Stabilization of the Virulence Plasmid pSLT of Typhimurium by Three Maintenance Systems and Its Evaluation by Using a New Stability Test.

Certain serovars belonging to subspecies I carry low-copy-number virulence plasmids of variable size (50-90 kb). All of these plasmids share the operon, which is important for systemic infection. Virulence plasmids are present at low copy numbers. Few copies reduce metabolic burden but suppose a risk of plasmid loss during bacterial division. This drawback is counterbalanced by maintenance modules that ensure plasmid stability, including partition systems and toxin-antitoxin (TA) loci. The low-copy number virulence pSLT plasmid of serovar Typhimurium encodes three auxiliary maintenance systems: one partition system () and two TA systems ( and ). The TA module has previously been shown to contribute to pSLT plasmid stability and to bacterial virulence. Here we describe a novel assay to measure plasmid stability based on the selection of plasmid-free cells following elimination of plasmid-containing cells by ParE toxin, a DNA gyrase inhibitor. Using this new maintenance assay we confirmed a crucial role of in pSLT maintenance. We also showed that , in addition to contribute to bacterial virulence, is important for plasmid stability. We have previously shown that encodes an inactive CcdB toxin. Using our new stability assay we monitored the contribution to plasmid stability of a variant containing a single mutation (R99W) that restores the toxicity of CcdB. The "activation" of CcdB (R99W) did not increase pSLT stability by . In contrast, behaves as a canonical type II TA system in terms of transcriptional regulation. Of interest, was shown to control the expression of a polycistronic operon in the pSLT plasmid. Collectively, these results show that the contribution of the CcdB toxin to pSLT plasmid stability may depend on its role as a co-repressor in coordination with CcdA antitoxin more than on its toxic activity.