Identification of an outer membrane protein of Escherichia coli, with a role in the coordination of deoxyribonucleic acid replication and cell elongation.

Protein G of molecular weight 15,000 is the fourth commonest protein in the outer membrane of Escherichia coli B/r. From experiments described here on the relationship of protein G production to cell elongation and septation, the hypothesis is proposed that protein G is a structural protein of cell elongation. Furthermore, a surplus of protein G is produced when deoxyribonucleic acid synthesis is arrested and septation is thereby prevented. Thus protein G may be an important coordination protein in E. coli for integration of deoxyribonucleic acid synthesis, cell envelope elongation, and septation. Inhibition of normal cell elongation in a rod configuration in E. coli B/r by the novel amidinopenicillanic acid FL1060 was accompanied by changes in the rate of appearance of protein G and several other outer membrane proteins. The rate of appearance of protein G decreased some 70% within 60 min, in parallel with termination of rounds of normal cell elongation. Filament-inducing concentrations of nalidixic acid increased dramatically the rate of appearance of protein G. After 30 min a plateau level some 250% higher than the control value was reached. Similar kinetics were observed in parallel with filament formation induced by incubation of a dnaB mutant of E. coli at the nonpermissive temperature. No change in the rate of appearance of protein G was observed during cephalexin- or benzylpenicillin-induced filament formation, indicating that increased protein G production was not a secondary consequence of filamentation. Cells treated with FL1060 lost their ability to be induced for protein G formation, with nalidixic acid, in parallel with their loss of ability to initiate rounds of normal cell elongation. A pulse-chase experiment demonstrated that the protein G appearing in the outer membrane as a consequence of inhibition of deoxyribonucleic acid synthesis was the result of de novo synthesis rather than of interconversion from previously synthesized protein species. A preliminary characterization of protein G revealed several similarities with the well-characterized lipoprotein of the outer membrane of E. coli. A comparison of the incorporation of several 14C-labeled amino acids into protein G and the lipoprotein revealed substantial differences, however, perhaps ruling out a simple relationship between these two proteins.     

Deoxyribonucleic Acid Synthesis During the Division Cycle of Escherichia coli: a Comparison of Strains B/r, K-12, 15, and 15T− Under Conditions of Slow Growth

The rates of deoxyribonucleic acid (DNA) synthesis during the division cycles of the Escherichia coli strains B/r, K-12 3000, 15T(-), and 15 have been measured in synchronous cultures, under several conditions of slow growth. These synchronous cultures were obtained by sucrose gradient centrifugation of exponentially growing cultures, after which the smallest cells were removed from the gradient and allowed to grow. Sucrose gradient centrifugation did not adversely affect the cell cycle, since an experiment in which an exponentially growing culture was pulsed with [(3)H]thymidine prior to the periodic separation and assay of the smallest cells resulted in the same conclusions, as given below. In the strains of E. coli that were studied, a decreased rate of [(3)H]thymidine incorporation was seen late in the cell cycle, prior to cell division. No decrease in the rate of [(3)H]thymidine incorporation was seen at or near the beginning of the cell cycle. Thus, all these strains appear to regulate DNA synthesis in a similar fashion during slow growth. In addition, a correlation between the appearance of cells with visible cross-walls and the start of a new round of DNA synthesis was seen, indicating that these two events might be related.     

Ribonucleotide reductase and the regulation of DNA replication: an old story and an ancient heritage

All organisms that synthesize their own DNA have evolved mechanisms for maintaining a constant DNA/cell mass ratio independent of growth rate. The DNA/cell mass ratio is a central parameter in the processes controlling the cell cycle. The co-ordination of DNA replication with cell growth involves multiple levels of regulation. DNA synthesis is initiated at specific sites on the chromosome termed origins of replication, and proceeds bidirectionally to elongate and duplicate the chromosome. These two processes, initiation and elongation, therefore determine the total rate of DNA synthesis in the cell. In Escherichia coli, initiation depends on the DnaA protein while elongation depends on a multiprotein replication factory that incorporates deoxyribonucleotides (dNTPs) into the growing DNA chain. The enzyme ribonucleotide reductase (RNR) is universally responsible for synthesizing the necessary dNTPs. In this review we examine the role RNR plays in regulating the total rate of DNA synthesis in E. coli and, hence, in maintaining constant DNA/cell mass ratios during normal growth and under conditions of DNA stress.     

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.     

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.     

Accumulation of a Protein Required for Division During the Cell Cycle of Escherichia coli

A heat-labile protein required for division accumulates during the duplication cycle of Escherichia coli. Its formation appears to commence shortly after the cell divides, and it reaches a maximal amount shortly before the next division. A plausible mechanism for timing cell division depends on building up the critical amount of this protein. Completion of deoxyribonucleic acid (DNA) replication is also necessary for division to occur, but it does not uniquely initiate division. The evidence for these conclusions comes from heat-shock experiments; heating to 45 C for 15 min delays division increasingly with the age of a cell. A heat shock given near the end of a cycle delays division for about 30 min, whereas at the beginning of the cycle it hardly affects division. The net result is synchronization of cell division. The effect of heat is increased in bacteria which have incorporated p-fluoro-phenylalanine into their proteins. When the incorporation is early and the heat shock is late in the cycle, division is delayed by about 30 min, indicating that the division protein is synthesized early even though its sensitivity is not observed until later. At any time in the cell cycle, heat shock simply delays total protein and DNA synthesis ((3)H-thymidine uptake) for approximately 14 min. DNA replication and cell division are thus discoordinated, since DNA replication is not synchronized by the treatment.     

The cell cycle in Escherichia coli B/r

Cell division and DNA synthesis were measured in synchronous cultures of E. coll B/r growing in glucose minimal medium at 37 °. The kinetic curves were analysed in order to find the variability of replication initiation, termination, and cell division events during the cell cycle. It is inferred that under the conditions used, cells begin to divide 17 min (D₀ = minimum D-period) after each termination of chromosome replication with a constant probability per unit of time (half-life = 4·5–6 min). This randomness produces an asymmetric frequency distribution of D-periods, similar but mirror-symmetric frequency distributions of initiation and termination periods, a symmetric, non-Gaussian distribution of interdivision intervals, and complex kinetic changes in the rate of DNA synthesis as a function of cell age. The results suggest that replication and division are precisely controlled with respect to mass accumulation, and the apparent variability of cell cycle events would only result from the use of the time of cell separation as a reference point for the definition of cell age rather than initiation or termination of replication.     

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.     

Chromosome replication during the division cycle in slowly growing, steady-state cultures of three Escherichia coli B/r strains.

The period of DNA synthesis C during the cell cycle was determined over a broad range of generation times in slowly growing, steady-state batch cultures in the exponential phase and in chemostat cultures of three strains of Escherichia coli, strains B/r A, B/r K, and B/r TT, utilizing measurements of average amounts of DNA per cell and cell survival after radioactive decay of 125I incorporated into the DNA of synthesizing cells. At each growth rate, values for cell survival and for C periods were the same within experimental errors for the three strains. The length of the DNA synthesis period increased linearly with generation (doubling) time T of the culture and approached a limiting value of C = 0.36T at very long generation times. In very slowly growing cultures, DNA replication was limited almost entirely to the final third of the cell cycle. D periods, between termination of DNA replication and cell division, were found to be relatively short at all growth rates for each strain. Average amounts of DNA per cell measured in slowly growing cultures of strains B/r A and B/r TT were indistinguishable from results for strain B/r K at the same growth rates. Amounts of DNA per cell calculated from the cell survival values alone are completely consistent with the measured DNA per cell.     

The effect of termination of membrane phospholipid synthesis on cell-dependent events in Caulobacter

Membrane phospholipid synthesis in Caulobacter crescentus has been shown to be related to the expression of specific cell cycle events. DNA synthesis was immediately inhibited if phospholipid synthesis was terminated either by glycerol starvation of a glycerol auxotroph or by treatment of mutant and wildtype cultures with cerulenin. Termination of phospholipid synthesis, by either method, resulted in the inhibition of stalk elongation, flagellum biogenesis and cell division. The inability to form a stalk appears to be directly due to the cessation of phospholipid synthesis, whereas the inhibition of flagella formation and cell division is likely a result of the secondary effect on DNA replication. Two cell cycle events, the ejection of the flagellum and stalk initiation, were shown to be independent of phospholipid synthesis and DNA replication.     

Regulation of Cell Division in Escherichia coli

The rate of cell division was measured in cultures of Escherichia coli B/r strain after periods of partial or complete inhibition of deoxyribonucleic acid (DNA) synthesis. The rate of DNA synthesis was temporarily decreased by removing thymidine from the growth medium or replacing it with 5-bromouracil. After restoration of DNA synthesis, a temporary period of accelerated cell division was observed. The results were consistent with the idea that chromosome replication begins when an initiator complement of fixed size accumulated in the cell. The increase in the potential for the initiation of new replication points during inhibition of DNA synthesis results in an increase in the rate of cell division after an interval which encompasses the time for the arrival of these replication points to the termini of the chromosomes and the time from this event to division.     

Initiation of deoxyribonucleic acid replication in Escherichia coli B: uncoupling from mass/deoxyribonucleic acid ratio.

In Escherichia coli growing at different rates, the ratio of cell mass to the number of chromosome origins tended to be constant at the time of the initiation of deoxyribonucleic acid (DNA) replication. This observation led to the assumption that the initiation event is controlled in some way by cell mass, e.g., by a growth-dependent synthesis of an initiator or dilution of a repressor. We have now found that the initiation of DNA synthesis can be uncoupled from cell mass. We used a synchronous culture of newly divided cells of E. coli B which was obtained by the membrane elution technique (C.E. Helmstetter, J. Mol. Biol. 24: 417-427, 1967) and was starved for an amino acid. Upon restoration of the amino acid, the cells not only divided at a size that was smaller than normal, but also initiated DNA replication long before they could increase their masses to reach the expected ratio of mass/DNA presumably required for initiation.     

Initiation of chromosome replication in dnaA and dnaC mutants of Escherichia coli B/r F.

Regulatory aspects of chromosome replication were investigated in dnaA5 and dnaC2 mutants of the Escherichia coli B/r F. When cultures growing at 25 degrees C were shifted to 41 degrees C for extended periods and then returned to 25 degrees C, the subsequent synchronous initiations of chromosome replication were spaced at fixed intervals. When chloramphenicol was added coincident with the temperature downshift, the extend of chromosome replication in the dnaA mutant was greater than that in the dnaC mutant, but the time intervals between initiations were the same in both mutants. Furthermore, the time interval between the first two initiation events was unaffected by alterations in the rate of rifampin-sensitive RNA synthesis or cell mass increase. In the dnaC2 mutant, the capacities for both initiations were achieved in the absence of extensive DNA replication at 25 degrees C as long as protein synthesis was permitted, but the cells did not progress toward the second initiation at 25 degrees C when both protein synthesis and DNA replication were prevented. Cells of the dnaA5 mutant did not achieve the capacity for the second initiation event in the absence of extensive chromosome replication, although delayed initiation may have taken place. A plausible hypothesis to explain the data is that the minimum interval is determined by the time required for formation of a supercoiled, membrane-attached structure in the vicinity of oriC which is required for initiation of DNA synthesis.     

Dependence of Cell Division on the Completion of Chromosome Replication in Caulobacter crescentus

The relationship between chromosome replication and cell division in the stalked bacterium Caulobacter crescentus has been investigated. Two compounds, hydroxyurea and mitomycin C, were found to inhibit completely deoxyribonucleic acid (DNA) synthesis while allowing continued cell growth and elongation. When these inhibitors were added to exponentially growing cultures, cell division stopped after 38 min when hydroxyurea was used and after 33 min when mitomycin C was used. The period of continued cell division corresponds closely to the period previously determined for the postsynthetic gap (G2) in the DNA cycle of this organism. These results indicate that cell division is coupled to the completion of chromosome replication in C. crescentus.     

Changes in cAMP levels in bacterial cells during the cell cycle

The changes in the cAMP level during the cell cycle in the synchronous cultures of E. coli were demonstrated. Two maxima in the cAMP level were revealed during each generation period. In the cell cycle of 40-45 min duration the first increase was observed approximately in the middle of the cycle, i. e., it was coincident with the initiation of DNA synthesis. Under these conditions the cAMP level increased 8-10 times (from 0.5 to 5.0 pmole per ml of cell suspension). The second, less pronounced increase in the cAMP level was observed immediately before or during the cell division and was probably related to the regulation of the cell wall formation.     

Effect of p-Fluorophenylalanine on Chromosome Replication in Escherichia coli

The effect of p-fluorophenylalanine (FPA) on deoxyribonucleic acid (DNA) synthesis and chromosome replication was studied in a thymine-requiring mutant of Escherichia coli. The rate and extent of chromosome replication were followed by labeling the DNA with isotopic thymine and a density marker, bromouracil. The DNA was extracted and analyzed by CsCl gradient centrifugation. The block in chromosome replication caused by high concentrations of FPA occurred at the same point on the chromosome as that caused by amino acid starvation. In a random culture, DNA in cells treated with FPA replicated only slightly slower than the DNA from cells that were not exposed to the analogue. In cultures which had been previously starved for thymine, however, the DNA from the cells treated with FPA showed a marked decrease in the rate and extent of replication. It was concluded that the E. coli cell is most sensitive to FPA when a new cycle of chromosome replication is being initiated at the beginning of the chromosome.     

Regulation of Deoxyribonucleic Acid Replication and Cell Division in Escherichia coli B/r

Synchronous cultures of Escherichia coli strain B/r were used to investigate the relationship between deoxyribonucleic acid (DNA) replication and cell division. We have determined that terminal steps in division can proceed in the absence of DNA synthesis. Inhibition of DNA replication with nalidixic acid prior to the start of a new round of replication does not stop cell division, which indicates that the start of the round is not essential in triggering cell division. Inhibition of DNA replication at any time prior to the termination of a round of replication completely blocks cell division, which suggests that there may be a link between the end of the replication cycle and the commitment of the cell to divide. Studies that use a temperature-sensitive mutant which is unable to synthesize DNA at the nonpermissive temperature are in complete agreement with those that use nalidixic acid to inhibit DNA synthesis. This adds support to the idea that the treatments employed limit their action to DNA synthesis. Investigation of minicell production indicates that the production of minicells is blocked when DNA synthesis is inhibited with nalidixic acid. Although nuclear segregation is not required for cell division, DNA synthesis is still required to trigger division. The evidence presented suggests strongly that (i) DNA synthesis is essential for cell division, (ii) the end of a round of replication triggers cell division, and (iii) there is considerable time lapse (one-half generation) between the completion of a round of DNA replication and physical separation of the cells.     

Control of membrane protein synthesis in bacillus subtilis

In synchronous cultures of Bacillus subtilis 168/S grown on succinate as a sole carbon source (mean generation time 115 min), chromosome initiation occurs at the beginning of the cell cycle but the rate of membrane protein synthesis doubles in mid-cycle more or less coincident with nuclear segregation. In glucose-grown cultures, the doubling in rate of membrane protein synthesis occurs at about the same time as nuclear segregation and DNA initiation at the beginning of the cycle. Control of the rate of membrane synthesis by the chromosome has been demonstrated by inhibiting DNA synthesis using thymine starvation and showing that membrane protein synthesis continues at a constant rate, whereas the rate of cytoplasmic protein synthesis almost doubles.I suggest that the replication of a region at or close to the chromosome terminus is required to allow the doubling in rate of membrane synthesis.