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Journal of Bacteriology, July 2001, p. 3833-3841, Vol. 183, No. 13
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.13.3833-3841.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Autoregulation of the dnaA-dnaN Operon and Effects of DnaA Protein Levels on Replication Initiation in Bacillus subtilis

Yoshitoshi Ogura, Yukiho Imai,dagger Naotake Ogasawara, and Shigeki Moriya*

Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan

Received 18 December 2000/Accepted 6 April 2001

In Escherichia coli, the DnaA protein level appears to play a pivotal role in determining the timing of replication initiation. To examine the effects on replication initiation in B. subtilis, we constructed a strain in which a copy of the dnaA gene was integrated at the purA locus on the chromosome under the control of an isopropyl-beta -D-thiogalactopyranoside (IPTG)-inducible promoter. However, increasing the DnaA level resulted in cell elongation and inhibition of cell growth by induction of the SOS response. Transcription of the native dnaA-dnaN operon was greatly reduced at high DnaA levels, but it was increased in a dnaA-null mutant, indicating autoregulation of the operon by DnaA. When a copy of the dnaN gene was added downstream of the additional dnaA gene at purA, the cells grew at high DnaA levels, suggesting that depletion of DnaN (beta  subunit of DNA polymerase III) within the cell by repression of the native dnaA-dnaN operon at high DnaA levels was the cause of the SOS induction. Flow cytometry of the cells revealed that the cell mass at initiation of replication increased at a lower DnaA level and decreased at DnaA levels higher than those of the wild type. Proper timing of replication initiation was observed at DnaA levels nearly comparable to the wild-type level. These results suggest that if the DnaA level increases with progression of the replication cycle, it could act as a rate-limiting factor of replication initiation in B. subtilis.

* Corresponding author. Mailing address: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0101, Japan. Phone: 81-743-72-5432. Fax: 81-743-72-5439. E-mail: moriya{at}bs.aist-nara.ac.jp.

dagger Present address: Genox Research, Inc., Kawasaki Laboratory, Teikyo Univ. Biotech. Center, 907 Nogawa, Miyamae-ku, Kawasaki, Kanagawa 216-0001, Japan.

Journal of Bacteriology, July 2001, p. 3833-3841, Vol. 183, No. 13
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.13.3833-3841.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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