Chromosomal gene capture mediated by the Pseudomonas putida TOL catabolic plasmid.

The Pseudomonas putida TOL plasmid pWW0 is able to mediate chromosomal mobilization in the canonical unidirectional way, i.e., from donor to recipient cells, and bidirectionally, i.e., donor-->recipient-->donor (retrotransfer). Transconjugants are recipient cells that have received DNA from donor cells, whereas retrotransconjugants are donor bacteria that have received DNA from a recipient. The TOL plasmid pWW0 is able to directly mobilize and retromobilize a kanamycin resistance marker integrated into the chromosome of other P. putida strains, a process that appears to involve a single conjugational event. The rate of retrotransfer (as well as of direct transfer) of the chromosomal marker is influenced by the location of the kanamycin marker on the chromosome and ranges from 10(-3) to less than 10(-8) retrotransconjugants per donor (transconjugants per recipient). The mobilized DNA is incorporated into the chromosome of the retrotransconjugants (transconjugants) in a process that seems to occur through recombination of highly homologous flanking regions. No interspecific mobilization of the chromosomal marker in matings involving P. putida and the closely related Pseudomonas fluorescens, which belongs to rRNA group I, was observed.     

Transposon mutagenesis analysis of meta-cleavage pathway operon genes of the TOL plasmid of Pseudomonas putida mt-2.

Hybrid plasmids containing the regulated meta-cleavage pathway operon of TOL plasmid pWWO were mutagenized with transposon Tn1000 or Tn5. The resulting insertion mutant plasmids were examined for their ability to express eight of the catabolic enzymes in Escherichia coli. The physical locations of the insertions in each of 28 Tn1000 and 5 Tn5 derivative plasmids were determined by restriction endonuclease cleavage analysis. This information permitted the construction of a precise physical and genetic map of the meta-cleavage pathway operon. The gene order xylD (toluate dioxygenase), L (dihydroxycyclohexidiene carboxylate dehydrogenase), E (catechol 2,3-dioxygenase), G (hydroxymuconic semialdehyde dehydrogenase), F (hydroxymuconic semialdehyde hydrolase), J (2-oxopent-4-enoate hydratase), I (4-oxalocrotonate decarboxylase), and H (4-oxalocrotonate tautomerase) was established, and gene sizes were estimated. Tn1000 insertions within catabolic genes exerted polar effects on distal structural genes of the operon, but not on an adjacent regulatory gene xylS.     

Genetic analysis of a relaxed substrate specificity aromatic ring dioxygenase, toluate 1,2-dioxygenase, encoded by TOL plasmid pWW0 of Pseudomonas putida

Summary Toluate 1,2-dioxygenase is the first enzyme of a meta-cleavage pathway for the oxidative catabolism of benzoate and substituted benzoates to Krebs cycle intermediates that is specified by TOL plasmid pWW0 of Pseudomonas putida. A collection of derivatives harbouring Tn1000 insertions and defective in toluate dioxygenase have been isolated from pPL392, a pBR322-based hybrid plasmid carrying the TOL plasmid meta-cleavage pathway operon. In parallel, a series of N-methyl-N-nitro-N-nitrosoguanidine-induced mutant plasmids defective in this enzyme activity were isolated from pNM72, a pKT231-based hybrid plasmid carrying the same operon. Pairs of mutant plasmids, consisting of one Tn1000 derivative and one nitrosoguanidine-induced derivative, were used for complementation analysis of toluate dioxygenase in Escherichia coli recA bacteria, in which the formation of 2-hydroxymuconic semialdehyde from benzoate was examined. Four cistrons for toluate 1,2-dioxygenase were thus identified. DNA fragments containing nitrosoguanidine-induced mutant cistrons plus the other meta-cleavage operon genes were cloned into pOT5, an R388-based vector, and complementation tests between different nitrosoguanidine-induced mutant cistrons were carried out in Pseudomonas putida cells, this time scoring for growth on p-toluate. This analysis also identified four cistrons. Examination of the products of these cistrons, by means of E. coli minicells containing pPL392 or its Tn1000 insertion derivatives, indicated that the first two cistrons of the operon comprise a single gene, xylX, which encodes a 57 kilodalton protein, and that the third cistron, xy/Y, encodes a 20 kilodalton protein.     

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.     

Codon usage patterns suggest independent evolution of two catabolic operons on toluene-degradative plasmid TOL pWW0 of Pseudomonas putida

TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes responsible for the degradation of toluene. The structural genes for these catobolic enzymes are clustered into two operons—namely, the xylCMAB and xylXYZLTEGFJQKIH operons. We examined the codon usage patterns of these catabolic genes by measuring the codon-usage distances between pairs of these catabolic genes. The codon-usage distance, d, between gene 1 and gene 2 was defined as d = [(p _j – q _j )²]^1/2, where p _j > and q _j are the frequencies of the j-th codon in gene 1 and 2, respectively, j being any one of the 64 possible codons. We found that the genes in the same operon exhibit similar codon-usage patterns while genes in the different operons exhibit different codon bias. This observation suggests that genes in the same operon have coevolved, and that the ancestors of the xylCMAB and xylXYZLTEGFJQKIH operons evolved in different organisms. Correspondence to: S. Harayama     

Gene order of the TOL catabolic plasmid upper pathway operon and oxidation of both toluene and benzyl alcohol by the xylA product.

TOL plasmid pWW0 specifies enzymes for the oxidative catabolism of toluene and xylenes. The upper pathway converts the aromatic hydrocarbons to aromatic carboxylic acids via corresponding alcohols and aldehydes and involves three enzymes: xylene oxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase. The synthesis of these enzymes is positively regulated by the product of xylR. Determination of upper pathway enzyme levels in bacteria carrying Tn5 insertion mutant derivatives of plasmid pWW0-161 has shown that the genes for upper pathway enzymes are organized in an operon with the following order: promoter-xylC (benzaldehyde dehydrogenase gene[s])-xylA (xylene oxygenase gene[s])-xylB (benzyl alcohol dehydrogenase gene). Subcloning of the upper pathway genes in a lambda pL promoter-containing vector and analysis of their expression in Escherichia coli K-12 confirmed this order. Two distinct enzymes were found to attack benzyl alcohol, namely, xylene oxygenase and benzyl alcohol dehydrogenase; and their catalytic activities were additive in the conversion of benzyl alcohol to benzaldehyde. The fact that benzyl alcohol is both a product and a substrate of xylene oxygenase indicates that this enzyme has a relaxed substrate specificity.     

Regulation of the degradative pathway enzymes coded for by the TOL plasmid (pWWO) from Pseudomonas putida mt-2

Pseudomonas putida mt-2 carries a plasmid (TOL, pWWO) which codes for a single set of enzymes responsible for the catabolism of toluene and m- and p-xylene to central metabolites by way of benzoate and m- and p-toluate, respectively, and subsequently by a meta cleavage pathway. Characterization of strains with mutations in structural genes of this pathway demonstrates that the inducers of the enzymes responsible for further degradation of m-toluate include m-xylene, m-methylbenzyl alcohol, and m-toluate, whereas the inducers of the enzymes responsible for oxidation of m-xylene to m-toluate include m-xylene and m-methylbenzyl alcohol but not m-toluate. A regulatory mutant is described in which m-xylene and m-methylbenzyl alcohol no longer induce any of the pathway enzymes, but m-toluate is still able to induce the enzymes responsible for its own degradation. Among revertants of this mutant are some strains in which all the enzymes are expressed constitutively and are not further induced by m-xylene. A model is proposed for the regulation of the pathway in which the enzymes are in two regulatory blocks, which are under the control of two regulator gene products. The model is essentially the same as proposed earlier for the regulation of the isofunctional pathway on the TOL20 plasmid from P. putida MT20.     

Adenosylcobalamin-mediated methyl transfer by toluate cis-dihydrodiol dehydrogenase of the TOL plasmid pWW0

We identified and characterized a methyl transfer activity of the toluate cis-dihydrodiol (4-methyl-3,5-cyclohexadiene-cis-1, 2-diol-1-carboxylic acid) dehydrogenase of the TOL plasmid pWW0 towards toluene cis-dihydrodiol (3-methyl-4,5-cyclohexadiene-cis-1, 2-diol). When the purified enzyme from the recombinant Escherichia coli containing the xylL gene was incubated with toluene cis-dihydrodiol in the presence of NAD+, the end products differed depending on the presence of adenosylcobalamin (coenzyme B12). The enzyme yielded catechol in the presence of adenosylcobalamin, while it gave 3-methylcatechol in the absence of the cofactor. Adenosylcobalamin was transformed to methylcobalamin as a result of the enzyme reaction, which indicates that the methyl group of the substrate was transferred to adenosylcobalamin. Other derivatives of the cobalamin such as aquo (hydroxy)- and cyanocobalamin did not mediate the methyl transfer reaction. The dehydrogenation and methyl transfer reactions were assumed to occur concomitantly, and the methyl transfer reaction seemed to depend on the dehydrogenation. To our knowledge, the enzyme is the first dehydrogenase that shows a methyl transfer activity as well.     

Molecular characterization of the Pseudomonas putida 2,3-butanediol catabolic pathway

Abstract The 2,3-butanediol dehydrogenase and the acetoin-cleaving system were simultaneously induced in Pseudomonas putida PpG2 during growth on 2,3-butanediol and on acetoin. Hybridization with a DNA probe covering the genes for the E1 subunits of the Alcaligenes eutrophus acetoin cleaving system and nucleotide sequence analysis identified acoA (975 bp), acoB (1020 bp), acoC (1110 bp), acoX (1053 bp) and adh (1086 bp) in a 6.3-kb genomic region. The amino acid sequences deduced from acoA , acoB , and acoC for E1α ( M _r 34639), E1β ( M _r 37268), and E2 ( M _r 39613) of the P. putida acetoin cleaving system exhibited striking similarities to those of the corresponding components of the A. eutrophus acetoin cleaving system and of the acetoin dehydrogenase enzyme system of Pelobacter carbinolicus and other bacteria. Strong sequence similarities of the adh translational product (2,3-butanediol dehydrogenase, M _r 38361) were obtained to various alcohol dehydrogenases belonging to the zinc- and NAD(P)-dependent long-chain (group I) alcohol dehydrogenases. Expression of the P. putida ADH in Escherichia coli was demonstrated. The aco genes and adh constitute presumably one single operon which encodes all enzymes required for the conversion of 2,3-butanediol to central metabolites.     

p-Cumate catabolic pathway in Pseudomonas putida Fl: cloning and characterization of DNA carrying the cmt operon.

Pseudomonas putida F1 utilizes p-cumate (p-isopropylbenzoate) as a growth substrate by means of an eight-step catabolic pathway. A 35.75-kb DNA segment, within which the cmt operon encoding the catabolism of p-cumate is located, was cloned as four separate overlapping restriction fragments and mapped with restriction endonucleases. By examining enzyme activities in recombinant bacteria carrying these fragments and sub-cloned fragments, genes encoding most of the enzymes of the p-cumate pathway were located. Subsequent sequence analysis of 11,260 bp gave precise locations of the 12 genes of the cmt operon. The first three genes, cmtAaAbAc, and the sixth gene, cmtAd, encode the components of p-cumate 2,3-dioxygenase (ferredoxin reductase, large subunit of the terminal dioxygenase, small subunit of the terminal dioxygenase, and ferredoxin, respectively); these genes are separated by cmtC, which encodes 2,3-dihydroxy-p-cumate 3,4-dioxygenase, and cmtB, coding for 2,3-dihydroxy-2,3-dihydro-p-cumate dehydrogenase. The ring cleavage product, 2-hydroxy-3-carboxy-6-oxo-7-methylocta-2,4-dienoate, is acted on by a decarboxylase encoded by the seventh gene, cmtD, which is followed by a large open reading frame, cmtI, of unknown function. The next four genes, cmtEFHG, encode 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase, 2-hydroxypenta-2,4-dienoate hydratase, 4-hydroxy-2-oxovalerate aldolase, and acetaldehyde dehydrogenase, respectively, which transform the decarboxylation product to amphibolic intermediates. The deduced amino acid sequences of all the cmt gene products except CmtD and CmtI have a recognizable but low level of identity with amino acid sequences of enzymes catalyzing analogous reactions in other catabolic pathways. This identity is highest for the last two enzymes of the pathway (4-hydroxy-2-oxovalerate aldolase and acetaldehyde dehydrogenase [acylating]), which have identities of 66 to 77% with the corresponding enzymes from other aromatic meta-cleavage pathways. Recombinant bacteria carrying certain restriction fragments bordering the cmt operon were found to transform indole to indigo. This reaction, known to be catalyzed by toluene 2,3-dioxygenase, led to the discovery that the tod operon, encoding the catabolism of toluene, is located 2.8 kb downstream from and in the same orientation as the cmt operon in P. putida F1.