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

Abstract

Comamonas testosteroni strain R5 expresses a higher level of phenol-oxygenating activity than any other bacterial strain so far characterized. The expression of the operon encoding multicomponent phenol hydroxylase (mPH), which is responsible for the phenol-oxygenating activity, is controlled by two transcriptional regulators, PhcS and PhcR, in strain R5. In this study, we identified a third transcriptional regulator for the mPH operon (PhcT) that belongs to the AraC/XylS family. While the disruption of phcT in strain R5 significantly reduced the expression of the mPH operon, it did not eliminate the expression. However, the disruption of phcT in strain R5 increased the expression of phcR. The phenol-oxygenating activity was abolished by the disruption of phcR, indicating that PhcT alone was not sufficient to activate the expression of the mPH operon. The disruption of phcS has been shown in our previous study to confer the ability of strain R5 to express the mPH operon in the absence of the genuine substrate for mPH. PhcT was not involved in the gratuitous expression. Strain R5 thus possesses a more elaborate mechanism for regulating the mPH operon expression than has been found in other bacteria.The expression of a bacterial catabolic pathway for aromatic compounds is often controlled by one or more transcriptional regulatory proteins (21), and sometimes, one transcriptional regulator controls the expression of another transcriptional regulator (24).The expression of multicomponent phenol hydroxylase (mPH) (9,11,12,19,20,36,37) is generally thought to be controlled by a regulator of the XylR/DmpR subclass within the NtrC-type family of transcriptional regulators, resulting in the expression of phenol-metabolizing enzymes only in the presence of the pathway substrate or its structural analog (2,12,14,18,19,22,26,(30)(31)(32)(33)37). The regulators of this subclass are activated by direct interaction with an effector molecule which is normally the substrate for the catabolic pathway the regulators control (29).Comamonas testosteroni R5 has been shown to exhibit an exceptionally high level of activity for phenol oxygenation (42). We have cloned a DNA fragment encoding mPH (phcKLM-NOP) and its cognate transcriptional activator (phcR) of the XylR/DmpR subclass from strain R5 (37). 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. For strain TA441, the regulator named AphS repressed the transcription of the mPH operon even in the presence of phenol, which pre...

Novel Regulator MphX Represses Activation of Phenol Hydroxylase Genes Caused by a XylR/DmpR-Type Regulator MphR in Acinetobacter calcoaceticus

“…Similar binding motifs were also found in the putative IHF-binding site of the phcR - phcK intergenic region [28]. Hence, it is suggested that these proteins could bind to the putative IHF binding sites and hamper the binding of IHF [15], [28], [48]. However, the IHF-binding consensus motif is absent in the A+T-rich sequence of the mph operon of strain PHEA-2.…”

“…One of the AphS binding sites (consensus motif, TATCGTAT) determined by gel-mobility shift assay is located around the putative IHF (Integration Host Factor)-binding site, but not within the putative UASs [7]. Similar binding motifs were also found in the putative IHF-binding site of the phcR - phcK intergenic region [28]. Hence, it is suggested that these proteins could bind to the putative IHF binding sites and hamper the binding of IHF [15], [28], [48].…”

“…The molecular mechanisms of xyl /Xyl and dmp /Dmp systems are well-characterized to be via activator binding to σ 54 -dependent promoter regions of respective target operons [14], [24], [25]. In addition to XylR/DmpR-type mediated regulation, co-regulation of mPH gene expression by different types of regulators has also been observed in Comamona s testosteroni strains TA441 and R5 [7], [23], [28], [29]. Furthermore, carbon catabolite repression and some host-dependent global regulations have been reported to affect the primary regulation by XylR/DmpR-type proteins [14], [30], [31].…”

Catabolism of Phenol and Its Derivatives in Bacteria

“…This third regulator, designated PhcT, belongs to the AraC/XylS-type regulators and was found to stimulate the expression of genes involved in phenol catabolism in the presence of the main PhcR activator (Teramoto, Ohnishi, Harayama, & Watanabe, 2002).…”

Bacterial Transcriptional Regulators for Degradation Pathways of Aromatic Compounds

“…Another example is provided by the phcT regulatory gene. PhcT is at the top of a regulatory cascade controlling phenol degradation (250). PhcT enhances transcriptional activation of the phc operon for phenol degra- …”