p-Cymene catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA encoding conversion of p-cymene to p-cumate.

Pseudomonas putida F1 utilizes p-cymene (p-isopropyltoluene) by an 11-step pathway through p-cumate (p-isopropylbenzoate) to isobutyrate, pyruvate, and acetyl coenzyme A. The cym operon, encoding the conversion of p-cymene to p-cumate, is located just upstream of the cmt operon, which encodes the further catabolism of p-cumate and is located, in turn, upstream of the tod (toluene catabolism) operon in P. putida F1. The sequences of an 11,236-bp DNA segment carrying the cym operon and a 915-bp DNA segment completing the sequence of the 2,673-bp DNA segment separating the cmt and tod operons have been determined and are discussed here. The cym operon contains six genes in the order cymBCAaAbDE. The gene products have been identified both by functional assays and by comparing deduced amino acid sequences to published sequences. Thus, cymAa and cymAb encode the two components of p-cymene monooxygenase, a hydroxylase and a reductase, respectively; cymB encodes p-cumic alcohol dehydrogenase; cymC encodes p-cumic aldehyde dehydrogenase; cymD encodes a putative outer membrane protein related to gene products of other aromatic hydrocarbon catabolic operons, but having an unknown function in p-cymene catabolism; and cymE encodes an acetyl coenzyme A synthetase whose role in this pathway is also unknown. Upstream of the cym operon is a regulatory gene, cymR. By using recombinant bacteria carrying either the operator-promoter region of the cym operon or the cmt operon upstream of genes encoding readily assayed enzymes, in the presence or absence of cymR, it was demonstrated that cymR encodes a repressor which controls expression of both the cym and cmt operons and is inducible by p-cumate but not p-cymene. Short (less than 350 bp) homologous DNA segments that are located upstream of cymR and between the cmt and tod operons may have been involved in recombination events that led to the current arrangement of cym, cmt, and tod genes in P. putida F1.     

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.     

Isopropylbenzene catabolic pathway in Pseudomonas putida RE204: nucleotide sequence analysis of the ipb operon and neighboring DNA from pRE4

Pseudomonas putida RE204 employs a set of plasmid-specified enzymes in the catabolism of isopropylbenzene (cumene) and related alkylbenzenes. A 21,768 bp segment of the plasmid pRE4, whose sequence is discussed here, includes the ipb (isopropylbenzene catabolic) operon as well as associated genetic elements. The ipb operon, ipbAaAbAcAdBCEGFHD, encodes enzymes catalyzing the conversion of isopropylbenzene to isobutyrate, pyruvate, and acetyl-coenzyme A as well as an outer membrane protein (IpbH) of uncertain function. These gene products are 75 to 91% identical to those encoded by other isopropylbenzene catabolic operons and are somewhat less similar to analogous proteins of related pathways for the catabolism of mono-substituted benzenes. Upstream of ipbAa, ipbR encodes a positive regulatory protein which has about 56% identity to XylS regulatory proteins of TOL (xylene/toluate) catabolic plasmids. This similarity and that of the DNA sequence in the proposed ipb operator-promoter region (ipbOP) to the same region of the xyl meta operon (xylOmPm) suggest that, although the IpbR and XylS regulatory proteins recognize very different inducers, their interactions with DNA to activate gene expression are similar. Upstream of ipbR is an 1196 bp insertion sequence, IS1543, related to IS52 and IS1406. Separating ipbR from ipbAa are 3 additional tightly clustered IS elements. These are IS1544, related to IS1543, IS52, and other members of the IS5 family; IS1545, related to IS1240; and IS1546, related to IS1491. Encompassing the ipb catabolic genes and the other genetic elements and separated from each other by 18,492 bp, are two identical, directly repeated 1007 bp DNA segments. Homologous recombination between these segments appears to be responsible for the occasional deletion of the intervening DNA from pRE4.     

D-lysine catabolic pathway in Pseudomonas putida: interrelations with L-lysine catabolism

The isolation of several mutant strains blocked in l-lysine degradation has permitted an assessment of the physiological significance of enzymatic reactions related to lysine metabolism in Pseudomonas putida. Additional studies with intact cells involved labeling of metabolic intermediates from radioactive l- or d-lysine, and patterns of enzyme induction in both wild-type and mutant strains. These studies lead to the conclusions that from l-lysine, the obligatory pathway is via delta-aminovaleramide, delta-aminovalerate, glutaric semialdehyde, and glutarate, and that no alternative pathways from l-lysine exist in our strain. A distinct pathway from d-lysine proceeds via Delta(1)-piperideine-2-carboxylate, l-pipecolate, and Delta(1)-piperideine-6-carboxylate (alpha-aminoadipic semialdehyde). The two pathways are independent in the sense that certain mutants, unable to grow on l-lysine, grow at wild-type rates of d-lysine, utilizing the same intermediates as the wild type, as inferred from labeling studies. This finding implies that lysine racemase in our strain, while detectable in cell extracts, is not physiologically functional in intact cells at a rate that would permit growth of mutants blocked in the l-lysine pathway. Pipecolate oxidase, a d-lysine-related enzyme, is induced by d-lysine and less efficiently by l-lysine. Aminooxyacetate virtually abolishes the inducing activity of l-lysine for this enzyme, suggesting that lysine racemase, although functionally inactive for growth purposes, may still have regulatory significance in permitting cross-induction of d-lysine-related enzymes by l-lysine, and vice versa. This finding suggests a mechanism in bacteria for maintaining regulatory patterns in pathways that may have lost their capacity to support growth. In addition, enzymatic studies are reported which implicate Delta(1)-piperideine-2-carboxylate reductase as an early step in the d-lysine pathway.     

Intermediates and enzymes between alpha-ketoarginine and gamma-guanidinobutyrate in the L-arginine catabolic pathway of Pseudomonas putida.

In Pseudomonas putida P2 grown on L-arginine as the sole source of carbon and nitrogen, catabolism of L-arginine forms of alpha-ketoarginine, gamma-guanidinobutyrate, and gamma-aminobutyrate. A previously undetected intermediate, gamma-guanidinobutyraldehyde, is identified as the product of alpha-ketoarginine decarboxylase. An 86-fold purification of this enzyme is described. Activity is thiamine pyrophosphate-dependent and cofactor reassociation is facilitated by divalent cations. The order of effectiveness is Mn-2+ greater than Mg-2+, Co-2+ greater than Ca-2+ greater than Ni-2+ greater than Zn-2+. An inducible enzyme that catalyzes conversion of gamma-guanidinobutyraldehyde to gamma-guanidinobutyrate has been studied in cell-free extracts. NAD-+, but no other cofactors, is required. By differential nutritional growth experiments, 4 regulatory units for the L-arginine pathway are proposed and inducers of 2 units are identified.     

Molecular characterization of the mde operon involved in L-methionine catabolism of Pseudomonas putida.

A 15-kb region of Pseudomonas putida chromosomal DNA containing the mde operon and an upstream regulatory gene (mdeR) has been cloned and sequenced. The mde operon contains two structural genes involved in L-methionine degradative metabolism: the already-identified mdeA, which encodes L-methionine gamma-lyase (H. Inoue, K. Inagaki, M. Sugimoto, N. Esaki, K. Soda, and H. Tanaka. J. Biochem. (Tokyo) 117:1120-1125, 1995), and mdeB, which encodes a homologous protein to the homodimeric-type E1 component of pyruvate dehydrogenase complex. A rho-independent terminator was present just downstream of mdeB, and open reading frames corresponding to other components of alpha-keto acid dehydrogenase complex were not found. When MdeB was overproduced in Escherichia coli, the cell extract showed the E1 activity with high specificity for alpha-ketobutyrate rather than pyruvate. These results suggest that MdeB plays an important role in the metabolism of alpha-ketobutyrate produced by MdeA from L-methionine. Accordingly, mdeB encodes a novel E1 component, alpha-ketobutyrate dehydrogenase E1 component, of an unknown alpha-keto acid dehydrogenase complex in P. putida. In addition, we found that the mdeR gene was located on the opposite strand and began at 127 bp from the translational start site of mdeA. The mdeR gene product has been identified as a member of the leucine-responsive regulatory protein (Lrp) family and revealed to act as an essential positive regulator allowing the expression of the mdeAB operon.     

Molecular cloning of 3-phenylcatechol dioxygenase involved in the catabolic pathway of chlorinated biphenyl from Pseudomonas putida and its expression in Escherichia coli.

Genes encoding 3-phenylcatechol dioxygenases were cloned from the chlorobiphenyl-degrading Pseudomonas putida strain OU83, using broad-host-range cosmid vector pCP13. Restriction enzyme analysis of DNA from 2,3-dioxygenase-positive chimeric cosmids showed DNA inserts ranging in size from 6.0 to 30 kilobases. The origin of the DNA insert in hybrid clones was established by using 32P-labeled hybrid clones (pOH101 and pOH810). A 2.3-kilobase HindIII fragment was common to two clones. The 2,3-dioxygenase from the parent P. putida strain, OU83, and the recombinant clones (pOH101 and pOH8101) showed similar characteristics as determined by isoelectric focusing and polyacrylamide gel electrophoresis. The 2,3-dioxygenase from the Escherichia coli recombinant cosmid showed a pI of 5.0, a Km of 14 microM, and broad substrate activity with catechol, 4-chlorocatechol, 4-methylcatechol, and 2,3-dihydroxybiphenyl.     

Molecular cloning of 3-phenylcatechol dioxygenase involved in the catabolic pathway of chlorinated biphenyl from Pseudomonas putida and its expression in Escherichia coli

Genes encoding 3-phenylcatechol dioxygenases were cloned from the chlorobiphenyl-degrading Pseudomonas putida strain OU83, using broad-host-range cosmid vector pCP13. Restriction enzyme analysis of DNA from 2,3-dioxygenase-positive chimeric cosmids showed DNA inserts ranging in size from 6.0 to 30 kilobases. The origin of the DNA insert in hybrid clones was established by using 32P-labeled hybrid clones (pOH101 and pOH810). A 2.3-kilobase HindIII fragment was common to two clones. The 2,3-dioxygenase from the parent P. putida strain, OU83, and the recombinant clones (pOH101 and pOH8101) showed similar characteristics as determined by isoelectric focusing and polyacrylamide gel electrophoresis. The 2,3-dioxygenase from the Escherichia coli recombinant cosmid showed a pI of 5.0, a Km of 14 microM, and broad substrate activity with catechol, 4-chlorocatechol, 4-methylcatechol, and 2,3-dihydroxybiphenyl.     

Identification and expression of the cym, cmt, and tod catabolic genes from Pseudomonas putida KL47: expression of the regulatory todST genes as a factor for catabolic adaptation

Pseudomonas putida KL47 is a natural isolate that assimilates benzene, 1-alkylbenzene (C(1)-C(4)), biphenyl, p-cumate, and p-cymene. The genetic background of strain KL47 underlying the broad range of growth substrates was examined. It was found that the cym and cmt operons are constitutively expressed due to a lack of the cymR gene, and the tod operon is still inducible by toluene and biphenyl. The entire array of gene clusters responsible for the catabolism of toluene and p-cymene/p-cumate has been cloned in a cosmid vector, pLAFR3, and were named pEK6 and pEK27, respectively. The two inserts overlap one another and the nucleotide sequence (42,505 bp) comprising the cym, cmt, and tod operons and its flanking genes in KL47 are almost identical (>99%) to those of P. putida F1. In the cloned DNA fragment, two genes with unknown functions, labeled cymZ and cmtR, were newly identified and show high sequence homology to dienelactone hydrolase and CymR proteins, respectively. The cmtR gene was identified in the place of the cmtI gene of previous annotation. Western blot analysis showed that, in strains F1 and KL47, the todT gene is not expressed during growth on Luria Bertani medium. In minimal basal salt medium, expression of the todT gene is inducible by toluene, but not by biphenyl in strain F1; however, it is constantly expressed in strain KL47, indicating that high levels of expression of the todST genes with one amino acid substitution in TodS might provide strain KL47 with a means of adaptation of the tod catabolic operon to various aromatic hydrocarbons.     

p-cymene pathway in Pseudomonas putida: initial reactions.

Initial reactions of the p-cymene pathway induced in Pseudomonas putida PL have been reinvestigated. Oxidation of the methyl group attached to the nucleus occurs in three steps to give p-cumic acid. The substrate for the ring cleavage of 2,3-dihydroxy-p-cumate is formed from p-cumate in two reactions via a dihydrodiol intermediate (2,3-dihydroxy-4-isopropylcyclohexa-4,6-dienoate) and not as previously postulated via 3-hydroxy-p-cumate. There are three pieces of evidence for the physiological role of the dihydrodiol intermediate. (i) a mutant of P. putida PL-pT-11/43, which is unable to grow with p-cumate, accumulates a compound from p-cumate, which was identified as 2,3-dihydroxy-4-isopropylcyclohexa-4,6-dienoate. (II) This metabolite is enzymically oxidized by a nicotinamide adenine dinucleotide-dependent dehydrogenase that is present in crude extracts of the wild type and a revertant strain (PL-pT-11/43-R1) but not in the mutant. (iii) 3-Hydroxy-p-cumate does not support growth of P . putida PL-W, and it is not oxidized by cells or extracts. 3-Hydroxy-p-cumate was readily isolated as before from culture supernatants, due to its ready formation from the dihydrodiol in acid solution. Mass spectral analysis of the dihydrodiol accumulated in 18O2-enriched atmospheres showed that both hydroxyl atoms are derived from the same molecule of O2. The formation and absorbance maxima of dihydrodiols that accumulated during the growth of the mutant PL-pT-11/43 in the presence of various benzoates (or toluenes) that have substituents at the carbon 4 atom also is reported.     

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.