Outgrowth and sporulation studies on Clostridium botulinum type E: influence of isoleucine

A defined medium (CDM) is described which supported growth and sporulation of type E strains of Clostridium botulinum, but not sporulation of other serotypes of C. botulinum or C. sporogenes. As compared to growth in complex medium, spore outgrowth was delayed and both the growth rate and the cell yield was reduced. However, efficiency of sporulation of the type E MSpt strain in a chemically defined medium (CDM) was the same as that in complex medium and, in fact, sporulation was nearly synchronous and completed within 3 h of the first appearance of phase-bright endospores, compared with completion in 9 h in TPGY. Growth studies with CDM, from which single amino acids were omitted, showed that isoleucine was essential for outgrowth of heat-activated spores of the MSp+ strain, whereas valine was required for that of the Ts-25 mutant. Radioactive isoleucine was incorporated by germinating MSp+ spores at an earlier stage and at a more rapid rate than labelled methionine or mixed amino acids. Uptake studies showed that isoleucine accumulated in a prominent acid-soluble pool during outgrowth, a period when its incorporation into protein was not evident. The results suggest that the isoleucine may be required for a purpose other than protein synthesis during outgrowth.