Materials and Methods

Discovery of new type I toxin–antitoxin systems adjacent to CRISPR arrays inClostridium difficile

MATERIALS AND METHODSPlasmid and bacterial strain construction and growth conditions C. difficile and E. coli strains and plasmids used in this study are presented in Supplementary Table S1. C. difficile strains were grown anaerobically (5% H2, 5% CO2 and 90% N2) in TY (36) or Brain Heart Infusion (BHI, Difco) media in an anaerobic chamber (Jacomex). When necessary, cefoxitin (Cfx; 25 μg/ml) and thiamphenicol (Tm; 15 μg/ml) were added to C. difficile cultures. E. coli strains were grown in LB broth (37), and when needed, ampicillin (100 μg/ml) or chloramphenicol (15 μg/ml) was added to the culture medium. The non-antibiotic analog anhydrotetracycline (ATc) was used for induction of the Ptet promoter of pRPF185 vector derivatives in C. difficile (38). Strains carrying pRPF185 derivatives were generally grown in TY medium in the presence of 250 ng/ml ATc and 7.5 μg/ml Tm for 7.5 h. Growth curves were obtained using a GloMax plate reader (Promega).All routine plasmid constructions were carried out using standard procedures (39). All primers used in this study are listed in Supplementary Table S2. For inducible expression of C. difficile genes, we used the pDIA6103 derivative of pRPF185 vector expression system lacking a gusA gene (15,38). The CD2517.1 gene (−89 to +178 relative to the translational start site), the CD2907.1 gene (−84 to +223 relative to the translational start site), CD2517.1-RCd8 TA region with RCd8 promoter (−306 to +504 relative to the translational start site of CD2517.1) and CD2907.1-RCd9 TA region with RCd9 promoter (−294 to +456 relative to the translational start site of CD2907.1) were amplified by PCR and cloned into StuI and BamHI sites of pDIA6103 vector under the control of the ATc-inducible Ptet promoter giving pDIA6319, pDIA6195, pDIA6202 and pDIA6196, respectively.The knockdown antisense system on pRPF185 vector derivative was used to deplete the C. difficile 630Δerm strain for the specific ribonucleases RNase III, RNase J and RNAse Y. The rncS gene fragment comprising part of 5′ untranslated region (UTR) and the beginning of the rncS coding part (−39 to +188 relative to the translational start site) was amplified by PCR on C. difficile 630Δerm strain genomic DNA and cloned into StuI and BamHI sites of pRPF185 vector in antisense orientation under the control of the ATc-inducible Ptet promoter giving pDIA6126. Similar strategy was used to construct plasmids pDIA5975 and pDIA5977 for inducible expression of antisense RNA for RNase J and RNase Y genes (+7 to +217 and +55 to +210 relative to the transcriptional start site (TSS) identified by deep sequencing, respectively).For subcellular localization of toxins we used reverse PCR approach to construct CD2517.1-HA and CD2907.1-HA-expressing plasmids on the basis of corresponding pDIA6103-derivatives with primers designed to introduce the HA-tag sequence at the C-terminal part of coding toxin regions, directly upstream the stop codon (Supplementary Table S2). To compare the action of short and long forms of antitoxins on cognate and non-cognate toxins when co-expressed either in cis or in trans (from a site distant from the vector MCS), we used reverse PCR approach and Gibson assembly to construct different plasmids on the basis of the corresponding pDIA6103-derivatives (pT) (Supplementary Tables S1, S2 and Supplementary methods). DNA sequencing was performed to verify plasmid constructs using pRPF185-specific primers IMV507 and IMV508. The resulting derivative pRPF185 plasmids were transformed into the E. coli HB101 (RP4) and subsequently mated with C. difficile 630Δerm (40) (Supplementary Table S1). C. difficile transconjugants were selected by sub-culturing on BHI agar containing Tm (15 μg/ml) and Cfx (25 μg/ml).Light microscopyFor light microscopy, bacterial cells were observed at 100× magnification on an Axioskop Zeiss Light Microscope. Cell length was estimated for more than 100 cells for each strain using ImageJ software (41).RNA extraction, quantitative real-time PCR, northern blot and 5′/3′RACETotal RNA was isolated from C. difficile strains grown 7.5 h in TY medium containing 7.5 μg/ml of Tm and 250 ng/ml of ATc as previously described (42). For biofilm samples C. difficile 630Δerm strain was grown for 72 h in TY medium using continuous-flow microfermentor culture system (43). The 24-h planktonic culture in TY medium was used for comparative analysis. The cDNA synthesis by reverse transcription and qRT-PCR analysis were performed as previously described (44). In each sample, the relative expression for a gene was calculated relatively to the 16S rRNA gene or dnaF gene (CD1305) encoding DNA polymerase III or ccpA gene encoding catabolite control protein. The relative change in gene expression was recorded as the ratio of normalized target concentrations (ΔΔCt) (45). Northern blot analysis and 5′/3′RACE experiments were performed as previously described (15).RNA band-shift assay and in vitro processing by RNase IIITemplates for the synthesis of RNA probes were obtained by PCR amplification using the Term and T7 oligonucleotides (Supplementary Table S2). RNAs were synthesized by T7 RNA polymerase with α-32P UTP as a tracer and were then gel purified. RNA concentrations were monitored by counting out the radioactivity and the RNA samples were stored until use (46). This gives a 298-nt long CD2907.1 and a 132-nt long RCd9 transcripts with three additional G at the 5′ extremity. Just before use, RCd9 RNA was 5′-radiolabeled and incubated with increasing concentrations of CD2907.1 mRNA under two different conditions referred as N (Native) and F (Full RNA duplex) conditions, respectively. Radiolabeled RCd9 was incubated either alone or with the unlabeled CD2907.1 RNA to allow them to anneal in Tris-Mg-acetate-Na-acetate (TMN) buffer for 5 min at 37°C (20 mM Tris acetate, pH 7.5, 10 mM magnesium acetate, 100 mM sodium acetate). Alternatively, after denaturation at 90°C for 2 min, labeled RCd9 RNAs in 1×TE were incubated 30 min at 37°C with the unlabeled CD2907.1 to allow them to anneal. The complexes were immediately loaded on native polyacrylamide gels to control for hybridization efficiency (47) or submitted to in vitro processing by RNase III of E. coli. RNase III digestion of free or complexed RCd9 was performed at 37°C in TMN buffer containing 1 μg tRNA from 1 min to 15 min with 0.05 units of RNase III (Epicentre). After precipitation, addition of loading buffer and heat denaturation, samples were analyzed on 8% polyacrylamide-Urea gels.Subcellular localization of HA-tagged toxins by cell fractionation and western blottingThe C. difficile cultures were inoculated from overnight grown cells in 10 ml of TY medium at OD 600 nm of 0.05, allowed to grow for 3 h before addition of 250 ng/ml ATc and incubation for 90 min followed by centrifugation and protein extraction. Cell lysis, fractionation and protein analysis were performed as previously described (48). Coomassie staining was performed for loading and fractionation control. Western blotting was performed as previously described (49) with anti-HA antibodies.Measurement of RNA decay by rifampicin assayFor determination of toxin and antitoxin RNA half-lives the C. difficile strains were grown in TY medium supplemented with 250 ng/ml ATc and 7.5 μg/ml Tm for 7.5 h at 37°C. Samples were taken at different times after addition of 200 μg/ml rifampicin (0, 2, 5, 10, 20, 40, 60 and 120 min) and subjected to RNA preparation and northern blotting. In silico screening for potential new TA genes and CRISPR arrays co-localizationThe raw sequencing read data of 2,584 C. difficile strains were downloaded for this genomic analysis (16,50). For each strain, we realized an assembly with Spades (51) and an automatic annotation using PROKKA (52). Then we selected small proteins from 40 to 60 amino acids in length, adjacent to CRISPR arrays and performed an orthology analysis using proteinortho5 (53). Multiple alignment was done using ClustalW (54).

Article TitleDiscovery of new type I toxin–antitoxin systems adjacent to CRISPR arrays inClostridium difficile


Clostridium difficile, a major human enteropathogen, must cope with foreign DNA invaders and multiple stress factors inside the host. We have recently provided an experimental evidence of defensive function of theC. difficileCRISPR (clusteredregularlyinterspacedshortpalindromicrepeats)-Cas (CRISPR-associated) system important for its survival within phage-rich gut communities. Here, we describe the identification of type I toxin–antitoxin (TA) systems with the first functional antisense RNAs in this pathogen. Through the analysis of deep-sequencing data, we demonstrate the general co-localization with CRISPR arrays for the majority of sequencedC. difficilestrains. We provide a detailed characterization of the overlapping convergent transcripts for three selected TA pairs. The toxic nature of small membrane proteins is demonstrated by the growth arrest induced by their overexpression. The co-expression of antisense RNA acting as an antitoxin prevented this growth defect. Co-regulation of CRISPR-Cas and type I TA genes by the general stress response Sigma B and biofilm-related factors further suggests a possible link between these systems with a role in recurrentC. difficileinfections. Our results provide the first description of genomic links between CRISPR and type I TA systems within defense islands in line with recently emerged concept of functional coupling of immunity and cell dormancy systems in prokaryotes.

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