Autoregulation of the dnaA-dnaN operon and effects of DnaA protein levels on replication initiation in Bacillus subtilis

Citation
Y. Ogura et al., Autoregulation of the dnaA-dnaN operon and effects of DnaA protein levels on replication initiation in Bacillus subtilis, J BACT, 183(13), 2001, pp. 3833-3841
Citations number
48
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Microbiology
Journal title
JOURNAL OF BACTERIOLOGY
ISSN journal
0021-9193 → ACNP
Volume
183
Issue
13
Year of publication
2001
Pages
3833 - 3841
Database
ISI
SICI code
0021-9193(200107)183:13<3833:AOTDOA>2.0.ZU;2-2
Abstract
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)-ind ucible promoter. However, increasing the DnaA level resulted in cell elonga tion and inhibition of cell growth by induction of the SOS response. Transc ription of the native dnaA-dnaN operon was greatly reduced at high DnaA lev els, 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, sugg esting that depletion of DnaN (beta subunit of DNA polymerase III) within t he cell by repression of the native dnaA-dnaN operon at high DnaA levels wa s 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 ti ming of replication initiation was observed at DnaA levels nearly comparabl e to the wild-type level. These results suggest that if the DnaA level incr eases with progression of the replication cycle, it could act as a rate-lim iting factor of replication initiation in B. subtilis.