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1.
The cell cycle kinetics of Escherichia coli B/r A and B/r K cells were studied by flow cytometry. Three-dimensional histograms of cell cultures show the number of cells as a function of cellular DNA and protein contents and give detailed pictures of the cell cycle distribution with regard to these parameters. Histograms of slowly growing chemostat cultures showed that cell cycle periods B and C + D increase with a decreasing growth rate and that the B period occupies an increasing fraction of the cycle. The DNA replication patterns of B/r A and K were found to be quite similar. At extremely low growth rates (doubling time [T] = 17 h), B/r A cells had a B period of 0.8 T, a C period of 0.1 T, and a D period of 0.1 T, and B/r K cells (T = 16 h) had a B period of 0.6 T, a C period of 0.15 T, and a D period of 0.25 T. Mass increase, i.e., essentially protein synthesis, was seen in all three periods of the cell cycle. For B/r A cells, the average rate of mass increase was 11 times greater in the D period than in the B period, whereas for B/r K cells the rate of mass increase was twice as great in the D period as in the B period. The DNA and cell size distributions of batch cultures in exponential growth were found to vary with time, indicating that such cultures are not suitable for studies of cell cycle kinetics.  相似文献   

2.
3.
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.  相似文献   

4.
A computer simulation routine was used to calculate the DNA distributions of exponentially growing cultures of Escherichia coli K-12. Simulated distributions were compared with distributions obtained experimentally by flow cytometry. Durations of the DNA replication period (C) and the postreplication period (D) were found by minimizing the difference between theoretical and experimental DNA histograms. It was demonstrated that the K-12 strains AB1157 and CM735 had C and D periods that differed widely from each other and from those of the previously measured strain B/rA, while strain MC1000 was shown to have the same durations of the C and D periods as strain B/rA. The variation between K-12 strains may explain the divergence in the literature regarding their C and D periods. Strains W3110 and AB1157 recA1 had DNA histograms that could not be adequately simulated by the classical Cooper-Helmstetter model, which is consistent with the asymmetrically located origin and terminus for W3110 and the asynchrony of initiation for AB1157 recA1.  相似文献   

5.
The relationship between the DNA content of an average bacterial cell in an exponential culture, the velocity of chromosome rePlication (C), the time between replication termination and cell division (D), and the doubling time (τ), originally derived by Cooper and Helmstetter, is shown to be independent of two assumptions made by those authors. That is, it is not necessary to assume an ideal age distribution of cells in an exponential culture, and replication need not initiate synchronously at every DNA origin sequence within the cell. This implies that the relationship has a more general validity than has been previously supposed, and that agreement of observations on exponential cultures with the Cooper-Helmstetter theory cannot be taken to prove the assumptions on which that theory was originally based.  相似文献   

6.
Many studies are performed on chromosome replication and segregation in Escherichia coli and other bacteria capable of complex replication with C phases spanning several generations. For such investigations an understanding of the replication patterns, including copy numbers of origins and replication forks, is crucial for correct interpretation of the results.Flow cytometry is an important tool for generation of experimental DNA distributions of cell populations. Here, a Visual Basic based simulation program was written for the computation of theoretical DNA distributions for different choices of cell cycle parameters (C and D phase durations, doubling time etc). These cell cycle parameters can be iterated until the best fit between the experimental and theoretical DNA histograms is obtained. The Excel file containing the simulation software is attached as supporting information.Cultures of Escherichia coli were grown at twelve different media and temperature conditions, with following measurements by flow cytometry and simulation of the DNA distributions. A good fit was found for each growth condition by use of our simulation program. The resulting cell cycle parameters displayed clear inter-media differences in replication patterns, but indicated a high degree of temperature independence for each medium. The exception was the poorest medium (acetate), where the cells grew with overlapping replication cycles at 42 °C, but without at the lower temperatures.We have developed an easy-to-use tool for determination of bacteria's cell cycle parameters, and consequently the cells' chromosome configurations. The procedure only requires DNA distribution measurements by flow cytometry. Use of this simulation program for E. coli cultures shows that even cells growing quite slowly can have overlapping replication cycles. It is therefore always important not only to assume cells' replication patterns, but to actually determine the cell cycle parameters when changing growth conditions.  相似文献   

7.
The theory describing the effect of inhibition of initiation of rounds of deoxyribonucleic acid (DNA) replication on the accumulation of DNA is derived, and an analysis is presented which allows the determination of the time C taken to replicate the bacterial chromosome from the kinetic changes in the accumulation of DNA. This analysis is applied to experiments in which inhibition of initiation was achieved by inhibiting protein or protein and ribonucleic acid synthesis with chloramphenicol or rifampin. The results for both antibiotics are identical and indicate that there is a delay of 6 to 11 min in the effect of the antibiotics on initiation of rounds of replication. If this delay is taken into account, then the value of the C period estimated from such experiments agrees with values obtained by other methods, whereas by conventional data evaluation of such experiments the C period would be overestimated. In the low thymine-requiring derivative of Escherichia coli B/r ATCC 12407 used here, the C period was found to be between 38 and 41 min for cultures growing with a mass doubling time of 29 min in glucose-amino acids medium, supplemented with 20 micrograms of thymine/ml.  相似文献   

8.
The macromolecular composition and a number of parameters affecting chromosome replication were examined over a range of exponential growth rates in two common Escherichia coli strains, B/r and K-12 AB1157. Based on improved measurements of DNA after treatment of exponential cultures with rifampin, the cell mass per chromosomal replication origin (initiation mass) and the time required to replicate the chromosome from origin to terminus (C period) were determined. For these two strains, the initiation mass approached values of 8 × 10−10 and 10 × 10−10 units of optical density (at 460 nm) of culture mass per oriC, respectively, at growth rates above 1 doubling/h (at 37°C). The amount of protein per oriC decreased with increasing growth rate for AB1157 and remained nearly constant for the B/r strain. The C period decreased for both strains in an essentially identical manner from about 70 min at 0.6 doublings/h to about 33 min at 3 doublings/h. From the initiation mass and C period, relative or absolute copy numbers for genes with known map locations can be accurately determined at different growth rates. At growth rates above 2 doublings/h, when chromosomes are highly branched, genes near the origin are about threefold more prevalent than genes near the terminus. At a growth rate of 0.6 doubling/h, this ratio is only about 1.7, which reflects the lower degree of chromosome branching.  相似文献   

9.
Koppes LJ  Woldringh CL  Nanninga N 《Biochimie》1999,81(8-9):803-810
The active replication forks of E. coli B/r K cells growing with a doubling time of 210 min have been pulse-labeled with [(3)H] thymidine for 10 min. By electron-microscopic autoradiography the silver grains have been localized in the various length classes. From the known pattern of the DNA replication period in the cell cycle at slow growth and from the average position of grains per length class it was deduced that DNA replication starts in the cell center and that it remains there for a substantial part of the DNA replication period. This suggests the occurrence of a centrally located DNA replication compartment.  相似文献   

10.
Morphological parameters of the cell division cycle have been examined in Escherichia coli B/r A and K. Whereas the shape factor (length of newborn cell/width) of the two strains was the same at rapid growth (doubling time, tau, less than 60 min), with decreasing growth rate the dimensions of the two strains did change so that B/r A cells became more rounded and B/r K cells became more elongated. The process of visible cell constriction (T period) lasted longer in B/r A than in B/r K during slow growth, reaching at tau = 200 min values of 40 and 17 min, respectively. The time between termination of chromosome replication and cell division (D period) was found to be longer in B/r A than in B/r K. As a result, in either strain completion of chromosome replication seemed always to occur before initiation of cell constriction. Nucleoplasmic separation did not coincide with termination as during rapid growth but occurred in both strains within the T period, about 10 min before cell division.  相似文献   

11.
R James  J Y Haga    A B Pardee 《Journal of bacteriology》1975,122(3):1283-1292
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.  相似文献   

12.
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.  相似文献   

13.
Length growth of synchronized Escherichia coli B/r substrain A (ATCC 12407) and B/r substrain F26 (Thy his) was followed with an electron microscope. Cells were grown with doubling times (tau) of 60 min (B/rA) and of 82 and 165 min (B/rF26). Different length growth patterns were found for the two substrains. In B/rF, the length growth rate increased about midway in the cell cycle. For tau = 165 min, the rate increase was preceded by a short period of slow growth. For B/r A (r = 60 min), this period seemed to occur at the beginning of the cell cycle. The possibility is raised that the different length growth patterns are related to different deoxyribonucleic acid replication patterns of the respective strains.  相似文献   

14.
An Escherichia coli HF4704S mutant temperature sensitive in deoxyribonucleic acid (DNA) synthesis and different from any previously characterized mutant was isolated. The mutated gene in this strain was designated dnaH. The mutant could grow normally at 27 C but not at 43 C, and DNA synthesis continued for an hour at a decreasing rate and then ceased. After temperature shift-up, the increased amount of DNA was 40 to 50%. When the culture was incubated at 43 C for 70 min and then transferred to 27 C, DNA synthesis resumed after about 50 min, initiating synchronously at a fixed region on the bacterial chromosome. The initiation step in DNA replication sensitive to 30 mug of chloramphenicol per ml occurs synchronously before the resumption of DNA replication after the temperature shift-down, being completed about 30 min before the start of DNA replication. When the cells incubated at 27 C in the presence of 30 mug of chloramphenicol per ml after the temperature shift-down to 27 C were transferred to 43 C with simultaneous removal of the antibiotic, no resumption of DNA replication was observed. When the culture was returned to 43 C after being released from high-temperature inhibition at 30 min before the start of DNA replication, no recovery replication was observed; whereas at 20 min, the recovery of replication was observed. These results indicated that HF4704S was temperature sensitive in the initiation of DNA replication. Analysis of HF4704S, by an interrupted conjugation experiment, indicated that gene dnaH was located at about 64 min on the E. coli C linkage map. In E. coli S1814 (a K-12 derivative), which was a dnaH(ts) transductant from HF4704S (C strain) with phage P1, the mutated gene (dnaH) was demonstrated to be closely linked to the thyA marker by conjugation and P1 transduction experiments and to be distinct from genes dnaA through dnaG.  相似文献   

15.
The accumulation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein was followed in cultures of Escherichia coli B/r during exponential growth in different media and for 2 h after a nutritional shift-up from succinate minimal medium (growth rate [mu1] = 0.67 doublings per h) to glucose plus amino acids medium (mu2 = 3.14 doublings per h). During postshift growth of the culture, the amounts of RNA (R), DNA (D), and protein (P) increased such that the ratios of the increments (delta R/delta P; delta D/delta P) were constants (k1, k2). This implies that the rates of accumulation of nuclei1:k2:1. These constants change from their preshift value to their final postshift value (i.e., k1 and k2) within a few minutes after the shift. k1 is a function of the activity of ribosomes, whereas k2 is related to the initiation of rounds of DNA replication. These parameters and the observed change in the doubling time of RNA (= mu2/mu1) were used to derive kinetic equations that describe the accumulation of DNA, RNA, protein, and cell mass during the 2- to 3-h transition period after a shift-up. The calculated kinetics agree closely with the observed kinetics.  相似文献   

16.
Chromosome Replication and the Division Cycle of Escherichia coli B/r   总被引:22,自引:16,他引:6       下载免费PDF全文
The average amount of deoxyribonucleic acid (DNA) per cell was measured in steady-state cultures of Escherichia coli B/r grown at 37 C in glucose-limited chemostats or in batch cultures in the exponential growth phase as maintained with one of several carbon sources. Within experimental errors, DNA content was dependent only on growth rate and independent of the type of culture, the carbon source, or the addition of growth factors. The amount of DNA per cell increased continuously with growth rate over the range of 0.02 to 3 divisions per hour. The data over the entire range of growth rates are in agreement with a constant time for a single replication point to traverse the entire genome, 47 min, and with cell division following 25 min after termination of replication. The measured amount of DNA per genome was 4.2 x 10(-15) g (or 2.5 x 10(9) daltons).  相似文献   

17.
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.  相似文献   

18.
The theoretical distributions of cell masses in exponential cultures of bacteria were derived for both total cells and cells having formed a constriction in preparation for division. The parameters used for this derivation include the mass doubling time, tau, the T-period, and 3 statistical parameters (h, sigma 1, sigma 2) which describe the variability of the cell cycle. The theoretical distributions were compared with observed distributions from E. coli B/rA growing in glucose minimal medium (45 min doubling time) to determine whether a stochastic process in the division pathway affects the time of initiation of constriction or the duration of the constriction process. The results indicate that the stochastic process determines the onset rather than the completion of constriction and that the timing of this process is coupled (6% variability, = sigma 1) to a given cell mass. The values obtained for the duration of the T-period, T = 9.3 min, and for a half-life parameter associated with the stochastic process, h = 4.3 min, agree with previously reported data.  相似文献   

19.
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.  相似文献   

20.
The theoretical relations between replication, initiation, termination, and deoxyribonucleic acid (DNA) accumulation were derived for experiments in which the length of the time required for the replication of the bacterial chromosome (C period) can be varied. This theory enables one to determine absolute values of the C period from kinetics of DNA accumulation after a "stepup" with thymine-requiring bacteria that are subjected to a sudden increase in the exogenous thymine concentration. Application of this method of data evaluation to an observed step-up experiment with a thy-derivative of Escherichia coli B/r (ATCC 12407) indicated that the theory describes the observed post-step accumulation of DNA accurately within experimental errors. It is also concluded that changes in the replication velocity (C) do not measurably affect the timing of initiation events in a culture.  相似文献   

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