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

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.

Spread and Persistence of Virulence and Antibiotic Resistance Genes: A Ride on the F Plasmid Conjugation Module

“…As with some bacterial chromosomes, multiple type II TA systems of different superfamilies can be present on the same plasmid. This is, for instance, the case with the MOB F12 A plasmid pECC-1470_100 (100 kbp, R1 subgroup, CP010345) that not only harbors three replicons but, in addition to the CcdAB TA system, carries a VapBC TA pair where VapC is a toxin that inhibits translation and cell growth by cleavage of initiator tRNA (56). The large F-like virulence plasmid pSLT (NC_ 003277) which is highly prevalent in pathogenic Salmonella enterica serovar Typhimurium strains also harbors these type II TA systems that were designated CcdAB ST and VapBC2 ST , respectively (57).…”

The Importance of the Expendable: Toxin–Antitoxin Genes in Plasmids and Chromosomes

“…Two recent articles published in Frontiers ( Chan and Espinosa, 2016 ; Lobato-Márquez et al, 2016b ) underline the concept that phenotypes associated to plasmid- or to chromosomally encoded TAs do overlap because independent of their location, toxins target similar functions and the TA operons are regulated and induced by similar conditions. The first example is provided by the pneumococcal pezAT operon ( Khoo et al, 2007 ; Chan and Espinosa, 2016 ).…”

“…The second example is related to the role of TAs of Salmonella enterica serovar Typhimurium carrying the virulence plasmid pLST during bacterial infection ( Lobato-Márquez et al, 2016b ). One of the two TAs encoded by plasmid pLST is vapBC ST .…”

“…This particular TA contributes to the successful colonization of recipient cells during infection, in conjunction with other type I and type II TAs encoded by the Salmonella chromosome ( Lobato-Márquez et al, 2015 ). In addition to its role during infection, the plasmidic copy of vapBC ST contributes to the maintenance of the plasmid ( Lobato-Márquez et al, 2016b ). Interestingly, the chromosomal copy of this particular TA seems to be inactive, so that the role in infection was taken up by the plasmid-encoded copy.…”

Genetic control of ColE1 plasmid stability that is independent of plasmid copy number regulation

“…Plasmid copy number, replication efficiency, post-segregational killing, and an effective decrease in partition units due to concatemerization or clustering are all variables that have been considered (Bergstrom et al, 2000; Cooper et al, 1987; De Gelder et al, 2007; Lau et al, 2013; Lili et al, 2007; San Millan et al, 2014; Werbowy et al, 2017). Given that naturally-occurring plasmids typically experience fluctuating selective pressures and would be susceptible to loss over long periods of time, these plasmids typically have mechanisms modulating one or more of these variables to enhance their stability (Li et al, 2015; Lobato-Marquez et al, 2016; Werbowy and Kaczorowski, 2016). These stabilizing mechanisms include plasmid addiction systems that kill plasmidless daughter cells (Kroll et al, 2010; Li et al, 2015; Mruk and Kobayashi, 2014; Werbowy and Kaczorowski, 2016), multimer resolution systems to suppress the formation of concatemers by homologous recombination (Summers et al, 1993; Werbowy et al, 2015), and the presence of multiple redundant mechanisms for replication initiation (Cesareni et al, 1991).…”