Dataset Information

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.

SUBMITTER: Lobato-Marquez D

PROVIDER: S-EPMC5065971 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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

Lobato-Márquez Damián D Molina-García Laura L Moreno-Córdoba Inma I García-Del Portillo Francisco F Díaz-Orejas Ramón R

Frontiers in molecular biosciences 20161017

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 an ...[more]

PMID: 27800482

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Project description:Bacterial small RNAs (sRNAs) are regulatory molecules playing relevant roles in response to environmental changes, stressful conditions and pathogenesis. The intracellular bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) is known to regulate expression of some sRNAs during colonization of fibroblasts. Here, we characterize a previously unknown sRNA encoded in the S. Typhimurium pSLT virulence plasmid that is specifically up-regulated by non-growing dormant bacteria persisting inside fibroblasts. This sRNA was inferred in microarray expression analyses, which unraveled enhanced transcriptional activity in the PSLT047- PSLT046 (mig5) intergenic region. The sRNA transcript was further identified as a 597-nucleotide molecule, which we named IesR-1, for 'Intracellular-expressed-sRNA-1'. IesR-1 expression is low in bacteria growing in axenic cultures across a variety of experimental conditions but displays a marked increase (∼200-300 fold) following bacterial entry into fibroblasts. Remarkably, induction of IesR-1 expression is not prominent in bacteria proliferating within epithelial cells. IesR-1 deletion affects the control of bacterial growth in defined fibroblast cell lines and impairs virulence in a mouse infection model. Expression analyses performed in the PSLT047-iesR-1-PSLT046 (mig5) region support a cis-acting regulatory mechanism of IesR-1 as antisense RNA over the PSLT047 transcript involving interaction at their respective 3' ends and modulation of PSLT047 protein levels. This model is sustained by the scarce production of PSLT047 protein observed in non-growing intracellular bacteria and the high amount of PSLT047 protein produced by bacteria carrying a truncated IesR-1 version with separated 5' and 3' regions. Taken together, these data reveal that S. Typhimurium sRNAs encoded in the pSLT virulence plasmid respond to a state of persistence inside the host cell. As exemplified by IesR-1, some of these sRNAs may contribute to diminish the relative levels of proteins, such as PSLT047, which are probably dispensable for the intracellular lifestyle.

Project description:The virulence plasmid pSLT as exemplified by the 94 Kb plasmid in Salmonella Typhimurium strain LT2 is only found in isolates of serovar Typhimurium. While it occurs commonly among such isolates recent genotyping methods have shown that it is mostly confined to certain genotypes. Although pSLT plasmids are capable of self-transmissibility under experimental conditions their confinement to certain host genotypes suggests that in practice they are maintained by vertical rather than by horizontal transmission. This would imply that evolution of the pSLT plasmid proceeds in parallel with evolution of its host. The development of a phylogenetic evolutionary framework for genotypes of S. Typhimurium based on single-nucleotide-polymorphism (SNPs) typing provided an opportunity to test whether the pSLT plasmid coevolves with its host genotype. Accordingly SNPs analysis was applied to the pSLT plasmids from 71 strains S. Typhimurium of Australian and international origins representing most of the genotypes which commonly have a pSLT. The phylogenetic tree showed that pSLT sequences clustered into almost the same groups as the host chromosomes so that each pSLT genotype was associated with a single host genotype. A search for tandem repeats in pSLT plasmids showed that a 9 bp VNTR in the traD gene occurred in the pSLT from all isolates belonging to Clade II but not from isolates belonging to Clade I. Another 9 bp repeat occurred only in three Clade I genotypes with a recent common ancestor. The evidence relating to both of these VNTRs supports the proposition that the pSLT plasmid is only transmitted vertically. Some isolates belonging to one S. Typhimurium genotype were found to have pSLTs which have lost a large block of genes when a resistance gene cassette has been acquired. Examples were found of pSLT plasmids which have recombined with other plasmids to form fusion plasmids sometimes with loss of some pSLT genes. In all cases the underlying genotype of the modified pSLT was the same as the genotype of regular pSLTs with the same host genotype implying that these changes have occurred within the host cell of the pSLT plasmid.

Dataset Information

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

Publications

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

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