THE EFFECTS OF TUNICAMYCIN ON SECRETION, ADHESION AND ACTIVITIES OF THE CELLULASE COMPLEX OF CLOSTRIDIUM-CELLULOLYTICUM, ATCC-35319

The effects of tunicamycin, an inhibitor of N-asparagine-linked glycos ylation, on the secretion, adhesion and activities of the cellulase co mplex produced by Clostridium cellulolyticum have been studied. Tunica mycin at 0.1 mu g/ml slightly inhibited growth on cellobiose. Endogluc anase, p-nitrophenylcellobiosidase and avicelase activities of the ''A vicel''-adsorbed fraction from a culture grown with this drug were dec reased 4.4-, 1.4- and 12.2-fold, respectively. During growth on cellul ose, tunicamycin considerably inhibited growth and adhesion of cells o n their substrate (only 28% of the cells were bound to cellulose). SDS -PAGE mobilities of some proteins excreted during growth with the drug were different from those of proteins from control cultures; the nati ve Avicel-adsorbed fraction (P-H2O) consisted of three major component s of molecular weights about 135, 90 and 68 kDa, whereas in the presen ce of tunicamycin (0.1 mu g/ml), the Avicel-adsorbed fraction (PH2OT) contained only a major band of 105 kDa, and the proteins of 135 and 68 kDa appeared weakly. By using the ''Dig Glycan Detection'' kit, some proteins appeared to be glycosylated, such as the 135-, 95-, 47- and 4 0-kDa proteins. Moreover, the affinity for Avicel and the avicelase ac tivity decreased dramatically for the Avicel-adsorbed fraction from a culture grown with the drug. The remaining avicelase activity of the P -H2O fraction in the presence of specific P135 antiserum was 50% of th e initial activity, whereas CMCase and pNPCbase were not affected. The glycosylated protein of 135 kDa played a prominent role in the adhesi on and avicelase activity of C. cellulolyticum. Moreover, the endogluc anase activity in a culture broth from tunicamycin-grown cells was mor e thermolabile and protease-sensitive than that from control cultures.