19 research outputs found

    Regulatory crosstalk between type I and type II toxin-antitoxin systems in the human pathogen <i>Enterococcus faecalis</i>

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    <p>We discovered a chromosomal locus containing 2 toxin-antitoxin modules (TAs) with an antisense transcriptional organization in the E. faecalis clinical isolate V583. These TAs are homologous to the type I txpA-ratA system and the type II mazEF, respectively. We have shown that the putative MazF is toxic for E. coli and triggers RNA degradation, and its cognate antitoxin MazE counteracts toxicity. The second module, adjacent to mazEF, expresses a toxin predicted to belong to the TxpA type I family found in Firmicutes, and the antisense RNA antidote, RatA. Genomic analysis indicates that the cis-association of mazEF and txpA-ratA modules has been favored during evolution, suggesting a selective advantage for this TA organization in the E. faecalis species. We showed regulatory interplays between the 2 modules, involving transcription control and RNA stability. Remarkably, our data reveal that MazE and MazEF have a dual transcriptional activity: they act as autorepressors and activate ratA transcription, most likely in a direct manner. RatA controls txpA RNA levels through stability. Our data suggest a pivotal role of MazEF in the coordinated expression of mazEF and txpA-ratA modules in V583. To our knowledge, this is the first report describing a crosstalk between type I and II TAs.</p

    Interaction between <i>E. faecalis</i> pp1 and pp7 (EfCIV583).

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    <p>(A) Semi-quantitative PCR detection of EfCIV583 circular forms (<i>attP</i>) and excision sites (<i>attB</i>) in wild-type (WT) and strains <i>pp1<sup>−</sup></i>, <i>pp1<sup>+</sup> pp7<sup>+</sup></i> and <i>pp7<sup>+</sup></i>. Excision and circularization products probed by semi-quantitative PCR on 100, 10 and 1 pg of total bacterial DNA prepared from cultures of WT and strains <i>pp1<sup>−</sup></i>, <i>pp1<sup>+</sup> pp7<sup>+</sup></i> and <i>pp7<sup>+</sup></i> induced for 2 h with 2 µg/ml of ciprofloxacin at 37°C. Twenty cycles were used to amplify products of pp1 and EfCIV583. These results are representative of two independent experiments. (B) Prophage DNA extracted from precipitated phage particles obtained from lysates of WT and strains <i>pp1<sup>−</sup></i>, <i>pp1</i><sup>+</sup><i>pp7</i><sup>+</sup> and <i>pp7</i><sup>+</sup> was separated by FIGE and analyzed by Southern-blot and hybridized sequentially using specific probes for pp1 and EfCIV583 genomes. The approximately 38.2 kb and 12 kb band corresponds to P1 and EfCIV583 genome, respectively. As ascertained by pp1-specific hybridization, migration of P1 DNA was delayed in lane <i>pp1</i><sup>+</sup> and <i>pp7</i><sup>−</sup> compared to lanes WT and <i>pp1</i><sup>+</sup><i>pp7</i><sup>+</sup>. Lambda DNA mono-cut mix (NEB) was run next to the samples to validate band sizes. (C) Scanning electron microscopy images of bacterial cells from strains <i>pp<sup>−</sup></i> and <i>pp1</i><sup>+</sup><i>pp7</i><sup>+</sup> after ciprofloxacin treatment. (D) Transmission electron microscopy images of phages produced by strain <i>pp1</i><sup>+</sup><i>pp7</i><sup>+</sup> after ciprofloxacin treatment. White and black arrows indicate big and small sized particles attributed to P1 and EfCIV583, respectively. Enlarged images of EfCIV583 and P1 (renamed vB_EfaS_V583-P1) are shown on the right.</p

    Prophage excision and replication.

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    <p>Agarose gel analysis of prophage excision and circularization products corresponding to <i>attB</i> and <i>attP</i> regions, respectively, probed by PCR. (A) Experimental approach: two sets of primers were used to detect prophage excision from the chromosome. The first set in red targets the excision site on the chromosome (<i>attB</i>) and the second set in green targets prophage circular forms (<i>attP</i>). (B) Prophage excision products corresponding to <i>attB</i> region were probed by PCR in WT cultures induced with 2 µg/ml of mitomycin C (M), or ciprofloxacin (C) or uninduced (−) at 28, 37 and 42°C. Ch corresponds to amplification of a strain specific chromosomal gene. (C) Prophage excision products in strain <i>pp3<sup>−</sup> pp5<sup>−</sup></i> at 37°C. (D–F) Excision and circularization products probed by semi-quantitative PCR on 100, 10 and 1 pg of total bacterial DNA prepared from cultures of WT (D) and strains <i>pp3<sup>−</sup> pp5<sup>−</sup></i> (E) <i>pp4<sup>+</sup></i> (F) induced for 2 h with 2 µg/ml of ciprofloxacin at 37°C except for detection of pp4 products in the WT strain that were obtained from DNA prepared after induction at 42°C. Twenty and 32 PCR cycles were used to amplify products of pp1 and pp7 and products of pp3, pp4 and pp5, respectively. These results are representative of three independent experiments.</p

    Genomic organization of <i>E. faecalis</i> V583 prophages.

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    <p>Open-reading frames are indicated by arrows. Only genes encoding predicted function are annotated. Colors correspond to the seven functional modules of temperate phages as depicted at the bottom right corner.</p

    DNA of pp1 and EFCIV583 are packaged in separated capsids.

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    <p>Presentation of two working hypotheses for pp1 and EFCIV583 DNA packaging in a dilysogen strain and experimental results corroborating one of them. On the left, DNAs are packaged inside the same capsid. The resulting virions are predicted to deliver both DNA during infection and to form plaques containing both P1 and EFCIV583 virions since pp1 is required for formation of EFCIV583 virions on indicator strains <i>pp<sup>−</sup></i> and <i>pp1</i><sup>− </sup><i>pp7</i><sup>−</sup>, both deleted for pp1 and EFCIV583. On the right, pp1 and EFCIV583 DNAs are packaged separately in two different capsids. The resulting virions would deliver either pp1 or EFCIV583 DNA during infection of strains <i>pp<sup>−</sup></i> and <i>pp1</i><sup>− </sup><i>pp7</i><sup>−</sup>, and would form only P1 plaques since pp1 is required for formation of EFCIV583 virions and co-infection by two particles is highly improbable. However, EFCIV583 virions would be detected on the indicator strain <i>pp7<sup>−</sup></i>, which harbors pp1. Lysates of strain <i>pp1<sup>+</sup> pp7<sup>+</sup></i> were tested on indicator strains <i>pp</i><sup>−</sup>, <i>pp1</i><sup>− </sup><i>pp7</i><sup>−</sup> and <i>pp7<sup>−</sup></i> and the resulting plaques were identified by pp1- and EFCIV583-specific PCRs. Our results strongly support that P1 and EfCIV583 genomes are packaged in two different capsids since plaques formed by <i>pp1<sup>+</sup> pp7<sup>+</sup></i> lysates on indicator strains <i>pp<sup>−</sup></i> and <i>pp1<sup>−</sup> pp7<sup>−</sup></i> were identified as P1 plaques only, while EFCIV583 virions were detected on indicator strain <i>pp7<sup>−</sup></i>.</p

    Detection of prophage packaged DNA.

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    <p>Encapsidated prophage DNA recovered from supernatant of WT cultures obtained at different temperatures 2 h after ciprofloxacin treatment at 2 µg/ml and detected by southern-blot hybridization with prophage specific probes. Non-treated and ciprofloxacin treated cultures correspond to lanes (−) and (C), respectively. DNA of P1, P5 and P7 is encapsidated at all temperatures under inducing conditions.</p

    Impact of prophages on <i>E. faecalis</i> adhesion to human platelets.

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    <p>The values shown are normalized to the percentage of adhesion to platelets of the WT strain. Data are expressed as mean ± SD. Platelet binding assays were performed in platelets from three different donors. P value between WT strain and adherent strains (<i>pp1<sup>+</sup></i>, <i>pp4<sup>+</sup></i> and <i>pp6<sup>+</sup></i>) is indicated.</p

    Model of P1/EfCIV583 interplay.

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    <p>Infectious P1 and EfCIV583 particles are produced by strains WT and <i>pp1<sup>+</sup> pp7<sup>+</sup></i> whereas no particles are produced in the absence of pp1 (strains <i>pp1<sup>−</sup></i> and <i>pp7<sup>+</sup></i>), showing that pp1 is required to form EfCIV583 virions. This hijacking phenomenon impairs the production of P1 particles in favor of EfCIV583. As observed by SEM and TEM, strain <i>pp1<sup>+</sup> pp7<sup>+</sup></i> produces two different sizes of phage particles: the biggest package most probably P1 DNA and the smallest EfCIV583 DNA. In the absence of EfCIV583 (strains <i>pp7<sup>−</sup></i> and <i>pp1<sup>+</sup></i>), P1 virions are produced at higher titer.</p

    Impact of Rho inactivation on swarming motility of <i>B</i>. <i>subtilis</i> cells.

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    <p>(A) Motility defect of the NCIB 3610 RM cells can be partially suppressed by the deletion of <i>slrR</i> and ectopic expression of <i>flhO-flhP</i> genes. Bacterial cultures were grown to an OD<sub>600</sub> 0.5, concentrated and spotted on the plate as described (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006909#sec019" target="_blank">Materials and methods</a>). The images were acquired after 20 hours of incubation at 37°C. Each icon represents top-grown image of centrally inoculated Petri plate (diameter 9 cm) containing LB and 0.7% of agar. Relevant genotypes are indicated on the side of each image. The repaired back to the wild type NCIB 3610 RM is denoted as <i>rho</i> wt*. The experiment was reproduced at least five times and included three biological replicas for each strain. The results from the representative experiment are presented. (B) Quantitative swarming assay of the indicated NCIB 3610 (blue lines) and isogenic NCIB 3610 RM (red lines) derivative strains. Values represent the mean of at least five experiments. (C) Impact of <i>rho</i> deletion on sense and antisense transcription of the <i>flhO-flhP</i> operon in the <i>B</i>. <i>subtilis</i> 1012 cells. Expression profiles are from the <i>B</i>. <i>subtilis</i> expression data browser (<a href="http://genome.jouy.inra.fr/cgi-bin/seb/index.py" target="_blank">http://genome.jouy.inra.fr/cgi-bin/seb/index.py</a>). Vertical bar on the top line indicates position of predicted putative terminator (shown in D). Sections show annotated genome (top) and expression profiles on the (+) and (–) strands (mid and bottom sections). Wild type (black) and RM (red) profiles are shown. (D) MFOLD [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006909#pgen.1006909.ref083" target="_blank">83</a>] predicted secondary structure (ΔG = −16, 30) within <i>flhP</i> asRNA.</p
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