A novel small acid soluble protein variant is important for spore resistance of most Clostridium perfringens food poisoning isolates

Abstract

Clostridium perfringens is a major cause of food poisoning (FP) in developed countries. C. perfringens isolates usually induce the gastrointestinal symptoms of this FP by producing an enterotoxin that is encoded by a chromosomal (cpe) gene. Those typical FP strains also produce spores that are extremely resistant to food preservation approaches such as heating and chemical preservatives. This resistance favors their survival and subsequent germination in improperly cooked, prepared, or stored foods. The current study identified a novel alpha/beta-type small acid soluble protein, now named Ssp4, and showed that sporulating cultures of FP isolates producing resistant spores consistently express a variant Ssp4 with an Asp substitution at residue 36. In contrast, Gly was detected at Ssp4 residue 36 in C. perfringens strains producing sensitive spores. Studies with isogenic mutants and complementing strains demonstrated the importance of the Asp 36 Ssp4 variant for the exceptional heat and sodium nitrite resistance of spores made by most FP strains carrying a chromosomal cpe gene. Electrophoretic mobility shift assays and DNA binding studies showed that Ssp4 variants with an Asp at residue 36 bind more efficiently and tightly to DNA than do Ssp4 variants with Gly at residue 36. Besides suggesting one possible mechanistic explanation for the highly resistant spore phenotype of most FP strains carrying a chromosomal cpe gene, these findings may facilitate eventual development of targeted strategies to increase killing of the resistant spores in foods. They also provide the first indication that SASP variants can be important contributors to intra-species (and perhaps inter-species) variations in bacterial spore resistance phenotypes. Finally, Ssp4 may contribute to spore resistance properties throughout the genus Clostridium since ssp4 genes also exist in the genomes of other clostridial species.

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 ,,.

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.

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 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.