Characterization of the Escherichia coli membrane domain responsible for binding oriC DNA

Abstract

It has previously been shown that hemimethylated DNA from the Escherichia coli replication origin (oriC) binds with high specificity to membrane fractions isolated from disrupted cells. In this article, the membrane localization of oriC-binding activity was studied by subjecting crude membrane preparations to successive cycles of sedimentation and flotation gradient analysis. This revealed that approximately two-thirds of the membrane-associated oriC-binding activity of the cell was not associated with the outer membrane fraction as previously suggested but was recovered instead in a unique membrane fraction (OCB1) whose buoyant density and protein profile differed from those of both inner and outer membranes. The specific activity of oriC binding in OCB1 was approximately fivefold higher than the activity of the isolated outer membrane peak. It is likely that membrane fraction OCB1 includes the membrane domain responsible for the binding of hemimethylated oriC to the cell envelope in intact cells.