Phospholipid synthesis during the cell division cycle of Escherichia coli.

Stepwise changes in the rate of phosphatidylethanolamine and phospholipid synthesis during the cell division cycle of Escherichia coli B/r were observed. The cell ages at the increases were found to be a function of the growth rate. At each growth rate, the increase occurred around the time new rounds of chromosome replication were inaugurated in the cycle.     

Changes in cell diameter during the division cycle of Escherichia coli

Extensive measurements of steady-state populations of several Escherichia coli strains have consistently indicated that cell diameter decreases with increasing cell length. This was observed both after electron microscopy of air-dried cells and after phase-contrast microscopy of living cells. The analysis was made by considering separately the unconstricted cells and three classes (slight, medium, and deep) of constricted cells in the population. During slow growth, cells with the average newborn length were up to 8% thicker than unconstricted cells twice as long. This decrease in diameter is less at higher growth rates. Despite the small changes and the large variation of the diameter in any particular length class, significant negative correlations between diameter and length were obtained. Cell diameter increases again at the end of the cell cycle as indicated by an increase of average diameter in the three consecutive classes of constriction.     

Inhibition of lateral wall elongation by mecillinam stimulates cell division in certain cell division conditional mutants of Escherichia coli.

The effect of mecillinam, a beta-lactam antibiotic that specifically binds penicillin-binding protein 2 of Escherichia coli, causes transition from rod to coccal shape, and inhibits cell division in sensitive cells, has been tested on three different E. coli temperature-sensitive cell division mutants. At the nonpermissive temperature, the antibiotic allows an increase in cell number for strains BUG6 and AX655 but not for AX621. In strain AX655, the cell division stimulation was observed only if the antibiotic was added immediately after shifting to the nonpermissive temperature, whereas in BUG6, the rise in cell number was observed also when mecillinam was added after 90 min of incubation at the nonpermissive temperature. In all cases, cell division began occurring 30 min after addition of the antibiotic. Mecillinam had no effect on division of dnaA, dnaB temperature-sensitive mutants or on division of BUG6 derivatives made resistant to this antibiotic. Other beta-lactam antibiotics such as penicillin, ampicillin, cephalexin, and piperacillin and non beta-lactam antibiotics such as fosfomycin, teichomycin, and vancomycin that inhibit cell wall synthesis did not show any effect on cell division for any of the mutants. The response of the three cell division mutants to mecillinam is interpreted in terms of a recently proposed model for shape regulation in bacteria.     

Dynamic localization cycle of the cell division regulator MinE in Escherichia coli

The MinC protein directs placement of the division septum to the middle of Escherichia coli cells by blocking assembly of the division apparatus at other sites. MinD and MinE regulate MinC activity by modulating its cellular location in a unique fashion. MinD recruits MinC to the membrane, and MinE induces MinC/MinD to oscillate rapidly between the membrane of opposite cell halves. Using fixed cells, we previously found that a MinE-green fluorescent protein fusion accumulated in an annular structure at or near the midcell, as well as along the membrane on only one side of the ring. Here we show that in living cells, MinE undergoes a rapid localization cycle that appears coupled to MinD oscillation. The results show that MinE is not a fixed marker for septal ring assembly. Rather, they support a model in which MinE stimulates the removal of MinD from the membrane in a wave-like fashion. These waves run from a midcell position towards the poles in an alternating sequence such that the time-averaged concentration of division inhibitor is lowest at midcell.     

Phospholipid turnover during the division cycle of Escherichia coli.

The turnover of phospholipids in Escherichia coli B/r was analyzed in synchronously growing populations. The turnover of presynthesized phosphatidyl-glycerol and cardiolipin continued at a constant exponential rate throughout the division cycle.     

Increase in cell mass during the division cycle of Escherichia coli B/rA.

Increase in the mean cell mass of undivided cells was determined during the division cycle of Escherichia coli B/rA. Cell buoyant densities during the division cycle were determined after cells from an exponentially growing culture were separated by size. The buoyant densities of these cells were essentially independent of cell age, with a mean value of 1.094 g ml-1. Mean cell volume and buoyant density were also determined during synchronous growth in two different media, which provided doubling times of 40 and 25 min. Cell volume and mass increased linearly at both growth rates, as buoyant density did not vary significantly. The results are consistent with only one of the three major models of cell growth, linear growth, which specifies that the rate of increase in cell mass is constant throughout the division cycle.     

ftsZ is an essential cell division gene in Escherichia coli.

The ftsZ gene is thought to be an essential cell division gene in Escherichia coli. We constructed a null allele of ftsZ in a strain carrying additional copies of ftsZ on a plasmid with a temperature-sensitive replication defect. This strain was temperature sensitive for cell division and viability, confirming that ftsZ is an essential cell division gene. Further analysis revealed that after a shift to the nonpermissive temperature, cell division ceased when the level of FtsZ started to decrease, indicating that septation is very sensitive to the level of FtsZ. Subsequent studies showed that nucleoid segregation was normal while FtsZ was decreasing and that ftsZ expression was not autoregulated. The null allele could not be complemented by lambda 16-2, even though this bacteriophage can complement the thermosensitive ftsZ84 mutation and carries 6 kb of DNA upstream of the ftsZ gene.     

Synchronization of cell division in Escherichia coli by amino acid starvation: strain specificity.

Synchronization of cell division by amino acid starvation can be induced in strains B and K-12 of Escherichia coli but not in strain B/r.     

Plasmolysis during the division cycle of Escherichia coli

Cells of Escherichia coli were plasmolyzed with sucrose. They were classified according to length by way of electron micrographs taken from samples prepared by agar filtration. The percentage of plasmolyzed cells increased about two- and threefold between mean cell sizes of newborn and separating cells. However, dividing cells were less frequently plasmolyzed than nondividing cells of the same length class. Analysis of cell halves (prospective daughters) in dividing cells showed that they behaved as independent cellular units with respect to plasmolysis. The results indicate that compressibility of the protoplast (given a certain plasmolysis space) is inversely related to cell size. That a dividing cell does not react as one osmotic compartment to osmotic stress may suggest that cell size-dependent strength of the cell membrane-cell wall association, rather than variation in turgor, plays a role during the cell division cycle.     

A new Escherichia coli cell division gene, ftsK.

A mutation in a newly discovered Escherichia coli cell division gene, ftsK, causes a temperature-sensitive late-stage block in division but does not affect chromosome replication or segregation. This defect is specifically suppressed by deletion of dacA, coding for the peptidoglycan DD-carboxypeptidase, PBP 5. FtsK is a large polypeptide (147 kDa) consisting of an N-terminal domain with several predicted membrane-spanning regions, a proline-glutamine-rich domain, and a C-terminal domain with a nucleotide-binding consensus sequence. FtsK has extensive sequence identity with a family of proteins from a wide variety of prokaryotes and plasmids. The plasmid proteins are required for intercellular DNA transfer, and one of the bacterial proteins (the SpoIIIE protein of Bacillus subtilis) has also been implicated in intracellular chromosomal DNA transfer.     

Control of cell division in Escherichia coli: effect of amino acid starvation.

The effect of amino acid starvation on cell division was studied in cells of Escherichia coli B. In this bacterial strain, deprivation of a required amino acid resulted in synchronous cell division upon restoration of the amino acid. This synchronization was apparently due to a shift forward in the cell cycle during the starvation. As a consequence, the cells divided at a size that was smaller than normal.     

Morphological analysis of nuclear separation and cell division during the life cycle of Escherichia coli.

Quantitative electron microscope observations were performed on Escherichia coli B/r after balanced growth with doubling times (tau) of 32 and 60 min. The experimental approach allowed the timing of morphological events during the cell cycle by classifying serially sectioned cells according to length. Visible separation of the nucleoplasm was found to coincide with the time of termination of chromosome replication as predicted by the Cooper-Helmstetter model. The duration of the process of constrictive cell division (10 min) appeared to be independent of the growth rate for tau equals 60 min or less but to increase with increase doubling time in more slowly growing cells. Physiological division, i.e., compartmentalization prior to physical separation of the cells, was only observed to occur in the last minute of the cell cycle. The morphological results indicate that cell elongation continues during the division process in cells with tau equals 32 min, but fails to continue in cells with tau equals 60 min.     

Cyclic AMP and cell division in Escherichia coli.

We examined several aspects of cell division regulation in Escherichia coli which have been thought to be controlled by cyclic AMP (cAMP) and its receptor protein (CAP). Mutants lacking adenyl cyclase (cya) or CAP (crp) were rod shaped, not spherical, during exponential growth in LB broth or glucose-Casamino Acids medium, and lateral wall elongation was normal; in broth, stationary-phase cells became ovoid. Cell mass was smaller for the mutants than for the wild type, but it remained appropriate for their slower growth rate and thus probably does not reflect early (uncontrolled) septation. The slow growth did not seem to reflect a gross metabolic disorder, since the mutants gave a normal yield on limiting glucose; surprisingly, however, the cya mutant (unlike crp) was unable to grow anaerobically on glucose, suggesting a role for cAMP (but not for CAP) in the expression of some fermentation enzyme. Both cya and crp mutants are known to be resistant to mecillinam, an antibiotic which inhibits penicillin-binding protein 2 (involved in lateral wall elongation) and also affects septation. This resistance does not reflect a lack of PBP2. Furthermore, it was not simply the result of slow growth and small cell mass, since small wild-type cells growing in acetate remained sensitive. The cAMP-CAP complex may regulate the synthesis of some link between PBP2 and the septation apparatus. The ftsZ gene, coding for a cell division protein, was expressed at a higher level in the absence of cAMP, as measured with an ftsZ::lacZ fusion, but the amount of protein per cell, shown by others to be invariable over a 10-fold range of cell mass, was independent of cAMP, suggesting that ftsZ expression is not regulated by the cAMP-CAP complex.     

Correlation between size and age at different events in the cell division cycle of Escherichia coli.

The variability of (i) the B period between birth and initiation of chromosome replication, (ii) the U period between initiation of chromosome replication and initiation of cell constriction, and (iii) the interdivision period (tau) have been estimated for slowly growing Escherichia coli B/r F. Cultures synchronized by the membrane elution technique were pulse-labeled with [3H]thymidine or continuously labeled with [3H]thymine. After fixation, the pattern of deoxyribonucleic acid replication was analyzed by electron microscopic radioautography. Cell length was found to increase exponentially with age at two different slow growth rates. The coefficient of variation of the B period was estimated to be 60%, that of the U period was 29%, and that of the interdivision period was 12%. From these values and the coefficient of variation of length at different cell cycle events were calculated a negative correlation between the B and U period (r = -0.9) and a positive correlation between length at birth and cell separation (r = 0.6). Initiation of chromosome replication and cell constriction were strictly correlated both with respect to age (r = 0.7) and length (r = 0.8). On the other hand, length at initiation of chromosome replication was distantly correlated with age (r = 0.1) or length at birth (r = 0.3). This low correlation excludes a model in which chromosome initiation is controlled by a random event in the B period. It favors a model in which chromosome initiation occurs at a particular distributed size independent of cell division.     

FtsZ regulates frequency of cell division in Escherichia coli.

Cell division is regulated so that it occurs only once per cell cycle. In Escherichia coli, a rod-shaped bacterium, division normally takes place at the center of the long axis of the cell; however, in the minicell mutant, division can also take place at the cell pole. Such divisions take place at the expense of normal divisions, resulting in an overall increase in nucleated cell length. We report here that increasing the level of FtsZ can completely suppress the cell length of the minicell mutant by increasing the frequency at which cell division events take place. This result suggests that the level of FtsZ controls the frequency of cell division in E. coli.     

Low-temperature conditional cell division mutants of Escherichia coli.

Fifteen low-temperature conditional division mutants of Escherichia coli K-12 was isolated. They grew normally at 39 degrees C but formed filaments at 30 degrees C. All exhibited a coordinated burst of cell division when the filaments were shifted to the permissive temperature (39 degrees C). None of the various agents that stimulate cell division in other mutant systems (salt, sucrose, ethanol, and chloramphenicol) was very effective in restoring colony-forming ability at 25 degrees C or in stimulating cell division in broth. One of these mutants, strain JS10, was found to have an altered cell envelope as evidenced by increased sensitivity to deoxycholate and antibiotics, as well as leakage of ribonulcease I, a periplasmic enzyme. This mutant had normal rates of DNA synthesis, RNA synthesis, and phospholipid synthesis at both the nonpermissive and permissive temperatures. However, strain JS10 required new protein synthesis in the apparent absence of new RNA synthesis for division of filaments at the permissive temperature. The division of lesion in strain JS10 is cotransducible with malA, aroB, and glpD and maps within min 72 to 75 on the E. coli chromosome.     

Cell elongation and division probability during the Escherichia coli growth cycle.

A new method is presented for determining the growth rate and the probability of cell division (separation) during the cell cycle, using size distributions of cell populations grown under steady-state conditions. The method utilizes the cell life-length distribution, i.e., the probability that a cell will have any specific size during its life history. This method was used to analyze cell length distributions of six cultures of Escherichia coli, for which doubling times varied from 19 to 125 min. The results for each culture are in good agreement with a single model of growth and division kinetics: exponential elongation of cells during growth phase of the cycle, and normal distributions of length at birth and at division. The average value of the coefficient of variation was 13.5% for all strains and growth rates. These results, based upon 5,955 observations, support and extend earlier proposals that growth and division patterns of E. coli are similar at all growth rates and, in addition, identify the general growth pattern of these cells to be exponential.