The positioning of constrictions in Escherichia coli filaments pinching off anucleate cells was analyzed by fluorescence microscopy of dnaX(Ts), dnaX(Ts) sfiA, dnaA46(Ts), gyrA(Am) supF(Ts), and gyrB(Ts) mutants. In filaments with actively replicating nucleoids, constrictions were positioned close to the nucleoid, whereas in nonreplicating filaments, positioning of constrictions within the anucleate region was nearly random. We conclude that constriction positioning depends in an unknown way on nucleoid replication activity.Cell division in Escherichia coli involves the tight coordination in time and space of the processes of cell growth, DNA replication, and cell constriction. Concomitant with the increase in cell mass and DNA replication, the daughter chromosomes are segregated into the cell halves. After termination of DNA replication and completion of segregation, the cell is constricted between the nucleoids in the cell center. Constriction requires discontinuation of enzyme activity for cell wall synthesis at the constriction site (1,21). This may be achieved either by local activation of enzymes or by positioning of specific enzymes at the constriction site. Although termination of DNA replication has been suggested to signal the cell to initiate a constriction (11), the mechanism is still not well understood. So far, factors that determine the positioning of a constriction site have not been identified.According to the concept of zonal growth in bacterial cells (5,8), the site of constriction is determined by growth zones that occur in the lateral cell wall. It has also been suggested that "zones of adhesion" between the cell membrane and the peptidoglycan layer may be involved in the positioning of constrictions (16). Both ideas imply that constriction sites are predetermined within the cell envelope in such a way that the specific enzymes for constriction are concentrated at potential division sites, which are placed at regular distances from the cell pole. Experiments with cells carrying the temperature-sensitive DNA initiation mutation dnaA46(Ts) have been interpreted in terms of predetermined division sites (2). At the restrictive temperature, the SOS response and the related cell division inhibition are not induced in this mutant (for a review, see reference 12), which thus continues to divide, pinching off DNA-less cells. At 42°C, the dnaA46(Ts) mutant was reported to pinch off DNA-less cells of uniform length similar to the newborn cell length (5, 10), suggesting that the cells can measure the distance between constriction and pole.Contrary to the results of Hirota et al. (5), observations on other DNA-less cell-forming mutants suggest the absence of regularly spaced, predetermined constriction sites in the lateral cell wall. For instance, the dnaB mutation, by which DNA replication and constriction initiation are also uncoupled, produces DNA-less cells that are not uniform in size (7). Anucleate cells with a broad range of cell lengths are also pinched off from filaments of the dnaX(Ts) mutant r...

No abstract

No abstract

Nucleoid segregation in the Escherichia coli minB mutant and in cells that over-produce minB gene products appeared defective as measured from fluorescence micrographs. Electrophoretic resolution of topoisomers of plasmid isolates from the minB strain revealed a decreased level of negative supercoiling; in addition, multimerization was observed. Over-production of the minB gene product also resulted in a decreased level of negative supercoiling. This phenotype is typical of the gyrB(ts) mutant, which is known to be affected in chromosome decatenation and supercoiling. We propose that the minB mutation and over-production of the minB gene products cause a defect in nucleoid segregation, which may be related to the decrease in negative supercoiling. As in the gyrB(ts) mutant, retardation of nucleoid segregation is proposed to inhibit constriction initiation in the cell centre and to give rise to nucleoid-free cell poles. As a consequence, these cells divide between nucleoid and cell pole, resulting in minicell and (sometimes) in anucleate cell formation.

Rothfield, Proc. Natl. Acad. Sci. USA 84:7144-7148, 1987) that these zones, called periseptal annuli, play a role in determining the division site, we analyzed the positions of these zones by phase-contrast and electron microscopy. In situ treatment of cells grown in agar showed that the youngest cell pole was the most susceptible to plasmolysis, whereas the constriction site was resistant. Lateral bays occurred only at some distance from a polar bay or a resistant constriction site. Orienting cells with their most prominently plasmolyzed polar bay in one direction showed that the lateral bays were always displaced away from the polar bay at about half the distance to the other cell pole. Even in a simple organism like Escherichia coli, there must be a mechanism to ensure that cell division always occurs between the segregated daughter chromosomes. Several proteins have been implicated to be specifically involved in the division process (5), but the positioning mechanism itself has remained elusive. Two opposing models for positioning of division sites have been proposed: in one model, a newborn cell already contains a preexisting division site formed by replication and lateral displacement of an annular envelope structure (2,3,13) and in the other model, the site is generated only after chromosome duplication and nucleoid segregation (9,19,20).In the first model, the growing envelope contains concentric rings of adhesion (so-called periseptal annuli) between the plasma membrane and the cell wall (peptidoglycan layer and outer membrane). The periseptal annulus model assumes that each newborn cell contains a pair of such annuli in its center. During subsequent cellular growth, new pairs of annuli are generated at both sides of the central structure and are laterally displaced in a gradual way to 1/4 and 3/4 positions of the cell length. The central annuli are used for septum formation in the periseptal domain between the annuli (14). After division, each newborn cell inherits an annulus at its pole and a central pair of annuli as a preseptation structure. This model predicts preexisting division sites in newborn cells and a commitment to division in the cell center independent of the DNA replication cycle.In the second model, it is the presence of the nucleoid which directs the division site by influencing the rate of peptidoglycan synthesis in two opposing ways. The nucleoid inhibits cell wall synthesis in its vicinity, as has been demonstrated by autoradiography (10). This inhibiting effect * Corresponding author. Electronic mail address: a430coli@ diamond.sara.nl. 2241has been called nucleoid occlusion (1). The opposing effect is activation of peptidoglycan synthesis in the region of termination of DNA replication, which usually occurs in the cell center. This activation, which has been shown to be necessary for initiation of constriction (16,18), is supposed to occur at a position along the cell envelope, which has been abandoned by the segregating nucleoids, thus alleviating their occlusion effect.The first...