Formation of DNA methylation patterns: nonmethylated GATC sequences in gut and pap operons.

Abstract

Most of the adenine residues in GATC sequences in the Escherichia coli chromosome are methylated by the enzyme deoxyadenosine methyltransferase (Dam). However, at least 20 GATC sequences remain nonmethylated throughout the cell cycle. Here we examined how the DNA methylation patterns of GATC sequences within the regulatory regions of the pyelonephritis-associated pilus (pap) operon and the glucitol utilization (gut) operon were formed. The results obtained with an in vitro methylation protection assay showed that the addition of the leucine-responsive regulatory protein (Lrp) to pap DNA was sufficient to protect the two GATC sequences in the pap regulatory region, GATC-I and GATC-II, from methylation by Dam. This finding was consistent with previously published data showing that Lrp was essential for methylation protection of these DNA sites in vivo. Methylation protection also occurred at a GATC site (GATC-44. 5) centered 44.5 bp upstream of the transcription start site of the gutABD operon. Two proteins, GutR and the catabolite gene activator protein (CAP), bound to DNA sites overlapping the GATC-44. 5-containing region of the gutABD operon. GutR, an operon-specific repressor, was essential for methylation protection in vivo, and binding of GutR protected GATC-44.5 from methylation in vitro. In contrast, binding of CAP at a site overlapping GATC-44.5 did not protect this site from methylation. Mutational analyses indicated that gutABD gene regulation was not controlled by methylation of GATC-44.5, in contrast to regulation of Pap pilus expression, which is directly controlled by methylation of the pap GATC-I and GATC-II sites.