Figure 1.
Ssp4 alignment versus other C. perfringens SASPs (Panel A) or Ssp4 homologues in other Clostridium spp. (Panel B).
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 [12],[16],[19].
Table 1.
Isolates used in this study.
Figure 2.
Expression of the ssp4 gene by wild-type F4969 or SM101 during vegetative growth and sporulation.
Panel A, RT-PCR analyses of SM101 ssp4, SM101 plc, or F4969 ssp4 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.
Table 2.
Spore resistance to heat (100°C) and sodium nitrite (nitrous acid).
Figure 3.
Intron-based mutagenesis to create SM101 and F4969 ssp4 null mutants.
Panel A, ssp4-specific PCR results for: wild-type SM101; SM101::ssp4, the SM101 ssp4 null mutant; or SM101::ssp4 transformed with pJIR751, pJIR751 carrying the SM101 ssp4 gene, or pJIR751 carrying the F4969 ssp4 gene; a blank lane; wild-type F4969; F4969::ssp4, the F4969 ssp4 null mutant; F4969::ssp4 transformed with the pJIR751 shuttle plasmid, pJIR751 carrying the F4969 ssp4 gene, or pJIR751 carrying the SM101 ssp4 gene. Presence of the larger (∼1.2 kb) PCR product reflects intron disrupted ssp4 gene, as depicted in Panel C. Panel B shows cpe genotyping PCR [8] results confirming that all F4969 or SM101 derivatives still carry, respectively, a plasmid cpe gene or a chromosomal cpe 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.
Figure 4.
Southern blot analysis of wild-type, ssp4 null mutants and complementing strains of F4969 or SM101.
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 ssp4 probe for panel B. Panel C shows an overlay of the A and B blots. DNA size markers are shown at left.
Figure 5.
Expression of the ssp4 gene and Ssp4 production by wild-type, ssp4 null mutants, and complementing strains of F4969 or SM101.
Panel A, RT-PCR analyses for ssp4 expression by SM101, SM101::ssp4, and complementing strains grown for 6 h in DS mediuim. Panel B, RT-PCR analyses of ssp4 expression by F4969, F4969::ssp4, 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, ssp4 null mutants or complementing strains of F4969 or SM101.
Figure 6.
DNA binding properties of purified recombinant His6-tagged rSsp4.
Panel A, purity and stability of Coomassie blue (top panel) or His6-tag Western blotted, purified His6-tagged rSsp4 proteins used in Fig. 6 B and C DNA binding experiments. Panel B, EMSA analysis of purified F4969, SM101 and 01E809 rSsp4 binding to C. perfringens DNA. Lane 1, free biotin-labeled C. perfringens DNA; lanes 2–4, indicated amounts of purified F4969 rSsp4 incubated with C. perfringens biotin-labeled DNA; lanes 5–7, indicated amounts of purified SM101 rSsp4 incubated with C. perfringens biotin-labeled DNA; and lanes 8–10, indicated amounts of purified 01E809 rSsp4 incubated with C. perfringens 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.