Alterations in the outer wall architecture caused by the inhibition of mycoside C biosynthesis inMycobacterium avium

Radiolabeled amino acids (l-U[C¹⁴]alanine,d-U[C¹⁴]alanine,l-U[C¹⁴]threonine, andl-U[C¹⁴]phenylalanine) were exponentially incorporated into the trichloroacetic acid (TCA)-insoluble material (whole cells) ofMycobacterium avium during the first 30–60 min of labeling. Bacteria labeled for 48 h were extracted with chloroform-methanol (21 vol/vol). The thin layer chromatography (TLC) analysis of native lipids showed that mycoside C was labeled by the amino acids used.d-cycloserine (d-CS) and other amino acid analogs were examined as potential inhibitors of mycoside C biosynthesis. It was found thatd-CS caused about 27% inhibition, whereaso-,p-, andm-fluoro-dl-phenylalanine (Fl-phe) caused 80%–90% inhibition of the mycoside C biosynthesis. Judging from the data on inhibition experiments, it was concluded that the mycoside C biosynthesis started from the fatty acyl end and proceeded by the stepwise addition ofd-phenylalanine,d-allo-threonine, andd-alanine. Thed-alanyl-d-alanine peptidoglycan intermediate did not seem to serve as a donor ofd-alanine for mycoside C biosynthesis. Ultrastructural observation of the bacteria treated withd-CS showed only partial alteration of the outer wall layer, whereasm-Fl-phe treatment caused profound alterations. Successive transfers of the bacteria in growth medium supplemented withm-Fl-phe resulted in extensive disorganization of the outer layer.