Process of cellular division in Escherichia coli: physiological study on thermosensitive mutants defective in cell division

Thermosensitive fts mutants of Escherichia coli belonging to seven previously identified genetic classes (ftsA to ftsG) were studied from a physiological standpoint. These mutants immediately stopped dividing and formed multinucleated filaments when the temperature was shifted to 41 C. Macromolecular syntheses (deoxyribonucleic acid), ribonucleic acid, cell mass, and murein) continued exponentially for at least 40 to 120 min. The number of surviving bacteria remained constant during the time of incubation, and this number began to decrease exponentially, as the rate of cell mass increase leveled off from the initial rate. The recovery of cell division at 30 C in these filamentous cells was studied after 60 min of incubation at 41 C. The existence of three types of mutants was shown. The ftsA and ftsE mutants resumed cell division without new protein synthesis; ftsD mutants resumed cell division only if new protein synthesis occured, while ftsB, C, F and G mutants did not resume cell division at all. No alteration in the cell envelope was detected by the method used here, although the ftsA, B, D, F and G mutations, in contrast with ftsC and E, caused an increased resistance to penicillin G. It was also shown that the recA mutation did not suppress the effect of the fts mutations and that none of the lysogenic fts mutants induced prophage multiplication while forming filaments. The effects of osmotic pressure and salts which rescue the mutant phenotype is described.     

Characterization of a thermosensitive Escherichia coli aspartyl-tRNA synthetase mutant.

The Escherichia coli tls-1 strain carrying a mutated aspS gene (coding for aspartyl-tRNA synthetase), which causes a temperature-sensitive growth phenotype, was cloned by PCR, sequenced, and shown to contain a single mutation resulting in substitution by serine of the highly conserved proline 555, which is located in motif 3. When an aspS fragment spanning the codon for proline 555 was transformed into the tls-1 strain, it was shown to restore the wild-type phenotype via homologous recombination with the chromosomal tls-1 allele. The mutated AspRS purified from an overproducing strain displayed marked temperature sensitivity, with half-life values of 22 and 68 min (at 42 degrees C), respectively, for tRNA aminoacylation and ATP/PPi exchange activities. Km values for aspartic acid, ATP, and tRNA(Asp) did not significantly differ from those of the native enzyme; thus, mutation Pro555Ser lowers the stability of the functional configuration of both the acylation and the amino acid activation sites but has no significant effect on substrate binding. This decrease in stability appears to be related to a conformational change, as shown by gel filtration analysis. Structural data strongly suggest that the Pro555Ser mutation lowers the stability of the Lys556 and Thr557 positions, since these two residues, as shown by the crystallographic structure of the enzyme, are involved in the active site and in contacts with the tRNA acceptor arm, respectively.     

Autolysis of a division mutant of Escherichia coli

The pleiotropic character of the envC chain-forming mutant of Escherichia coli was found to include leakage of periplasmic enzymes and an abnormal tendency to autolyse. Washed suspensions of envC cells released murein fragments into the supernatant, and cell extracts from the mutant were richer than those of wild type in exo-beta-N-acetylglucosaminidase (187% of the wild-type value) and in soluble endopeptidase (256%) activities, but n-acetylmuramoylamidase, D,D-carboxypeptidase, L,Dj-carboxypeptidase and transglycosylase were not markedly different. When envC cells were grown in medium containing 0.58 M-sucrose, the chains broke up into rods, the L,D-carboxypeptidase activity increased about sixfold and D,Dj-carboxypeptidase 1B about twofold. It is suggested that L,D-carboxypeptidase is involved in septum splitting. The results suggest that the triggering of autolysis in E. coli envC depends on the alteration of envelope constituents rather than on an enhanced activity of murein hydrolases.     

Induction of cell division in a temperature-sensitive division mutant of Escherichia coli by inhibition of protein synthesis.

Synchronous cells of the thermosensitive division-defective Escherichia coli strain MACI (divA) divided at the restrictive temperature (42 degrees C) if they were allowed to grow at 42 degrees C for a certain period before protein synthesis was inhibited by adding chloramphenicol (CAP) or rifampicin. The completion of chromosome replication was not required for such divA-independent division. Synchronous cells of strain MACI divided in the presence of an inhibitor of DNA synthesis, nalidixic acid, if they were shifted to 42 degrees C and CAP or rifampicin was added after some time; cells of the parent strain MC6 (div A+) treated in the same way did not divide. These data suggest that coupling of cell division to DNA synthesis depends on the divA function. The ability to divide at 42 degrees C, whether or not chromosome termination was allowed, was directly proportional to the mean cell volume of cultures at the time of CAP addition, suggesting that cells have to be a certain size to divide under these conditions. The period of growth required for CAP-induced division had to be at the restrictive temperature; when cells were grown at 30 degrees C, in the presence of nalidixic acid to prevent normal division, they did not divide on subsequent transfer to 42 degrees C followed, after a period, by protein synthesis inhibition. A model is proposed in which the role of divA as a septation initiator gene is to differentiate surface growth sites by converting a primary unregulated structure, with the capacity to make both peripheral wall and septum, to a secondary structure committed to septum formation.     

Escherichia coli mutant Y16 is a double mutant carrying thermosensitive ftsH and ftsI mutations.

The Escherichia coli mutant Y16, which shows thermosensitive colony formation and filamentation with reduced amounts of penicillin-binding protein 3 (PBP3), has mutations in the ftsI gene encoding PBP3 and in the ftsH gene. The ftsI mutation markedly reduces the amount of PBP3 at 42 degrees C, whereas the amount of the ftsH single mutant is slightly reduced.     

An Escherichia coli mutant defective in lipid export

Escherichia coli phospholipids and lipopolysaccharide, made on the inner surface of the inner membrane, are rapidly transported to the outer membrane by mechanisms that are not well characterized. We now report a temperature-sensitive mutant (WD2) with an A270T substitution in a trans-membrane region of the ABC transporter MsbA. As shown by (32)P(i) and (14)C-acetate labeling, export of all major lipids to the outer membrane is inhibited by approximately 90% in WD2 after 30 min at 44 degrees C. Transport of newly synthesized proteins is not impaired. Electron microscopy shows reduplicated inner membranes in WD2 at 44 degrees C, consistent with a key role for MsbA in lipid trafficking.     

Relationship between chromosome replication and cell division in a thymineless mutant of Escherichia coli B/r.

The relationship between chromosome replication and cell division was investigated in a thymineless mutant of Escherichia coli B/r. Examination of the changes in average cell mass and DNA content of exponential cultures resulting from changes in the thymine concentration in the growth medium suggested that as the replication time (C) is increased there is a decrease in the period between termination of a round of replication and the subsequent cell division (D). Observations on the pattern of DNA synthesis during the division cycle were consistent with this relationship. Nevertheless, the kinetics of transition of exponential cultures moving between steady states of growth with differing replication velocities provided evidence to support the view that the time of cell division is determined by termination of rounds of replication under steady-state conditions.     

Unusual properties of a new division mutant of Escherichia coli

The properties of a division mutant of Escherichia coli were investigated. At 42 degrees C, this mutant forms nonseptate, multinucleate, filamentous cells typical of division mutants, and at the permissive temperature, is sensitive to ultraviolet (UV) irradiation. Temperature and UV sensitivities are probably due to a single mutation. The mutant phenotype is dominant to wild type. The mutant cells make DNA nearly as effectively as control cells at 42 degrees C or following UV irradiation. They exhibit normal host-cell reactivation capacities and can express all manifestations of the SOS response with the exception of Weigle reactivation. The genetic lesion which mediates this pleiotropic effect is located very close to the leu locus of the linkage map.     

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.     

Isolation and characterization of thermosensitive Escherichia coli mutants defective in deoxyribonucleic acid replication

Thermosensitive deoxyribonucleic acid replication-defective mutants have been isolated by using an autoradiographic selection method. The mutants have been analyzed genetically and biochemically. Some of the mutants show thermosensitivity of in vitro deoxyribonucleic acid replication. These can be classified into three groups according to their behavior in in vitro complementation assays. This classification is congruent with that obtained by genetic mapping by using cotransduction frequencies with selected markers in P1 transduction analysis.     

Cell division of the Escherichia coli lon- mutant

Summary Escherichia coli lon ^-cells were subjected to treatments which produced a decrease in the DNA/mass ratio of the cell. Thymine starvation, a shift-up from minimal medium to rich medium, and exposure to BUdR each caused greater inhibition of cell division in lon ^-cells than in lon ⁺cells. DNA metabolism was found to be the same in both lon ⁺and lon ^-cells during these treatments. The results are consistent with the hypothesis that the lon ^-defect leads to inhibition of cell division under conditions which produce a decreased DNA/mass ratio.     

Replication of ColE1 plasmid deoxyribonucleic acid in a thermosensitive dnaA mutant of Escherichia coli.

The replication of ColE1 deoxyribonucleic acid (DNA) took place at the restrictive temperature in a dnaA mutant, dnaA167(Ts). It proceeded at a constant rate at 42 degrees C for at least 3 h. The replication was insensitive to rifampin, which blocked replication at the permissive temperature or in the presence of chloramphenicol, even at the restrictive temperature. A linear DNA strand of ColE1 longer than unit genome size was synthesized. The structure of the replicating molecules observed by electron microscopy was mostly sigma shaped, composed of a circle of a unit genome length with a double-stranded tail. These observations suggest that the replication of ColE1 DNA proceeds via a rolling-circle type of structure in the absence of dnaA function.     

A pleiotropic defect of membrane synthesis in a thermosensitive mutant tsC42 of Escherichia coli.

Synthesis of membrane proteins in a thermosensitive mutant of Escherichia coli K12, tsC42, that has a defect in a mechanism of cell cycle-dependent duplication of membrane enzymes was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The cells were labeled differentially with [14C]- and [3H]arginine and the membrane proteins synthesized at nonpermissive and permissive temperatures were compared. The results showed that at the nonpermissive temperature, the syntheses of cytoplasmic membrane proteins and outer membrane proteins were reduced more than 70% and 50%, respectively. No significant accumulation of precursor molecules of membrane proteins at the nonpermissive temperature was detected in pulse-chase experiments. It is therefore assumed that the mutant has a defect in a gene that regulates the biosynthesis of many membrane proteins.     

Isolation of an Escherichia coli mutant defective in cytochrome biosynthesis

Abstract A pleiotropic mutant of Escherichia coli affected in cytochrome biosynthesis was detected by anaerobic screening on a solid medium containing triphenyltetrazolium. When grown anaerobically on glycerol, nitrate and Casamino acids, this mutant exhibited a level of soluble cytochrome c ₅₅₂ which was ten times higher than that found in wild-type cells. The level of membrane-bound cytochrome b and the activity of nitrate reductase were about half the normal level. The mutant grew aerobically on succinate or d,l -lactate at a greatly reduced rate. The mutation impairing the growth ability at the locus sox (succinate oxidation) is also responsible for the deficiency of cytochrome b , nitrate reductase and formate dehydrogenase. Mapping by transduction placed sox at 86.7 min on the chromosome, very close to the glnA locus. Genetic analysis also indicated that the elevated level of cytochrome c ₅₅₂ was the result of a separate mutation, the location of which is yet to be determined.     

Novel Escherichia coli mutant, dnaR, thermosensitive in initiation of chromosome replication.

A newly isolated Escherichia coli mutant thermosensitive in DNA synthesis had an allele named dnaR130, which was located at 26.3 minutes on the genetic map. The mutant was defective in initiation of chromosome replication but not in propagation at a high temperature. This mutant was capable of growing in the absence of the rnh function at the high temperature by means of a dnaA-independent replication mechanism. In the mutant exposed to the high temperature, an oriC plasmid was able to replicate, although at a lower rate than at the low temperature. The plasmid replication at the high temperature depended on the dnaA function essential for the initiation of replication from oriC. The mutant lacking the rnh function persistently maintained the oriC plasmid at the high temperature in a dnaA-dependent manner. Thus, the dnaR function was required for initiation of replication of the bacterial chromosome from oriC but not the oriC plasmid. This result reveals that a dnaR-dependent initiation mechanism that is dispensable for oriC plasmid replication operates in the bacterial chromosome replication.