2-hydroxymuconic Semialdehyde Dehydrogenase Research Articles

Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters.

Pseudomonas veronii 1YdBTEX2, a benzene and toluene degrader, and Pseudomonas veronii 1YB2, a benzene degrader, have previously been shown to be key players in a benzene-contaminated site. These strains harbor unique catabolic pathways for the degradation of benzene comprising a gene cluster encoding an isopropylbenzene dioxygenase where genes encoding downstream enzymes were interrupted by stop codons. Extradiol dioxygenases were recruited from gene clusters comprising genes encoding a 2-hydroxymuconic semialdehyde dehydrogenase necessary for benzene degradation but typically absent from isopropylbenzene dioxygenase-encoding gene clusters. The benzene dihydrodiol dehydrogenase-encoding gene was not clustered with any other aromatic degradation genes, and the encoded protein was only distantly related to dehydrogenases of aromatic degradation pathways. The involvement of the different gene clusters in the degradation pathways was suggested by real-time quantitative reverse transcription PCR.

Deepening TOL and TOU catabolic pathways of Pseudomonas sp. OX1: Cloning, sequencing and characterization of the lower pathways

Pseudomonas sp. OX1 is able to metabolize toluene and o-xylene through the TOU catabolic pathway, whereas its mutant M1 strain was found to be able to use m- and p-xylene as carbon and energy source, using the TOL catabolic pathway. Here we report the complete nucleotide sequence of the phe lower operon of the TOU catabolic pathway, and the sequence of the last four genes of the xyl-like lower operon of the TOL catabolic pathway. DNA sequence analysis shows the gene order within the operons to be pheCDEFGHI (phe operon) and xyl-likeQKIH (xyl-like operon), identical to the order found for the isofunctional genes of meta operons in the toluene/xylene pathway of TOL plasmid pWW0 from Pseudomonas putida mt-2 and the phenol/methylphenol pathway of pVIl50 from Pseudomonas sp. CF600. The nucleotide and the deduced amino acid sequences are homologous to the equivalent gene and enzyme sequences from other Pseudomonas meta pathways. Recombinant 2-hydroxymuconic semialdehyde dehydrogenase (HMSD) and 2-hydroxymuconic semialdehyde hydrolase (HMSH), coded by pheCD genes, respectively, and ADA and HOA enzymes from both phe and xyl operons were expressed in E.coli and steady-state kinetic analysis was carried out. The analysis of the kinetic parameters of HMSD and HMSH showed that the enzymes from Pseudomonas sp. OX1 are more specialized to channel metabolites into the two branches of the lower pathway than homologous enzymes from other pseudomonads. The kinetics parameters of recombinant ADA from phe and xyl-like operon were found to be similar to those of homologous enzymes from other Pseudomonas strains. In addition, the enzyme from xyl-like operon showed a substrate affinity three times higher than the enzyme from phe operon.

Degradation of chloroaromatics by Pseudomonas putida GJ31: assembled route for chlorobenzene degradation encoded by clusters on plasmid pKW1 and the chromosome

Pseudomonas putida GJ31 has been reported to grow on chlorobenzene using a meta-cleavage pathway with chlorocatechol 2,3-dioxygenase (CbzE) as a key enzyme. The CbzE-encoding gene was found to be localized on the 180 kb plasmid pKW1 in a cbzTEXGS cluster, which is flanked by transposases and encodes only a partial (chloro)catechol meta-cleavage pathway comprising ferredoxin reductase, chlorocatechol 2,3-dioxygenase, an unknown protein, 2-hydroxymuconic semialdehyde dehydrogenase and glutathione S-transferase. Downstream of cbzTEXGS are located cbzJ, encoding a novel type of 2-hydroxypent-2,4-dienoate hydratase, and a transposon region highly similar to Tn5501. Upstream of cbzTEXGS, traNEOFG transfer genes were found. The search for gene clusters possibly completing the (chloro)catechol metabolic pathway of GJ31 revealed the presence of two additional catabolic gene clusters on pKW1. The mhpRBCDFETP cluster encodes enzymes for the dissimilation of 2,3-dihydroxyphenylpropionate in a novel arrangement characterized by the absence of a gene encoding 3-(3-hydroxyphenyl)propionate monooxygenase and the presence of a GntR-type regulator, whereas the nahINLOMKJ cluster encodes part of the naphthalene metabolic pathway. Transcription studies supported their possible involvement in chlorobenzene degradation. The upper pathway cluster, comprising genes encoding a chlorobenzene dioxygenase and a chlorobenzene dihydrodiol dehydrogenase, was localized on the chromosome. A high level of transcription in response to chlorobenzene revealed it to be crucial for chlorobenzene degradation. The chlorobenzene degradation pathway in strain GJ31 is thus a mosaic encoded by four gene clusters.

Novel organization of catechol meta pathway genes in the nitrobenzene degrader Comamonas sp. JS765 and its evolutionary implication

The catechol meta cleavage pathway is one of the central metabolic pathways for the degradation of aromatic compounds. A novel organization of the pathway genes, different from that of classical soil microorganisms, has been observed in Sphingomonas sp HV3 and Pseudomonas sp. DJ77. In a Comamonas sp. JS765, cdoE encoding catechol 2,3-dioxygenase shares a common ancestry only with tdnC of a Pseudomonas putida strain, while codG encoding 2-hydroxymuconic semialdehyde dehydrogenase shows a higher degree of similarity to those genes in classical bacteria. Located between cdoE and cdoG are several putative genes, whose functions are unknown. These genes are not found in meta pathway operons of other microorganisms with the exception of cdoX2, which is similar to cmpX in strain HV3. Therefore, the gene cluster in JS765 reveals a third type of gene organization of the meta pathway.

Novel organization of catechol meta-pathway genes in Sphingomonas sp. HV3 pSKY4 plasmid.

Sphingomonas sp. strain HV3 (formerly Pseudomonas sp. HV3), which degrades aromatics and chloroaromatics, harbors a mega-plasmid, pSKY4. A sequenced 4 kb fragment of the plasmid reveals a novel gene organization for catechol meta-pathway genes. The putative meta operon starts with the cmpF gene encoding a 2-hydroxymuconic semialdehyde hydrolase. The gene has a 6 bp overlap with the previously characterized ring-cleavage gene, catechol 2,3-dioxygenase, cmpE. Downstream of cmpE is a 429 bp open reading frame of unknown function. Gene cmpC, encoding a 2-hydroxymuconic semialdehyde dehydrogenase, starts 44 bp further downstream. It has the highest homology to 2-hydroxymuconic semialdehyde dehydrogenases of dmp and xyl pathways and to XylC from the marine oligotroph Cycloclasticus oligotrophus. The gene organization is different from other known meta pathways. This is the first report of organization of plasmid-encoded meta-pathway genes in the genus Sphingomonas.

Microorganisms degrading chlorobenzene via a meta-cleavage pathway harbor highly similar chlorocatechol 2,3-dioxygenase-encoding gene clusters.

Pseudomonas putida GJ31 harbors a degradative pathway for chlorobenzene via meta-cleavage of 3-chlorocatechol. Pseudomonads using this route for chlorobenzene degradation, which was previously thought to be generally unproductive, were isolated from various contaminated environments of distant locations. The new isolates, Pseudomonas fluorescens SK1 (DSM16274), Pseudomonas veronii 16-6A (DSM16273), Pseudomonas sp. strain MG61 (DSM16272), harbor a chlorocatechol 2,3-dioxygenase (CbzE). The cbzE-like genes were cloned, sequenced, and expressed from the isolates and a mixed culture. The chlorocatechol 2,3-dioxygenases shared 97% identical amino acids with CbzE from strain GJ31, forming a distinct family of catechol 2,3-dioxygenases. The chlorocatechol 2,3-dioxygenase, purified from chlorobenzene-grown cells of strain SK1, showed an identical N-terminal sequence with the amino acid sequence deduced from cloned cbzE. In all investigated chlorobenzene-degrading strains, cbzT-like genes encoding ferredoxins are located upstream of cbzE. The sequence data indicate that the ferredoxins are identical (one amino acid difference in CbzT of strain 16-6A compared to the others). In addition, the structure of the operon downstream of cbzE is identical in strains GJ31, 16-6A, and SK1 with genes cbzX (unknown function) and the known part of cbzG (2-hydroxymuconic semialdehyde dehydrogenase) and share 100% nucleotide sequence identity with the entire downstream region. The current study suggests that meta-cleavage of 3-chlorocatechol is not an atypical pathway for the degradation of chlorobenzene.

Kinetic property and phylogenic relationship of 2-hydroxymuconic semialdehyde dehydrogenase encoded in tomC gene of Burkholderia cepacia G4.

2-Hydroxymuconic semialdehyde (2-HMS) dehydrogenase catalyzes the conversion of 2-HMS to 4-oxalocrotonate, which is a step in the meta cleavage pathway of aromatic hydrocarbons in bacteria. A tomC gene that encodes 2-HMS dehydrogenase of Burkholderia cepacia G4, a soil bacterium that can grow on toluene, cresol, phenol, or benzene, was overexpressed into E. coli HB101, and its gene product was characterized in this study. 2-HMS dehydrogenase from B. cepacia G4 has a high catalytic efficiency in terms of Vmax/Km towards 2-hydroxy-5-methylmuconic semialdehyde followed by 2-HMS but has a very low efficiency for 5-chloro-2-hydroxymuconic semialdehyde. However, the enzyme did not utilize 2-hydroxy-6-oxo-hepta-2,4-dienoic acid and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid as substrates. The molecular weight of 2-HMS dehydrogenase from B. cepacia G4 was predicted to be 52 kDa containing 485 amino acid residues from the nucleotide sequence of the tomC gene, and it exhibited the highest identity of 78% with the amino acid sequence of 2-HMS dehydrogenase that is encoded in the aphC gene of Comamonas testosteroni TA441. 2-HMS dehydrogenase from B. cepacia G4 showed a significant phylogenetic relationship not only with other 2-HMS dehydrogenases, but also with different dehydrogenases from evolutionarily distant organisms.

Meta-pathway degradation of phenolics by thermophilic Bacilli

Thermophilic bacteria capable of degrading phenol as the sole carbon source were isolated from sewage effluent. The isolates were aerobic, sporulating, motile rod-shaped bacteria characterized as Bacillus species with growth temperature optima of 50–60°C. The enzyme catalyzing the second step in the phenol degradation meta-cleavage pathway, catechol-2,3-dioxygenase, was detected in all isolates grown in the presence of phenol. One strain, designated Bacillus strain Cro3.2, was capable of degrading phenol, o-, m-, and p-cresol via the meta-pathway and tolerated phenol at concentrations up to 0.1% (w/v) without apparent inhibition of growth. Phenol degradation activities in strain Cro3.2 were induced 3–5 h after supplementation by phenol, orcinol, and the cresols but not by halo- or nitro-substituted phenols. Maximal rates of phenol degradation in stirred bioreactors (10 μmol/min −1/g −1 cells) were achieved at an O 2 delivery rate of 1.0 vvm and temperatures of 45–60°C; however, catechol-2,3-dioxygenase (but not 2-hydroxymuconic semialdehyde dehydrogenase) was rapidly inactivated at high oxygen concentrations. Whole cells of Bacillus strain Cro3.2 entrapped in calcium alginate, polyacrylamide, and agarose gels showed widely different rates of phenol degradation. In calcium alginate gels, rapid loss of phenol-degrading activity was attributed to calcium-induced inactivation of catechol-2,3-dioxygenase. No stabilization with respect to oxygen-induced inactivation was observed under any of the immobilization conditions. It is concluded that the counteractive effects of oxygen limitation at low dO 2 and inactivation of catechol-2,3-dioxygenase at high dO 2 levels pose a significant impediment to the use of resting thermophile cells in the treatment of phenolic waste streams.

Genetic Structure of thebphGGene Encoding 2-Hydroxymuconic Semialdehyde Dehydrogenase ofAchromobacter xylosoxidansKF701

2-Hydroxymuconic semialdehyde dehydrogenase catalyzes the conversion of 2-hydroxymuconic semialdehyde (HMS) to an enol form of 4-oxalocrotonate which is a step in the catecholmeta-cleavage pathway. AbphGgene encoding HMS dehydrogenase ofA. xylosoxidansKF701, a soil bacterium degrading biphenyl, was identified at between catechol 2,3-dioxygenase gene and HMS hydrolase gene, and its sequence was analyzed. An open reading frame (ORF) corresponding tobphGgene was consisted of 1461 nucleotides with ATG initiation codon and TGA termination codon. The ORF exhibited 66% of G + C content, and a putative ribosome-binding sequence, AGAGA, was identified at about 10 nucleotides upstream initiation codon of thebphGgene. ThebphGgene can encode a polypeptide of molecular weight 52 kDa containing 486 amino acid residues. A deduced amino acid sequence of HMS dehydrogenase encoded inbphGgene fromA. xylosoxidansKF701 exhibited the highest 94% homology with that of corresponding enzyme encoded inxylGfromP. putidamt-2, 63% to 90% homology with those of other reported HMS dehydrogenases, and 29% to 42% homology with those of betaine aldehyde dehydrogenase, 5-carboxy-HMS dehydrogenase, aldehyde dehydrogenase, indole-3-acetaldehyde dehydrogenase, succinic semialdehyde dehydrogenase, methylmalonate semialdehyde dehydrogenase, and succinylglutamate 5-semialdehyde dehydrogenase. From an alignment of amino acid sequence of HMS dehydrogenase fromA. xylosoxidansKF701 with other reported dehydrogenases, putative cofactor NAD+-binding regions and catalytic residues were identified.

Nucleotide Sequence of thePseudomonassp. DJ77phnGGene Encoding 2-Hydroxymuconic Semialdehyde Dehydrogenase

The nucleotide sequence of a 1520 bp region, spanning the coding region for themeta-cleavage pathway enzyme, 2-hydroxymuconic semialdehyde dehydrogenase, was determined. This enzyme, encoded by thephnG,is the first of three sequential enzymes required for conversion of 2-hydroxymuconic semialdehyde, which is produced from catechol by the PhnE catechol 2,3-dioxygenase, to 2-hydroxypent-2,4-dienoate in the dehydrogenative branch of the pathway. The deduced protein sequence is 484 amino acid residues long with aMrof 51504. ThephnGhas a high degree of homology with genes encoding isofunctional proteins from otherPseudomonasstrains. We now show that the relative position of thephnGdehydrogenase gene in thephnoperon is unique compared to the othermeta-cleavage operons which have a dehydrogenative branch of the pathway.

Degradation of 4-Chlorophenol via the meta Cleavage Pathway by Comamonas testosteroni JH5

Comamonas testosteroni JH5 used 4-chlorophenol (4-CP) as its sole source of energy and carbon up to a concentration of 1.8 mM, accompanied by the stoichiometric release of chloride. The degradation of 4-CP mixed with the isomeric 2-CP by resting cells led to the accumulation of 3-chlorocatechol (3-CC), which inactivated the catechol 2,3-dioxygenase. As a result, further 4-CP breakdown was inhibited and 4-CC accumulated as a metabolite. In the crude extract of 4-CP-grown cells, catechol 1,2-dioxygenase and muconate cycloisomerase activities were not detected, whereas the activities of catechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, 2-hydroxymuconic semialdehyde hydrolase, and 2-oxopent-4-enoate hydratase were detected. These enzymes of the meta cleavage pathway showed activity with 4-CC and with 5-chloro-2-hydroxymuconic semialdehyde. The activities of the dioxygenase and semialdehyde dehydrogenase were constitutive. Two key metabolites of the meta cleavage pathway, the meta cleavage product (5-chloro-2-hydroxymuconic semialdehyde) and 5-chloro-2-hydroxymuconic acid, were detected. Thus, our previous postulation that C. testosteroni JH5 uses the meta cleavage pathway for the complete mineralization of 4-CP was confirmed.

Novel organization of catechol meta-pathway genes in Sphingomonas sp. HV3 pSKY4 plasmid

Sphingomonas sp. strain HV3 (formerly Pseudomonas sp. HV3), which degrades aromatics and chloroaromatics, harbors a mega-plasmid, pSKY4. A sequenced 4 kb fragment of the plasmid reveals a novel gene organization for catechol meta-pathway genes. The putative meta operon starts with the cmpF gene encoding a 2-hydroxymuconic semialdehyde hydrolase. The gene has a 6 bp overlap with the previously characterized ring-cleavage gene, catechol 2,3-dioxygenase, cmpE. Downstream of cmpE is a 429 bp open reading frame of unknown function. Gene cmpC, encoding a 2-hydroxymuconic semialdehyde dehydrogenase, starts 44 bp further downstream. It has the highest homology to 2-hydroxymuconic semialdehyde dehydrogenases of dmp and xyl pathways and to XylC from the marine oligotroph Cycloclasticus oligotrophus. The gene organization is different from other known meta pathways. This is the first report of organization of plasmid-encoded meta-pathway genes in the genus Sphingomonas.

Overlapping substrate specificities of benzaldehyde dehydrogenase (the xylC gene product) and 2-hydroxymuconic semialdehyde dehydrogenase (the xylG gene product) encoded by TOL plasmid pWW0 of Pseudomonas putida

Two aldehyde dehydrogenases involved in the degradation of toluene and xylenes, namely, benzaldehyde dehydrogenase and 2-hydroxymuconic semialdehyde dehydrogenase, are encoded by the xylC and xylG genes, respectively, on TOL plasmid pWW0 of Pseudomonas putida. The nucleotide sequence of xylC was determined in this study. A protein exhibiting benzaldehyde dehydrogenase activity had been purified from cells of P. putida (pWW0) (J. P. Shaw and S. Harayama, Eur. J. Biochem. 191:705-714, 1990); however, the amino-terminal sequence of this protein does not correspond to that predicted from the xylC sequence but does correspond to that predicted from the xylG sequence. The protein purified in the earlier work was therefore 2-hydroxymuconic semialdehyde dehydrogenase (the xylG gene product). This conclusion was confirmed by the fact that this protein oxidized 2-hydroxymuconic semialdehyde (kcat/Km = 1.6 x 10(6) s-1 M-1) more efficiently than benzaldehyde (kcat/Km = 3.2 x 10(4) s-1 M-1). The xylC product, the genuine benzaldehyde dehydrogenase, was purified from extracts of P. putida (pWW0-161 delta rylG) which does not synthesize 2-hydroxymuconic semialdehyde dehydrogenase. The amino-terminal sequence of the purified protein corresponds to the amino-terminal sequence deduced from the xylC sequence. This enzyme efficiently oxidized benzaldehyde (kcat/Km = 1.7 x 10(7) s-1 M-1) and its analogs but did not oxidize 2-hydroxymuconic semialdehyde or its analogs.

CoA-dependent methylmalonate-semialdehyde dehydrogenase, a unique member of the aldehyde dehydrogenase superfamily. cDNA cloning, evolutionary relationships, and tissue distribution.

Three overlapping cDNA clones encoding methylmalonate-semialdehyde dehydrogenase (MMSDH; 2-methyl-3-oxopropanoate:NAD+ oxidoreductase (CoA-propanoylating); EC 1.2.1.27) have been isolated by screening a rat liver lambda gt 11 library with nondegenerate oligonucleotide probes synthesized according to polymerase chain reaction-amplified portions coding for the N-terminal amino acid sequence of rat liver MMSDH. The three clones cover a total of 1942 base pairs of cDNA, with an open reading frame of 1569 base pairs. The authenticity of the composite cDNA was confirmed by a perfect match of 43 amino acids known from protein sequencing. The composite cDNA predicts a 503 amino acid mature protein with M(r) = 55,330, consistent with previous estimates. Polymerase chain reaction was used to obtain the sequence of the 32 amino acids corresponding to the mitochondrial entry peptide. Northern blot analysis of total RNA from several rat tissues showed a single mRNA band of 3.8 kilobases. Relative mRNA levels were: kidney greater than liver greater than heart greater than muscle greater than brain, which differed somewhat from relative MMSDH protein levels determined by Western blot analysis: liver = kidney greater than heart greater than muscle greater than brain. A 1423-base pair cDNA clone encoding human MMSDH was isolated from a human liver lambda gt 11 library. The human MMSDH cDNA contains an open reading frame of 1293 base pairs that encodes the protein from Leu-74 to the C terminus. Human and rat MMSDH share 89.6 and 97.7% identity in nucleotide and protein sequence, respectively. MMSDH clearly belongs to a superfamily of aldehyde dehydrogenases and is closely related to betaine aldehyde dehydrogenase, 2-hydroxymuconic semialdehyde dehydrogenase, and class 1 and 2 aldehyde dehydrogenases.

DNA sequence determination of the TOL plasmid (pWW0) xylGFJ genes of Pseudomonas putida: implications for the evolution of aromatic catabolism

The meta operon of the Pseudomonas putida TOL plasmid (pWWO) encodes all enzymes of a meta-cleavage pathway for the metabolism of benzoic acids to Krebs-cycle intermediates. We have determined and analysed the nucleic acid sequence of a 3442 bp region of the meta operon containing the xyl-GFJ genes whose products are involved in the post meta-ring fission transformation of catechols. Homology analysis of the xylGFJ gene products revealed evidence of biochemical relatedness, suggested enzymatic mechanisms, and permitted us to propose evolutionary events which may have generated the current variety of aromatic degradative pathways. The xylG gene, which specifies 2-hydroxymuconic semialdehyde dehydrogenase (HMSD), was found to encode a protein of 51.7 kDa. The predicted protein sequence exhibits significant homology to eukaryotic aldehyde dehydrogenases (ADHs) and to the products of two other Pseudomonas catabolic genes, i.e. xylC and alkH. Expansion of the ADH superfamily to include these prokaryotic enzymes permitted a broader analysis of functionally critical ADH residues and phylogenetic relationships among superfamily members. The importance of three regions of these enzymes previously thought to be critical to ADH activity was reinforced by this analysis. However glutamine-487, also thought to be critical, is less well conserved. The revised ADH phylogeny proposed here suggests early catabolic ADH divergence with subsequent interkingdom gene exchange. The xylF gene, which specifies 2-hydroxymuconic semialdehyde hydrolase (HMSH), was delineated by N-terminal sequence analysis of the purified gene product and is shown to encode a protein of 30.6 kDa. Homology analysis revealed sequence similarity to a chromosomally encoded serine hydrolase, especially in the region of the previously identified active-site serine residue, suggesting that HMSH may also possess a serine hydrolytic enzymatic mechanism. Likewise, the xylJ gene, which specifies 2-hydroxy-pent-2,4-dienoate hydratase (HPH), was delineated by N-terminal sequence analysis of purified HPH, and was found to encode a 23.9 kDa protein. Sequence comparisons revealed that both HMSH and HPH have analogues in the tod gene cluster, which specifies a toluene/benzene degradative pathway. Although the newly identified todF and todJ genes had been at least partially sequenced (Zylstra and Gibson, 1989), the open reading frames had not been positively identified. The presence of todJ provides strong evidence that the reactions following ring fission in the tod pathway are identical to those of the TOL pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Nucleotide sequences of the meta-cleavage pathway enzymes 2-hydroxymuconic semialdehyde dehydrogenase and 2-hydroxymuconic semialdehyde hydrolase from Pseudomonas CF600

The nucleotide sequence of a 2493 base pair (bp) region, spanning the coding regions for the meta-cleavage pathway enzymes 2-hydroxymuconic semialdehyde dehydrogenase (HMSD) and 2-hydroxymuconic semialdehyde hydrolase (HMSH), was determined. The deduced protein sequence for HMSD is 486 amino acid residues long with an M r of 51 682. HMSD has homology with a number of aldehyde dehydrogenases from various eukaryotic sources. The deduced protein sequence for HMSH is 283 amino acids long with an M r of 30 965. The amino acid composition of this enzyme is similar to that of isofunctional enzymes from toluene and m-cresol catabolic pathways.

The meta cleavage operon of TOL degradative plasmid pWWO comprises 13 genes

The meta-cleavage operon of TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes which transform benzoate/toluates to Krebs cycle intermediates via extradiol (meta-) cleavage of (methyl)catechol. The genetic organization of the operon was characterized by cloning of the meta-cleavage genes into an expression vector and identification of their products in Escherichia coli maxicells. This analysis showed that the meta-cleavage operon contains 13 genes whose order and products (in kilodaltons) are xylX(57)-xylY(20)-xylZ(39)-xylL(28)-xylT(1 2)-xylE(36)-xylG(60)-xylF(34)- xylJ(28)-xylQ(42)-xylK(39)-xylI(29)-xylH(4 ). The xylXYZ genes encode three subunits of toluate 1,2-dioxygenase. The xylL, xylE, xylG, xylF, xylJ, xylK, xylI, and xylH genes encode 1,2-dihydroxy-3,5-cyclohexadiene-1-carboxylate dehydrogenase, catechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, 2-hydroxymuconic semialdehyde hydrolase, 2-oxopent-4-enoate hydratase, 4-hydroxy-2-oxovalerate aldolase, 4-oxalocrotonate decarboxylase and 4-oxaloccotonate tautomerase, respectively. The functions of xylT and xylQ are not known at present. The comparison of the coding capacity and the sizes of the products of the meta-cleavage operon genes indicated that most of the DNA between xylX and xylH consists of coding sequences.

A New Class of TOL Plasmid Deletion Mutants in Pseudomonas putida MT15 and Their Reversion by Tandem Gene Amplification

Pseudomonas putida MT15 contains a large plasmid, pWW15, of about 250 kbp, which encodes the genes for toluene and xylene catabolism. Growth on benzoate selects strongly against the wild-type and results in the segregation of three phenotypically distinguishable mutant types. (1) B1 mutants, which have lost the complete plasmid. (2) B3 mutants, in which the plasmid has undergone a large deletion of about 90 kbp which appears to affect the regulation of the catabolic enzymes; these mutants retain the ability to grow on m-xylene and toluene (Mxy+ Tln+) but no longer grow on the metabolite of m-xylene, m-toluate (Mtol–). (3) A novel class not previously described, the B5 mutants, which still grow well on toluene but grow very poorly on m-xylene and do not grow on m-toluate (Mxy– Tln+ Mtol–). The B5 mutants appear to share the regulatory lesion of the B3 mutants but in addition do not express the xylF and xylG gene products, 2-hydroxymuconic semialdehyde hydrolase and 2-hydroxymuconic semialdehyde dehydrogenase. The plasmids in the B5 mutants have also undergone a deletion of about 90 kbp similar to, but distinguishable from, that in the B3 mutants. Both B3 and B5 mutants can revert to growth on m-toluate. The revertants all show elevated constitutive levels of catechol 2,3-oxygenase, 2-hydroxymuconic semialdehyde dehydrogenase and 2-hydroxymuconic semialdehyde hydrolase which are not further induced by m-toluate. The reversion is accompanied by the tandem amplification of a region of 23–28 kbp on either side of the original deletion. As a result of Southern hybridizations, it was shown that the amplified region contains the structural genes of some of the enzymes which metabolize m-toluate but not the enzymes which convert m-xylene to m-toluate.

Naphthalene plasmids in pseudomonads.

A rapid method beginning with the direct lysis of bacteria in alkaline sodium dodecyl sulfate was used to detect naphthalene plasmids in pseudomonads. The strains NCIB 9816, PG, ATCC 17483, and ATCC 17484, which can grow on naphthalene as the sole source of carbon and energy, were examined. All except ATCC 17483 contained more than one plasmid. ATCC 17483 did not contain any plasmids. The largest pair of plasmids found in each of NCIB 9816 and PG(NAH2 and NAH3, respectively) determined naphthalene metabolism and could be transferred by conjugation. This also transferred the unusually regulated meta pathway enzymes for catechol metabolism. NAH2 determines the constitutive production of low concentrations of catechol 2,3-dioxygenase and 2-hydroxymuconic acid semialdehyde dehydrogenase, and NAH3 determines the constitutive production of high concentration of these. NAH2 and NAH3 gave identical fragments on digestion with BamHI or HindIII, but these were quite different from those of NAH. Nonetheless, NAH2 and NAH3 hybridized with NAH.

Molecular cloning of gene xylS of the TOL plasmid: evidence for positive regulation of the xylDEGF operon by xylS.

The xylDEGF operon and the regulatory gene xylS of the TOL plasmid found in Pseudomonas putida mt-2 were cloned onto Escherichia coli vector plasmids. A 9.5-kilobase fragment, derived from the TOL segment of pTN2 deoxyribonucleic acid, carried the xyl genes D, E, G, and F, which encode toluate oxygenase, catechol 2,3-oxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, and 2-hydroxymuconic semialdehyde hydrolase, respectively. The enzymes were noninducible unless a 3-kilobase PstI fragment, derived also from the TOL segment, was provided in either cis or trans. The PstI fragment appeared to contain the regulatory gene xylS, which produced a positive regulator. The regulator was activated by m-toluate or benzoate, but not by m-xylene or m-methylbenzyl alcohol. the map positions of xylG and xylF were also determined.