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

• Published in: Molecular microbiology 2000-09, Vol.37 (5), p.1087-1093
• Main Authors: Von Freiesleben, Ulrik, Rasmussen, Knud V., Atlung, Tove, Hansen, Flemming G.
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - physiology; Chromosomes, Bacterial; DNA Replication; DNA, Bacterial - biosynthesis; DNA-Binding Proteins - physiology; DnaA protein; Drug Resistance, Microbial; Escherichia coli - drug effects; Escherichia coli - genetics; integration host factor; Integration Host Factors; Replication Origin; Rifampin - pharmacology; RNA, Bacterial - biosynthesis
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1046/j.1365-2958.2000.02060.x

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.