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Effect of Escherichia coli IHF mutations on plasmid p15A copy number
1Department of Microbiology, Technical University of Gdańsk, Poland.
Abstract:
Four isogenic strains (himAhimD double mutant, himA and himD single mutants, and their wild type counterpart) harboring orip15A plasmid (pACYC184 or pACYC184Amp or pACYC177) show different copy numbers of that plasmid in the early stationary phase of cultivation. The copy number of orip15A plasmid increases about four times in the himAhimD double (65-70 copies per cell) and himD single mutant cells (50-56 copies per cell) and was almost the same in himA mutant (17-18 copies per cell) and wild type cells (14-16 copies per cell). The results suggest that HimD can form homodimers, which are functionally competent for the regulation of orip15A plasmid copy number. Complementation experiments of himAhimD double mutant cells using plasmid carrying himA and himD genes (pPLhiphimA-5) confirm the effect of integration host factor (IHF) absence on increasing the copy number of orip15A plasmid (plasmid producing IHF complemented the defect of IHF mutant). The absence of IHF (using himAhimD double mutant as host) had no effect on the copy number of the pBR322 (oripMB1) plasmid.
Insights
The himD mutation significantly increases the copy number of the orip15A plasmid, suggesting HimD protein homodimers regulate plasmid levels. Integration host factor (IHF) absence also elevates orip15A plasmid copy number.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- The himA and himD genes encode subunits of the Integration Host Factor (IHF), a critical bacterial protein involved in DNA replication and recombination.
- Plasmid copy number is tightly regulated and essential for maintaining genetic elements within host cells.
Purpose of the Study:
- To investigate the role of IHF, specifically the HimA and HimD subunits, in regulating the copy number of the orip15A plasmid.
- To determine if HimD protein can function as a homodimer in regulating plasmid replication.
Main Methods:
- Construction and analysis of isogenic bacterial strains with defined mutations in himA and himD.
- Cultivation of bacterial strains harboring orip15A plasmids under specific conditions.
- Quantification of plasmid copy numbers using cell counts.
- Complementation experiments to confirm the role of IHF.
Main Results:
- The himAhimD double mutant and himD single mutant strains exhibited a fourfold increase in orip15A plasmid copy number compared to wild-type cells.
- The himA single mutant showed a plasmid copy number similar to wild-type cells.
- Complementation with a plasmid expressing himA and himD restored normal orip15A plasmid copy number in the himAhimD double mutant.
- Absence of IHF did not affect the copy number of the pBR322 (oripMB1) plasmid.
Conclusions:
- The HimD protein, potentially as a homodimer, plays a significant role in the negative regulation of orip15A plasmid copy number.
- IHF is crucial for maintaining the normal copy number of the orip15A plasmid, but not the pBR322 plasmid.
- These findings elucidate a specific regulatory mechanism for orip15A plasmid maintenance in bacteria.