Anne Olliver

Chiara Saggioro

John Herrick

Bianca Sclavi

John Herrick #

molecular switch to control levels of ribonucleotide reductase expression in Escherichia coli

Keywords: ribonucleotide reductase, DnaA, transcription regulation, cell cycle, DNA replication Transcription regulation DNA replication DnaA

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DnaA-ATP acts as a molecular switch to control levels of ribonucleotide reductase expression in Escherichia coli.

Introduction

Ribonucleotide reductase (RNR) is an essential enzyme for the growth and reproduction of all DNA-dependent living organisms [START_REF] Nordlund | Ribonucleotide reductases[END_REF]. This enzyme is responsible for the last step in the synthesis of the deoxynucleotides (dNTPs) necessary for DNA replication. Imbalances in the amounts of dNTPs in the cell cause an increase in the rate of mutagenesis [START_REF] Stubbe | Ribonucleotide reductases: the link between an RNA and a DNA world?[END_REF][START_REF] Gon | A novel regulatory mechanism couples deoxyribonucleotide synthesis and DNA replication in Escherichia coli[END_REF][START_REF] Wheeler | Stimulation of mutagenesis by proportional deoxyribonucleoside triphosphate accumulation in Escherichia coli[END_REF][START_REF] Mathews | DNA precursor metabolism and genomic stability[END_REF] and double strand breaks [START_REF] Guarino | Double-strand break generation under deoxyribonucleotide starvation in Escherichia coli[END_REF], therefore the activity and the expression of the RNR enzyme must be tightly regulated as a function of the cell's replicative state. The activity of the enzyme is influenced by negative feedback by dNTPs, by the [START_REF] Nordlund | Ribonucleotide reductases[END_REF]. In Escherichia coli (E. coli) the concentration of the enzyme in the cell is mainly regulated at the level of transcription whereas in yeast and higher eukaryotes it is also regulated by proteolysis (reviewed in [START_REF] Herrick | Ribonucleotide reductase and the regulation of DNA replication: an old story and an ancient heritage[END_REF]) and compartmentalization [START_REF] Lee | Dif1 is a DNA-damage-regulated facilitator of nuclear import for ribonucleotide reductase[END_REF].

In E. coli, the principal aerobic RNR enzyme is encoded by the nrdAB operon. The work of James Fuchs and coworkers has characterized several aspects of the control of its expression. Their work established that nrdAB expression peaks to coincide with initiation of DNA replication and is induced following fork arrest by DNA replication inhibitors or by thymine starvation [START_REF] Sun | Escherichia coli ribonucleotide reductase expression is cell cycle regulated[END_REF]. Recent evidence suggests that the expression of the enzyme is regulated by negative feedback [START_REF] Gon | A novel regulatory mechanism couples deoxyribonucleotide synthesis and DNA replication in Escherichia coli[END_REF], probably through the NrdR transcription factor that represses transcription when bound to dATP [START_REF] Rodionov | Identification of a bacterial regulatory system for ribonucleotide reductases by phylogenetic profiling[END_REF][START_REF] Torrents | NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes[END_REF][START_REF] Grinberg | Functional analysis of the Streptomyces coelicolor NrdR ATP-cone domain: role in nucleotide binding, oligomerization, and DNA interactions[END_REF].

The E. coli nrdAB operon is expressed from a complex promoter containing binding sites for several transcription factors including FIS, IciA and DnaA [START_REF] Han | Effect of IciA protein on the expression of the nrd gene encoding ribonucleoside diphosphate reductase in E. coli[END_REF][START_REF] Augustin | Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion[END_REF]. Moreover, an inverted repeat (IR45) and two AT-rich sequences are found upstream of the core promoter region; their presence has been shown to be important for the timing of expression as a function of initiation of DNA replication [START_REF] Jacobson | A 45 bp inverted repeat is required for cell cycle regulation of the Escherichia coli nrd operon[END_REF] (Figure 1).

In addition to being a transcription factor for several genes, the DnaA protein is best known for its key regulatory role in the initiation of DNA replication [START_REF] Kaguni | DnaA: Controlling the Initiation of Bacterial DNA Replication and More[END_REF]. Binding of DnaA to a set of high and low affinity sites found at the origin (oriC) results in DNA melting at the AT-rich region, thus allowing for assembly of the replication forks [START_REF] Speck | Mechanism of origin unwinding: sequential binding of DnaA to doubleand single-stranded DNA[END_REF][START_REF] Grimwade | Mutational analysis reveals Escherichia coli oriC interacts with both DnaA-ATP and DnaA-ADP during pre-RC assembly[END_REF]. The amount of DnaA in the cell has been shown to regulate both the timing and the synchrony of initiation at the multiple origins that may be present in the cell [START_REF] Skarstad | Timing of initiation of chromosome replication in individual Escherichia coli cells[END_REF]. The activity of DnaA is dependent on its nucleotide bound state [START_REF] Speck | ATP-and ADP-dnaA protein, a molecular switch in gene regulation[END_REF]. When DnaA is produced it is under the ATP-bound form.

DnaA-ATP, contrary to DnaA-ADP, binds with high affinity to its binding sites and is able to form oligomeric structures [START_REF] Sekimizu | ATP activates dnaA protein in initiating replication of plasmids bearing the origin of the E. coli chromosome[END_REF][START_REF] Simmons | DnaA Protein of Escherichia coli: oligomerization at the E. coli chromosomal origin is required for initiation and involves specific N-terminal amino acids[END_REF][START_REF] Erzberger | Structural basis for ATP-dependent DnaA assembly and replication-origin remodeling[END_REF]. The rate of ATP hydrolysis is significantly enhanced by the formation of a complex with the Hda protein and the beta clamp during ongoing DNA replication [START_REF] Katayama | The initiator function of DnaA protein is negatively regulated by the sliding clamp of the E. coli chromosomal replicase[END_REF]. Thus the ratio of DnaA-ATP to DnaA-ADP in the cell is a complex function of the number of replication forks, the rate of DNA replication and the rate of DnaA-ATP production [START_REF] Kurokawa | Replication cycle-coordinated change of the adenine nucleotide-bound forms of DnaA protein in Escherichia coli[END_REF][START_REF] Donachie | Coupling the initiation of chromosome replication to cell size in Escherichia coli[END_REF]. The rate of production of DnaA-ATP is in part regulated by autorepression of transcription of the dnaA gene in addition to possible regeneration of DnaA-ATP from DnaA-ADP [START_REF] Polaczek | Regulation of expression of the dnaA gene in Escherichia coli: role of the two promoters and the DnaA box[END_REF], Fujimitsu et al., 2009). It has been proposed that the cyclic changes in the DnaA-ATP to DnaA-ADP ratio contributes to the timing of initiation of DNA replication [START_REF] Donachie | Coupling the initiation of chromosome replication to cell size in Escherichia coli[END_REF].

Fuchs and coworkers identified two binding sites for the DnaA protein upstream of the -35 region of the nrdAB promoter [START_REF] Augustin | Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion[END_REF]. Their work showed that DnaA acts as a transcriptional activator of nrdAB expression but does not influence its cell-cycle dependent expression (Sun et al., 1994). Later studies by Beckwith and coworkers highlighted a negative role of DnaA-ATP in nrdAB expression, and proposed that the decrease in DnaA-ATP by Hda-dependent ATP hydrolysis following replication initiation results in the cell cycle control of nrdAB expression [START_REF] Gon | A novel regulatory mechanism couples deoxyribonucleotide synthesis and DNA replication in Escherichia coli[END_REF]. From an analysis of the DnaA binding sites found at the nrdAB promoter we previously proposed that DnaA-ATP activates transcription at low concentrations and represses at high concentrations [START_REF] Herrick | Ribonucleotide reductase and the regulation of DNA replication: an old story and an ancient heritage[END_REF]. The aim of this work is to test this hypothesis and to elucidate the role of DnaA in the By the use of quantitative footprinting coupled with in vivo measurements of promoter activity we have characterized the binding of RNA polymerase (RNAP) and of DnaA at the nrdAB promoter and DnaA's role in its transcription regulation. Here we show that RNAP directly interacts with the AT-rich regions and the inverted-repeat region upstream of the promoter, previously identified as important for cell-cycle control (Sun et al., 1994[START_REF] Jacobson | A 45 bp inverted repeat is required for cell cycle regulation of the Escherichia coli nrd operon[END_REF] and that DnaA does not affect these interactions. In addition we have found that DnaA can act both as an activator and a repressor, depending on the changing concentration of DnaA-ATP during the cell cycle. These results allow us to propose a model for nrdAB gene regulation by which DnaA can influence the total amount of RNR expression but not its timing as a function of DNA replication.

Results

DnaA-ATP binds with higher affinity than DnaA-ADP to the DnaA binding sites box1 and box2

The two DnaA binding sites found upstream of -35 on the nrdAB promoter (box1 and box2) are highly homologous to the 9-bp DnaA-binding sequences found at the origin of replication, oriC, with box1 being exactly homologous to R4 [START_REF] Samitt | In vivo studies of DnaA binding to the origin of replication of Escherichia coli[END_REF] (Figure 1).

We used in vitro DNase I footprinting experiments to measure the affinity of binding of DnaA-ATP and DnaA-ADP to the nrdAB promoter. The DNA template used was the pGEMT-Easy plasmid carrying the 631 bp PCR fragment containing the entire nrdAB promoter from -444 to +175 (pGEM-Pnrd plasmid). Sun and Fuchs have shown that the activity of the nrdAB promoter is significantly increased on a negatively supercoiled plasmid (Sun & Fuchs, 1994), we thus decided to carry out these experiments on a negatively the pGEM-Pnrd plasmid described above. Figure 3 shows the footprinting titration pattern of DnaA-ATP on the non-template strand of the nrdAB promoter on linear and supercoiled DNA (Figure 3a and 3b respectively). In both cases, DnaA-ATP shows protection of box1 and box2, together with exposure of hypersensitive sites at -59 and -46 as in Figure 2. The nucleotides protected by the protein are approximately the same as those that are protected on the template strand as shown in Figure 2, ie -58 to -48 (box1) and -45 to -30 (box2) for both linear and supercoiled DNA.

The quantification and profiles of the titration experiments are shown in Figure 3 c andd.

Nonlinear least squares fitting of the data to the binding equation (see Experimental Procedures) yields the dissociation constant, Kd, and Hill coefficient, n. These values are summarized in Table 1. The Kd is around 20 nM and the Hill coefficient is about 2 for DnaA-ATP binding to either linear or supercoiled DNA, indicating that DnaA-ATP binds to these two sites in a cooperative manner on both linear and supercoiled templates.

Interestingly, however, at the higher concentrations of DnaA-ATP, protection of the downstream promoter region is observed on linear DNA from -29 to +15 (Figure 3a and Figure 6S of Supporting Information). In this region a possible DnaA binding site is identified using the Virtual Footprint software [START_REF] Münch | Virtual Footprint and PRODORIC: an integrative framework for regulon prediction in prokaryotes[END_REF] according to the DnaA consensus binding sequence (TT A / T TNCACA). The protection of bases -4 to +1 appears with a Kd of 103 nM of DnaA-ATP (Table 1), and is not observed with DnaA-ADP within this concentration range (data not shown). Protection at this site was also observed with supercoiled DNA (data not shown); however, it is significantly weaker and thus it is not possible to properly quantify this binding. Together these results show that DnaA-ATP binds to two high affinity sites upstream of -35 and to lower affinity sites overlapping the nrdAB core promoter region. The DnaA protein in its ATP-bound form thus forms different In order to determine how DnaA-ATP binding may affect the activity of RNA polymerase at the nrdAB promoter we carried out DNase I footprinting experiments with RNAP and/or DnaA-ATP on the supercoiled promoter. The results presented in Figure 4 show the protection patterns of both proteins at a 50 nM concentration on the template strand.

RNAP can bind to the promoter in the absence of other proteins and protects a large region of DNA within the core and upstream regions of the promoter, in agreement with previous results showing that RNAP can transcribe from this promoter in the absence of additional transcription factors (Sun & Fuchs, 1994[START_REF] Gon | A novel regulatory mechanism couples deoxyribonucleotide synthesis and DNA replication in Escherichia coli[END_REF].

Most interestingly, RNAP binding results in the protection of the AT-rich regions and the upstream half of the inverted repeat (IR45) sequence identified by Fuchs and coworkers as being important for cell cycle control of expression (Sun et al., 1994[START_REF] Jacobson | A 45 bp inverted repeat is required for cell cycle regulation of the Escherichia coli nrd operon[END_REF]. A protection of the regions upstream of -140 is also observed in the presence of RNAP. In an experiment when the nrdAB core promoter sequence is disrupted (data not shown) the protections of the core and upstream regions are lost except for those upstream of -140. We have assigned this protection to a second RNAP binding site that could correspond to the Px promoter previously identified by in vitro transcription assays (Sun & Fuchs, 1994).

It has been shown that Px transcription is repressed by FIS binding (Sun & Fuchs, 1994).

Whether transcription from the Px promoter occurs in vivo remains to be determined.

When both RNAP and DnaA-ATP are incubated with the promoter region we observe the sum of the footprints of both proteins almost covering the entire promoter region, but no [START_REF] Schaper | Interaction of the initiator protein DnaA of Escherichia coli with its DNA target[END_REF][START_REF] Speck | ATP-and ADP-dnaA protein, a molecular switch in gene regulation[END_REF]. DNA bending by DnaA may act to stabilize the upstream contacts by RNAP. These results show that DnaA is not necessary to recruit RNAP to the promoter in vitro, however the close proximity of the DnaA binding sites to the -35 region suggest that in vivo it may play a stabilizing role in the presence of possible transcription repressors, such as NrdR, that can compete with RNAP for promoter binding [START_REF] Torrents | NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes[END_REF].

Quantitative DNase I footprinting of DnaA binding shows decreased affinity for mutated DnaA sites on the promoter.

In order to study the effect of DnaA binding on the activity of the promoter in vivo we carried out directed mutagenesis to its different binding sites. Based on our in vitro footprinting results and on previous work by Augustin et al [START_REF] Augustin | Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion[END_REF] 2S of Supporting Information. The first observation is that the mutation in the DnaA box near the -10 region (M12-14GG) does not affect DnaA binding to the upstream regions in box1 or box2. The mutation altering the DnaA box 2 (M40-42box2) decreases, as expected, the affinity of DnaA-ATP for DnaA box2 (by a factor of 2), and to a less extent DnaA binding to box1. Finally, the mutation altering DnaA box1 (M49box1) significantly decreases the affinity of DnaA-ATP for both DnaA box1 (by a factor of 4) and for DnaA box2 (by a factor of 3). The same is observed with the other mutant altering DnaA box1 (M45-47box1). The resulting affinity was so low that we could not properly quantify DnaA-ATP binding to this mutated DNA.

Affinity of DnaA-ATP for downstream DNA (DnaA-ATP boxDown) was assessed by DNaseI footprinting on linear DNA, as the protection was easier to observe in these conditions. The DnaA-ATP protection pattern on wild type and mutant M12-14GG is shown in Figure 5b. A decreased affinity of DnaA-ATP for the binding sites overlapping the core promoter (boxDown) in the mutant M12-14GG is clearly evident. This agrees with the presence of an oligomeric form of DnaA-ATP that is dependent on the presence of downstream low affinity sites. In order to determine whether the binding of DnaA-ATP to the downstream sites is dependent on the presence of the high affinity sites we carried out DNase I footprinting experiments on linear DNA in the presence of the M45-47box1 and M49box1 mutations (Figure 6S of Supporting Information). These results show that the protection of the promoter region due to the oligmer is not significantly affected by the absence of DnaA binding to the high affinity sites.

DnaA binding to the high affinity sites is required for activation of nrdAB transcription in vivo, while binding to the downstream low affinity site represses its transcription. In order to determine the effects on nrdAB expression in vivo of DnaA binding to its different sites we measured the rate of change in the cellular level of the GFP protein ((dGFP/dt)/OD) expressed under control of the nrdAB promoter and its mutants in the low copy pKK plasmid by growing the cells in a 96-well fluorimeter [START_REF] Zaslaver | A comprehensive library of fluorescent transcriptional reporters for Escherichia coli[END_REF]. Cells carrying the different plasmids containing either the wild type or mutant promoter were diluted 1 to 10000 from an overnight culture and grown at 30°C in M9 minimal medium supplemented with 0.4 % glucose or M9 minimal medium (Figure 6) with 0.4% glucose and 0.5% casamino acids (Figure 3S). The results shown in Figure 6a show the rate of change in the cellular level of the GFP protein ((dGFP/dt)/OD), a measure of the promoter activity, as a function of time. The OD 600 curve provides a simultaneous measure of the growing cell culture. In this experiment cells grow at a constant rate with a generation time of 69 minutes up to 800 minutes when the growth rate begins to decrease, as the culture enters the transition to stationary phase (see Figure 3SB in Supporting Information for the plots of the growth rate as a function of time). The rate of expression from the nrdAB promoter increases to peak before the decrease in growth rate. The changing activity of the promoter suggests that even though the cells are growing at a constant rate they are no longer in balanced growth. This may be due to the threshold for the measurement of the optical density in the fluorimeter resulting in the measurement of the cells only after early exponential phase, when the cellular metabolism has begun to respond to changes in the growth environment.

The increased level of nrdAB expression in mutants M12-14GG and M12-14AG can be considered to be due to the loss of the low affinity DnaA-ATP boxDown repressing site, whereas the decreased level of nrdAB expression in the mutants M40-42box2, M45-47box1 and M49box1 can be considered to be due to the loss of the high affinity DnaA box1 and box2 activating sites. Thus the change of nrdAB promoter activity induced by the mutations of The M12-14GG mutation substitutes a cytosine with a guanine at both positions -12 and -14 to create a thymine-guanine step at -15 and -14 upstream of the transcription initiation site. This new sequence resembles an "extended -10" promoter configuration and could stabilize interactions with RNAP and explain the enhanced nrdAB expression observed [START_REF] Mitchell | Identification and analysis of 'extended -10' promoters in Escherichia coli[END_REF]. To control for this possible effect, we constructed mutant M12-14AG which substitutes a cytosine with an adenine at position -14 (Figure 1b). Expression was similar for both mutants, confirming that the increase in expression is not due to a stabilization of RNAP on the promoter. In addition in vitro transcription experiments show that the M12-14GG mutations has a small positive effect on transcription from the nrdAB promoter, which however cannot account for the three-fold increase of expression in vivo. The presence of the other mutations on the promoter region does not significantly influence RNA polymerase activity in the absence of DnaA (Figure 7S of Supporting Information).

The M12-14GG mutations also overlaps one of the two possible binding sites for the NrdR protein, albeit the lower affinity site [START_REF] Torrents | NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes[END_REF]. NrdR has been proposed to be a repressor of nrdAB transcription as a function of its nucleotide bound state. This could result in a mechanism for the observed negative feedback of nrdAB expression as a function of dNTP concentration [START_REF] Torrents | NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes[END_REF][START_REF] Gon | A novel regulatory mechanism couples deoxyribonucleotide synthesis and DNA replication in Escherichia coli[END_REF][START_REF] Grinberg | Functional analysis of the Streptomyces coelicolor NrdR ATP-cone domain: role in nucleotide binding, oligomerization, and DNA interactions[END_REF]. In order to determine whether the effect observed in the presence of this mutation is due to the loss of binding of NrdR we carried out in vivo fluorescence measurements with a strain lacking the NrdR protein (BW25113 ∆nrdR) (Figure 6b). In this genetic background the M12-14GG mutation still has a significant positive effect on the level of transcription, confirming that the loss of repression is due to the decrease in binding affinity for DnaA. While the increase in expression due to the M12-14GG mutation does not affect the pattern of expression in response to a change in the growth rate, the pattern of expression in the absence of NrdR is significantly different (Figure 6b). These results show that NrdRdependent negative feedback dependent on dNTP accumulation occurs throughout the growth curve. However it plays a more significant role upon entry into stationary phase in accordance with the results of Torrents et al [START_REF] Torrents | NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes[END_REF].

These results clearly show that DnaA binding upstream of the promoter is required for transcription activation while its oligomerisation to the downstream sites results in transcription repression. This indicates that DnaA has both a positive and a negative effect on the level of in vivo nrdAB expression. The same amplitude of the effect of the mutations is observed for the two growth conditions tested (Figure 6 and Figure 3S of Supporting Information). In addition the same "bell type" pattern of expression is observed for both wild type and mutated nrdAB promoters, suggesting that this expression profile is independent of DnaA binding to the promoter.

The timing of expression of nrdAB with replication initiation is only partly affected by

DnaA activation and independent of DnaA repression.

In order to determine the effects of DnaA activation and repression on the cell-cycle dependent expression of nrdAB we carried out flow cytometry measurements of the amount of GFP per cell as a function of DNA content (Figure 7). The strains containing a pKK plasmid with one of the different promoters regulating gfp expression were grown at 30°C in M9 minimal media. When the cultures reached an OD 450 of about 0.15, rifampicin and cephalexin were added to stop re-initiation and cell division (see Experimental Procedures). The active replication forks continue replication to termination while all ongoing transcription will stop due to the presence of rifampicin. Flow cytometry analysis of DNA content shows three peaks corresponding to 2, 4 and 8 chromosomes, with 4 chromosomes being the most populated (Figure 7a). The presence of a small percentage of cells with 8 chromosomes indicates that the initiation interval in these growth conditions spans the time of cell division and that there may be a fraction of the population that initiates DNA replication just before division, instead of right after cell birth. From the percentage of cells in each of the DNA peaks one can estimate the initiation age [START_REF] Skarstad | Escherichia coli DNA distributions measured by flow cytometry and compared with theoretical computer simulations[END_REF] (Experimental Procedures). The initiation age varies between 3 and 10 minutes with a doubling time of 45 to 50 minutes (Figure 4S).

Flow cytometry quantification of GFP content (Figure 7b) largely supports the results obtained on the effect of the mutations on gene expression by the measurement of the GFP expression rate (Figure 6a). This corresponds to the amount of GFP present at the time of antibiotic addition. The two dimensional dot plot shows the GFP content for each of the DNA peaks (Figure 7c). The median of GFP content for each two-dimensional peak was used to determine the ratios of GFP for 4 and 2 chromosomes (GFP_4/2) and 8 and 4 chromosomes (GFP_8/4) (Figure 8A andB).

From the initiation age one can estimate the amount of GFP that has accumulated during that time for a specific pattern of expression. For example, for a stochastically expressed gene that doubles the amount of expressed protein during each cell cycle, the value for GFP_4/2 and GFP_8/4 for the whole population will be at around 1.47 and 1.35 respectively, with a variation of ± 0.02 to take into account the range of initiation age values (Experimental Procedures). For a gene that is expressed only at the time of initiation however, the value of this ratio will be higher, due to the increase in expressed protein taking place at the same time for every cell in the population, and will approach 2 for a gene whose expression in addition follows a step function (Figure 8C). We have compared the GFP_4/2 and GFP_8/4 values for the wild type and mutant nrdAB promoters characterized above to those measured from the GFP content in a strain containing the uninduced lac promoter on the pKK plasmid (Figure 8A). The uninduced lac promoter is a control for a gene that is expressed stochastically throughout the cell cycle [START_REF] Yu | Probing gene expression in live cells, one protein molecule at a time[END_REF].

The nrdAB wild type promoter construct results in a value of GFP_4/2 that is consistent with a cooperative activation of transcription at the time of replication initiation, 1.91 ± 0.14.

The M12-14GG mutation increases the rate of expression by relieving the repression by DnaA-ATP (Figure 6, Figure 7b). In this case the pattern of expression however, is not very different from the wild type promoter as measured by a GFP_4/2 value of 1.75 ± 0.14. DnaA repression thus plays a role in limiting the amount of transcript produced at the nrdAB operon but not its timing. The M45-47box1 mutation results in a decrease of the GFP_4/2 value, 1.64 ± 0.06, which however is still higher than the control lac promoter, 1.37 ± 0.10, indicating that activation by DnaA may influence the cooperativity of induction as well as the overall expression level of the nrdAB gene. The values for the GFP_8/4 are lower than those of GFP_4/2, probably due to the short amount of time between replication initiation, and thus gene induction, and cell division, not being sufficient to contain the full peak of transcription activity (Figure 5S in Supporting Information). Together these results show that the coupling of nrdAB expression with the time of replication initiation is not determined by DnaA alone.

Discussion

DnaA is both an activator and a repressor of RNR expression.

In 2006 Gon et al. showed that DnaA represses transcription from the nrdAB promoter controlling expression of the major aerobic ribonucleotide reductase gene in Escherichia coli [START_REF] Gon | A novel regulatory mechanism couples deoxyribonucleotide synthesis and DNA replication in Escherichia coli[END_REF] while earlier the Fuchs laboratory had shown an activating role for DnaA at the same promoter [START_REF] Augustin | Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion[END_REF]. Here we demonstrate that both DnaA-ADP and DnaA-ATP can bind to the nrdAB promoter at two activating sites, and that the DnaA protein in its ATP-bound form can form oligomers at higher concentrations that result in repression of Our in vitro footprinting results show that DnaA interacts with the promoter to form two different structures depending on its nucleotide-bound form and its concentration. At low concentrations, DnaA-ATP binds upstream of the -35 region to two DnaA high affinity sites, box1 and box2, and enhances nrdAB expression, as shown by the in vivo GFP expression results in agreement with previous work [START_REF] Augustin | Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion[END_REF]. Here we also show that in vitro RNAP can also bind to the promoter in the absence of additional factors and protects a large region up to -125 base pairs upstream of the transcription initiation site. Furthermore, due to the high affinity of RNAP for this promoter we do not observe significant RNAP recruitment by DnaA in vitro by DNaseI footprinting (data not shown). The footprinting pattern obtained in the presence of both proteins appears to be the sum of the individual proteins' patterns, indicating that DnaA binding does not significantly affect the structure of the RNAP-promoter complex (Figure 4).

Stabilization of the RNAP-promoter complex by DnaA and upstream contacts however, may be more significant for RNAP recruitment in vivo than in our in vitro system.

In the cell, RNAP is competing with other proteins for access to the promoter, notably NrdR [START_REF] Torrents | NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes[END_REF], DnaA-ATP itself [START_REF] Gon | A novel regulatory mechanism couples deoxyribonucleotide synthesis and DNA replication in Escherichia coli[END_REF] and possibly abundant nucleoid proteins. The position of the DnaA binding sites immediately upstream of the -35 sequence suggests a class II mode of activation i.e. via a direct interaction with region 4 of the σ subunit and the α-NTD of RNAP bound to this promoter element [START_REF] Dove | Region 4 of sigma as a target for transcription regulation[END_REF].

Recently, the presence of DnaA bound to the upstream sites of the dnaA P2 promoter has been shown to stabilize the activity of RNAP in the presence of the transcription inhibitor rifampicin [START_REF] Flåtten | DnaA protein interacts with RNA polymerase and partially protects it from the effect of rifampicin[END_REF]. In a similar fashion, at the acs promoter, for example, the binding of CRP to similarly placed activating sites stabilizes RNAP binding in the presence of nucleoid proteins such as FIS and IHF [START_REF] Sclavi | The multiple roles of CRP at the complex acs promoter depend on activation region 2 and IHF[END_REF].

The combination of our in vitro and in vivo results shows that, when the concentration of DnaA-ATP increases, the protein can bind to downstream low affinity sites overlapping with promoter's -10 region and can thus directly inhibit RNA polymerase binding. At these higher DnaA-ATP concentrations we observe protection of bases located between position -29 and +15. In contrast to what is observed at oriC, binding of DnaA at the high affinity sites does not appear to significantly decrease the concentration at which the oligomer is formed [START_REF] Miller | Bacterial origin recognition complexes direct assembly of higher-order DnaA oligomeric structures[END_REF]. The structure of the DnaA oligomer published by Berger and coworkers shows a right-handed superhelix that stabilizes a positive supercoil [START_REF] Erzberger | Structural basis for ATP-dependent DnaA assembly and replication-origin remodeling[END_REF].

This may be a reason for the weaker protections observed on a negatively supercoiled plasmid compared to linear DNA at high DnaA-ATP concentrations (Figure 3), and implies that the degree of negative supercoiling in vivo may affect repression by DnaA-ATP. The absence of downstream protection at high concentrations of DnaA-ADP (data not shown) is due to its inability to form oligomeric structures [START_REF] Erzberger | Structural basis for ATP-dependent DnaA assembly and replication-origin remodeling[END_REF]. This does not exclude the possibility that DnaA-ADP may, however, act as an activator from the upstream sites, albeit at high DnaA-ADP/DnaA-ATP ratios (Figure 2).

Oscillation in DnaA-ATP as a function of DNA replication activates and represses transcription from the nrdAB promoter but does not determine the timing of expression as a function of DNA replication initiation.

James Fuchs and coworkers have shown that nrdAB expression coincides with initiation of DNA synthesis [START_REF] Sun | Escherichia coli ribonucleotide reductase expression is cell cycle regulated[END_REF]. This could act to supply the newly assembled replication forks with the required dNTPs. However, the mechanism by which gene expression can be precisely timed in a prokaryotic cell to coincide with a specific cell cycle event, such as replication initiation, in the absence of a well-defined checkpoint control remains to be identified. Given the positive and negative roles that DnaA-ATP has on transcription regulation at the nrdAB promoter, one could propose that the timing of nrdAB expression could result from the oscillation in DnaA-ATP to DnaA-ADP ratio during the cell cycle ( [START_REF] Fujimitsu | Specific genomic sequences of E. coli promote replicational initiation by directly reactivating ADP-DnaA[END_REF] and reviewed by [START_REF] Katayama | Feedback controls restrain the initiation of Escherichia coli chromosomal replication[END_REF][START_REF] Kaguni | DnaA: Controlling the Initiation of Bacterial DNA Replication and More[END_REF]) and thus mirror DnaA's interactions with the origin of DNA replication, oriC [START_REF] Gon | A novel regulatory mechanism couples deoxyribonucleotide synthesis and DNA replication in Escherichia coli[END_REF]. While Fuchs and coworkers had shown that DnaA activation did not influence the pattern of nrdAB expression as a function of the cell cycle [START_REF] Augustin | Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion[END_REF], the role of DnaA-dependent transcription repression remains to be established.

The current models for the timing of replication initiation propose that at oriC DnaA is bound at the high affinity R sites throughout the cell cycle. These sites act to stabilize a higher order oligomeric structure of DnaA resulting from the increase in DnaA-ATP at the time of initiation. This structure in turn induces DNA melting at the AT-rich region creating a substrate for assembly of the replication forks [START_REF] Erzberger | Structural basis for ATP-dependent DnaA assembly and replication-origin remodeling[END_REF][START_REF] Ozaki | A common mechanism for the ATP-DnaA-dependent formation of open complexes at the replication origin[END_REF][START_REF] Grimwade | Mutational analysis reveals Escherichia coli oriC interacts with both DnaA-ATP and DnaA-ADP during pre-RC assembly[END_REF][START_REF] Miller | Bacterial origin recognition complexes direct assembly of higher-order DnaA oligomeric structures[END_REF][START_REF] Riber | Loss of Hda activity stimulates replication initiation from I-box, but not R4 mutant origins in Escherichia coli[END_REF]. Following initiation the hydrolysis of the DnaA-bound ATP by the Hda-dependent RIDA process enhanced by ongoing DNA replication and the synthesis of new datA loci result in a decrease in free DnaA-ATP that contributes to the eclipse period (reviewed in [START_REF] Nielsen | Once in a lifetime: strategies for preventing re-replication in prokaryotic and eukaryotic cells[END_REF]).

The observation that the effect of the mutations of the activating DnaA sites on promoter activity in vivo reflects the changes in binding affinity measured in vitro (Figures 5 and6) indicates that the concentration of DnaA-ATP in the cell is within the range of its affinity constant for these upstream sites, at least for the period during which RNAP is recruited to the promoter. In addition, the 6-fold difference between the affinity of DnaA-ATP for its high and low affinity sites indicates that repression of the nrdAB promoter does not occur until the proportion of DnaA-ATP reaches its maximum at the time of initiation in agreement with the known oscillations in the DnaA-ATP to DnaA-ADP ratio (Figure 8D). This mode of nrdAB transcription regulation however would seem to be incompatible with NrdAB expression coinciding with replication initiation [START_REF] Sun | Escherichia coli ribonucleotide reductase expression is cell cycle regulated[END_REF].

In order to test whether DnaA activation and repression of the nrdAB promoter influences its timing of expression we have measured the effect of relieving DnaA-dependent repression or activation on the pattern of gene transcription. In order to measure changes in the level of gene expression as a function of the DNA replication status in an unsynchronized population we measured the amount of GFP per cell as a function of its DNA content by flow cytometry (Figures 7 and8). By the addition of rifampicin and cephalexin, transcription, replication initiation and cell division are blocked, while ongoing replication is allowed to reach termination. The cells will thus contain an integral number of chromosomes, and the amount of GFP that was present at the time of antibiotic addition. We thus obtain a measure of the median amount of GFP before and after replication initiation has taken place.

The experimental values we obtain for the GFP ratios for wild type nrdAB promoter and the uninduced lac promoter are in agreement with their expected pattern of expression, i.e. a gene that is expressed to coincide with the time of initiation [START_REF] Sun | Escherichia coli ribonucleotide reductase expression is cell cycle regulated[END_REF]) and a gene that is expressed stochastically, at a different time for each cell in the population [START_REF] Yu | Probing gene expression in live cells, one protein molecule at a time[END_REF] (Figure 8). The values obtained from GFP expression under control of the mutant promoters show that even though the M12-14GG mutation results in a 3 fold increase in expression rate, it has only a small effect on the timing of expression. These results are consistent with transcription repression by DnaA-ATP taking place at the time of replication initiation to limit the height of the peak of expression after the nrdAB promoter has been 8C,D). This would result in the amount of RNR produced being proportional to the amount of free DnaA-ATP, consistent with the results of Beckwith and coworkers on the effect of DnaA mutations in vivo [START_REF] Gon | A novel regulatory mechanism couples deoxyribonucleotide synthesis and DNA replication in Escherichia coli[END_REF]).

The M45-47box1 mutation on the other hand results in a more significant change in the GFP ratio of 4 and 2 chromosomes cells. Here we show that DnaA oligomerisation can take place independently of DnaA binding to the high affinity sites. This mutation therefore affects activation and not repression by DnaA-ATP. Augustin et al have shown that the timing of the peak in nrdAB expression with replication initiation is not affected by the presence of mutations to the DnaA high affinity sites [START_REF] Augustin | Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion[END_REF]. The effect of these mutations on the GFP ratio observed here confirms their results and suggests that the presence of DnaA at the high affinity sites influences the cooperativity and synchrony of activation by insuring that all the copies of the nrdAB promoter are occupied by RNAP at the time of induction (Figure 8C). The fact that we can measure these differences in cells where GFP is expressed from a promoter-gfp construct on a plasmid, shows that the timing of nrdAB expression is independent of its position on the chromosome and the timing of gene doubling, in agreement with the results of Fuchs and coworkers [START_REF] Sun | Escherichia coli ribonucleotide reductase expression is cell cycle regulated[END_REF]. DnaA binding alone therefore does not suffice to control the timing of nrdAB expression.

The upstream region of the promoter responsible for cell-cycle dependent expression is contacted directly by RNA polymerase.

Fuchs and coworkers identified an upstream region of the promoter containing a 45 base pair inverted repeat flanked by two AT-rich regions as being necessary for the timing of RNR expression (Sun et al., 1994[START_REF] Jacobson | A 45 bp inverted repeat is required for cell cycle regulation of the Escherichia coli nrd operon[END_REF]. Here we show that RNAP can directly contact these sites. The C-terminal domains of the α subunits of RNAP (α-CTDs), connected to the core of the enzyme through flexible linkers, have been shown to have a high affinity for the long AT stretches that make up the regulatory region known as the UP element found upstream of several promoters from -40 up to -120 [START_REF] Pemberton | FIS modulates the kinetics of successive interactions of RNA polymerase with the core and upstream regions of the tyrT promoter[END_REF][START_REF] Aiyar | Upstream A-tracts increase bacterial promoter activity through interactions with the RNA polymerase alpha subunit[END_REF]. These upstream interactions induce DNA wrapping around the enzyme [START_REF] Cellai | Upstream promoter sequences and alphaCTD mediate stable DNA wrapping within the RNA polymerase-promoter open complex[END_REF] and DNA bending by DnaA may further increase the stability of the nucleo-protein complex by decreasing the energy required by RNAP to wrap the DNA. Stable RNAPpromoter complexes have been identified on several genes' regulatory regions in both prokaryotes and eukaryotes, indicating that the transition into transcription elongation is a common target for regulation of gene expression (reviewed in [START_REF] Wade | The transition from transcriptional initiation to elongation[END_REF]). At the time of replication initiation a local change in DNA topology could affect the binding of the RNAP to the upstream region of the promoter to decrease the stability of the RNAP-promoter complex, facilitating promoter escape and gene expression. The latter is in agreement with the known effects of DNA supercoiling on promoter escape [START_REF] Hsu | Promoter clearance and escape in prokaryotes[END_REF] and with the results published by Fuchs and coworkers where a change in the distance between the promoter core region and the upstream sites resulted in both a loss of supercoiling dependence of expression in vitro and of cell cycle dependent expression in vivo of the nrdAB operon (Sun & Fuchs, 1994).

Together these results allow us to propose a model for the role of DnaA in the regulation of RNR expression (Figure 8D). DnaA bound to the high affinity sites, in its ATP or ADP-bound form, stabilizes RNAP binding at the nrdAB promoter. The α-CTDs of the RNAP form contacts with the upstream AT-rich region, contributing to the stability of the nucleoprotein complex. The presence of a stable RNAP-promoter complex protects it from inhibition by DnaA-ATP as its concentration increases prior to initiation. We propose that transcription is induced at the time of replication initiation by a local change in DNA topology releasing the upstream contacts of RNAP. At this time the concentration of DnaA-ATP has rebinding. The level of nrdAB expression is thus limited by the amount of free DnaA-ATP at initiation. Once DNA replication elongation has begun, the combined effects of RIDA and the increase in DnaA binding sites available on the chromosome result in a decrease in free DnaA-ATP, allowing RNAP to bind again to the nrdAB promoter prior to the following initiation event. The amount of NrdAB enzyme and dNTPs can affect both the frequency of initiation and the rate of DNA replication [START_REF] Fujimitsu | Modes of over-initiation, dnaA gene expression and the inhibition of cell division in a novel cold-sensitive hda mutant in Escherichia coli[END_REF][START_REF] Odsbu | A reduction in ribonucleotide reductase activity slows down the chromosome replication fork but does not change its localization[END_REF]. While transcription regulation by DnaA-ATP does not play a significant role in the timing of cell cycle dependent RNR expression, it may play a more important role in maintaining the amount of RNR proportional to the number of replication forks. Finally, DnaA's double role, at the origin and as a transcription factor of several genes, may allow it to act as a general mediator in the coupling of DNA replication initiation and elongation [START_REF] Messer | DnaA initiator--also a transcription factor[END_REF][START_REF] Goranov | A transcriptional response to replication status mediated by the conserved bacterial replication protein DnaA[END_REF][START_REF] Ishikawa | Distribution of stable DnaA-binding sites on the Bacillus subtilis genome detected using a modified ChIP-chip method[END_REF].

Experimental procedures

Bacterial strains, plasmids, and growth conditions E.coli K-12 strains BW25113 [rrnB3 ∆ lacZ4787 hsdR514 ∆(araBAD)567 ∆ (rhaBAD)568 rph-1], referred to as the wild type, and its BW25113 yabD (nrdR) derivative JWKO0403 (yabD knock-out, referred to as BW25113 ∆nrdR) were used for the fluorimeter experiments [START_REF] Baba | Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection[END_REF]. E. coli JM109 strain was used for classical cloning (Promega). E. coli WM2287 strain carrying the pdnaA116 plasmid was used for DnaA purification [START_REF] Schaper | Interaction of the initiator protein DnaA of Escherichia coli with its DNA target[END_REF]. Plasmid pGEMT-Easy (Promega) was used for classical cloning procedures. The medium-to-low copy plasmid pKK-gfp was used for (Qbiogene) plasmid by digestion with BglII and HindIII restriction enzymes, and inserted in pKK232-8 (GE Healthcare) (see [START_REF] Bouffartigues | H-NS cooperative binding to high-affinity sites in a regulatory element results in transcriptional silencing[END_REF] for details). Strains were grown routinely in Luria-Bertani (LB) medium at 37°C, and antibiotic selection was maintained either with ampicillin at 100 µg/ml, kanamycin at 50 µg/ml or chloramphenicol at 30 µg/ml.

For in vivo measurements of GFP expression in Pnrd-gfp fusions, strains were grown in M9 minimal medium (Na 2 HPO 4 •2H 2 O 8.9 g/L; KH 2 PO 4 3 g/L; NH 4 Cl 1 g/L; NaCl 0.5 g/L; CaCl 2 0.0001 M; MgSO 4 0.002 M; Tryptophane 0.004 g/L; Tymidine 0.005 g/L) supplemented or not with glucose 0.4 % and/or casamino acids 0.5 %.

DNA manipulation

Standard methods were used for DNA manipulation as PCR, DNA analysis, cloning, and chemical transformation. Plasmid DNA was isolated using the NucleoSpin plasmid kit (Macherey-Nagel). PCR fragments were purified with the QIAquick PCR purification kit (Qiagen), and with the QIAquick Gel Extraction Kit (Qiagen) when isolated from a 0.8 % agarose gel. DNA digestions and ligations were carried out according to the manufacturer's instructions (Ozyme).

Oligonucleotides

The oligonucleotides used in this study are listed in Table 1S in Supporting Information.

Plasmid construction

The nrdAB promoter sequence (Pnrd) was amplified by PCR using E. coli MG1655 chromosomal DNA as template, and PnrdR and PnrdF primers that introduce a XbaI and a

Mutagenesis

Based on our in vitro footprinting results and on previous work done by [START_REF] Augustin | Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion[END_REF], the nrdAB promoter carried by the pGEM-Pnrd plasmid was mutated in the DnaA binding sites by introducing different point mutations. Oligonucleotides were designed to incorporate these point mutations within the promoter (Table 1S). Two complementary mutated oligonucleotides were used for the construction of each mutant. In two separate reactions, fragments upstream and downstream of the point mutation were PCRamplified using one of the flanking primers (PnrdF or PnrdR) and one of the complementary oligonucleotides containing the point mutation to be introduced. This PCR was performed at 50°C, a low enough temperature allowing mismatches at positions to be mutated, and with the PfuUltra High-Fidelity DNA Polymerase (Stratagene). We thus created two fragments of Pnrd carrying the mutation at one end, that we gel-purified. By an additional PCR at 53°C using only the flanking primers (PnrdF or PnrdR), we fused these two fragments in one, thus creating a mutated promoter. This amplified fragment was purified and cloned into pGEMT-Easy vector. The latter was used to transform JM109 strains. Sequencing was performed by Genome express (Cogenics, genome express, Grenoble, France). Six different mutated Pnrd promoters carried by the pGEM plasmid were created: pGEM-PnrdM12-14GG (C to G at -12 and -14), pGEM-PnrdM12-14AG (C to G at -12 and C to A at -14), pGEM-PnrdM40-42box2 (A to T and T to A at -42 and -40), pGEM-PnrdM45-47box1 (C to G at -45 and -47), pGEM-PnrdM49box1 (T to A at position -49), and pGEM-PnrdMoff (T to G at -7 and -9) (Figure 1b). These mutated pGEM-Pnrd vectors were then digested by XmaI and XbaI, and the mutated Pnrd fragments extracted were subcloned into pKK-gfp plasmid in order to measure GFP expression in living cells. Plasmids pKK-PnrdM12-14GG-gfp, pKK-PnrdM12-14AGgfp, pKK-PnrdM40-42box2-gfp, pKK-PnrdM45-47box1-gfp, pKK-PnrdM49box1-gfp and pKK-PnrdMoff-gfp were thus created.

DnaA purification

Native DnaA protein was overexpressed from the E. coli WM2287 strain carrying the pdnaA116 plasmid. Protein production was induced by 0.3 mM of isopropyl β-Dthiogalactopyranoside (IPTG). The purification was performed according to Walter Messer's protocol [START_REF] Messer | DnaA as a transcription regulator[END_REF] except minor changes to the protocol. After precipitation with 28 % (NH 4 ) 2 SO 4 , the proteins from the pellet were resuspended and desalted using a 5 ml HiTrap fast desalting column (GE Healthcare). The protein solution was applied to a 1 ml MonoS column (HR 5/5, Amersham, Pharmacia), the pooled fractions were concentrated using an Ultra-15 centrifugal filter device of 30 KDa exclusion limit (Amicon), and loaded onto a 24 ml gel filtration column (Superdex 75 HR 10/30, Amersham Biosciences). Note that all along the purification, 5 % glycerol was used instead of 20 % sucrose in the buffers that otherwise were as described by [START_REF] Messer | DnaA as a transcription regulator[END_REF]. The purified protein was stored in aliquots at -20°C in LG-buffer (45 mM Hepes/KOH pH 7.6, 600 mM K-glu, 1 mM DTT, 10 mM Mg-acetate, 0.5 mM EDTA, and 20 % glycerol).

Dialysis of DnaA

According to the method of Speck et al [START_REF] Speck | ATP-and ADP-dnaA protein, a molecular switch in gene regulation[END_REF], we dialyzed the protein against ATP or ADP in order to work with an "exclusive" nucleotide state of DnaA (DnaA-ATP-or DnaA-ADP). This was obtained by an overnight incubation at 4°C of 5 µM of DnaA with 3 mM of ATP or ADP in a total volume of 20 µl of LG-buffer (45 mM HEPES-KOH pH 7,6 ; 600 mM K-glu ; 10 mM Mg-acetate ; 1 mM DTT ; 0.5 mM EDTA ; 5 % glycerol), followed by dialysis against the same buffer containing 3 mM of the corresponding nucleotide: the 20 µl of protein were placed onto a 0.025 µm MF-Millipore membrane (Millipore) floating on the buffer solution and dialyzed for 40 min at room temperature.

RNA polymerase purification

RNA polymerase was purified as described earlier [START_REF] Sclavi | Real-time characterization of intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase at the T7A1 promoter[END_REF]

DNase I Protection assay

Protection patterns of the nrdAB promoter region by DnaA and/or RNA polymerase (RNAP) were assayed by in vitro DNase I footprinting. Template DNA was linear (a 226 bp PCR fragment using primers PeF and PeR, one or the other being labeled) or supercoiled (plasmid pGEM-Pnrd).

DNA labeling: Primers PeF and PeR were 5' labeled with γ 32 P-ATP (GE Healthcare) and T4 polynucleotide kinase (Fermentas). They were quickly purified with the Bio-Spin 6 Protection assay. Binding reactions were carried out with 1 nM of DNA (linear or supercoiled), and 0 to 500 nM of dialysed DnaA-ATP or DnaA-ADP, and/or 0 to 100 nM of RNAP, in 10 µl of binding buffer (1 mM DTT; 0.5 mg/ml Ac-BSA, 20 mM Hepes pH 7.6, 50 mM K-glu and 5 mM MgCl 2 ) and 3 mM of ATP or ADP (Sigma-Aldrich). Samples were incubated for 20 min at 37°C. When DnaA and RNAP were used in the same binding reaction, DnaA was added first and incubated for 15 min, and RNAP was added in second time for an extra 10 min. DNA was then cut by 4 µg/ml of DNase I diluted in a digestion buffer (25 mM Tris pH 7; 5 mM MgCl 2 ; 1 mM CaCl 2 ; 2 mM DTT; 100 mM KCl) during 25 seconds at 37°C. When using "cold" supercoiled plasmid DNA, the reaction was stopped by addition of 10 µl of a primer extension mix containing a labeled primer (PeF or PeR). A classical PCR was run out to extend from the labeled fragment on the cut DNA, during 30 cycles at 53°C. When using labeled linear PCR fragments of DNA, the reaction was stopped by addition of 50 µl of a STOP solution (0.2 µg/ml of glycogen; 0.04 mg/ml of calf thymus non specific DNA; and TE buffer). In both cases, DNA was then precipitated with ethanol and resuspended in 12 µl of a gel loading buffer (90 % of formamide; 0.025 % of bomophenol blue; 0.025 % of xylene cyanol). The DNA fragments were visualized on sequencing gels. Samples were incubated for 5 min at 95°C and loaded onto 6 % acrylamide gels in denaturing conditions (8 M of urea; 1 X TBE buffer). After electrophoresis, gels were dried and autoradiographed. A G+A-specific DNA sequencing reaction was realized for each DNase I experiment in order to localize the protected regions [START_REF] Maxam | A new method for sequencing DNA[END_REF]. [START_REF] Brenowitz | DNase I footprint analysis of protein-DNA binding[END_REF]. Nonlinear least square curve fitting of the data was carried out using the following binding equation:

y = L+(U-L)*(K n *x n /(1+K n *x n ))
with L: lower y value; U: upper y value, K: affinity constant, and n: Hill coefficient using the Origin software (OriginLab). The values obtained for the U and L were used to normalize the data from 1 to 0 to obtain the value for the normalized band intensity shown in the plots in Figure 3 and Figure 1S in Supporting Information. This value is inversely proportional to the fractional saturation of the site by the protein. The results of at least three independent experiments were combined and fit again to obtain the values for the affinity constant and the Hill coefficient shown in Table 1 and Figure 5 and Table 2S of Supporting Information.

In vivo measurement of Pnrd expression.

We used the fluorescence of the GFP protein expressed under control of the nrdAB promoter in the medium-to-low copy pKK plasmid to follow wild type and mutated Pnrd activity. The rate of GFP production, divided by the OD, provides a measure of the promoter activity [(dGFP/dt)/OD] as a function of time [START_REF] Zaslaver | A comprehensive library of fluorescent transcriptional reporters for Escherichia coli[END_REF]. Cells carrying these different plasmids were diluted 1:10000 from an overnight culture in M9 minimal medium were grown in 96-wells plates, in a WallacVictor3 multiwell fluorimeter (Perkin-Elmer) set with an automatically repeating protocol of shaking and OD 600 and fluorescence readings. medium. The data analysis was carried out as described by [START_REF] Zaslaver | A comprehensive library of fluorescent transcriptional reporters for Escherichia coli[END_REF]) using a program written specifically for these experiments to be described in a separate publication.

Flow Cytometry

Cells were grown overnight in M9 minimal media supplemented with 0.2 % glucose and 0.5 % casamino acids. Following a 1:1000 dilution in the same medium the cells were grown at 30°C up to an OD 450 of 0.15-0.2. Rifampicin and cephalexin (300 µg/ml and 36 µg/ml final concentrations respectively) were added to block transcription and cell division.

After 2 hours, 2 ml of the cells were harvested on ice, spun 3 minutes at 3000 rpm, resuspended in 400 µl of PBS and an equal volume of 2X fixing solution (4.8 % paraformaldehyde and 0.03 % gluteraldehyde) was added as described by [START_REF] Maithreye | Delay-induced transient increase and heterogeneity in gene expression in negatively auto-regulated gene circuits[END_REF]. After 15 minutes at room temperature and 30 minutes on ice the cells were washed by three consecutive cycles of spin (3 minutes at 3000 rpm) and wash with 1 ml of PBS. Finally cells were resuspended in 500 µl of PBS. These samples can be stored at 4°C for up to two months. The night prior to the flow cytometer measurements 500 µl of Hoechst 33342 (Molecular Probes, Eugene, OR) in PBS (1.5 µg/ml final) was added to the cells to label the DNA overnight. The measurements were obtained on a Influx500 (BDBiosciences) flow cytometer at the Flow Cytometry Platform of the Institute Jacques Monod, Paris, France.

The initiation age, a i , was determined from the fraction (F) of cells with the number of genome equivalents present prior to initiation as determined by the flow cytometry DNA plots, such as the one in Figure 7 [START_REF] Skarstad | Escherichia coli DNA distributions measured by flow cytometry and compared with theoretical computer simulations[END_REF],

F = 2 (1-ai) a i = τ -τ * (log (2 -F)) / log(2))
with, in this case, a doubling time, τ, of 50 minutes. We carried out a simulation of a population of cells doubling the amount of GFP gene at random times throughout the cell cycle, such as the example shown in Figure 8B, by using Microsoft Excel. We then used the measured initiation age to estimate the average amount of GFP in the population before and after initiation has taken place (simulation value shown in Figure 8 A andB). IciA, Fis, DnaA and NrdR [START_REF] Han | Effect of IciA protein on the expression of the nrd gene encoding ribonucleoside diphosphate reductase in E. coli[END_REF][START_REF] Augustin | Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion[END_REF][START_REF] Rodionov | Identification of a bacterial regulatory system for ribonucleotide reductases by phylogenetic profiling[END_REF][START_REF] Torrents | NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes[END_REF]. The two AT-rich sequences (AT) flanking a 45-bp inverted repeat (IR45) are represented as grey boxes, and a region required for nrd cell cycle dependent expression is indicated as a hatched box (Sun et al., 1994[START_REF] Jacobson | A 45 bp inverted repeat is required for cell cycle regulation of the Escherichia coli nrd operon[END_REF]. Positions indicated are based on the +1 start site of transcription of Pnrd 110 bp upstream of the ATG codon of nrdA [START_REF] Tuggle | Regulation of the operon encoding ribonucleotide reductase in Escherichia coli: evidence for both positive and negative control[END_REF]. The start site of transcription of Px (Sun & Fuchs, 1994) is also represented. The main primers used in this study (PnrdF, PnrdR, PeF and PeR) are represented as grey arrows and detailed in supplementary Table 1. [START_REF] Münch | Virtual Footprint and PRODORIC: an integrative framework for regulon prediction in prokaryotes[END_REF] and two NrdR binding sites found at the nrd promoter (light grey boxes) [START_REF] Rodionov | Identification of a bacterial regulatory system for ribonucleotide reductases by phylogenetic profiling[END_REF][START_REF] Torrents | NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes[END_REF] DnaA bound at the high affinity sites (grey ovals), in either its ATP or ADP-bound form, binds to the upstream, high affinity, sites (a) and stabilizes RNAP binding (white oval).

RNAP forms an interaction to the upstream AT-rich region via its α-CTDs (small white [START_REF] Han | Effect of IciA protein on the expression of the nrd gene encoding ribonucleoside diphosphate reductase in E. coli[END_REF][START_REF] Augustin | Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion[END_REF][START_REF] Rodionov | Identification of a bacterial regulatory system for ribonucleotide reductases by phylogenetic profiling[END_REF][START_REF] Torrents | NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes[END_REF]. The two AT-rich sequences (AT) flanking a 45-bp inverted repeat (IR45) are represented as grey boxes, and a region required for nrd cell cycle dependent expression is indicated as a hatched box (Sun et al., 1994[START_REF] Jacobson | A 45 bp inverted repeat is required for cell cycle regulation of the Escherichia coli nrd operon[END_REF]. Positions indicated are based on the +1 start site of transcription of Pnrd 110 bp upstream of the ATG codon of nrdA [START_REF] Tuggle | Regulation of the operon encoding ribonucleotide reductase in Escherichia coli: evidence for both positive and negative control[END_REF]. The start site of transcription of Px (Sun & Fuchs, 1994) is also represented. The main primers used in this study (PnrdF, PnrdR, PeF and PeR) are represented as grey arrows and detailed in supplementary Table 1.

(b) Sequence surrounding the DnaA binding sites. The -35 and -10 regions are underlined by a dotted line. The two nine-base sequences with homology to the DnaA consensus binding sequence, box1 and box2, are indicated as open boxes. A putative DnaA binding site identified by the Virtual Footprint software (dotted line box) [START_REF] Münch | Virtual Footprint and PRODORIC: an integrative framework for regulon prediction in prokaryotes[END_REF] and two NrdR binding sites found at the nrd promoter (light grey boxes) [START_REF] Rodionov | Identification of a bacterial regulatory system for ribonucleotide reductases by phylogenetic profiling[END_REF][START_REF] Torrents | NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes[END_REF]

  and by the availability of reducing equivalents

  nrdAB expression and its possible effect on the timing of expression as a function of the cell cycle.

  promoter depending on its concentration and, as the evidence here suggests, the topological state of the DNA.RNAP binding results in an extended protection on the nrdAB promoter independently of DnaA.

  observed in either the extent or the pattern of protection under these conditions. It has been shown that one DnaA monomer bound to a DnaA box induces a bend of 42°; this distortion of the double helix in turn results in the increased accessibility to DNaseI cleavage observed here and at the dnaA promoter

  , the nrdAB promoter was mutated by introducing different point mutations. These mutations alter DnaA box1, DnaA box2, and the downstream DnaA binding site. Four different mutated nrdAB promoters carried by the PGEM plasmid were analyzed: M12-14GG (C to G at positions -12 and -14), M40-42box2 (A to T and T to A at positions -42 and -40), M45-47box1 (C to G at positions -45 and -47) and M49box1 (T to A at position -49) (Figure 1b). Affinity of DnaA-ATP for wild type and mutated DnaA box1 and box2 promoters was measured by quantitative DNase I footprinting experiments on supercoiled DNA. In each case, the dissociation constant Kd is measured, as shown in the bar graph in Figure 5a. Representative gels are shown in Figure 1S of Supporting Information. The quantification of the DnaA titration on the wild type and mutant promoters is presented in Figure 2S. The

  dissociation constant (Kd) and the Hill coefficient (n) are summarized in Table

  sites is directly proportional to the changes in DnaA binding affinity by these same mutations as measured in vitro.

  new information can help to explain the seemingly inconsistent results observed previously and opens the possibility that the change in DnaA-ATP to DnaA-ADP ratio at the time of replication initiation may contribute to the cell cycle dependent pattern of NrdAB expression.

  oligomerize and occupy the low affinity sites, inhibiting RNAP

  Pnrd-gfp fusions. The gfp gene was obtained from the pQBIT7GFP

  XmaI restriction sites. Plasmid pGEM-Pnrd carrying this Pnrd PCR fragment of 631 bp (including the XmaI and XbaI sites) was constructed using classical cloning methods according to pGEMT-Easy protocol (Promega), and transformed in E. coli JM109 strains. This Pnrd sequence was then cloned in the pKK-gfp plasmid in order to obtain fusions where the fluorescent protein GFP is expressed under the nrdAB promoter. The PnrdF-PnrdR DNA fragment was extracted from pGEM-Pnrd by XmaI and XbaI digestion, and ligated into the same sites of pKK-gfp to create plasmid pKK-Pnrd-gfp. Plasmid pKK-Pnrd-gfp was transformed in BW25113 strains. A 215 bp PCR fragment containing the lac promoter sequence (Plac) was cloned according to the same protocols in pGEMT-Easy and in pKK-gfp plasmids using primers PlacR and PlacF, and used as a control for fluorimeter and flow cytometry experiments. Plasmid pKK-gfp carrying no promoter was used to measure background fluorescence for both fluorimeter and flow cytometry experiments. Universal primer T7ter was used in combination with an internal primer to verify plasmid constructions.

  ) according to the manufacturer instructions. Purified oligonucleotides were then used for primer extension of supercoiled plasmid cut by DNase I, or for amplification of PCR fragments used as linear DNA. Both were carried out by a standard PCR with 30 cycles of amplification at 53°C.

  . The intensities of the bands in the gels were quantified using the ImageQuant 5.0 software. The band intensities were normalized with the intensity of a reference band that does not change as a function of DnaA concentration and with the intensity of the band in the absence of protein

  GFP fluorescence was measured at 535 nm after excitation at 485 nm. Background fluorescence was determined from the fluorescence of cells bearing a promoterless GFP vector and background absorbance was determined from wells containing only the growth

Fig 1 .

 1 Fig 1. Schematic representation of the E. coli nrdAB promoter (Pnrd). (a) Pnrd

  Sequence surrounding the DnaA binding sites. The -35 and -10 regions are underlined by a dotted line. The two nine-base sequences with homology to the DnaA consensus binding sequence, box1 and box2, are indicated as open boxes. A putative DnaA binding site identified by the Virtual Footprint software (dotted line box)

  are also shown. The site directed mutations in the DnaA binding sites constructed in this study are shown. M49box1: T to A at position -49; M45-47box1: C to G at positions -45 and -47; M40-42box2: A to T and T to A at positions -42 and -40; M12-14GG:C to G at positions -12 and -14; M12-14AG: C to A at -12 and C to G at -14, and Moff: T to G at positions -7 and -9.

Fig 2 .

 2 Fig 2. DnaA-ATP and DnaA-ADP binding to the nrdAB promoter. DNase I protection

Fig 3 .

 3 Fig 3. Titration of DnaA-ATP binding to the nrdAB promoter. (a) DNase I protection

  binding of the protein. Lane 1: free DNA, lane 2 to lane 9: DNA with 20, 30, 50, 100, 200, 300 and 500 nM respectively. (b) DNase I protection pattern on the non template strand of the nrdAB promoter on a supercoiled plasmid. Lane 1: free DNA, lane 2 to lane 9: DNA with DnaA-ATP at 5, 10, 20, 30, 50, 100, 150 and 200 nM respectively. Two different preparations of DnaA were used in the experiments shown in (A) and (B), explaining the different range in DnaA concentrations. (c) Quantification of DnaA-ATP binding to box1, box2 and boxDown regions on the linear DNA. (d) Quantification of DnaA-ATP binding to box1 and box2 regions on supercoiled DNA. The values obtained from the nonlinear least square fitting of the data (dissociation constant and Hill coefficient) are summarized in Table 1. Results are shown from at least three independent experiments.

Fig 4 .

 4 Fig 4. RNA polymerase binding to the nrdAB promoter. DNase I protection pattern of

Fig 5 .Figure

 5 Fig 5. Effect of nrdAB mutations on binding of DnaA-ATP. (a) Comparison of DnaA-

Fig 6 .

 6 Fig 6. GFP expression rate from Pnrd-gfp plasmid fusions measured in living cells.

Fig. 7 .

 7 Fig.7. Flow cytometry measurements of DNA and GFP content in the strains

Fig. 8 .

 8 Fig. 8. (A) Ratio of the GFP content in 4 and 2 chromosomes cells and (B) in 8 and 4

  b). At the time of replication initiation a change in local DNA topology releases RNAP to transcribe the nrdAB operon (c-d). The high concentration of DnaA-ATP at the time of initiation results in an oligomeric DnaA structure covering the core promoter sequences and inhibiting RNAP rebinding (e). Following replication initiation the combination of RIDA and the synthesis of new DnaA binding sites on the chromosome result in a decrease in DnaA-ATP and loss of the oligomeric structure (a) allowing RNAP to bind again.

Fig 1 .

 1 Fig 1. Schematic representation of the E. coli nrdAB promoter (Pnrd). (a) Pnrd contains multiple transcription factor binding sites. Here are represented binding sites for IciA, Fis, DnaA and NrdR[START_REF] Han | Effect of IciA protein on the expression of the nrd gene encoding ribonucleoside diphosphate reductase in E. coli[END_REF][START_REF] Augustin | Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion[END_REF][START_REF] Rodionov | Identification of a bacterial regulatory system for ribonucleotide reductases by phylogenetic profiling[END_REF][START_REF] Torrents | NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes[END_REF]. The two AT-rich sequences (AT) flanking a 45-bp inverted repeat (IR45) are represented as grey boxes, and a region required for nrd cell cycle dependent expression is indicated as a hatched box(Sun et al., 1994[START_REF] Jacobson | A 45 bp inverted repeat is required for cell cycle regulation of the Escherichia coli nrd operon[END_REF]. Positions indicated are based on the +1 start site of transcription of Pnrd 110 bp upstream of the ATG codon of nrdA[START_REF] Tuggle | Regulation of the operon encoding ribonucleotide reductase in Escherichia coli: evidence for both positive and negative control[END_REF]. The start site of transcription of Px(Sun & Fuchs, 1994) is also represented. The main primers used in this study (PnrdF, PnrdR, PeF and PeR) are represented as grey arrows and detailed in supplementary Table1.(b) Sequence surrounding the DnaA binding sites. The -35 and -10 regions are underlined by a dotted line. The two nine-base sequences with homology to the DnaA consensus binding sequence, box1 and box2, are indicated as open boxes. A putative DnaA binding site identified by the Virtual Footprint software (dotted line box)[START_REF] Münch | Virtual Footprint and PRODORIC: an integrative framework for regulon prediction in prokaryotes[END_REF] and two NrdR binding sites found at the nrd promoter (light grey boxes)[START_REF] Rodionov | Identification of a bacterial regulatory system for ribonucleotide reductases by phylogenetic profiling[END_REF][START_REF] Torrents | NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes[END_REF] are also shown. The site directed mutations in the DnaA binding sites constructed in this study are shown. M49box1: T to A at position -49; M45-47box1: C to G at positions -45 and -47; M40-42box2: A to T and T to A at positions -42 and -40; M12-14GG: C to G at positions -12 and -14; M12-14AG: C to A at -12 and C to G at -14, and Moff: T to G at positions -7 and -9. 169x106mm (600 x 600 DPI)

Fig 3 .Fig 5 .

 35 Fig 1. Schematic representation of the E. coli nrdAB promoter (Pnrd). (a) Pnrd contains multiple transcription factor binding sites. Here are represented binding sites for IciA, Fis, DnaA and NrdR[START_REF] Han | Effect of IciA protein on the expression of the nrd gene encoding ribonucleoside diphosphate reductase in E. coli[END_REF][START_REF] Augustin | Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion[END_REF][START_REF] Rodionov | Identification of a bacterial regulatory system for ribonucleotide reductases by phylogenetic profiling[END_REF][START_REF] Torrents | NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes[END_REF]. The two AT-rich sequences (AT) flanking a 45-bp inverted repeat (IR45) are represented as grey boxes, and a region required for nrd cell cycle dependent expression is indicated as a hatched box(Sun et al., 1994[START_REF] Jacobson | A 45 bp inverted repeat is required for cell cycle regulation of the Escherichia coli nrd operon[END_REF]. Positions indicated are based on the +1 start site of transcription of Pnrd 110 bp upstream of the ATG codon of nrdA[START_REF] Tuggle | Regulation of the operon encoding ribonucleotide reductase in Escherichia coli: evidence for both positive and negative control[END_REF]. The start site of transcription of Px(Sun & Fuchs, 1994) is also represented. The main primers used in this study (PnrdF, PnrdR, PeF and PeR) are represented as grey arrows and detailed in supplementary Table1.(b) Sequence surrounding the DnaA binding sites. The -35 and -10 regions are underlined by a dotted line. The two nine-base sequences with homology to the DnaA consensus binding sequence, box1 and box2, are indicated as open boxes. A putative DnaA binding site identified by the Virtual Footprint software (dotted line box)[START_REF] Münch | Virtual Footprint and PRODORIC: an integrative framework for regulon prediction in prokaryotes[END_REF] and two NrdR binding sites found at the nrd promoter (light grey boxes)[START_REF] Rodionov | Identification of a bacterial regulatory system for ribonucleotide reductases by phylogenetic profiling[END_REF][START_REF] Torrents | NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes[END_REF] are also shown. The site directed mutations in the DnaA binding sites constructed in this study are shown. M49box1: T to A at position -49; M45-47box1: C to G at positions -45 and -47; M40-42box2: A to T and T to A at positions -42 and -40; M12-14GG: C to G at positions -12 and -14; M12-14AG: C to A at -12 and C to G at -14, and Moff: T to G at positions -7 and -9. 169x106mm (600 x 600 DPI)

Table 1 .

 1 Affinity (1/Kd) and cooperativity (n) values of DnaA-ATP binding to the nrdAB promoter on linear and supercoiled DNA as obtained from the analysis shown in Figure3.

	DNA	Region	Dissociation constant (Kd, nM)	Hill coefficient (n)
	_________________________________________________________________________
	Linear	DnaA box 1		21 ± 3	1,8 ± 0,4
		DnaA box 2		28 ± 4	1,8 ± 0,5
		DnaA-ATP box Down	103 ± 22	1,8 ± 0,6
	Supercoiled DnaA box 1		19 ± 2	2,0 ± 0,4
		DnaA box 2		23 ± 2	2,7 ± 0,6
	_________________________________________________________________________

Molecular Microbiology

Acknowledgments

This work was supported by the Agence Nationale pour la Recherche (projet ANR-JCJC-0113-01) and by the Association pour la Recherche sur le Cancer (ARC). We thank Ulisse Ferrari and Marco Cosentino Lagomarsino for the program for data analysis of the fluorimeter experiments and for useful discussions, Kirsten Skarstad for providing us with the DnaA-expressing strain WM2287, Nicole Boggetto of the Plate-forme Cytométrie en Flux of the Institut Jacques Monod, Paris, France, for technical assistance for the flow cytometry experiments.