A. Gehin et H. Petitdemange, THE EFFECTS OF TUNICAMYCIN ON SECRETION, ADHESION AND ACTIVITIES OF THE CELLULASE COMPLEX OF CLOSTRIDIUM-CELLULOLYTICUM, ATCC-35319, Research in microbiology, 146(3), 1995, pp. 251-262
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.