Transcriptional repression mediated by LysR-type regulator CatR bound at multiple binding sites

Abstract

The catBCA operon of Pseudomonas putida encodes enzymes involved in the catabolism of benzoate. Transcription of this operon requires the LysR-type transcriptional regulator CatR and an inducer molecule, cis,cis-muconate. Previous gel shift assays and DNase I footprinting have demonstrated that CatR occupies two adjacent sites proximal to the catBCA promoter in the presence of the inducer. We report the presence of an additional binding site for CatR downstream of the catBCA promoter within the catB structural gene. This site, called the internal binding site (IBS), extends from +162 to +193 with respect to the catB transcriptional start site and lies within the catB open reading frame. Gel shift analysis and DNase I footprinting determined that CatR binds to this site with low affinity. CatR binds cooperatively with higher affinity to the IBS in the presence of the two upstream binding sites. Parallel in vivo and in vitro studies were conducted to determine the role of the internal binding site. We measured beta-galactosidase activity of catB-lacZ transcriptional fusions in vivo. Our results suggest a probable cis-acting repressor function for the internal binding site. Site-directed mutagenesis of the IBS verified this finding. The location of the IBS within the catB structural gene, the cooperativity observed in footprinting studies, and phasing studies suggest that the IBS likely participates in the interaction of CatR with the upstream binding sites by looping out the intervening DNA.

FIG. 1

Sequences and organization of the RBS, ABS, and IBS. The −35 and −10 consensus promoter sequences are underlined. The G and A residues of the G-N₁₁-A motif are in boldface.

FIG. 1

Sequences and organization of the RBS, ABS, and IBS. The −35 and −10 consensus promoter sequences are underlined. The G and A residues of the G-N₁₁-A motif are in boldface.

FIG. 2

Comparison of sequences of the IBS and the RBS. The IBS sequences protected by CatR from DNase I digestion are bracketed. The interrupted inverted repeat involved in sequence specific recognition by CatR is underlined. The G and A nucleotides of the binding motif are indicated by asterisks.

FIG. 3

Structures and in vivo activities of the catBCA constructs used in this study. Each arrow indicates the transcriptional start site (+1) of catB. All constructs harbor the RBS, the ABS, and the −35 and −10 consensus promoter sequences. Plasmid pCH2Z1 includes a part of the catB structural gene, up to the IBS, cloned upstream of the lacZ gene in pKRZ1; however, this construct lacks the IBS. Plasmid pCH3Z1 was constructed similarly; however, it extends further into the catB gene to include the IBS. Plasmids p171GT, p173CA, and p184TG are similar to pCH3Z1 except for the introduced point mutations as indicated. The β-galactosidase specific activities of each of the lacZ fusions in PRS2000 are displayed at the right.

FIG. 4

CatR binding to the wild-type and mutant IBS. (A) Gel shift assay demonstrating binding of purified CatR in the absence of inducer to a 321-bp DNA fragment containing the wild-type IBS. Lane 1 contains free, unbound DNA. Lanes 2 to 5 contain reaction mixtures with the following concentrations of purified CatR: 1.3 × 10⁻⁷, 1.7 × 10⁻⁷, 3.4 × 10⁻⁷, and 1.3 × 10⁻⁶ M. Lanes 1 to 5 in panels B to D correspond exactly to lanes 1 to 5 in panel A except that they represent assays performed with the IBS mutant probes 171GT, 173CA, and 184TG, respectively.

FIG. 5

DNase I footprinting of CatR binding to the IBS. (A) A 321-bp end-labeled DNA fragment containing the IBS but not the RBS or ABS was incubated with different amounts of CatR and subjected to digestion with DNase I. Lane G+A shows a Maxam-Gilbert reaction. Lanes 1 to 5 contain the following concentrations of CatR: 0 (free DNA), 8.5 × 10⁻⁸, 5.1 × 10⁻⁷, 1.3 × 10⁻⁶, and 5.1 × 10⁻⁶ M. The inducer did not alter the protection profile. (B) Lanes 1 to 4 contain a 340-bp end-labeled DNA fragment harboring the RBS/ABS and the IBS digested with the following concentrations of CatR present in the footprinting reactions: 0 (free DNA), 8.5 × 10⁻⁸, 3.4 × 10⁻⁷, and 8.5 × 10⁻⁷ M. The protected regions corresponding to the RBS and the IBS are indicated on the right.