FIG 4.

Glycine-alanine fermentation of C. difficile growing in the absence of proline and leucine. Cells obtained from ADM cultures grown to an OD₆₀₀ of ca. 0.6 were used to inoculate glycine-alanine (Gly/Ala) medium lacking proline and leucine and in which glycine was the only remaining Stickland acceptor (see Materials and Methods). The growth substrates available in Gly/Ala medium were 5 mM glucose, 10 mM glycine, 10 mM alanine, and low concentrations of isoleucine, valine, methionine, and tryptophan. In addition, 2 mM sodium acetate and 3.9 mM NH₄Cl were present. Triplicate growths were monitored, and amino acids and glucose were assayed as indicated for Fig. 3. OD₆₀₀ (*, □, and ×) and concentrations of glucose (Glc), glycine (Gly), alanine (Ala), and ammonia (NH₃) are shown.