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
.