Improved site-specific mutagenesis in Rhodococcus opacus using a novel conditional suicide plasmid

Area of Science:

• Microbiology
• Synthetic Biology
• Biotechnology

Background:

• Rhodococcus opacus PD630 is a versatile bacterium used in bioremediation, metal recovery, and triacylglycerol storage.
• Genetic manipulation of R. opacus via homologous recombination is challenging due to low DNA transfer and recombination frequencies.

Purpose of the Study:

• To develop an improved vector system for efficient homologous recombination in R. opacus.
• To investigate the essentiality of mycolic acids for R. opacus viability using the developed vector.

Main Methods:

• Construction and evaluation of a conjugative, conditional suicide plasmid based on the Corynebacterium glutamicum plasmid pGA1 replicon.
• Utilized a dual inducible and repressible promoter system for replication control and sacB as a counterselection marker.
• Employed the vector to attempt deletion of the accD3 gene, essential for mycolic acid synthesis.

Main Results:

• The developed plasmid derivative enabled efficient homologous recombination in R. opacus, overcoming previous limitations.
• Deletion of the accD3 gene was only possible in strains harboring a plasmid-encoded copy, indicating its essentiality.
• Mycolic acid synthesis is crucial for the viability of R. opacus, similar to its role in Mycobacterium species.

Conclusions:

• A novel conditional suicide plasmid vector significantly enhances homologous recombination in R. opacus.
• Mycolic acids are essential for R. opacus survival, highlighting a conserved characteristic within certain bacterial genera.
• The developed vector and approach are applicable for genetic engineering in Rhodococcus and related bacteria, and for plasmids requiring plasmid-encoded proteins for replication.