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

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.

Genome streamlining and chemical defense in a coral reef symbiosis

“…Much like symbionts with extremely reduced genomes (1), Ca. E. faulkneri lacks both dnaA and ftsZ, which are involved in chromosomal replication and cell division, respectively (29,30). These genes are maintained even in the free-living bacterium with the smallest genome, Mycoplasma genitalium (27).…”

Suppression of initiation defects of chromosome replication in Bacillus subtilis dnaA and oriC-deleted mutants by integration of a plasmid replicon into the chromosomes

“…B. subtilis cells growing exponentially were collected by centrifugation, and cell lysates were prepared as described previously (25). Cell lysates containing approximately equal amounts of total protein (20 g) were applied to each lane of a sodium dodecyl sulfate-polyacrylamide gradient (10 to 20%) gel, and resolved proteins were transferred to a nitrocellulose membrane.…”

“…Cell lysates containing approximately equal amounts of total protein (20 g) were applied to each lane of a sodium dodecyl sulfate-polyacrylamide gradient (10 to 20%) gel, and resolved proteins were transferred to a nitrocellulose membrane. DnaA protein was detected by using the ECL Western blotting detection reagents (Amersham) after the nitrocellulose membrane was treated with antiDnaA rabbit antiserum (25) followed by treatment with a second antibody (goat anti-rabbit immunoglobulin G-horseradish peroxidase conjugate) (Bio-Rad Laboratories, Richmond, Calif.). To know the limit of detection for the DnaA protein in this immunoblot assay, cell lysate from a dnaA-null mutant was mixed with a cell lysate from the wild-type strain in various proportions, and an equal amount of total protein (40 g) was resolved and analyzed as described above.…”

Control of Initiation of Sporulation by Replication Initiation Genes in Bacillus subtilis

“…These include the status of the tricarboxylic acid cycle (11) and chromosome integrity, which consists of chromosome organization (10, 24), DNA damage (8), and initiation of DNA replication (9). It is presumed that chromosome integrity is monitored to ensure that cells do not initiate sporulation unless both the mother cell and forespore receive a complete genome.In B. subtilis, three genes are known to be required for initiation, but not elongation, of DNA replication: dnaA, dnaB, and dnaD (12,20,21). DnaA is the highly conserved DNA replication initiator in bacteria.…”

“…In B. subtilis, three genes are known to be required for initiation, but not elongation, of DNA replication: dnaA, dnaB, and dnaD (12,20,21). DnaA is the highly conserved DNA replication initiator in bacteria.…”