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Identification and characterization of the nitrobenzene catabolic plasmids pNB1 and pNB2 in Pseudomonas putida HS12.

ABSTRACT: Pseudomonas putida HS12, which is able to grow on nitrobenzene, was found to carry two plasmids, pNB1 and pNB2. The activity assay experiments of wild-type HS12(pNB1 and pNB2), a spontaneous mutant HS121(pNB2), and a cured derivative HS124(pNB1) demonstrated that the catabolic genes coding for the nitrobenzene-degrading enzymes, designated nbz, are located on two plasmids, pNB1 and pNB2. The genes nbzA, nbzC, nbzD, and nbzE, encoding nitrobenzene nitroreductase, 2-aminophenol 1,6-dioxygenase, 2-aminomuconic 6-semialdehyde dehydrogenase, and 2-aminomuconate deaminase, respectively, are located on pNB1 (59.1 kb). Meanwhile, the nbzB gene encoding hydroxylaminobenzene mutase, a second-step enzyme in the nitrobenzene catabolic pathway, was found in pNB2 (43.8 kb). Physical mapping, cloning, and functional analysis of the two plasmids and their subclones in Escherichia coli strains revealed in more detail the genetic organization of the catabolic plasmids pNB1 and pNB2. The genes nbzA and nbzB are located on the 1.1-kb SmaI-SnaBI fragment of pNB1 and the 1.0-kb SspI-SphI fragment of pNB2, respectively, and their expressions were not tightly regulated. On the other hand, the genes nbzC, nbzD, and nbzE, involved in the ring cleavage pathway of 2-aminophenol, are localized on the 6.6-kb SnaBI-SmaI fragment of pNB1 and clustered in the order nbzC-nbzD-nbzE as an operon. The nbzCDE genes, which are transcribed in the opposite direction of the nbzA gene, are coordinately regulated by both nitrobenzene and a positive transcriptional regulator that seems to be encoded on pNB2.

SUBMITTER: Park HS

PROVIDER: S-EPMC94317 | biostudies-literature | 2000 Feb

REPOSITORIES: biostudies-literature

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Identification and characterization of the nitrobenzene catabolic plasmids pNB1 and pNB2 in Pseudomonas putida HS12.

Park H S HS Kim H S HS

Journal of bacteriology 20000201 3

Pseudomonas putida HS12, which is able to grow on nitrobenzene, was found to carry two plasmids, pNB1 and pNB2. The activity assay experiments of wild-type HS12(pNB1 and pNB2), a spontaneous mutant HS121(pNB2), and a cured derivative HS124(pNB1) demonstrated that the catabolic genes coding for the nitrobenzene-degrading enzymes, designated nbz, are located on two plasmids, pNB1 and pNB2. The genes nbzA, nbzC, nbzD, and nbzE, encoding nitrobenzene nitroreductase, 2-aminophenol 1,6-dioxygenase, 2- ...[more]

PMID: 10633088

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Project description: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.

Dataset Information

Identification and characterization of the nitrobenzene catabolic plasmids pNB1 and pNB2 in Pseudomonas putida HS12.

Publications

Identification and characterization of the nitrobenzene catabolic plasmids pNB1 and pNB2 in Pseudomonas putida HS12.

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