binding region, we are able to express functional 4-OT in yields of at least 10 mg of pure enzyme/liter of culture. 4-OT has been previously characterized and shown to be an extremely efficient catalyst (Whitman, C. P., Aird, B. A., Gillespie, W. R., and Stolowich, N. J. (1991) J. Am. Chem. Soc. 113, 3154-3162). Kinetic and physical characterization of the E. coli-expressed protein show that it is identical with that of the 4-OT isolated from P. putida. The functional unit is apparently a pentamer of identical subunits, each consisting of only 62 amino acid residues. This is the smallest enzyme subunit reported to date. The amino acid sequence, determined in part from automated Edman degradation and also deduced from the primary sequence of xylH, did not show homology with any of the sequences in the current data bases nor with any of the sequences of enzymes that catalyze similar reactions. We propose that the active site of 4-OT may be established by an overlap of subunits and comprised of amino acid residues belonging to several, if not all, of the subunits.     

Duplication of both xyl catabolic operons on TOL plasmid pWW15.

Pseudomonas fluorescens MT15 is the host of the large (250 kbp) TOL plasmid pWW15. We have shown by a combination of hybridization, molecular cloning and enzyme assay that pWW15 carries two distinct regions which share homology with the upper pathway operons (xylCMABN) of other TOL plasmids and two distinct regions which are homologous to the meta pathway operons (xylXYZLTEGFJQKIH) of other TOL plasmids. Both the areas of homology to the upper pathway operons appear to carry all of the structural genes for the three catabolic enzymes of the operon. One of the regions of meta pathway operon homology encodes a complete functional pathway, but the second is incomplete and appears to carry only the genes from xylF downstream.     

Hybrid pathway for chlorobenzoate metabolism in Pseudomonas sp. B13 derivatives.

Derivatives of Pseudomonas sp. B13 which had acquired the capability to utilize 4-chloro- and 3,5-dichlorobenzoate as a consequence of the introduction of genes of the TOL plasmid of Pseudomonas putida mt-2 were studied. The utilization of these substrates, a property not shared by the parent strains, was shown to depend upon the combined activities of enzymes from the donor and from the recipient. During growth on 3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate, predominantly the toluate 1,2-deoxygenase and both dihydrodihydroxybenzoate dehydrogenases of the parent strains were induced. On the other hand, no catechol 2,3-dioxygenase from P. putida mt-2 was detectable, so that degradation of chlorocatechols by the nonproductive meta-cleavage pathway was avoided. Instead of that, chlorocatechols were subject to ortho cleavage and totally degraded by the preexisting enzymes of Pseudomonas sp. B13.