Single-channel recordings with HlyA_Δ158–167 and HlyA_Δ180–203.

<p>Single-channel recordings of asolectin membranes were performed in the presence of 100 ng/ml HlyA_Δ158–167 (upper trace) and 150 ng/ml HlyA_Δ180–203 (lower trace). The aqueous phase contained 150 mM KCl (pH 6). The applied membrane potential was 20 mV; T = 20°C. The transient conductance steps in the upper trace (HlyA_Δ158–167) had a conductance of about 600 to 700 pS. The mutant HlyA_Δ180–203 produced under the given conditions only current noise (fuzzy channels) and no defined conductance states.</p

Extracellular secretion and hemolytic activity of <i>E. coli</i> HlyA and of HlyA mutants.

<p>(A) SDS-PAGE of extracellular proteins from <i>E. coli</i> 5K containing different plasmids. Lane 1, molecular mass markers given in kDa; lane 2, <i>E. coli</i> 5K/pACYC184 (vector control); lane 3, <i>E. coli</i> 5K/pANN202–312* overproducing HlyA; lane 4, isogenic strain overproducing HlyA_Δ71–110; lane 5, isogenic strain overproducing HlyA_Δ158–167; lane 6, isogenic strain overproducing HlyA_Δ180–203; lane 7, isogenic strain overproducing HlyA_Δ264–286. The proteins in cell-free culture supernatants (harvested in the late log phase) were precipitated by addition of ice-cold trichloroacetic acid (final concentration, 10%), pelleted by centrifugation at 12,000×g, washed with acetone, dried under vacuum, and dissolved in sample buffer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112248#pone.0112248-Ludwig2" target="_blank">[11]</a>. Proteins from 100 µl culture supernatant were separated on the gel and visualized by silver staining. (B) Hemolytic phenotype of <i>E. coli</i> 5K/pANN202–312* overproducing HlyA and of isogenic strains overproducing the HlyA mutants with the indicated deletions. Bacteria from individual colonies were picked onto a sheep blood/Cm agar plate that was subsequently incubated for 24 hours at 37°C.</p

Calculation of the channel diameters of HlyA and HlyA_Δ71–110 from the single-channel conductance.

<p>The single-channel conductance data of HlyA and HlyA_Δ71–110 were fitted by using the Renkin correction factor multiplied by the aqueous diffusion coefficients of the different cations. The single-channel conductance for the different cations taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112248#pone-0112248-t002" target="_blank">Table<u> 2</u></a> was normalized to that observed for Rb⁺ (hydrated ion radius  = 0.105 nm), which was set to 1.0, and plotted versus the hydrated ion radii taken from Table 3 of Maier <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112248#pone.0112248-Maier1" target="_blank">[60]</a>. The points correspond to the single-channel conductance observed with Li⁺, Na⁺, K⁺, Cs⁺, N(CH₃)₄⁺, N(C₂H₅)₄⁺, and Tris⁺, which were all used for the pore diameter estimation (see Discussion). (A) The fit (solid lines) is shown for wildtype HlyA channels with <i>r</i> = 1.6 nm (upper line) and <i>r</i> = 1.0 nm (lower line). The best fit was achieved with <i>r</i> = 1.3 nm (diameter = 2.6 nm), which corresponds to the broken line. (B) The fit (solid lines) is shown for the HlyA_Δ71–110 channels with <i>r</i> = 1.1 nm (upper line) and <i>r</i> = 0.7 nm (lower line). The best fit of all data was achieved with <i>r</i> = 0.9 nm (diameter = 1.8 nm), which corresponds to the broken line.</p

Results of osmotic protection experiments with aerolysin of <i>A. sobria</i>.

<p>Sheep erythrocytes in saline solution (control) or in saline solution supplemented with 30 mM of different carbohydrates (arabinose, cellobiose, and melezitose, with diameters of 0.62, 0.92, and 1.14 nm, respectively) were incubated with the toxin at 37°C for 30 min (A) and 90 min (B). Erythrocyte lysis was determined as a function of increasing aerolysin concentrations.</p

Average single-channel conductance, <i>G</i>, of HlyA_Δ71–110 and HlyA_Δ264–286 in different salt solutions.^a.

a<p>The membranes were formed from 1% (mass/volume) asolectin dissolved in n-decane. The aqueous solutions were unbuffered and had a pH of 6. The applied voltage was 20 mV, and the temperature was 20°C. The average single-channel conductance, <i>G</i> (i.e. current divided by voltage), was calculated from at least 80 single events. The standard deviation of the single-channel conductance was generally below ±15%. <i>c</i> is the concentration of the aqueous salt solutions. The single-channel conductance of wildtype HlyA of <i>E. coli</i> is given for comparison <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112248#pone.0112248-Benz3" target="_blank">[28]</a>. The values denoted with an asterix were measured during this study with purified HlyA. n.m. means not measured.</p><p>Average single-channel conductance, <i>G</i>, of HlyA_Δ71–110 and HlyA_Δ264–286 in different salt solutions.^a.</p

Single-channel recordings with HlyA_Δ158–167 and HlyA_Δ180–203.

<p>Single-channel recordings of asolectin membranes were performed in the presence of 100 ng/ml HlyA_Δ158–167 (upper trace) and 150 ng/ml HlyA_Δ180–203 (lower trace). The aqueous phase contained 150 mM KCl (pH 6). The applied membrane potential was 20 mV; T = 20°C. The transient conductance steps in the upper trace (HlyA_Δ158–167) had a conductance of about 600 to 700 pS. The mutant HlyA_Δ180–203 produced under the given conditions only current noise (fuzzy channels) and no defined conductance states.</p

Primers used for site-directed mutagenesis to study the effect of deletions of different stretches of amino acids of HlyA on hemolytic activity and properties of the HlyA channels.

a<p>The 5′- and 3′-terminal halves of the primers represent the nucleotide sequences of <i>hlyA</i> flanking the desired deletions on both sides. The deletion site is indicated by a vertical bar (|).</p><p>Primers used for site-directed mutagenesis to study the effect of deletions of different stretches of amino acids of HlyA on hemolytic activity and properties of the HlyA channels.</p

Effect of charges in wildtype and mutant HlyA on the single-channel conductance.

<p>The single-channel conductance of the HlyA, HlyA_Δ71–110, and HlyA_Δ264–286 channels is shown as a function of the KCl concentration in the aqueous phase. The solid lines represent the fit of the single-channel conductance data with eqn. <i>G(c) = G₀⋅c₀⁺</i> (a combination of eqs. (3–5 and 7) assuming the presence of negative point charges within the channel (for HlyA: 2.3 negative charges, <i>q = </i>−3.68×10^–19 As; for HlyA_Δ71–110∶1.7 negative charges, <i>q</i> = -2.72×10^–19 As; for HlyA_Δ264–286∶2 negative charges, <i>q = </i>-3.2×10^–19 As) and assuming a channel diameter of 2 nm, 1.4 nm, and 1.6 nm for HlyA, HlyA_Δ71–110, and HlyA_Δ264–286, respectively. <i>c</i>, concentration of the KCl solution in M (molar); <i>G</i>, average single-channel conductance in nS (nano Siemens, 10^–9 S); <i>G₀</i>, specific single-channel conductance in the absence of negative point charges given in pS/M. The broken, dotted, and fractured (straight) lines show the single-channel conductance of the HlyA, HlyA_Δ264–286, and HlyA_Δ71–110 channels in the absence of point charges and correspond to linear functions between channel conductance and bulk aqueous concentration (eqn. (7); <i>G(c) = G₀⋅c</i>).</p

Results of osmotic protection experiments with HlyA, HlyA_Δ71–110, and HlyA_Δ264–286.

<p>Sheep erythrocytes were incubated with the toxins at 37°C for 60 min in saline solution (control) or in saline solution supplemented with 30 mM of PEGs of different molecular masses (PEG 400, 600, 1000, 2000, 3000, and 4000, with diameters of 1.07, 1.32, 1.72, 2.47, 3.05, and 3.54 nm, respectively). The concentration of HlyA was 0.5 µg/ml and that of HlyA_Δ71–110 and HlyA_Δ264–286 2.5 µg/ml. The degree of hemolysis is shown as a function of the molecular mass of the PEGs.</p

Single-channel recordings with <i>E. coli</i> HlyA, HlyA_Δ71–110, and HlyA_Δ264–286.

<p>Single-channel recordings of asolectin membranes were performed in the presence of 50 ng/ml HlyA (left side, upper trace), 50 ng/ml HlyA_Δ71–110 (left side, lower trace), and 50 ng/ml HlyA_Δ264–286 (right side). The aqueous phase contained 150 mM KCl (pH 6). The applied membrane potential was 20 mV; T = 20°C. The average single-channel conductance was 520 pS for HlyA, 150 pS for HlyA_Δ71–110, and 320 pS for HlyA_Δ264–286.</p