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

Abstract

Certain <i>Salmonella enterica</i> serovars belonging to subspecies I carry low-copy-number virulence plasmids of variable size (50-90 kb). All of these plasmids share the <i>spv</i> 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 <i>Salmonella enterica</i> serovar Typhimurium encodes three auxiliary maintenance systems: one partition system (<i>parAB</i>) and two TA systems (<i>ccdAB</i>_ST and <i>vapBC2</i>_ST). The TA module <i>ccdAB</i>_ST has previously been shown to contribute to pSLT plasmid stability and <i>vapBC2</i>_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 <i>parAB</i> in pSLT maintenance. We also showed that <i>vapBC2</i>_ST, in addition to contribute to bacterial virulence, is important for plasmid stability. We have previously shown that <i>ccdAB</i>_ST encodes an inactive CcdB_ST toxin. Using our new stability assay we monitored the contribution to plasmid stability of a <i>ccdAB</i>_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 <i>ccdAB</i>_ST. In contrast, <i>ccdAB</i>_ST behaves as a canonical type II TA system in terms of transcriptional regulation. Of interest, <i>ccdAB</i>_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.