1. The growth characteristics of nine micro-organisms on complex broth and defined media, usually with a single nitrogen source (other than vitamins), were examined as a necessary step before growth of cells for enzyme assays. Six of these bacteria gave a positive colour test with a creatine–potassium hydroxide reagent, indicating the presence of acetoin, which other investigators have shown is formed via the intermediate, α-acetolactate. 2. Cell-free extracts of exponential-phase cells of Bacillus subtilis, Staphylococcus aureus, Proteus morganii, Acetobacter rancens (two strains), A. kuetzingianus, A. acetosus, Acetomonas (Acetobacter) melanogenus and Acetomonas (Acetobacter) suboxydans (A.T.C.C. no. 621) were found to contain the enzyme, dihydroxy acid dehydratase (2,3-dihydroxy acid hydro-lyase). 3. The specific activity of the dehydratase from organisms grown on valine- and isoleucine-deficient media was greater than those grown on a complex broth or media containing complete amino acid mixtures. The omission of valine plus isoleucine from a medium containing 19 amino acids caused an increase in the dehydratase specific activity of Staphylococcus aureus and Proteus morganii. 4. The rate of keto acid formation from αβ-dihydroxyisovalerate by extracts of six of the above-named organisms was faster than, but somewhat proportional to, the similar rate from αβ-dihydroxy-β-methyl-n-valerate as substrate. 5. These findings may be related to acetolactate synthesis, acetoin formation and valine–isoleucine biosynthesis in the above-mentioned micro-organisms.
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