Comparative study of aromatic ring meta-cleavage enzymes in Pseudomonas strains with plasmid and chromosomal genetic control of the catabolism of biphenyl and m-toluate

It was shown that two different enzymes of aromatic ring oxidative meta-cleavage (2,3-dihydroxybiphenyl-1,2-dioxygenase), DBO and catechol-2,3-dioxygenase, C230) function in Pseudomonas strains with a plasmid and chromosomal genetic control of biphenyl and toluate catabolism. A comparative analysis of DBO's and C230's expressed by the pBS241 biphenyl degradative plasmid in P. putida BS893, pBS311 in P. putida U83, chromosomal genes in P. putida BF and C230 from P. putida PaW160 (pWWO) was carried out. It was found that the DBO's of all strains under study are highly specialized enzymes in respect of 2,3-dihydroxybiphenyl cleavage and are also able to cleave 3-methyl-catechol and catechol (but not 4-methylcatechol) at low rates. In contrast with DBO's, in Pseudomonas strains the substrate specificities of all C230's are variable. The C230's expressed by the D-plasmids pBS241 and pBC311 have a moderate affinity for catechol, 3-methyl- and 4-methylcatechol, but are unable to cleave 2,3-dihydroxybiphenyl. The C230 which is encoded by the chromosomal structure gene from P. putida BF is very similar to C230 which codes for the TOL-plasmid pWWO. These plasmid differ from C230's expressed by biphenyl D-plasmids due to their capability to cleave 2,3-dihydroxybiphenyl in addition to catechol cleavage. All DBO's and C230's under study possess a number of properties that are typical for the enzymes having an oxidative meta-cleaving effect. The different roles of these enzymes in biphenyl and toluate catabolism in Pseudomonas strains are 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.     

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.     

Conjugal transfer of a TOL-like plasmid and extension of the catabolic potential of Pseudomonas putida F1

Strain mX was isolated from a petrol-contaminated soil, after enrichment on minimal medium with 0.5% (v/v) meta-xylene as a sole carbon source. The strain was tentatively characterized as Pseudomonas putida and harboured a large plasmid (pMX) containing xyl genes involved in toluene or meta-xylene degradation pathways via an alkyl monooxygenase and a catechol 2,3-dioxygenase. This new TOL-like plasmid was stable over two hundred generations and was self-transferable. After conjugal transfer to P. putida F1, which possesses the Tod chromosomal toluene biodegradative pathway, the transconjugant P. putida F1(pMX) was able to grow on benzene, toluene, meta-xylene, para-xylene, and ethylbenzene compounds as the sole carbon sources. Catechol 2,3-dioxygenases of the transconjugant cells presented a more relaxed substrate specificity than those of parental cells (strain mX and 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.     

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.     

Lignin catabolic pathways reveal unique characteristics of dye-decolorizing peroxidases in Pseudomonas putida

Lignin is one of the largest carbon reservoirs in the environment, playing an important role in the global carbon cycle. However, lignin degradation in bacteria, especially non-model organisms, has not been well characterized either enzymatically or genetically. Here, a lignin-degrading bacterial strain, Pseudomonas putida A514, was used as the research model. Genomic and proteomic analyses suggested that two B subfamily dye-decolorizing peroxidases (DypBs) were prominent in lignin depolymerization, while the classic O₂-dependent ring cleavage strategy was utilized in central pathways to catabolize lignin-derived aromatic compounds that were funnelled by peripheral pathways. These enzymes, together with a range of transporters, sequential and expression-dose dependent regulation and stress response systems coordinated for lignin metabolism. Catalytic assays indicated these DypBs show unique Mn²⁺ independent lignin depolymerization activity, while Mn²⁺ oxidation activity is absent. Furthermore, a high synergy between DypB enzymes and A514 cells was observed to promote cell growth (5 × 10¹² cfus/ml) and lignin degradation (27%). This suggested DypBs are competitive lignin biocatalysts and pinpointed limited extracellular secretion capacity as the rate-limiting factor in bacterial lignin degradation. DypB production was, therefore, optimized in recombinant strains and a 14,141-fold increase in DypB activity (56,565 U/l) was achieved, providing novel insights for lignin bioconversion.     

Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440

Analysis of the catabolic potential of Pseudomonas putida KT2440 against a wide range of natural aromatic compounds and sequence comparisons with the entire genome of this microorganism predicted the existence of at least four main pathways for the catabolism of central aromatic intermediates, that is, the protocatechuate (pca genes) and catechol (cat genes) branches of the beta-ketoadipate pathway, the homogentisate pathway (hmg/fah/mai genes) and the phenylacetate pathway (pha genes). Two additional gene clusters that might be involved in the catabolism of N-heterocyclic aromatic compounds (nic cluster) and in a central meta-cleavage pathway (pcm genes) were also identified. Furthermore, the genes encoding the peripheral pathways for the catabolism of p-hydroxybenzoate (pob), benzoate (ben), quinate (qui), phenylpropenoid compounds (fcs, ech, vdh, cal, van, acd and acs), phenylalanine and tyrosine (phh, hpd) and n-phenylalkanoic acids (fad) were mapped in the chromosome of P. putida KT2440. Although a repetitive extragenic palindromic (REP) element is usually associated with the gene clusters, a supraoperonic clustering of catabolic genes that channel different aromatic compounds into a common central pathway (catabolic island) was not observed in P. putida KT2440. The global view on the mineralization of aromatic compounds by P. putida KT2440 will facilitate the rational manipulation of this strain for improving biodegradation/biotransformation processes, and reveals this bacterium as a useful model system for studying biochemical, genetic, evolutionary and ecological aspects of the catabolism of aromatic compounds.     

Chromosomal location of TOL plasmid DNA in Pseudomonas putida.

The soil isolate Pseudomonas putida MW1000 can grow on toluene and other hydrocarbons; in this respect it is similar to strains of Pseudomonas which carry the TOL plasmid. By conjugation experiments, the genes conferring these growth abilities have been shown to be located on the bacterial chromosome, linked to vil and catB. A 56-kilobase segment of the bacterial chromosome of MW strains carrying the TOL genes can transpose to the IncP-1 plasmid R18-18. Physical analysis of these TOL R18-18 hybrids has shown that the TOL segment is almost identical to the same region found in the TOL plasmid pWW0.     

Physical map of the DNA of bacteriophage tf of Pseudomonas putida

A physical map has been constructed for P. putida bacteriophage tf DNA containing single-strand breaks (nicks). Localization of cleavage sites for EcoRI, HindIII, HpaI ClaI, BamHI, SalI, XbaI and XhoI restriction endonucleases was determined. Position of single-strand breaks was mapped by electrophoretic analysis of denatured tf DNA and electron microscopy of partially denatured DNA samples. The tf genome is characterized by the presence of two classes of nicks differing in the frequency of their presence in population of bacteriophage DNA molecules.     

Mutants defective in isomerase and decarboxylase activities of the 4-hydroxyphenylacetic acid meta-cleavage pathway in Pseudomonas putida.

Degradation of 2-hydroxy-5-carboxymethylmuconic semialdehyde, the ring fission product of the 4-hydroxyphenylacetate meta-cleavage pathway, by mutant strains P23X19 and P23X16 of Pseudomonas putida NCI B 9865 was studied. Both mutants were unable to grow on either 4-hydroxyphenylacetate of 3,4-dihydroxyphenylacetate. Cell extracts of P23X19, grown in the presence of 3,4-dihydroxyphenylacetate, degraded the ring fission product to a compound that accumulated and had maximum UV absorption at 300 nm, pH 7.4, and 345 nm, pH 12. These are the spectral characteristics of 2-keto-5-carboxymethylhex-3-ene-1,6-dioate, the substrate for the decarboxylase in this pathway. This observation is consistent with P23X19's being decarboxylase defective. Cell extracts of P23X16, grown in the presence of 3,4-dihydroxyphenylacetate, degraded the ring fission product to a compound that accumulated and has maximum UV absorption at 295 nm, pH 7.4, and 345 nm, pH 12. This compound spontaneously degraded to a compound with the spectral properties of the decarboxylase substrate. The compound accumulated by P23X16 was also obtained when the decarboxylase substrate was treated with borate. It is suggested that the compound accumulated by P23X16 is the substrate of an isomerase. The results are consistent with P23X16's being unable to synthesize a functional isomerase while retaining decarboxylase activity and establish the physiological importance of an enzyme-catalyzed isomerization in the meta-cleavage degradation of 4-hydroxyphenylacetate.     

A cleavage map of the TOL plasmid of Pseudomonas putida mt-2.

Summary A cleavage map of the TOL plasmid pWWO has been determined for the restriction endonucleases HindIII and XhoI. A number of techniques were employed including (i) digestion of purified cleavage products with a second enzyme; (ii) hybridisation of purified XhoI fragments to Southern blots of HindIII digest products and (iii) analysis of a number of deletion mutants.     

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.