Regulation of the acetate operon in Escherichia coli: purification and functional characterization of the IclR repressor.

Abstract

Growth of Escherichia coli on acetate requires operation of the anaplerotic sequence known as the glyoxylate bypass. In this pathway three different enzymes are activated: malate synthase, isocitrate lyase and isocitrate dehydrogenase kinase/phosphatase which are encoded by genes aceB, aceA and aceK, respectively. These three genes are clustered, in that order, in the same acetate (ace) operon whose expression is under the transcriptional control of the iclR gene located downstream from aceK. We have cloned the iclR gene in the pKK233–2 vector which allows optimization of both transcription and translation initiation. The IclR repressor has been overproduced, then purified to homogeneity in a one‐step procedure by cation exchange chromatography after ammonium sulfate fractionation. Its specific interaction with the operator/promoter region of the ace operon has been analyzed by gel retardation and DNase I footprinting experiments. The IclR repressor has been shown to recognize a 35 bp palindromic sequence which largely overlaps the ‐35 recognition site of RNA polymerase. Moreover, the formation of the complex between IclR and the operator/promoter region has been found to be impaired by phosphoenol pyruvate but insensitive to acetate, acetyl‐CoA, pyruvate, and oxaloacetate. These results are discussed in terms of primary regulation of the expression of the ace operon.

Preparation and properties of pure, full‐length Ic1R protein of escherichia coli. Use of time‐of‐flight mass spectrometry to investigate the problems encountered

“…2). The open reading frame of the IclR gene (Sunnarborg et al, 1990;Negre et al, 1991) predicts a protein with a mass of 29,739.3 u, or 29,608.1 if the initial methionine has been removed (see Fig. I), so it was clear that the protein we had isolated was truncated in some way.…”

“…Figure 7 shows that, under these conditions, IclR (0.2-1 mg/mL) elutes from Sephadex G-100 (20 mM Tris-C1, pH 7.8, 0.4 M KCI, 1 mM EDTA, 50 mM &ME) as a tetramer (M, -117,000). Negre et al (1991) found that their IclR preparation sedimented in a sucrose density gradient with a sedimentation coefficient consistent with a dimer (M, -60,000). Together these several results show that freshly prepared lclR is a tetramer at 1 9 0 2 0 0 2 1 0 2 2 0 230 2 4 0 2 5 0 2 6 0 wavelength (nrn) complexes by Orchard and May (1993).…”

“…It has been demonstrated that IclR protein expressed from the cloned iclR gene interacts specifically with a small operator-like region of DNA in the promoter region of the aceBAK operon (Cortay et al, 1991;Nkgre et al, 1991Nkgre et al, , 1992.…”

Autoregulation of iclR, the gene encoding the repressor of the glyoxylate bypass operon

“…Binding of IclR to sequences upstream of iclR was tested by gel shift analysis. IclR was overexpressed and purified by a modification of the method described by Cortay et al (4). The probe (1 ng) included sequences from Ϫ152 to ϩ3 relative to the translational start site and was labeled with [␥-32 P]ATP and T4 polynucleotide kinase.…”

“…The IclR binding site was identified by DNase I footprint analysis. IclR was overexpressed and purified by a modification of the method described by Cortay et al (4). DNase I footprint analysis of the IclR-DNA complex was carried out by a modification of the method described by Shih and Towle (27).…”