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Integration and excision of pMC7105 in Pseudomonas syringae pv. phaseolicola: involvement of repetitive sequences
Abstract:
The site for integration of pMC7105 into the chromosome of Pseudomonas syringae pv. phaseolicola has been mapped to a 2.6-kilobase-pair (kb) Bg/II-EcoRI fragment on this 150-kb indigenous plasmid. Selected excision plasmids resulting from imprecise excision of pMC7105 were used to identify one of the plasmid-chromosome juncture fragments and to characterize the mechanism of recombination from the chromosome. A 14.2-kb BamHI plasmid-chromosome juncture fragment has been identified in pEX8060 (234 kb), an excision plasmid which carries approximately 90 kb of chromosomal sequences to the left of the site of integration. This fragment contains a portion of the 2.6-kb Bg/II-EcoRI fragment as well as chromosomal sequences. Blot hybridization with a probe made from selected fragments of pMC7105 revealed three distinct repetitive sequences, RS-I, RS-II, and RS-III, on this plasmid. The 2.6-kb fragment, to which the site of integration maps, also contains RS-II. Five copies of RS-II are present in pMC7105, and more than 20 copies are present in the chromosome. Eight small excision plasmids were shown to result from recombination among fragments of pMC7105 that contain common repetitive sequences. The results indicate that integration and excision of pMC7105 occur through general recombination at homologous repetitive sequences.
Insights
The integration and excision of the pMC7105 plasmid in Pseudomonas syringae pv. phaseolicola are facilitated by general recombination. This process occurs at homologous repetitive sequences, specifically RS-II, found on both the plasmid and the chromosome.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The pMC7105 plasmid integrates into the Pseudomonas syringae pv. phaseolicola chromosome.
- Understanding the mechanism of this integration and subsequent excision is crucial for studying plasmid-chromosome interactions.
Purpose of the Study:
- To map the integration site of pMC7105 on the bacterial chromosome.
- To characterize the recombination mechanism involved in plasmid integration and excision.
Main Methods:
- Restriction enzyme digestion (Bg/II, EcoRI, BamHI) to map DNA fragments.
- Construction and analysis of excision plasmids.
- Blot hybridization using probes from pMC7105 fragments.
- Identification and characterization of repetitive sequences (RS-I, RS-II, RS-III).
Main Results:
- The integration site of pMC7105 was mapped to a 2.6-kb Bg/II-EcoRI fragment.
- A 14.2-kb BamHI plasmid-chromosome juncture fragment was identified in an excision plasmid, containing chromosomal sequences.
- Three repetitive sequences (RS-I, RS-II, RS-III) were found on pMC7105, with RS-II also present on the integration fragment.
- RS-II is highly repeated on both the plasmid (5 copies) and the chromosome (>20 copies).
- Small excision plasmids resulted from recombination between common repetitive sequences.
Conclusions:
- Integration and excision of pMC7105 occur via general recombination.
- Homologous repetitive sequences, particularly RS-II, mediate the recombination process between the plasmid and the chromosome.