A defined medium was devised for use in washed-cell experiments with post-exponential-phase cultures of Bacillus amyloliquefaciens. The medium allowed alpha-amylase to be secreted, bacterial concentration to increase and l-[U-(14)C]valine to be incorporated into protein at a linear rate, which was the same as in a post-exponential-phase culture, for up to 6h. 2. Determination of the specific radioactivity of l-[U-(14)C]valine in the medium, the intracellular amino acid pool, the cellular protein and the isolated alpha-amylase, after a 3h incubation of washed cells in the defined medium, showed that at least 76% of the alpha-amylase secreted was synthesized de novo. 3. By isolating the alpha-amylase formed during a 6h incubation in the presence of l-[U-(14)C]valine it was shown that the specific radioactivity of the N-terminal valine, within the limits of experimental error, was the same as that of the total valine residues from the complete alpha-amylase molecule. 4. A consideration of these results in relation to the whole literature on the subject strongly supports the idea that there is no reason to suppose that extracellular alpha-amylase is formed from a high-molecular-weight precursor in B. amyloliquefaciens and closely related organisms with identical characteristics of exoenzyme secretion.
