Interactions of the AtoC/antizyme with regulatory elements of the Escherichia coli atoDAEB operon

Department of Pharmaceutical Sciences; Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, 54124, Greece, and The National Hellenic Research Foundation, 48 Vas. Constantinou, Athens 11636, Greece

^* To whom correspondence should be addressed. Email: pchristo{at}pharm.auth.gr.

	Abstract

AtoC has a dual function as both the post-translational inhibitor of polyamine biosynthetic enzymes (antizyme, Az) and the transcriptional regulator of genes involved in short-chain fatty acid catabolism (atoDAEB operon). We have previously shown that AtoC/Az is the response regulator of the AtoS-AtoC two-component signal transduction system that activates atoDAEB when Escherichia coli is exposed to acetoacetate. Here we show that the same cis-elements control both promoter inducibility and AtoC/Az binding. Chromatin immunoprecipitation experiments confirmed the acetoacetate-inducible binding of AtoC/Az to the predicted DNA region in vivo. DNAse I protection footprinting analysis revealed that AtoC/Az binds two 20 bp stretches, constituting an inverted palindrome, that are located at -146 to -107 relative to the transcription initiation site. Analysis of promoter mutants obtained by in vitro chemical mutagenesis of the atoDAEB promoter verified both the importance of AtoC/Az binding for the inducibility of the promoter by acetoacetate and the ⁵⁴-dependence of the atoDAEB expression. Integration host factor was also identified as a critical component of the AtoC/Az-mediated induction of atoDAEB.