Novel system for recognizing and eliminating foreign DNA in Pseudomonas putida.

Area of Science:

• Molecular Biology
• Microbial Genetics
• Bacterial Plasmid Stability

Background:

• Plasmids are extrachromosomal DNA elements crucial for bacterial adaptation and gene transfer.
• Plasmid stability within host cells is essential for maintaining genetic traits and experimental reproducibility.
• Cross-species plasmid behavior can vary significantly due to species-specific genetic mechanisms.

Purpose of the Study:

• To investigate the stability of hybrid plasmids containing foreign DNA in different bacterial species.
• To elucidate the mechanism underlying plasmid instability observed in Pseudomonas putida.
• To determine if foreign DNA influences the stability of unrelated plasmids in the same host.

Main Methods:

• Construction of hybrid plasmids by cloning Escherichia coli and Pseudomonas putida chromosomal DNA fragments into pQSR49.
• Assessment of plasmid stability and copy number in E. coli and P. putida.
• Introduction of a second plasmid (RK2) into P. putida harboring unstable hybrid plasmids to evaluate its stability.

Main Results:

• Hybrid plasmids with E. coli DNA were unstable in P. putida but stable in E. coli.
• Hybrid plasmids with P. putida DNA were stable in both species.
• Plasmid instability in P. putida resulted from plasmid loss, not cell toxicity, and was not linked to reduced replication.
• The unrelated plasmid RK2 also became unstable in P. putida when co-existing with unstable hybrids carrying foreign DNA.

Conclusions:

• Pseudomonas putida possesses an inducible mechanism that actively eliminates foreign DNA carried on plasmids.
• This mechanism leads to the instability of hybrid plasmids containing non-native DNA sequences.
• The findings suggest a defense system in P. putida against foreign genetic elements.