Differential DNA bending introduced by the Pseudomonas putida LysR‐type regulator, CatR, at the plasmid‐borne pheBA and chromosomal catBC promoters

Abstract

The plasmid-borne pheBA operon of Pseudomonas putida strain PaW85 allows growth of the host cells on phenol. The promoter of this operon is activated by the chromosomally encoded LysR-type regulator CatR, in the presence of the inducer cis,cis-muconate. cis,cis-muconate is an intermediate of catechol degradation by the chromosomally encoded ortho or beta-ketoadipate pathway. The catBC operon encodes two enzymes of the beta-ketoadipate pathway and also requires CatR and cis,cis-muconate for its expression. The promoters of the pheBA and catBC operons are highly homologous, and since both respond to CatR, it is likely that the pheBA promoter was recruited from the ancestral catBC promoter. Gel shift assays and DNase I footprinting have shown that the pheBA promoter has a higher binding affinity for CatR than the catBC promoter. Like the catBC promoter, the pheBA promoter forms two complexes (C1 and C2) with CatR in the absence of cis,cis-muconate, but only forms a single complex (C2) in the presence of cis,cis-muconate. Like the catBC promoter CatR repression binding site (RBS) and activation binding site (ABS) arrangement, the pheBA promoter demonstrates the presence of a 26 bp segment highly homologous to the RBS that is protected by CatR from DNase I digestion in the absence of the inducer. An additional 16 bp sequence, similar to the catBC promoter ABS, is protected only when the inducer cis-cis-muconate is present. The binding of CatR in absence of cis,cis-muconate bends the catBC and pheBA promoter regions to significantly different degrees, but CatR binding in the presence of cis,cis-muconate results in a similar degree of DNA bending. The evolutionary implications of the interactions of CatR with these two promoters are discussed.

Synergistic transcriptional activation by one regulatory protein in response to two metabolites

“…Regulators similar to BenM include CatR and ClcR of Pseudomonas putida which regulate the catabolism of catechol͞phenol and 3-chlorocatechol, respectively (6)(7)(8). They are Class I transcriptional activators that contact a C-terminal region of the ␣-subunit of RNA polymerase (9).…”

Critical nucleotides in the interaction of CatR with the pheBA promoter: conservation of the CatR-mediated regulation mechanisms between the pheBA and catBCA operons

“…CCM allows CatR to bind to a second site, the activation binding site (ABS) and to activate transcription from the promoter. Binding of CatR to the ABS of the catBCA or the pheBA promoter is cooperative, requiring the presence of an intact RBS (Kasak et al, 1993 ;Parsek et al, 1992Parsek et al, , 1995. Footprinting studies revealed that the ABS overlaps the k35 hexamer of the promoters (Chugani et al, 1997 ;Parsek et al, 1992Parsek et al, , 1995.…”

“…A comparison of the nucleotide residues in the pheBA and catBCA promoters suggests that the two promoters are highly conserved and the DNase I protection patterns of CatR on these two promoter sequences are very similar (Chugani et al, 1997 ;Parsek et al, 1995) (Fig. 1b).…”

“…1b). CatR binds to the pheBA and the catBCA promoters in both the presence and absence of the inducer but activates transcription only in the presence of the inducer CCM (Parsek et al, 1995 ;Rothmel et al, 1991). In the absence of CCM, CatR binds to the recognition binding site (RBS).…”

“…The genes pheB and pheA are flanked by two IS elements, IS1472 and IS1411 (Kallastu et al, 1998). The right-end sequences of the transposon Tn4652 (Tsuda et al, 1989) (Parsek et al, 1995 ;Chugani et al, 1997). The protected regions are designated as RBS and ABS.…”