In synchronized culture of Escherichia coli, the specific arrest of phospholipid synthesis (brought about by glycerol starvation in an appropriate mutant) did not affect the rate of ongoing DNA synthesis but prevented the initiation of new rounds. The initiation block did not depend on cell age at the time of glycerol removal, which could be before, during, or after the doubling in the rate of phospholipid synthesis (DROPS) and as little as 10 min before the expected initiation. We conclude that the initiation of DNA replication is not triggered by the preceding DROPS but requires active phospholipid synthesis. Conversely, when DNA replication initiation was specifically blocked in a synchronized culture of a dnaC(Ts) mutant, two additional DROPS were observed, after which phospholipid synthesis continued at a constant rate for at least 60 min. Similarly, when DNA elongation was blocked by thymine starvation of a synchronized culture, one additional DROPS was observed, followed by linear phospholipid accumulation. Control experiments showed that specific inhibition of cell division by ampicillin, heat shock, or induction of the SOS response did not affect phospholipid synthesis, suggesting that the arrest of DROPS observed was due to the DNA replication block. The data are compatible with models in which the DROPS is triggered by an event associated with replication termination or chromosome segregation.It has been speculated that in bacteria the cell membrane may play a role in chromosome segregation (6,7,19). Subsequent work with Escherichia coli has provided evidence for attachment of the membrane to DNA or to oriC, the replication origin (5,13,15,20), and a membrane protein with a specific binding site in oriC has been described (8, 9). However, little is known about possible regulatory interconnections between membrane synthesis and DNA replication (4,18,21).In the bacterial cell cycle a small number of events have been characterized as occurring only at a precise cell age. All of these events involve either the chromosome or the cell envelope. On the one hand there are initiation and termination of chromosome replication and nucleoid segregation, and on the other hand there are cell septation, the doubling of the rate of protein insertion into the outer membrane (2), and the doubling in the rate of phospholipid synthesis (DROPS; 10, 16, 17). The latter event involves both phosphatidylethanolamine and phosphatidylglycerol, whose rates of synthesis are constant with an abrupt doubling at a particular moment of the cell cycle (10).We previously looked for a temporal relation between the DROPS and replication initiation or cell division in several E. coli strains growing in different media. The timing of these events was found to vary with the growth conditions (10). The work was done with cultures synchronized by repeated phosphate starvation (11, 12), giving more than three cycles of synchronous growth for all strains and media used.In the present study, again using synchronized cultures, we analyzed (i) the...

Synchronized cells of Escherichia coli were pulse-labeled with [3H]leucine and subjected to membrane fractionation to determine whether a fraction that is enriched for membrane-murein adhesion zones (fraction OML) was preferentially generated at specific times during the cell cycle, as previously suggested from studies of ikyD and cha mutants. Contrary to this prediction, the experiments showed that OML was formed continuously during the division cycle.Escherichia coli and other gram-negative bacteria are surrounded by a cell envelope that includes two membranes, the cytoplasmic (inner) membrane and the outer membrane. The region between the two membranes (the periplasmic space) contains the rigid murein sacculus that determines cell shape. It was shown by Bayer (1) that the inner membrane is closely associated with the murein-outer membrane layer at numerous sites within the cell envelope, forming zones of adhesion that can be seen in thin-section electron micrographs. The adhesion zones have been implicated in several cellular processes, including the export of proteins and lipopolysaccharides from inner membrane to outer membrane (reviewed in reference 2).Membrane-murein attachments also appear to play a role in the cell division process, as shown by the observation that the division site is flanked by two circumferential zones of adhesion, the periseptal annuli, that extend completely around the cell cylinder (7, 10). The periseptal annuli appear at the division site before the onset of septal invagination (6). Their formation represents the earliest detectable morphological event in the differentiation of these sites.Membrane isolation procedures have been described that permit the isolation of a fraction (OML [8]) that appears to be enriched in membrane-murein adhesion zones (3,8). In vivo and in vitro studies have indicated that the OML fraction participates in lipopolysaccharide export to the outer membrane and in the incorporation of new murein subunits into the sacculus (8), supporting its assignment as an adhesion zone fraction. It is possible that this fraction includes the periseptal annuli, but there is no direct evidence for this.It was previously shown that the OML fraction accumulates to high levels in certain cell division mutants of E. coli and Salmonella typhimurium (cha and lkyD, respectively) that are blocked at a late stage of the division process (5). This finding suggested a relationship between OML and cell division. DNA synthesis was required for the accumulation of OML in the mutant strains, further linking OML to the division cycle. In one suggested model to explain these observations, the membrane junctions contained in OML are assembled at a specific stage of the cell cycle (5). In this model, the lkyD+ and cha+ gene products are required for progression to the next stage of the cycle, which is normally associated with a transient cessation in the formation of * Corresponding author.OML. This would explain the accumulation of OML in Chaand LkyD-cells. This model predicts a disconti...