Intron-based mutagenesis to create SM101 and F4969 <i>ssp4</i> null mutants.

<p>Panel A, <i>ssp4</i>-specific PCR results for: wild-type SM101; SM101::<i>ssp4</i>, the SM101 <i>ssp4</i> null mutant; or SM101::<i>ssp4</i> transformed with pJIR751, pJIR751 carrying the SM101 <i>ssp4</i> gene, or pJIR751 carrying the F4969 <i>ssp4</i> gene; a blank lane; wild-type F4969; F4969::<i>ssp4</i>, the F4969 <i>ssp4</i> null mutant; F4969::<i>ssp4</i> transformed with the pJIR751 shuttle plasmid, pJIR751 carrying the F4969 <i>ssp4</i> gene, or pJIR751 carrying the SM101 <i>ssp4</i> gene. Presence of the larger (∼1.2 kb) PCR product reflects intron disrupted <i>ssp4</i> gene, as depicted in Panel C. Panel B shows <i>cpe</i> genotyping PCR <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000056#ppat.1000056-Miyamoto1" target="_blank">[8]</a> results confirming that all F4969 or SM101 derivatives still carry, respectively, a plasmid <i>cpe</i> gene or a chromosomal <i>cpe</i> gene. The left-pointing arrow for F4969 depicts an antisense intron insertion while the right-pointing arrow for SM101 depicts a sense intron insertion. Bars underneath i, ii, and iii of Panel C indicate expected PCR product sizes using B1F and B1R primers.</p

Southern blot analysis of wild-type, <i>ssp4</i> null mutants and complementing strains of F4969 or SM101.

<p>Panel A shows Southern blot hybridization of a DIG-labeled intron probe. The Southern blot was then stripped and re-probed with a DIG-labeled <i>ssp4</i> probe for panel B. Panel C shows an overlay of the A and B blots. DNA size markers are shown at left.</p

Expression of the <i>ssp4</i> gene by wild-type F4969 or SM101 during vegetative growth and sporulation.

<p>Panel A, RT-PCR analyses of SM101 <i>ssp4</i>, SM101 <i>plc</i>, or F4969 <i>ssp4</i> expression using cultures grown for 2–10 h in TGY (for vegetative growth) or Duncan-Strong sporulation medium (DS). Panel B and C show post-inculation change in optical density (OD₆₀₀) for cultures of SM101 or F4969 growing in, respectively, TGY or DS medium.</p

Spore resistance to heat (100°C) and sodium nitrite (nitrous acid).

1<p>pCS, pCF and pCO are the shuttle plasmid pJIR751 carrying the cloned <i>ssp4</i> gene (upstream sequence and ORF) from, respectively, SM101, F4969 or 01E809.</p

DNA binding properties of purified recombinant His₆-tagged rSsp4.

<p>Panel A, purity and stability of Coomassie blue (top panel) or His₆-tag Western blotted, purified His₆-tagged rSsp4 proteins used in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000056#ppat-1000056-g006" target="_blank">Fig. 6 B and C</a> DNA binding experiments. Panel B, EMSA analysis of purified F4969, SM101 and 01E809 rSsp4 binding to <i>C. perfringens</i> DNA. Lane 1, free biotin-labeled <i>C. perfringens</i> DNA; lanes 2–4, indicated amounts of purified F4969 rSsp4 incubated with <i>C. perfringens</i> biotin-labeled DNA; lanes 5–7, indicated amounts of purified SM101 rSsp4 incubated with <i>C. perfringens</i> biotin-labeled DNA; and lanes 8–10, indicated amounts of purified 01E809 rSsp4 incubated with <i>C. perfringens</i> biotin-labeled DNA. Panel C, effects of NaCl on binding of rSsp4 to DNA. Beads (100 µg) containing calf thymus DNA (Sigma) were incubated with 100 ng of rSsp4 from the indicated strain. After washing with 0, 0.25 M, 0.50 M, 0.75 M or 1.0 M NaCl, the beads were boiled and then analyzed by SDS-PAGE. Lane 1, input protein; lane 2, 100 µg DNA-free beads incubated with 100 ng purified rSsp4 from SM101, 01E809 or F4969; lanes 3–7, 100 µg DNA-containing beads incubated with 100 ng purified rSsp4 from SM101, 01E809 or F4969 and washed with indicated concentrations of NaCl.</p

Expression of the <i>ssp4</i> gene and Ssp4 production by wild-type, <i>ssp4</i> null mutants, and complementing strains of F4969 or SM101.

<p>Panel A, RT-PCR analyses for <i>ssp4</i> expression by SM101, SM101::<i>ssp4</i>, and complementing strains grown for 6 h in DS mediuim. Panel B, RT-PCR analyses of <i>ssp4</i> expression by F4969, F4969::<i>ssp4</i>, and complementing strains grown for 6 h in DS medium. Lane 1 shows size markers. Lanes labeled “+” were from samples receiving reverse transcriptase, while lanes labeled “-“ lacked reverse transcriptase to show the absence of DNA contamination. Panel C, Western blot analyses for Ssp4 production by overnight DS cultures of wild-type, <i>ssp4</i> null mutants or complementing strains of F4969 or SM101.</p

Isolates used in this study.

<p>All isolates classify as type A (data not shown).</p>1<p>This study and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000056#ppat.1000056-Sarker2" target="_blank">[9]</a>.</p

Ssp4 alignment versus other <i>C. perfringens</i> SASPs (Panel A) or Ssp4 homologues in other <i>Clostridium</i> spp. (Panel B).

<p>Box shows the conserved region common to all SASPs. Bold residues represent the variant residues in Ssp4 of F4969 and SM101. Sequences were obtained from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000056#ppat.1000056-Setlow1" target="_blank">[12]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000056#ppat.1000056-Pathema1" target="_blank">[16]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000056#ppat.1000056-Bettegowda1" target="_blank">[19]</a>.</p

Cells expressing XS-O mutants have reduced ability to bind PAC-1 and fibrinogen.

<p><b>A. Left</b>, PAC-1 binding of cells expressing XS-O mutants 321/358 and 321/360 the absence of treatment (control; CON) or in the presence of mAb PT25-2, DTT, or DTT+PT25-2. FITC-PAC-1 was added at 5 µg/ml and binding was assessed via flow cytometry. Binding is expressed as net normalized fluorescence intensity (NNFI), in which the geometric mean fluorescence intensity after subtracting nonspecific binding is divided by the relative surface receptor expression as judged by the binding of mAb 10E5. Data expressed as mean ± SD; n = 5. <b>Right</b>, Fibrinogen binding using Alexa488-fibringen (200 µg/ml) as indicated above in “<b>A</b>” for PAC-1 (mean ± SD; n = 6). <b>B.</b> The αIIb F992A/F993A activating mutations fail to rescue PAC-1 and fibrinogen binding in the XS-O mutants.</p

Overlapping PCR assay analysis of <i>cpe</i> locus diversity amongst type C isolates.

<p>An overlapping PCR assay specific for amplification of the type C isolate CN2078 <i>cpe</i> locus region (R1 to R8) was performed using the primer battery shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010932#pone-0010932-t002" target="_blank">Table 2</a>. (A) Map depicting the relationship between CN2078 <i>cpe</i> locus ORFs and reactions comprising this overlapping PCR battery. (B) PCR products produced by these reactions using DNA from type C isolates: CN2078, CN3753 and CN3748. (C) PCR products produced by these reactions using DNA from type C isolates: CN2076, CN3758 and CN3763. Numbers at left of each gel indicate migration of size markers in kb.</p