Multiple Regulatory Systems Coordinate DNA Replication with Cell Growth in Bacillus subtilis

In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s) that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes.     

Cell cycle parameters of slowly growing Escherichia coli B/r studied by flow cytometry

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.     

The DnaA protein determines the initiation mass of Escherichia coli K-12

DNA replication was studied in a dnaA(Ts) strain containing a plasmid with the dnaA+ gene under plac control. At 42 degrees C, initiation of DNA replication was totally dependent upon the gratuitous inducer isopropyl beta-D-thiogalactopyranoside (IPTG). Flow cytometric measurements showed that at 13% induction of the lac promoter the growth rate, cell size, DNA content, and timing of initiation of DNA replication were indistinguishable from those observed in a wild-type control cell. Higher levels of induction resulted in initiations earlier in the cell cycle and a corresponding increase in the time from initiation to termination. We conclude that the concentration of DnaA protein determines the time of initiation and thereby the initiation mass. With an induction level equal to or above 13%, the synchrony of multiple initiations within one cell was close to that found in a wild-type control cell, showing that a cyclic variation in DnaA content is not necessary for a high degree of synchrony.     

Regulation of the replication initiator DnaA in Caulobacter crescentus

The decision to initiate DNA replication is a critical step in the cell cycle of all organisms. In nearly all bacteria, replication initiation requires the activity of the conserved replication initiation protein DnaA. Due to its central role in cell cycle progression, DnaA activity must be precisely regulated. This review summarizes the current state of DnaA regulation in the asymmetrically dividing α-proteobacterium Caulobacter crescentus, an important model for bacterial cell cycle studies. Mechanisms will be discussed that regulate DnaA activity and abundance under optimal conditions and in coordination with the asymmetric Caulobacter cell cycle. Furthermore, we highlight recent findings of how regulated DnaA synthesis and degradation collaborate to adjust DnaA abundance under stress conditions. The mechanisms described provide important examples of how DNA replication is regulated in an α-proteobacterium and thus represent an important starting point for the study of DNA replication in many other bacteria. This article is part of a Special Issue entitled: Dynamic gene expression, edited by Prof. Patrick Viollier.     

Timely binding of IHF and Fis to DARS2 regulates ATP–DnaA production and replication initiation

In Escherichia coli, the ATP-bound form of DnaA (ATP–DnaA) promotes replication initiation. During replication, the bound ATP is hydrolyzed to ADP to yield the ADP-bound form (ADP–DnaA), which is inactive for initiation. The chromosomal site DARS2 facilitates the regeneration of ATP–DnaA by catalyzing nucleotide exchange between free ATP and ADP bound to DnaA. However, the regulatory mechanisms governing this exchange reaction are unclear. Here, using in vitro reconstituted experiments, we show that two nucleoid-associated proteins, IHF and Fis, bind site-specifically to DARS2 to activate coordinately the exchange reaction. The regenerated ATP–DnaA was fully active in replication initiation and underwent DnaA–ATP hydrolysis. ADP–DnaA formed heteromultimeric complexes with IHF and Fis on DARS2, and underwent nucleotide dissociation more efficiently than ATP–DnaA. Consistently, mutant analyses demonstrated that specific binding of IHF and Fis to DARS2 stimulates the formation of ATP–DnaA production, thereby promoting timely initiation. Moreover, we show that IHF–DARS2 binding is temporally regulated during the cell cycle, whereas Fis only binds to DARS2 in exponentially growing cells. These results elucidate the regulation of ATP–DnaA and replication initiation in coordination with the cell cycle and growth phase.     

Cell size and the initiation of DNA replication in bacteria

In eukaryotes, DNA replication is coupled to the cell cycle through the actions of cyclin-dependent kinases and associated factors. In bacteria, the prevailing view, based primarily from work in Escherichia coli, is that growth-dependent accumulation of the highly conserved initiator, DnaA, triggers initiation. However, the timing of initiation is unchanged in Bacillus subtilis mutants that are ~30% smaller than wild-type cells, indicating that achievement of a particular cell size is not obligatory for initiation. Prompted by this finding, we re-examined the link between cell size and initiation in both E. coli and B. subtilis. Although changes in DNA replication have been shown to alter both E. coli and B. subtilis cell size, the converse (the effect of cell size on DNA replication) has not been explored. Here, we report that the mechanisms responsible for coordinating DNA replication with cell size vary between these two model organisms. In contrast to B. subtilis, small E. coli mutants delayed replication initiation until they achieved the size at which wild-type cells initiate. Modest increases in DnaA alleviated the delay, supporting the view that growth-dependent accumulation of DnaA is the trigger for replication initiation in E. coli. Significantly, although small E. coli and B. subtilis cells both maintained wild-type concentration of DnaA, only the E. coli mutants failed to initiate on time. Thus, rather than the concentration, the total amount of DnaA appears to be more important for initiation timing in E. coli. The difference in behavior of the two bacteria appears to lie in the mechanisms that control the activity of DnaA.     

Cell cycle regulation and the timing of chromosome replication in a marine synechococcus (cyanobacteria) during light- and nitrogen-limited growth

Cell cycle behavior in the marine Synechococcus strain WH8101 was examined in detail over a wide range of light- and nitrogen-limited growth rates. The presence of bimodal DNA frequency distributions under all conditions confirms that the overlapping rounds of DNA replication that characterize E. coli and other fast-growing prokaryotes are not present in this organism. Although chromosome replication time, C , was constrained to a fairly narrow range of values overall, it nevertheless did vary with growth rate and limiting factor. Light-limited cells growing at moderate rates had higher C values than did N-limited cells growing at comparable rates (by as much as a factor of 2). As these cells became light saturated, however, C decreased sharply to the level observed under N limitation. The post-replication period, D , decreased monotonically with growth rate under both light and N limitation, approaching a constant value at moderate to high growth rates. Average cell volume at the time of initiation of DNA replication was calculated from the values of C and D , combined with directly measured mean cell volume, and was found to be constant at all growth rates above ∼0.7 d⁻¹. This pattern was confirmed by estimates of initiation volume based on flow cytometric light scatter measurements, and suggests that as has been found in other prokaryotic systems, cell mass may play an important role in regulating the timing of chromosome replication in cyanobacteria. Furthermore, because the magnitude of C + D influences average cell mass (given a constant mass at initiation), changes in these parameters (particularly C ) may be responsible for the previously reported nonlinear relationship between light-limited growth rate and both RNA cell⁻¹ and average cell volume.     

Coupling the initiation of chromosome replication to cell size in Escherichia coli

Bacterial cells change size dramatically with change in growth rate, but the ratio between cell volume and the number of copies of the origin of chromosome replication (oriC) is roughly constant at the time of initiation of DNA replication at almost all growth rates. Recent research on the inactivation of initiator protein (DnaA) and depletion of DnaA pools by the high-affinity DnaA-binding locus datA allows us to propose a simple model to explain the long-standing question of how Escherichia coli couples DNA replication to cell size.     

The Escherichia coli SeqA protein destabilizes mutant DnaA204 protein

In wild-type Escherichia coli cells, initiation of DNA replication is tightly coupled to cell growth. In slowly growing dnaA204 (Ts) mutant cells, the cell mass at initiation and its variability is increased two- to threefold relative to wild type. Here, we show that the DnaA protein concentration was two- to threefold lower in the dnaA204 mutant compared with the wild-type strain. The reason for the DnaA protein deficiency was found to be a rapid degradation of the mutant protein. Absence of SeqA protein stabilized the DnaA204 protein, increased the DnaA protein concentration and normalized the initiation mass in the dnaA204 mutant cells. During rapid growth, the dnaA204 mutant displayed cell cycle parameters similar to wild-type cells as well as a normal DnaA protein concentration, even though the DnaA204 protein was highly unstable. Apparently, the increased DnaA protein synthesis compensated for the protein degradation under these growth conditions, in which the doubling time was of the same order of magnitude as the half-life of the protein. Our results suggest that the DnaA204 protein has essentially wild-type activity at permissive temperature but, as a result of instability, the protein is present at lower concentration under certain growth conditions. The basis for the stabilization in the absence of SeqA is not known. We suggest that the formation of stable DnaA-DNA complexes is enhanced in the absence of SeqA, thereby protecting the DnaA protein from degradation.     

Deletion of the datA site does not affect once-per-cell-cycle timing but induces rifampin-resistant replication

In Escherichia coli, three mechanisms have been proposed to maintain proper regulation of replication so that initiation occurs once, and only once, per cell cycle. First, newly formed origins are inactivated by sequestration; second, the initiator, DnaA, is inactivated by the Hda protein at active replication forks; and third, the level of free DnaA protein is reduced by replication of the datA site. The datA site titrates unusually large amounts of DnaA and it has been reported that reinitiation, and thus asynchrony of replication, occurs in cells lacking this site. Here, we show that reinitiation in ΔdatA cells does not occur during exponential growth and that an apparent asynchrony phenotype results from the occurrence of rifampin-resistant initiations. This shows that the datA site is not required to prevent reinitiation and limit initiation of replication to once per generation. The datA site may, however, play a role in timing of initiation relative to cell growth. Inactivation of active ATP-DnaA by the Hda protein and the sliding clamp of the polymerase was found to be required to prevent reinitiation and asynchrony of replication.     

The DnaA Protein Is Not the Limiting Factor for Initiation of Replication in Escherichia coli

The bacterial replication cycle is driven by the DnaA protein which cycles between the active ATP-bound form and the inactive ADP-bound form. It has been suggested that DnaA also is the main controller of initiation frequency. Initiation is thought to occur when enough ATP-DnaA has accumulated. In this work we have performed cell cycle analysis of cells that contain a surplus of ATP-DnaA and asked whether initiation then occurs earlier. It does not. Cells with more than a 50% increase in the concentration of ATP-DnaA showed no changes in timing of replication. We suggest that although ATP-DnaA is the main actor in initiation of replication, its accumulation does not control the time of initiation. ATP-DnaA is the motor that drives the initiation process, but other factors will be required for the exact timing of initiation in response to the cell’s environment. We also investigated the in vivo roles of datA dependent DnaA inactivation (DDAH) and the DnaA-binding protein DiaA. Loss of DDAH affected the cell cycle machinery only during slow growth and made it sensitive to the concentration of DiaA protein. The result indicates that compromised cell cycle machines perform in a less robust manner.     

Effects of Chromosome Underreplication on Cell Division in Escherichia coli

The key processes of the bacterial cell cycle are controlled and coordinated to match cellular mass growth. We have studied the coordination between replication and cell division by using a temperature-controlled Escherichia coli intR1 strain. In this strain, the initiation time for chromosome replication can be displaced to later (underreplication) or earlier (overreplication) times in the cell cycle. We used underreplication conditions to study the response of cell division to a delayed initiation of replication. The bacteria were grown exponentially at 39°C (normal DNA/mass ratio) and shifted to 38 and 37°C. In the last two cases, new, stable, lower DNA/mass ratios were obtained. The rate of replication elongation was not affected under these conditions. At increasing degrees of underreplication, increasing proportions of the cells became elongated. Cell division took place in the middle in cells of normal size, whereas the longer cells divided at twice that size to produce one daughter cell of normal size and one three times as big. The elongated cells often produced one daughter cell lacking a chromosome; this was always the smallest daughter cells, and it was the size of a normal newborn cell. These results favor a model in which cell division takes place at only distinct cell sizes. Furthermore, the elongated cells had a lower probability of dividing than the cells of normal size, and they often contained more than two nucleoids. This suggests that for cell division to occur, not only must replication and nucleoid partitioning be completed, but also the DNA/mass ratio must be above a certain threshold value. Our data support the ideas that cell division has its own control system and that there is a checkpoint at which cell division may be abolished if previous key cell cycle processes have not run to completion.     

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.     

Bacillus subtilis Cell Cycle as Studied by Fluorescence Microscopy: Constancy of Cell Length at Initiation of DNA Replication and Evidence for Active Nucleoid Partitioning

Fluorescence microscopic methods have been used to characterize the cell cycle of Bacillus subtilis at four different growth rates. The data obtained have been used to derive models for cell cycle progression. Like that of Escherichia coli, the period required by B. subtilis for chromosome replication at 37°C was found to be fairly constant (although a little longer, at about 55 min), as was the cell mass at initiation of DNA replication. The cell cycle of B. subtilis differed from that of E. coli in that changes in growth rate affected the average cell length but not the width and also in the relative variability of period between termination of DNA replication and septation. Overall movement of the nucleoid was found to occur smoothly, as in E. coli, but other aspects of nucleoid behavior were consistent with an underlying active partitioning machinery. The models for cell cycle progression in B. subtilis should facilitate the interpretation of data obtained from the recently introduced cytological methods for imaging the assembly and movement of proteins involved in cell cycle dynamics.     

DnaA protein overproduction abolishes cell cycle specificity of DNA replication from oriC in Escherichia coli.

Initiation of DNA replication from oriC in Escherichia coli takes place at a specific time in the cell division cycle, whether the origin is located on a chromosome or a minichromosome, and requires participation of the product of the dnaA gene. The effects of overproduction of DnaA protein on the cell cycle specificity of the initiation event were determined by using minichromosome replication as the assay system. DnaA protein was overproduced by inducing the expression of plasmid-encoded dnaA genes under control of either the ptac or lambda pL promoter. Induction of DnaA protein synthesis caused a burst of minichromosome replication in cells at all ages in the division cycle. The magnitude of the burst was consistent with the initiation of one round of replication per minichromosome in all cells. The replication burst was followed by a period of reduced minichromosome replication, with the reduction being greater at 30 than at 41 degrees C. The results support the idea that the DnaA protein participates in oriC replication at a stage that is limiting for initiation. Excess DnaA protein enabled all cells to achieve the state required for initiation of DNA polymerization by either effecting or overriding the normal limiting process.     

Effects of (p)ppGpp on the Progression of the Cell Cycle of Caulobacter crescentus

Bacteria must control the progression of their cell cycle in response to nutrient availability. This regulation can be mediated by guanosine tetra- or pentaphosphate [(p)ppGpp], which are synthesized by enzymes of the RelA/SpoT homologue (Rsh) family, particularly under starvation conditions. Here, we study the effects of (p)ppGpp on the cell cycle of Caulobacter crescentus, an oligotrophic bacterium with a dimorphic life cycle. C. crescentus divides asymmetrically, producing a motile swarmer cell that cannot replicate its chromosome and a sessile stalked cell that is replication competent. The swarmer cell rapidly differentiates into a stalked cell in appropriate conditions. An artificial increase in the levels of (p)ppGpp in nonstarved C. crescentus cells was achieved by expressing a truncated relA gene from Escherichia coli, encoding a constitutively active (p)ppGpp synthetase. By combining single-cell microscopy, flow cytometry approaches, and swarming assays, we show that an increase in the intracellular concentration of (p)ppGpp is sufficient to slow down the swarmer-to-stalked cell differentiation process and to delay the initiation of chromosome replication. We also present evidence that the intracellular levels of two master regulators of the cell cycle of C. crescentus, DnaA and CtrA, are modulated in response to (p)ppGpp accumulation, even in the absence of actual starvation. CtrA proteolysis and DnaA synthesis seem indirectly inhibited by (p)ppGpp accumulation. By extending the life span of the motile nonreproductive swarmer cell and thus promoting dispersal and foraging functions over multiplication under starvation conditions, (p)ppGpp may play a central role in the ecological adaptation of C. crescentus to nutritional stresses.     

Comparison among patterns of macromolecular synthesis in Escherichia coli B/r at growth rates of less and more than one doubling per hour at 37 degrees C.

In Escherichia coli B/r, the relationship between the patterns of chromosome replication and of synthesis of envelope components differs at various growth rates. At growth rates greater than 1.0 doubling per h at 37 degrees C, the average mass and age at initiation of rounds of chromosome replication are similar to those at increase in incorporation of precursors into a major outer membrane protein and phosphatidylethanolamine. At growth rates less than 1.0 doubling per h at 37 degrees C the average mass and age at increase in the synthesis of these envelope components differ from those at initiation of chromosome replication. The average cell mass per chromosomal origin at initiation of rounds of chromosome replication is not a constant and varies between growth rates greater and less than 1.0 doubling per h.     

Linear Cell Growth in Escherichia coli

Growth was studied in synchronous cultures of Escherichia coli, using three strains and several rates of cell division. Synchrony was obtained by the Mitchison-Vincent technique. Controls gave no discernible perturbation in growth or rate of cell division. In all cases, mean cell volumes increased linearly (rather than exponentially) during the cycle except possibly for a small period near the end of the cycle. Linear volume growth occurred in synchronous cultures established from cells of different sizes, and also for the first volume doubling of cells prevented from division by a shift up to a more rapid growth rate. As a model for linear kinetics, it is suggested that linear growth represents constant uptake of all major nutrient factors during the cycle, and that constant uptake in turn is established by the presence of a constant number of functional binding or accumulation sites for each growth factor during linear growth of the cell.