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

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.The work in RD and FG's laboratories is supported by grants BFU2011-25939 (RD), CSD2008-00013 (RD and FG), and BIO2013-46281-P/BIO2015-69085-REDC (FG) from the Spanish Ministry of Economy and Competitiveness.Peer reviewedPeer Reviewe

Interactions of Kid–Kis toxin–antitoxin complexes with the parD operator-promoter region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid–Kis oligomers

The parD operon of Escherichia coli plasmid R1 encodes a toxin–antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly understood aspect of the kid–kis system is its autoregulation at the transcriptional level. Using macromolecular (tandem) mass spectrometry and DNA binding assays, we here demonstrate that Kis pilots the interaction of the Kid–Kis complex in the parD regulatory region and that two discrete Kis-binding regions are present on parD. The data clearly show that only when the Kis concentration equals or exceeds the Kid concentration a strong cooperative effect exists between strong DNA binding and Kid(2)–Kis(2)–Kid(2)–Kis(2) complex formation. We propose a model in which transcriptional repression of the parD operon is tuned by the relative molar ratio of the antitoxin and toxin proteins in solution. When the concentration of the toxin exceeds that of the antitoxin tight Kid(2)–Kis(2)–Kid(2) complexes are formed, which only neutralize the lethal activity of Kid. Upon increasing the Kis concentration, (Kid(2)–Kis(2))(n) complexes repress the kid–kis operon

Coupling between the Basic Replicon and the Kis-Kid Maintenance System of Plasmid R1: Modulation by Kis Antitoxin Levels and Involvement in Control of Plasmid Replication

kis-kid, the auxiliary maintenance system of plasmid R1 and copB, the auxiliary copy number control gene of this plasmid, contribute to increase plasmid replication efficiency in cells with lower than average copy number. It is thought that Kis antitoxin levels decrease in these cells and that this acts as the switch that activates the Kid toxin; activated Kid toxin reduces copB-mRNA levels and this increases RepA levels that increases plasmid copy number. In support of this model we now report that: (i) the Kis antitoxin levels do decrease in cells containing a mini-R1 plasmid carrying a repA mutation that reduces plasmid copy number; (ii) kid-dependent replication rescue is abolished in cells in which the Kis antitoxin levels or the CopB levels are increased. Unexpectedly we found that this coordination significantly increases both the copy number of the repA mutant and of the wt mini-R1 plasmid. This indicates that the coordination between plasmid replication functions and kis-kid system contributes significantly to control plasmid R1 replication.This study was founded by projects CSD2008-00013 and BFU2011-25939 of the Spanish Ministry of Science and Innovation. Discussions related to this work with members of the group are kindly acknowledged. The comments and corrections to the manuscript of Elizabeth Diago-Navarro are kindly acknowledged.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

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

Toxin–antitoxin (TA) genes were first reported in plasmids and were considered expendable genetic cassettes involved in the stable maintenance of the plasmid replicon by interfering with growth and/or viability of bacteria in which the plasmid was lost. TAs were later found in bacterial chromosomes and also in integrated mobile genetic elements; they were proposed to be involved in the bacterial response to stressful situations. At present, 100s of TAs have been identified and classified in up to six families (I to VI), with those belonging to the type II (constituted by two protein components) being the most studied. Based on well-characterized examples of several type II TAs, we discuss in this review that irrespective of their locations in plasmids or chromosomes, TAs functionally overlap as indicated by: (i) in both locations they can mediate the maintenance of genetic elements to which they are physical linked, and (ii) they can induce persistence or virulence in response to stress situations. Examples of functional confluences in homologous TA systems with different locations are also given. We also consider whether the physiological role of TAs is due to their genetic organization as operons or to their inherent properties, like the short lifespan of the antitoxin component

Methods employing bacterial ParD kis/ParD kid toxin-antitoxin system for killing eukaryotic cells

Filing Date: 2000-07-17.-- Priority Data: GB 9916810 (1999-07-16).The present invention relates to killing cells, or at least impeding cell cycle progression. More particularly it relates to methods and means for attacking eukaryotic cells, such as tumour cells, with cytostatic, cytotoxic and/or cytopathic agents. Specifically, the present invention employs the ParD kid toxin and ParD kis antitoxin under appropriate control for selective cell cycle inhibition and/or killing of target cells

Bacteriophage P1 Ban protein is a hexameric DNA helicase that interacts with and substitutes for Escherichia coli DnaB

11 p.-9 fig.-1 tab.Since the ban gene of bacteriophage P1 suppresses a number of conditionally lethal dnaB mutations in Escherichia coli, it was assumed that Ban protein is a DNA helicase (DnaB analogue) that can substitute for DnaB in the host replication machinery. We isolated and sequenced the ban gene, puri®ed the product, and analysed the function of Ban protein in vitro and in vivo. Ban hydrolyses ATP, unwinds DNA and forms hexamers in the presence of ATP and magnesium ions. Since all existing conditionally lethal dnaB strains bear DnaB proteins that may interfere with the protein under study, we constructed a dnaB null strain by using a genetic set-up designed to provoke the conditional loss of the entire dnaB gene from E.coli cells. This novel tool was used to show that Ban restores the viability of cells that completely lack DnaB at 30oC, but not at 42oC. Surprisingly, growth was restored by the dnaB252 mutation at a temperature that is restrictive for ban and dnaB252 taken separately. This indicates that Ban and DnaB are able to interact in vivo. Complementary to these results, we demonstrate the formation of DnaB±Ban hetero-oligomers in vitro by ion exchange chromatography. We discuss the interaction of bacterial proteins and their phageencoded analogues to ful®l functions that are essential to phage and host growth.This work was supported by grants from the European Commission grant QLRT-1999-30634 to R.D.-O. and E.L. R.D.-O. acknowledges support by the `Programa de Grupos Estrategicos' (C.A.M.) 2000±2003 and grant BIO99-0859-CO1 from the Spanish MEC.Peer reviewe

Replicación , rango de huésped y mantenimiento de plásmidos en bacterias Gram-negativas: Estudios con pPS10, un plásmido de pseudomonas con R1, un factor de resistencia a antibióticos de enterobacterias

Peer reviewe

Distinct type I and type II toxin-antitoxin modules control Salmonella lifestyle inside eukaryotic cells

10 p.-5 fig.-2 tab.Toxin-antitoxin (TA) modules contribute to the generation of non-growing cells in response to stress. These modules abound in bacterial pathogens although the bases for this profusion remain largely unknown.Using the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium as a model, here we show that a selected group of TA modules impact bacterial fitness inside eukaryotic cells. We characterized in this pathogen twenty-seven TA modules, including type I and type II TA modules encoding antisense RNA and proteinaceous antitoxins, respectively. Proteomic and gene expression analyses revealed that the pathogen produces numerous toxins of TA modules inside eukaryotic cells. Among these, the toxins HokST,LdrAST, and TisBST, encoded by type I TA modules and T4ST and VapC2ST, encoded by type II TA modules,promote bacterial survival inside fibroblasts. In contrast, only VapC2ST shows that positive effect in bacterial fitness when the pathogen infects epithelial cells. These results illustrate howS. Typhimurium uses distinct type I and type II TA modules to regulate its intracellular lifestyle in varied host cell types. This function specialization might explain why the number of TA modules increased in intracellular bacterial pathogens.This study was supported by grants BIO2013-46281-P (to F.G.-d.P.),BFU2011-25939 (to R. D.-O.), and CSD2008/00013 (INTERMODS, Consolider Program)(to F.G.-d.P. and R. D.-O.), funded by the Spanish Ministry of Economy and Competitiveness.Peer reviewe

DnaA dependent replication of plasmid R1 occurs in the presence of point mutations that disrupt the dnaA box of oriR

We have found that DnaA dependent replication of R1 still occurred when 5 of the 9 bases in the dnaA box present in oriR were changed by site directed mutagenesis although the replication efficiency decreased to 20% and 70% of the wild-type origin in vitro and in vivo respectively. Additional mutation of a second dnaA box, 28bp upstream oriR, that differs in only one base from the consensus sequence, did not affect the level of replication whereas polyclonal antibodies against DnaA totally abolished in vitro replication in the absence of the dnaA box. Wild-type RepA as well as a RepA mutant, RepA2623, that binds to oriR but that is inactive in promoting in vitro replication of plasmid R1, induce efficient binding of DnaA to the dnaA box. However, specific binding of DnaA to oriR was not detected by DNase I protection experiments in the absence of the dnaA box. These results suggest that the entrance of the DnaA protein in oriR is promoted initially by interactions with a RepA-oriR pre-initiation complex and that, in the absence of the dnaA box, these interactions can support, with reduced efficiency, DnaA dependent replication of plasmid R1.Peer Reviewe

Genetic and functional analysis of the basic replicon of pPS10, a plasmid specific for pseudomonas isolated from Pseudomonas syringae patovar savastanoi

The sequence of a 1823 base-pair region containing the replication functions of pPS10, a narrow host-range plasmid isolated from a strain of Pseudomonas savastanoi, is reported. The origin of replication, oriV, or pPS10 is contained in a 535 base-pair fragment of this sequence that can replicate in the presence of trans-acting function(s) of the plasmid. oriV contains four iterons of 22 base-pairs that are preceded by G + C-rich and A + T-rich regions. A dnaA box located adjacent to the repeats of the origin is dispensable but required for efficient replication of pPS10; A and T are equivalent bases at the 5′ end of the box. repA, the gene of a trans-acting replication protein of 26,700 Mr has been identified by genetic and functional analysis, repA is adjacent to the origin of replication and is preceded by the consensus sequences of a typical σ70 promoter of Escherichia coli. The RepA protein has been identified, using the minicell system of E. coli, as a polypeptide with an apparent molecular mass of 26,000. A minimal pPS10 replicon has been defined to a continuous 1267 base-pair region of pPS10 that includes the oriV and repA sequences.This work was supported by grants BI088-0294 and BT-42-84 from the Spanish CICYT and CSIC. C.N. and R.G. were supported by fellowships from the Spanish MEC