Reversibility of SOS-associated division inhibition in Escherichia coli.

In Escherichia coli the SOS response, induced by DNA-damaging treatments, includes two systems of cell division inhibition, SfiA and SfiC, which are thought to prevent cell division by interacting with the division protein FtsZ. It is shown here that SfiA-mediated division inhibition is readily reversible, even in the absence of de novo protein synthesis, suggesting that functional FtsZ molecules can be recovered from SfiA-FtsZ complexes. The action of SfiC, on the other hand, is essentially irreversible; induction by expression of the recA (Tif) mutation for 60 min results in division inhibition that continues for at least 180 min after the end of the induction period. An excess of the presumed target molecule FtsZ, furnished by a multicopy plasmid, suppresses the action of SfiA but not SfiC. Simultaneous induction of SfiA and SfiC results in irreversible division inhibition, showing that SfiC is epistatic to SfiA. The irreversibility of SfiC action is most readily accounted for by assuming that the SfiC product, unlike SfiA, is stable. The reversibility of SfiA action is slower in a lon mutant, in which the SfiA protein is partially stabilized. From the kinetics of division resumption in the absence of protein synthesis, we estimated the in vivo half-life of the SfiA protein to be 10 min in a lon+ strain and 170 min in a lon mutant.     

Are there DNA damage checkpoints in E. coli?

The concept of regulatory ‘checkpoints’ in the eukaryotic cycle has proved to be a fruitful one. Here, its applicability to the bacterial cell cycle is examined. A primitive DNA damage checkpoint operates in E. coli such that, after exposure to ultraviolet light, while excision repair occurs, chromosome replication continues very slowly with the production of discontinuous daughter strands. The slower the rate of excision of photoproducts, the greater the delay before the normal rate of DNA replication is restored, the additional time for repair ensuring that normal survival is maintained. A model is proposed in which replication rate is controlled by the ratio of RecAcoated to uncoated single stranded regions of DNA in the replication fork. There are also two cell division inhibitors SulA (=SfiA) and SfiC under the control of the SOS system and sensitive to DNA damage, but they are irrelevant to the survival of wild-type bacteria under normal conditions. In strains where SulA and SfiC do not operate, inhibition is not influenced by the rate of excision repair and so fails one of the criteria for a DNA damage checkpoint, namely the monitoring of the DNA for the level of residual damage.     

Inhibition of cell division in hupA hupB mutant bacteria lacking HU protein.

Escherichia coli hupA hypB double mutants that lack HU protein have severe cellular defects in cell division, DNA folding, and DNA partitioning. Here we show that the sfiA11 mutation, which alters the SfiA cell division inhibitor, reduces filamentation and production of anucleate cells in AB1157 hupA hupB strains. However, lexA3(Ind-) and sfiB(ftsZ)114 mutations, which normally counteract the effect of the SfiA inhibitor, could not restore a normal morphology to hupA hupB mutant bacteria. The LexA repressor, which controls the expression of the sfiA gene, was present in hupA hupB mutant bacteria in concentrations half of those of the parent bacteria, but this decrease was independent of the specific cleavage of the LexA repressor by activated RecA protein. One possibility to account for the filamentous morphology of hupA hupB mutant bacteria is that the lack of HU protein alters the expression of specific genes, such as lexA and fts cell division genes.     

Repair on the Go: E. coli Maintains a High Proliferation Rate while Repairing a Chronic DNA Double-Strand Break

DNA damage checkpoints exist to promote cell survival and the faithful inheritance of genetic information. It is thought that one function of such checkpoints is to ensure that cell division does not occur before DNA damage is repaired. However, in unicellular organisms, rapid cell multiplication confers a powerful selective advantage, leading to a dilemma. Is the activation of a DNA damage checkpoint compatible with rapid cell multiplication? By uncoupling the initiation of DNA replication from cell division, the Escherichia coli cell cycle offers a solution to this dilemma. Here, we show that a DNA double-strand break, which occurs once per replication cycle, induces the SOS response. This SOS induction is needed for cell survival due to a requirement for an elevated level of expression of the RecA protein. Cell division is delayed, leading to an increase in average cell length but with no detectable consequence on mutagenesis and little effect on growth rate and viability. The increase in cell length caused by chronic DNA double-strand break repair comprises three components: two types of increase in the unit cell size, one independent of SfiA and SlmA, the other dependent of the presence of SfiA and the absence of SlmA, and a filamentation component that is dependent on the presence of either SfiA or SlmA. These results imply that chronic checkpoint induction in E. coli is compatible with rapid cell multiplication. Therefore, under conditions of chronic low-level DNA damage, the SOS checkpoint operates seamlessly in a cell cycle where the initiation of DNA replication is uncoupled from cell division.     

Control of cell division by sex factor F in Escherichia coli. II. Identification of genes for inhibitor protein and trigger protein on the 42.84-43.6 F segment

The genetic structure of the 42.84-43.6 F (BamHI-PstI) segment of the F plasmid, which contains all the F DNA sequences necessary for coupling cell division of F+ bacteria with plasmid DNA replication, was analyzed by isolating a series of amber mutants. Two cistrons were found in this region and they were designated letA and letD (an abbreviation for lethal mutation). The letA and letD cistrons were mapped on the 42.84-43.35 F (BamHI- XmaI ) segment and the 43.07-43.6 F (HincII-PstI) segment, respectively, and are presumed to correspond to the first (43.04-43.26 F) and second (43.26-43.57 F) open reading frames, respectively, which were found in this region by nucleotide sequencing. The letD gene product acts to inhibit cell division of the host bacteria and to induce prophages in lysogenic bacteria, whereas the letA gene product acts to suppress the activity of the letD gene product. Taking into consideration the fact that the 42.84-43.6 F segment carries all the F plasmid genes necessary for coupling cell division with plasmid DNA replication, and that the expression of the genes is likely to be controlled by plasmid DNA replication, we constructed the following hypothesis. Before completion of plasmid DNA replication, LetD protein acts to prevent cell division of the host bacteria. When plasmid DNA replication is completed, synthesis of LetA protein (and also LetD protein) takes place and the LetA protein synthesized acts to suppress the activity of LetD protein and make the cell ready for cell division. Actual cell division will take place when replication of both chromosomal and plasmid DNA is completed and the termination protein of the chromosome and the LetA protein of F plasmid are both synthesized. When cell division takes place LetA protein is consumed, and as a result LetD protein becomes active and prevents cell division until the next round of DNA replication is completed.     

Involvement of FtsZ protein in shift-up-induced division delay in Escherichia coli.

A nutritional shift-up from glucose minimal medium to LB broth was previously shown to cause a division delay of about 20 min in synchronized cultures of Escherichia coli, and a similar delay was observed after a nutritional pulse (a shift-up followed rapidly by a return to glucose minimal medium). Using synchronized cultures, we show here that the pulse-induced division delay does not require protein synthesis during the period in LB broth, suggesting that a nonprotein signal is generated by the shift-up and transmitted to the cell division machinery. The cell division protein FtsZ, target of the SOS-associated division inhibitor SfiA (or SulA), seems to be involved in the postshift division delay. Mutants in which the FtsZ-SfiA interaction is reduced, either sfiA (loss of SfiA) or ftsZ(SfiB) (modification of FtsZ), have a 50- to 60-min division delay after a shift-up. Furthermore, after a nutritional pulse, the ftsZ(SfiB) mutant had only a 10- to 16-min delay. These results suggest that the FtsZ protein is the target element of the cell division machinery to which the shift-up signal is transmitted.     

Anucleate cell production and surface extension in a temperature-sensitive chromosome initiation mutant of Bacillus subtilis.

At 45 C, in a temperature-sensitive initiation mutant (TsB134) of Bacillus subtilis 168 Thy- tryp-, growing in a glucose-arginine minimal medium, chromosome completion occurred over a period of 80 to 90 min, after which there was no further nuclear division. Normal symmetrical cell divisions continued for a generation afterwards, so that nuclei were segregated into separate cells. During this period asymmetric divisions started to occur. Septa appeared at 25 to 30% from one end of the cell, giving a small anucleate cell and a larger nucleate cell. During inhibition of deoxyribonucleic acid (DNA) synthesis by thymine starvation under the restrictive conditions, asymmetrical division also occurred until there was approximately one nucleus per cell (about one generation time). Asymmetric division, giving anucleate cells, then occurred. Similar results were obtained when DNA synthesis was inhibited by nalidixic acid. After 3 h at 45 C, the rate of anucleate cell production in the presence and absence of thymine was constant at one division per 85 min per chromosome terminus present when DNA synthesis stopped. In the absence of DNA synthesis (during thymine starvation) at 35 C, growth in cell length was linear (i.e., the rate was constant), but at 45 C during thymine starvation the rate gradually increased by more than twofold. It is suggested that this was due to the establishment of new sites of growth associated with anucleate cell production. In the presence of thymine at 45 C, the rate of length extension increased by more than fourfold, which it is suggested was caused by the appearance of new growth zones as a result of chromosome termination and a contribution associated with anucleate cell production. If the mutant was incubated at 45 C for 90 min, both in the presence and absence of thymine, then anucleate cell formation could continue on restoration to 35 C in the absence of thymine...     

Synthesis of high mobility group proteins in regenerating rat liver.

Incorporation of [3H]lysine into the non-histone chromosomal proteins HMG1, HMG2, and HMG17 and into each of the five major classes of histones was measured in rat liver at various times after partial hepatectomy. Histone synthesis was closely coupled temporally to that of DNA, although a small amount of histone was shown to be produced before DNA replication began. In contrast, the incorporation curves for the high mobility group (HMG) proteins showed little correlation with that for DNA. At 4 h after partial hepatectomy, protein synthesis had virtually ceased. Thereafter, the rates of synthesis of the HMG proteins rose steadily so that by 12 h, well before the onset of DNA replication they had reached about two-thirds of the maximum rates attained during the first cell division cycle. Histones had only reached about one-sixth of their maximum rates at this time. The lack of coupling betweeen the synthesis of the HMG proteins and DNA was confirmed by experiments with inhibitors of DNA replication. Reduction of DNA synthesis to less than 10% of the uninhibited rate had little or no effect on incorporation into the HMG proteins, whereas, under similar conditions, the rate of synthesis of histones was reduced by more than 50%.     

Inhibition of an early event in the cell division cycle of Escherichia coli by FL1060, an amidinopenicillanic acid.

Analysis of exponential and synchronous cultures of Escherichia coli B/r after the addition of FL1060 indicates a block point for division by this agent some 15 to 20 min before the end of the preceding cell division cycle, a time corresponding to the beginning of the C period of the cell division cycle. Morphological examination of FL1060-treated synchronous cultures of E. coli /r was consistent with inhibition by FL1060 of a very early event in the cell division cycle. This event appears to be essential for normal cell surface elongation in a rod configuration. Temporary treatment of synchronous cultures of E. coli B/r with FL1060 resulted in division delay, the extent of which was a function of the duration of exposure to FL1060. However, even after relatively long times of FL1060 treatment the delayed divisions were still synchronous. Although FL1060 had no direct effect on deoxyribonucleic acid (DNA) synthesis, the synchronous delayed division occuring after temporary treatment with FL1060 were accompanied by a delay in the attainment of resistance of cell division to inhibitors of DNA, ribonucleic acid, and protein synthesis. These results suggest aht an FL1060-sensitive event initiates at the beginning of the C period of the cell division cycle of E. coli and is responsible for normal cell elongation. This cell elongation pathway procedes independently of DNA synthesis, but there is an interaction between this pathway and termination of a round of DNA replication in which a normal rod configuration is necessary to allow a signal for cell division to be generated upon completion of DNA replication.     

dnaT, dominant conditional-lethal mutation affecting DNA replication in Escherichia coli.

Normally, bacteria cease DNA replication in the absence of protein synthesis. A variety of treatments, such as thymine starvation or a shift-up to rich medium, lead to continued DNA replication in the absence of protein synthesis. Mutants are described which always terminate replication under these conditions. These conditional lethal mutants, dnaT1 and dnaT2, contransduce with serB and dnaC. The mutation also affects cell division. All aspects of the mutant phenotype (obligatory termination of replication, temperature sensitivity of DNA replication and growth, and aberrant cell division at permissive growth temperatures) were transdominant to the wild-type phenotype. Episomes carrying the dnaT mutation appeared to be unstable. The existence of such a dominant mutation was predicted by a model of chromosome termination proposed by Kogoma and Lark (J. Mol. Biol. 94:243-256, 1975).     

Linkages between deoxyribonucleic acid synthesis and cell division in Myxococcus xanthus.

Addition of chloramphenicol or 0.5 M glycerol to growing Myxococcus xanthus resulted in an immediate cessation of cell division and 40% net increase in deoxyribonucleic acid (DNA). Although the chloramphenicol-treated cells divided in the presence of nalidixic acid after chloramphenicol was removed, glycerol-induced myxospores required DNA synthesis for subsequent cell division. Myxospores prepared from chloramphenicol-treated cells lost this potential to divide in the presence of nalidixic acid. The "critical period" of DNA synthesis necessary for cell division after germination overlapped in time (3 to 5 h) with initiation of net DNA synthesis. The length of the critical period of DNA synthesis was estimated at 12 min, or 5% of the M. xanthus chromosome. The requirement for cell division during germination also involved ribonucleic acid and protein synthesis after DNA synthesis. The data suggest that replication at or near the origin of the chromosome triggers the formation of a protein product that is necessary but not sufficient for subsequent cell division; DNA termination is also required. During myxospore formation, the postulated protein is destroyed, thereby reestablishing and making apparent this linkage between early DNA synthesis and cell division.     

EVIDENCE FOR A TEMPORAL INCOMPATIBILITY BETWEEN DNA REPLICATION AND DIVISION DURING THE CELL CYCLE OF TETRAHYMENA

The mechanism of coordination between DNA replication and cell division was studied in Tetrahymena pyriformis GL-C by manipulation of the timing of these events with heat shocks and inhibition of DNA synthesis. Preliminary experiments showed that the inhibitor combination methotrexate and uridine (M + U) was an effective inhibitor of DNA synthesis. Inhibition of the progression of DNA synthesis with M + U in exponentially growing cells, in which one S period usually occurs between two successive divisions, or in heat-shocked cells, when successive S periods are known to occur between divisions, resulted in the complete suppression of the following division. In further experiments in which the division activities were reassociated with the DNA synthetic cycle by premature termination of the heat-shock treatment, it was shown that (a) the completion of one S period during the treatment was sufficient for cell division, (b) the beginning of division events suppressed the initiation of further S periods, and (c) if further S periods were initiated while the heat-shock treatment was continued, division preparations could not begin until the necessary portion of the S period was completed, even though DNA had previously been duplicated. It was concluded that a temporal incompatibility exists between DNA synthesis and division which may reflect a coupling mechanism which insures their coordination during the normal cell cycle.     

Novel mechanism of cell division inhibition associated with the SOS response in Escherichia coli.

Certain Escherichia coli strains were shown to possess a novel system of cell division inhibition, called the SfiC+ phenotype. SfiC+ filamentation had a number of properties similar to those of sfiA-dependent division inhibition previously described: (i) both are associated with the SOS response induced by expression of the recA(Tif) mutation, (ii) both are associated with cell death, (iii) both are amplified in mutants lacking the Lon protease, and (iv) both are suppressed by sfiB mutations. SfiC+ filamentation and sfiA-dependent division inhibition differed in (i) the physiological conditions under which loss of viability is observed, (ii) the extent of amplification in lon mutants, (iii) their genetic regulation (SfiC+ filamentation is not under direct negative control of the LexA repressor), and (iv) their genetic determinants (SfiC+ filamentation depends on a locus, sfiC+, near 28 min on the E. coli map and distinct from sfiA).     

Lethality of bypass polymerases in Escherichia coli cells with a defective clamp loader complex of DNA polymerase III

Escherichia coli DNA polymerase III (Pol III) is one of the best studied replicative DNA polymerases. Here we report the properties of an E. coli mutant that lacks one of the subunits of the Pol III clamp loader complex, Psi (psi), as a result of the complete inactivation of the holD gene. We show that, in this mutant, chronic induction of the SOS response in a RecFOR-dependent way leads to lethality at high temperature. The SOS-induced proteins that are lethal in the holD mutant are the specialized DNA polymerases Pol II and Pol IV, combined with the division inhibitor SfiA. Prevention of SOS induction or inactivation of Pol II, Pol IV and SfiA encoding genes allows growth of the holD mutant, although at a reduced rate compared to a wild-type cell. In contrast, the SOS-induced Pol V DNA polymerase does not participate to the lethality of the holD mutant. We conclude that: (i) Psi is essential for efficient replication of the E. coli chromosome; (ii) SOS-induction of specialized DNA polymerases can be lethal in cells in which the replicative polymerase is defective, and (iii) specialized DNA polymerases differ in respect to their access to inactivated replication forks.     

Model scenarios for evolution of the eukaryotic cell cycle

Progress through the division cycle of present day eukaryotic cells is controlled by a complex network consisting of (i) cyclin-dependent kinases (CDKs) and their associated cyclins, (ii) kinases and phosphatases that regulate CDK activity, and (iii) stoichiometric inhibitors that sequester cyclin-CDK dimers. Presumably regulation of cell division in the earliest ancestors of eukaryotes was a considerably simpler affair. Nasmyth (1995) recently proposed a mechanism for control of a putative, primordial, eukaryotic cell cycle, based on antagonistic interactions between a cyclin-CDK and the anaphase promoting complex (APC) that labels the cyclin subunit for proteolysis. We recast this idea in mathematical form and show that the model exhibits hysteretic behaviour between alternative steady states: a Gl-like state (APC on, CDK activity low, DNA unreplicated and replication complexes assembled) and an S/M-like state (APC off, CDK activity high, DNA replicated and replication complexes disassembled). In our model, the transition from G1 to S/M ('Start') is driven by cell growth, and the reverse transition ('Finish') is driven by completion of DNA synthesis and proper alignment of chromosomes on the metaphase plate. This simple and effective mechanism for coupling growth and division and for accurately copying and partitioning a genome consisting of numerous chromosomes, each with multiple origins of replication, could represent the core of the eukaryotic cell cycle. Furthermore, we show how other controls could be added to this core and speculate on the reasons why stoichiometric inhibitors and CDK inhibitory phosphorylation might have been appended to the primitive alternation between cyclin accumulation and degradation.     

Partitioning, Movement, and Positioning of Nucleoids in Mycoplasma capricolum

The nucleoids in Mycoplasma capricolum cells were visualized by phase-combined fluorescence microscopy of DAPI (4', 6-diamidino-2-phenylindole)-stained cells. Most growing cells in a rich medium had one or two nucleoids in a cell, and no anucleate cells were found. The nucleoids were positioned in the center in mononucleoid cells and at one-quarter and three-quarters of the cell length in binucleoid cells. These formations may have the purpose of ensuring delivery of replicated DNA to daughter cells. Internucleoid distances in binucleoid cells correlated with the cell lengths, and the relationship of DNA content to cell length showed that cell length depended on DNA content in binucleoid cells but not in mononucleoid cells. These observations suggest that cell elongation takes place in combination with nucleoid movement. Lipid synthesis was inhibited by transfer of cells to a medium lacking supplementation for lipid synthesis. The transferred cells immediately stopped dividing and elongated while regular spaces were maintained between the nucleoids for 1 h. After 1 h, the cells changed their shapes from rod-like to round, but the proportion of multinucleoid cells increased. Inhibition of protein synthesis by chloramphenicol induced nucleoid condensation and abnormal positioning, although partitioning was not inhibited. These results suggest that nucleoid partitioning does not require lipid or protein synthesis, while regular positioning requires both. When DNA replication was inhibited, the cells formed branches, and the nucleoids were positioned at the branching points. A model for the reproduction process of M. capricolum, including nucleoid migration and cell division, is discussed.     

THE EFFECT OF HYDROXYUREA AND FLUORODEOXYURIDINE ON CELL CYCLE EVENTS IN THE CHLOROCOCCAL ALGA SCENEDESMUS QUADRICAUDA (CHLOROPHYTA)1

The effect of hydroxyurea and 5-fluorodeoxyuridine (FdUrd) on the course of growth (RNA and protein synthesis) and reproductive (DNA replication and nuclear and cellular division) processes was studied in synchronous cultures of the chlorococcal alga Scenedesmus quadricauda (Turp.) Bréb. The presence of hydroxyurea (5 mg·L^?1)from the beginning of the cell cycle prevented growth and further development of the cells because of complete inhibition of RNA synthesis. In cells treated later in the cell cycle at the time when the cells were committed to division, hydroxyurea present in light affected the cells in the same way as a dark treatment without hydroxyurea; i. e. RNA synthesis was immediately inhibited followed after a short time period by cessation of protein synthesis. Reproductive processes including DNA replication to which the commitment was attained, however, were initiated and completed. DNA synthesis continued until the constant minimal ratio of RNA to DNA was reached. FdUrd (25 mg·L^?1) added before initiation of DNA replication in control cultures prevented DNA synthesis in treated cells. Addition of FdUrd at any time during the cell cycle prevented or immediately stopped DNA replication. However, by adding excess thymidine (100 mg·L^?1), FdUrd inhibition of DNA replication could be prevented. FdUrd did not affect synthesis of RNA, protein, or starch for at least one cell cycle. After removal of FdUrd, DNA synthesis was reinitiated with about a 2-h delay. The later in the cell cycle FdUrd was removed, the longer it took for DNA synthesis to resume. At exposures to FdUrd longer than two or three control cell cycles, cells in the population were gradually damaged and did not recover at all.     

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.