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

Abstract

AtoC has a dual function as both an antizyme, the posttranslational inhibitor of polyamine biosynthetic enzymes, and the transcriptional regulator of genes involved in short-chain fatty acid catabolism (the atoDAEB operon). We have previously shown that AtoC 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 binding. Chromatin immunoprecipitation experiments confirmed the acetoacetate-inducible binding of AtoC to the predicted DNA region in vivo. DNase I protection footprinting analysis revealed that AtoC binds two 20-bp stretches, constituting an inverted palindrome, that are located at -146 to -107 relative to the transcription initiation site. Analyses of promoter mutants obtained by in vitro chemical mutagenesis of the atoDAEB promoter verified both the importance of AtoC binding for the inducibility of the promoter by acetoacetate and the sigma54 dependence of atoDAEB expression. The integration host factor was also identified as a critical component of the AtoC-mediated induction of atoDAEB.

FIG. 1.

Mapping of the acetoacetate-responsive cis element within the atoDAEB promoter. (A) Organization of the E. coli genomic sequences originally cloned into pUC-Az (2). The restriction enzyme sites used in the generation of reporter constructs are indicated. (B and C) E. coli Top10 cells carrying the indicated reporter constructs were grown in modified M9 mineral medium (28) in the absence (−; empty bars) or presence (+; filled bars) of 10 mM acetoacetate, and β-galactosidase (LacZ) activity in permeabilized cells was measured as described previously (20). (B) Susceptibility of reporter constructs carrying various regions of the atoDAEB promoter fused to the lacZ reporter gene to induction by acetoacetate. (C) Effects of acetoacetate on the activity of the atoS, atoC, and rcsC promoters.

FIG. 2.

Mapping of the AtoC binding site(s) within the atoDAEB promoter. (A) The binding of AtoC (35 nM) to atoDAEB promoter regions atoD1 (−206 to +73), atoD2 (−120 to +73), and atoD3 (−47 to +73) was analyzed by nonradioactive EMSAs. (B) The DNA binding reactions were performed in the presence of 10 ng of the atoD1 probe (approximately 3 nM) and increasing amounts (5-, 10-, and 20-fold molar excesses) of either unlabeled specific (atoD1) or nonspecific (atoD2) competitors (see Materials and Methods) and analyzed by EMSAs. The AtoC-DNA complexes and the free probes are indicated by arrows, whereas the protein-independent bands are indicated by dots. +, present; −, absent.

FIG. 3.

DNase I footprinting analysis of the AtoC binding sites within the atoDAEB promoter regulatory region. (A) DNase I footprinting was performed with increasing concentrations of rAtoC and promoter fragments labeled in the coding (upper) or noncoding (lower) strand. Boxes indicate the protected areas, and arrowheads indicate the hypersensitivity positions. The gray boxes indicate the inverted palindrome protected by AtoC (see the text). (B) The atoDAEB sequence (−186 to +54) and its regulatory elements are shown. Note that the transcription initiation site shown in this figure is predicted and has not been validated experimentally. Shaded sequences represent the AtoC-protected areas, and arrowheads indicate AtoC-induced hypersensitivity. The positions of some predicted functional elements, such as the promoter region from −24 to −12 (in ovals), the putative ribosome binding site (boxed), and the initiator ATG (bold), are indicated. The positions of the hydroxylamine-induced mutations that render the promoter inactive and/or unresponsive to acetoacetate (Fig. 5 and 6) are marked by dots.

FIG. 4.

ChIP analysis of AtoC binding to atoDAEB promoter fragments in vivo. The binding of AtoC to plasmid-borne atoDAEB promoter fragments that either carry (atoD1) or lack (atoD2) the predicted binding site of AtoC (filled oval) in the presence (+) and absence (−) of acetoacetate (inducer) was determined. The atoSC⁺ strain BW25113 and its ΔatoSC isogenic counterpart BW28878, carrying either the plasmid pMLB-atoD1 or pMLB-atoD2 (Table 1), were used in this experiment. Immunoprecipitations were performed in the absence (−) or presence of anti-AtoC (α-AtoC/Az) antibodies, and they were followed by PCR amplification of the immunoprecipitated DNA fragments by using one atoDAEB-specific (U2patoD [Table 1]) and one plasmid-specific (universal M13 forward) primer. Chromatin input controls are indicated.

FIG. 5.

Acetoacetate inducibility of atoDAEB promoter mutants obtained by chemical mutagenesis. E. coli Top10 cells carrying wild-type or mutant atoDAEB promoters fused to the lacZ reporter gene were grown in modified M9 mineral medium (28) in the absence (−; empty bars) or presence (+; filled bars]) of 10 mM acetoacetate, and β-galactosidase (LacZ) activity in permeabilized cells was measured as described previously (20). The position of each mutation is indicated on the left. wt, wild type.

FIG. 6.

In vitro binding of AtoC to wild-type and mutant atoDAEB promoter fragments. The binding of purified rAtoC (8 and 16 nM) to wild-type or mutant atoDAEB promoter (−206-to-+73) probes was analyzed by EMSAs. The mutations are identified at the top of each lane. AtoC-DNA complexes and free probes are indicated by arrows, whereas the positions of protein-independent bands are indicated by dots. wt, wild type.

FIG. 7.

Effect of the IHF on the acetoacetate inducibility of wild-type and mutant atoDAEB promoters. A pair of isogenic wild-type (N99 [wt]) or IHF-negative (N7193 [IHF−]) E. coli strains (Table 1) were used to monitor the activities of the wild-type atoDAEB promoter (atoD1) and mutant atoDAEB promoters (with a mutation at position −53 or −140) in the absence (empty bars) or presence (filled bars) of 10 mM acetoacetate. The promoters had been fused to the lacZ reporter gene, and the activity of β-galactosidase (LacZ) in permeabilized cells was measured as described previously (20).