DnaA Activity Research Articles

Dispensability of extrinsic DnaA regulators in Escherichia coli cell-cycle control

Investigating a long-standing conceptual question in bacterial physiology, we examine why DnaA, the bacterial master replication initiator protein, exists in both ATP and ADP forms, despite only the ATP form being essential for initiation. We engineered the Δ4 Escherichia coli strain, devoid of all known external elements facilitating the DnaA-ATP/ADP conversion and found that these cells display nearly wild-type behaviors under nonoverlapping replication cycles. However, during rapid growth with overlapping cycles, Δ4 cells exhibit initiation instability. This aligns with our model predictions, suggesting that the intrinsic ATPase activity of DnaA alone is sufficient for robust initiation control in E. coli and the DnaA-ATP/ADP conversion regulatory elements extend the robustness to multifork replication, indicating an evolutionary adaptation. Moreover, our experiments revealed constant DnaA concentrations during steady-state cell elongation in both wild-type and Δ4 cells. These insights not only advance our understanding of bacterial cell-cycle regulation and DnaA but also highlight a fundamental divergence from eukaryotic cell-cycle controls, emphasizing protein copy-number sensing in bacteria versus programmed protein concentration oscillations in eukaryotes.

Direct single-cell observation of a key Escherichia coli cell-cycle oscillator.

Initiation of DNA replication in Escherichia coli is coupled to cell size via the DnaA protein, whose activity is dependent on its nucleotide-bound state. However, the oscillations in DnaA activity have never been observed at the single-cell level. By measuring the volume-specific production rate of a reporter protein under control of a DnaA-regulated promoter, we could distinguish two distinct cell-cycle oscillators. The first, driven by both DnaA activity and SeqA repression, shows a causal relationship with cell size and divisions, similarly to initiation events. The second one, a reporter of DnaA activity alone, loses the synchrony and causality properties. Our results show that transient inhibition of gene expression by SeqA keeps the oscillation of volume-sensing DnaA activity in phase with the subsequent division event and suggest that DnaA activity peaks do not correspond directly to initiation events.

Bacillus subtilis remains translationally active after CRISPRi-mediated replication initiation arrest.

Initiation of bacterial DNA replication takes place at the origin of replication (oriC), a region characterized by the presence of multiple DnaA boxes that serve as the binding sites for the master initiator protein DnaA. This process is tightly controlled by modulation of the availability or activity of DnaA and oriC during development or stress conditions. Here, we aimed to uncover the physiological and molecular consequences of stopping replication in the model bacterium Bacillus subtilis. We successfully arrested replication in B. subtilis by employing a clustered regularly interspaced short palindromic repeats interference (CRISPRi) approach to specifically target the key DnaA boxes 6 and 7, preventing DnaA binding to oriC. In this way, other functions of DnaA, such as a transcriptional regulator, were not significantly affected. When replication initiation was halted by this specific artificial and early blockage, we observed that non-replicating cells continued translation and cell growth, and the initial replication arrest did not induce global stress conditions such as the SOS response.IMPORTANCEAlthough bacteria constantly replicate under laboratory conditions, natural environments expose them to various stresses such as lack of nutrients, high salinity, and pH changes, which can trigger non-replicating states. These states can enable bacteria to (i) become tolerant to antibiotics (persisters), (ii) remain inactive in specific niches for an extended period (dormancy), and (iii) adjust to hostile environments. Non-replicating states have also been studied because of the possibility of repurposing energy for the production of additional metabolites or proteins. Using clustered regularly interspaced short palindromic repeats interference (CRISPRi) targeting bacterial replication initiation sequences, we were able to successfully control replication initiation in Bacillus subtilis. This precise approach makes it possible to study non-replicating phenotypes, contributing to a better understanding of bacterial adaptive strategies.

Linking DNA replication to the Z ring.

To grow and divide properly, bacteria need to coordinate DNA replication with cell division. A recent study identifies CcrZ as a protein from Streptococcus pneumoniae that links these two processes by interacting with FtsZ, the orchestrator of cell division, and modulating the activity of DnaA, the initiator of DNA replication.

CcrZ is a pneumococcal spatiotemporal cell cycle regulator that interacts with FtsZ and controls DNA replication by modulating the activity of DnaA

Most bacteria replicate and segregate their DNA concomitantly while growing, before cell division takes place. How bacteria synchronize these different cell cycle events to ensure faithful chromosome inheritance by daughter cells is poorly understood. Here, we identify Cell Cycle Regulator protein interacting with FtsZ (CcrZ) as a conserved and essential protein in pneumococci and related Firmicutes such as Bacillus subtilis and Staphylococcus aureus. CcrZ couples cell division with DNA replication by controlling the activity of the master initiator of DNA replication, DnaA. The absence of CcrZ causes mis-timed and reduced initiation of DNA replication, which subsequently results in aberrant cell division. We show that CcrZ from Streptococcus pneumoniae interacts directly with the cytoskeleton protein FtsZ, which places CcrZ in the middle of the newborn cell where the DnaA-bound origin is positioned. This work uncovers a mechanism for control of the bacterial cell cycle in which CcrZ controls DnaA activity to ensure that the chromosome is replicated at the right time during the cell cycle.

The DnaA Cycle in Escherichia coli: Activation, Function and Inactivation of the Initiator Protein.

This review summarizes the mechanisms of the initiator protein DnaA in replication initiation and its regulation in Escherichia coli. The chromosomal origin (oriC) DNA is unwound by the replication initiation complex to allow loading of DnaB helicases and replisome formation. The initiation complex consists of the DnaA protein, DnaA-initiator-associating protein DiaA, integration host factor (IHF), and oriC, which contains a duplex-unwinding element (DUE) and a DnaA-oligomerization region (DOR) containing DnaA-binding sites (DnaA boxes) and a single IHF-binding site that induces sharp DNA bending. DiaA binds to DnaA and stimulates DnaA assembly at the DOR. DnaA binds tightly to ATP and ADP. ATP-DnaA constructs functionally different sub-complexes at DOR, and the DUE-proximal DnaA sub-complex contains IHF and promotes DUE unwinding. The first part of this review presents the structures and mechanisms of oriC-DnaA complexes involved in the regulation of replication initiation. During the cell cycle, the level of ATP-DnaA level, the active form for initiation, is strictly regulated by multiple systems, resulting in timely replication initiation. After initiation, regulatory inactivation of DnaA (RIDA) intervenes to reduce ATP-DnaA level by hydrolyzing the DnaA-bound ATP to ADP to yield ADP-DnaA, the inactive form. RIDA involves the binding of the DNA polymerase clamp on newly synthesized DNA to the DnaA-inactivator Hda protein. In datA-dependent DnaA-ATP hydrolysis (DDAH), binding of IHF at the chromosomal locus datA, which contains a cluster of DnaA boxes, results in further hydrolysis of DnaA-bound ATP. SeqA protein inhibits untimely initiation at oriC by binding to newly synthesized oriC DNA and represses dnaA transcription in a cell cycle dependent manner. To reinitiate DNA replication, ADP-DnaA forms oligomers at DnaA-reactivating sequences (DARS1 and DARS2), resulting in the dissociation of ADP and the release of nucleotide-free apo-DnaA, which then binds ATP to regenerate ATP-DnaA. In vivo, DARS2 plays an important role in this process and its activation is regulated by timely binding of IHF to DARS2 in the cell cycle. Chromosomal locations of DARS sites are optimized for the strict regulation for timely replication initiation. The last part of this review describes how DDAH and DARS regulate DnaA activity.

Countermeasures to survive excessive chromosome replication in Escherichia coli.

In Escherichia coli, like all organisms, DNA replication is coordinated with cell cycle progression to ensure duplication of the genome prior to cell division. Chromosome replication is initiated from the replication origin, oriC, by the DnaA protein associated with ATP. Initiations take place once per cell cycle and in synchrony at all cellular origins. DnaA also binds ADP with similar affinity as ATP and in wild-type cells the majority of DnaA molecules are ADP bound. In cells where the DnaAATP/DnaAADP ratio increases or in cells where DnaAATP has increased access to oriC, premature initiations take place, often referred to as overinitiation. Overinitiating cells are generally characterized by their slow growth and in the most severe cases lethal accumulation of DNA strand breaks. Here, we review the different strategies adopted by E. coli to survive overinitiation. We propose a unifying model where all mutations that suppress overinitiation keep replication forks separated in time and, thereby, reduce the formation of strand breaks. One group of mutations does so by lowering the activity of oriC and/or DnaA to reduce the frequency of initiations to an acceptable level. In the other group of mutations, replication forks are kept apart by preventing formation of damages that would otherwise cause replication blocks, by allowing bypass of replication blocks and/or by slowing down replication forks. This group of suppressors restores viability despite excessive chromosome replication and provides new insights into mechanisms that safeguard DNA integrity.

The Role of the N-Terminal Domains of Bacterial Initiator DnaA in the Assembly and Regulation of the Bacterial Replication Initiation Complex.

The primary role of the bacterial protein DnaA is to initiate chromosomal replication. The DnaA protein binds to DNA at the origin of chromosomal replication (oriC) and assembles into a filament that unwinds double-stranded DNA. Through interaction with various other proteins, DnaA also controls the frequency and/or timing of chromosomal replication at the initiation step. Escherichia coli DnaA also recruits DnaB helicase, which is present in unwound single-stranded DNA and in turn recruits other protein machinery for replication. Additionally, DnaA regulates the expression of certain genes in E. coli and a few other species. Acting as a multifunctional factor, DnaA is composed of four domains that have distinct, mutually dependent roles. For example, C-terminal domain IV interacts with double-stranded DnaA boxes. Domain III drives ATP-dependent oligomerization, allowing the protein to form a filament that unwinds DNA and subsequently binds to and stabilizes single-stranded DNA in the initial replication bubble; this domain also interacts with multiple proteins that control oligomerization. Domain II constitutes a flexible linker between C-terminal domains III–IV and N-terminal domain I, which mediates intermolecular interactions between DnaA and binds to other proteins that affect DnaA activity and/or formation of the initiation complex. Of these four domains, the role of the N-terminus (domains I–II) in the assembly of the initiation complex is the least understood and appears to be the most species-dependent region of the protein. Thus, in this review, we focus on the function of the N-terminus of DnaA in orisome formation and the regulation of its activity in the initiation complex in different bacteria.

Insufficient levels of the nrdAB-encoded ribonucleotide reductase underlie the severe growth defect of the Δhda E. coli strain.

The ATP-bound form of the Escherichia coli DnaA replication initiator protein remodels the chromosomal origin of replication, oriC, to load the replicative helicase. The primary mechanism for regulating the activity of DnaA involves the Hda and β clamp proteins, which act together to dramatically stimulate the intrinsic DNA-dependent ATPase activity of DnaA via a process termed Regulatory Inactivation of DnaA. In addition to hyperinitiation, strains lacking hda function also exhibit cold sensitive growth at 30°C. Strains impaired for the other regulators of initiation (i.e., ΔseqA or ΔdatA) fail to exhibit cold sensitivity. The goal of this study was to gain insight into why loss of hda function impedes growth. We used a genetic approach to isolate 9 suppressors of Δhda cold sensitivity, and characterized the mechanistic basis by which these suppressors alleviated Δhda cold sensitivity. Taken together, our results provide strong support for the view that the fundamental defect associated with Δhda is diminished levels of DNA precursors, particularly dGTP and dATP. We discuss possible mechanisms by which the suppressors identified here may regulate dNTP pool size, as well as similarities in phenotypes between the Δhda strain and hda+ strains exposed to the ribonucleotide reductase inhibitor hydroxyurea.

ClpAP is an auxiliary protease for DnaA degradation in Caulobacter crescentus.

The Clp family of proteases is responsible for controlling both stress responses and normal growth. In Caulobacter crescentus, the ClpXP protease is essential and drives cell cycle progression through adaptor-mediated degradation. By contrast, the physiological role for the ClpAP protease is less well understood with only minor growth defects previously reported for ΔclpA cells. Here, we show that ClpAP plays an important role in controlling chromosome content and cell fitness during extended growth. Cells lacking ClpA accumulate aberrant numbers of chromosomes upon prolonged growth suggesting a defect in replication control. Levels of the replication initiator DnaA are elevated in ΔclpA cells and degradation of DnaA is more rapid in cells lacking the ClpA inhibitor ClpS. Consistent with this observation, ClpAP degrades DnaA in vitro while ClpS inhibits this degradation. In cells lacking Lon, the protease previously shown to degrade DnaA in Caulobacter, ClpA overexpression rescues defects in fitness and restores degradation of DnaA. Finally, we show that cells lacking ClpA are particularly sensitive to inappropriate increases in DnaA activity. Our work demonstrates an unexpected effect of ClpAP in directly regulating replication through degradation of DnaA and expands the functional role of ClpAP in Caulobacter.

Multiple DNA Binding Proteins Contribute to Timing of Chromosome Replication in E. coli.

Chromosome replication in Escherichia coli is initiated from a single origin, oriC. Initiation involves a number of DNA binding proteins, but only DnaA is essential and specific for the initiation process. DnaA is an AAA+ protein that binds both ATP and ADP with similar high affinities. DnaA associated with either ATP or ADP binds to a set of strong DnaA binding sites in oriC, whereas only DnaAATP is capable of binding additional and weaker sites to promote initiation. Additional DNA binding proteins act to ensure that initiation occurs timely by affecting either the cellular mass at which DNA replication is initiated, or the time window in which all origins present in a single cell are initiated, i.e. initiation synchrony, or both. Overall, these DNA binding proteins modulate the initiation frequency from oriC by: (i) binding directly to oriC to affect DnaA binding, (ii) altering the DNA topology in or around oriC, (iii) altering the nucleotide bound status of DnaA by interacting with non-coding chromosomal sequences, distant from oriC, that are important for DnaA activity. Thus, although DnaA is the key protein for initiation of replication, other DNA-binding proteins act not only on oriC for modulation of its activity but also at additional regulatory sites to control the nucleotide bound status of DnaA. Here we review the contribution of key DNA binding proteins to the tight regulation of chromosome replication in E. coli cells.

Spatiotemporal choreography of chromosome and megaplasmids in the Sinorhizobium meliloti cell cycle.

A considerable share of bacterial species maintains multipartite genomes. Precise coordination of genome replication and segregation with cell growth and division is vital for proliferation of these bacteria. The α-proteobacterium Sinorhizobium meliloti possesses a tripartite genome composed of one chromosome and the megaplasmids pSymA and pSymB. Here, we investigated the spatiotemporal pattern of segregation of these S. meliloti replicons at single cell level. Duplication of chromosomal and megaplasmid origins of replication occurred spatially and temporally separated, and only once per cell cycle. Tracking of FROS (fluorescent repressor operator system)-labelled origins revealed a strict temporal order of segregation events commencing with the chromosome followed by pSymA and then by pSymB. The repA2B2C2 region derived from pSymA was sufficient to confer the spatiotemporal behaviour of this megaplasmid to a small plasmid. Altering activity of the ubiquitous prokaryotic replication initiator DnaA, either positively or negatively, resulted in an increase in replication initiation events or G1 arrest of the chromosome only. This suggests that interference with DnaA activity does not affect replication initiation control of the megaplasmids.

Control regions for chromosome replication are conserved with respect to sequence and location among Escherichia coli strains.

In Escherichia coli, chromosome replication is initiated from oriC by the DnaA initiator protein associated with ATP. Three non-coding regions contribute to the activity of DnaA. The datA locus is instrumental in conversion of DnaAATP to DnaAADP (datA dependent DnaAATP hydrolysis) whereas DnaA rejuvenation sequences 1 and 2 (DARS1 and DARS2) reactivate DnaAADP to DnaAATP. The structural organization of oriC, datA, DARS1, and DARS2 were found conserved among 59 fully sequenced E. coli genomes, with differences primarily in the non-functional spacer regions between key protein binding sites. The relative distances from oriC to datA, DARS1, and DARS2, respectively, was also conserved despite of large variations in genome size, suggesting that the gene dosage of either region is important for bacterial growth. Yet all three regions could be deleted alone or in combination without loss of viability. Competition experiments during balanced growth in rich medium and during mouse colonization indicated roles of datA, DARS1, and DARS2 for bacterial fitness although the relative contribution of each region differed between growth conditions. We suggest that this fitness advantage has contributed to conservation of both sequence and chromosomal location for datA, DARS1, and DARS2.

The DnaA N‐terminal domain interacts with Hda to facilitate replicase clamp‐mediated inactivation of DnaA

DnaA activity for replication initiation of the Escherichia coli chromosome is negatively regulated by feedback from the DNA-loaded form of the replicase clamp. In this process, called RIDA (regulatory inactivation of DnaA), ATP-bound DnaA transiently assembles into a complex consisting of Hda and the DNA-clamp, which promotes inter-AAA+ domain association between Hda and DnaA and stimulates hydrolysis of DnaA-bound ATP, producing inactive ADP-DnaA. Using a truncated DnaA mutant, we previously demonstrated that the DnaA N-terminal domain is involved in RIDA. However, the precise role of the N-terminal domain in RIDA has remained largely unclear. Here, we used an in vitro reconstituted system to demonstrate that the Asn-44 residue in the N-terminal domain of DnaA is crucial for RIDA but not for replication initiation. Moreover, an assay termed PDAX (pull-down after cross-linking) revealed an unstable interaction between a DnaA-N44A mutant and Hda. In vivo, this mutant exhibited an increase in the cellular level of ATP-bound DnaA. These results establish a model in which interaction between DnaA Asn-44 and Hda stabilizes the association between the AAA+ domains of DnaA and Hda to facilitate DnaA-ATP hydrolysis during RIDA.

Regulating DNA Replication in Bacteria

The replication origin and the initiator protein DnaA are the main targets for regulation of chromosome replication in bacteria. The origin bears multiple DnaA binding sites, while DnaA contains ATP/ADP-binding and DNA-binding domains. When enough ATP-DnaA has accumulated in the cell, an active initiation complex can be formed at the origin resulting in strand opening and recruitment of the replicative helicase. In Escherichia coli, oriC activity is directly regulated by DNA methylation and specific oriC-binding proteins. DnaA activity is regulated by proteins that stimulate ATP-DnaA hydrolysis, yielding inactive ADP-DnaA in a replication-coupled negative-feedback manner, and by DnaA-binding DNA elements that control the subcellular localization of DnaA or stimulate the ADP-to-ATP exchange of the DnaA-bound nucleotide. Regulation of dnaA gene expression is also important for initiation. The principle of replication-coupled negative regulation of DnaA found in E. coli is conserved in eukaryotes as well as in bacteria. Regulations by oriC-binding proteins and dnaA gene expression are also conserved in bacteria.

The Primosomal Protein DnaD Inhibits Cooperative DNA Binding by the Replication Initiator DnaA in Bacillus subtilis

DnaA is an AAA+ ATPase and the conserved replication initiator in bacteria. Bacteria control the timing of replication initiation by regulating the activity of DnaA. DnaA binds to multiple sites in the origin of replication (oriC) and is required for recruitment of proteins needed to load the replicative helicase. DnaA also binds to other chromosomal regions and functions as a transcription factor at some of these sites. Bacillus subtilis DnaD is needed during replication initiation for assembly of the replicative helicase at oriC and during replication restart at stalled replication forks. DnaD associates with DnaA at oriC and at other chromosomal regions bound by DnaA. Using purified proteins, we found that DnaD inhibited the ability of DnaA to bind cooperatively to DNA and caused a decrease in the apparent dissociation constant. These effects of DnaD were independent of the ability of DnaA to bind or hydrolyze ATP. Other proteins known to regulate B. subtilis DnaA also affect DNA binding, whereas much of the regulation of Escherichia coli DnaA affects nucleotide hydrolysis or exchange. We found that the rate of nucleotide exchange for B. subtilis DnaA was high and not affected by DnaD. The rapid exchange is similar to that of Staphylococcus aureus DnaA and in contrast to the low exchange rate of Escherichia coli DnaA. We suggest that organisms in which DnaA has a high rate of nucleotide exchange predominantly regulate the DNA binding activity of DnaA and that those with low rates of exchange regulate hydrolysis and exchange.

Evidence for roles of the Escherichia coli Hda protein beyond regulatory inactivation of DnaA

The ATP-bound form of the Escherichia coli DnaA protein binds 'DnaA boxes' present in the origin of replication (oriC) and operator sites of several genes, including dnaA, to co-ordinate their transcription with initiation of replication. The Hda protein, together with the β sliding clamp, stimulates the ATPase activity of DnaA via a process termed regulatory inactivation of DnaA (RIDA), to regulate the activity of DnaA in DNA replication. Here, we used the mutant dnaN159 strain, which expresses the β159 clamp protein, to gain insight into how the actions of Hda are co-ordinated with replication. Elevated expression of Hda impeded growth of the dnaN159 strain in a Pol II- and Pol IV-dependent manner, suggesting a role for Hda managing the actions of these Pols. In a wild-type strain, elevated levels of Hda conferred sensitivity to nitrofurazone, and suppressed the frequency of -1 frameshift mutations characteristic of Pol IV, while loss of hda conferred cold sensitivity. Using the dnaN159 strain, we identified 24 novel hda alleles, four of which supported E. coli viability despite their RIDA defect. Taken together, these findings suggest that although one or more Hda functions are essential for cell viability, RIDA may be dispensable.

Cell Size Control in Bacteria

Like eukaryotes, bacteria must coordinate division with growth to ensure cells are the appropriate size for a given environmental condition or developmental fate. As single-celled organisms, nutrient availability is one of the strongest influences on bacterial cell size. Classic physiological experiments conducted over four decades ago first demonstrated that cell size is directly correlated with nutrient source and growth rate in the Gram-negative bacterium Salmonella typhimurium. This observation subsequently served as the basis for studies revealing a role for cell size in cell cycle progression in a closely related organism, Escherichia coli. More recently, the development of powerful genetic, molecular, and imaging tools has allowed us to identify and characterize the nutrient-dependent pathway responsible for coordinating cell division and cell size with growth rate in the Gram-positive model organism Bacillus subtilis. Here, we discuss the role of cell size in bacterial growth and development and propose a broadly applicable model for cell size control in this important and highly divergent domain of life.

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.

DnaA and the timing of chromosome replication in Es-cherichia coli as a function of growth rate

BackgroundIn Escherichia coli, overlapping rounds of DNA replication allow the bacteria to double in faster times than the time required to copy the genome. The precise timing of initiation of DNA replication is determined by a regulatory circuit that depends on the binding of a critical number of ATP-bound DnaA proteins at the origin of replication, resulting in the melting of the DNA and the assembly of the replication complex. The synthesis of DnaA in the cell is controlled by a growth-rate dependent, negatively autoregulated gene found near the origin of replication. Both the regulatory and initiation activity of DnaA depend on its nucleotide bound state and its availability.ResultsIn order to investigate the contributions of the different regulatory processes to the timing of initiation of DNA replication at varying growth rates, we formulate a minimal quantitative model of the initiator circuit that includes the key ingredients known to regulate the activity of the DnaA protein. This model describes the average-cell oscillations in DnaA-ATP/DNA during the cell cycle, for varying growth rates. We evaluate the conditions under which this ratio attains the same threshold value at the time of initiation, independently of the growth rate.ConclusionsWe find that a quantitative description of replication initiation by DnaA must rely on the dependency of the basic parameters on growth rate, in order to account for the timing of initiation of DNA replication at different cell doubling times. We isolate two main possible scenarios for this, depending on the roles of DnaA autoregulation and DnaA ATP-hydrolysis regulatory process. One possibility is that the basal rate of regulatory inactivation by ATP hydrolysis must vary with growth rate. Alternatively, some parameters defining promoter activity need to be a function of the growth rate. In either case, the basal rate of gene expression needs to increase with the growth rate, in accordance with the known characteristics of the dnaA promoter. Furthermore, both inactivation and autorepression reduce the amplitude of the cell-cycle oscillations of DnaA-ATP/DNA.