Plasmids for making multiple knockouts in a radioresistant bacterium Deinococcus radiodurans

Area of Science:

• Microbiology
• Molecular Biology
• Bacterial Genetics

Background:

• Gene knockouts are typically achieved via homologous recombination, replacing target genes with selection markers.
• Deinococcus radiodurans, a radioresistant bacterium, has limitations in maintaining standard ColE1 origin-based plasmids.

Purpose of the Study:

• To construct and validate plasmids for generating gene disruption mutants in D. radiodurans.
• To investigate the effects of single and multiple gene disruptions on D. radiodurans growth.

Main Methods:

• Construction of plasmids with expressing cassettes for various antibiotic resistance markers (nptII, aadA, cat, aac(3)).
• Generation of single, double, and triple gene disruption mutants in D. radiodurans using homologous recombination.
• Assessment of mutant growth under normal conditions and in combination with a Topoisomerase II inhibitor.

Main Results:

• Successfully generated single and multiple gene disruption mutants in D. radiodurans.
• Double and triple mutants displayed significant growth retardation compared to single mutants.
• Synergistic growth inhibition was observed between multiple mutants and a Topoisomerase II inhibitor.

Conclusions:

• The developed plasmids are effective tools for creating multiple gene deletions in D. radiodurans.
• Multiple gene disruptions impact bacterial growth and can synergize with specific inhibitors.
• These plasmids offer a valuable approach for genetic manipulation in bacteria incompatible with ColE1 plasmids.