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Originally published In Press as doi:10.1074/jbc.M402762200 on August 23, 2004

J. Biol. Chem., Vol. 279, Issue 44, 45546-45555, October 29, 2004
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DiaA, a Novel DnaA-binding Protein, Ensures the Timely Initiation of Escherichia coli Chromosome Replication*

Takuma Ishida{ddagger}, Nobuyoshi Akimitsu{ddagger}§, Tamami Kashioka{ddagger}, Masakazu Hatano{ddagger}||, Toshio Kubota**, Yasuyuki Ogata{ddagger}{ddagger}, Kazuhisa Sekimizu§, and Tsutomu Katayama§§

From the Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

Received for publication, March 11, 2004 , and in revised form, August 18, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The DnaA protein is the initiator of Escherichia coli chromosomal replication. In this study, we identify a novel DnaA-associating protein, DiaA, that is required for the timely initiation of replication during the cell cycle. DiaA promotes the growth of specific temperature-sensitive dnaA mutants and ensures stable minichromosome maintenance, whereas DiaA does not decrease the cellular DnaA content. A diaA::Tn5 mutation suppresses the cold-sensitive growth of an overinitiation type dnaA mutant independently of SeqA, a negative modulator of initiation. Flow cytometry analyses revealed that the timing of replication initiation is disrupted in the diaA mutant cells as well as wild-type cells with pBR322 expressing the diaA gene. Gel filtration and chemical cross-linking experiments showed that purified DiaA forms a stable homodimer. Immunoblotting analysis indicated that a single cell contains about 280 DiaA dimers. DiaA stimulates minichromosome replication in an in vitro system especially when the level of DnaA included is limited. Moreover, specific and direct binding between DnaA and DiaA was observed, which requires a DnaA N-terminal region. DiaA binds to both ATP- and ADP-bound forms of DnaA with a similar affinity. Thus, we conclude that DiaA is a novel DnaA-associating factor that is crucial to ensure the timely initiation of chromosomal replication.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In Escherichia coli, the timing of initiation of chromosomal replication during the cell cycle is strictly regulated (1). Only a few protein factors related to this regulation have been identified to date. The DnaA protein is the initiator of replication, and its nucleotide-bound forms change in coordination with the replication cycle (2–4). The cellular content of ATP-bound DnaA increases temporarily around the time of replication initiation (5). DnaA in its active ATP-bound form unwinds duplex in the initiation complex, which includes the origin (oriC) and DnaA multimers, so that DnaB helicase, DnaG primase, and DNA polymerase III holoenzyme can assemble replisomes on single-stranded DNA (4, 6). The level of DnaA-ATP decreases in a DNA replication-dependent manner due to hydrolysis of DnaA-bound ATP. The resultant ADP-DnaA is inactive. The DNA-loaded sliding clamp subunit of DNA polymerase III holoenzyme and Hda protein functionally interact with ATP-DnaA to induce hydrolysis (7, 8). This system for regulatory inactivation of DnaA is designated "RIDA1" and limits initiation to once per oriC per cell cycle. In dnaA mutant cells, the timing of initiation at oriC is disrupted during the cell cycle, even under conditions that allow the cells to grow (9, 10). This finding indicates that timely initiation requires strict regulation of DnaA activity

In E. coli, when the growth rate of cells is above a certain level, the next round of replication is initiated while the present round is still ongoing (1). These cells contain two or four copies, or four or eight copies of oriC, depending on the growth conditions. In wild-type cells, the initiation steps at multiple oriC sites are synchronized (9). In seqA mutant cells, this synchrony is disturbed, resulting in odd numbers of oriC copies in a cell (11). The dam gene product, Dam, methylates the adenine residue in the palindromic sequence GATC that is repeated 11 times within the minimal oriC region (12). SeqA preferentially binds to these hemimethylated GATC sequences, which inhibits re-initiation at oriC (11, 13–16). Semi-conservative replication of fully methylated oriC DNA yields hemimethylated oriC, which is sustained for a while by SeqA until Dam re-methylates these sites. Lack of SeqA accelerates the timing of re-methylation of oriC (11). Asynchronous initiation is also observed in dam mutants (17). Moreover, cells expressing mutants of dnaC and Histone-like proteins, HU, IHF, and FIS, display an asynchronous replication phenotype during exponential growth (18–21). These histone-like proteins may participate in initiation complex formation at oriC (21–23).

As specified above, strict regulation of oriC and DnaA functions is necessary to ensure precise initiation timing. In this study, we isolated a suppressor gene from an overinitiation-type dnaA mutant, dnaAcos (24–27). Our data show that this previously uncharacterized gene is required to ensure the timely initiation of chromosomal replication. A mutant bearing an inactive form of this gene displays an asynchronous replication phenotype. Moreover, the encoded protein directly and specifically binds DnaA. We designate this novel gene diaA, representing "DnaA initiator-associating factor."


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains, Plasmids, Media, and Buffer—E. coli K-12 strains used for genetic experiments are described in Table I. The recA::Tn10 mutation was introduced via P1 transduction. Transductants of recipient strains bearing wild-type diaA and diaA::Tn5 mutations were obtained with similar efficiency. The recA mutation was confirmed by measuring its sensitivity to UV. pKP1673, a gift from Dr. Takeyoshi Miki, is a low copy mini-R vector containing genes for active partition and chloramphenicol resistance (30). For plasmid complementation experiments, a 3.3-kb HpaI fragment bearing the diaA gene (yraO) was isolated from a Kohara {lambda} phage #518 (31) and inserted into the StuI site of pKP1673, resulting in pNA095. A 1.2-kb HpaI-BssHII fragment containing the entire diaA gene was similarly isolated, the BssHII end was filled in, and the resultant fragment was inserted to the StuI site of pKP1673, generating pNA102. The diaA gene within pNA102 was digested with MluI, filled in, and self-ligated to produce pNA121. The 1.2-kb HpaI-BssHII fragment was similarly filled in and inserted into the HincII site of pUC19. From the resultant plasmid (pNA159), a 1.2-kb EcoRI-PstI fragment containing diaA was isolated and inserted into the corresponding restriction sites of pT7-5, generating pKA251. For protein purification, BL21(DE3) cells containing pKA251 were used for the overproduction of DiaA. The filled-in 1.2-kb HpaI-BssHII fragment was additionally inserted into the EcoRV site of pBR322 and designated pNA135. pTOA5 (2.7 kb) and pTOA24 (6.1 kb) minichromosomes bearing oriC-mioC and gidA-oriC-mioC regions, respectively, in addition to the {beta}-lactamase gene, were kindly provided by Dr. Tohru Ogawa (32). The media used are described in a previous report (24, 26, 33). M9 medium was supplemented with 0.2% glucose, 2 µg/ml thiamine, 20 µg/ml tyrosine, and 40 µg/ml each of isoleucine, valine, threonine, methionine, and tryptophan. Unless otherwise indicated, the medium was supplemented with 50 µg/ml thymine. M13oriCE10 and pBSoriC (or pTB101) are derivatives of M13mp10 and pBluescript vectors, respectively, containing a minimal oriC region (34). Buffer C contained 40 mM Hepes-KOH (pH 7.6), 1 mM EDTA, 2 mM dithiothreitol, and 15% glycerol. Buffer C' represents buffer C containing 10% glycerol. Buffer C'' contained 50 mM Hepes-KOH (pH 7.6), 1 mM EDTA, 2 mM dithiothreitol, and 20% sucrose.


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  		TABLE I
Strains used in this study

 
Isolation of dnaAcos Suppressors Using Transposon Mutagenesis and Determination of the Insertion Site—A previously described procedure was employed (26, 27). Briefly, defective {lambda} phage containing Tn5 ({lambda}467) was introduced into NA001 (dnaAcos) at 42 °C, and Tn5 was transposed onto the chromosome. After the strain was cured of {lambda} phage, kanamycin-resistant colonies were isolated and purified at 30 °C. Tn5 in isolated suppressor mutants was transduced back into NA001 using P1 phage for confirmation of growth at 30 °C. One of the resultant transductants was designated NA026. The BamHI fragment of NA026 chromosomal DNA, which includes part of Tn5 encoding the kanamycin-resistant gene and the border region between Tn5 and the chromosome, was cloned into pUC18. The border region was sequenced, as described previously (26, 27).

Immunoblotting Analysis—Immunoblot analysis for DiaA was basically similar to that for DnaA (25), except that polyclonal rabbit anti-DiaA antiserum and NA141 (diaA::Tn5) were used. Briefly, cells were grown exponentially at 37 °C in LB medium. At an optical density (A595) of 0.8, 1-ml aliquots were withdrawn, and 5% trichloroacetic acid immediately added. Precipitates formed on ice were collected by brief centrifugation and used for SDS-polyacrylamide (12%) gel electrophoresis. Separated proteins were blotted onto Immobilon-P membrane (Millipore) and detected using polyclonal rabbit anti-DiaA antiserum and alkaline phosphate-conjugated anti-rabbit antiserum (Bio-Rad).

Flow Cytometry Analysis—Flow cytometry was performed according to an earlier report (26, 27, 33). Briefly, cells were grown exponentially for about 10 generations at 30 °C in the indicated media with serial dilutions until an optical density (A660) of 0.2. Incubation was further continued in the presence of rifampicin (150 µg/ml) and cephalexin (10 µg/ml) for 4 h at the same temperature. Cells were collected by brief centrifugation and suspended in cold 70% ethanol. After washing and suspension of cells in cold buffer containing 10 mM Tris-HCl (pH 7.5) and 20 mM magnesium sulfate, chromosomal DNA was stained with mithramycin (27 µg/ml) and ethidium bromide (5 µg/ml). Cells were analyzed with a BRYTE-HS flow cytometer (Bio-Rad). dnaA46 cells incubated at 42 °C and containing one or two chromosomes per cell were used to calibrate the chromosome number.

Overproduction and Purification of DiaA—BL21(DE3) cells bearing pKA251 were grown at 37 °C in LB medium (4.8 liters) containing ampicillin (50 µg/ml). Isopropyl-1-thio-{beta}-D-galactosidase (1 mM) was added at an optical density (A660) of 0.8, and incubation continued for 4 h. Cells were harvested by centrifugation at 4 °C, resuspended to an optical density (A595) of 200 in cold buffer containing 25 mM Hepes-KOH (pH7.6), 1 mM EDTA, and 2 mM dithiothreitol, and frozen in liquid nitrogen for storage at –80 °C. Frozen cells were thawed at 4–8 °C, incubated on ice for 30 min in the presence of 100 mM KCl and 300 µg/ml lysozyme, and frozen in liquid nitrogen. All the procedures described below were performed at 0–6 °C. Frozen cell paste was thawed, and supernatant fractions obtained by centrifugation in a Beckman type 50.2 Ti rotor at 40,000 rpm for 20 min. Proteins in the supernatant (fraction I, 21.5 ml) were precipitated with 0.22 g/ml ammonium sulfate, collected by centrifugation, and resuspended in chilled buffer C (fraction II, 1.5 ml). The solution was dialyzed against buffer C and diluted in the same buffer to 2 mg protein/ml. Supernatant (44 ml) was obtained by centrifugation, and a 40-ml aliquot was loaded onto a hydroxylapatite column (18 ml) equilibrated with buffer C at a flow rate of 18 ml/h. The column was washed with three volumes of buffer C, three volumes of buffer C containing 1 M KCl, and two volumes of buffer C containing 1 mM potassium phosphate. Proteins were eluted with a linear gradient (180 ml) from 1 to 300 mM potassium phosphate in buffer C. DiaA was detected in fractions containing 10–20 mM potassium phosphate (fraction III, 10 ml). Next, fraction III was dialyzed against buffer C containing 50 mM KCl and loaded onto a Mono Q column (bed volume of 1 ml, Amersham Biosciences) equilibrated with the same buffer at a flow rate of 0.2 ml/min. The column was washed with three volumes of buffer C containing 50 mM KCl, followed by five volumes of buffer C containing 200 mM KCl. Proteins were eluted with a linear gradient (10 ml) from 200 to 600 mM KCl in buffer C. DiaA protein was eluted at around 350 mM KCl (fraction IV, 1.6 ml).

Chemical Cross-linking of DiaA—DiaA (5 µg) was incubated at room temperature for 3 h in buffer (25 µl) containing 40 mM Hepes-KOH (pH7.6), 40 mM KCl, 1 mM EDTA, 2 mM dithiothreitol, 20% sucrose, and the indicated amounts of glutaraldehyde. Protein was precipitated by incubation on ice in the presence of 10% trichloroacetic acid. Precipitates were collected, dissolved in 40 µl of standard SDS sample buffer, and incubated at 100 °C for 5 min. Aliquots (10 µl) were subjected to SDS-polyacrylamide (12%) gel electrophoresis, and proteins were stained with Coomassie Brilliant Blue.

Gel Filtration of DiaA Protein—DiaA protein (200 µl of fraction IV, 3.1 mg/ml) was loaded on a Superdex 75 HR 10/30 column (Amersham Biosciences) equilibrated with buffer C containing 150 mM KCl at 4–6 °C at a flow rate of 0.3 ml/min. Using the same buffer and flow rate, DiaA was eluted. The elution profile was monitored by absorbance at 280 nm.

In Vitro Minichromosome Replication and RIDA Assay—A replicative crude extract was prepared from WM433 (dnaA204) and TK24 (WM433 diaA::Tn5), using the method described previously (7, 25, 28). Using the indicated amounts of extracts and DnaA protein, M13oriCE10 minichromosome RFI (200 ng; 600 pmol as nucleotide) was incubated in reaction (25 µl), as described previously (25, 28, 35). The RIDA reaction was promoted as above, except that [{alpha}-32P]dTTP was excluded and [{alpha}-32P]ATP-DnaA was added (7, 8, 35). Nucleotide-bound forms of DnaA were assessed by immunoprecipitation and thinlayer chromatography, in accordance with earlier reports (7, 8).

Assays for Specific Binding of DiaA to DNA-bound DnaA—Gel filtration experiments were performed as follows: DnaA protein (11.5 pmol) and pBSoriC (0.43 pmol) were incubated for 5 min at 30 °C in buffer C (25 µl) containing 50 mM KCl. Fraction II (1 µg) prepared from DiaA-overproducing cells was added. After further incubation for 5 min at 30 °C, the reaction was loaded onto a Microspin S-400HR spin column (Amersham Biosciences) equilibrated with the same buffer, and the void fraction was obtained by centrifugation (3000 rpm, 2 min) at room temperature. Control experiments in which DnaA, pBSoriC, or DiaA were excluded from the reaction, were additionally performed. Eluted proteins were analyzed by SDS-polyacrylamide (12%) gel electrophoresis and silver staining. Similar results were obtained in the presence of 150 mM KCl.

A pull-down assay using a biotin-conjugated polynucleotide was performed as we described previously (36), except that DiaA was included in the binding reaction. Briefly, the polynucleotide used was doublestrand DNA (29-mer) containing DnaA box R1 with a 5'-biotin-conjugated 30-mer T-stretch tag. DnaA protein (10 pmol) and biotin-tagged DnaA box DNA (5 pmol) were incubated for 5 min on ice in buffer C' containing 100 mM KCl (50 µl). DiaA protein (2.4 pmol of dimer or the indicated amounts) was added, and incubation continued for 5 min on ice. Streptavidin-coated magnetic beads in suspension (50 µl; Promega) were washed twice in buffer C' and resuspended in an equal volume of buffer C' containing 100 mM KCl and bovine serum albumin (100 µg/ml), then added to the reaction mixture, and incubation continued for 1 h at 4 °C with gentle rotation. Streptavidin-coated beads and bound materials were collected using magnetic force, washed in buffer C' containing 100 mM KCl and 100 µg/ml bovine serum albumin, and suspended in standard SDS sample buffer (20 µl). Beads were excluded as above, and proteins were analyzed using SDS-polyacrylamide gel electrophoresis. The buffer for washing included the same nucleotide at a concentration of 1 µM when nucleotide-bound DnaA was used.

When a cleared soluble extract (fraction I) prepared from WM433 was used instead of DiaA, the above method was applied, except for the addition of buffer C'' containing 150 mM KCl (instead of buffer C' containing 100 mM KCl). Briefly, DnaA protein (20 pmol) and biotin-tagged DnaA box DNA (20 pmol) were incubated for 5 min on ice in buffer C'' (200 µl) containing 150 mM KCl. DnaA bound to DNA was isolated using streptavidin beads as above, suspended in buffer C'' (160 µl) containing 400 µg of WM433 fraction I and 40 mM KCl, and incubated for 1 h at 4 °C with gentle rotation. Beads bearing bound materials were collected, washed in 160 µl of buffer C'' containing 150 mM KCl and bovine serum albumin (100 µg/ml), and suspended in SDS sample buffer (80 µl). Aliquots (20 and 50 µl) of this sample were subjected to SDS-polyacrylamide gel electrophoresis as above and immunoblotted using anti-DiaA antiserum, respectively. The WM433 fraction I was obtained with the method of Fuller et al. (28), except that buffer C'' containing 250 mM KCl, 20 mM spermidine-HCl, and 300 µg/ml lysozyme was employed for cell lysis.

Construction of Overproducing Plasmids of Biotin-tagged DnaA Proteins—The full-length DnaA-coding region (1.4 kb) was amplified by PCR from pKA234 (29) using primers, 5'-GCGGATCCGTGTCACTTTCGCTTTG (TAKU5) and 5'-GCGGTACCCTTACGATGACAATGTTCTG (TAKU6). The resultant fragment was ligated to pAN6 vector (Avidity) using the BamHI and KpnI sites, resulting in pTKM12. A 1.5-kb region was amplified by PCR from pTKM12 using primers, 5'-CTAGTCTAGATTTAAGAAGGAGATATACATATGTCCGGCCTGAACGAC (KWdN5XSA) and 5'-CTTCTCTCATCCGCCAAAACAG (sim16). The resultant fragment was ligated to pBAD18 using the XbaI and HindIII sites, resulting in pST11-2, which was an overproducer of the DnaA conjugated with the biotin ligase recognition peptide (bioDnaA).

A 0.45-kb region was amplified by PCR from pST11-2 using primers, KWdN5XSA, and 5'-CGGAATTCTTAACGATAGGTCGGTTCTGCC (TAKU12). The resultant fragment was digested with XbaI and ligated to pBAD18 using the NheI and SmaI sites, resulting in pTKM13, which was an overproducer of the DnaA domains I–II conjugated with the biotin ligase recognition peptide (bioDAD I–II).

A 5.6-kb region was amplified by PCR from pST11-2 using primers, 5'-GGATCCTCGAGCTCCCGGCG (TAKU68) and 5'-TCTAACGTAAACGTCAAACACACG (TAKU69). The resultant fragment was self-ligated, resulting in pTKM14, which was an overproducer of the DnaA domains III–IV conjugated with the biotin ligase recognition peptide (bioDAD III-IV). All constructions were confirmed by nucleotide sequencing.

Overproduction and Purification of Biotin-tagged DnaA Proteins— MC1061 cells bearing pBirA (Avidity) and pST11-2 were grown at 37 °C in 2.4 liter of LB medium containing ampicillin (100 µg/ml) and chloramphenicol (20 µg/ml). When an optical density (A660) of the culture reached 0.4–0.6, 1% L(+)-arabinose, 1 mM isopropyl-1-thio-{beta}-D-galactosidase, and 50 µM (+)-biotin were included and incubation was further continued for 3 h. Cells were harvested by centrifugation and resuspended in buffer C'' as described previously for purification of DnaA (37, 38). MC1061 cells bearing pBirA and pTKM13 or pTKM14 were similarly used.

BioDnaA and bioDAD III–IV were purified from the overproducing cells, using the same procedures as those of the wild-type DnaA that we previously described (38) except that proteins in fraction I were precipitated in buffer containing 0.21 g/ml and 0.28 g/ml ammonium sulfate for bioDnaA and bioDAD III–IV, respectively.

For purification of bioDAD I–II, cleared lysates (fraction I) were prepared from MC1061 cells bearing pBirA and pTKM13, as described for purification of wild-type DnaA (37, 38). Proteins in fraction I were loaded at on a column (3-ml bed volume) containing SoftLink-SoftRelease-Avidin Resin (Promega) pre-equilibrated with buffer C'' containing 250 mM KCl, using a flow rate of 4 ml/h. Flow-through fractions were reloaded on the column. These loading procedures were repeated twice more. After the column was washed using 33 ml of buffer C'' containing 250 mM KCl, bioDAD I–II was eluted using buffer C'' containing 250 mM KCl and 5 mM (+)-biotin. Fractions containing bioDAD I–II were collected, and bioDAD I–II was further purified using a Superose 12 HR10/30 gel-filtration column pre-equilibrated with buffer D (36). All procedures using columns were done at 4–6 °C. Purity of DnaA proteins was >90% as judged by SDS-polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining.

DiaA-bioDnaA Binding Assay—DiaA (420 ng, 10 pmol as a dimer) was incubated for 5 min on ice in buffer C'' (150 µl) containing 100 mM KCl, 100 µg/ml bovine serum albumin, and 1 µM ATP in the presence or absence of bioDnaA (2.2 µg, 40 pmol), bioDAD I–II (680 ng, 40 pmol), or bioDAD III–IV (1.6 µg, 40 pmol). The mixture was further incubated for 1 h at 4 °C with gentle rotation in the presence of the streptavidin-conjugated magnetic beads (Promega) equilibrated in the same buffer (50 µl) as above. The beads and bound materials were collected using magnetic force and washed three times using buffer C'' (100 µl) containing 100 mM KCl. Protein bound to biotinylated proteins was eluted in 10 µl of standard SDS sample buffer and analyzed using SDS-polyacrylamide (13%) gel electrophoresis and Coomassie Brilliant Blue staining.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of a Novel Gene That Suppresses dnaAcos— The dnaAcos cells induce overreplication of chromosomal DNA at 30 °C, resulting in inhibition of colony formation (24, 25). We previously isolated a number of cold-resistant suppressor mutants from a dnaAcos strain (NA001) by random transposon mutagenesis (26). In this study, the Tn5 transposon in a suppressor mutant was inserted into a functionally uncharacterized ORF, yraO (b3149 or o196) (Fig. 1A). For identification of the ORF, the Tn5-chromosome boundary region was sequenced. When the transposon was introduced back into the parental dnaAcos mutant by P1 phage-mediated transduction, all resultant transductants grew at 30 °C and 42 °C on LB agar medium with similar colony forming efficiencies as well as the original suppressor mutant. One of these transductants was designated NA026, and used for further experiments.



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  		FIG. 1.
Identification of the diaA gene. A, chromosome-derived regions cloned in pKP1673 (a mini-R vector) are depicted as thick bars. ORF regions and direction of transcription are represented by open arrows. A part of the nucleotide sequence of diaA with the site of Tn5 insertion is shown. A restriction map is additionally included below. "+" indicates that dnaAcos cold sensitivity is suppressed by introduction of the indicated plasmid. "–" indicates that cold sensitivity is not affected. Cs, cold-sensitive; Cr, cold-resistant. B, cells were grown overnight in LB medium containing chloramphenicol (12.5 µg/ml) at a permissive temperature (30 °C or 42 °C), diluted in saline to 107–8 cells/ml, and streaked on LB agar plates with (LB+Cm) or without (LB) chloramphenicol (12.5 µg/ml). Next, plates were incubated for 25 h at 30 °C or 20 h at 42 °C. The position of each strain on the plates is indicated. KH5402-1 (wild-type); NA001 (dnaAcos); NA026 (dnaAcos diaA::Tn5); pKP1673 (mini-R vector); pNA102 (pKP1673-wild-type diaA); pNA121 (pKP1673-diaA bearing a frameshift mutation).

 
Next, we performed the plasmid complementation test using a mini-R plasmid-derivative low copy number vector (pKP1673) to confirm the gene responsible for suppression of dnaAcos cold sensitivity (Fig. 1, A and B). NA026 cells containing the pKP1673 vector grew on LB plates at 30 °C, whereas growth of NA026 cells containing intact yraO-containing plasmid (pNA102) was severely inhibited at 30 °C on LB plates with and without chloramphenicol (Fig. 1B). The introduction of mutagenized yraO (pNA121) did not affect growth of NA026 at 30 °C, similar to data observed with the pKP1673 vector. Using the maxicell method (39), we observed that yraO indeed encodes a 21-kDa protein (data not shown). Based on these findings, we conclude that yraO is required to support dnaAcos cold sensitivity. The novel gene is designated "diaA."

DiaA Stimulates Initiation at oriC—When dnaAcos cells induce overreplication of chromosomal DNA at 30 °C, cell division is arrested, resulting in filamentous cells and inhibition of colony formation (24, 25, 33). Suppressor genes isolated from the dnaAcos mutant can be classified into two groups. One group blocks overinitiation of replication, thereby promoting cell division (26, 27), whereas the other group allows cells to divide without repressing chromosomal overreplication (33).

To determine whether diaA::Tn5 inhibits initiation at oriC, we investigated whether the mutant suppresses the growth of temperature-sensitive dnaA mutants at intermediate temperatures between those that are permissive and restrictive (Table II). Temperature-sensitive dnaA mutants used in this study are initiation-defective, and colony formation is inhibited at 39 °C or higher temperatures. Upon P1 transduction, diaA::Tn5 was introduced at 30 °C into dnaA mutants and the parental wild-type dnaA strain with similar efficiency, as determined using plaque-forming unit of the phage lysate. Colony formation of the dnaA46 mutant was severely inhibited at 35 °C and 37 °C, following the introduction of diaA::Tn5. Moreover, this inhibition was totally complemented by a low copy plasmid bearing the wild-type diaA gene, pNA102, indicating that diaA itself is required for growth at intermediate temperatures. Similar inhibition was clearly observed for the dnaA5 and dnaA601 mutants, whereas the growth of the dnaA508 and dnaA167 mutants was not significantly suppressed. Thus, diaA::Tn5-dependent inhibition of colony formation of dnaA mutants was observed in a dnaA allele-specific manner.


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  		TABLE II
The diaA gene stimulates growth of temperature-sensitive dnaA mutants in an allele-specific manner

Cells of each strain grown overnight at 30 °C in LB medium were plated on LB plates, and incubated for 20 h at 30 °C or 20 h at 42 °C. Chloramphenicol was included in the medium when plasmid was present. Followings are mutation sites in dnaA alleles (40): dnaA46 (A184V, H252Y), dnaA5 (A184V, G426S), dnaA601 (A184V, P296Q), dnaA508 (P28L, T80I), and dnaA167 (V157E).

 
Next, we analyzed whether the diaA mutation impairs initiation, even in cells bearing the wild-type dnaA gene, by assessing the stability of minichromosome maintenance (Table III). The minichromosomes employed in our study are pTOA5 and pTOA24 (32), which bear the bla gene and the oriC region (see "Experimental Procedures"). Plasmids were transformed at 30 °C into wild-type diaA and diaA::Tn5 strains bearing the recA mutation that inhibits homologous recombination between plasmid and the chromosome. Transformation efficiency of these plasmids into diaA::Tn5 (NA173) was similar to that observed with the wild-type strain (NA169), as compared using pBR322, as a standard. However, upon further incubation of single transformant colonies in LB medium overnight in the absence of ampicillin, only less than 1% of cells of the diaA mutant strain contained minichromosomes, whereas the wild-type diaA strain relatively stably maintained minichromosomes (Table III). This low stability of minichromosomes in the diaA mutant was effectively complemented by a mini-R plasmid bearing the wild-type diaA gene. The data confirm that the diaA itself is required for stable minichromosome maintenance.


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  		TABLE III
DiaA is required for stable maintenance of minichromosomes

Transformants of minichromosomes were obtained at 37 °C on LB plates containing ampicillin (20 µg/ml) and purified in the same medium. Cells containing the indicated plasmids were grown overnight at 37 °C in LB medium (5 ml) without ampicillin, plated on LB plates with (+) or without (-) ampicillin, and incubated for 20 h at 37 °C. Chloramphenicol (12.5 µg/ml) was constantly included in the above media.

 
We further investigated whether the reduced initiation potential of the diaA mutant can be attributed to a decrease in the cellular DnaA content. Cells were grown at 30 °C in Tryptone medium. Immunoblotting data revealed that the DnaA content was not decreased by the diaA mutation (Table IV). The diaA mutant and its parental wild-type strain grew at similar rates. Moreover, relative cell sizes between the two strains were similar.


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  		TABLE IV
Growth and cellular DnaA content of the diaA mutant

Cells were exponentially grown at 30 °C in Tryptone medium and were harvested when the optical density (A660) reached 0.1. Immunoblotting analysis for DnaA was performed using a previously described method (26). The DnaA amount shown is the mean of independent two measurements.

 
Suppression of dnaAcos Is Independent of seqA—SeqA binds preferentially to hemimethylated oriC DNA and inhibits remethylation and replicational initiation of oriC (2). We thus speculated whether the SeqA function is abnormally facilitated by the lack of diaA, which results in the inhibition of initiation at oriC and suppression of dnaAcos. However, suppression of cold-sensitive growth was still observed upon introduction of a seqA-null mutation into the dnaAcos diaA double mutant (Table V).


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  		TABLE V
diaA suppression of dnaAcos is independent of seqA

P1 transduction was performed using P1vir phage propagated in MM294 containing seqA::Tn10 (26). Transductants were obtained on LB plates containing tetracycline (12.5 µg/ml).

 
DiaA Is Required for the Timely Initiation of Chromosomal Replication—Next, we determined whether diaA functions in replication control, using flow cytometry. To analyze the oriC gene dosage in each growing cell, rifampicin and cephalexin were included to allow ongoing replication forks to complete the entire replication cycle in each cell (26, 27, 33, 41). The wild-type parental strain (KH5402-1) and its diaA derivative (NA141) grown at 30 °C in various media displayed similar doubling times, specifically, about 60 min in Tryptone medium, 145 min in M9 medium supplemented with glucose, and 50 min in LB medium containing glucose. Furthermore, the average cell sizes of the two strains were similar in each medium.

Flow cytometry data revealed that wild-type cells grown in Tryptone medium contained two or four oriCs per cell (Fig. 2A), whereas a considerable proportion of the diaA::Tn5 mutant cells displayed odd numbers of oriC per cell (Fig. 2B). Similar results were obtained when cells were grown in supplemented M9 medium (Fig. 2, C and D). Moreover, in the mutant strain, cells bearing four origins were decreased, and those containing fewer origins were relatively increased. Results obtained using LB medium containing glucose were also well consistent with these data (Fig. 2, E and F).



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  		FIG. 2.
Timely initiation of replication is inhibited in the diaA mutant. KH5402-1 (wild-type) (A, C, and E) and NA141 (diaA::Tn5) cells (B, D, and F) were grown exponentially at 30 °C in Tryptone medium (A and B), supplemented M9 medium (C and D) and LB medium containing 0.2% glucose (E and F). At an optical density (A660) of 0.2, rifampicin and cephalexin were added to the cultures, and incubation was further continued for 4 h. Cell size and DNA content were quantified with a flow cytometer, as described under "Experimental Procedures." At least 20,000 cells were surveyed. TR, Tryptone medium; M9, supplemented M9 medium; LBG, LB medium containing glucose.

 
The abnormal initiation mode in the diaA mutant was repressed through complementation of the wild-type diaA gene by a mini-R plasmid (Fig. 3). The results collectively indicate that timely initiation at multiple oriCs in a single cell requires diaA.



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  		FIG. 3.
DiaA is required for the timely initiation of chromosomal replication. Cells of NA141 (diaA::Tn5) containing the pKP1673 mini-R vector (A, C, and E) or pNA102, a pKP1673-derivative carrying wild-type diaA (B, D, and F) were grown exponentially at 30 °C in Tryptone medium (A and B), LB medium (C and D), or LB medium containing 0.2% glucose (E and F). Cells were analyzed by flow cytometry, as described for Fig. 2. TR, Tryptone medium; LB, LB medium; LBG, LB medium containing glucose; vector, pKP1673; pdiaA+, pNA102 (pKP1673 bearing the wild-type diaA).

 
We further analyzed whether overexpression of DiaA affects the initiation timing of chromosomal replication. When cells bearing the diaA gene cloned into pBR322 were similarly analyzed by flow cytometry, we observed inhibition of synchronous initiation at oriC (Fig. 4).



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  		FIG. 4.
DiaA oversupply disrupts timely initiation. Cells of KH5402-1 (wild-type) containing the pBR322 vector (A and C) or pNA135, a pBR322 derivative containing wild-type diaA (B and D) were exponentially grown at 30 °C in Tryptone medium (A and B), or LB medium containing 0.2% glucose (C and D). Cells were analyzed by flow cytometry, as described for Fig. 2.

 
Purification of DiaA Protein, Homodimer Formation, and Cellular Content—To analyze the function of DiaA in vitro, we overexpressed and purified the protein (Fig. 5A and Table VI). Using the Edman degradation method and purified DiaA protein, the N-terminal amino acid sequence was determined as "MQERIKA," identical to that of the yraO/b3149 ORF. Results of gel filtration and chemical cross-linking experiments consistently indicate that DiaA forms a homodimer in solution. The molecular mass of DiaA protein estimated by gel filtration was about 45 kDa (Fig. 5B). Following incubation of purified DiaA in the presence of a cross-linking agent, SDS-polyacrylamide gel electrophoresis revealed a complex with a molecular mass of about 40 kDa in addition to monomeric DiaA protein (21 kDa) (Fig. 5C). Complexes with higher masses (corresponding to oligomers) were not observed.



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  		FIG. 5.
Purification and dimer formation of DiaA. A, overexpressed DiaA protein was purified as described in "Experimental Procedures." Proteins (3 µg) in fraction II (ammonium sulfate precipitates), fraction III (hydroxylapatite purification step), and fraction IV (Mono Q purification step) were analyzed by SDS-polyacrylamide (12%) gel electrophoresis and Coomassie Brilliant Blue staining. B, DiaA protein was analyzed using a Superdex 75 gel-filtration column. Eluted DiaA is represented by a broken line, and the corresponding molecular size is signified by an arrow. The molecular size markers (MW) used are: phosphorylase B (97 kDa), bovine serum albumin (68 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa). C, DiaA protein was incubated in buffer containing the indicated amounts of glutaraldehyde (see "Experimental Procedures"). Protein was precipitated in 10% trichloroacetic acid and analyzed by SDS-polyacrylamide (12%) gel electrophoresis and Coomassie Brilliant Blue staining. D, the cellular content of DiaA protein was quantified by immunoblotting. Cells of KH5402-1 (wild-type) and NA141 (diaA::Tn5) were grown in LB medium at 30 °C until an optical density (A595) of 0.8, chilled immediately on ice, and fixed in 10% trichloroacetic acid. Proteins corresponding to 1-ml aliquots of the cultures (including 3.0 x 108 cells) were analyzed by SDS-polyacrylamide (12%) gel electrophoresis and immunoblotting, using rabbit polyclonal anti-DiaA antiserum. The indicated amounts of purified DiaA protein were also used as a quantitative standard. The number of DiaA dimers in cells was calculated using cell numbers.

 


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  		TABLE VI
Purification of DiaA protein

DiaA protein was overexpressed and purified, as described under "Experimental Procedures." One unit of DiaA activity promoted the incorporation of 1 pmol of nucleotide/min in an in vitro minichromosome replication system, as described in Fig. 6.

 
Next, we estimated the amounts of DiaA in exponentially growing cells at 30 °C in LB medium. Immunoblotting analysis revealed a band corresponding to DiaA protein in the wild-type strain, but not the diaA::Tn5 mutant (Fig. 5D). By comparison with the amounts of purified DiaA, the number of DiaA dimers per cell was estimated at about 280.

DiaA Stimulates Replication of Minichromosomes in Vitro— Using the diaA::Tn5 mutant (TK24) and its parental strain bearing wild-type diaA (WM433), we prepared a crude protein fraction that was active for minichromosome replication in a DnaA-dependent manner (28). The replicative fraction obtained from the diaA mutant was consistently about 50% less active for oriC replication, compared with that from the wild-type parent strain (Fig. 6, A and B). Furthermore, addition of DiaA restored the replicative activity of the diaA mutant extract almost to wild-type levels, in a dose-dependent manner. However, the activity of the wild-type extract was not stimulated by additional DiaA protein (Fig. 6C and Table VI). This DiaA stimulation was especially prominent within a range in which levels of DnaA added are limited and replication activity is responsible to the amount of DnaA protein added to the extract (Fig. 6D). These results suggest that DiaA plays a stimulatory role in replication from oriC, in agreement with the in vivo data.



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  		FIG. 6.
DiaA stimulates DnaA-dependent replication of minichromosome in vitro. Replicative crude extracts prepared from WM433 (dnaA204) and TK24 (dnaA204 diaA::Tn5), a derivative of WM433, were used for replication of a minichromosome (M13oriCE10) in the presence of the indicated amounts of DnaA and DiaA (A–D). Hydrolysis of DnaA-bound ATP observed in this reaction system was additionally assessed (E and F) (see "Experimental Procedures"). A, the indicated amounts of replicative extracts prepared from WM433 or TK24 were used. Reactions were incubated for 20 min at 30 °C in the presence of 0.5 pmol of DnaA. B, the time course of reaction was analyzed. The reaction contained 1 pmol of DnaA, and 300 µg of replicative extracts prepared from WM433 or TK24. C, the reaction was incubated for 20 min at 30 °C in the presence of 1 pmol of DnaA, 250 µg of the replicative extract prepared from WM433 or TK24, and the indicated amounts of DiaA. D, the reaction was incubated for 20 min at 30 °C in the presence of 0.23 pmol of DiaA dimer and the indicated amounts of DnaA. E, [{alpha}-32P]ATP-bound DnaA (1 pmol) was incubated for 20 min at 30 °C in the presence of the indicated amounts of replicative extracts prepared from WM433 or TK24. Nucleotide-bound DnaA was isolated by immunoprecipitation, and bound nucleotides were analyzed by polyethyleneimine thin-layer chromatography (7, 8). F, the time course of experiments shown in panel E was analyzed using 300µg of replicative extracts.

 
The stimulation of oriC replication may additionally be attributed to the inhibition of RIDA in the diaA mutant extract. In the replication system used here, DnaA-bound ATP is hydrolyzed by RIDA to yield ADP-DnaA (7, 8, 34). However, DnaA-ATP hydrolysis by RIDA was not significantly inhibited in diaA mutant extracts under the conditions used for oriC replication (Fig. 6, E and F).

Direct and Specific Interactions between DnaA and DiaA— Using gel filtration experiments and a pull-down assay, we investigated whether DiaA directly binds to DnaA. Plasmid bearing oriC was incubated in the presence or absence of DnaA protein and partially purified DiaA fraction. Proteins that bound the plasmid were isolated using a gel filtration spin column and analyzed by SDS-polyacrylamide gel electrophoresis. The results indicate that, under the conditions that DnaA protein was eluted in an oriC plasmid-dependent manner, DiaA elution was dependent on both DnaA protein and oriC plasmid (Fig. 7A).



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  		FIG. 7.
DiaA binds to DnaA complexed with DnaA box. Specific binding of DiaA to DnaA was analyzed using gel filtration (A) and pull-down methods (B and C). For details of conditions, see "Experimental Procedures." A, reactions (25 µl) were incubated for 5 min at 30 °C in the presence (+) or absence (–) of pBSoriC (0.43 pmol), DnaA (11.5 pmol), and fraction I proteins (1 µg) obtained from DiaA-overexpressed cells (DiaA), and loaded onto an S-400HR Microspin column. Proteins bound to pBSoriC were isolated by brief centrifugation and analyzed by SDS-polyacrylamide (12%) gel electrophoresis and silver-staining. Proteins detected on the gel are specified. B, reactions were incubated on ice in the presence (+) or absence (–) of biotin-tagged DnaA box DNA (DnaA box; 5 pmol), purified DiaA protein (2.4 pmol as dimer), and the indicated forms of DnaA (10 pmol). Materials bound to DnaA box-bearing DNA were isolated using streptavidin-conjugated magnetic beads. Isolated proteins were analyzed as above. Bovine serum albumin (BSA) was included in the reaction and washing buffer. C, experiments similar to B were performed using the indicated amounts of DiaA. Isolated proteins were estimated on the gel using quantitative standards. The ratio of DiaA dimer to recovered DnaA is shown. D, experiments similar to those of panel B were performed using a soluble protein extract (fraction I) prepared from WM433 (dnaA204), instead of purified DiaA. Proteins co-isolated with beads were analyzed as above (PAGE). Results of immunoblotting experiments are additionally depicted (IMM). The migration position of DiaA is specified with an asterisk.

 
Next, we performed a pull-down assay using streptavidin-conjugated magnetic beads and a biotin-tagged DNA fragment containing DnaA box R1 (36). In all cases where nucleotide-unbound, ATP-bound, and ADP-bound DnaA were used, DiaA was precipitated in a DnaA-dependent manner (Fig. 7B). To determine the affinity of DiaA for each nucleotide-bound form of DnaA, we estimated the number of DiaA molecules that co-precipitated with DnaA, using quantitative standards of DnaA and DiaA proteins (Fig. 7C). Recovery of similar number of DiaA was observed in each form of DnaA. In these experiments, even though excessive amounts of bovine serum albumin were included in binding reaction and washing buffer, co-isolation of DnaA and DiaA was observed.

Using a similar pull-down method, DiaA protein was specifically recovered in a DnaA-dependent manner from soluble cell lysates obtained from a wild-type diaA strain (Fig. 7D). The results indicate specific binding between DiaA and DnaA complexed with the DnaA box.

Direct Binding of DiaA to the DnaA N-terminal Domain—To ask if DiaA directly binds to DnaA in the absence of DnaA box, we used a biotin-tagged form of DnaA (bioDnaA) for a pull-down method (42). The biotinylated DnaA was incubated in buffer containing DiaA and excessive amounts of bovine serum albumin. Materials bound to streptavidin-conjugated beads were recovered, and DnaA-bound protein was eluted in the presence of SDS. DnaA virtually remains bound to the beads because of extremely tight affinity between biotin and streptavidin. The results indicated that DiaA specifically bound to DnaA in a DNA-independent manner (Fig. 8). Activities of the biotinylated DnaA in binding to ATP and initiation of minichromosomal replication in vitro were comparable to those of the non-tagged form.



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  		FIG. 8.
DiaA directly binds to the DnaA N-terminal region. A, DiaA (10 pmol as dimer) was incubated on ice in buffer containing 100 µg/ml bovine serum albumin in the absence (no DnaA) or presence of 40 pmol of bioDnaA, bioDnaA domains I–II (bioDAD I–II), or bioDnaA domains III–IV (bioDAD III–IV), as indicated. Protein bound to the biotinylated DnaA proteins were isolated using streptavidin-conjugated magnetic beads, eluted in the presence of 1% SDS, and analyzed by SDS-polyacrylamide (13%) gel electrophoresis and Coomassie Brilliant Blue staining. See "Experimental Procedures" for details. Migration positions of proteins are indicated by arrowheads. I, input proteins; E, eluted proteins; MW, molecular size markers (Bio-Rad). B, experiments similar to A were performed using the indicated amounts of DiaA in the absence or presence of 40 pmol of bioDnaA (), bioDAD I–II ({blacktriangleup}), and bioDAD III–IV ({square}). Amounts of isolated DiaA were estimated, and the ratio of isolated DiaA to DnaA or DADs included in binding mixture is shown (DiaA dimer/input DnaA). No DiaA was isolated when binding mixture included 10–40 pmol of DiaA dimer in the absence of bioDnaA.

 
DnaA protein is structurally subdivided to four domains (4, 42–45). The N-terminal domains I–II (amino acids 1–129) contains regions for associating with DnaB and DnaA itself. Domain III (amino acids 130–350) that contains an ATP-binding/hydrolysis module also plays a role in associating with DnaB and DnaA. The C-terminal domain IV (amino acids 351–467) is the DNA-binding region that contains a helix-turn-helix motif (4, 36, 46). We constructed and purified biotin-tagged forms of truncated DnaA that contains domains I–II (bioDAD I–II) or domains III–IV (bioDAD III–IV). Using the same pull-down method as that used above, binding of DiaA to the DnaA domains I–II, but not to domains III–IV, was observed (Fig. 8). These results are well consistent with binding of DiaA to DnaA bound to the DnaA box (Fig. 7). The biotinylated DnaA domains III–IV was active in binding to ATP and a DnaA box R1 at a level comparable to the wild-type DnaA. Our results collectively indicate that DiaA binds directly and specifically to DnaA.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we show that a previously uncharacterized gene, diaA, encodes a protein required for the timely initiation of chromosomal replication. Most importantly, DiaA binds directly and specifically to the DnaA initiator protein. DiaA forms a stable complex with DnaA, even when the two proteins coexist in crude extracts (Fig. 7). Therefore, we propose that this novel protein plays an important role in regulating the initiation of chromosomal replication via direct interactions with the DnaA initiator.

To date, DnaB helicase is the only E. coli genome-encoded protein that has been identified as a direct DnaA binding factor (42–44). This interaction is part of the mechanism of initiation at oriC. During initiation at oriC, part of the duplex DNA in the oriC-DnaA complex is unwound, and DnaB within the DnaB-DnaC complex is loaded onto the single-stranded region. At this loading step, DnaA interacts directly with DnaB (44). In the RIDA process, Hda and the DNA-loaded {beta} clamp are required for DnaA-ATP hydrolysis (7, 8, 47). These physical interactions with DnaA in this process are currently under investigation.

Lack of the diaA gene impairs maintenance of the minichromosome and inhibits the growth of specific dnaA temperature-sensitive mutants (Tables II and III). Consistent with this finding, DiaA stimulates replication in an in vitro minichromosome replication system. Moreover, flow cytometry experiments (Fig. 2) reveal that DiaA stimulates initiation at oriC. For example, in LB medium containing glucose (Fig. 2, E and F), the number of cells bearing eight oriC copies decreased, and those bearing much fewer oriC copies increased in the absence of diaA. The results consistently suggest that DiaA plays a stimulatory role in initiation. In addition, when DiaA was overexpressed from the pBR322 vector, asynchronous pheno-type of replication was observed (Fig. 4). Quantitative control of the DiaA amount in cells is presumably important to maintain the precise replication cycle.

Although overproduced DiaA induced asynchronous initiations in vivo (Fig. 4), excessive amounts of DiaA did not inhibit replication of minichromosome in the in vitro system (Fig. 6C). Because maintenance of synchronous initiation requires the concerted action of HU, IHF, and FIS (18–21), we can not exclude a possibility that DiaA directly or indirectly interferes with one of these factors in vivo when overexpressed.

In the in vitro replication system, hydrolysis of DnaA-bound ATP was not inhibited by DiaA (Fig. 6, E and F). In addition, we examined if DiaA might facilitate the exchange of ADP bound to DnaA with ATP, but this nucleotide exchange was not promoted by the protein in vitro (data not shown). We speculate that DiaA stimulates initiation by another, novel mechanism. The protein may facilitate formation of initiation complex or unwinding of DNA in this complex. Alternatively, DiaA might recruit other replicative proteins to initiation complex. Especially in vivo, DiaA might be related to control for subcellular localization of DnaA molecules or DnaA-oriC complex, which might result in facilitation of timely initiation. At least, subcellular localization of oriC copies in a cell is changed in a replication cycle-dependent manner (48).

DiaA contains a SIS domain (sugar isomerase) that is a common motif among the phosphosugar isomerases observed in archeal, prokaryotic, and eukaryotic cells (49, 50). This domain participates in the formation of a specific protein structure required for binding to various phosphosugars. For example, E. coli gmhA, a member of the SIS family, encodes phosphoheptose isomerase that catalyzes the conversion of sedoheptulose 7-phosphate to D-glycero-D-manno-heptose 7-phosphate (51). This reaction is the first step in the synthesis of ADP-L-glycero-D-mannose, which produces lipopolysaccharide, an inner membrane component. In addition to phosphosugar isomerases, this domain is observed in specific transcriptional regulators (50). In these cases, the factors may bind specific phosphosugars to regulate the expression of downstream genes. For example, the RpiR protein contains an SIS domain in addition to a DNA-binding domain. RpiR regulates the transcription of ribB encoding an enzyme that interconverts ribulose 5-phosphate and ribose 5-phosphate (52). RibR may bind to ribose phosphate to regulate ribB transcription (50). Several genes have DnaA boxes in the promoter-flanking regions (4). DnaA bound to these sites plays a positive or negative role in gene transcription. DiaA might modulate this role for DnaA by interacting with DnaA bound to the box. Moreover, DiaA might interact with DnaA complexed with oriC in vivo. Effects of phospho-sugar on DiaA function remain to be elucidated.


   FOOTNOTES
 
* This work was supported, in part, by research grants from the Inamori Foundation and the Ministry of Education, Culture, Sports, Science and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} These authors contributed equally to this work. Back

§ Present address: Graduate School of Pharmaceutical Sciences, University of Tokyo 113-0033, Japan. Back

Present address: Institute for Chinese Medicine, Nakamura City Hospital, Kochi 787-0023, Japan. Back

|| Present address: Santen Pharmaceutical Co., Nara 630-0101, Japan. Back

** Present address: Dept. of Pharmacy, Kyushu University Hospital, Fukuoka 812-8582, Japan. Back

{ddagger}{ddagger} Present address: Radioisotope Center, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan. Back

§§ To whom correspondence should be addressed. Tel.: 81-92-642-6641; Fax: 81-92-642-6646; E-mail: katayama{at}phar.kyushu-u.ac.jp.

1 The abbreviations used are: RIDA, regulatory inactivation of DnaA; ORF, open reading frame; DAD, DnaA domain. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Sota Hiraga for suggestions on flow cytometry analysis and to Dr. Tohru Ogawa and Dr. Takeyoshi Miki for plasmids.



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