Cell Division During Inhibition of Deoxyribonucleic Acid Synthesis in Escherichia coli

When cultures of Escherichia coli B/r growing at various rates were exposed to ultraviolet light, mitomycin C, or nalidixic acid, deoxyribonucleic acid (DNA) synthesis stopped but cell division continued for at least 20 min. The chromosome configurations in the cells which divided were estimated by determining the rate of DNA synthesis during the division cycle. The cultures were pulse-labeled with (14)C-thymidine, and the amount of label incorporated into cells of different ages was found by measuring the radioactivity in cells born subsequent to the labeling period. The cells which divided in the absence of DNA synthesis were those which had completed a round of chromosome replication prior to the treatments. It was concluded that completion of a round of replication is a necessary and sufficient condition of DNA synthesis for cell division.     

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.     

Control of Cell Division in Escherichia coli: Experiments with Thymine Starvation

This paper describes the kinetics of cell division in populations of cells which have been grown first under conditions which specifically inhibit deoxyribonucleic acid (DNA) synthesis (in the absence of thymine or the presence of nalidixic acid) and subsequently under conditions which allow DNA synthesis to recommence. Cell division does not take place during inhibition of DNA synthesis. There is a delay between recommencement of DNA synthesis and recommencement of cell division. The length of this delay increases as a function of the length of the preceding period of inhibition of DNA synthesis. The first division after this delay is partly synchronous, but all subsequent division is asynchronous. These observations are explained in terms of a model which supposes that the formation of initiator of chromosome replication during a period when DNA synthesis is inhibited results in a block to cell division. Division does not then occur until this "extra" round of DNA synthesis is completed.     

Modulation of cell wall synthesis by DNA replication in Escherichia coli during initiation of cell growth.

Resting cells of Escherichia coli are able to initiate growth and murein biosynthesis in the presence of beta-lactam antibiotics binding to penicillin-binding proteins (PBPs) 1a and 1b (E. J. de la Rosa, M. A. de Pedro, and D. Vázquez, Proc. Natl. Acad. Sci. USA 82:5632-5635, 1985). Under these conditions, cells elongate normally until they approach the first doubling in mass, the time at which cell lysis starts. Assuming that coupling between DNA replication and cell division both in cells starting growth and in growing cells is essentially similar, triggering of the lytic response in the beta-lactam-treated cells coincides with the termination of the first round of DNA replication. This coincidence suggests that both events are interrelated. We investigated this possibility by studying the initiation of growth in cultures of wild-type strains and in cell division mutants treated with beta-lactams inhibiting PBPs 1a and 1b and with the DNA replication inhibitor nalidixic acid. Addition of nalidixic acid, even late in the first cell cycle, prevented the lytic response of the cells to the blockade of PBPs 1a and 1b. The effect of nalidixic acid is more likely due to its action on DNA replication itself than to its indirect inhibitory effect on cell division or to its ability to induce the SOS system of the cell. These observations favor the idea that the cell wall biosynthetic machinery might be modulated by DNA replication at precise periods during cell growth.     

On the termination of the DNA replication cycle in Escherichia coli.

The initiation of the DNA replication cycle in Escherichia coli requires protein synthesis. Marunouchi &; Messer (1973) have hypothesized that an additional protein synthesis step is required for the replication of the terminal segment of the chromosome, and that replication of this segment is a prerequisite for subsequent cell division. We have not confirmed the existence of a unique terminal segment using a protocol designed to label the hypothesized segment with [³H]dThd². Our protocol avoids the increased incorporation of [³H]dThd into DNA caused by abrupt increases in temperature, a complication implicit in the technique of Marunouchi &; Messer (1973).Treatment with nalidixic acid (an inhibitor of semiconservative DNA synthesis) in sufficient concentration to prevent replication of the postulated terminal segment prevents cell division but also causes loss of viability. This makes it difficult to correlate the effect of nalidixic acid on cell division with DNA synthesis inhibition alone.     

Resporulation of outgrowing Bacillus subtilis spores.

Germinated spores of Bacillus subtilis were incubated in outgrowth medium and tested periodically for capacity to sporulate when suspended in sporulation medium. Concurrent measurements were made of deoxyribonucleic acid (DNA) content and numbers of cell division septa and nucleoids. Sporulation potential is shown to reach a peak at about 110 min at which time the chromosomes are probably well into the second round of replication. Experiments with nalidixic acid show that sporulation potential can be generated in the outgrowth medium even when DNA synthesis is largely prevented. Further experiments show that nalidixic acid apparently does not prevent the formation of DNA initiation complexes, which can subsequently function after resuspension in the sporulation medium. The results support those previously obtained with a temperature-sensitive DNA mutant which indicated that sporulation could only be induced at a specific stage of chromosome replication, and then only if the cells are in a state of nutritional "step-down".     

Regulation of ribonucleoside diphosphate reductase synthesis in Escherichia coli: increased enzyme synthesis as a result of inhibition of deoxyribonucleic acid synthesis.

Inhibition of deoxyribonucleic acid (DNA) synthesis in Escherichia coli by chemical inhibitors or by shifting cultures of temperature-sensitive elongation (dnaE and dnaB) or initiation (dnaA) mutants to nonpermissive conditions led to greatly increased synthesis of the enzyme ribonucleoside diphosphate reductase, which catalyzes the first reaction unique to the pathway leading to DNA replication. In contrast to the Gudas and Pardee proposed model for control of the synthesis of DNA repair enzymes, in which both DNA inhibition and DNA degradation are involved, DNA synthesis inhibition in recA, recB, recC, or lex strains results in increased synthesis of ribonucleotide reductase, which suggests that DNA degradation is not required. We propose that inhibition of DNA synthesis causes a cell to accumulate an unknown compound that stimulates the initiation of a new round of DNA replication, and that this same signal is used to induce ribonucleotide reductase synthesis.     

Defective cell division in thermosensitive mutants of Salmonella typhimurium

Summary Two temperature-sensitive mutants ofSalmonella typhimurium defective in cell division (divA anddivC) have been isolated. Cell division in these mutants is arrested at elevated temperature while DNA and protein synthesis are unaffected. This results in formation of long filaments. Filaments returned to permissive temperature divide after a short lag. Inhibition of DNA synthesis by nalidixic acid does not block these divisions. This suggests that the thermosensitive step is required late in the division cycle. Chloramphenicol prevents the division of filaments shifted back to permissive temperature in one of these mutants (divA) and allows limited division to take place in the other mutant (divC). The arrest of cell division at elevated temperature may be phenotypically cured by high osmolarity of the medium. The mutationdivA has been mapped betweenrha andmetB and the mutationdivC betweenleu andaziA.If the filaments ofdivA are starved for thymine and then returned to permissive temperature with the simultaneous restoration of thymine the start of their division is delayed in comparison with the division of the control (unstarved) filaments. The argument is raised that a proper ratio of terminated chromosomes to cell mass must be attained to allow division.     

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.     

Deoxyribonucleic Acid Replication and Cell Division in Escherichia coli at 33 C

At 33 C (60-min generation time) the time required to replicate the chromosome is C = 60 min. The time between the end of a round of replication and cell division is D = 20 min, as at 37 C. Nalidixic acid and a temperature shift in a dnaB mutant give identical results for the determination of the end of a round of replication.     

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.     

Temporal sequence of events during the initiation process in Escherichia coli deoxyribonucleic acid replication: roles of the dnaA and dnaC gene products and ribonucleic acid polymerase.

Three thermosensitive deoxyribonucleic acid (DNA) initiation mutants of Escherichia coli exposed to the restrictive temperature for one to two generations were examined for the ability to reinitiate DNA replication after returning to the permissive temperature in the presence of rifampin, chloramphenicol, or nalidixic acid. Reinitiation in the dnaA mutant was inhibited by rifampin but not by chloramphenicol, whereas renitiation was not inhibited by rifampin but not by chloramphenicol, whereas reinitiation was not inhibited in two dnaC mutants by either rifampin or chloramphenicol. To observe the rifampin inhibition, the antibiotic must be added at least 10 min before return to the permissive temperature. The rifampin inhibition of reinitiation was not observed when a rifampin-resistant ribonucleic acid ((RNA) polymerase gene was introduced into the dnaA mutant, demonstrating that RNA polymerase synthesizes one or more RNA species required for the initation of DNA replication (origin-RNA). Reinitiation at 30 degrees C was not inhibited by streptolydigin in a stretolydigin-sensitive dnaA muntant. Incubation in the presence of nalidixic acid prevented subsequent reinitiation in the dnaC28 mutant but did not inhibit reinitiation in the dnaA5 muntant. These results demonstrate that the dnaA gene product acts before or during the synthesis of an origin-RNA, RNA polymerase synthesizes this origin RNA, and the dnaC gene product is involved in a step after this RNA synthesis event. Furthermore, these results suggest that the dnaC gene product is involved in the first deoxyribounucleotide polymerization event wheareas the dnaA gene product acts prior to this event. A model is presented describing the temporal sequence of events that occur during initiation of a round of DNA replication, based on results in this and the accompanying paper.     

Deoxyribonucleic Acid Synthesis in Synchronously Growing Mycoplasma gallisepticum

Early log-phase cells of Mycoplasma gallisepticum A5969 were synchronized by holding in Eagle minimal essential medium (MEM) for 2 h. When transferred out of MEM into tryptose medium, the cells exhibited synchronous growth. Deoxyribonucleic acid (DNA) synthesis proceeded continuously during this growth but stopped during the period of cell division. One round of DNA replication was observed per cell doubling, and a unique region of DNA was found to be permanently bound to the membrane.     

Replication of bacteriophage M13. 8. Differential effects of rifampicin and nalidixic acid on the synthesis of the two strands of M13 duplex DNA

Replication of bacteriophage M13 replicative forms is inhibited by rifampicin, an antibiotic that specifically inhibits the Escherichia coli RNA polymerase, and by nalidixic acid, an inhibitor of phage and bacterial DNA replication. Synthesis of the M13 complementary strand during RF³ replication was at least tenfold more sensitive to inhibition by rifampicin and by nalidixic acid than was that of the viral strand. Since M13 complementary strand synthesis is relatively insensitive to chloramphenicol, an inhibitor of protein synthesis, its inhibition by rifampicin suggests that complementary strands are initiated during RF replication by an RNA priming mechanism similar to that involved in parental RF formation. The nalidixic acid-sensitivity of complementary strand synthesis during RF replication clearly distinguishes this process from the nalidixic acid-resistant formation of the parental complementary strand in the conversion of the infecting single strand to RF.Production of progeny viral strands is indirectly affected by rifampiein in two ways. It prevents the conversion of supercoiled RF (RFI) to the open form (RFII), an essential step both in RF replication and in single-strand synthesis. In addition, rifampiein interferes with the expression of gene 5, an M13 gene function required for the accumulation of progeny viral strands.     

Replication of Deoxyribonucleic Acid During the Division Cycle of Salmonella typhimurium

The rate of thymidine incorporation into cells of Salmonella typhimurium growing in different media has been measured. In glucose-minimal medium, deoxyribonucleic acid (DNA) replication occurs during the first two-thirds of the division cycle; the final one-third of the division cycle was devoid of DNA replication. The measured doubling time of S. typhimurium in this medium is approximately 48 min, indicating that C (the time for a round of replication) and D (the time between termination and cell division) are approximately 32 and 16 min, respectively. At slower growth rates the pattern of replication is the same as glucose minimal medium. At faster growth rates the "gap" in DNA synthesis disappears. At rapid growth rates evidence for multiple forks is obtained.     

Coordinating DNA replication with cell division: lessons from outgrowing spores

Progress in solving the long-standing puzzle of how a cell coordinates chromosome replication with cell division is significantly aided by the use of synchronous cell populations. Currently three systems are employed for obtaining such populations: the Escherichia coli 'baby machine', the developmentally-controlled cell cycle of Caulobacter crescentus, and Bacillus subtilis germinated and outgrowing spores. This review examines our current understanding of the relationship between replication and division and how the use of B. subtilis outgrowing spores and, more recently its combination with immunofluorescence microscopy, has contributed significantly to this important area of biology. About 20 years ago, and also more recently, this system was used to show convincingly that termination of DNA replication is not essential for a central septum to form, raising the possibility that the early stages of division occur well before termination. It has also been demonstrated that there is no major synthesis of the division initiation proteins, FtsZ and DivIB, linked to initiation, progression or completion of the first round of chromosome replication accompanying spore outgrowth. This has led to the suggestion that the primary link between chromosome replication and cell division at midcell is not likely to occur through a control over the levels of these proteins. Very recent work has employed a combination of the use of B. subtilis outgrowing spores with immunofluorescence microscopy to investigate the relationship between midcell Z ring assembly and the round of chromosome replication linked to it. The results of this work suggest a role for initiation and progression into the round of replication in blocking midcell Z ring formation until the round is complete or almost complete, thereby ensuring that cell division occurs between two equally-partitioned chromosomes.