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Microbiology 153 (2007), 3370-3379; DOI  10.1099/mic.0.2007/009001-0
© 2007 Society for General Microbiology

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

Yan Li{dagger}, Kenji Kurokawa, Luzia Reutimann{ddagger}, Hikaru Mizumura, Miki Matsuo§ and Kazuhisa Sekimizu

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

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{alpha}. 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.

Abbreviations: EMS, ethylmethane sulfonate

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

{ddagger}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.






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Copyright © 2007 Society for General Microbiology.