Genomic Studies with Escherichia coli MelR Protein: Applications of Chromatin Immunoprecipitation and Microarrays

Abstract

Escherichia coli MelR protein is a transcription activator that is essential for melibiose-dependent expression of the melAB genes. We have used chromatin immunoprecipitation to study the binding of MelR and RNA polymerase to the melAB promoter in vivo. Our results show that MelR is associated with promoter DNA, both in the absence and presence of the inducer melibiose. In contrast, RNA polymerase is recruited to the melAB promoter only in the presence of inducer. The MelR DK261 positive control mutant binds to the melAB promoter but cannot recruit RNA polymerase. Further analysis of immunoprecipitated DNA, by using an Affymetrix GeneChip array, showed that the melAB promoter is the major, if not the sole, target in E. coli for MelR. This was confirmed by a transcriptomics experiment to analyze RNA in cells either with or without melR.Expression of the Escherichia coli melAB genes, which encode proteins necessary for transport and metabolism of the disaccharide melibiose, is dependent on the transcription activator, MelR, encoded by the adjacent melR gene (13). Previous studies have shown that transcription from the melAB promoter is activated by MelR and have focused on using biochemistry to understand the mechanism of activation (1,4,7,12). Recent work has shown that MelR activates transcription by direct interaction with the RNA polymerase subunit via residue D261 (5). Although activation requires the inducer melibiose, in vitro studies have shown that MelR can bind to the melAB promoter both in the presence and absence of melibiose (1). In the experiments presented here, we have exploited novel chromatin immunoprecipitation (ChIP) and microarray technologies to study the interactions of MelR in vivo. ChIP has been used to investigate MelR and RNA polymerase binding to the melAB regulatory region in vivo, while microarrays have been used to show that the melAB promoter is the principal target for MelR in E. coli. MATERIALS AND METHODSE. coli strains, plasmids, and oligonucleotides. Bacterial strains, plasmids, and oligonucleotides used in this work are listed in Table 1. In all experiments, E. coli strains WAM131, WAM132, or MG1655, carrying plasmids as appropriate, were grown to mid-exponential phase (optical density at 650 nm of 0.4 to 0.6) in minimal M63 medium, supplemented with fructose and amino acids, either with or without melibiose, according to the same method used previously in studies of the regulation of the E. coli mel operon (13).ChIP. In all experiments, in vivo cross-linking of bacterial nucleoprotein was initiated by the addition of formaldehyde (final concentration of 1%) to cultures. After 20 min, cross-linking was quenched by the addition of glycine (final concentration of 0.5 M). Typically, cells were then harvested from 10 ml of culture by centrifugation, washed twice with Tris-buffered-saline (pH 7.5), resuspended in 1 ml of lysis buffer (10 mM Tris [pH 8.0], 20% sucrose, 50 mM NaCl, 10 mM EDTA, 10 mg of lysozyme per ml) and incubated at 37°C for 30 min. Following lysis, 4 ml of immuno...

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