Abstract
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 (ccdABST and vapBC2ST). The TA module ccdABST has previously been shown to contribute to pSLT plasmid stability and vapBC2ST 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 vapBC2ST, in addition to contribute to bacterial virulence, is important for plasmid stability. We have previously shown that ccdABST encodes an inactive CcdBST toxin. Using our new stability assay we monitored the contribution to plasmid stability of a ccdABST variant containing a single mutation (R99W) that restores the toxicity of CcdBST. The “activation” of CcdBST (R99W) did not increase pSLT stability by ccdABST. In contrast, ccdABST behaves as a canonical type II TA system in terms of transcriptional regulation. Of interest, ccdABST was shown to control the expression of a polycistronic operon in the pSLT plasmid. Collectively, these results show that the contribution of the CcdBST toxin to pSLT plasmid stability may depend on its role as a co-repressor in coordination with CcdAST antitoxin more than on its toxic activity.
Highlights
During evolution bacterial pathogens acquire new genes dedicated to manipulate host processes
Typhimurium possesses at least three main mechanisms to ensure its stable maintenance in the cell:(i) a copy number control of replication mediated by repB and repC replicons; (ii) the parAB partition system; and (iii) the TA systems ccdABST and vapBC2ST
In accordance with the literature, we show that the ParAB partition system stabilizes the pSLT plasmid very efficiently
Summary
During evolution bacterial pathogens acquire new genes dedicated to manipulate host processes. Serovar Typhimurium, together with a few other serovars of subspecies I, possesses a virulence plasmid (Jones et al, 1982) These plasmids have a variable size of 50–90 kb and share common features such as low copy number (1-2 plasmids per chromosome), a similar repC replicon (similar to the repFIB family) and a conserved set of virulence genes encoding toxins and fimbrial proteins (including spv and pef operons) (Bäumler et al, 1998; Rotger and Casadesús, 1999). If the TAencoding plasmid is lost, the antitoxin cannot be replenished and the free toxin eliminates or reduces the growth of daughter cells diluting plasmid-free cells in the population (Yamaguchi and Inouye, 2011) This phenomenon is called post-segregational killing (Gerdes et al, 1986)
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