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PMID: 9573187 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

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

Journal of bacteriology ·Vol. 180 ·No. 9 ·1998-05-00 ·Pages 2367-72

Chugani SA, Parsek MR, Chakrabarty AM

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.

MeSH Terms
Bacterial Proteins Base Sequence Binding Sites DNA Footprinting DNA-Binding Proteins/metabolism Gene Expression Regulation, Bacterial Genes, Bacterial Intramolecular Lyases/biosynthesis,genetics Molecular Sequence Data Operon Protein Binding Pseudomonas putida/genetics Repressor Proteins/metabolism Transcription Factors/metabolism
Chemicals
Bacterial Proteins DNA-Binding Proteins Repressor Proteins Transcription Factors CatR protein, bacteria Intramolecular Lyases muconate cycloisomerase
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Chugani S A
Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago 60612, USA.
Parsek M R
Chakrabarty A M
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/JB.180.9.2367-2372.1998
Published
1998-05-00
Pages
2367-72
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC107177
Subset
IM
Grants
NIEHS NIH HHS · R01 ES004050 · United States
NIEHS NIH HHS · ES04050-12 · United States
External Links
PubMed source DOI: 10.1128/JB.180.9.2367-2372.1998 PMC Full Text View journal details
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