Rifampicin‐resistant initiation of chromosome replication from oriC in ihf mutants

Abstract

IHF (integration host factor) mutants exhibit asynchronous initiation of chromosome replication from oriC as determined from flow cytometric analysis of cultures where RNA synthesis was inhibited with rifampicin. However, the run‐out kinetics of chromosome replication in ihf mutants shows that they continue to produce oriCs for some time in the absence of RNA synthesis resulting in a twofold increase in the oriC per mass ratio. An ihf dnaA double mutant did not exhibit this continued increase of the oriC per mass ratio. This indicates that ihf mutants can initiate replication from oriC in a rifampicin‐resistant initiation mode but requires fully functional DnaA protein. The origin per mass ratio, determined by a quantitative Southern blotting technique, showed that the ihf mutants had an origin per mass ratio that was 60% of the wild type although it had a normal DnaA protein concentration. This shows that the initiation mass was substantially higher in the ihf mutants. The oriC per terminus ratio, which was also determined by Southern blotting, was very low in the ihf mutant, although it grew with the same doubling times as the wild‐type strain. This indicates that cells lacking IHF replicate their chromosome(s) very fast.

DnaA binding locus datA promotes DnaA-ATP hydrolysis to enable cell cycle-coordinated replication initiation

“…We observed that datA efficiently hydrolyzed ATP bound to DnaA in a manner dependent on both IHF and Arg285 of the DnaA Argfinger, suggesting that specific DnaA multimer formation is important for DDAH. Deletion of either datA or ihf increased cellular ATP-DnaA levels in a RIDA-independent manner, consistent with the fact that untimely initiations occur in datA and IHF mutant cells (23)(24)(25)27). Because IHF binding to datA in cells was detected specifically at the postinitiation stage, datA function is likely to be temporally regulated.…”

“…datA-deleted cells, like seqA-deleted cells, perform untimely initiations at a level that does not inhibit cell growth (23). Furthermore, deletion of datA or ihf causes rifampicin-resistant initiations at oriC (26,27). The Bacillus subtilis and Streptomyces coelicolor genomes also have DnaA box clusters analogous to datA that can repress untimely initiations (28,29).…”

Relationship between Fork Progression and Initiation of Chromosome Replication in E. coli

“…Reduction of chromosome replication overlaps, with the associated lowering of the ori/ter ratio, together with an increased replication rate, have been also found in strains containing different defective dnaA alleles, such as dnaA204 (Torheim et al, 2001), dnaA46, dnaA174, and dnaA345 (Gon et al, 2006;Morigen et al, 2009), as well as in wild type cells containing extra copies of datA sequence which is believed to reduce the availability of DnaA protein (Morigen et al, 2003). Similar results have also been obtained in studies of hns (Atlung & Hansen, 2002) and ihf mutants (von Freiesleben et al, 2000). Additionally, we have also shown by growing the nrdA101 mutant in poor media at 30ºC, that the improvement in the replication fork progression is accompanied by a decrease in replication overlap.The correlation between these effects has been well established but the mechanism remains elusive.…”

Precise determinations of C and D periods by flow cytometry in Escherichia coli K-12 and B/r

“…We have not investigated this phenomenon in more detail. However, it is known that insertional inactivation of the ihfA or ihfB alleles (von Freiesleben et al, 2000) and of the hns gene (Atlung & Hansen, 2002) can decrease the C period. Although the DNA distributions of the two cultures look very different, the durations of the cell cycle periods do fall within the general scatter in Fig.…”