Inorganic Phosphate Induces Spore Morphogenesis and Enterotoxin Production in the Intestinal Pathogen Clostridium perfringens

Efficiency of spore formation under P_i-buffered or Tris-buffered conditions. Cultures of the wild-type strain SM101 were grown in DSMM supplemented with different amounts of 1 M Tris-HCl, pH 8.0, or 1 M Na₂HPO₄ for 20 h at 37°C. The final pH after growth and the final cellular yield (circles) and spore efficiency (triangles) under each growth condition are indicated. Open symbols, Tris-buffered cultures; filled symbols, P_i-buffered cultures. The results shown are the average of five independent experiments; bars indicate standard errors. Similar effects of P_i on spore efficiency were obtained with other enterotoxigenic C. perfringens human isolates other than SM101 (data not shown). Essentially the same results as the ones observed in DSMM-Tris were obtained when the Tris buffer was replaced by MOPS buffer (DSMM-MOPS; data not shown).

cpe expression and CPE production under different growth conditions. (A) Wild-type cultures of a derivative SM101 strain carrying a cpe-gusA plasmid were grown in DSMM with or without the addition of P_i (35 mM) and/or glucose (1%). Samples were removed at the indicated times and analyzed for β-glucuronidase activity as indicated (30). ○, DSMM plus P_i; ▵, DSMM alone; ⋄, DSMM plus P_i and glucose; and □, DSMM plus glucose. (B) Cultures carrying the cpe-gusA reporter were developed in three different media able to support the growth of C. perfringens: DSMM, TY, and FT medium with or without the addition of P_i (35 mM; see text for details). The bars indicate the level of β-glucuronidase activity accumulated by 1 h after the end of the exponential phase of growth. (C) Western blotting experiment using anti-CPE antibodies (20) of C. perfringens cultures grown in the indicated medium with or without supplementation with inorganic phosphate. The cellular pellet from the culture grown in DSMM with P_i was diluted 100-fold, while all the other pellets were undiluted. Data from representative experiments are shown.

Cell phenotypes of C. perfringens grown in the presence or absence of P_i signaling. (A to D) Phase-contrast photomicrographs of Spo0A-proficient (SM101) and Spo0A-deficient cells (IH101) grown for 5 h in DSMM with or without added P_i. Wild-type cells (SM101) grown in the presence of added P_i displayed polar prespores (A), while the non-P_i-supplemented culture (B) showed cells with a cellular phenotype resembling spo0A mutants (11) blocked at stage zero of sporulation, regardless of whether inorganic phosphate was added or not (C and D). After the overnight growth of the wild-type cultures, no prespores were observed in the non-P_i-supplemented culture, while in the P_i-supplemented culture a great proportion of mature and free spores (more than 60%) were observed (data not shown). (E to H) Fluorescent photomicrographs with DAPI (specific for DNA staining) and FM 4-64 (specific for membrane lipid staining) of wild-type cells (SM101) grown for 5 h in DSMM with or without supplementation with P_i.