Replication cycle dependent association of SeqA to the outer membrane fraction of E-coli

The hemimethylated oriC binding activity of the E. coli heavy density membr ane fraction (outer membrane) was investigated by DNase I footprinting expe riments using membranes obtained from different replication stages of PC-2 (dnaCts) cells. The maximal binding activity was found at the beginning of replication cycle and then decreased gradually. The same pattern of variati on was observed with SeqA protein detected in the membranes by immunoblotti ng. Both binding activity and the presence of SeqA were conserved in the ou ter membrane even after floating centrifugation of the heavy density membra ne fraction in a sucrose gradient, indicating that SeqA in fact can associa te with the membrane and that this association varies according to replicat ion cycle. Site specific binding to hemimethylated oriC, of the heavy densi ty membrane obtained from seqA mutant, could be restored by addition of a l ow amount of His-tagged SeqA protein. (C) 1999 Societe francaise de biochim ie et biologie moleculaire/Editions scientifiques et medicales Elsevier SAS .