Binding of the CPA at the C-terminal sugar binding motifs, C1 and C2, is critical to pyocin L1 cytotoxicity.

<p>ITC binding isotherms of (A) wild-type (B) D180A (C) D150A and (D) D150A/D180A pyocin L1 all at (100 µM) titrated into isolated LPS-derived polysaccharide (1 mg ml⁻¹) from wild-type <i>P. aeruginosa</i> PAO1. Fit to a single binding site model is shown. (E) Spot tests to determine cytotoxic activity of wild-type and pyocin L1 variants against of <i>P. aeruginosa</i> PAO1. Purified protein (starting concentration 400 µg ml⁻¹ with 2-fold sequential dilutions) was spotted onto a growing lawn of <i>P. aeruginosa</i> PAO1. Clear zones indicate pyocin L1 cytotoxicity.</p

Pyocin L1 binds strongly to CPA from <i>P. aeruginosa</i> PAO1.

<p>(A) ITC binding isotherm of pyocin L1 (150 µM) titrated into isolated LPS-derived polysaccharide (1 mg ml⁻¹) from wild-type <i>P. aeruginosa</i> PAO1. Strong, saturable heats were observed indicative of a strong interaction. Curve fitted with a single binding site model. (B) ITC isotherm of pyocin L1 (150 µM) titrated into isolated LPS-derived polysaccharide (1 mg ml⁻¹) from PAO1 <i>wzt</i>. No saturable binding isotherm was observed.</p

Pyocin L1 shows specificity for d-rhamnose compared with d-mannose.

<p>(A) ITC binding isotherm of d-rhamnose (50 mM) titrated into pyocin L1 (100 µM). Weakly saturable heats were observed, indicative of binding with modest affinity (Kd ∼5–10 mM). (B) ITC binding isotherm of d-mannose (50 mM) titrated into pyocin L1 (100 µM). Small-weakly saturable heats were observed, indicative of very weak interaction (Kd ∼50 mM). Titration of monomeric sugars into ¹⁵N-labelled pyocin L1, monitored using ¹H-¹⁵N HSQC NMR spectroscopy. Shifts within spectra were converted to chemical shift perturbation (CSP) values using equation Δ_ppm = √ [Δδ_HN+(Δδ_N*α_N)²]. CSP values are plotted against sugar concentration in (C) and (E) and visualised in (D) and (F). Peak positions, which correspond to backbone amide signals, at selected sugar concentrations (blue: no sugar, green: 60 mM, red: 100 mM) are shown. Perturbation of peak position (ppm) is indicative of association between ligand and protein molecules in solution.</p

C-terminal MMBL-sugar binding motifs of pyocin L1 bind d-rhamnose and d-mannose.

<p>Electron density (at 1.3 σ) with fitted stick model of pyocin L1 MMBL-sugar binding site C1 with: (A) d-rhamnose (XXR), (C) d-mannose (BMA), (E) no bound sugar, and sugar binding site C2 with: (B) d-rhamnose, (D) d-mannose, (F) no bound sugar. For clarity, electron density is clipped to within 1.5 Å of visible atoms.</p

Putidacin L1 binds strongly to LPS-derived polysaccharides from susceptible but not tolerant or resistant <i>P. syringae</i> isolates.

<p>ITC isotherm of LPS-derived polysaccharides (3 mg ml⁻¹) from strains highly sensitive to putdacin L1: (A) <i>P. syringae</i> LMG 2222, (B) <i>P. syringae</i> LMG 5456 titrated into putidacin L1 (60 µM). Large, saturable heats are indicative of binding. LPS-derived polysaccharides (3 mg ml⁻¹) from strains non-sensitive to putidacin L1: (C) <i>P. syringae</i> NCPPB 2563, (D) <i>P. syringae</i> DC3000, or highly tolerant (E) <i>P. syringae</i> LMG 1247 to putidacin L1, show no heats of binding when titrated into putidacin L1 (60 µM).</p

CPA production correlates with pyocin L1 killing.

<p>(A) Inhibition of growth of <i>P. aeruginosa</i> E2 and tolerant mutants M4 and M11 by pyocin L1, as shown by a soft agar overlay spot-test. 5 µl of purified pyocin L1 (1.5 mg ml⁻¹) was spotted onto a growing lawn of cells. Clear zones indicate cell death. (B) Expression of CPA by <i>P. aeruginosa</i> E2 and tolerant mutants, visualised by immunoblotting with the CPA specific antibody N1F10. (C) Inhibition of growth of <i>P. aeruginosa</i> PAO1 and PAO1 <i>wzm</i> and <i>wzt</i> mutants by pyocin L1 (details as for A). (D) Expression of CPA by PAO1 and <i>wzm</i> and <i>wzt</i> strains (details as for B).</p

Crystal structure of pyocin L1 reveals tandem MMBL domains and sugar-binding motifs.

<p>(A) Ribbon diagram of structure of pyocin L1 in complex with α-d-rhamnose, amino acids 2-256. N-terminal domain (green), C-terminal domain (pink), C-terminal extension (red), α-d-rhamnose (spheres) and sugar binding sites containing the conserved or partially conserved QxDxNxVxY motif are highlighted (blue) and are designated N1, N2 and C1, C2 according to order of appearance in the primary sequence of the N- and C-terminal domains, respectively. Pyocin L1 residues involved in hydrogen bonding with α-d-rhamnose are shown in stick representation. (B) Sequence and secondary structure (β-sheets = arrows, α-helices = coils) of pyocin L1 with colours corresponding to the structure in (A). Residues conserved in sugar binding motifs are shown in bold. (C) Structural alignment of pyocin L1 (green) and putidacin L1 (blue) based on N-terminal MMBL domain in wall-eyed stereo. (D) Structural alignment of pyocin L1 (green) and <i>Allium sativum</i> agglutinin (1BWU) (pink) based on N-terminal MMBL domain in wall-eyed stereo.</p