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. 2000 Nov;182(22):6339-46.
doi: 10.1128/jb.182.22.6339-6346.2000.

BenR, a XylS Homologue, Regulates Three Different Pathways of Aromatic Acid Degradation in Pseudomonas Putida

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Free PMC article

BenR, a XylS Homologue, Regulates Three Different Pathways of Aromatic Acid Degradation in Pseudomonas Putida

C E Cowles et al. J Bacteriol. .
Free PMC article

Abstract

Pseudomonas putida converts benzoate to catechol using two enzymes that are encoded on the chromosome and whose expression is induced by benzoate. Benzoate also binds to the regulator XylS to induce expression of the TOL (toluene degradation) plasmid-encoded meta pathway operon for benzoate and methylbenzoate degradation. Finally, benzoate represses the ability of P. putida to transport 4-hydroxybenzoate (4-HBA) by preventing transcription of pcaK, the gene encoding the 4-HBA permease. Here we identified a gene, benR, as a regulator of benzoate, methylbenzoate, and 4-HBA degradation genes. A benR mutant isolated by random transposon mutagenesis was unable to grow on benzoate. The deduced amino acid sequence of BenR showed high similarity (62% identity) to the sequence of XylS, a member of the AraC family of regulators. An additional seven genes located adjacent to benR were inferred to be involved in benzoate degradation based on their deduced amino acid sequences. The benABC genes likely encode benzoate dioxygenase, and benD likely encodes 2-hydro-1,2-dihydroxybenzoate dehydrogenase. benK and benF were assigned functions as a benzoate permease and porin, respectively. The possible function of a final gene, benE, is not known. benR activated expression of a benA-lacZ reporter fusion in response to benzoate. It also activated expression of a meta cleavage operon promoter-lacZ fusion inserted in an E. coli chromosome. Third, benR was required for benzoate-mediated repression of pcaK-lacZ fusion expression. The benA promoter region contains a direct repeat sequence that matches the XylS binding site previously defined for the meta cleavage operon promoter. It is likely that BenR binds to the promoter region of chromosomal benzoate degradation genes and plasmid-encoded methylbenzoate degradation genes to activate gene expression in response to benzoate. The action of BenR in repressing 4-HBA uptake is probably indirect.

Figures

FIG. 1
FIG. 1
Initial steps for the ortho and meta cleavage pathways used by P. putida to degrade 4-HBA, benzoate, and methylbenzoates. The meta cleavage pathway is encoded by the TOL catabolic plasmid. The methyl group can be present in either the 3 or 4 position of the ring.
FIG. 2
FIG. 2
Amino acid sequence alignment of BenR and XylS proteins. Identical residues are outlined in black. Similar residues are shaded gray.
FIG. 3
FIG. 3
(A) Map of chromosomally encoded benzoate degradation genes from P. putida. (B) Map of the benA promoter region, diagram of the region incorporated to construct the benA-lacZ fusion plasmid, pCCH101, and nucleotide sequence of the benA promoter region. The putative BenR binding sites are heavily underlined. Introduced restriction sites are lightly underlined. The transcriptional and translational start sites are in bold. (C) Determination of the 5′ end of the benA transcript by primer extension. RNA was isolated from glucose-grown cells (lane 1) and glucose-benzoate-grown cells (lane 2) as described in the text. A sequence ladder was generated with the same primer (lanes C, T, A, and G). The first nucleotide in the transcript is shown in bold.
FIG. 4
FIG. 4
(A) β-Galactosidase activities of the benA-lacZ fusion (pCCH101) in wild-type (PRS2000) and benR mutant (PRS4157) cells. Cells were grown on succinate (white), succinate plus benzoate (black), or succinate plus catechol (gray) and assayed as described in Materials and Methods. (B) β-Galactosidase activities of E. coli cells harboring the benA-lacZ fusion (pCCH101) and/or the BenR expression construct (pCCH106) in the absence (white) or presence (black) of 1 mM benzoate. β-Galactosidase activity was assayed as described in Materials and Methods.
FIG. 5
FIG. 5
β-Galactosidase activities of benR mutant (PRS4157) cells carrying the benA-lacZ fusion plasmid (pCCH101) and the TOL plasmid. Cells were grown on succinate, succinate plus 4-hydroxybenzoate, succinate plus benzoate, or succinate plus 3-methylbenzoate, as indicated, and assayed as described in Materials and Methods.
FIG. 6
FIG. 6
(A) Map of the chromosomally encoded pcaRKF cluster of 4-HBA degradation genes. (B) Map of the pcaK-lacZ fusion, pHNN216. The putative PcaR binding site is heavily underlined. Restriction sites introduced by cloning are lightly underlined. The transcriptional and translational start sites are in bold (36). (C) β-Galactosidase activities of the pcaK-lacZ fusion in wild-type (PRS2000) and benR mutant (PRS4157) cells. Cells were grown on succinate (white), 4-HBA (black), or 4-HBA plus benzoate (gray), and β-galactosidase activity was assayed as described in Materials and Methods.
FIG. 7
FIG. 7
Representative 4-HBA uptake assay of wild-type (PRS2000) and benR mutant (PRS4157) cells grown on 4-HBA or 4-HBA plus benzoate as indicated. Assays were performed as described in Materials and Methods.

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