Integration and excision of plasmid DNA in Lactococcus lactis subsp. lactis
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Abstract:
The capacity of the 75-kb lactose-proteinase plasmid pCI301 from Lactococcus lactis subsp. lactis UC317 to recombine with the lactococcal chromosome was examined. Low-frequency integration of pCI301 sequences was detected following protoplast transformation of strain MG136Sm with total plasmid DNA from strain UC317. Excision of integrated sequences was subsequently observed at a low level. Excised sequences were rescued through recombination with and mobilization by the conjugative enterococcal plasmid pAMB1. Transconjugants harboring novel recombinant pCI301::pAMB1 plasmids, both pAMB1 and a pCI301 derivative, and pAMB1 only were isolated. The latter represents a class of transconjugant in which an elevated level of reintegration of pCI301 DNA in the recipient chromosome has occurred.
Insights
The lactose-proteinase plasmid pCI301 from Lactococcus lactis can integrate into the bacterial chromosome. This integrated DNA can be excised and mobilized by other plasmids, creating novel recombinant plasmids.
Area of Science:
- Microbiology
- Molecular Biology
- Bacteriology
Background:
- Lactococcus lactis is a key lactic acid bacterium used in dairy fermentation.
- Plasmids play crucial roles in bacterial adaptation and genetic exchange.
- The lactose-proteinase plasmid pCI301 is important for specific traits in Lactococcus lactis.
Purpose of the Study:
- To investigate the recombination capacity of the Lactococcus lactis plasmid pCI301 with the host chromosome.
- To explore the mechanisms of integration and excision of pCI301 sequences.
- To analyze the mobilization and recombination of integrated pCI301 DNA by other plasmids.
Main Methods:
- Protoplast transformation of Lactococcus lactis strain MG136Sm with total plasmid DNA from strain UC317.
- Detection and analysis of integrated pCI301 sequences in the recipient chromosome.
- Conjugation experiments using the enterococcal plasmid pAMB1 to mobilize excised pCI301 sequences.
Main Results:
- Low-frequency integration of pCI301 sequences into the Lactococcus lactis chromosome was observed.
- Excision of integrated pCI301 sequences occurred at a low frequency.
- Recombination with and mobilization by the conjugative plasmid pAMB1 resulted in novel recombinant plasmids and strains with altered pCI301 integration levels.
Conclusions:
- The Lactococcus lactis plasmid pCI301 exhibits the capacity for chromosomal integration and subsequent excision.
- Conjugative plasmids like pAMB1 can facilitate the mobilization and genetic manipulation of integrated plasmid sequences.
- These findings contribute to understanding plasmid-chromosome interactions and developing tools for genetic engineering in Lactococcus lactis.