The binding of hemimethylated oriC to Escherichia coli membranes has been implicated in the prevention of premature reinitiation at newly replicated chromosomal origins in a reaction that involves the SeqA protein. We describe the resolution of the membrane-associated oriCbinding activity into two fractions, both of which are required for the high-affinity binding of hemimethylated oriC. The active component in one fraction is identified as SeqA. The active component of the second fraction is a previously undescribed protein factor, SeqB. The reconstituted system reproduced the salient characteristics of the membraneassociated binding activity, suggesting that the membraneassociated oriC-binding machinery of E. coli is likely to be a multiprotein system that includes the SeqA and SeqB proteins.In Escherichia coli and most other bacterial species, chromosome replication is initiated at a specific point during the cell cycle (1). It is clear that a mechanism exists to prevent premature reinitiation until the proper time in the cell cycle is reached (2). Several factors have been proposed to play a role in the sequestration of newly replicated origins to prevent premature reinitiation during the eclipse period.The methylation state of the chromosomal origin has been implicated both in the binding of oriC to membranes and in the prevention of premature reinitiation at newly replicated origins. In E. coli, adenine residues within chromosomal GATC sequences are methylated in a reaction catalyzed by Dam methylase. Because the methylation reactions occur after replication, adenine residues within GATC sequences in the newly synthesized strand remain unmethylated for a period of time. The oriC region contains a high density of GATC sites, and methylation of adenine residues at several of these sites is delayed significantly relative to the time of methylation at GATC sites elsewhere in the chromosome (3, 4). As a result, the oriC region of newly replicated chromosomes remains hemimethylated for about 30-40% of the cell cycle.Evidence that hemimethylated oriC does not serve as an effective template for initiation of replication in vivo came from the finding of Russell and Zinder (5) that a fully methylated oriC plasmid was unable to replicate in a Dam

Bifunctional cross-linking reagents were used to identify cell envelope proteins that interacted with the murein sacculus. This revealed that a number of [3H]leucine-labeled proteins and [3H]palmitate-labeled lipoproteins were reproducibly cross-linked to the sacculus in plasmolyzed cells. The results suggested that most of the cell envelope lipoproteins, and not only the murein lipoprotein, mediate interactions between the murein sacculus and the inner and/or outer membrane of the cell.

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