Incorporation of D-alanine into the membrane of Streptococcus pyogenes and its stabilized L-form

A principal aim of this study was to explain our earlier finding of a lack of d-alanine in the glycerol teichoic acid from the membrane of a stabilized L-form of Streptococcus pyogenes (B. M. Slabyj and C. Panos, 1973. J. Bacteriol. 114:934-942). It was found that the incorporation of d-alanine into the membrane teichoic acid of S. pyogenes requires either supernatant fraction or two enzymes from supernatant fraction, stimulator (d-alanine activating enzyme) and d-alanine:membrane acceptor ligase, plus membrane fragments, ATP and Mg(2+). A similar system from the L-form is inoperative. Also, no incorporation is observed with L-form or coccal supernatant fractions when L-form membranes are used. However, d-alanine incorporation is observed when L-form enzymes are used with membrane fragments from the parental streptococcus. Thus, the L-form possesses the required soluble components for d-alanine incorporation but the L-form membrane cannot function as acceptor even though it contains d-alanine-deficient membrane teichoic acid. These results suggest that a defect has occurred in the membrane of this stabilized L-form for d-alanine incorporation into membrane teichoic acid.