Characterization of the Escherichia coli modified cytosine restriction (mcrB) gene

Abstract

The McrB restriction system of Escherichia coli K-12 is responsible for the inactivation of 5-methylcytosine-containing DNA. The mcrB mutation of E. coli strain K802 was complemented by hybrid plasmid pUC9-14 which consists of a 5.5-kb Bg/II-Eco RI fragment from the E. coli K-12 chromosome cloned in pUC9 (Ross and Braymer, 1987). The limits of the mcrB gene within the 5.5-kb insert were defined by deleting portions the fragment and assaying for McrB restriction of M. AluI-methylated DNA. A 51-kDa polypeptide was identified as the mcrB gene product based on an analysis of maxicell-labeled polypeptides from pUC9-14 and deletion derivatives of this plasmid. Deletion analyses and transcription initiation assays enabled us to determine the direction of transcription and translation of mcrB. Transcription initiates approx. 710 bp beyond the end of the hsdS gene, and proceeds in the same direction as the transcription of the hsdR, hsdM, and hsdS genes, which is clockwise on the conventional E. coli map.