Cell cycle analysis of F''lac replication in Escherichia coli B/r.

The timing and control of replication of an F'lac plasmid was investigated in two substrains of Escherichia coli B/r lac/F'lac growing at a variety of rates. The cellular content of covalently closed circular F'lac deoxyribonucleic acid and the cellular mass at the time of F'lac replication both increased as a function of growth rate. The timing of plasmid replication during the division cycle was determined by measuring the inducibility of beta-galactosidase in cells of different ages in exponentially growing cultures. At all growth rates, the rate of induced beta-galactosidase synthesis increased in a step-wise fashion during the division cycle, indicating that the F'lac plasmid replicated at a discrete time in the cycle. At growth rates greater than one doubling per h, the cell age at F'lac replication was indistinguishable from the cell age at chromosomal lac+ replication in an isogenic F- parent. The ratio of plasmids to chromosomal origins decreased from about 0.7 to 0.4 between growth rates of 1.0 to 2.5 doublings per h. These observations are all consistent with replication of F'lac at about the same time in the division cycle as replication of the homologous chromosomal region at these growth rates. This similarity in timing of replication of homologous deoxyribonucleic acid regions was not evident in slower-growing cells.     

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.     

Use of Fluorouracil-Uracil Combinations to Study Growth Accompanied by Insufficient Deoxyribonucleic Acid Synthesis in Bacillus cereus

5-Fluorouracil (FU) at a concentration of 16 muM almost totally inhibited deoxyribonucleic acid (DNA) synthesis and cell division by Bacillus cereus, whereas growth continued at an exponential rate (25% of control for at least 3 h). In cultures simultaneously given 160 muM uracil (U) along with the FU, DNA synthesis still stopped, but cell division continued for one generation at the control rate and at a much slower rate beyond that; in the meantime, cell mass continued to increase at an essentially normal rate. The cells in cultures treated with FU or FU plus U were elongated and contained about half of the control content of DNA, with one nuclear area per cell instead of two. Loss of cloning ability, unlike mass increase, was always correlated with the continuing inhibition of DNA synthesis, in either FU- or U plus FU-treated cultures.     

Balanced macromolecular biosynthesis in "protoplasts" of Streptococcus faecalis

Osmotically fragile forms of Streptococcus faecalis 9790 were grown in 0.5 m sucrose- or 0.5 m NH(4)Cl-stabilized medium. The "protoplast" cultures exhibit an average growth rate constant of 0.66 to 0.94 mass doublings/hr. In a variety of experiments, turbidity and the net content of protein, ribonucleic acid (RNA) and deoxyribonucleic acid increase at the same rate, indicating balanced macromolecular biosynthesis. A total of two to three mass doublings was obtained, with no evidence of cell division. After osmotic shock, "protoplast" cultures released 93 to 94% of their RNA content in a form not sedimentable at 12,800 x g for 15 min, in contrast to streptococci, which released 7% of their RNA content after the same treatment.     

Reinitiation of deoxyribonucleic acid synthesis by deoxyribonucleic acid initiation mutants of Escherichia coli: role of ribonucleic acid synthesis, protein synthesis, and cell division.

The dnaA and dnaC genes are thought to code for two proteins required for the initiation of chromosomal deoxyribonucleic acid replication in Escherichia coli. When a strain carrying a mutation in either of these genes is shifted from a permissive to a restrictive temperature, chromosome replication ceases after a period of residual synthesis. When the strains are reincubated at the permissive temperature, replication again resumes after a short lag. This reinitiation does not require either protein synthesis (as measured by resistance to chloramphenicol) or ribonucleic acid synthesis (as measured by resistance to rifampin). Thus, if there is a requirement for the synthesis of a specific ribonucleic acid to initiate deoxyribonucleic acid replication, this ribonucleic acid can be synthesized prior to the time of initiation and is relatively stable. Furthermore, the synthesis of this hypothetical ribonucleic acid does not require either the dnaA of dnaC gene products. The buildup at the restrictive temperature of the potential to reinitiate deoxyribonucleic acid synthesis at the permissive temperature shows rather complex kinetics the buildup roughly parallels the rate of mass increase of the culture for at least the first mass doubling at the restrictive temperature. At later times there appears to be a gradual loss of initiation potential despite a continued increase in mass. Under optimal conditions the increase in initiation potential can equal, but not exceed, the increase in cell division at the restrictive temperature. These results are most easily interpreted according to models that postulate a relationship between the initiation of deoxyribonucleic acid synthesis and the processes leading to cell division.     

Amber mutation affecting the length of Escherichia coli cells.

An amber mutation in a newly found gene (wee) of Escherichia coli has been isolated from strain OV-2, which harbors a temperature-sensitive suppressor. At 42 degrees C cells of the mutant, OV-25, increased in mass and deoxyribonucleic acid content and divided at normal rates, compared with the wild type under the same growth conditions. Total cell length increased under the restrictive conditions, although at a slightly lower rate. Values of mean cell length and cell volume, contrary to what would be expected from the increment in the rate of increase in particles, mass, and deoxyribonucleic acid, became at 42 degrees C smaller than those found in the wild type. A parallel increase in protein content per length and cell density and a loss of viability were found to occur after four generations at the restrictive temperature. The behavior of strain OV-25 in the absence of the wee gene product could be interpreted in terms of either a faulty regulation of the elongation processes or their abnormal coordination with the cell cycle. The genetic location of the wee gene has been found to be at 83.5 min on the E. coli genetic map.     

Unidirectional Growth and Branch Formation of a Morphological Mutant, Agrobacterium tumefaciens

Morphological characteristics of thermoconditional mutant Agrobacterium tumefaciens F-502 were investigated in relation to growth, division, and synthesis of cellular components. As a result of a shift from 27 to 37 C, mutant cells altered their morphology from short rods to elongated and branched forms; in addition, division and deoxyribonucleic acid synthesis were inhibited at 37 C. At 37 C unidirectional cell growth and branch formation occurred at one end of a cell, and the elongation rate of a cell was proportional to cell length. A hypothetical model for branch formation is presented in which the maximal elongation rate, 1.8 mum/h, at one end of a cell is an essential factor for initiation of branch formation.     

Stalk Formation and Its Inhibition in Caulobacter crescentus

Estimates of average rates of stalk formation over several generations of growth in Caulobacter crescentus showed that long-stalked Sk1 mutant and phosphate-starved wild-type cultures produce stalk material at about twice the rate of wild-type C. crescentus grown with adequate nutrients. Thus, the long stalks of Sk1 or phosphate-starved caulobacters are not merely a function of their longer doubling times. Inhibition of cell division of Sk1 418 with mitomycin C (MC) caused production of cellular filaments and resulted in inhibition of stalk formation. There was no appreciable decrease in total cell mass or in rates of ribonucleic acid and protein synthesis in the MC-treated cultures as compared with controls, but stalk formation, which is normally dependent on these processes, was severely retarded. Average stalk lengths in MC-treated Sk1 cultures were 30% of those found in control cultures. MC-produced cellular filaments were also subjected to deoxyribonucleic acid analysis and ultrastructural examination. The deoxyribonucleic acid content of MC-treated bacteria was about 50 to 60% that of untreated bacteria. Hydroxyurea also was found to produce some cellular filaments and shorter stalks, but with accompanying decreases in growth rate and yield.     

Control of cell length in Bacillus subtilis.

During inhibition of deoxyribonucleic acid synthesis in Bacillus subtilis 168 Thy-minus Tryp-minus, the rate of length extension is constant. A nutritional shift-up during thymine starvation causes an acceleration in the linear rate of length extension. During a nutritional shift-up in the presence of thymine, the rate of length extension gradually increases, reaching a new steady state at about 50 min before the new steady-state rate of cell division is reached. The steady-state rates of nuclear division and length extension are reached at approximately the same time. The ratio of average cell length to numbers of nuclei per cell in exponential cultures is constant over a fourfold range of growth rates. These observations are consistent with: (i) surface growth zones which operate at a constant rate of length extension under any one growth condition, but which operate at an absolute rate proportional to the growth rate of the culture, (ii) a doubling in number of growth zones at nuclear segregation, and (iii) a requirement for deoxyribonucleic acid replication for the doubling in a number of sites.     

Chromosome replication and cell division in plasmid-containing Escherichia coli B/r.

The kinetics of chromosome replication and cell division have been examined in recA mutants of Escherichia coli B/r containing F' plasmids of various sizes. Plasmid-mediated alterations in growth properties were detected only with the presence of the larger F' plasmids, and were reflected in decreased mean cell sizes and growth rates. The lengths of C and D in all plasmid-containing strains were in accord with the values for plasmid-free parental strains growing with similar generations times. The findings were consistent with an absence of competition between the chromosomal and extrachromosomal replicons for rate-limiting components involved in the initiation of deoxyribonucleic acid synthesis or in the elongation of deoxyribonucleic acid chains.     

Macromolecular synthesis in synchronized cultures of Anacystis nidulans.

Synchronous culture of Anacystis nidulans has been induced by the light-dark-light regimen. At various time intervals during synchronous growth, samples were pulsed with radioactive labels to determine phospholipid, protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA) syntheses within the cell division cycle. A temporal order of protein, RNA, and DNA syntheses occurred within the cell division cycle, whereas phospholipid was characteristically synthesized during midcycle (during cell enlargement) and during the time of cell division. Chemically determined protein, RNA, and DNA syntheses were found to support the schedule of these macromolecules in cultures growing at an 8-h doubling time.     

Early Initiation of Deoxyribonucleic Acid Replication and Shortening of Generation Time Associated with Inhibition of Lateral Wall Formation by Mecillinam

The effects of mecillinam on the growth of rods of the pH-conditional morphology mutant MirM7 was studied. It has been found that mecillinam causes, coincident with transition to coccal shape, a balanced rise in the rate of viable count increase and the rate of macromolecular synthesis which lasts either until the cells enter a stationary growth phase or indefinitely, in the case of continuously diluted cultures. When the antibiotic is removed from cells which have already become coccoid, cells continue to grow at a faster rate until they resume the rod shape. No change in the per-cell rate of protein synthesis has been seen in untreated or mecillinam-treated cells before or after the change in growth rate. Studies with synchronously growing cells have shown that the antibiotic causes a shortening in the I period (initiation of deoxyribonucleic acid replication). Evaluation of the residual divisions in nalidixic acid-treated, exponential-phase cells has shown that mecillinam also shortens the D period (cell division). It is proposed that, in strain MirM7, inhibition of lateral wall elongation by the antibiotic allows the initiation of a new septum, though inhibition is still in progress. The initiation of a new septum is, in turn, responsible for both the early inibition of deoxyribonucleic acid replication and accelerated division. In the parental strain, MirA12, as well as in other sensitive gram-negative rods which divide, become cocci, and stop dividing after addition of the antibiotic, inhibition of lateral wall formation activates a feedback mechanism which prevents insertion of new septa (Satta et al., J. Bacteriol. 142:43-51, 1980). Consequently, no early initiation of deoxyribonucleic acid replication is observed, and the last division allowed by the antibiotic occurs in due time. This negative control is missing in MirM7.     

Macromolecular Synthesis in Saccharomyces cerevisiae in Different Growth Media

Synthesis of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein was determined in Saccharomyces cerevisiae during amino acid and pyrimidine starvation and during shift-up and shift-down conditions. During amino acid starvation, cell mass, cell number, and RNA continued to increase for varying periods. During amino acid and pyrimidine starvation, cell mass and RNA showed little increase, whereas total DNA increased 11 to 17%. After a shift from broth medium to a minimal defined medium, increase in RNA and protein remained at the preshift rate before assuming a lower rate. DNA increase remained at an intermediate rate during shift-down, and then dropped to a low rate. During shift-up from minimal to broth medium, increase in cell number, protein, and DNA showed varying lag periods before increasing to the new rate characteristic of broth medium; each of these quantities exhibited a step sometime in the first 2 hr after transfer to rich medium, suggesting a partial synchronous division. Immediately after shift-up, RNA synthesis assumed a high rate, and then dropped to a rate characteristic of growth in the rich medium after about 1 hr.     

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

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

Chromosome Replication and the Division Cycle of Escherichia coli B/r

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).     

Division Cycle of Myxococcus xanthus III. Kinetics of Cell Growth and Protein Synthesis

The kinetics of cell growth and protein synthesis during the division cycle of Myxococcus xanthus was determined. The distribution of cell size for both septated and nonseptated bacteria was obtained by direct measurement of the lengths of 8,000 cells. The Collins-Richmond equation was modified to consider bacterial growth in two phases: growth and division. From the derived equation, the growth rate of individual cells was computed as a function of size. Nondividing cells (growth phase) comprised 91% of the population and took up 87% of the time of the division cycle. The absolute and specific growth rates of nondividing cells were observed to increase continually throughout the growth phase; the growth rate of dividing cells could not be determined accurately by this technique because of changes in the geometry of cells between the time of septation and physical separation. The rate of protein synthesis during the division cycle was measured by pulselabeling an exponential-phase culture with radio-active valine or arginine and then preparing the cells for quantitative autoradiography. By measuring the size of individual cells as well as the number of grains, the rate of protein synthesis as a function of cell size was obtained. Nondividing cells showed an increase in both the absolute and specific rates of protein synthesis throughout the growth phase; the specific rate of protein synthesis for dividing cells was low when compared to growthphase cells. Cell growth and protein synthesis are compared to the previously reported kinetics of deoxyribonucleic acid and ribonucleic acid synthesis during the division cycle.     

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.     

Medium-dependent variation of deoxyribonucleic acid segregation in Escherichia coli.

The degree to which deoxyribonucleic acid segregates nonrandomly has been investigated for Escherichia coli B/r growing in different media. The degree of nonrandom segregation observed is dependent on the medium, with segregation becoming less random as the growth rate decreases. This indicates that there must be some varying probabilistic component to the segregation process. A probabilistic modification of the Pierucci-Zuchowski model is proposed as well as a probabilistic model, in which it is proposed that deoxyribonucleic acid strands segregate, with a probability greater than 0.5, in the same direction (toward the same pole) as at the previous cell division.     

Growth rate-dependent control of chromosome replication initiation in Escherichia coli.

The initiation mass, defined as cell mass per origin of deoxyribonucleic acid replication (optical density units at 460 nm of culture/origins per milliliter of culture), reflects the intracellular concentration or activity of a hypothetical factor that controls initiation of chromosome replication in bacteria. In Escherichia coli B/r, the initiation mass was found to increase about twofold with increasing growth rate between 0.6 and 1.6 doublings per h; at higher growth rates it remained essentially constant (measured up to 2.4 doublings per h). A low-thymine-requiring (thyA deoB) derivative of E. coli B/r, strain TJK16, was found to have a 60 to 80% greater initiation mass than B/r which was independent of the replication velocity and not related to the thyA and deoB mutations. It is suggested that TJK16 had acquired, during its isolation, a mutation in a gene affecting the initiation of deoxyribonucleic acid replication. The initiation age was not altered by this mutation, but other parameters, including deoxyribonucleic acid concentration and cell size, were changed in comparison with the B/r parent, as expected from theoretical considerations.     

Relationship between the DNA content and mesosome number in cells of Bacillus.

In cells of Bacillus there is evidence that deoxyribonucleic acid forms an association with some membranous structure within the cell, possibly mesosomes. Cells of varieties of Bacillus cereus and Bacillus subtilis were examined to see if any quantitative relationship existed between the numbers of mesosomes and DNA content. No direct relationship could be domonstrated. However, cells of Bacillus cereus var. alesti A(-) maintained a characteristic and constant DNA content and number of mesosomes regardless of growth conditions. During sporulation, a variant of A(-), termed A(-)3, SEQUESTERS ITS DNA at both ends of the cell, leaving a small amount of DNA but no mesosomes in the center compartment. Since this center compartment is capableof growth and division upon replacement in fresh medium (rejuventation) it was examinedfor mesosome content as DNA synthesis and division were initiated. In most cells, acentral mesosome was formed at the site of cell septum formation; however, the presenceof a mesosome was not an absolute prerequisite for cell division. We propose that atthe onset of cell growth, mesosomes primarily function in the process of cell septum formation. As growth and division proceed, mesosomes are produced in characteristicnumbers and may act as the site of DNA synthesis and (or) segregation.