Inhibition of Bacterial Growth by β-Chloro-D-Alanine

Abstract

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

Characterization of Escherichia coli d -Cycloserine Transport and Resistant Mutants

“…DadA has been shown to convert D-chloroalanine to ␤-chloropyruvate, which inhibits alanine racemase activity (72,73). In addition, ␤-chloropyruvate inhibits solute transport in E. coli by a process that has not been fully characterized, but the strong alkylating properties of this molecule are believed to be responsible for this effect (73)(74)(75).…”

Specificity of peptide transport systems in Lactococcus lactis: evidence for a third system which transports hydrophobic di- and tripeptides

“…Approximately 10 8 CFU of an exponentially growing CDM culture were spread on a 1.2% (wt/vol) agar plate containing a complete amino acid mixture. After plating was performed, a single crystal of the toxic peptide analog L-phenylalanyl-␤-chloro-L-alanine (FCA) (13) was put in the center of each plate. Growth inhibition was observed after 48 h of incubation at 30ЊC.…”

Dehalogenation and Deamination ofl-2-Amino-4-chloro-4-pentenoic Acid byProteus mirabilis

“…10 ) This result suggests that the metabolic enzymes of L-amino acids are inhibited by L-ACP as reported for 3-chloroalanine. 6 ) Growth inhibition of P. mirabilis in the presence of L-ACP was observed. However, the suicide inactivation of the enzyme was not observed in the a,y-elimination reaction of L-ACP.…”

“…4 ,5) Manning et al have reported that the D-and L-isomers of synthetic 3-chloroalanine inhibit the growth of Diplococcus pneumoniae, Streptococcus pyogenes, Bacillus subtilis, and Escherichia coli. 6 ) They demonstrated that the antibacterial action of 3-chloroalanine was caused by the inactivation of both n-amino acid transaminase and alanine racemase. The physiological effects of both isomers of this amino acid have been investigated and demonstrated as a suicide substrate 7 ) or an inhibitor of an active transport system.…”