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Journal of Bacteriology, July 2001, p. 3833-3841, Vol. 183, No. 13
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-
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

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