E. Pas

Topography of peptidoglycan synthesis during elongation and polar cap formation in a cell division mutant of Escherichia coli MC4100

SUMMARY: A cell division mutant of Escherichia coli K12 lysA, the temperature sensitive ftsZ strain, was pulse-labelled with [3H]diaminopimelic acid (DAP) during growth in minimal salts medium both at the permissive (28°C) and restrictive (42°C) temperature. In contrast to other known cell division mutants, ftsZ filaments obtained during growth at 42°C show no sign of persisting or newly initiated constrictions. The location of the incorporated DAP in dividing cells and in filaments was analysed with an improved autoradiographic method in which preparations of well-spread sacculi are covered with a dry emulsion. From the populations of sacculi complete distributions were obtained, which compared well with those of the intact cells. The grain-density distributions of cells dividing at 28°C showed that the rate of surface synthesis was strongly increased at the site of constriction at the expense of the activity in the lateral wall, suggesting a redistribution of surface synthesis activity. In individual filaments elongating at 42°C no indication for the existence of narrow or broad growth zones was found, suggesting a dispersed mode of lateral wall synthesis. These observations are in accordance with theoretical predictions on the rate of surface synthesis during the constriction period in cells which elongate at a constant diameter.

R174 of Escherichia coli FtsZ is involved in membrane interaction and protofilament bundling, and is essential for cell division

We investigated the interaction between FtsZ and the cytoplasmic membrane using inside‐out vesicles. Comparison of the trypsin accessibility of purified FtsZ and cytoplasmic membrane‐bound FtsZ revealed that the protruding loop between helix 6 and helix 7 is protected from trypsin digestion in the latter. This hydrophobic loop contains an arginine residue at position 174. To investigate the role of R174, this residue was replaced by an aspartic acid, and FtsZ‐R174D was fused to green fluorescent protein (GFP). FtsZ‐R174D‐GFP could localize in an FtsZ and in an FtsZ84(Ts) background at both the permissive and the non‐permissive temperature, and it had a reduced affinity for the cytoplasmic membrane compared with wild‐type FtsZ. FtsZ‐R174D could also localize in an FtsZ depletion strain. However, in contrast to wild‐type FtsZ, FtsZ‐R174D was not able to complement the ftsZ84 mutation or the depletion strain and induced filamentation. In vitro polymerization experiments showed that FtsZ‐R174D is able to polymerize, but that these polymers cannot form bundles in the presence of 10 mM CaCl2. This is the first description of an FtsZ mutant that has reduced affinity for the cytoplasmic membrane and does not support cell division, but is still able to localize. The mutant is able to form protofilaments in vitro but fails to bundle. It suggests that neither membrane interaction nor bundling is a requirement for initiation of cell division.

The Escherichia cohi minB mutation resembles gyrB in defective nucleoid segregation and decreased negative supercoiling of plasmids

SummaryNucleoid 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.

Perpendicular planes of FtsZ arcs in spheroidal Escherichia coli cells.

Division planes in Escherichia coli, usually restricted to one dimension of the rod-shaped cell, were induced at all possible planes by transforming the cells to spheroids with mecillinam (inactivating PbpA). Such cells displayed many nucleoids and arcs of FtsZ, genetically tagged to green fluorescent protein, that developed to rings at constriction sites all around their surface. These observations are consistent with the view (Woldringh et al., J. Bacteriol. 176 (1994) 6030-6038) that nucleoids, forced during replication to segregate in the length axis of the cell by the rigid bacillary envelope, induce assembly of FtsZ to division rings in between them.