A toxin-antitoxin system confers stability to the IncP-7 plasmid pCAR1

Toxin-antitoxin (TA) systems were initially discovered as plasmid addiction systems. Previously, our studies implied that the high stability of the IncP-7 plasmid pCAR1 in different Pseudomonas spp. hosts was due to the presence of a TA system on the plasmid. Bioinformatics approaches suggested that ORF174 and ORF175 could constitute a type II TA system, a member of the RES-Xre family, and that these two open reading frames (ORFs) constitute a single operon. As expected, the ORF175 product is a toxin, which decreases the viability of the host, P. resinovorans, while the ORF174 product functions as an antitoxin that counteracts the effect of ORF175 on cell growth. Based on these findings, we renamed ORF174 and ORF175 as prcA (antitoxin gene) and prcT (toxin gene), respectively. The prcA and prcT genes were cloned into the unstable plasmid vector pSEVA644. The recombinant vector was stably maintained in P. resinovorans and Escherichia coli cells under nonselective conditions following 6 days of daily subculturing. The empty vector (without the prcA and prcT genes) could not be maintained, which suggested that the PrcA/T system can be used as a tool to improve the stability of otherwise unstable plasmids in P. resinovorans and E. coli strains.