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Comparative qualitative analyses of hydrolysis products of extracellular polysaccharides 1972

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COMPARATIVE QUALITATIVE ANALYSES OF HYDROLYSIS PRODUCTS OF EXTRACELLULAR POLYSACCHARIDES PRODUCED BY SOME YEASTS AND YEAST-LIKE FUNGI by P a t r i c i a E. M. Flodin B.Sc, University of B r i t i s h Columbia, 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Botany We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1972 In p resen t i ng t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree tha t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and s tudy. I f u r t h e r agree t h a t permiss ion f o r ex tens ive copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head o f my Department o r by h is r e p r e s e n t a t i v e s . I t i s understood tha t copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in sha l l not be a l lowed w i t h o u t my w r i t t e n pe rmiss ion . Department o f The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada D a t e ftfy£- /3 } )9 7^L i ABSTRACT The objective of the experiments was to compare ' q u a l i t a t i v e l y the monosaccharides i n the hydrolysis products of the e x t r a c e l l u l a r polysaccharides of several yeasts and ye a s t - l i k e fungi. S p e c i f i c a l l y , the study was aimed at finding s i m i l a r i t i e s and differences that might be useful i n suggesting and supporting taxonomic r e l a t i o n - ships. Gas chromatography and paper chromatography were used as methods of analyses i n an e f f o r t to f i n d out what method i s s u f f i c i e n t at the q u a l i t a t i v e l e v e l for dist i n g u i s h i n g some genera of yeasts and y e a s t - l i k e fungi; and what method would be best at the quantitative l e v e l for dist i n g u i s h i n g amongst some species of the same genus. From the a n a l y t i c a l r e s u l t s i t was found that paper chromatography using the solvents ethyl acetate: pyridine: water, (8:2:2) was s u f f i c i e n t for q u a l i t a t i v e determination of the monosaccharides i n the e x t r a c e l l u l a r polysaccharide hydro- l y s i s products. However, indications were that quantitative analyses by gas chromatography, using the t r i m e t h y l s i l y l derivatives of the monosaccharides would have been successful in d i s t i n g u i s h i n g among species of the same genus. Two groups were formed on the bases of the q u a l i t a t i v e r e s u l t s . Group I contained two subgroups. Subgroup I encompassed those yeasts and ye a s t - l i k e fungi with the monosaccharides galactose, glucose, mannose, xylose present i n the hydrolysis products of t h e i r e x t r a c e l l u l a r polysaccharides. Included i n t h i s Subgroup I are: Cryptococcus l a u r e n t i i , Tremella mesenterica, B u l l e r a alba, Sporobolomyces odorus, Sporobolomyces s i n g u l a r i s , and Rhodotorula g l u t i n i s . Sub- group II i s Ustilago hordei only, with the monosaccharides galactose, glucose, mannose, and lacking xylose. Group II contains Taphrina populina only, with glucose and mannose present and both galactose and xylose absent. The two groups formed support some of the taxonomic r e l a t i o n s h i p s that have already been suggested. The Tremella - Cryptococcus taxonomic r e l a t i o n s h i p that had previously been postulated on the basis of s i m i l a r i t i e s i n e x t r a c e l l u l a r polysaccharide hydrolysis products, morphology, carbon a s s i m i l a t i o n patterns, enzymatic x y l o s y l a t i o n reaction, and starch formation was supported. Secondly, the Cryptococcus-Bullera r e l a t i o n s h i p that had been suggested on the basis of i n o s i t o l a s s i m i l a t i o n , lack of pseudomycelium, and s i m i l a r i t i e s i n starch synthesis, was supported by the q u a l i t a t i v e analysis of the monosaccharides present i n the e x t r a c e l l u l a r polysaccharide hydrolysis products. The i i i monosaccharides found i n both Cryptococcus l a u r e n t i i and B u l l e r a a l b a e x t r a c e l l u l a r p o l y s a c c h a r i d e s were the same q u a l i t a t i v e l y . D u a l i t y amongst s p e c i e s of Sporobolomyces might be supported with f u r t h e r work using q u a n t i t a t i v e gas chromatographic a n a l y s e s . T h i s d u a l i t y had been p o s t u l a t e d on account of the d u a l i t y shown i n a n t i g e n i c a n a l y s e s and percent G+C base a n a l y s e s of DNA. T a p h r i n a p o p u l i n a can be d i s t i n g u i s h e d from Rhodotorula g l u t i n i s and Cryptococcus l a u r e n t i i . C ryptococcus l a u r e n t i i produces s t a r c h and a s s i m i l a t e s i n o s i t o l : Rho d o t o r u l a g l u t i n i s a s s i m i l a t e s i n o s i t o l but does not produce s t a r c h ; and T a p h r i n a p o p u l i n a produces s t a r c h but does not a s s i m i l a t e i n o s i t o l . Two monosaccharides p r e s e n t i n the e x t r a c e l l u l a r p o l y s a c c h a r i d e h y d r o l y s i s products of both C r y p t o - coccus l a u r e n t i i and R h o d o t o r u l a g l u t i n i s are g a l a c t o s e and x y l o s e whereas T a p h r i n a p o p u l i n a l a c k s these two monosaccharides. R e s u l t s o b t a i n e d from the q u a l i t a t i v e a n a l y ses of the e x t r a c e l l u l a r p o l y s a c c h a r i d e s produced by f u n g i may be important t a x o n o m i c a l l y . T h i s i s because the q u a l i t a t i v e i n f o r m a t i o n may be used when d e c i d i n g on P e r f e c t - I m p e r f e c t f u n g a l r e l a t i o n s h i p s . However, t h i s i n f o r m a t i o n should be c o n s i d e r e d along w i t h data from other f i e l d s such as morphology, c y t o l o g y , and g e n e t i c s b e f o r e h y p o t h e s i z i n g on a taxonomic r e l a t i o n s h i p . V Contents Page Abstract i Contents v Tables v i Figures v i i Acknowledgements x Introduction 1 Li t e r a t u r e Review 3 Materials and Methods 12 Results 29 Discussion 48 References 58 Appendix 62 v i T a b l e s T a b l e Page I The h o s t , l o c a t i o n , c o l l e c t i o n date, and c o l l e c t o r of each fungus used i n the experiments (when known). 13 I I Paper Chromatography r e s u l t s 31 I I I Gas Chromatography r e s u l t s 35 IV Gas cflaxomatography-peak Areas 63 V Gas chromatography-percent monosaccharide 67 v i i F i g u r e s F i g u r e Page 1 Procedure f o r the p r o d u c t i o n , i s o l a t i o n and a n a l y s i s of the e x t r a c e l l u l a r p o l y s a c c h a r i d e 14 2 Apparatus f o r the IR-120 c a t i o n exchange r e s i n 23 3 T r e m e l l a m e s e n t e r i c a and B u l l e r a a l b a . S e p a r a t i o n of products using descending paper chromatography w i t h s o l v e n t s e t h y l a c e t a t e : p y r i d i n e : water, (8:2:2) 32 4 Sporobolomyces s i n g u l a r i s , Sporobolomyces odorus (#949) , Sporobolomyces odorus (#981) , Cryptococcus l a u r e n t i i . S e p a r a t i o n of the products u s i n g descending paper chromatography with s o l v e n t s e t h y l a c e t a t e : p y r i d i n e : water, (8:2:2). 33 U s t i l a g o h o r d e i , R h o d o t o r u l a g l u t i n i s , T a p h r i n a p o p u l i n a . S e p a r a t i o n of products u s i n g descending paper chroma- tography with s o l v e n t s e t h y l a c e t a t e : p y r i d i n e : water, (8:2:2). T r e m e l l a m e s e n t e r i c a . S e p a r a t i o n of products as t r i m e t h y l s i l y l d e r i v a t i v e s Cryptococcus l a u r e n t i i . S e p a r a t i o n o f products as t r i m e t h y l s i l y l d e r i v a t i v e s . B u l l e r a a l b a . S e p a r a t i o n of p r o d u c t s as t r i m e t h y l s i l y l d e r i v a t i v e s . Sporobolomyces odorus (#949). Separ- a t i o n of the products as t r i m e t h y l s i l y l d e r i v a t i v e s . Sporobolomyces odorus (#981). Separ- a t i o n of the products as t r i m e t h y l s i l y l d e r i v a t i v e s . Sporobolomyces s i n g u l a r i s . S e p a r a t i o n o f the products as t r i m e t h y l s i l y l d e r i v a t i v e s . IX F i g u r e Page 12 Rhodotorula g l u t i n i s . S e p a r a t i o n of the products as t r i m e t h y l s i l y l d e r i v a t i v e s . 45 13 U s t i l a g o h o r d e i . S e p a r a t i o n of the products as t r i m e t h y l s i l y l d e r i v a t i v e s . 46 14 T a p h r i n a p o p u l i n a . S e p a r a t i o n of the products as t r i m e t h y l s i l y l d e r i v a t i v e s . 47 X ACKNOWLEDGEMENTS F i r s t , I would l i k e t o thank Dr. R. J . Bandoni, Department of Botany, U.B.C. f o r h i s c o n s t r u c t i v e c r i t i c i s m s on t h i s t h e s i s and f o r h e l p f u l s u g g e s t i o n s concerning the d i r e c t i o n o f my r e s e a r c h work. Second, I would l i k e t o thank Dr. G. G. S. Dutton, Department o f Chemistry, U.B.C. f o r the use o f the gas chromatograph machine, chromatograph paper, s o l v e n t s , and t a n k s . A l s o f o r h e l p f u l a d v i c e concerning chromatography I would l i k e t o thank Dr. G. G. S. Dutton, Mr. R. H. Walker, and Dr. S. K a b i r . T h i r d , I would l i k e t o thank my husband Bruce f o r h i s p a t i e n c e and understanding w h i l e work f o r t h i s degree was i n p r o g r e s s . INTRODUCTION In g e n e r a l , the purpose of the experiments was t o gather v a l u a b l e i n f o r m a t i o n t h a t may a i d i n the s y n t h e s i s o f a c l a s s i f i c a t i o n scheme. The d a t a o b t a i n e d from the q u a l i - t a t i v e a n a l y s e s of the e x t r a c e l l u l a r p o l y s a c c h a r i d e s o f some y e a s t s and y e a s t - l i k e f u n g i may g i v e support t o some of the a l r e a d y p o s t u l a t e d P e r f e c t - I m p e r f e c t f u n g a l r e l a t i o n s h i p s . T h i s process o f c r i t i c a l l y re-examining, adding t o , and c o r r e l a t i n g the r e s u l t s of p r e v i o u s workers i s a n e c e s s a r y p a r t o f any r e s e a r c h . S p e c i f i c a l l y , t h e o b j e c t i v e s o f the experiments were: 1. To determine q u a l i t a t i v e l y the h y d r o l y s i s p r o d u c t s o f the e x t r a c e l l u l a r p o l y s a c c h a r i d e s produced by s e v e r a l y e a s t s and y e a s t - l i k e f u n g i . 2. To employ both gas chromatography and paper chromato- graphy as methods o f a n a l y s i s and t o determine: (a) which method i s b e s t a t the q u a l i t a t i v e l e v e l , (b) which method i s bes t f o r s e p a r a t i n g genera o n l y , and s p e c i e s o f the same genus. 3. To compare and c o n t r a s t the q u a l i t a t i v e r e s u l t s with those o f p r e v i o u s workers. 1 To compare and contrast the yeasts and y e a s t - l i k e fungi investigated with respect to the monosaccharides i n t h e i r e x t r a c e l l u l a r polysaccharide hydrolysis products. To arrange the yeasts and y e a s t - l i k e fungi investigated into groups on the basis of q u a l i t a t i v e s i m i l a r i t i e s or differences i n the monosaccharides i n t h e i r e x t r a c e l l u l a r polysaccharide hydrolysis products. To suggest taxonomic re l a t i o n s h i p s considering the above formed groups i n conjunction with data supplied from other f i e l d s such as morphology and genetics. To examine the v a l i d i t y of using c e r t a i n biochemical analyses, i n p a r t i c u l a r , those of the e x t r a c e l l u l a r polysaccharides i n the synthesis of a c l a s s i f i c a t i o n scheme. LITERATURE REVIEW For convenience, the y e a s t s and y e a s t - l i k e f u n g i used i n the experiments w i l l be reviewed i n groups. Group I Subgroup I c o n t a i n s T r e m e l l a m e s e n t e r i c a F r . , Cryptococcus l a u r e n t i i ( K u f f e r a t h ) Skinner, B u l l e r a a l b a (Hanna) Derx., Sporobolomyces s i n g u l a r i s P h a f f e t Do Carmo Sousa, Sporobolomyces odorus Derx., and Rhodotorula g l u t i n i s (Fres.) H a r r i s o n . Group I Subgroup I I i s U s t i l a g o h o r d e i (Pers.) L a g e r h . Group I I i s T a p h r i n a p o p u l i n a F r . Group I Subgroup I The e x t r a c e l l u l a r p o l y s a c c h a r i d e s of T r e m e l l a m e s e n t e r i c a and Cryptococcus l a u r e n t i i have been i n v e s t i g a t e d e x t e n s i v e l y . The polymer produced by T r e m e l l a m e s e n t e r i c a c o n t a i n s both an a c i d i c h e t e r o p o l y s a c c h a r i d e and a n e u t r a l g l u c a n . S l o d k i , i n 1966 (43), i d e n t i f i e d the a c i d h y d r o l y s i s products of the a c i d i c h e t e r o p o l y s a c c h a r i d e . These products were O - a c e t y l , x y l o s e , mannose, and g l u c u r o n i c a c i d i n the r a t i o o f 0.5 : 4.4 : 3.8 : 1 r e s p e c t i v e l y . F r a s e r and Jennings, i n 1971 (19), i d e n t i f i e d the n e u t r a l p o r t i o n of the e x t r a c e l l u l a r p o l y s a c c h a r i d e . I t was a n e u t r a l glucan, l i n e a r i n s t r u c t u r e , composed of 200«C-D-glucopyranose u n i t s linkedoCl-6 and «Cl-4 i n the r a t i o of 1:2 r e s p e c t i v e l y . S i m i l a r l y , the e x t r a c e l l u l a r p o l y s a c c h a r i d e produced by Cryptococcus l a u r e n t i i c o n t a i n s an a c i d i c and a n e u t r a l 3 4 f r a c t i o n . Abercrombie et a l , i n 1960 (1), found the a c i d i c f r a c t i o n t o be composed o f D-Mannose, D-Xylose and D- G l u c u r o n i c a c i d . However, s t u d i e s by Jeanes e t a l , i n 1964 (24) i n d i c a t e d the presence of D-Mannose, D-Xylose and D- G l u c u r o n i c a c i d . O - A c e t y l was determined t o be seven p e r c e n t . They a l s o found some t r a c e s o f g a l a c t o s e and g l u c o s e . S t r u c t u r a l s t u d i e s showed t h a t the "backbone" was composed of D-Mannose and the end groups were D-Xylose and D - G l u c u r o n i c a c i d . The n e u t r a l p o r t i o n o f the e x t r a c e l l u l a r p o l y s a c c h a r i d e c o n t a i n e d D-Glucose l i n k e d 1-3, 1-4, 1-2 and/or 1-6 (24, 47) . The biosyn'theses o f the car b o h y d r a t e s found i n the a c i d i c f r a c t i o n were a l s o i n v e s t i g a t e d by Abercrombie e t a l , 1960 ( 2 ) . D-Mannose and D- G l u c u r o n i c a c i d were formed from the hexoses without any a p p r e c i a b l e breakdown of the hexose s k e l e t o n . D-Xylose was formed from the hexoses mainly by a process i n v o l v i n g the l o s s o f carbon s i x ; and D-Xylose and L-Arabinose were both c o n v e r t e d t o D-Mannose, D-Xylose, and D-Gl u c u r o n i c a c i d w i t h rearrangement o f the pentose s k e l e t o n t h a t may have i n v o l v e d the a c t i o n o f t r a n s a l d o l a s e s and t r a n s k e t o l a s e s . The c o n d i t i o n s f o r maximum p r o d u c t i o n o f the e x t r a c e l l u l a r p o l y s a c c h a r i d e s of Cry p t o c o c c u s l a u r e n t i i f o r i n d u s t r i a l purposes were i n v e s t i g a t e d by Cadmus e t a l , 1962 (12) . The l i t e r a t u r e suggests t h a t evidence e x i s t s f o r 5 p o s t u l a t i n g f i r s t , i n g e n e r a l , Cryptococcus-Basidiomycetous r e l a t i o n s h i p s and secondly, more s p e c i f i c a l l y , C r y p t o c o c c u s - T r e m e l l a r e l a t i o n s h i p s . F i r s t , the Cryptococcus-Basidiomyce- tous r e l a t i o n s h i p s were formulated on the b a s i s of DNA base ana l y s e s , S t o r c k , i n 1956 (48), found the percent C+G content i n some Basidiomycetes t o be f i f t y p e r c e n t . He a l s o determined Cryptococcus a l b i d u s G+C p e r c e n t t o be f i f t y - f i v e . Nakase and Komagata, i n 1968 (33), found t h a t f i v e Cryptococcus s p e c i e s out of one hundred and f o r t y y e a s t s t e s t e d had a G+C content of f o r t y - s i x t o f i f t y - s i x p e r c e n t . Second, the C r y p t o c o c c u s - T r e m e l l a r e l a t i o n s h i p s were brought t o l i g h t by s e v e r a l workers (28, 41). S l o d k i e t a l , i n 1966 (41), proposed a p o s s i b l e taxonomic r e l a t i o n s h i p because o f s i m i l a r i t i e s between some members of Cryptococcus and T r e m e l l a s p e c i e s . I t was found t h a t s p e c i e s of T r e m e l l a t h a t produced polymers s i m i l a r t o those produced by Cryptococcus a l s o had s i m i l a r carbon a s s i m i l a t i o n p a t t e r n s . A l l s t r a i n s l i s t e d by S l o d k i e t a l (41) a s s i m i l a t e d glucose, x y l o s e , D-Arabinose, mannitol, a d o n i t o l , and t r e h a l o s e . Both genera have the a b i l i t y t o symthesize s t a r c h at a pH of 5.0 or lower (32, 41). The ready removal by a c i d h y d r o l y s i s of the x y l o s e 6 r e s i d u e s , perhaps owing t o t h e i r apparent p e r i p h e r a l l o c a t i o n r e l a t i v e t o the mannose-glucuronic a c i d "backbone" (1, 24) has l e d t o the p r e p a r a t i o n of a u s e f u l a c c e p t o r f o r the study of enzymatic x y l o s y l a t i o n r e a c t i o n s ( 1 3 ) . Cryptococcus i s a source of enzymes c a t a l y z i n g the b i o s y n t h e s i s of x y l o s y l donor n u c l e o t i d e , UDP-xylose ( 3 ) . E x t r a c t s from Cryptococcus l a u r e n t i i and T r e m e l l a m e s e n t e r i c a are n o n s p e c i f i c w i t h r e s p e c t t o c a t a l y s i s of x y l o s y l t r a n s f e r from UDP-xylose t o p a r t i a l l y d e x y l o s y l a t e d a c c e p t o r polymers b e l o n g i n g t o e i t h e r organism ( 4 1 ) . Under c e r t a i n c o n d i t i o n s b a s i d i o s p o r e s o f some T r e m e l l a l e s bud t o produce y e a s t - l i k e c o l o n i e s i n c u l t u r e t h a t are s i m i l a r t o those o f Cryptococcus ( 2 8 ) . F r a s e r and Jennings p r e s e n t evidence a g a i n s t the C r y p t o c o c c u s - T r e m e l l a r e l a t i o n s h i p s ( 1 9 ) . The n e u t r a l g l u c a n o f T r e m e l l a m e s e n t e r i c a has not been shown t o c o n t a i n theOCl-3 l i n k a g e s t h a t are prese n t i n the Cryptococcus l a u r e n t i i n e u t r a l glucan (1, 4 7 ) . The T r e m e l l a m e s e n t e r i c a n e u t r a l g l u c a n i s s a i d t o resemble s t r u c t u r a l l y the n e u t r a l g l u c a n produced by P u l l u l a r i a s p e c i e s . The reasons f o r the resemblance are thed.1-4, oC 1-6 l i n k e d glucopyranose u n i t s i n both (8, 9, 11, 19, 35, 45, 53, 54) . 7 Some s p e c i e s of Cryptococcus and B u l l e r a c o u l d be t a x o n o m i c a l l y r e l a t e d ( 3 2 ) . They a s s i m i l a t e i n o s i t o l and break the e c - g l u c o s i d i c bonds of m e l i b i o s e , m e l e z i t o s e , and m e t h y l - D - g l u c o s i d e . Among o t h e r s , they a s s i m i l a t e sucrose, l a c t o s e , c e l l o b i o s e , and s e v e r a l p e n t o s e s . S t a r c h s y n t h e s i s i s p r e s e n t and pseudomycelium i s absent. Some s t r a i n s of B u l l e r a t h a t have l o s t t h e i r a b i l i t y t o produce and d i s c h a r g e b a l l i s t o s p o r e s might be c l a s s e d with the genus Cryptococcus (32) . The e x t r a c e l l u l a r p o l y s a c c h a r i d e s of Rho d o t o r u l a g l u t i n i s , Sporobolomyces s i n g u l a r i s , and Sporobolomyces s p e c i e s have a l s o been i n v e s t i g a t e d . G o r i n e t a l ( 2 0 ) , d e s c r i b e d the e x t r a c e l l u l a r p o l y s a c c h a r i d e of Rh o d o t o r u l a g l u t i n i s as a s t r a i g h t c h a i n mannan, composed of at l e a s t n i n e t y u n i t s of a l t e r n a t e l y l i n k e d 1-3, {$ 1-4 D-mannopy- ranose r e s i d u e s . They a l s o r e p o r t e d a hexose and a methyl pentose among h y d r o l y s i s p r o d u c t s . S l o d k i , i n 1966 (43), r e p o r t e d the e x t r a c e l l u l a r p o l y s a c c h a r i d e of two u n i d e n t i f i e d Sporobolomyces s p e c i e s t o be a phosph o r y l a t e d g a l a c t a n of eCl-3, o< 1-6 l i n k e d u n i t s i n approximately equal p r o p o r t i o n s . D-Galactose and 8 D-Galactose-6-phosphate were the o n l y components found on h y d r o l y s i s . G o r i n e t a l (21) s t a t e d t h a t Sporobolomyces s i n g u l a r i s produced a t r i s a c c h a r i d e , g a l a c t o s y l - l a c t o s e and a t e t r a - s a c c a r i d e , g a l a c t o b i o s y l l a c t o s e from l a c t o s e . The D- G a l a c t o p y r a n o s y l u n i t s were/?l-4 l i n k e d . P h a f f and Spencer, i n 1969 (36) r e p o r t e d an e x t r a c e l l u l a r mannan from S p o r o b o l o - myces ros e u s and Sporobolomyces s i n g u l a r i s t h a t was s i m i l a r t o the e x t r a c e l l u l a r mannan r e p o r t e d by G o r i n e t a l (20) t o be produced by Rh o d o t o r u l a g l u t i n i s . S e v e r a l c h a r a c t e r i s t i c s o f R h o d o t o r u l a and Sporobolomyces s p e c i e s s e t them a p a r t from the m a j o r i t y of y e a s t s . A n t i g e n i c a n a l y s e s by T s u c h i y a e t a l (51) i n d i c a t e d t h a t s p e c i e s of R h o d o t o r u l a and Sporobolomyces had no common a n t i g e n . A l s o f i v e groups were s y n t h e s i z e d t h a t d i d not g i v e c r o s s r e a c t i o n s w i t h each o t h e r . R h o d o t o r u l a and Sporobolomyces s p e c i e s were not c o n t a i n e d i n the group t h a t c o n s i s t e d of the m a j o r i t y of y e a s t s . The c e l l w a l l s of Sporobolomyces and Rhodoto r u l a s p e c i e s seem t o be s i m i l a r i n composition y e t d i f f e r e n t from most other y e a s t s . They are s a i d t o have a low gl u c o s e content, (or l a c k i t completely) and a h i g h c h i t i n c o n t e n t . A mannan t h a t i s capable o f g i v i n g a p r e c i p i t a t e with F e h l i n g ' s s o l u t i o n i s absent (15, 30, 46) . 9 The r e l a t i o n s h i p s of Rhodotorula and Sporobolomyces s p e c i e s t o the Basidiomycetes has been put forward by s e v e r a l workers. S t o r c k , i n 1966 (48), working on DNA base analyses found a d u a l i t y i n both the Sporobolomyces and the Rhodotorula s p e c i e s . T h i s d u a l i t y was r e f l e c t e d i n the a n t i g e n i c work of T s u c h i y a mentioned p r e v i o u s l y ( 5 1 ) . Sporobolomyces roseus was found t o have a C+G r a t i o o f f i f t y p e r c e n t and Sporobolomyces s a l m o n i c o l o r and R h o d o t o r u l a m u c i l a g i n o s a both had a C+G r a t i o of s i x t y - f i v e p e r c e n t . A l l t h r e e of these C+G r e a d i n g s are w i t h i n the range expressed f o r B a s i d i o m y c e t e s . Nakase and Komagata, i n 1968 (33), found t h a t s p e c i e s w i t h s t r o n g urease a c t i v i t y had a h i g h G+C content, i m p l y i n g a Basidiomycetous r e l a t i o n s h i p . In g e n e r a l , e l e v e n R h o d o t o r u l a s p e c i e s out of one hundred f o r t y s p e c i e s o f y e a s t s t e s t e d had 47.5 t o 65 p e r c e n t G+C c o n t e n t . F u r t h e r i n f o r m a t i o n on the r e l a t i o n s h i p of Rhodotorula and Sporobolomyces s p e c i e s t o the Basidiomycetes i s concerned w i t h t h e i r l i f e c y c l e s . Rhodotorula g l u t i n i s was demonstrated t o be the i m p e r f e c t stage of the U s t i l a g e n a c e o u s Rhodosporidium t o r u l o i d e s by Banno, i n 1967 ( 4 ) . Newell and F e l l , i n 1970, (34), found t h a t the h a p l o i d mating type s t r a i n s o f Rhodosporidium sphaerocarpum were i d e n t i c a l w i t h s t r a i n s o f 10 Rhodotorula g l u t i n i s . Lodder and Kreger van R i j (31) and Lodder et a l (14) suggested that the genus Rhodotorula i s an imperfect or degenerate Basidiomycete. They stated that some of the species of the genus Rhodotorula, were to be considered asporogenous Sporobolomyces species, since Sporobolomyces without b a l l i s t o s p o r e production would be indistinguishable from Rhodotorula species. Kluyver and van N i e l (27) f i r s t r a i s e d the p o s s i b i l i t y that the genus Sporobolomyces might be of Basidiomycetous o r i g i n . This was because the ejaculation mechanism of the b a l l i s t o s p o r e s of Sporobolomyces i s the same as the ejac u l a t i o n mechanism of basidiospores. van der Walt and Pitout (52) submitted evidence based on the DNA analysis for the existence of 2N (diploid) and N (haploid) generations of Sporobolomyces salmonicolor. However, because the ba l l i s t o s p o r e s of d i p l o i d colonies produce only a diplophase and the b a l l i s t o s p o r e s of haploid colonies produce only a haplophase, the p o s s i b i l i t y of the balli s t o s p o r e s being basidiospores (meiospores) as suggested by S a i n c l i v i e r (39, 40) i s ruled out. Banno, 1967 (4), found no conjugation between Rhodotorula and Sporobolomyces stra i n s investigated, and he therefore stated that there was no possible r e l a t i o n s h i p between b a l l i s t o s p o r e production by 11 Sporobolomyces and the sexual c y c l e concerned w i t h R h o d o t o r u l a . Group I Subgroup II The e x t r a c e l l u l a r p o l y s a c c h a r i d e of U s t i l a g o h o r d e i has not been i n v e s t i g a t e d . Group I I M o r p h o l o g i c a l l y , c u l t u r e s of the y e a s t stage of T a p h r i n a p o p u l i n a were s a i d t o resemble Cryptococcus and R h o d o t o r u l a (32). However, T a p h r i n a does not a s s i m i l a t e i n o s i t o l , t h e r e f o r e d i s t i n g u i s h i n g i t from C r y p t o c o c c u s ; i t does produce s t a r c h which d i s t i n g u i s h e s i t from R h o d o t o r u l a . Wickerham, i n 1952 (55) suggested a r e l a t i o n s h i p between Lipomyces and T a p h r i n a based on s t a r c h p r o d u c t i o n and m u l t i s p o r e d a s c i found i n both genera. However, Kramer, i n 1960 (29) found t h a t the ascus development i n T a p h r i n a i s a l t o g e t h e r d i f f e r e n t from Lipomyces. T h i s f a c t would make any r e l a t i o n s h i p between T a p h r i n a and Lipomyces d o u b t f u l . MATERIALS AND METHODS For convenience, M a t e r i a l s and Methods are d i s c u s s e d i n t h r e e s e c t i o n s . S e c t i o n A covers maintenance o f c u l t u r e s f o r p o l y s a c c h a r i d e p r o d u c t i o n , S e c t i o n B d e a l s w i t h the i s o l a t i o n o f the e x t r a c e l l u l a r p o l y s a c c h a r i d e and S e c t i o n C d e a l s w i t h the a n a l y s i s of the crude e x t r a c e l l u l a r polysaccharide« Fungi were o b t a i n e d from the Mycology L a b o r a t o r y , Department of Botany, U n i v e r s i t y of B r i t i s h Columbia, Vancouver, B . C . 12 T a b l e I 13 Fungus Host L o c a t i o n C o l l e c t i o n C o l l e c t o r Date Cryptococcus l a u r e n t i i NRRLYB-4920 (U.B.C.#8114) B u l l e r a a l b a Goldstream 18/4/60 R.J.Bandoni (Hanna) Derx. P r o v i n c i a l U.B.C. #983 Park, B. C. Sporobolomyces s o i l Harrapa, 2/69 M.Rafiq odorus West U.B.C. #949 P a k i s t a n Sporobolomyces Smut N i c e , 1930 H.G.Derx odorus. Derx. i n f e c t e d France U.B.C. #981 C i t r u s l e a v e s Sporobolomyces i n s e c t Oregon, 1962 s i n g u l a r i s f r a s s U.S.A. P h a f f e t Do from Carmo-Sousa dead U.B.C. #8018 Tsuqa. R h o d o t o r u l a g l u t i n i s U.B.C. #940 (I.F.O.#0559) T r e m e l l a m e s e n t e r i c a R.J.B.#2259-6 on f a l l e n A l nus U.B .C. Endowment Lands 14/10/61 R.J.Bandoni U s t i l a g o h o r d e i U.B.C. #570 (C .Person E3(-)) Hordeum 1964 P .L .Thomas T a p h r i n a p o p u l i n a U.B.C. #249 Poplus l e a f U.B.C. 7/68 S.Reid Endowment Lands 14 Figure 1 Procedure for the production, i s o l a t i o n , and analysis of the e x t r a c e l l u l a r polysaccharide. SLANTS PLATES MYP MYP 20OC 25°C±2°C FLASKS CH5J& 125 ML., 250 ML., 7-14 DAYS, 2 5 0 C ± 2 0 C , SHAKE **CENTRIFUCED l̂ .OOOxG., 30 MIN., 0°C. CELLS DISCARDED CELL-FREE MEDIUM RETAINED E CONCENTRATED CELL-FREE MEDIUM ON FLASH EVAPORATOR. E ETHANOL-POTASSIUM ACETATE PRECIPITATION. K ETHANOL SOLUBLE IMPURITIES DISCARDED. WHITE PRECIPITATE RETAINED. E FREEZE-DRIED. E ACID HYDROLYSIS, IN H2S04, 2k HOURS, 9 0 O C , IN SEALED GLASS PYREX TUBES. I BARIUM CARBONATE NEUTRALIZATION. Bag0u RESIDUE DISCARDED, IR-120 DEI0NI2ATI0N. £ NEUTRALIZED, DEIONIZED, HYDROLIZED SOLUTION CONCENTRATED ON THE FLASH EVAPORATOR. CONCENTRATED TO A SYRUP I E CONCENTRATED TO DRYNESS PAPER CHROMATOGRAPHY ETHYL ACETATE:PYRIDINE:WATER (8:2:2) PREPARED TMS DERIVATIVES #10 MG. SAMPLE, 1 ML. PYRIDINE, .5 ML. HMDS, .25 ML. TMS. E GAS CHROMATOGRAPHY S e c t i o n A Maintenance of C u l t u r e s f o r P o l y s a c c h a r i d e P r o d u c t i o n Fungi were maintained on MYP medium i n c u l t u r e tubes at 20°C. The f u n g i were t r a n s f e r r e d with a s m a l l s t e r i l e (flamed and cooled) wire loop from the MYP tube c u l t u r e s t o MYP p l a t e s . Contaminants were r e v e a l e d by m i c r o s c o p i c examination of the c u l t u r e s . T e t r a c y c l i n e was used t o combat b a c t e r i a . MYP Medium used f o r tubes and p l a t e s . 1. I n g r e d i e n t s : Malt E x t r a c t 15 grams Bacto M a l t E x t r a c t , D i f c o L a b o r a t o r i e s Yeast E x t r a c t 0.5 grams Bacto Yeast E x t r a c t , D i f c o L a b o r a t o r i e s Soytone or Peptone 2.5 grams Bacto Peptone, Bacto Soytone, D i f c o L a b o r a t o r i e s D i s t i l l e d water 1,000 m i l l i l i t r e s Agar 15 grams T e t r a c y c l i n e 8 m l . / l i t r e N u t r i t i o n a l of medium B i o c h e m i c a l as r e q u i r e d . 2. P r e p a r a t i o n of the Medium P l a t e s : The medium was a u t o c l a v e d f o r twenty minutes a t 15 p s i . , c o o l e d , poured, s o l i d i f i e d . P l a t e s were i n o c u l a t e d as d e s c r i b e d above, and unused p l a t e s were s t o r e d i n the r e f r i g e r a t o r . Tubes: S l a n t s were prepared by f i l l i n g tubes with the r e q u i r e d amount of molten agar. The agar was allowed t o s o l i d i f y with the tube r e s t i n g a t an a n g l e . Unused s l a n t s were s t o r e d i n the r e f r i g e r a t o r . The tubes were i n o c u l a t e d as d e s c r i b e d above. 3. B u i l d up of inoculum Pure c u l t u r e s on MYP agar were used t o b u i l d up a l a r g e amount of inoculum. C u l t u r e s were cut lengthwise or scraped from the agar, and t r a n s f e r r e d t o l i q u i d C a s e i n H y d r o l y s a t e 5% Glucose Medium (CH5%G). L i q u i d CH5%G was i n c r e a s e d g r a d u a l l y as the f u n g a l inoculum b u i l t up. L i q u i d c u l t u r e s were shaken r e c i p r o c a l l y at 25°C +_ 2°C f o r seven to f o u r t e e n days. 4. C a s e i n H y d r o l y s a t e 5% Glucose Medium (1, 19) ( f o r t e n 2 , 8 0 0 ml. f l a s k s ) I n g r e d i e n t s Medium I D i s t i l l e d water 4 l i t r e s C a s e i n H y d r o l y s a t e 15 grams - v i t a m i n Free, S a l t Free, N u t r i t i o n a l B i o c h e m i c a l C o r p o r a t i o n , C l e v e l a n d , Ohio, U.S.A. 17 KN03 KH2PO4 K2HPO4 MgS04 . 7H20 Thiamine H y d r o c h l o r i d e Trace Elements S o l u t i o n 5 grams 20 grams 15 grams 2.5 grams 5 m i l l i g r a m s 25 m i l l i l i t r e s I n g r e d i e n t s Trace Elements S o l u t i o n ZnSO^ . 7H2O CuS0 4 . 5H20 MnSQ 4 . H 20 FeSO, 7H 20 D i s t i l l e d Water I n g r e d i e n t s Medium I I 2.0 grams 0.1 grams 1.5 grams 2 .0 grams 1 l i t r e D i s t i l l e d water 1 l i t r e G lucose 250 grams T e t r a c y c l i n e 8 ml. / l i t r e as nece s s a r y ^ 5. P r e p a r a t i o n and I n o c u l a t i o n o f CH5%G Medium Medium I was d i v i d e d among ten c o n i c a l 2,800 ml. f l a s k s . F l a s k tops were covered w i t h f o i l . Media I and I I were a u t o c l a v e d f o r twenty minutes a t 15 p s i . Under s t e r i l e c o n d i t i o n s , 100 ml. a l i q u o t s of Medium I I were added t o each o f the t e n f l a s k s . Each f l a s k was i n o c u l a t e d w i t h a suspension of yeast and s t e r i l e d i s t i l l e d water. L i q u i d c u l t u r e s were shaken on a r e c i p r o c a l shaker at 2 5°C + 2°C f o r seven t o f o u r t e e n days. C a s e i n H y d r o l y s a t e 5% Glucose Medium was chosen because i t was used f o r polymer p r o d u c t i o n by both Cryptococcus l a u r e n t i i (1), and T r e m e l l a m e s e n t e r i c a (19) and because i t was more c h e m i c a l l y d e f i n e d than e i t h e r MY Broth with 5% Glucose (22, 41, 44) or the A u t o l y z e d Brewer's Yeast Medium with 5% Glucose (12, 24) . 19 S e c t i o n B E x t r a c t i o n and I s o l a t i o n of the E x t r a c e l l u l a r P o l y s a c c h a r i d e s 1. C e n t r i f u g a t i o n of the l i q u i d c u l t u r e and recovery of the c e l l f r e e medium. A f t e r two weeks growth, the l i q u i d c u l t u r e was c e n t r i f u g e d at 14,000 g., f o r t h i r t y minutes, at zero degrees c e n t i g r a d e . The c e l l f r e e medium was r e t a i n e d and the c e l l s were d i s c a r d e d . Samples of the c e l l f r e e medium were checked m i c r o s c o p i c a l l y t o make sure t h a t a l l the c e l l s had been removed during c e n t r i f u g a t i o n . 2. Ethanol-Potassium Acetate p r e c i p i t a t i o n of the crude . e x t r a c e l l u l a r p o l y s a c c h a r i d e from the c e l l f r e e medium. A sm a l l sample of the c e l l f r e e medium was concentrated to about one-half i t s o r i g i n a l volume of the f l a s h evaporator. A measured amount of the concentrated medium was added slowly, w i t h shaking, to a known q u a n t i t y of c o l d e t h a n o l . Small amounts of potassium acetate were added t o f a c i l i t a t e the p r e c i p i t a t i o n of the p o l y s a c c h a r i d e . When a white p r e c e i p i t a t i o n appeared the q u a n t i t y of concentrated medium t h a t had been added t o the known q u a n t i t y of ethanol was recorded. The p r e c i p i t a t i o n was then c a r r i e d out on a lar g e s c a l e . Dry i c e was added t o the ethanol t o a g i t a t e and keep i t c o l d d u r i n g the p r e c i p i t a t i o n . The f i b r o u s white p r e c i p i t a t e was r e t a i n e d by c e n t r i f u g i n g or d e c a n t i n g o f f the e t h a n o l and e t h a n o l s o l u b l e i m p u r i t i e s . The white p r e c i p i t a t e was d i s s o l v e d i n a s m a l l amount of d i s t i l l e d water and the r e s u l t i n g aqueous s o l u t i o n was added t o e t h a n o l as b e f o r e . The white p r e c i p i t a t e was c e n t r i f u g e d down, r e t a i n e d , then d i s s o l v e d i n a s m a l l amount of d i s t i l l e d water. The crude e x t r a c e l l u l a r p o l y s a c c h a r i d e was poured i n t o s e v e r a l round bottom f l a s k s and f r e e z e - d r i e d o v e r n i g h t . The crude, f l u f f y , white, f r e e z e - d r i e d product was weighed and b o t t l e d . P a r a f i l m was used t o keep moisture out o f the b o t t l e s . 3 . H y d r o l y s i s of the crude e x t r a c e l l u l a r p o l y s a c c h a r i d e . A p o r t i o n o f the f r e e z e - d r i e d crude e x t r a c e l l u l a r p o l y s a c c h a r i d e was h y d r o l y z e d w i t h IN H 2 S 0 4 f o r twenty-four hours a t 100°C i n s e a l e d g l a s s Pyrex t u b e s . 4. N e u t r a l i z a t i o n with Barium Carbonate and d e i o n i z a t i o r f w i t h IR-120 c a t i o n exchange r e s i n . (a) N e u t r a l i z a t i o n with BaCO-3 A s l u r r y of barium carbonate and d i s t i l l e d water was prepared i n a beaker i n the fume hood. The s l u r r y was added, a s m a l l amount a t a time, t o a f l a s k c o n t a i n i n g the 21 s o l u t i o n of a c i d h y d r o l y z e d m a t e r i a l . The f l a s k was s w i r l e d v i g o r o u s l y and n e u t r a l i z a t i o n was determined w i t h l i t m u s paper. The r e s u l t i n g mixture was f i l t e r e d through a f u n n e l ; the c l e a r e d , n e u t r a l i z e d s o l u t i o n was r e t a i n e d and the Baso^ r e s i d u e was d i s c a r d e d . (b) P r e p a r a t i o n of the IR-120 c a t i o n exchange r e s i n column Regeneration of IR-120 c a t i o n exchange r e s i n . The r e s i n was p l a c e d i n a beaker a t l e a s t four times the volume of the r e s i n . The r e s i n was washed s e v e r a l times with water, d e c a n t i n g a l l the water o f f each t i m e . Enough 2N NaOH was added t o the r e s i n so t h a t a b a s i c r e a c t i o n on l i t m u s paper was o b t a i n e d a f t e r the r e s i n and 2N NaOH had been w e l l s t i r r e d and l e f t f o r at l e a s t t e n minutes. The r e s i n was washed co m p l e t e l y f r e e of NaOH. 2N HC1 was added t o the r e s i n u n t i l a v e r y p o s i t i v e a c i d pH was r e a c h e d . The r e s i n and the 2N HC1 were s t i r r e d v e r y w e l l . The r e s i n was then washed r e l a t i v e l y f r e e o f a c i d . The a c i d treatment was r e p e a t e d and the r e s i n was then washed s e v e r a l times with water. The washed r e s i n was t r a n s f e r r e d t o a g l a s s column and washed w i t h water u n t i l a n e g a t i v e c h l o r i d e t e s t was o b t a i n e d ( 1 6 ) . P r e p a r a t i o n of the IR-120 column f o r d e i o n i z a t i o n of the BaC0 3 n e u t r a l i z e d p o l y s a c c h a r i d e A g l a s s column of s l u r r i e d IR-120 was prepared as i n F i g u r e 2. A p l u g of g l a s s wool was i n s e r t e d using a g l a s s rod at the bottom of the column. The r e q u i r e d amount of IR-120 c a t i o n exchange r e s i n was s l u r r i e d w i t h d i s t i l l e d water and poured i n t o the column along a g l a s s r o d . The rod was removed and the r e s i n allowed t o s e t t l e . Another s m a l l g l a s s wool p l u g was i n s e r t e d a t the top of the column of IR-120. A p i e c e of rubber t u b i n g and a screw clamp were att a c h e d to the bottom of the g l a s s column. The column was washed s e v e r a l times with d i s t i l l e d water, then the n e u t r a l i z e d s o l u t i o n of h y d r o l y s i s products was poured i n t o the column. T h i s m a t e r i a l was washed through w i t h d i s t i l l e d water t i l l t he M o l i s c h t e s t f o r carbohydrate m a t e r i a l was n e g a t i v e . An Erlenmeyer f l a s k was used t o c o l l e c t the d e i o n i z e d s o l u t i o n o f n e u t r a l i z e d h y d r o l y s i s p r o d u c t s . The M o l i s c h t e s t f o r Carbohydrates (18, 26, 56) In g e n e r a l , the M o l i s c h t e s t i s the a c t i o n of s t r o n g s u l p h u r i c a c i d on sugars and subsequent r e a c t i o n of the products formed with p h e n o l i c s u b s t a n c e s . S p e c i f i c a l l y , i n t h i s t e s t , i f performed i n a t e s t tube: t o approximately 0.05 grams of carbohydrate i n 1 ml. of water was added one t o two drops of f i f t e e n p e r c e n t a l c o h o l s o l u t i o n of n a p h t h o l . Concentrated s u l p h u r i c a c i d was added by p o u r i n g i t s l o w l y down the s i d e of the t e s t tube t o form a l a y e r under the sugar 23 F i g u r e 2 Apparatus f o r the IR-120 c a t i o n exchange r e s i n . ring stand with screw clamp glass column glass wool plug •IR-120 cation exchange resin glass wool plug Erlenmeyer flask —deionized solution 24 s o l u t i o n . In the presence of carbohydrate m a t e r i a l a v i o l e t c o l o r appeared a t the i n t e r f a c e of the two l i q u i d s because o f the f o r m a t i o n o f f u r f u r a l d e r i v a t i v e s and t h e i r r e a c t i o n with naphthol ( 1 8 ) . I t i s b e l i e v e d (26) t h a t the mechanism o f the r e a c t i o n proceeds under the i n f l u e n c e o f 15M s u l p h u r i c a c i d from the glucopyranose form t o the s t r a i g h t c h a i n aldehyde form with subsequent d e h y d r a t i o n and r i n g c l o s u r e t o 5 - h y d r o x y m e t h y l f u r f u r a l . T h i s i s f o l l o w e d by the h y d r o l y t i c s c i s s i o n o f the 5- h y d r o x y l m e t h y l group w i t h the p r o d u c t i o n of formaldehyde and f u r f u r a l , f u r f u r a l d e g r a d a t i o n products, or f u r f u r a l polymers. Important P r o p e r t i e s of A m b e r l i t e IR-120. The c a t i o n exchange r e s i n i s s t r o n g l y a c i d i c , s u l f o n a t e d , p o l y s t y r e n e type of medium p o r o s i t y . The apparent d e n s i t y (average) i s 0.77g/ml.; the mesh s i z e (wet) i s 20-50 mesh; the v o i d volume, 35-40%; degree of r e g e n e r - a t i o n i s 98% (minimum). Mo i s t u r e h o l d i n g c a p a c i t y i s 49-55% and the t o t a l exchange c a p a c i t y i s , by volume, 1.75 meq./ml. min. and by weight ( d r y ) , 5.0 meq./g.min. c 25 S e c t i o n C Q u a l i t a t i v e A n a l y s i s of the Crude E x t r a c e l l u l a r P o l y s a c c h a r i d e H y d r o l y s i s Products 1. Gas Chromatography 2 . Paper Chromatography 1. Gas Chromatography P r e p a r a t i o n of the t r i m e t h y l s i l y l d e r i v a t i v e s of the n e u t r a l i z e d , d e i o n i z e d h y d r o l y s i s p r o d u c t s . A p o r t i o n of the n e u t r a l i z e d , d e i o n i z e d , h y d r o l y z e d e x t r a c e l l u l a r p o l y s a c c h a r i d e was evaporated t o dryness i n a s m a l l round bottom f l a s k on the f l a s h e v a p o r a t o r . The p r e - p a r a t i o n of the TMS d e r i v a t i v e s was c a r r i e d out i n the fume hood. To approximately 10 mg. of sample was added 1 ml. o f anhydrous p y r i d i n e (kept over KOH p e l l e t s ) . To the s o l u t i o n or suspension of m a t e r i a l i n p y r i d i n e was added 0.5 m l . HMDS ( h e x a m e t h y l d i s i l a z a n e ) , and 0.25 m l . of TMS ( t r i m e t h y l - c h l o r o s i l a n e ) . The s o l u t i o n became clou d y on the a d d i t i o n of TMS, presumably because o f the p r e c i p i t a t e ammonium c h l o r i d e . The f l a s k was stoppered immediately w i t h a ground g l a s s stopper or a covered cork and shaken v i g o r o u s l y f o r about t h i r t y seconds, then allowed to stand f o r 5 minutes at room temperature. Formation of the TMS d e r i v a t i v e s o c c u r r e d r a p i d l y at room temperature. A l l f r e e h y d r o x y l groups were s i l y l a t e d and the y i e l d of the TMS d e r i v a t i v e s was q u a n t i t a t i v e ( 4 9 ) . In g e n e r a l , the s i l y l a t i o n r e a c t i o n as presented by H e n g l e i n and S c h e i n o s t (23) i s : v » v I - S i - CI + HO-C- + Base—» - Si-O-C- + Base * HC1 / T * | The s i l y l a t e d m a t e r i a l was i n j e c t e d w i t h a g l a s s s y r i n g e i n t o the i n j e c t o r p o r t of the gas chromatography machine. Chromatography was c a r r i e d out on an F and M 720 d u a l column in s t r u m e n t . The two columns were 8 f t . x 0.25 i n . c o i l e d copper columns packed with e q u a l weights ( t o w i t h i n 20 mg.) of 20% SF 96 on 60-80 mesh D i a t o p o r t S. The columns were h e l d i s o t h e r m a l l y a t 190° f o r approximately t h r e e minutes and then programmed a t 2° per minute t o h o l d a t 220° . Helium flow was approximately 88 ml. per minute (6.8 seconds f o r 10 ml.) . 2. Paper Chromatography A p o r t i o n of the h y d r o l y z e d , n e u t r a l i z e d and d e i o n i z e d p o l y s a c c h a r i d e was f l a s h evaporated t o a s y r u p . Small amounts of the d i l u t e d (H2O) syrup were a p p l i e d w i t h a flamed, c o o l e d , wire loop t o 24 i n . by 7.4 i n . Whatman #1 Chromatography Paper. At the same time, spots were a l s o made of known monosaccharides. Descending paper chromatography was c a r r i e d out u s i n g the s o l v e n t s e t h y l a c e t a t e : p y r i d i n e : water i n the r a t i o s o f 8:2:2, i n an e q u i l i b r a t e d g l a s s chromatography tank f o r approximately 48 h o u r s . The chromatograms were removed from the tank, d r i e d , then developed w i t h AgNC>3 d i p (50) . P r e p a r a t i o n of the AgNC-3 d i p The p r i n c i p l e behind the AgN03 d i p pro c e s s i s based on the T o l l e n ' s s i l v e r m i r r o r t e s t f o r c a r b o h y d r a t e s . The s i l v e r i s reduced by aldehydes and sugars c o n t a i n i n g f r e e aldehyde or ketone groups ( 8 ) . I n g r e d i e n t s (50) 1. Acetone-AgN03 S o l u t i o n T h i s reagent s o l u t i o n was prepared by d i l u t i n g 0.1 ml. of s a t u r a t e d aqueous s i l v e r n i t r a t e s o l u t i o n t o 20 ml. with acetone, and adding water dropwise, w i t h shaking, u n t i l the s i l v e r n i t r a t e which s e p a r a t e s on the a d d i t i o n o f acetone has r e d i s s o l v e d . Spreading o f the spots i s l i m i t e d because of the s p a r i n g s o l u b i l i t y o f sugars i n acetone. (0.014% at 23°C f o r c r y s t a l l i n e glucose.) 2. E t h a n o l - NaOH S o l u t i o n The 0.5N s o l u t i o n o f NaOH i n aqueous e t h a n o l was made by d i l u t i n g s a t u r a t e d aqueous NaOH s o l u t i o n with e t h a n o l . 28 3 . 6N Ammonium hydr o x i d e Procedure (50) The d r i e d paper chromatogram s t r i p was passed r a p i d l y through the AgN03-acetone reagent s o l u t i o n twice, d r y i n g a f t e r each run through. The d r y paper was then passed once through the ethanol-NaOH s o l u t i o n . Brown s i l v e r o xide was immediately produced. Reducing sugars formed dense b l a c k spots of s i l v e r at room temperature. When r e d u c t i o n was judged complete, excess s i l v e r oxide was d i s s o l v e d by immersion of the s t r i p i n 6N ammonium hydroxide f o r a few minutes, a f t e r which i t was washed with water and d r i e d . The spots c o u l d have been rendered j e t b l a c k by momentary exposure t o H2S. The i d e n t i t y of the spots was determined by comparing t h e p o s i t i o n s of the unknown spots w i t h those of the s t a n d a r d s . T h i s method was more a c c u r a t e than c a l c u l a t i n g the R F v a l u e s . S i n c e some of the chromatograms were run f o r 48 hours and the s o l v e n t f r o n t had run o f f the paper, i t was c o n s i d e r e d u n r e l i a b l e t o c a l c u l a t e the r a t i o of the movement of a spot t o the movement o f the s o l v e n t f r o n t . 29 RESULTS The r e s u l t s are presented i n two s e c t i o n s . F i r s t , those o b t a i n e d from paper chromatography. Second, those o b t a i n e d from gas chromatography. The q u a l i t a t i v e r e s u l t s o b t a i n e d from paper chromatography are shown i n T a b l e I I . F i g u r e s 3, 4,and 5 are r e p r e s e n t a t i o n s of the a c t u a l paper chromatograms. The t e n t a t i v e q u a l i t a t i v e r e s u l t s f o r gas chromatography are shown i n T a b l e I I I . F i g u r e s 6-14 are r e p r e s e n t a t i o n s of the a c t u a l gas chromatograms. Because t h e r e s u l t s o b t a i n e d from paper chromatography were s u f f i c i e n t f o r q u a l i t a t i v e d e t e r m i n a t i o n s of the monosaccharides i n t h e e x t r a c e l l u l a r p o l y s a c c h a r i d e h y d r o l y s a t e s , not much emphasis was p l a c e d on the gas chromatography. Only p r e - l i m i n a r y gas chromatography experiments were c a r r i e d o u t . The i d e n t i t i e s of the peaks were determined t e n t a t i v e l y by f i r s t , assuming t h a t the peaks r e p r e s e n t e d the monosaccharides t h a t were shown t o be p r e s e n t by the paper chromatographic examinations of the h y d r o l y s a t e s . Secondly, the i n d i v i d u a l peak r e t e n t i o n times were compared with the r e t e n t i o n times o f known monosaccharides chromatographed under s i m i l a r c o n d i t i o n s (17, 37, 4 9 ) . However, more p o s i t i v e i d e n t i f i - c a t i o n c o u l d have been o b t a i n e d by experiments u s i n g the 30 peak enhancement t e c h n i q u e . T h i s i n v o l v e s adding a known monosaccharide to the h y d r o l y s a t e and then o b s e r v i n g which peaks are enhanced on the gas chromatograms. The peaks enhanced would most l i k e l y be those of the known mono- s a c c h a r i d e t h a t was added. T h i s process would be c a r r i e d out f o r a l l the monosaccharides thought t o be presen t i n t h e h y d r o l y s a t e . The gas and paper chromatography r e s u l t s appear t o be the same. But, u n t i l t h i s s y s t e m a t i c k i n d o f i d e n t i f i c a t i o n i n v o l v i n g peak enhancement i s c a r r i e d out the gas chromatography r e s u l t s a re i n c o n c l u s i v e . However, the gas chromatograms should remain i n the t h e s i s . Re- se a r c h e r s doing q u a n t i t a t i v e gas chromatography on the e x t r a c e l l u l a r p o l y s a c c h a r i d e s produced by some y e a s t s and y e a s t - l i k e f u n g i may want t o r e f e r t o and i n t e r p r e t f u r t h e r some of the gas chromatograms. (See Appendix page 62.) Table II PAPER CHROMATOGRAPHY RESULTS 31 Gr oup Fungus Monosaccharides i n the Ex t r a - c e l l u l a r P o l y s a c c h a r i d e H y d r o l y s i s Products Group I Sub- group I Cryptococcus l a u r e n t i i NRRL YB-4920 (UBC #8114) G a l a c t o s e Glucose Mannose X y l o s e T r e m e l l a m e s e n t e r i c a RJB #2259-6 B u l l e r a + + + + a l b a (Hanna) Derx UBC #983 Sporobolomyces + + + + odorus UBC #949 Sporobolomyces + + + + odorus UBC #981 Rhod o t o r u l a + + + + g l u t i n i s UBC #940 S por ob o 1 omy ce s + + + + s i n g u l a r i s UBC #8018 Group I U s t i l a g o h o r d e i + + + Sub UBC #570 group I I (C.Person E3(-)) Group I I T a p h r i n a p o p u l i n a UBC #249 + + F i g u r e 3 T r e m e l l a m e s e n t e r i c a and B u l l e r a a l b a . S e p a r a t i o n o f products u s i n g descending paper chromatography and s o l v e n t s e t h y l a c e t a t e : p y r i d i n e : water, (8:2:2). Tremella mesenterica Xylose in a a: s z Arabinose X Mannose Glucose Galactose * OO CO Bullera alba Q a: < a z Xylose Arabinose Mannose Glucose Galactose C O cO F i g u r e 4 Sporobolomyces s i n g u l a r i s , Sporobolomyces odorus (#949), Sporobolomyces odorus (#981) Cryptococcus l a u r e n t i i . S e p a r a t i o n of products u s i n g descending paper chromatography and s o l v e n t s e t h y l a c e t a t e : p y r i d i n e : water, (8:2:2).  F i g u r e 5 U s t i l a g o h o r d e i , R h o d o t o r u l a g l u t i n i s , Sporobolomyces s i n g u l a r i s , T a p h r i n a p o p u l i n a . S e p a r a t i o n o f products u s i n g descending paper chromatography and s o l v e n t s e t h y l a c e t a t e : p y r i d i n e : water, (8:2:2) . Mannose Glucose Xylose Galactose Arabinose Taphrina populina 249 Ustilaqo hordei 570 Sporobolomyces singuloris o o Xylose Arabinose Mannose Glucose Galactose Rhodotorula glutinis CD 35 Ta b l e I I I Gas Chromatography R e s u l t s Fungus Peak R e t e n t i o n Time (Minutes) T e n t a t i v e I d e n t i f i c a t i o n T r e m e l l a m e s e n t e r i c a 1 2 3 4 5 6 7 8 2.9 S o l v e n t 13.8 X y l o s e 17.5 X y l o s e 19.8 X y l o s e 22.6 G a l a c t o s e , Mannose 25.7 G a l a c t o s e 27.4 G a l a c t o s e , Glucose, Mannose 30.5 Glucose Cryptococcus l a u r e n t i i 1 2 3 4 19.7 X y l o s e 23.0 X y l o s e 26.6 G a l a c t o s e , Mannose 31.8 G a l a c t o s e , Glucose, Mannose 37.0 Glucose T a b l e I I I (Continued) Gas Chromatography R e s u l t s 36 Fungus Peak R e t e n t i o n Time (Minutes) T e n t a t i v e I d e n t i f i c a t i o n B u l l e r a a l b a 1 2 3 4 5 6 7 8 2.9 s o l v e n t 4.5 s o l v e n t 6.2 s o l v e n t 18.0 X y l o s e 20.4 X y l o s e 23.2 G a l a c t o s e , Mannose. 25.9 G a l a c t o s e 28.7 G a l a c t o s e , Glucose, Mannose 33.4 Glucose Sporobolomyces odorus (#949) 1 2 3 4 5 6 7 8 17.4 X y l o s e 19.1 X y l o s e 21.2 X y l o s e 24.0 G a l a c t o s e , Mannose 26.6 G a l a c t o s e , Mannose 29.3 G a l a c t o s e 31.9 G a l a c t o s e , Glucose, Mannose 37.0 Glucose T a b l e I I I (Continued) Gas Chromatography R e s u l t s 37 Fungus Peak R e t e n t i o n Time (Minutes) T e n t a t i v e I d e n t i f i c a t i o n Sporobolomyces odorus (#981) 1 2 3 4 5 6 17.8 19.6 22.0 28.1 31.0 33.8 38.7 X y l o s e X y l o s e X y l o s e G a l a c t o s e , Mannose G a l a c t o s e G a l a c t o s e , Glucose, Mannose Glucose Sporobolomyces s i n g u l a r i s 1 2 3 18.8 X y l o s e 28.2 G a l a c t o s e , Mannose 30.2 G a l a c t o s e , Glucose, Mannose 34.2 Glucose R h o d o t o r u l a g l u t i n i s 1 2 3 4 5 6 3.0 S o l v e n t 15.3 X y l o s e 17.3 X y l o s e 22.2 G a l a c t o s e , Mannose 24.8 G a l a c t o s e 26.4 G a l a c t o s e , Glucose, Mannose 30.4 Glucose Table I I I (Continued) Gas Chromatography Results 38 Fungus Peak Retention Time (Minutes) Tentative I d e n t i f i c a t i o n Ustilago hordei 1 2 23.5 25.0 30.2 36.2 Galactose, Mannose Galactose Galactose, Glucose, Mannose Glucose Taphrina populina 1 2 3 4 5 14.7 Mannose 23.5 Mannose 24.3 Glucose 29.0 Glucose 50.3 u n i d e n t i f i e d F i g u r e 6 T r e m e l l a m e s e n t e r i c a . S e p a r a t i o n of p r o d u c t s as t r i m e t h y l s i l y l d e r i v a t i v e s . i DETECTOR RESPONSE F i g u r e 7 Cryptococcus l a u r e n t i i . S e p a r a t i o n o f p roducts as t r i m e t h y l s i l y l derivatives„ DETECTOR RESPONSE F i g u r e 8 B u l l e r a a l b a . S e p a r a t i o n of products as t r i m e t h y l s i l y l derivatives„ DETECTOR RESPONSE F i g u r e 9 Sporobolomyces odorus (#949). S e p a r a t i o n of products as t r i m e t h y l s i l y l derivatives« DETECTOR RESPONSE I f F i g u r e 10 Sporobolomyces odorus (#981). S e p a r a t i o n o f p r o d u c t s as t r i m e t h y l s i l y l d e r i v a t i v e s . DETECTOR RESPONSE 0) CD 00 F i g u r e 11 Sporobolomyces s i n g u l a r i s . S e p a r a t i o n of products as t r i m e t h y l s i l y l d e r i v a t i v e s . Sporobolomyces singularis T^JTQ ftp 1 ' CVJ $> RETENTION TIME (MINUTES) F i g u r e 12 R h o d o t o r u l a g l u t i n i s . S e p a r a t i o n o f products as t r i m e t h y l s i l y l d e r i v a t i v e s DETECTOR RESPONSE F i g u r e 13 U s t i l a g o h o r d e i . S e p a r a t i o n o f p r o d u c t s as t r i m e t h y l s i l y l d e r i v a t i v e s .  F i g u r e 14 T a p h r i n a p o p u l i n a . S e p a r a t i o n of products as t r i m e t h y l s i l y l d e r i v a t i v e s 1 1 1 1 1 1 1 1 . i n ro Q ro Ul ro CD Q St KJ cu co in RETENTION TIME (MINUTES) 48 DISCUSSION The d i s c u s s i o n r e l a t e s my r e s u l t s t o those of p r e v i o u s workers. I t a l s o makes some l o g i c a l d e d u c t i o n s . For convenience, the y e a s t s and y e a s t - l i k e f u n g i i n v e s t i g a t e d were a s s i g n e d t o two groups. Group I was d i v i d e d i n t o two subgroups. Fungi i n Subgroup I were: Cr y p t o c o c c u s l a u r e n t i i T r e m e l l a mesenterica, B u l l e r a a l b a , Sporobolomyces odorus, Sporobolomyces s i n g u l a r i s and R h o d o t o r u l a g l u t i n i s . The monosaccharides g a l a c t o s e , g l u c o s e , mannose and x y l o s e were pre s e n t i n t h e h y d r o l y s a t e s o f the e x t r a c e l l u l a r p o l y - s a c c h a r i d e s produced by the f u n g i i n Subgroup I . Subgroup I I i n c l u d e d U s t i l a g o h o r d e i w i t h g a l a c t o s e , g l u c o s e and mannose p r e s e n t ; x y l o s e a b s e n t . Group I I c o n s i s t e d of T a p h r i n a p o p u l i n a w i t h g l u c o s e and mannose p r e s e n t ; g a l a c t o s e and x y l o s e absent. I n t e r e s t i n g l y , the two groups formed above c o n t a i n some f u n g i t h a t have a l r e a d y been suggested as t a x o n - o m i c a l l y r e l a t e d . A C r y p t o c o c c u s - T r e m e l l a taxonomic r e l a t i o n - s h i p was f i r s t put forward by Kobayashi and Tubaki ( 2 8 ) . Members of the two genera are s i m i l a r i n e x t r a c e l l u l a r p o l y - s a c c h a r i d e p r o d u c t i o n , i s o l a t i o n and a n a l y s i s (1, 19, 24, 4 4 ) . They a l s o have s i m i l a r carbon a s s i m i l a t i o n p a t t e r n s and produce 49 s t a r c h (32, 41). S i m i l a r morphology and enzymatic x y l o s y l - a t i o n r e a c t i o n s are a l s o important (3, 13, 28, 32, 41) . My r e s u l t s present b i o c h e m i c a l i n f o r m a t i o n t h a t may support the r e l a t i o n s h i p a l r e a d y suggested between Cryptococcus l a u r e n t i i and T r e m e l l a m e s e n t e r i c a ( 4 1 ) . I found the monosaccharides g a l a c t o s e , g l u c o s e , mannose and x y l o s e t o be present i n the h y d r o l y s a t e s o f the e x t r a c e l l u l a r p o l y s a c c h a r i d e s produced by both Cryptococcus l a u r e n t i i and T r e m e l l a m e s e n t e r i c a . The presence of g l u c o s e , mannose, and x y l o s e i s i n agreement wi t h the r e s u l t s o f p r e v i o u s workers who found both an a c i d i c xylomannan and a n e u t r a l g l u c a n t o be p r e s e n t i n the e x t r a c e l l u l a r p o l y s a c c h a r i d e h y d r o l y s a t e of both Cryptococcus l a u r e n t i i and T r e m e l l a m e s e n t e r i c a (1, 19, 24, 41, 44) . G a l a c t o s e had been r e p o r t e d o n l y i n t r a c e amounts from Cryptococcus l a u r e n t i i (24) and had not been r e p o r t e d p r e v i o u s l y from T r e m e l l a m e s e n t e r i c a . G a l a c t o s e may have been present as a contaminant from c e l l w a l l or e n d o c e l l u l a r m a t e r i a l from y e a s t c e l l s t h a t might have broken down d u r i n g c u l t u r i n g or c e n t r i f u g a t i o n . Or, p o s s i b l y , g a l a c t o s e i s more t i g h t l y bound to the c e l l w a l l than the other monosaccharides and under c e r t a i n c o n d i t i o n s has not been p r e v i o u s l y i s o l a t e d and a n a l y z e d . Perhaps an e l e c t r o n m i c r o s c o p i s t working on f u n g a l c e l l w a l l s and u s i n g 50 r a d i o a c t i v e t r a c e r s would be a b l e t o r e s o l v e t h i s . Though T r e m e l l a m e s e n t e r i c a has not y e t been shown t o c o n t a i n 1-3 l i n k a g e s i n i t s e x t r a c e l l u l a r p o l y s a c c h a r i d e (19), as has C ryptococcus l a u r e n t i i t h e r e i s s t i l l s u b s t a n t i a l e v i d e n c e t o support a r e l a t i o n s h i p between these two s p e c i e s . Members of the genera B u l l e r a and C ryptococcus have been suggested as being t a x o n o m i c a l l y r e l a t e d on the bases o f s i m i l a r g e n e r a l morphology and l a c k o f pseudomycelium; s t a r c h p r o d u c t i o n and carbon a s s i m i l a t i o n . Both genera a s s i m i l a t e i n o s i t o l and break the o C - g l u c o s i d i c bonds of m e l i b i o s e , m e l e z i t o s e , and methyl«C-D-glucoside . They a l s o both produce s t a r c h and l a c k pseudomycelium. I f B u l l e r a c u l t u r e s had not been known t o produce b a l l i s t o s p o r e s , some of them might have been i n c l u d e d i n t h e genus Cryp t o c o c c u s (32 ) . My f i n d i n g s t h a t the monosaccharides o f B u l l e r a a l b a are q u a l i t a t i v e l y the same as t h o se o f Cryptococcus l a u r e n t i i may g i v e added support t o the B u l l e r a - C ryptococcus r e l a t i o n s h i p s i n g e n e r a l . However, the r e s u l t s p e r t a i n more d i r e c t l y t o g i v i n g b i o c h e m i c a l support t o a B u l l e r a a l b a and C ryptococcus l a u r e n t i i r e l a t i o n s h i p . As shown i n my r e s u l t s , both Sporobo1omyces s i n g u l a r i s and Sporobolomyces odorus produced an e x t r a c e l l u l a r p o l y - s a c c h a r i d e with th e monosaccharides g a l a c t o s e , g l u c o s e , mannose 51 and x y l o s e i n the h y d r o l y s a t e . The presence o f g a l a c t o s e and glucose i n the Sporobolomyces s i n g u l a r i s e x t r a c e l l u l a r p o l y s a c c h a r i d e h y d r o l y s a t e i s i n agreement w i t h the r e s u l t s of G o r i n e t a l ( 2 1 ) . The f i n d i n g of mannose i n the e x t r a - c e l l u l a r p o l y s a c c h a r i d e h y d r o l y s a t e of Sporobolomyces s i n g u l a r i s i s i n agreement wi t h the r e s u l t s of P h a f f and Spencer ( 3 6 ) . X y l o s e had not been r e p o r t e d p r e v i o u s l y t o be p r e s e n t i n the Sporobolomyces s i n g u l a r i s h y d r o l y s a t e . The e x t r a c e l l u l a r p o l y s a c c h a r i d e produced by Sporobolomyces odorus had not been p r e v i o u s l y a n a l y z e d . The monosaccharides t h a t I found t o be pr e s e n t i n the h y d r o l y s a t e of the e x t r a c e l l u l a r p o l y s a c c h a r i d e produced by Rhodotorula g l u t i n i s were g a l a c t o s e , g l u c o s e , mannose and x y l o s e . Mannose may be p a r t o f a mannan s i m i l a r t o those mannans produced by both Rhodotorula g l u t i n i s and Sporo b o l o - myces s i n g u l a r i s (20, 36). G o r i n e t a l s t a t e d t h a t i n a d d i t i o n t o the mannan, Rhodotorula g l u t i n i s produced a hexose and a methyl pentose. Perhaps e i t h e r g a l a c t o s e or glu c o s e t h a t I determined t o be pr e s e n t i n the Rhodoto r u l a g l u t i n i s e x t r a c e l l u l a r p o l y s a c c h a r i d e h y d r o l y s a t e c o u l d be the same as the u n i d e n t i f i e d hexose of G o r i n e t a l ( 2 0 ) . A l s o , x y l o s e , the pentose t h a t I found t o be prese n t i n the Rhodotorula g l u t i n i s e x t r a c e l l u l a r h y d r o l y s a t e c o u l d be a 52 d e r i v a t i v e of the u n i d e n t i f i e d methyl pentose of G o r i n e t a l (20) . The q u a l i t a t i v e r e s u l t s f o r the U s t i l a g o h o r d e i e x t r a c e l l u l a r p o l y s a c c h a r i d e are new. The monosaccharides p r e s e n t were: g a l a c t o s e , glucose and mannose. X y l o s e was absent. I n d i c a t i o n s were t h a t q u a n t i t a t i v e , i n s t e a d o f q u a l i t a t i v e a n a l y s e s u s i n g gas chromatography might be more important i n d e t e r m i n i n g what s p e c i e s of Sporobolomyces and Rhodoto r u l a are most c l o s e l y r e l a t e d . A d u a l i t y amongst some members of both Rhodotorula and Sporobolomyces t h a t was suggested by a n t i g e n i c and p e r c e n t G+C a n a l y s e s (32, 33, 48, 51), may be supported by q u a n t i t a t i v e gas chromatographic a n a l y s e s . The r e s u l t s f o r T a p h r i n a p o p u l i n a are new. i n t h i s case the q u a l i t a t i v e a n a l y s e s may prove t o be another u s e f u l means of d i s t i n g u i s h i n g T a p h r i n a s p e c i e s from those o f Rhodotorula and C r y p t o c o c c u s . However, more a n a l y s e s o f other s p e c i e s are necessary t o determine whether t h i s i s s o . My r e s u l t s can o n l y d i s t i n g u i s h T a p h r i n a p o p u l i n a from Rhodotorula g l u t i n i s and Cryptococcus l a u r e n t i i . Some of the known t e s t s f o r d i s t i n g u i s h i n g these genera at the moment are carbon a s s i m i l a t i o n and o b s e r v a t i o n of s t a r c h 53 p r o d u c t i o n . T a p h r i n a i s i n o s i t o l n e g a t i v e and produces s t a r c h ; Rhodotorula i s s t a r c h n e g a t i v e ; and Cryptococcus i s i n o s i t o l p o s i t i v e ( 3 2 ) . T a p h r i n a and Lipomyces s p e c i e s have been p o s t u l a t e d as t a x o n o m i c a l l y r e l a t e d because o f s i m i l a r i t i e s i n s t a r c h p r o d u c t i o n , and m u l t i s p o r e s a s c i found i n both genera ( 5 5 ) . However, the development o f the a s c i i n T a p h r i n a was shown by Kramer (29) t o be a l t o g e t h e r d i f f e r e n t from those i n Lipomyces. My r e s u l t s showed t h a t T a p h r i n a p o p u l i n a produced an e x t r a c e l l u l a r p o l y s a c c h a r i d e with the monosaccharides g l u c o s e and mannose presen t i n the h y d r o l y s i s p r o d u c t . But S l o d k i and Wickerham showed t h a t the Lipomyces e x t r a - c e l l u l a r p o l y s a c c h a r i d e was composed of mannose o n l y ( 4 2 ) . The Lipomyces-Taphrina r e l a t i o n s h i p seems d o u b t f u l a t t h i s p o i n t . B a r n e t t , i n 1957 (5), s t a t e d t h a t t h e r e were fo u r major unsolved problems of y e a s t taxonomy. These were t h a t : (1) There i s a need t o d e v i s e d i f f e r e n t s e t s of b i o c h e m i c a l t e s t s f o r d i f f e r e n t groups of y e a s t s . (2) I t would be h e l p f u l t o have a q u a n t i t a t i v e e v a l u a t i o n of how many t e s t s are necessary to separate d i f f e r e n t s t r a i n s i n t o a l r e a d y named groups, say s p e c i e s . (3) The b i o c h e m i c a l a c t i v i t y d e s c r i b e d should be c o n s i d e r e d q u a n t i t a t i v e l y . 54 (4) The c o n d i t i o n s f o r each of these t e s t s needs t o be assessed c r i t i c a l l y and l a b i l e c h a r a c t e r s should be a v o i d e d . In 1961, B a r n e t t (6) suggested t h a t a c l a s s i f i c a t i o n should be developed i n which t h e r e are no s p o r u l a t i o n t e s t s , m o r p h o l o g i c a l c r i t e r i a are giv e n g r e a t e r p r e c i s i o n , and b i o c h e m i c a l t e s t s are designed t o g i v e more i n f o r m a t i o n . He s t a t e d t h a t the Dutch c l a s s i f i c a t i o n has t h r e e major weaknesses. These are: (1) S p o r u l a t i o n t e s t s a re g i v e n supreme importance, a r r a y s of media are used, and a search f o r spores i s n e c e s s a r y . Negative t e s t s are e q u i v o c a b l e . (2) M o r p h o l o g i c a l c r i t e r i a are i l l d e f i n e d and d i f f i c u l t t o s p e c i f y p r e c i s e l y . (3) B i o c h e m i c a l t e s t s are cr u d e . I n 1966, B a r n e t t (7) s t a t e d t h a t : (1) the presence of enzymes i n some y e a s t s and absence i n o t h e r s can l e a d t o c o r r e l a t i o n s between the r e s u l t s o f many other t e s t s . (2) a n a l y s e s of the r e s u l t s o f l a r g e numbers of t e s t s c o u l d be used t o make e v i d e n t the b i o c h e m i c a l f e a t u r e s t h a t are l i k e l y t o u n d e r l i e those c o r r e l a t i o n s . With r e f e r e n c e t o my experiments, i t i s e v i d e n t t h a t a q u a n t i t a t i v e a n a l y s i s u s i n g GLC would have been b e t t e r f o r d i s t i n g u i s h i n g between s p e c i e s of the same genus, e.g. Sporobolomyces s i n g u l a r i s and Sporobolomyces odorus. I t i s a l s o apparent t h a t t o be s t a t i s t i c a l l y c o r r e c t i n im p l y i n g 55 taxonomic r e l a t i o n s h i p s , r e s e a r c h e r s should use the same methods and techniques f o r c u l t u r i n g the f u n g i and f o r i s o l a t i n g and a n a l y z i n g the e x t r a c e l l u l a r p o l y s a c c h a r i d e from those f u n g i t h a t are t o be compared. V a r i a t i o n s i n the p r o p e r t i e s o f the p o l y s a c c h a r i d e may be dependent on the f e r m e n t a t i v e c o n d i t i o n s . These v a r i a t i o n s are r e l a t e d t o the c o n s t i t u t i o n and mol e c u l a r s i z e of the polymer ( 2 4 ) . A s h o r t d i s c u s s i o n o f f e r m e n t a t i v e c o n d i t i o n s t h a t are o f importance i n t h i s r e s p e c t i s r e l e v a n t h e r e . F i r s t , c o n s i d e r t h e growth p e r i o d . A c i d i c xylomannan was produced by T r e m e l l a m e s e n t e r i c a a t seven days (19), and by Cryptococcus l a u r e n t i i at four (44), f i v e (24) and s i x days ( 1 2 ) . N e u t r a l glucan, was produced o n l y a f t e r f o u r t e e n days growth by both Cryptococcus l a u r e n t i i and T r e m e l l a m e s e n t e r i c a (1, 1 9 ) . Sporobolomyces s p e c i e s produced p h o s p h o r y l a t e d g a l a c t a n s at twelve days ( 4 3 ) . Secondly, the pH of the medium i s an important f a c t o r t o c o n s i d e r . An i n i t i a l pH of 6.4 t o 6.8 was s u i t a b l e f o r polymer p r o d u c t i o n by Cryptococcus l a u r e n t i i (1, 12) and a pH of 6.0 was r e q u i r e d f o r phosph o r y l a t e d g a l a c t a n p r o d u c t i o n by Sporobolomyces s p e c i e s (43) . Other workers found t h a t Sporobolomyces s i n g u l a r i s had good growth a t pH 6.0 but no p o l y s a c c h a r i d e p r o d u c t i o n . Y i e l d s of t r i s a c c h a r i d e and t e t r a s a c c h a r i d e i n c r e a s e d a t pH 3.75. Above pH 4.0 no o l i g o s a c c h a r i d e was formed (21) . Temperature i s a t h i r d c o n d i t i o n of importance i n e x t r a c e l l u l a r p o l y - s a c c h a r i d e p r o d u c t i o n . Maximum polymer p r o d u c t i o n by Cryptococcus l a u r e n t i i o c c u r r e d a t 25°C (12); by S p o r o b o l o - myces s i n g u l a r i s a t 22°C (21); and by R h o d o t o r u l a g l u t i n i s at 20°C (12); by Sporobo1omyces s i n g u l a r i s at 22°C (21); and by Rhodotorula g l u t i n i s at 20°C and 24°C ( 2 0 ) . F o u r t h l y , a e r a t i o n and other f a c t o r s such as t r a c e s of MnS04 i n c r e a s e d the p o l y s a c c h a r i d e p r o d u c t i o n by Cryptococcus l a u r e n t i i ( 1 2 ) . I n most cases the medium, temperature, pH, a e r a t i o n , and growth p e r i o d were kept constant so t h a t a n a l y t i c a l r e s u l t s might be s t a t i s t i c a l l y comparable. Because the s t r u c t u r e and contents of the e x t r a c e l l u l a r p o l y s a c c h a r i d e s are so c l o s e l y governed by the c u l t u r a l c o n d i t i o n s i t i s apparent t h a t the a n a l y s e s of the e x t r a c e l l u l a r p o l y s a c c h a r i d e s may not be one of the best parameters t o use t a x o n o m i c a l l y . Perhaps a study encompassing q u i t e a few b i o c h e m i c a l parameters would be v a l u a b l e . T h i s i s because the r e s e a r c h e r would be a b l e t o s e l e c t those parameters t h a t were l e a s t v a r i a b l e and use them t a x o n o m i c a l l y . For i n s t a n c e , the q u a l i t a t i v e or q u a n t i t a t i v e a n a l yses of the c e l l w a l l amino a c i d s might be more u s e f u l than the a n a l y s e s of the e x t r a c e l l u l a r p o l y s a c c h a r i d e s . With r e g a r d t o the C r y p t o c o c c u s - T r e m e l l a r e l a t i o n s h i p s should the f a c t t h a t T r e m e l l a m e s e n t e r i c a has not been shown t o have 1-3 l i n k a g e s i n i t s e x t r a c e l l u l a r p o l y s a c c h a r i d e as has Cryptococcus l a u r e n t i i (19) be weighted h e a v i l y a g a i n s t the proposed taxonomic r e l a t i o n s h i p ? I n other words, how many t e s t s are nece s s a r y to prove the r e l a t i o n s h i p between Cryptococcus l a u r e n t i i and T r e m e l l a mesenterica? P e r s o n a l l y I don't t h i n k t h a t the f a c t t h a t T r e m e l l a m e s e n t e r i c a has not been shown t o have 1-3 l i n k a g e s i n i t s e x t r a c e l l u l a r p o l y s a c c h a r i d e as has Cryptococcus l a u r e n t i i s h o u l d be weighted h e a v i l y a g a i n s t t h i s r e l a t i o n s h i p . Johnson made a statement t h a t I t h i n k i s q u i t e f i t t i n g . He s a i d t h a t no o p t i m a l c l a s s i f i c a t i o n can be d e f i n e d , but improvement i s p o s s i b l e up t o the p o i n t o f i n h e r e n t i n s t a b i l i t y (25) . 58 REFERENCES 1. Abercrombie, M. J . , Jones, J . K. N., Perry, M. B., Lock, M. V., Stoodley, R. J . 1960. Can. J . Chem. 38, 1617. 2. Abercrombie, M. J . , Jones, J . K. N., P e r r y , M. B. 1960. Ccin. J . Chem. 38, 2007. 3. Ankel H., F e i n g o l d , D. S. 1964. I n t e r n . Congr. Biochem., 6th Congr., New York. A b s t r . VT, p. 502. 4. Banno, I . 1967. J . Gen. A p p l . 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A p p l . M i c r o b i o l . 3_, 1961. 23. H e n g l e i n , Von F. A., S c h e i n o s t , K. 1956. Makromol. Chem. 21_, 59. 24. Jeanes, A., P i t t s l e y , J . E., Watson, P. R. 1964. J . A p p l . Polymer S c i . 8., 2775. 25. Johnson, L . A. S. 1968. Proceedings o f the Linnean S o c i e t y of New South Wales. 93, p a r t 1, page 8„ 26. K l e i n , B., Weissman, M. 1953. A n a l . Chem. 2_5, 771. 27. K l u y v e r , A. J . , van N e i l , C. B. 1924. Z e n t r . Bakt. P a r a s i t e n k . A b t . I I , 63_, 1. 28. Kobayashi, Y., Tubaki, K. 1965. T r a n s . M y c o l . Soc. Japan 6_, 29. 29. Kramer, C. L . 1960. Myc o l o g i a 52., 295. 30. Kreger, D. R. 1954. Biochim. B i o p h y s . A c t a . 13, 1. 60 31. Lodder, J . , Kreger van R i j , N. J . W. 1952. The Ye a s t s , A Taxonomic Study, North H o l l a n d P u b l i s h i n g Co. Amsterdam. 32. Lodder, J . 1970. The Yeasts, A Taxonomic Study. Second r e v i s e d and e n l a r g e d e d i t i o n . 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Wallenfels, K., Bender, H., K e i l i c h G., Bechtler, G. 1965. Biochem. Z i e t . 341, 433. 55. Wickerham. L. J . 1952 . Ann. Rev. M i c r o b i o l . 6_, 317. 56. Wolfrom, M., Schultz, R. D., C a v a l i e r i , L. F. 1948. J . Am. Chem. Soc. 70, 514. APPENDIX The appendix presents quantitative r e s u l t s that were not included i n the main part of the t h e s i s . F i r s t , quantitative gas chromatography r e s u l t s . The fact that the i d e n t i t i e s of the peaks i n the gas chromatograms are only tentative should he taken into consideration. Table T_V shows the areas of the peaks on the gas chromatograms. Table V shows the percent monosaccharide of the t o t a l sugar. These percentages were calculated using correction factors f o r the monosaccharides i n equilibrium solutions (17»37»^9)» Second, some mention of quantitative y i e l d s of crude e x t r a c e l l u l a r polysaccharides should be made. Tremella mesenterica, cryptococcus l a u r e n t i i , B u l l e r a alba, sporobolomyces odorus, sporobolomyces s i n g u l a r i s , and Rhodotorula g l u t i n i s a l l produced approximately one to two grams of crude polysaccharide i n approximately s i x l i t r e s of culture medium. Taphrina populina and Ustilago horde1 both produced les s than one gram of crude polysaccharide i n s i x l i t r e s of culture medium. 62 Table IV 63 Fungus Peak Number Tentative I d e n t i f i c a t i o n Peak Area Tremella mesenterica 2 3 4 5 6 7 8 Solvent Xylose Xylose Xylose Galactose, Mannose Galactose 235 7, 040 7, 847 11,610 1,241 Galactose, Glucose, Mannose 10,594 Glucose T o t a l Area 1, 414 39,981 Cryptococcus l a u r e n t i i 1 2 Xylose Xylose Galactose, Mannose Galactose, Glucose, Mannose Glucose Total Area 376 7,943 40,763 251,730 266,866 567,678 Table IV (continued) Fungus Peak Number Tentative I d e n t i f i c a t i o n 6k Peak Area B u l l e r a alba 1 2 3 4 5 6 7 8 9 Total Area Solvent Solvent Solvent Xylose Xylose Galactose, Mannose Galactose Glucose 2, 701 3, 548 13,568 495 Galactose, Glucose, Mannose 5,564 1, 412 27,288 Sporobolomyces odorus (#949) ] 3 4 5 6 7 8 Xylose Xylose Xylose Galactose, Mannose 389 2,439 3,953 71 Galactose, Mannose 22,724 Galactose 12,306 Galactose, Glucose, Mannose 36,460 Glucose 15,072 To t a l Area 93,414 Table IV (continued) * 5 Fungus Peak Number Tentative I d e n t i f i c a t i o n Peak Area Sporobolomyces odorus 1 (#981) 4 5 6 7 To t a l Area Xylose Xylose Xylose Galactose, Mannose Galactose 1, 007 4, 691 8,074 27,010 19,782 Galactose, Glucose, Mannose 50,024 Glucose 6, 766 117,354 Sporobolomyces s i n g u l a r i s 1 2 3 4 Total Area Rhodotorula g l u t i n i s 1 2 3 4 5 6 Total Area Xylose Galactose, Mannose Glucose Solvent Xylose Xylose Galactose, Mannose Galactose 287 6, 357 Galactose, Glucose, Mannose 1,095 1,471 9,210 688 1,378 9, 724 1, 537 Galactose, Glucose, Mannose 14,368 Glucose 11,709 1 Q . A D A Table IV (continued) 66 Fungus Peak T e n t a t i v e Peak Number I d e n t i f i c a t i o n Area U s t i l a g o h o r d e i 1 G a l a c t o s e , Mannose 527 2 G a l a c t o s e 190 3 G a l a c t o s e , Glucose, Mannose 1,233 4 Glucose 653 T o t a l Area 2,603 Ta p h r i n a p o p u l i n a 1 Mannose 3,469 2 Mannose 2 55 3 Glucose 1,374 4 Glucose 1,567 T o t a l Area 6, 665 Tab l e V 67 Fungus Mono sa c cha r i d e p e r c e n t o f T o t a l Suga r T r e m e l l a m e s e n t e r i c a x y l o s e 38.00 G a l a c t o s e 9*50 Mannose 46 . 9 0 G l u c o s e 5«60 C r y p t o c o c c u s l a u r e n t i i X y l o s e G a l a c t o s e , K a n n o s e G l u co se 1.40 22.40 76.20 B u l l e r a a l b a Sp o r ob o l oinyc e s odo rus — mm X y l o s e G a l a c t o s e Mannose G l u c o s e X y l o s e G a l a c t o s e Mannose G l u c o s e 23.00 6.16 62.4-0 8.44 7.24 45.10 21.36 26.30 Sporobo lomyces odo rus G F W T ) X y l o s e G a l a c t o s e Mannose G l u c o s e 11.70 57.50 21.39 9.41 Table v (continued) 68 Fungus Monosaccharide Percent of Total Sugar Sporobolomyces s i n g u l a r l s Xylose 3»30 Galactose, Mannose 70.76 Glucose 25.94 Rhodotorula g l u t i n l s Xylose 5.20 Galactose 13*34 Mannose 33 . 16 Glucose 48.30 Tjstllago horde! Galactose 25.00 Mannose 34 . 3 0 Glucose 40 . 7 0 Taphrina populina Mannose Glucose 56.00 44.00

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