Analysis of PorACj purification.

<p>(A) Western blot analysis illustrating IMAC purification of his-tagged PorACj protein. The protein was expressed in <i>C. glutamicum</i> ATCC13032 <i>ΔporHΔporA</i> and purified by Ni²⁺ affinity from the supernatant of detergent extracted whole cells. CMDIE represents chloroform-methanol treated cells in which the crude protein content was concentrated around 8 fold by diethyl-ether precipitation of pXJK0268His transfected (+) or non-transfected (−) <i>C. glutamicumΔporHΔporA</i> cells. Subsequent to tricine (12%)-SDS-PAGE the gel was blotted on a nitrocellulose membrane and PorACj-His was visualized by Anti-His antibodies and a chemiluminescent reaction. All samples were boiled for 5 minutes in Redmix before loading. (B) Silver stained tricine (16.5%)-SDS-PAGE of Ni²⁺-purified and factor Xa digested PorACj-His protein. Lanes: 1, 3 units of protease Xa (control); 2, 10 µl of three pooled Ni-NTA elution containing PorACj-His; 3, 10 µl of protease Xa treated and purified PorACj protein (for details see text). The dot blot immunoassay pictures underneath lanes 2 and 3 show cleavage of the histidine tail using anti-his antibody of 5 µl of the corresponding protein samples. Before loading all samples were boiled for 5 minutes in Redmix.</p

Analysis of secondary structure of PorACj usinf CD-spectrometry.

<p>A: CD spectra of recombinant PorACj (69 µM) and PorA-His₈ (12 µM) solubilized in 0.5% Genapol, 100 mM NaCl, 50 mM TrisHCl and 1 mM CaCl₂, pH 8 measured at room temperature. B: CD-spectra of the same protein samples as in (A). The aqueous solutions of the proteins was supplemented with 4 M urea to destroy the secondary structure of the proteins.</p

Conductance (G) at a given membrane potential (V_m) divided by the conductance at 10

<p> <b>mV (G₀) expressed as a function of the membrane potential.</b> The symbols represent the mean (± SD) of six measurements, in which pure PorACj protein was added to the <i>cis</i>-side of the membranes. The aqueous phase contained 1 M KCl and 100 ng/ml porin. The membranes were formed from PC/<i>n</i>-decane at a temperature of 20°C.</p

Analysis of PorACj secondary structure.

<p>(A) The panel shows the hydrophobicity indices of the individual amino acids of PorACj according to ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075651#pone.0075651-Kyte1" target="_blank">[80]</a>. (B) The secondary structure of PorACj was predicted using a consensus method [83] at the Pole Bioinformatique Lyonnaise network (<a href="http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_seccons.html" target="_blank">http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_seccons.html</a>); the protein was suggested to form α-helices. Amino acid residues arranged on basis of heptameric repeats (a–g) showing distinct separation in a hydrophobic domain supposable surrounded by lipid molecules (dark grey) while the hydrophilic domain (light grey) is suggested to represent the component orientated to the water-filled lumen in the presumed oligomeric PorACj.</p

Investigation of the voltage-dependence of PorACj in single-channel experiments.

<p>A: The purified protein was added to the <i>cis</i>-side of a PC membrane (10 ng/ml) and the reconstitution of channels was followed until about 10 PorACj-channels inserted into the membrane. Then 40 mV were applied to the <i>cis</i>-side of the membrane, and the membrane current was measured as a function of time. The aqueous phase contained 1M KCl; T = 20°C. B: Histogram of 56 closing events of the experiment in A and and similar experiments. The closing events were plotted in a bargraph as a function of the conductance of the closing events. ! M KCl; T = 20°C. Note that the PorACj channels closed in two distinct conductance values of 1 and 2 nS.</p

Dendrogram representing the phylogenetic relationships of PorA and PorH of different <i>Corynebacterium</i> species obtained by the neighbor-joining method.

<p>The tree was derived from the alignments of corresponding gene sequences. The support of each branch, as determined from 1,000 bootstrap samples, is indicated by the value at each node (in percent).The software used to construct alignment and tree was MEGA5.1.The sequence was aligned by ClustalW. Parameters: Multiple Alignment: Gap Opening Penalty: 10; Gap Extension Penalty: 0.2; Protein Weight Matrix: Gonnet; Gap Separation Distance: 4; Delay Divergent Cutoff (%): 30; The phylogenetic tree of corynebacterial species was constructed using the Maximum Likelihood statistical method; Substitution Model; Substitutions Type: Amino acid; Model/Method: Jones-Taylor-Thornton (JTT) model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075651#pone.0075651-Tamura1" target="_blank">[81]</a>.</p

Minimum inhibitory concentration (MIC) and diameters of inhibition zones of antimicrobial agents for <i>C. glutamicum</i> ΔHA pXjk0268His and <i>C. glutamicum</i> ΔHA as control.

<p>NI means no inhibition of growth, i.e. no growth inhibition zone.</p

Investigation of the voltage-dependence of PorACj in a multi-channel experiment.

<p>The purified protein was added to the <i>cis</i>-side of a PC membrane (100 ng/ml) and the reconstitution of channels was followed until equilibrium. Then increasing positive (upper traces) and negative voltages (lower traces) were applied to the <i>cis</i>-side of the membrane, and the membrane current was measured as a function of time. The aqueous phase contained 1 M KCl; T = 20°C.</p

Study of pore-forming capacity of purified PorACj.

<p>(A) Single-channel recording of a PC/<i>n</i>-decane membrane in the presence of pure PorACj. The aqueous phase contained 1 M KCl, pH 6 and 10 ng/ml protein. The applied membrane potential was 20 mV; T = 20^°C. (B) Histogram of the probability P(G) for the occurrence of a given conductivity unit observed with membranes formed of 1% PC dissolved in <i>n</i>-decane. It was calculated by dividing the number of fluctuations with a given conductance rise by the total number of conductance fluctuations in the presence of pure PorACj. Two frequent conductive units were observed for 295 single events taken from eight individual membranes. The average conductance of the steps corresponding to the left-side maximum was 1.25 nS and that of the right-side maximum was 2.5 nS. The aqueous phase contained 1 M KCl, pH 6 and 10 ng/ml protein, the applied membrane potential was 20 mV, T = 20°C.</p

Fit of the single-channel conductance data of PorACj by using the Renkin correction factor times the aqueous diffusion coefficients of the different anions

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075651#pone.0075651-Trias3" target="_blank">[<b>67</b>]</a><b>.</b> The product of both numbers was normalized to 1 for a = 1.05 nm (Br⁻). Single-channel conductance was normalized to the one of 0.1 M KBr and plotted versus the hydrated ion radii taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075651#pone-0075651-t003" target="_blank">Table 3</a>. The single-channel conductance correspond to Br⁻, Cl⁻, NO₃⁻, ClO₃⁻, F⁻, HCOO⁻ and CH₃COO⁻ which were all used for the pore diameter estimation. The fit (solid lines) is shown for r = 0.5 nm (lower line) and r = 0.9 nm (upper line). The best fit was achieved with r = 0.7 nm (diameter = 1.4 nm) corresponding to the broken line.</p