Pyruvate was shown to be the precursor of diacetyl and acetoin in Streptococcus diacetilactis, but dialyzed cell-free extracts of S. diacetilactis and Leuconostoc citrovorum that had been treated with anion-exchange resin to remove coenzyme A (CoA) formed only acetoin from pyruvate in the presence of thiamine pyrophosphate (TPP) and Mg++ or Mn++ ions. The ability to produce diacetyl was restored by the addition of acetyl-CoA. Acetyl-phosphate did not replace the acetyl-CoA. Neither diacetyl nor acetoin was formed when the otherwise complete reaction system was modified by using boiled extract or by omitting the extract, pyruvate, TPP, or the metal ions. Free acetaldehyde was not involved in the biosynthesis of diacetyl or acetoin from pyruvate, dialyzed cell-free extracts of the bacteria produced only acetoin (besides C02) from a-acetolactate, and acetoin was not involved in the biosynthesis of diacetyl. Only one of the optical isomers present in racemic a-acetolactate was attacked by the extracts, and there was no appreciable spontaneous decarboxylation of the a-acetolactate at the pH (4.5) used in experiments. 174 Vol. 95, No. I

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Aliphatic chain compounds at least four carbons long with vicinal carbonyl groups in the 2,3 positions were detected by the Westerfeld test. Acetoin, which has one carbonyl group and an adjacent hydroxyl group, gave positive results, but methyl acetoin (3-hydroxy-3-methyl-2-butanone) was negative, and subsequent tests supported the conclusion that acetoin is oxidized to diacetyl by ax-naphthol during the Westerfeld test in the absence or presence of air. 2,3-Pentanedione and 2,3-heptanedione gave positive results, but equimolar concentrations of these compounds gave maximal absorbancy readings that were only 35% (2,3-pentanedione) and 31% (2,3-heptanedione) of those obtained with diacetyl or acetoin. Negative results were obtained with pyruvic acid, 2,3-butylene glycol, and carbon ring compounds (1,2-cyclohexanedione, alloxan, and 3,4-dihydroxy-3-cyclobutene-1,2-dione). a-Naphthol could not be replaced in the test by ,-naphthol, 1,2,3,4-tetrahydroxy-1-naphthol, or 5,6,7,8-tetrahydroxy-1-naphthol. Creatine could not be replaced by arginine, guanidine HCl, or guanidinoacetic acid.