Surface GAPDH promotes binding to C1q.

<p>Bacterial culture was withdrawn at mid-exponential growth phase (OD_600nm 0.3). FITC-labeled bacteria were incubated for 1 h at 4°C on 1 μg of C1q coated on 96-wells plate. After five washes, the fluorescence of FITC was measured. A representative experiment of 3 independent experiments is shown including the standard deviation of triplicate points. Significance was determined by t-test analysis on 3 independent experiments.</p

GAPDH increases C1q deposition on pneumococcal cell wall.

<p>Pneumococcal sacculi containing only peptidoglycan were deposited on 96-wells plate and incubated with or without purified GAPDH. Normal human serum (NHS) was added and subsequent C1q and C4 deposition was measured using anti-C1q and anti-C4 antibodies. A representative experiment of 2 independent experiments is shown including the standard deviation of triplicate points.</p

Pneumococcal lysis induced by LytA promotes GAPDH surface localization.

<p>(A) Growth profiles of the R6 strain in CY and CY + 1% Cho and of the R6 Δ<i>lytA</i> mutant in CY medium. (B) Bacterial suspensions of the R6 strain grown in CY and in CY + 1% Cho and the R6 Δ<i>lytA</i> mutant grown in CY were treated by alkaline buffer to release surface-associated proteins. Proteins present in the pellet (P) fraction corresponding to the cytoplasmic extract and in the alkaline supernatant fraction, which contains proteins detached from the cell surface, were analyzed by Western blot using appropriate polyclonal antibodies. Samples were analyzed on the same polyacrylamide gel. Left panel: detection of FtsZ (44.4 kDa) used to monitor the non-lytic effect of the alkaline treatment. Right panel: detection of GAPDH (38 kDa). Equivalent amount of loading material was determined based on OD_600nm values and gel scanning quantification and not on CFU measurements since the chaining morphology of the R6 Δ<i>lytA</i> mutant and the one induced by the presence of 1% Cho alters colony counting [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125377#pone.0125377.ref065" target="_blank">65</a>]. This procedure was also applied in experiments showed in Figs 1C, 1D and 1E. (C) Quantification of pneumococcal GAPDH associated to the bacterial surface in the R6 strain grown in CY and in CY + 1% Cho and in the R6 Δ<i>lytA</i> mutant grown in CY. GAPDH was detected by Western blot and quantification of the signal was performed. The average of three independent experiments is shown. (D) Same protocol as 1C. Amount of GAPDH associated to the cell wall fraction at different stages of growth. (E) Same protocol as 1C. Amount of GAPDH associated to the cell wall fraction analyzed by subcellular fractionation.</p

Western blot analysis of membrane-bound BlaR1 produced by <i>B. subtilis</i> strains carrying plasmids harboring <i>blaR</i>1 mutants.

<p>Membrane proteins from induced (+) or uninduced (-) cultures were separatedon on SDS-PAGE. Purified BlaR-CTD antibodies were used for the Western blotting. Pre-stained protein molecular weight markers were used (M). The pinpoints an intense and non-specific band (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036400#pone-0036400-g006" target="_blank">Figure 6</a>). BlaR1 and BlaR1* point out, respectively, the full size and the activated <i>B. licheniformis</i> BlaR1 receptor. Except for mutant E²⁷⁴A, all other mutants exhibit non-inducible β-lactamase phenotype. The E²⁷⁴A mutant has the same profile as the wild-type (to compare see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036400#pone-0036400-g006" target="_blank">Figure 6</a>). For details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036400#pone-0036400-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036400#pone-0036400-t002" target="_blank">2</a> and Experimental procedures.</p

Induction of BlaPβ-lactamase of the BlaR1 L3 loop mutants.

*<p>The induction factor corresponds to the ratio between the β-lactamase quantity/A⁶⁰⁰ for induced culture and the β-lactamase quantity/A⁶⁰⁰ for uninduced culture.</p

Membrane topology of the penicillin-sensory transducer, BlaR1.

<p>This receptor contains two domains, an extracellular domain: BlaR-CTD and a transmembrane domain: BlaR-NTD. BlaR-CTD exhibits the three motifs of the penicillin binding protein family (S*⁴⁰²TYK, Y⁴⁷⁶GN, K⁵³⁹TG, where S⁴⁰² is the active serine). BlaR-NTD includes four transmembrane segments (TM1, TM2, TM3, TM4) connected by three loops (L1, L2, L3). The cytoplasmic L3 loop contains the H²¹²EXXH motif, characteristic to zinc-metalloproteases.</p

Oligonucleotides used in this study.

<p>Modified codons are underlined and mutagenised bases are highlighted.</p

Bacterial strains and plasmids used in this study.

<p>Ap^r: ampicillin resistance.</p><p>Cm^r: chloramphenicol resistance.</p

Coomassie Blue-stained SDS-PAGE of partially purified inclusion bodies of wild-type and L3 loop mutants (A) and Zinc blot analysis (B).

<p>In A and B: 1: bovine carbonic anhydrase (AC) (∼30 kDa; 5 µg), was used as positive control and 2 to 6 are, respectively, wild type (WT), E²¹³A, H²¹²A/H²¹⁶A, D²⁵⁷ A, D²²¹ A and E²⁵³ A L3 loop mutants. For each gel 40 µg of inclusion bodies were loaded. The fluorographies were exposed at -70°C for 72 hours. M: molecular weight marker. The arrow indicates L3 loops.</p

Western blot analysis of membranes of <i>B. subtilis</i> transformed with pMK4 (negative control), pDML995 (wild type), pDML1269 (E²¹³A mutant) or pDML3045 (R³⁰⁴A/R³⁰⁵A mutant).

<p>Membrane proteins from induced (+) or uninduced (−) cultures were separatedon on SDS-PAGE. Purified BlaR-CTD antibodies were used for the Western blotting. Pre-stained protein molecular weight markers were used (M). The indicates an intense and non-specific band. BlaR1 and BlaR1* highlight, respectively, the full size and the activated <i>B. licheniformis</i> BlaR1 receptor.</p