Macromolecular composition of bacteria

Equations are presented that describe the macromolecular composition in exponential bacterial cultures as functions of five parameters: doubling time of the culture (τ), protein per origin of replication (P₀), chromosome replication time (C-period), peptide chain elongation rate (c_p), and the time between termination of replication and cell division (D-period). Implicit in the value for some of these parameters is a specific macromolecular control system: the control of the growth rate (τ), the timing of initiation of rounds of chromosome replication (P₀), and the regulation of cell division (D). The utility of these relations is illustrated by using updated measurements of the macromolecular composition of E. coli B/r to calculate values for the fundamental parameters and to predict the composition of a mutant which has a defect in the control of DNA replication. Furthermore, the meaning of several often-cited physiological parameters (RNA/protein, RNA/cell and RNA/genome) is examined. The relations presented here show that these parameters and their variation with growth rate are not directly relevant to arguments about control of ribosome synthesis or culture growth.     

Intensive DNA Replication and Metabolism during the Lag Phase in Cyanobacteria

Unlike bacteria such as Escherichia coli and Bacillus subtilis, several species of freshwater cyanobacteria are known to contain multiple chromosomal copies per cell, at all stages of their cell cycle. We have characterized the replication of multi-copy chromosomes in the cyanobacterium Synechococcus elongatus PCC 7942 (hereafter Synechococcus 7942). In Synechococcus 7942, the replication of multi-copy chromosome is asynchronous, not only among cells but also among multi-copy chromosomes. This suggests that DNA replication is not tightly coupled to cell division in Synechococcus 7942. To address this hypothesis, we analysed the relationship between DNA replication and cell doubling at various growth phases of Synechococcus 7942 cell culture. Three distinct growth phases were characterised in Synechococcus 7942 batch culture: lag phase, exponential phase, and arithmetic (linear) phase. The chromosomal copy number was significantly higher during the lag phase than during the exponential and linear phases. Likewise, DNA replication activity was higher in the lag phase cells than in the exponential and linear phase cells, and the lag phase cells were more sensitive to nalidixic acid, a DNA gyrase inhibitor, than cells in other growth phases. To elucidate physiological differences in Synechococcus 7942 during the lag phase, we analysed the metabolome at each growth phase. In addition, we assessed the accumulation of central carbon metabolites, amino acids, and DNA precursors at each phase. The results of these analyses suggest that Synechococcus 7942 cells prepare for cell division during the lag phase by initiating intensive chromosomal DNA replication and accumulating metabolites necessary for the subsequent cell division and elongation steps that occur during the exponential growth and linear phases.     

The Relative Numbers of Different Genes in Exponential Microbial Cultures

It is shown that the results of the marker frequency analysis of Sueoka and Yoshikawa (1965) can be derived as very good approximations from a model where the rigid assumptions of their analysis are relaxed to take into account statistical variations in the timing of cell events. It is further shown that the expression for the amount of DNA per cell can be approximated by an elementary exponential function of the growth rate, and this result facilitates genetic mapping by DNA hybridization techniques. An analysis of recent data on gene frequencies in Escherichia coli corroborates a model of symmetric, bidirectional chromosome replication with a replication time of approximately thirty minutes.     

Changes of Escherichia coli cell cycle parameters during fast growth and throughout growth with limiting amounts of thymine

It is generally accepted that during fast growth of Escherichia coli, the time (D) between the end of a round of DNA replication and cell division is constant. This concept is not consistent with the fact that average cell mass of a culture is an exponential function of the growth rate, if it is also accepted that average cell mass per origin of DNA replication (M_i) changes with growth rate and negative exponential cell age distribution is taken into account. Data obtained from cell composition analysis of E. coli OV-2 have shown that not only (M_i) but also D varied with growth rate at generation times () between 54 and 30 min. E. coli OV-2 is a thymine auxotroph in which the replication time (C) can be lengthened, without inducing changes in , by growth with limiting amounts of thymine. This property has been used to study the relationship between cell size and division from cell composition measurements during growth with different amounts of thymine. When C increased, average cell mass at the end of a round of DNA replication also increased while D decreased, but only the time lapse (d) between the end of a replication round and cell constriction initiation appeared to be affected because the constriction period remained fairly constant. We propose that the rate at which cells proceed to constriction initiation from the end of replication is regulated by cell mass at this event, big cells having shorter d times than small cells.Abbreviations OD₄₅₀ and OD₆₃₀ Optical density at a given wavelength in nm Dedicated to Dr. John Ingraham to honor him for his many contributions to Science     

Cell division after inhibition of chromosome replication in Escherichia coli

The residual cell divisions after thymine starvation of exponential cultures of TJK16, a thymine-requiring derivative of Escherichia coli B/r, were evaluated. The results indicate that under the conditions used (glucose minimal medium 37 °), (1) only cells that had terminated a round of replication divided; (2) once termination had occurred, thymine starvation and replication no longer affected the time of cell division; (3) synchronously terminating subpopulations of cells began to divide about 17 min after termination; after that time, the rate of division decreased exponentially. The results confirm the previously inferred asymmetric distribution of D-periods in an exponential population of E. coli bacteria and suggest that an event associated with termination of replication is required for cell division. The method of data evaluation presented can be used to determine the duration of the D-period and to find the parameter values (halflife and onset) of the stochastic phase of the D-period in exponential cultures, eliminating the need for synchronization procedures.     

Phosphodiesterase in human colon carcinoma cell line CaCo-2 in culture

In a series of in vitro experiments we characterised the relationship between DNA distribution in the G1, S and G2/M phases of cell cycle and PDE and GST activity in CaCo-2 cells. The DNA distribution in CaCo-2 cells, was assessed by flow cytometry, with fluorescent dyes at different time points of culture. The exponential increase in cell number continued until day 10 when there was cell saturation. The effect of medium replacement on PDE activity was assayed in the first 10 h after medium replacement. The 6^th hour is the time at which PDE activity was found to be highest. We have assayed the PDE enzyme with cGMP and cAMP as substrates. Only cAMP was consumed from this enzyme. We found a very close correlation between the DNA distribution in the various phases of the cell cycle and the PDE activity. PDE activity was very high during the active replication phase, whereas GST activity was high after confluency.     

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.     

Sequential function of gene products relative to DNA synthesis in the yeast cell cycle

An experimental rationale for deciphering the relative dependence of steps in a developmental pathway (Jarvik & Botstein, 1973; Hereford & Hartwell, 1974) has been employed to determine the relationship between the hydroxyurea-sensitive step and various temperature-sensitive steps in the cell cycle of Saccharomyces cerevisiae. Since hydroxyurea inhibits DNA replication in yeast (Slater, 1973), the data identify gene products upon whose function DNA replication is dependent (cdc 4, 6, 7, 2, 8, 21) and gene products whose function or synthesis requires DNA replication (cdc 2, 8, 21, 9, 13, 16, 23, 5, 15). Other gene products (cdc 3, 11, 24) function independent of DNA replication. These results suggest that the events of the cell cycle occur in a proscribed order because many of the gene products that mediate these events arc restricted to a prescribed sequence of function.Mutations in two genes (cdc 2 and 6) result in cells that remain sensitive to hydroxyurea after an incubation at the restrictive temperature, despite the fact that both mutants incorporate radioactive precursors into DNA at the restrictive temperature (Hartwell, 1973). It is suggested that cdc 6 specifies a function that is necessary for the proper initiation of DNA replication, and cdc 2 a function that is necessary for correct DNA elongation, and that in the absence of either of these functions the DNA that is made is either faulty or incomplete.     

Mitochondrial DNA synthesis in cell cycle mutants of saccharomyces cerevisiae

Mitochondrial DNA replication was examined in mutants for seven different Saccharomyces cerevisiae genes which are essential for nuclear DNA replication. In cdc8 and cdc21, mutants defective in continued replication during the S phase of the cell cycle, mitochondrial DNA replication ceases at the nonpermissive temperature. Replication is temperature sensitive even when these mutants are arrested in the G1 phase of the cell cycle with α factor, a condition where mitochondrial DNA replication continues for the equivalent of several generations at the permissive temperature. Therefore the cessation of replication results from a defect in mitochondrial replication per se, rather than from an indirect consequence of cells being blocked in a phase of the cell cycle where mitochondrial DNA is not normally synthesized. Since the temperature-sensitive mutations are recessive, the products of genes cdc8 and cdc21 must be required for both nuclear and mitochondrial DNA replication. In contrast to cdc8 and cdc21, mitochondrial DNA replication continues for a long time at the nonpermissive temperature in five other cell division cycle mutants in which nuclear DNA synthesis ceases within one cell cycle: cdc4, cdc7, and cdc28, which are defective in the initiation of nuclear DNA synthesis, and cdc14 and cdc23, which are defective in nuclear division. The products of these genes, therefore, are apparently not required for the initiation of mitochondrial DNA replication.     

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.     

Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA

In rolling circle replication, a circular template of DNA is replicated as a long single-stranded DNA concatamer that spools off when a strand displacing polymerase traverses the circular template. The current view is that this type of replication can only produce single-stranded DNA, because the only 3′-ends available are the ones being replicated along the circular templates. In contrast to this view, we find that rolling circle replication in vitro generates large amounts of double stranded DNA and that the production of single-stranded DNA terminates after some time. These properties can be suppressed by adding single-stranded DNA-binding proteins to the reaction. We conclude that a model in which the polymerase switches templates to the already produced single-stranded DNA, with an exponential distribution of template switching, can explain the observed data. From this, we also provide an estimate value of the switching rate constant.     

Fluctuations du taux d'AMP cyclique et des activites adenylate cyclase et AMP cyclique-phosphodiesterase dans les cultures synchrones d'un procaryote, Nocardia restricta

Cultures of Nocardia restricta, a prokaryote from the group of Actinomycetes, can be synchronised by diluting, in a fresh growth medium, cells already in stationary phase. The synchronisation of the cultures is monitored by examining the synchrony of DNA replication.In these synchronised cultures, the intracellular cyclic AMP level exhibits rythmic oscillations with a period equal to the generation time of the culture. There is only one peak per generation. The average ratio of maximum to minimum concentrations is at least 3.Cyclic AMP accumulates also in the medium with a step pattern. It appears in the medium during maximum production of cyclic AMP in the cell.The specific activity of adenylate cyclase (EC 4.6.1.1) measured in the 30 000 × g pellet of cell-free extracts also oscillates and correlates well with fluctuations in the cyclic AMP level. At the end of exponential growth, cyclic-AMP phosphodiesterase (EC 3.1.4.17) is detectable in the cells. The specific activity of this enzyme measured in the 30 000 × g supernatant of cell-free extracts shows also an oscillating pattern.To our knowledge it is the first time that such oscillations in the metabolism of cyclic AMP are described among prokaryotes. It is now possible to look at a link between this phenomenon and the cell cycle of the organism.     

The human GINS complex associates with Cdc45 and MCM and is essential for DNA replication

The GINS complex, originally discovered in Saccharomyces cerevisiae and Xenopus laevis, binds to DNA replication origins shortly before the onset of S phase and travels with the replication forks after initiation. In this study we present a detailed characterization of the human GINS (hGINS) homolog. Using new antibodies that allow the detection of endogenous hGINS in cells and tissues, we have examined its expression, abundance, subcellular localization and association with other DNA replication proteins. Expression of hGINS is restricted to actively proliferating cells. During the S phase, hGINS becomes part of a Cdc45–MCM–GINS (CMG) complex that is assembled on chromatin. Down-regulation of hGINS destabilizes CMG, causes a G1–S arrest and slows down ongoing DNA replication, effectively blocking cell proliferation. Our data support the notion that hGINS is an essential component of the human replisome.     

Variation in periodic replication of the chromosome in Escherichia coli B/rTT.

A method using 5-bromouracil photolysis induction with 313 nm radiation was employed to estimate the variation in the period between successive rounds of DNA replication in rapidly growing cultures of Escherichia coli$\text{B}\text{rTT}$ The coefficient of variation of this period was 9.3%, which is significantly less than the corresponding value of about 20% reported for variation in the cell interdivision period. Thus chromosome replication is much more tightly controlled than is cell division. The reduced variability of the DNA replication cycle indicates that the period (D) between termination of a round of DNA replication and cell division and the following period ending in initiation of the next round of DNA replication (B) are riot independent of each other but tend to have compensatory variations. The results suggest that other events in the cell cycle are related more closely to DNA replication rather than to the much less regular event of cell division.     

The dynamics of genome replication using deep sequencing

Eukaryotic genomes are replicated from multiple DNA replication origins. We present complementary deep sequencing approaches to measure origin location and activity in Saccharomyces cerevisiae. Measuring the increase in DNA copy number during a synchronous S-phase allowed the precise determination of genome replication. To map origin locations, replication forks were stalled close to their initiation sites; therefore, copy number enrichment was limited to origins. Replication timing profiles were generated from asynchronous cultures using fluorescence-activated cell sorting. Applying this technique we show that the replication profiles of haploid and diploid cells are indistinguishable, indicating that both cell types use the same cohort of origins with the same activities. Finally, increasing sequencing depth allowed the direct measure of replication dynamics from an exponentially growing culture. This is the first time this approach, called marker frequency analysis, has been successfully applied to a eukaryote. These data provide a high-resolution resource and methodological framework for studying genome biology.     

Cell Reproduction and Morphological Changes in Mycoplasma capricolum

The cell reproduction of Mycoplasma capricolum was studied. The velocity of DNA replication fork progression was about 6 kb/min, which is 10 times slower than that of Escherichia coli. The time required for one round of DNA replication accorded with the doubling time. The origin/terminus ratio was 2.0. M. capricolum cell morphology was classified into two types, rod and branched. In the ordinary-growth phase, the rod cells accounted for about 90% of the total population, with branched cells comprising the remaining 10%. The proportion of branched cells increased to 90% following inhibition of DNA replication by nucleoside starvation. An increase in the proportion of branched cells was induced by transfer of a temperature-sensitive mutant deficient in DNA replication to the restrictive temperature. The rod cells had a regular structure, a fixed cell length, and constrictions in the center. The DNA contents of individual rod cells were distributed with a standard deviation of 0.40 of average. The branched cells had irregular structures and a wide distribution of DNA contents. Counting of viable cells revealed that the cells ceased division upon cell type conversion; however, branched cells maintained a reproductive capacity. A model for the reproduction process is proposed.Mycoplasmas are parasitic bacteria that have extremely low G+C contents and small genomes (9). Their morphology is irregular because of the lack of a peptidoglycan layer.In Escherichia coli, initiation of chromosomal DNA replication occurs once during the cell’s replicative cycle, and the nucleoids partition before cell division (13). The chromosomal replication of E. coli initiates in a small region and proceeds in both directions. It is mainly controlled by the timing and frequency of initiation, while the velocity of replication is constant.In mycoplasmas, chromosome replication also starts at a fixed site, followed by bidirectional progression (19–21, 25, 40). As in many eubacteria (36), the dnaA gene is expressed and plays important roles in the initiation of replication (35). These observations suggest that the outline of chromosome replication of mycoplasmas is similar to that of E. coli. However, the process of mycoplasma cell reproduction has not been clarified. Moreover, the cell division cycle of E. coli cannot be simply applied to mycoplasmas because of their irregular cell morphology (4). A model has been suggested for the cell cycle of Mycoplasma mycoides (6, 30, 31), which is closely related to Mycoplasma capricolum (39). According to this model, an elementary rounded body grows into a filamentous form and then new elementary rounded bodies are developed within this filament and released, but this model has not been adequately substantiated.In this study, we analyzed the process of DNA replication, cell morphology, and viability under various conditions of M. capricolum and proposed a model of cellular reproduction for this bacterium.     

Selection and timing of replication of plasmids R1drd-19 and F'lac in Escherichia coli.

The selection and timing of plasmid replication was studied in exponentially growing cultures of Escherichia coli K-12 carrying the plasmid R1drd-19 and E. coli strains B/r A and B/r F carrying the plasmid F′lac. In all cases plasmid replication was studied by analysis of covalently closed circular (CCC) DNA. The turnover time of replicating plasmid DNA into CCC-DNA was found to be less than 4 min. Density shift experiments (from ¹⁵NH₄⁺, D₂O to ¹⁴NH₄⁺, H₂O) showed that plasmids R1drd-19 and F′lac are selected randomly for replication. This means that one of the plasmid copies in a cell is selected and replicated. There is no further plasmid replication in the cell until all plasmid copies, including the newly formed ones, have the same probability of being selected for replication. The early kinetics of the appearance of light plasmid DNA after the density shift showed that the time interval between successive replications of plasmids R1drd-19 and F′lac is $\text{τ}\text{n}$, where τ is the generation time and n is the average number of plasmid replications per cell and cell cycle. In a second type of experiment, exponentially growing cells were separated into a series of size classes by low-speed centrifugation in sucrose step gradients. Replication of plasmids R1drd-19 and F′lac was equally frequent in all size classes. This result is in accordance with the results of the density shift experiment. It can therefore be concluded that replication of plasmids R1drd-19 and F′lac is evenly spread over the whole cell cycle, which means that one plasmid replication occurs every time the cell volume has increased by one initiation mass.