Related Experiment Videos
Imprecise excision of plasmid pE194 from the chromosomes of Bacillus subtilis pE194 insertion strains
B Conrad1, V I Bashkirov, J Hofemeister
1Institut für Pflanzengenetik und Kulturpflanzenforschung, Gatersleben, Germany.
Abstract:
Plasmid pE194 has been shown to be rescued by integration after cultivation of infected Bacillus subtilis recE4 cells at a restrictive high temperature. The plasmid is also spontaneously excised from the chromosome at a low frequency by precise or imprecise excision (J. Hofemeister, M. Israeli-Reches, and D. Dubnau, Mol. Gen. Genet. 189:58-68, 1983). We have investigated nine excision plasmids, carrying insert DNA 1 to 6 kbp in length, either in a complete pE194 or in a partially deleted pE194 copy. Type 1 (additive) excision plasmids have the left- and right-junction DNAs preserved as 13-bp direct repeats (5'-GGGGAGAAAACAT-3') corresponding to the region between positions 864 and 876 in pE194. In type 2 (substitutive) excision plasmids, a conserved 13-bp sequence remains only at the right junction while the left junction has been deleted during the excision process. The type 3 excision plasmid carries at each junction the tetranucleotide 5'-TCCC-3', present in pE194 between positions 1995 and 1998. Although we isolated the excision plasmids from different integration mutants, the insert DNAs of eight independently isolated plasmids showed striking sequence homology, suggesting that they originated from one distinct region of the B. subtilis chromosome. Thus, we postulate that imprecise excision of pE194 occurs most frequently after its translocation from the original insertion site into a preferred excision site within the host chromosome. The imprecise excision from this site occurs at excision breakpoints outside the pE194-chromosome junctions in a chromosomal region which remains to be investigated further.
Insights
Plasmid pE194 excision from Bacillus subtilis chromosomes was studied. Imprecise excision likely occurs after translocation to a preferred chromosomal site, involving specific DNA repeat sequences at junctions.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Plasmid pE194 can be rescued by integration in Bacillus subtilis recE4 cells at high temperatures.
- Spontaneous excision of pE194 from the chromosome occurs at low frequencies, either precisely or imprecisely.
Purpose of the Study:
- To investigate the mechanisms and characteristics of pE194 excision plasmids.
- To analyze the DNA sequences involved in precise and imprecise excision events.
Main Methods:
- Cultivation of infected Bacillus subtilis recE4 cells at restrictive high temperatures.
- Isolation and characterization of nine excision plasmids with varying insert lengths.
- DNA sequence analysis of junction regions in different types of excision plasmids.
Main Results:
- Identified three types of excision plasmids based on junction DNA sequences: type 1 (additive), type 2 (substitutive), and type 3.
- Type 1 plasmids feature direct repeats of a 13-bp sequence at both junctions.
- Type 2 plasmids retain the 13-bp repeat at the right junction only, with left junction deletion.
- Type 3 plasmids have a conserved tetranucleotide sequence at each junction.
- Eight independently isolated plasmids showed significant sequence homology in their insert DNAs, suggesting a common origin on the B. subtilis chromosome.
Conclusions:
- Imprecise pE194 excision is likely facilitated by translocation to a preferred chromosomal site.
- Excision breakpoints occur outside the pE194-chromosome junctions within a specific chromosomal region.
- Further investigation is needed to identify the chromosomal region involved in these preferred excision events.