PhcS Represses Gratuitous Expression of Phenol-Metabolizing Enzymes in Comamonas testosteroni R5

Abstract

We identified an open reading frame, designated phcS, downstream of the transcriptional activator gene (phcR) for the expression of multicomponent phenol hydroxylase (mPH) in Comamonas testosteroni R5. The deduced product of phcS was homologous to AphS of C. testosteroni TA441, which belongs to the GntR family of transcriptional regulators. The transformation of Pseudomonas aeruginosa PAO1c (phenol negative, catechol positive) with pROR502 containing phcR and the mPH genes conferred the ability to grow on phenol, while transformation with pROR504 containing phcS, phcR, and mPH genes did not confer this ability. The disruption of phcS in strain R5 had no effect on its phenol-oxygenating activity in a chemostat culture with phenol. The phenol-oxygenating activity was not expressed in strain R5 grown in a chemostat with acetate. In contrast, the phenol-oxygenating activity in the strain with a knockout phcS gene when grown in a chemostat with acetate as the limiting growth factor was 66% of that obtained in phenol-grown cells of the strain with a knockout in the phcS gene. The disruption of phcS and/or phcR and the complementation in trans of these defects confirm that PhcS is a trans-acting repressor and that the unfavorable expression of mPH in the phcS knockout cells grown on acetate requires PhcR. These results show that the PhcS protein repressed the gratuitous expression of phenol-metabolizing enzymes in the absence of the genuine substrate and that strain R5 acted by an unknown mechanism in which the PhcS-mediated repression was overcome in the presence of the pathway substrate.

Bacterial Transcriptional Regulators for Degradation Pathways of Aromatic Compounds

“…The dmpR gene itself is located directly upstream of and is divergently oriented from dmpK. A large number of highly similar regulators to DmpR have been found, which all control phenol degradation operons (Table 6) (8,153,158,177,229,249). A few other examples worth mentioning here are TbuT and TbmR, which control toluene monooxygenase gene expression in Burkholderia picketti PK01 (22) and Burkholderia sp.…”

“…In the presence of the pathway substrate, repression is released by interaction of the regulator with the aromatic compounds or one of its metabolites (62,149,165,249). This was interpreted as a loss of DNA binding affinity mediated by the chemical inducer (see below) (62,165).…”

“…The phcS and aphS genes both belong to a small operon (phcRS and aphRS), of which the second gene is coding for an NtrC/XylR-type regulator (see below) (7). The aphRS and phcRS operons are located upstream of and transcribed in the opposite direction from their target catabolic operons (8,249). The AphS protein acts as a factor for silencing the aph genes for phenol metabolism, whereas AphR is its transcription activator.…”

“…Since then, a few other GntR members have been found for catabolic pathways ( Table 4). The repressors PhcS and AphS regulate the expression of the phenol degradation pathway in C. testosteroni strains R5 and TA441 (7,249). The phcS and aphS genes both belong to a small operon (phcRS and aphRS), of which the second gene is coding for an NtrC/XylR-type regulator (see below) (7).…”

An AraC/XylS Family Member at a High Level in a Hierarchy of Regulators for Phenol-Metabolizing Enzymes inComamonas testosteroniR5

“…This work (37) and an electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (our unpublished data) indicated that the high activity of strain R5 was due to the high level of mPH expression, leading us to investigate its transcriptional mechanism. PhcR caused the expression of the mPH operon even in the absence of the genuine substrate for mPH, but this gratuitous expression was repressed by a member of the GntR family of transcriptional regulators named PhcS (38). This GntR family member for regulating the mPH operon has also been identified for C. testosteroni TA441.…”