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.     

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.     

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.     

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.     

Genetic, functional and sequence analysis of the xylR and xylS regulatory genes of the TOL plasmid pWW0

Mutant derivatives of a plasmid, pCF20, which carries the XhoI-D fragment of the TOL plasmid pWW0 have been isolated using Tn5 transposon mutagenesis. Insertion mutations of the xylR and xylS regulatory genes of the catabolic pathway have been isolated and characterized and their ability to induce catechol 2,3-oxygenase activity determined. Analysis of the insertion mutants and also segments of the XhoI-D fragment cloned into plasmid pUC8 in maxicells has identified a 68 kDa polypeptide product encoded by the xylR gene. No clear candidate for the xylS polypeptide was observed. The nucleotide sequence of the xylS region, the intergenic region and part of the xylR region has been determined and open reading frames (ORFs) assigned for both genes. The ORF designated xylS appears capable of encoding a polypeptide of approximately 37 kDa.     

Nucleotide sequence of xylE from the TOL pDK1 plasmid and structural comparison with isofunctional catechol-2,3-dioxygenase genes from TOL, pWW0 and NAH7.

Detailed restriction and nucleotide sequence analysis of the Pseudomonas putida TOL plasmid pDK1 xylE gene revealed significant homology with isofunctional xylE (81.5%) and nahH (78.0%) genes from the TOL pWW0 and NAH7 plasmids. The highest degrees of nucleotide and apparent amino acid conservation (82.2 and 86.4%, respectively) among all three genes were found to exist within a region comprising 264 nucleotides encoding the C terminus. A comparison of localized regions revealed significantly greater homology between xylEpWW0 and xylEpDK1 within the C-terminal region, whereas xylEpWW0 and nahH showed greater similarity at the N terminus. The possibility that xylEpWW0 may represent a genetic hybrid of xylEpDK1 and nahH is discussed.     

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 five genes in the upper-pathway operon of TOL plasmid pWW0 from Pseudomonas putida and identification of the gene products.

The upper operon of the TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes which transform toluene and xylenes to benzoate and toluates. The genetic organization of the operon was characterized by cloning of the upper operon genes into an expression vector and identification of their products in Escherichia coli maxicells. This analysis showed that the upper operon contains at least five genes in the order of xylC-xylM-xylA-xylB-xylN. Between the promoter of the operon and xylC, there is a 1.7-kilobase-long space of DNA in which no gene function was identified. In contrast, most of the DNA between xylC and xylN consists of coding sequences. The xylC gene encodes the 57-kilodalton benzaldehyde dehydrogenase. The xylM and xylA genes encode 35- and 40-kilodalton polypeptides, respectively, which were shown by genetic complementation tests to be subunits of xylene oxygenase. The structural gene for benzyl alcohol dehydrogenase, xylB, encodes a 40-kilodalton polypeptide. The last gene of this operon is xylN, which synthesizes a 52-kilodalton polypeptide of unknown function.     

Evolutionary conservation of chloroplast genes coding for the large subunits of fraction 1 protein

Crystalline fraction 1 protein, obtained from four species of Nicotiana, have identical polypeptide compositions and isoelectric points. However, the tryptic peptide map of the large subunit of this protein from N. knightiana and N. paniculata differs from that of N. tomentosa and N. tomentosiformis. Since the large subunits of fraction 1 protein are coded by chloroplast DNA, the difference in their primary structure reflects the structural changes of the chloroplast genes containing the coding information. This indicates that the rate of mutation of chloroplast DNA seems to be higher than predicated from the analysis of isoelectric points of this protein.     

Ubiquity of plasmids in coding for toluene and xylene metabolism in soil bacteria: evidence for the existence of new TOL plasmids.

Thirteen bacteria have been isolated from nine different soil samples by selective enrichment culture on m-toluate (m-methylbenzoate) minimal medium. Eight of these were classified as Pseudomonas putida, one as a fluorescent Pseudomonas sp., and four as nonfluorescent Pseudomonas sp. All 13 strains appeared to carry TOL plasmids superficially similar to that previously described in P. putida mt-2 in that: (i) all the wild-type strains could utilize toluene, m-xylene, and p-xylene as sole carbon and energy sources, (ii) these growth substrates were metabolized through the corresponding alcohols and aldehydes to benzoate, m-toluate, and p-toluate, respectively, and thence by the divergent meta (or alpha-ketoacid) pathway, and (iii) the isolates could simultaneously and spontaneously lose their ability to utilize the hydrocarbons, alcohols, aldehydes, and acids, particularly during growth on benzoate, giving rise to cured strains which could grow only on benzaldehyde and benzoate of the aromatic substrates by the alternative ortho (or beta-ketoadipate) pathway. Eight of the isolates were able to transfer their TOL plasmids into their own cured strains, but only five were able to transfer them in interstrain conjugation into the cured strains, but only five were able to transfer them in interstrain conjugation into the cured derivative of P. putida mt-2. However, P. putida mt-2 was able to transfer its TOL plasmid into 11 of the cured isolates, and eight of these were able to retransmit this foreign plasmid in intrastrain conjugation with their own cured derivatives. Three of the isolates, MT 14, MT 15, and MT 20, differed significantly from the others in that the wild-type strains dissimilated the p-methyl-substituted substrates poorly, and also, during growth on benzoate, in addition to the cured derivatives, they gave rise to derivatives with a phenotype intermediate between the cured and wild-type strains, the biochemical and genetic nature of which has not been elucidated.     

Identification and characterization of Tn4656, a novel class II transposon carrying a set of toluene-degrading genes from TOL plasmid pWW53

It has been reported that the toluene-degrading (xyl) genes from Pseudomonas putida plasmid pWW53 are able to translocate to broad-host-range drug resistance plasmid RP4, and pWW53-4 is one of the smallest RP4 derivatives (H. Keil, S. Keil, R. W. Pickup, and P. A. Williams, J. Bacteriol. 164:887-895, 1985). Our investigation of pWW53-4 in this study demonstrated that such a translocated region that is 39 kb long is a transposon. This mobile element, Tn4656, was classified as a class II transposon since its transposition occurred by a two-step process: transposase (TnpA)-mediated formation of the cointegrate and resolvase (TnpR)-mediated site-specific resolution of the cointegrate at the two copies of the res site. The Tn4656 TnpA and TnpR functions encoded in the rightmost 4-kb region were found to be exchangeable with those specified by other Tn1721-related class II transposons, including another toluene transposon, Tn4653. Sequence analysis of the transposition-related genes and sites of Tn4656 also supported the hypothesis that this transposon is closely related to the Tn1721-related transposons. The lower transposition frequency of Tn4656 has been suggested to be due to the unique nucleotide sequence of one of the terminal 39-bp inverted repeats.     

Construction of hybrid xylE genes between the two duplicate homologous genes from TOL plasmid pWW53: comparison of the kinetic properties of the gene products

The two xylE genes for catechol 2,3-oxygenase, encoded by TOL plasmid pWW53, carry a common SalI restriction site within the reading frame. Each gene was cut at the SalI site and the 5' end of each gene spliced to the 3' end of the other to form hybrid genes, from both of which catalytically active catechol 2,3-oxygenase activities were expressed. The kinetic parameters were determined for the gene products of both the hybrid and the wild-type xylE genes with catechol, 3-methylcatechol and 4-methylcatechol as substrates. Comparison of the results suggested firstly, that the C-terminal regions of the enzymes determined both the binding and the catalytic specificity, and, secondly, that the N-terminal region of one of the enzymic gene products contained a secondary binding site which caused inhibition by excess substrate for methylcatechol substrates but not for catechol. One of the wild-type enzymes appeared to have an intrinsically higher activity for all three substrates than the other. This higher activity depended on the presence of both its C- and N-terminal regions, and in both hybrid enzymes, which contained only one of these regions, activity was significantly reduced.     

Multiple genes coding for octopine-degrading enzymes in Agrobacterium.

Most biotype 2 strains of Agrobacterium tumefaciens and A. radiobacter which utilize nopaline also degrade octopine. In all such strains studied, the ability to degrade octopine did not appear to be transferred to plasmidless recipient cells under conditions of plasmid transfer in which the ability to utilize nopaline was transferred. An octopine-degrading mutant was isolated in a strain cured of its plasmid, suggesting that genes of octopine degradation may have a chromosomal location in some strains. In strains in which octopine utilization is coded by plasmid genes, octopine degradation was always inducible, whereas in strains which degrade both octopine and nopaline, octopine utilization was constitutive although nopaline degradation was inducible. When plasmids coding for octopine-utilizing ability were transformed into a strain containing either a nopaline- or null-type plasmid, transformants able to degrade octopine were either not observed or were unstable upon purification. All of these data suggest that plasmids associated with virulence are incompatible with one another, and therefore imply that the major groups of plasmids associated with virulence have a common origin.     

Potential DNA slippage structures acquired during evolutionary divergence of Acinetobacter calcoaceticus chromosomal benABC and Pseudomonas putida TOL pWW0 plasmid xylXYZ, genes encoding benzoate dioxygenases.

The xylXYZ DNA region is carried on the TOL pWW0 plasmid in Pseudomonas putida and encodes a benzoate dioxygenase with broad substrate specificity. The DNA sequence of the region is presented and compared with benABC, the chromosomal region encoding the benzoate dioxygenase of Acinetobacter calcoaceticus. Corresponding genes from the two biological sources share common ancestry: comparison of aligned XylX-BenA, XylY-BenB, and XylZ-BenC amino acid sequences revealed respective identities of 58.3, 61.3, and 53%. The aligned genes have diverged to assume G+C contents that differ by 14.0 to 14.9%. Usage of the unusual arginine codons AGA and AGG appears to have been selected in the P. putida xylX gene as it diverged from the ancestor it shared with A. calcoaceticus benA. Homologous A. calcoaceticus and P. putida genes exhibit different patterns of DNA sequence repetition, and analysis of one such pattern suggests that mutations creating different DNA slippage structures made a significant contribution to the evolutionary divergence of xylX.     

Substrate specificity of catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 of Pseudomonas putida and its relationship to cell growth.

Catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 of Pseudomonas putida consists of four identical subunits, each containing one ferrous ion. The enzyme catalyzes ring cleavage of catechol, 3-methylcatechol, and 4-methylcatechol but shows only weak activity toward 4-ethylcatechol. Two mutants of catechol 2,3-dioxygenases (4ECR1 and 4ECR6) able to oxidize 4-ethylcatechol, one mutant (3MCS) which exhibits only weak activity toward 3-methylcatechol but retained the ability to cleave catechol and 4-methylcatechol, and one phenotypic revertant of 3MCS (3MCR) which had regained the ability to oxidize 3-methylcatechol were characterized by determining their Km and partition ratio (the ratio of productive catalysis to suicide catalysis). The amino acid substitutions in the four mutant enzymes were also identified by sequencing their structural genes. Wild-type catechol 2,3-dioxygenase was inactivated during the catalysis of 4-ethylcatechol and thus had a low partition ratio for this substrate, whereas the two mutant enzymes, 4ECR1 and 4ECR6, had higher partition ratios for it. Similarly, mutant enzyme 3MCS had a lower partition ratio for 3-methylcatechol than that of 3MCR. Molecular oxygen was required for the inactivation of the wild-type enzyme by 4-ethylcatechol and of 3MCS by 3-methylcatechol, and the inactivated enzymes could be reactivated by incubation with FeSO4 plus ascorbic acid. The enzyme inactivation is thus most likely mechanism based and occurred principally by oxidation and/or removal of the ferrous ion in the catalytic center. In general, partition ratios for catechols lower than 18,000 did not support bacterial growth. A possible meaning of the critical value of the partition ratio is discussed.