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Requirement of the Escherichia coli dnaA gene function for ori-2-dependent mini-F plasmid replication
Abstract:
The mini-F plasmids pSC138, pKP1013, and pKV513 were unable to transform Escherichia coli cells with a dnaA-defective mutation under nonpermissive conditions. The dnaA defect was suppressed for host chromosome replication either by the simultaneous presence of the rnh-199 (amber) mutation or by prophage P2 sig5 integrated at the attP2II locus on the chromosome, both providing new origins for replication independent of dnaA function. The dnaA mutations tested were dnaA17, dnaA5, and dnaA46. dnaA5 and dnaA46 are missense mutations. dnaA17 is an amber mutation whose activity is controlled by the temperature-sensitive amber suppressor supF6. Under permissive conditions in which active DnaA protein was available, the mini-F plasmids efficiently transformed the cells. However, the transformants lost the plasmid as the cells multiplied under conditions in which DnaA protein was inactivated or its synthesis was arrested. As controls, plasmids pSC101 and pBR322 were examined along with mini-F; pSC101 behaved in the same manner as mini-F, showing complete dependence on dnaA for stable maintenance, whereas pBR322 was indifferent to the dnaA defect. Thus, ori-2-dependent mini-F plasmid replication seems to require active dnaA gene function. This notion was strengthened by the results of deletion analysis which revealed that integrity of at least one of the two DnaA boxes present as a tandem repeat in ori-2 was required for the origin activity of mini-F replication.
Insights
Mini-F plasmids require functional DnaA protein for replication in Escherichia coli. Suppressing dnaA defects restored replication, but stable plasmid maintenance still depended on active DnaA protein.
Area of Science:
- Molecular Biology
- Bacteriology
- Genetics
Background:
- The DnaA protein is essential for initiating DNA replication in Escherichia coli.
- Mini-F plasmids rely on specific replication origins for their maintenance.
Purpose of the Study:
- To investigate the role of the DnaA protein in the replication and stable maintenance of mini-F plasmids.
- To determine the specific requirements of mini-F plasmid replication origins for DnaA function.
Main Methods:
- Transformation of Escherichia coli strains with dnaA-defective mutations using mini-F plasmids (pSC138, pKP1013, pKV513).
- Utilizing suppressor mutations (rnh-199, supF6) and prophage integration (P2 sig5) to bypass dnaA defects.
- Assessing plasmid stability under permissive and non-permissive conditions for DnaA protein activity.
- Comparative analysis with control plasmids (pSC101, pBR322).
- Deletion analysis of the mini-F replication origin (ori-2) to identify essential DnaA binding sites.
Main Results:
- Mini-F plasmids failed to transform dnaA-defective E. coli under non-permissive conditions.
- Suppression of dnaA defects allowed initial transformation but not stable plasmid maintenance.
- Plasmid pSC101 showed similar dnaA dependence, while pBR322 was independent.
- Deletion analysis indicated that DnaA boxes within ori-2 are crucial for mini-F replication.
Conclusions:
- Active DnaA protein function is essential for ori-2-dependent mini-F plasmid replication.
- Stable maintenance of mini-F plasmids is contingent upon functional DnaA protein.
- The integrity of DnaA boxes in ori-2 is required for mini-F plasmid origin activity.