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

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-encodedmeta 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. AbenR 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. ThebenABC genes likely encode benzoate dioxygenase, andbenD 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-lacZreporter fusion in response to benzoate. It also activated expression of a meta cleavage operon promoter-lacZ fusion inserted in an E. coli chromosome. Third, benRwas required for benzoate-mediated repression of pcaK-lacZfusion 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.

Isolation and molecular characterization of a novel <i>pseudomonas putida</i> strain capable of degrading organophosphate and aromatic compounds

“…ben cluster consists of nine genes involved in benzoate uptake and degradation: transcriptional regulator (benR), three subunits of benzoate 1,2-dioxygenase (benABC), 1,6-dihydroxycyclohexa-2,4-diene-1-carboxylate dehydrogenase (benD), two putative benzoate transporters (benK, benE), catechol 1,2-dioxygenase (designated here as benG), and benzoate-specific outer membrane porin (benF). The enzymes encoded in ben cluster convert benzoate to cis,cis-muconate via 1,2-cis-dihydroxybenzoate and catechol (62). Thus, phenol, benzene, and benzoate upper degradation pathways converge at cis,cismuconate, which is further processed by the enzymes of catBCA operon and pca regulon to tricarboxylic acid cycle intermediate, succinyl-CoA (Figure 7).…”

Novel insights into the interplay between peripheral reactions encoded by xyl genes and the chlorocatechol pathway encoded by tfd genes for the degradation of chlorobenzoates by Ralstonia eutropha JMP134

“…Therefore, it is possible that in the chromosome of strain JMP134 a regulatory function is responding to CBs and is cross-activating the expression of the xylXYZL module. This kind of cross-regulation has been shown in P. putida for benzoate catabolism, between the pWW0-encoded proteins XylS and BenR, the regulator of the chromosomal benzoate-degradation pathway and both proteins belonging to the AraC\XylS family of regulators (Cowles et al, 2000 ;Jeffrey et al, 1992). To obtain further evidence for the presence of a putative AraC\XylS family regulator similar to XylS from pWW0 in the chromosome of R. eutropha, Southern hybridization of EcoRI-digested JMP134 and JMP222 genomic DNA was performed using the 725 bp BstEII-generated fragment containing part of xylS.…”

The benPK operon, proposed to play a role in transport, is part of a regulon for benzoate catabolism in Acinetobacter sp. strain ADP1

“…Porin-like genes similar to benP have been identified near several genetic regions involved in bacterial aromatic compound degradation (Cowles et al, 2000 ;Segura et al, 1999). For example, phaK, essential for the assimilation of phenylacetic acid in a Pseudomonas putida strain, appears to encode a specific channel-forming protein (Olivera et al, 1998).…”

“…PhaK was inferred to form an outer-membrane channel with narrow substrate specificity since inactivation of its gene prevents the use as a sole carbon source of phenylacetate but not 4-hydroxyphenylacetate or additional phenylacetaterelated compounds (Olivera et al, 1998). A predicted role for BenF in aromatic compound catabolism is based on the co-expression of benF with several genes in an operon for benzoate dissimilation (Cowles et al, 2000).…”