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dnaB and dnaI temperature-sensitive mutants of Staphylococcus aureus: evidence for involvement of DnaB and DnaI in synchrony regulation of chromosome replication

Yan Li

Luzia Reutimann

Miki Matsuo

Laboratory of Microbiology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Correspondence
Kenji Kurokawa
kurokawa{at}mol.f.u-tokyo.ac.jp

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
DnaB and DnaI proteins conserved in low-GC content Gram-positive bacteria are apparently involved in helicase loading at the replication initiation site and during the restarting of stalled replication forks. In this study, we found five novel dnaB mutants and three novel dnaI mutants by screening 750 temperature-sensitive Gram-positive Staphylococcus aureus mutants. All of the mutants had a single amino acid substitution in either DnaB or DnaI that controlled temperature-sensitive growth, as confirmed by transduction experiments using phage 80. DNA synthesis as measured by [³H]thymine incorporation, origin-to-terminus ratios and flow cytometric analysis revealed that the dnaB and dnaI mutants were unable to initiate DNA replication at restrictive temperatures, which is similar to previous findings in Bacillus subtilis. Furthermore, some of the mutants were found to exhibit asynchrony in the initiation of DNA replication. Also, a fraction of the dnaI mutant cells showed arrested replication, and the dnaI mutant tested was sensitive to mitomycin C, which causes DNA lesions. These results suggest that DnaB and DnaI are required not only for replication initiation and but also for regulation of its synchrony, and they provide support for the involvement of DnaI activity in the restart of arrested replication forks in vivo.

Present address: Division of Microbial Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan.

Present address: Institute of Microbiology, ETH Zurich, Wolfgang-Pauli-Str. 10, CH-8093 Zurich, Switzerland.

Present address: Department of Bacteriology, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
The timing and synchrony of bacterial chromosome replication is strictly regulated at the initiation step. Initiation occurs only when the bacterium reaches initiation mass, a specific cell mass per chromosomal origin. To allow initiation, multiple origins within a bacterial cell are activated to open simultaneously once per cell cycle.

DnaA initiates DNA replication, binds to oriC, and mediates opening of the DNA strand. Control of the activity and amount of DnaA plays a critical role in the regulation of replication timing in Escherichia coli (Messer, 2002). Synchrony in E. coli cells is regulated by genes whose products have the common ability to associate with oriC with involvement of dnaA, dnaC, dam, seqA, hobH and fis (Kornberg & Baker, 1992). Mutation of the recA and rpoC genes and the datA region for DnaA titration cause asynchrony.

There are significant differences in the sets of genes involved in replication initiation between Gram-positive Bacillales, such as Bacillus subtilis and Staphylococcus aureus, and the Gram-negative E. coli. Three priming proteins found in Bacillales, DnaD, DnaB and DnaI (which may load the DnaC helicase), have no homologues in E. coli (Bruand & Ehrlich, 1995; Bruand et al., 1995; Moriya et al., 1999; Ogasawara et al., 1986). In addition, the B. subtilis YabA protein, which acts as a negative regulator for initiation at the B. subtilis oriC, has no homologue in Gram-negative E. coli. Conversely, SeqA, Dam and Hda, three E. coli proteins that negatively regulate the initiation of DNA replication, are absent in the genomes of the Gram-positive bacteria B. subtilis and S. aureus. Thus, the Gram-positive bacteria possess an alternative system for initiation control, although the molecular details remain unknown.

Both DnaB and DnaI, which are encoded in a single operon (Bruand & Ehrlich, 1995), are essential for the initiation of DNA replication in B. subtilis (Karamata & Gross, 1970; Mendelson & Gross, 1967). These two proteins interact directly but independently with the replicative DnaC helicase and are thought to deliver DnaC helicase to targeted regions of DNA. At these sites, DnaI acts as a helicase loader, whereas DnaB serves as an accelerator of loading (Imai et al., 2000; Ioannou et al., 2006; Soultanas, 2002; Velten et al., 2003). In B. subtilis, DnaB and DnaI, together with PriA and DnaD, are required for primosome assembly, which is needed to restart replication (Bruand et al., 2001, 2005). The dnaB gene is unique because its temperature-sensitive mutants simultaneously lose the attachment of oriC to the cell membrane and the ability to initiate a new round of replication after an increase to the restrictive temperature (Rokop et al., 2004; Winston & Sueoka, 1980). DnaI is an AAA⁺ family protein (Neuwald et al., 1999), with an N-terminal domain that can interact with replicative helicases and a C-terminal domain that contains ATPase and cryptic DNA-binding activities (Ioannou et al., 2006). This protein is known to be a target of bacteriophage protein-mediated inhibition of DNA replication in S. aureus (Liu et al., 2004). Despite this information, the exact function of DnaB and DnaI in replication initiation and its control are uncertain.

S. aureus is a clinically important Gram-positive pathogenic bacterium. Comparison of the genomic sequences of S. aureus and B. subtilis reveals the presence of similar gene sets for DNA replication. In fact, many S. aureus plasmids can replicate in B. subtilis (Ehrlich, 1977). We have recently characterized several DNA replication mutants of S. aureus (Inoue et al., 2001; Kaito et al., 2002; Li et al., 2004; Murai et al., 2006). Herein, we report the isolation and characterization of five dnaB and three dnaI mutants in S. aureus. Our results suggest that DnaB and DnaI are required for the growth of staphylococcal cells. The results agree with the previous study in B. subtilis showing that these proteins are required for the initiation of chromosome replication. Our study suggests for the first time that dnaB and dnaI are involved in synchrony regulation of chromosome replication. The results also provide novel in vivo evidence that DnaI participates in the restart of replication from stalled replication forks.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Bacterial strains and plasmids.
The S. aureus strains used in this study are listed in Table 1. Temperature-sensitive strains of S. aureus were isolated by mutagenesis of RN4220 (Novick et al., 1993) with ethylmethane sulfonate (EMS; Sigma), as described previously (Inoue et al., 2001). Strains in which the erythromycin-resistant marker on pMutinT3 was integrated near the dnaB and dnaI operon were used as donors for phage transduction experiments (Table 1). In brief, the region between SA1505 and SA1506 (chromosomal location 1713953 to 1714765 on the S. aureus N315 genome) was amplified with primers 5'-GCCCGAATTCTCACGCATAATCAACCCTGT-3' and 5'-GCCCGGATCCTCAAACGCGTTGTAAACATG-3'. We used the complete sequence of the S. aureus N315 genome (Kuroda et al., 2001) registered in the Genome Information Broker program in the DNA Data Bank of Japan (http://gib.genes.nig.ac.jp/single/index.php?spid=Saur_N315). The amplified fragment was digested with EcoRI and BamHI, and cloned into the EcoRI and BamHI sites of pMutinT3, a suicide vector for S. aureus with a replication origin that functions in E. coli (Vagner et al., 1998). The resulting plasmid, pMBI, was integrated into parent or mutant strains via homologous recombination, as confirmed by Southern blotting. E. coli JM109 (Takara Bio) was used for subcloning. Plasmids pND50 and pKE515 were used as shuttle vectors between E. coli and S. aureus (Li et al., 2004; Yamagishi et al., 1996). An S. aureus DNA replication gene library was prepared by cloning 17 PCR-amplified putative replication genes into the SmaI site of pND50 based on the N315 sequence. Plasmid pSdnaB contained the dnaB gene region (1736 bp; chromosomal location 1717095 to 1718830 on the N315 genome described above) in pKE515, which was derived from the RN4220 genomic DNA library (Li et al., 2004) and was confirmed by sequencing analysis. Plasmid pSdnaI was pND50 with a 1155 bp portion of the dnaI gene (amplified using primers 5'-GGTCTTGATCCGGGTATTCA-3' and 5'-GCAAGATCGACAAGCATTTCT-3') inserted at the SmaI site. Each SmaI site on pKE515 and pND50 is the downstream position of the cat gene encoding chloramphenicol acetyltransferase and, in our experience, insertion of an open reading frame in the same direction as the cat gene is often critical for the cloned gene to function efficiently (data not shown). So, it is assumed that transcription from the cat gene on the pKE515 or pND50 vectors flows into the SmaI site and enables expression of an inserted open reading frame. This could allow the expression of DnaB or DnaI proteins from the pSdnaB or pSdnaI plasmids, respectively.

DNA sequencing.
DNA fragments containing the dnaB region were amplified by PCR from chromosomal DNA templates using primers 5'-CAAAGTCCTGCGACGTGTTA-3' and 5'-TCTTGTGAGAAACGACCAGTT-3', purified with QIAquick gel extraction kits (Qiagen), and used as sequencing reaction templates. Thermal sequencing reactions were performed with PRISM BigDye terminator kits and analysed with an ABI 373A automated DNA sequencer (Applied Biosystems). For the dnaI region, the same primers were used as for pSdnaI construction.

Transformation and phage transduction of S. aureus..
Competent cells for electroporation were prepared as described previously (Inoue et al., 2001). Briefly, plasmid DNA (100 ng) was delivered into competent cells by electroporation at 2.5 kV, 25 µF, and 100  in a 0.2 cm cuvette (Gene Pulser II; Bio-Rad). Electroporated cells were grown in BHI medium (Becton Dickinson) containing 10 % sucrose for 1 h at 30 °C and spread on LB agar plates containing antibiotics for selection. Transduction using phage 80 was performed as described by Novick (1991).

Search for replication mutants.
A pND50-based plasmid library, containing 17 putative DNA replication-related genes, was delivered by electroporation into the temperature-sensitive S. aureus mutants. Colonies grown at 43 °C on LB agar plates containing 12.5 µg chloramphenicol ml^–1 were isolated, and plasmid DNA was extracted. The nucleotide sequences of the DNA fragments inserted in the plasmids were determined.

Continuous labelling of DNA and protein synthesis in S. aureus..
To monitor DNA synthesis, overnight cultures were diluted 1000-fold in LB medium containing 50 µg unlabelled thymine ml^–1 and [³H]thymine and then grown at 30 °C until the OD₆₀₀ reached 0.1–0.15. The cells were then shifted to 43 °C or 30 °C and incubated for various amounts of time, after which a portion of the cells was treated with 10 % (w/v) TCA containing 0.1 M sodium pyrophosphate, 0.5 M NaCl and 25 µg thymine ml^–1. Acid-insoluble fractions were collected, and radioactivity was measured using a liquid scintillation counter. To monitor protein synthesis, [³H]thymine was replaced by [³⁵S]methionine.

Origin-to-terminus ratio.
Southern hybridization experiments were performed to determine the relative abundance of the chromosomal origin and terminus as described previously (Li et al., 2004). Overnight cultures were diluted 1000-fold in LB medium with 50 µg thymine ml^–1 and incubated at 30 °C until the OD₆₀₀ reached 0.3. Half of each culture was harvested as exponential-phase cells, and the rest was further incubated at 43 °C for 2 h. Genomic DNA was extracted, double-digested with EcoRI and EcoRV, separated by agarose (1.5 %) gel electrophoresis, and transferred to a nitrocellulose membrane. The position of each ³²P-labelled probe, as defined by the S. aureus N315 genome sequence, was 0.08 min for the origin and 50.03 min for the terminus. Each region cloned into the SmaI site of pND50 was excised by digestion with KpnI and BamHI, separated by agarose gel electrophoresis, extracted, and labelled with [-³²P]dCTP using a random primer DNA-labelling kit version 2 (Takara Bio). Hybridization intensities were determined using a BAS1500 image analyser (Fuji Film). The relative origin-to-terminus ratio was normalized using the intensity of the pSot plasmid, which contained both the origin and the terminus regions of the probe at the SmaI and HincII sites of pND50, respectively. EcoRI/HindIII/BamHI-digested pSot gave a theoretical molar ratio of 1.

Flow cytometric assay.
The dynamics of the chromosomal DNA replication process was analysed using flow cytometry as described previously (Li et al., 2004; Skarstad et al., 1995). Briefly, exponentially growing cells at an OD₆₀₀ of 0.15 were harvested and either treated with 50 µg rifampicin ml^–1 and 10 µg cephalexin ml^–1 at 30 °C for an additional 2 h, or incubated at 43 °C with or without cephalexin for an additional 2 h. Harvested cells were fixed in 75 % ethanol, treated with 20 µg RNaseA ml^–1 at 50 °C for 1 h, sonicated, washed, and stained with 1 µM SYTOXgreen (Molecular Probes). Samples were analysed using a FACSCalibur (Becton Dickinson). A single-cell suspension was generated by sonication as described previously (Li et al., 2004; Murai et al., 2006).

Mitomycin C sensitivity.
Exponentially growing S. aureus cells (OD₆₀₀ 0.1–0.15) at 30 °C were incubated at 43 °C for 2 h in the presence of 0, 50, 100, 200 or 300 ng mitomycin C ml^–1. The cells were then diluted and spread on LB agar plates, incubated at 30 °C for 36 h, and colony numbers were counted.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Isolation of temperature-sensitive dnaB and dnaI mutants of S. aureus
To identify the essential genes for S. aureus cell growth, we isolated more than 750 temperature-sensitive mutants that grew at 30 °C but not at 43 °C (Inoue et al., 2001; Li et al., 2004). In this study, we searched for replication mutants using a pND50-based plasmid library that contains 17 putative DNA replication-related genes. The transformants of 10 strains grown at 43 °C carried the plasmid pSdnaBI containing the dnaB and dnaI gene operon. To determine which gene is responsible for complementation, the strains were transformed by electroporation with a pSdnaB or pSdnaI plasmid in shuttle vector pKE515 or pND50, respectively. Of the 10 mutant strains, five carrying pSdnaB grew at 43 °C (TS2203, TS2831, TS4374, TS8410 and TS9803). An additional three strains carrying pSdnaI grew at 43 °C (TS6120, TS7302 and TS8651) (data not shown). The results suggest that these temperature-sensitive mutants are candidates for mutants of dnaB and dnaI, respectively.

Determination of mutation sites in the dnaB and dnaI genes
To confirm the mutations in the candidate dnaB or dnaI gene mutants, we determined the nucleotide sequences of their chromosomal dnaB or dnaI genes. We also compared them to the sequences from their parent strain, RN4220.

The dnaB gene of S. aureus is predicted to encode a 466 aa protein with a molecular mass of 54.5 kDa and 28 % amino acid sequence identity with the corresponding protein of B. subtilis strain 168 (Fig. 1a). The predicted amino acid sequence contains an N-terminal hydrophobic region, a putative DNA-binding region (Ogasawara et al., 1986) and a phage Rep-like motif in the middle of the C-terminal region (Rokop et al., 2004) (Fig. 1b). Two putative ATP-binding motifs deduced from the GKT motif in B. subtilis DnaB (Hoshino et al., 1987) were not detected in S. aureus DnaB. Each of the DnaB mutants had a single transition mutation (C113T, G268A, G275A, C931T and C1082T), resulting in single amino acid substitutions (P38L, A90T, G92E, P311S and P361L in TS4374, TS2203, TS2831, TS9803 and TS8410, respectively). The mutants were named dnaB4374, dnaB2203, dnaB2831, dnaB9803 and dnaB8410, respectively (Fig. 1b).

View larger version (24K): [in this window] [in a new window]   Fig. 1. Mutational sites in B. subtilis DnaB (a), and S. aureus DnaB (b) and DnaI (c). In (a), arrows indicate temperature-sensitive mutations and lines indicate suppressor mutations for the priA mutation. For the DnaI protein in (c), A and B in the highly conserved region represent Walker-type ATPase motifs A and B, respectively.

The mutations in the S. aureus dnaB mutants were located in the N- and C-terminal regions (Fig. 1b). This resembles the polarization of the B. subtilis dnaB mutations (Fig. 1a) and supports the hypothesis that DnaB contains two domains (Bruand et al., 2001; Ogasawara et al., 1986). Pro38, which was substituted in dnaB4347, is highly conserved among bacterial DnaB proteins and is located in the hydrophobic region (Fig. 1b), which may mediate association with and recruitment of oriC to the cell membrane. Thus, the Pro38 residue probably plays an important role in the membrane binding of DnaB by contributing hydrophobicity and by restricting the conformation of the membrane-binding domain. Both Ala90 and Gly92 were located in the turn portion of the helix–turn–helix motif, which participates in DNA binding (Fig. 1b). The alteration of the side-chains of these residues may affect the tertiary structure of the motif and reduce the ability of B. subtilis DnaB to bind single-stranded DNA (Bruand et al., 2005). Pro311 is not conserved amongst DnaB proteins, suggesting that it does not have a common function in DnaB proteins. The reduction of DnaB activity at 43 °C in the P311S mutant is probably due to a change in the protein's conformation. The dnaB8410 mutant has a P361L mutation, which corresponds to the S371 residue in B. subtilis DnaB. In B. subtilis, the S371P mutation of dnaB75 suppresses the temperature-sensitive phenotypes of the dnaD23 and dnaB134 mutations as well as the poor growth phenotype of the priA1 mutation. Furthermore, the S371P-mutated DnaB75 protein has a higher affinity for single-stranded DNA compared to wild-type DnaB (Bruand et al., 2001, 2005; Rokop et al., 2004). Thus, the Pro361 residue of S. aureus DnaB and the Ser371 residue of B. subtilis DnaB may be involved in a conformational change of the protein. The Gly159 and Gly222 residues, which are substituted in the dnaI mutation, are well conserved among DnaI proteins and are located in the nucleotide-binding motifs Walker A and Walker B, respectively (Fig. 1c). Therefore, these two mutations might affect control of DnaI protein function through the binding of adenine nucleotides.

Phage transduction experiments
Because EMS-treated cells are thought to have multiple mutations in their chromosomal DNA, we examined the role of the mutations in the temperature-sensitive phenotype by transduction using phage 80. This method is analogous to a system using phage P1 to transduce E. coli. These general transducing phages carry a fraction of DNA from their previous bacterial host (donor) and inject it in the same manner as phage DNA into the next host (recipient). The exogenous DNA can participate in homologous recombination events along with the endogenous DNA, resulting in its integration into the recipient's genome. In this study, a drug-resistance gene was integrated near the dnaBI operon of each mutant strain, and the resulting strains were used as donors. The phages from donors were used to infect temperature-resistant parent strains RN4220 or NI8, and cells undergoing homologous recombination were selected by growth in the presence of drugs. In this assay, if the dnaB or dnaI mutation is responsible for the temperature-sensitive phenotype, a certain portion of the drug-resistant recombinants will show temperature-sensitive cell growth because of genetic linkage. Transductants of thyA mutant NI8 or RN4220 were selected at 30 °C by erythromycin resistance based on the integration of suicide plasmid pMBI, a pMutinT3 derivative harbouring a 813 bp fragment near the dnaBI operon and the erythromycin-resistance gene. The temperature sensitivity of all five dnaB mutants and all three dnaI mutants was highly linked with erythromycin resistance and at similar frequencies (63–75 % and 77–84 %, respectively; Table 2). Consistent with this, pSdnaB or pSdnaI complemented the temperature sensitivity of the respective transductants (Table 3). These results suggest that the temperature sensitivity of these mutant strains is due to mutations in DnaB or DnaI, respectively.

View larger version (20K): [in this window] [in a new window]   Fig. 2. DNA and protein synthesis of dnaB and dnaI mutants. LYTBI, LYT2831 (dnaB2831) and LYT8651 (dnaI8651) cells were grown at 30 °C to an OD600 of 0.1–0.15 in LB medium (5 ml) containing 50 µg thymine ml–1 and [3H]thymine or [35S]methionine. Cultures were shifted to 43 °C (filled symbols) or left at 30 °C (open symbols) and then incubated for the indicated time. Incorporation of [3H]thymine (squares) or [35S]methionine (circles) into acid-insoluble fractions was measured. Incorporation at each time point is expressed relative to incorporation at time 0. Data are means±SE of at least three independent experiments (error bars not shown where smaller than symbols).

View larger version (35K): [in this window] [in a new window]   Fig. 3. Flow cytometric analyses of dnaB and dnaI mutants. Results are shown for exponentially growing RNBI (a, b, c, d), LY6120 (e, f, g, h), LY7302 (i, j, k, l), LY2831 (m, n, o, p) and LY8410 (q, r, s, t) cells grown at 30 °C in LB medium (OD600 0.15) and sampled directly for flow cytometry (a, e, i, m, q), after a 2 h incubation at 30 °C in the presence of rifampicin (Rfp) and cephalexin (Cex) (b, f, j, n, r), after a 2 h incubation at 43 °C in the presence of cephalexin (c, g, k, o, s), or after a 2 h incubation at 43 °C (d, h, l, p, t). Harvested cells were fixed, stained with SYTOXgreen, and analysed for chromosome content using a FACSCalibur. Data are representative of more than three independent experiments.

Mitomycin C sensitivity
It has been suggested that B. subtilis DnaI, together with DnaB and DnaD, functions in replication fork reassembly by a PriA-dependent and independent mechanism (Bruand et al., 2001; Marsin et al., 2001). Loss of PriA protein results in increased sensitivity to UV irradiation, suggesting that replication restart, which is mediated by PriA, participates in DNA repair (Kogoma et al., 1996; Polard et al., 2002). The dnaB75 mutation has been shown to suppress the effects of a B. subtilis priA deletion mutation (Bruand et al., 2001), and we previously observed that the S. aureus dnaD mutant is sensitive to mitomycin C and UV irradiation (Li et al., 2004). Therefore, S. aureus DnaI may also be involved in the restart of replication and DNA repair.

We found that LY6120 cells incubated for 2 h at 43 °C without antibiotics had a major 1N peak with a tail between the 1N and 2N peaks (Fig. 3h). Similar patterns were observed for LY7302 (Fig. 3l) and LY8651 cells (data not shown). These results suggest that the population includes cells that do not complete the elongation step of a round of DNA replication from 1N to 2N and thus represent replication arrests in the dnaI mutants. A similar phenotype was reported for an E. coli dnaC helicase loader mutant (Maisnier-Patin et al., 2001). Therefore, we examined whether the dnaI mutant has a defect in DNA repair. In LYT8651 cells, mitomycin caused a decrease in the viable cell number that was complemented by the pSdnaI plasmid (Fig. 4). This suggests that the dnaI8651 mutation is sensitive to mitomycin C, a compound known to cause DNA lesions. This finding suggests that DnaI participates in DNA repair.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Mitomycin C sensitivity of the LYT8651 mutant. Exponentially growing cells at 30 °C were incubated for 2 h at 43 °C in the presence of the indicated concentration of mitomycin C. Colony numbers of harvested cells were counted and are expressed relative to the number in the absence of mitomycin C. The data shown are representative of two independent experiments. The strains used were LYTBI (parent, ), LYT8651 (dnaI8651, ), LYT8651 harbouring pND50 () and LYT8651 harbouring pSdnaI ().

Flow cytometric analysis of the dnaB2831 and dnaI7302 mutant cells cultured at 30 °C in the presence of cephalexin and rifampicin showed 3N in addition to 2N and 4N peaks (Fig. 3). Bacterial cells contain 2n (n=0, 1, 2, etc.) origins because of the synchronous initiation at oriC (Skarstad et al., 1995). Thus, DnaB and DnaI appear to be involved in the synchrony of initiation of DNA replication. To our knowledge, this is the first time that this has been described in Gram-positive bacteria. How the synchrony of the initiation step is controlled is currently unknown. Because the period for replication initiation is strictly controlled, the lower ability of DnaB2831 and DnaI7302 to load the helicase at oriC could result in an asynchronous phenotype.

Finally, the present results (Fig. 3h and l, Fig. 4) support the involvement of DnaI in the restart of replication from arrested replication forks in vivo.

	ACKNOWLEDGEMENTS

 
We thank Drs N. Ogasawara and J. J. Ferretti for kindly providing pMutinT3 and pSF151, respectively. We also thank Makiko Miyatani, Hiromi Komaki and Kozue Saito for their technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, by the Industrial Technology Research Grant Program '04 from the New Energy and Industrial Technology Development Organization of Japan, and by grants from Shionogi & Co. Ltd and Genome Pharmaceuticals Co. Ltd.

Edited by: K. E. Weaver

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Received 12 April 2007; revised 19 June 2007; accepted 20 June 2007.

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