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 glycosylation, on the secretion, adhesion and activities of the cellulase complex produced by Clostridium cellulolyticum have been studied. Tunicamycin at 0.1 micrograms/ml slightly inhibited growth on cellobiose. Endoglucanase, p-nitrophenylcellobiosidase and avicelase activities of the "Avicel"-adsorbed fraction from a culture grown with this drug were decreased 4.4-, 1.4- and 12.2-fold, respectively. During growth on cellulose, tunicamycin considerably inhibited growth and adhesion of cells on 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 native Avicel-adsorbed fraction (PH2O) consisted of three major components of molecular weights about 135, 90 and 68 kDa, whereas in the presence of tunicamycin (0.1 micrograms/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 40-kDa proteins. Moreover, the affinity for Avicel and the avicelase activity decreased dramatically for the Avicel-adsorbed fraction from a culture grown with the drug. The remaining avicelase activity of the PH2O fraction in the presence of specific P135 antiserum was 50% of the initial activity, whereas CMCase and pNPCbase were not affected. The glycosylated protein of 135 kDa played a prominent role in the adhesion and avicelase activity of C. cellulolyticum. Moreover, the endoglucanase activity in a culture broth from tunicamycin-grown cells was more thermolabile and protease-sensitive than that from control cultures.
Gehin
A
A
Université de Nancy I, Laboratoire de Chimie biologique I, Vandoeuvre-lès-Nancy, France.
Petitdemange
H
H
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001422:Bacterial Adhesion / Q000187:drug effects*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002480:Cellulase / Q000737:chemistry / Q000378:metabolism*; D003013:Clostridium / Q000187:drug effects* / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D013696:Temperature; D014415:Tunicamycin / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D014415:Tunicamycin; D002480:Cellulase