i3-Chloroamino acids have been shown recently to bind efficiently to transaminases and decarboxylases and to undergo #3-elimination reactions in situ (7,8). In some cases the enzyme catalyzing the (3-elimination reaction is irreversibly inactivated presumably by reaction of the enzyme-bound aminoacrylic acid with some functional group of the protein (8). Accordingly, it has occurred to us that (3-chloro-D-alanine might inhibit irreversibly the pyridoxal phosphate enzymes responsible for the formation of D-alanine in bacterial cells and thereby preclude the synthesis of a cell-wall constituent necessary for bacterial growth. MATERIALSThe D-and -isomers of fl-chloroalanine were purchased from Cyclo as the hydrochloride salts. Elemental analysis of (3-chloro-D-alanine gave: C, 22. METHODSConditions for Growth of Bacteria. For periodic measurement of the effect of (3-chloro-D-alanine on the growth of pneumococcus, portions of cultures in the logarithmic phase of growth were added under sterile conditions to 9 volumes of a minimal medium devoid of alanine (9) in 18 X 150-mm tubes. The compounds to be tested (the i-and D-isomers of 0-chloroalanine, D-and L-alanine, and D-alanyl-D-alanine) were then added, and incubations were carried out at 37°. In these experiments bacterial growth was measured by nephelometry on a Coleman instrument periodically for 3-6 hr.For determination of enzyme activities and of free intracellular alanine, cultures of either E. coli or B. subtilis were grown aerobically at 370 in 25-ml Erlenmeyer flasks in yeast extract-supplemented medium [0.5% dialyzed Difco yeast extract added to the minimal medium of Frantz (10)] and nutrient broth, respectively. The cultures were treated with 3-4 mM fl-chloro-D-alanine while in the logarithmic phase of growth, and incubated for an additional 3-4 hr. The cells were collected by centrifugation at 23,000 X g at 40 for 10 min. After being washed in 5-10 ml of 0.85% NaCl, they were resuspended in 10 ml 0.1 M potassium phosphate, pH 7.0. The cells were ruptured by sonication for 1-2 min at 00 in a Branson 750-watt sonicator at a setting of 3; the sonicate was then centrifuged for 10 min at 27,000 X g and 4°. The concentration of protein in the extract was estimated by the absorbancies at 280 and 260 nm (11).Determination' of the Amounts and Configurations ofChloroalanine and Alanine. The amounts of fl-chloroalanine and alanine in the culture supernatants and extracts of E. coli and B. subtilis were determined by amino-acid analysis (12, 13). Under our experimental conditions, (3-chloroalanine eluted at 58 ml and alanine at 122 ml from the 0.9 X 50-cm