title: Stabilization of the Virulence Plasmid pSLT ofSalmonellaTyphimurium by Three Maintenance Systems and Its Evaluation by Using a New Stability Test.
abstract: CertainSalmonella entericaserovars belonging to subspecies I carry low-copy-number virulence plasmids of variable size (50-90 kb). All of these plasmids share thespvoperon, 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 ofSalmonella entericaserovar Typhimurium encodes three auxiliary maintenance systems: one partition system (parAB) and two TA systems (ccdABSTandvapBC2ST). The TA moduleccdABSThas previously been shown to contribute to pSLT plasmid stability andvapBC2STto 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 ofparABin pSLT maintenance. We also showed thatvapBC2ST, in addition to contribute to bacterial virulence, is important for plasmid stability. We have previously shown thatccdABSTencodes an inactive CcdBSTtoxin. Using our new stability assay we monitored the contribution to plasmid stability of accdABSTvariant containing a single mutation (R99W) that restores the toxicity of CcdBST. The "activation" of CcdBST(R99W) did not increase pSLT stability byccdABST. In contrast,ccdABSTbehaves as a canonical type II TA system in terms of transcriptional regulation. Of interest,ccdABSTwas shown to control the expression of a polycistronic operon in the pSLT plasmid. Collectively, these results show that the contribution of the CcdBSTtoxin to pSLT plasmid stability may depend on its role as a co-repressor in coordination with CcdASTantitoxin more than on its toxic activity.
authors: Section of Microbiology, Department of Medicine, Centre for Molecular Bacteriology and Infection, Imperial College London London, UK.
authors: Damián
authors: D
authors: Lobato-Márquez
authors: Department of Cell and Developmental Biology, University College London London, UK.
authors: Laura
authors: L
authors: Molina-García
authors: Departamento de Microbiología Molecular y Biología de las Infecciones, Centro de Investigaciones Biológicas-Spanish National Research Council Madrid, Spain.
authors: Inma
authors: I
authors: Moreno-Córdoba
authors: Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología-Spanish National Research Council Madrid, Spain.
authors: Francisco
authors: F
authors: García-Del Portillo
authors: Departamento de Microbiología Molecular y Biología de las Infecciones, Centro de Investigaciones Biológicas-Spanish National Research Council Madrid, Spain.
authors: Ramón
authors: R
authors: Díaz-Orejas
keywords: Salmonella Typhimurium; plasmid stability; toxin-antitoxin; transcriptional regulation; virulence plasmid