Stabilization of the Virulence Plasmid pSLT of Salmonella Typhimurium by Three Maintenance Systems and Its Evaluation...

Certain Salmonella enterica serovars belonging to subspecies I carry low-copy-number virulence plasmids of variable size (50-90 kb). All of these plasmids share the spv 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 Salmonella enterica serovar Typhimurium encodes three auxiliary maintenance systems: one partition system (parAB) and two TA systems (ccdAB_ST and vapBC2_ST). The TA module ccdAB_ST has previously been shown to contribute to pSLT plasmid stability and vapBC2_ST 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 parAB in pSLT maintenance. We also showed that vapBC2_ST, in addition to contribute to bacterial virulence, is important for plasmid stability. We have previously shown that ccdAB_ST encodes an inactive CcdB_ST toxin. Using our new stability assay we monitored the contribution to plasmid stability of a ccdAB_ST variant containing a single mutation (R99W) that restores the toxicity of CcdB_ST. The "activation" of CcdB_ST (R99W) did not increase pSLT stability by ccdAB_ST. In contrast, ccdAB_ST behaves as a canonical type II TA system in terms of transcriptional regulation. Of interest, ccdAB_ST was shown to control the expression of a polycistronic operon in the pSLT plasmid. Collectively, these results show that the contribution of the CcdB_ST toxin to pSLT plasmid stability may depend on its role as a co-repressor in coordination with CcdA_ST antitoxin more than on its toxic activity.