BIOCHEMICAL AND MICROCHEMICAL STUDY OF THE CELL WALLS OF RHIZIDIOMYCES SP.

FULLER, MELVIN S. (Brown U., Providence, R. I.) Biochemical and microchemical study of the cell walls of Rhizidiomyces sp. Amer. Jour. Bot. 47(10): 838-842. lllus. 1960.-The presence of chitin in the cell walls of the fungus Rhizidiomyces is demonstrated by qualitative analysis of enzymatic and hydrochloric-acid hydrolysates of partially cleaned cell walls. Qualitative examination of the enzymatic and acid hydrolysates did not, however, serve for the detection of cellulose present in the cell walls of Rhizidiomyces. With microchemical tests, both chitin and cellulose can be detected. These microchemical tests served to indicate the localization of the chitin and cellulose in the cell walls of mature plants before and during zoospore discharge. CONTROVERSY over the simultaneous occurrence of chitin and cellulose in fungus cell walls was reviewed recently by Fuller and Barshad (1960). These authors also demonstrated by X-ray diffraction analysis that the cell walls of the unicellular aquatic Phycomycete, Rhizidiomyces, contained both chitin and cellulose. Because the occurrence of chitin and cellulose in other fungi had been questioned (e.g., Young, 1958), it seemed advisable also to examine the cell walls of Rhizidiomyces with biochemical and microchemical methods. Further, the microchemical tests, if positive, should serve for localization of the chitin and cellulose in the cell walls. Although the sole use of microchemical methods for the detection of chitin and cellulose has been criticized (Frey, 1950; Roelofsen and Hoette, 1951), the writer believes their use for the localization of chitin and cellulose in the present study is justified by the results obtained in the previous X-ray diffraction analyses (Fuller and Barshad, 1960) of Rhizidiomyces cell walls. Therefore, the studies reported herein were undertaken to substantiate the X-ray demonstration of chitin and cellulose in Rhizidiomyces cell walls and to localize these 2 polysaccharides in the cell walls of plants prior to and during the discharge of zoospores. MATERIALS AND METHODS.-The growth of the fungus and preparation of the cell walls for studv, i.e., chemical extraction of the protoplasm with 4.5% KOH. has been described previously (Fuller and Barshad, 1960). The starting material for the studies to be related here was, therefore, a suspension of "chemically cleaned" cell walls. The details of the various methods of study used are best presented with the results in the following sections. 1 Received for publication February 29, 1960. 2This investigation was supported in part by a fellowship (EF-7364) from the National Institute of Allergy and Infectious Diseases, Public Health Service. Part of a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Department of Botany. University of California, Berkeley. The assistance of Professors Ralph Emerson and Leonard Machlis during the course of this study is gratefully acknowledged. BIOCHEMICAL STUDIES OF CHEMICALLY CLEANED CELL WALLS.-Hydrolysates of the cell walls were prepared with chitinase, cellulase and 6 N HCI. Chitiniase hydrolysis of the cell walls.-The chitinase preparation used in these studies was an ultrafilter concentrate of an exocellular enzyme produced by Streptomyces griseus. The steps in the preparation of the enzyme and a description of its properties and specificity have been made by Dr. D. M. Reynolds (1954) who very kindly supplied the necessary enzyme for the present studies. To test the effect of the chitinase on the cell walls of RPhizidionmyces, 5 samples with 1 ml. of a slurry of chemically cleaned walls (approx. 110 mg. of wall material expressed as dry weight) were incubated individually for 24 hr. at 37?C. with 9 ml. of the ultrafilter concentrated enzyme in 0.02 M phosphate buffer at pH 7. The reaction mixtures contained 10-4 M merthiolate to prevent microbial decomposition. Following the incubation period, the supernatant enzyme from each sample was collected and saved. Nine ml. of fresh enzyme was added to each lot of cell walls and the whole incubated for a second 24-hr. period. The liquid from the 2 incubations was combined and the total volume of each of the replicates made up to 25 ml. by adding distilled water. A second series of wall samples was incubated with 0.02 M phosphate buffer and 10-4 M merthiolate only to see if any reducing sugars were present in the absence of the enzyme. The liquid in these controls was also made up to 25 ml. after the two 24-hr. incubation periods. The liquid from the enzyme-treated wvall preparations and the controls was heated at 80'C. for 5 min. to inactivate any enzymes present. The diluted enzyme hydrolysates and the controls were next analyzed for reducing sugar by measuring their capacity to reduce the yellow ferricyanide ion according to the methods outlined by Berger and Reynolds (1958). The N-acetylglucosamine standards used had 15, 30, 45, 60, 75, 90, 105, 120, 135, and 150 yg. of the compound per ml. The optical densities of 1-ml. samples of the 5 enzyme hydrolysates indicated that 22.9-25.5 g. of reducing sugar had been hydrolyzed from each