NDLI: Isolation of a dnaA mutant of Bacillus subtilis defective in initiation of replication: amount of DnaA protein determines cells' initiation potential.

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Isolation of a dnaA mutant of Bacillus subtilis defective in initiation of replication: amount of DnaA protein determines cells' initiation potential.

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Isolation of a dnaA mutant of Bacillus subtilis defective in initiation of replication: amount of DnaA protein determines cells' initiation potential.

Content Provider	PubMed Central
Author	Moriya, S. Kato, K. Yoshikawa, H. Ogasawara, N.
Abstract	The dnaA gene is essential for initiation of chromosomal replication in Escherichia coli. A gene homologous with the E. coli dnaA was found in the replication origin region of the Bacillus subtilis chromosome. We have now isolated a temperature sensitive mutant of the B. subtilis dnaA by in vitro mutagenesis of the cloned gene. At a nonpermissive temperature, 49 degrees C, DNA replication stops completely after 60% increase in a rich medium, while cell mass continues to increase exponentially at 2.5 times the rate at 30 degrees C. A ratio of gene frequency between purA (origin marker) and metB (terminus marker) changes gradually from 2.7 at 30 degrees C to 1.0 in 45 min at 49 degrees C, indicating completion of the ongoing replication cycle. Upon the temperature shift down to 30 degrees C after the incubation at 49 degrees C for 60 min, DNA replication resumes without delay, and the purA/metB ratio increases rapidly to 6, i.e. consecutive initiation of more than two rounds of replication. Addition of chloramphenicol at the time of the temperature shift down did not inhibit the increase in the purA/metB ratio, while rifampicin inhibited the re-initiation completely. The mutation is a single base change from C to T in the dnaA gene resulting in an amino acid substitution from Ser to Phe in the DnaA protein. The mutation was responsible for both temperature sensitive growth and the defect in initiation of chromosomal replication. We observed a remarkable correlation between the amount of DnaA protein and the amount of initiation potential accumulated during incubation at the non-permissive temperature.
Starting Page	2905
File Format	PDF
ISSN	14602075
e-ISSN	14602075
Journal	The EMBO Journal
Issue Number	9
Volume Number	9
Language	English
Publisher Date	1990-09-01
Access Restriction	Open
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Neuroscience Immunology and Microbiology Medicine Molecular Biology

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