Engineering the bacterium Comamonas testosteroni CNB-1: Plasmid curing and genetic manipulation

Comamonas species are attractive microbial hosts for bioremediation engineering due to their versatile metabolic capacity and diverse niches inhabited. To establish Comamonas testosteroni CNB-1—a common Comamonas strain—as a programmable cellular chassis, its indigenous 91-kb-long plasmid, pCNB1, must be eliminated; in addition, the organism has to exhibit feasiblity for effective genetic manipulation. Here we present a novel plasmid curing strategy involving rare-cutting homing endonuclease and a selection and counter-selection system. With a self-eliminating helper plasmid, we successfully removed the target plasmid pCNB1, along with the helper plasmid itself, in a single step at an efficiency of 64%. By enhancing the homing endonuclease expression, we were able to further improve the efficiency to almost 100%. The curing of pCNB1 provided a plasmid-free strain for hosting an IncP-type replicon based shuttle vehicle which can be utilized for gene circuit engineering. Additionally, using a PCR-based Cre-loxP system, we demonstrated an increased ability to genetically manipulate the chromosome of C. testosteroni CNB-1. This study provides a rapid and effective solution to eliminate indigenous plasmids, and also facilitates the development of C. testosteroni as a promising chassis for future remediation applications.