CELL WALL COMPOSITION AND PAPILLA BIOSYNTHESIS IN BLASTOCLADIELLA EMERSONII

WILLIAM DIRK SIKKEMA, Purdue University

Abstract

Cell walls from Blastocladiella emersonii were isolated by repeated washing and centrifugation. Purity of cell walls was assessed by light microscopy, electron microscopy, and chemical reproducibility. Examination of cell walls by electron microscopy indicated that Blastocladiella cell walls consist of an inner microfibrillar network and an outer amorphous layer. The results of physical analysis by X-ray and infra red spectroscopy were consistent with chitin as the major cell wall component. Gross chemical analysis indicated that Blastocladiella cell walls were composed to (as % dry weight): 74.7% amino sugar (as N-acetyl hexosamine), 10.7% neutral sugar (as hexose), 0.8% uronic acid, 10.6% protein, and 4.2% lipid. Analysis of the neutral sugars showed that isolated cell walls contain 1.5% mannose, 3.0% galactose, and 3.0% glucose. Isolated cell walls were fractionated using a hot sodium dodecyl sulfate (SDS) extraction followed by either a Pronase digestion or a hot potassium hydroxide extraction. The hot SDS extract was shown to contain two polymer types, a glucan/galactan and a glycoprotein. However, the residue from the hot SDS extraction still contained most of the neutral sugars and protein that were present in the isolated walls. Both Pronase digestion and the hot potassium hydroxide extraction removed all of the neutral sugars except glucose. Based on the cell wall fractionation studies, it was suggested that the amorphous layer contained a glucose/galactan polymer and a glycoprotein, while the microfibrillar component contained a glucan in addition to the chitin. The discharge papilla of Blastocladiella is a small plug that replaces a localized region of the cell wall during sporulation. Previous work by others had suggested that the papilla might contain a glycoprotein. This possibility was examined by an investigation of {('3)H}-mannose incorporation during early and late sporulation. The results indicated that {('3)H}-mannose is incorporated at a substantially greater rate during late sporulation. In contrast, the {('3)H}-leucine incorporation pattern remained unchanged over similar labeling periods. Autoradiography of cells labeled with {('3)H}-mannose during late sporulation indicate that a major portion of the label was being incorporated into the papillate region of the cell. When cells labeled with {('3)H}-mannose during early and late sporulation were extracted with hot SDS and the extract passed over a Con A-Sepharose column, a larger amount of the material from cells labeled during late sporulation was bound to the column. When the material from the late labeling period was eluted from the Con A-column and run on polyacrylamide gels, a single large peak of approximately 70,000 daltons was obtained. Papilla biosynthesis was further investigated using various inhibitors. 2-Deoxy-D-glucose, bacitracin, and tunicamycin, all reported inhibitors of glycosylation, were tested for their ability to inhibit papilla formation. All were found to inhibit papilla formation, but 2-deoxy-D-glucose and bacitracin exhibited various other nonspecific effects. Tunicamysin was shown to inhibit both {('3)H}-mannose incorporation and papilla formation without affecting {('3)H}-leucine incorporation. Also, the time of inhibition relative to the induction of sporulation coincided with the increased rate of {('3)H}-mannose incorporation found coincident with late sporulation. The effects of the microtubule inhibitor colchicine and the microfilament inhibitor cytochalasin B on papilla formation were also investigated. Both were found to inhibit papilla formation. However, colchicine was shown to exhibit nonspecific effects. In contrast, cytochalasin B, while inhibiting papilla formation, had no apparent effect on either {('3)H}-leucine incorporation or {('3)H}-mannose incorporation. The results suggest that microfilaments may be involved in papilla formation.

Degree

Ph.D.

Subject Area

Microbiology

COinS