Isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli were compared with their parent strain in temperature shift experiments. To improve detection of phenotypic differences in division behavior and cell shape, the strains were grown in glucose-minimal medium with a decreased osmolality (about 100 mosM). Already at the permissive temperature, all mutants, particularly the pbpB and ftsQ mutants,showed an increased average cell length and cell mass. The pbpB and ftsQ mutants also exhibited a prolonged duration of the constriction period. All strains, except ftsZ, continued to initiate new constrictions at 42rC, suggesting the involvement of FtsZ in an early step of the constriction process. The new constrictions were blunt in ftsQ and more pronounced in ftsA and pbpB filaments, which also had elongated median constrictions. Whereas the latter strains showed a slow recovery of cell division after a shift back to the permissive temperature, ftsZ and ftsQ filaments recovered quickly. Recovery of filaments occurred in all strains by the separation of newborn cells with an average length of two times L0, the length of newborn cells at the permissive temperature. The increased size of the newborn cells could indicate that the cell division machinery recovers too slowly to create normal-sized cells. Our results indicate a phenotypic resemblance between ftsA and pbpB mutants and suggest that the cell division gene products function in the order FtsZ-FtsQ-FtsA, PBP3. TheftsE mutant continued to constrict and divide at 42°C, forming short filaments, which recovered quickly after a shift back to the permissive temperature. After prolonged growth at 42°C, chains of cells, which eventually swelled up, were formed. Although theftsE mutant produced filaments in broth medium at the restrictive temperature, it cannot be considered a cell division mutant under the presently applied conditions. Escherichia coli is one of the very few organisms in which the genetics of the regulation of cell division can be studied: a number of specific conditional mutants that are affected in cell division have been described (for a review, see reference 11), and an enzymatic activity of one of the gene products has been demonstrated (18). Cell division in E. coli occurs by a seemingly simple ingrowth of the envelope layers without the help of additional structures like external wall bands (15) or internal skeletal compounds (30). In spite of this simplicity, it has appeared difficult to determine the specific role of the various cell division genes. First, the composition of the envelope layers at the division site has not yet been shown to differ chemically from the rest of the envelope. As a result, we have to describe the process in morphological terms and distinguish visual stages like initiation of cell wall ingrowth, formation of polar caps, and cell separation. The second reason is that inhibition of cell division can be the result of even slight perturbations of DNA synthesis (the SOS response), protein synthes...
A cell division mutant of Escherichia coli K12 IysA, the temperature sensitive frsZ 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 graindensity 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.Abbreoiation : DAP, diaminopimelic acid. 0001-3573 0 1987 SGM
SummaryWe 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 wildtype 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 CaCl 2 . 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.
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.
The rate at which the peptidoglycan precursor meso-diaminopimelic acid (DAP) is incorporated into the cell wall of Escherichia coli cells was determined by pulse-label experiments. For different E. coli strains, the incorporation rate was compared with the rate of uptake of DAP into the cell. With E. coli W7, a dap lys mutant generally used in this kind of studies, steady-state incorporation was reached only after about 0.75 of the doubling time. This lag period can be ascribed to the presence of a large internal DAP pool in the cells. An E. coli K-12 lysA strain was constructed which could be grown without DAP in its medium. Consequently, due to the higher specific activity of the added [3H]DAP, faster incorporation and higher levels of radioactivity in the peptidoglycan layer were observed in the K-12 lysA strain than in the W7 strain. In addition, uptake and incorporation were faster in steady state (within about 0.2 of the doubling time), indicating a smaller DAP pool. The lag period could be further diminished and the incorporation rate could be increased by feedback inhibition of the biosynthetic pathway to DAP with threonine and methionine. These results make MC4100 lysA a suitable strain for studies on peptidoglycan synthesis. To explain our observations, we suggest the existence of an expandable pool of DAP in E. coli which varies with the DAP concentration in the growth medium. With
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