NMS profile of the interactions of Kid with CcdA proteins at different toxin-antitoxin ratios.

<p>(A), Mass spectrum obtained at a Kid∶CcdA ratio of 2∶1. (B), Mass spectrum obtained at a Kid∶CcdA ratio of 1∶1.(C) Mass spectrum obtained at a Kid∶CcdA ratio of 1∶2. All the protein mixtures were performed in 100 mM ammonium acetate pH 6.7. The lowest protein concentration was 10 µM in all cases. CcdA protein is represented with maroon circles and Kid protein is represented with blue squares. Each complex found is represented with the appropriate combination of squares and circles.</p

Effects of Kid/Kis and CcdB/A on protein synthesis in <i>E. coli</i> cell extracts.

<p>(A) Coupled transcription/translation assay with <i>E. coli</i> S30 extracts using a luciferase-encoding plasmid as template. ³⁵S-labelled products translated in the absence or presence of purified Kid, Kis, CcdB and CcdA proteins were analysed using 15% SDS-PAGE. Tracks: 1, C+ control sample treated with chloramphenicol (30 µg/mL); 2, C−, assay run in the absence of toxin/antitoxin proteins; 3 and 4, effects of adding Kid (0.075, 0.3 µM); 5, neutralisation of Kid by Kis (0.3 µM each); 6, 7, 8, assays run in the presence of Kid (0.075 µM) and CcdA at 0.075, 0.037, 0.018 µM respectively; 9, 10, CcdA alone at 0.018, 0.075 µM. (B) Bar graph representation and quantitative analysis of data in (A). The signals of the labelled proteins were scanned and an integrated value calculated for each condition using a Quantity One informatics program. The percentage of synthesis of total proteins in each track was referred to the value obtained in the untreated sample. The bars above the different values represent the standard deviation calculated from three independent experiments. A Student's t-test indicated that the differences between values in bars linked with a bracket were significant (p-value = 0.0217). * Represents a p-value≤0.05.</p

<i>ccd</i> and <i>parD</i> toxin-target and toxin-antitoxin interactions.

<p>√ indicates the toxin has the activity or the neutralisation of the toxin by the antitoxin.</p><p>X indicates not effect.</p>*<p>indicates antitoxin stimulates toxin activity.</p>1<p>Data from ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046499#pone.0046499-SantosSierra2" target="_blank">[38]</a>.</p

DNA gyrase-mediated cleavage of DNA, stimulated by CcdB.

<p>A. Negatively supercoiled pBR322 (3.5 nM) was incubated with 30 nM gyrase, 1.4 mM ATP and various concentrations of CcdB between 0.001–10 µM, as indicated, for 1 h at 25°C. Cleaved DNA was revealed by the addition of SDS and proteinase K. After incubation for 30 mins at 37°C, reactions were stopped with STEB and DNA was subjected to phenol extraction, and samples were analysed on a 1% agarose gel. N, nicked DNA; L, linear DNA; SC, supercoiled DNA. (B) CcdA inhibits the action of CcdB. Negatively supercoiled pBR322 was incubated with gyrase as described in (A) in the presence of 1.4 mM ATP, with 1 µM CcdB and various concentrations of CcdA between 0.1–10 µM as indicated. N, nicked DNA; L, linear DNA; SC, supercoiled DNA.</p

NMS profile of the interactions of CcdB and His₆Kis proteins at different toxin-antitoxin ratios.

<p>(A) Mass spectrum obtained at a CcdB∶His₆Kis molar ratio of 2∶1. (B) Mass spectrum obtained at a CcdB∶His₆Kis molar ratio of 1∶1. (C) Mass spectrum otained at a CcdB∶His₆Kis molar ratio of 1∶2. (D) Mass spectrum obtained at a CcdB∶His₆Kis molar ratio of 1∶4. All the protein mixtures were performed in 100 mM ammonium acetate pH 6.7. The lowest protein concentration was 10 µM in all cases except the 1∶4 ratio that was 5 µM. CcdB protein is represented with green squares and His₆Kis with yellow circles. Each complex found is represented with the appropriate combination of squares and circles.</p

<i>In vitro</i> analysis of RNA cleavage by Kid/Kis and CcdB/CcdA.

<p>(A) 5′ ³²P-labelled CopT RNA was incubated for 2 min at 37°C in the presence of Kid or CcdB prior to separation on 8% polyacrylamide gels in the presence of urea. Tracks: 1, C−, untreated full-length (FL) RNA; 2, RNA treated with 0.2 µM Kid; 3, RNA treated with 0.2 µM Kid and 0.2 µM Kis; 4, RNA treated with 0.2 µM Kid and 0.2 µM CcdA; 5, RNA treated with 0.2 µM CcdA; 6, RNA treated with 0.2 µM CcdB; 7, RNA treated with 0.2 µM CcdA and 0.2 µM CcdB; 8, RNA treated with 0.2 µM Kid and 1.5 µM BSA; 9, RNA treated with 0.2 µM Kid and 0.2 µM CcdB. (B) Bar graph representation and quantitative analysis of data in (A): Total RNA (uncleaved and cleaved products) was calculated for each track scanning the different bands using the Quantity One® program (Bio-Rad). RNase activity was calculated as the percentage of full length RNA substrate. The average value for each condition was calculated from three independent experiments. The 100% value indicates absence of RNase activity. The standard deviation is indicated above the different bars. A Student's t-test indicated that the differences between values in lanes 2 and 3, linked with a bracket, were significant (p-value = 0.0067). ** Represents a p-value≤0.01.</p

Theoretical and observed molecular masses of the toxins, antitoxins and their complexes.

<p>Molecular mass is expressed in Daltons.</p><p>E: Theoretical molecular mass expected for a protein or complex.</p><p>O: Molecular mass observed by Nanoflow Electrospray Ionization Mass Spectrometry. The standard deviation is calculated from at least three different experiments.</p

Evaluation of DNA gyrase activity in the presence of different toxins and antitoxins.

<p>(A) Kid does not inhibit the action of DNA gyrase. Relaxed pBR322 (21 nM) was incubated with 30 nM gyrase, ATP (1.4 mM) and various concentrations of CFX (ciprofloxacin; 0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 µM) and Kid (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 µM) as indicated, for 1 h at 37°C. Assays were either (a) stopped or (b) cleaved DNA was revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 mins. Reactions were stopped with 40% sucrose, 100 mM Tris·HCl (pH 7.5), 100 mM EDTA, 0.5 mg/ml bromophenol blue, and samples were subjected to phenol extraction and analysed on a 1% agarose gel (a) run in the absence of ethidium bromide or (b) run in the presence of ethidium bromide (1 µg/mL). N, nicked DNA; L, linear DNA; SC, supercoiled DNA; R, relaxed DNA. (B) Kis, but not MazE, can inhibit the action of CcdB. Relaxed pBR322 (21 nM) was incubated with 30 nM DNA gyrase, ATP (1.4 mM) and various concentrations of CcdB (2 µM), CcdA (1, 2 & 4 µM), Kis (2, 20 & 40 µM) or MazE (2, 20 & 40 µM) as indicated, at 37°C for 1 h. Toxin-antitoxin ratios were 1∶0.5, 1∶1, 1∶2 in Tracks 3–5 and 1∶1, 1∶10, 1∶20 in Tracks 6–8 & 9–11. Antitoxin only controls were at highest concentration used (Tracks 12–14). Cleaved DNA was revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 mins. Reactions were stopped with STEB, and samples were subjected to phenol extraction and analysed on a 1% agarose gel run in the presence of ethidium bromide. N, nicked DNA; L, linear DNA; SC, supercoiled DNA.</p

Kid–Kis complexes (molar ratio of 1:1) interact tightly with region I

<p><b>Copyright information:</b></p><p>Taken from "Interactions of Kid–Kis toxin–antitoxin complexes with the operator-promoter region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid–Kis oligomers"</p><p></p><p>Nucleic Acids Research 2007;35(5):1737-1749.</p><p>Published online 21 Feb 2007</p><p>PMCID:PMC1865072.</p><p>© 2007 The Author(s)</p> Macromolecular native mass spectrometry was performed on Kid–Kis and on Kid–Kis– DNA complexes in ammonium acetate (50 mM), pH 5.8. () Mass spectrum of a mixture of Kid:Kis at a molar ratio of 1:1 (Kis 15 μM) and () and () mass spectra of Kid:Kis: DNA mixtures at molar ratios of 40:40:1 and 10:10:1 (Kis 15 μM), respectively. Kid and Kis are indicated with blue rectangles and orange ellipses, respectively, and the DNA fragment with double strand. Each complex is represented by an appropriate combination of rectangles, ellipses and/or DNA double strand. Molecular masses and relative amounts of complexes are shown in Supplementary Tables 1 and 2, respectively

Macromolecular tandem mass spectrometry reveals topology of Kid–Kis– DNA region I complexes

<p><b>Copyright information:</b></p><p>Taken from "Interactions of Kid–Kis toxin–antitoxin complexes with the operator-promoter region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid–Kis oligomers"</p><p></p><p>Nucleic Acids Research 2007;35(5):1737-1749.</p><p>Published online 21 Feb 2007</p><p>PMCID:PMC1865072.</p><p>© 2007 The Author(s)</p> Tandem mass spectrometry was performed on Kid–Kis and Kid–Kis– DNA in ammonium acetate (50 mM), pH 5.8. () Tandem mass spectra of Kis–Kid–Kid–Kis after selection of the 20 ion and () tandem mass spectra of Kis–Kid–Kid–Kis– DNA complex after selection of the 21 ion. Acceleration voltages varied between 25 and 65 V. Kid and Kis are indicated with blue rectangles and orange ellipses, respectively and the DNA fragment with double strand. Each complex is represented by an appropriate combination of rectangles, ellipses and/or DNA double strand