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Biochemical changes during sporulation and spore germination of macroconidia in Microsporum gypseum Leighton, Terrance James 1970

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BIOCHEMICAL CHANGES DURING SPORULATION AND SPORE GERMINATION OF MACROCONIDIA IN MICROSPORUM GYPSEUM by TERRANCE JAMES LEIGHTON B.Sc, Oregon State University, 1966 A THESIS SUBMITTED IN THE REQUIREMENTS DOCTOR OF PARTIAL FULFILMENT OF FOR THE DEGREE OF PHILOSOPHY In the Department of M tcrobiology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1970 In presenting t h i s thesis in p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make i t f r e e l y available for reference and study. I further agree tha permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of t h i s thesis for f i n a n c i a l gain shall not be allowed without my written permission. Department of Microbiology  The University of B r i t i s h Columbia Vancouver 8, Canada Date 19 May 1970 ABSTRACT A method f o r obtaining p u r i f i e d ungerminated macroconidia' is described, and a technique for obtaining 85 to 30% germination 2 of macroconidia under normal n u t r i t i o n a l conditions is presented. Macroconidia of Microsporum gypseum release free amino acids into the medium during germination. A single a l k a l i n e protease is also found in the germination supernatant f r a c t i o n . The p u r i f i e d protease is capable of hydrolyzing isolated spore coats in v?tro. Phenyl methyl sulfonyl f l u o r i d e (PMSF) is an e f f e c t i v e i n h i b i t o r of the protease. Incorporation of PMSF at 10 M into the germ-ination system i n h i b i t s spore 'germination and the release of free amino nitrogen. Addition of PMSF a f t e r germ tube emergence is completed has no e f f e c t on subsequent outgrowth. The addition of exogenous p u r i f i e d protease to quiescent spores results in more than a 2 .5~fold increase ih germinated spores. It is concluded that spore coat prot e o l y s i s is an ess e n t i a l event in the germination of dermatophyte macroconidia. A model system to explain macro-conidia] germination response to i n h i b i t i o n , temperature s h i f t , and addition of protease is presented. Microsporum gypseum macroconidia germinated at 37 C possessed from one to eight nuclei per germinated spore compartment. The d i s t r i b u t i o n of nuclei per spore compartment was the result of a random packaging of nuclei from the a v a i l a b l e nuclear population. i i f P a r t i a l germination i n h i b i t i o n by incubation at 25 C or at 37 £ in the presence of 10 M PMSF resulted in an enrichment of germinated spores containing high numbers of nuclei per compart-ment. The s e l e c t i o n of higher nuclear numbers was s t a t i s t i c a l l y s i g n i f i c a n t . Compartments possessing high numbers of nuclei appeared to be pre-committed for spore germination and were therefore not s e n s i t i v e to germination i n h i b i t i o n . The r e l a t i o n s h i p of the germination response to temperature s h i f t is discussed with respect to the organism's natural environment. Biochemical events which occur during macroconidial germination have been studied in M_. gypseum. The s p e c i f i c a c t i v i t y levels of various metabolic enzymes have been assayed during germination time periods. The accumulated levels of several of these enzymes, as a function of exogenous carbohydrate source, has been i n v e s t i -gated. M_. gypseum was found to possess a c o n s t i t u t i v e glyoxalate shunt, a c o n s t i t u t i v e glucokinase, a fructose PEP transferase and a mannitol PEP transferase. The integration of endogenous reserve u t i l i z a t i o n during germination is discussed. The a s s i m i l a t i o n and conversion of C glucose, C amino 14 acids and C u r a c i l into TCA-precipitable material has been studied during early germination time periods. The time course of pool accumulations is also presented. The de novo synthesis of sporulation and spore germination proteins during spore germi-nation is described. The integration of metabolite a s s i m i l a t i o n and d i f f e r e n t i a l synthesis is discussed. Developmental mutants affected in either sporulation or spore germination have been isolated from M_. gypseum with the aid of nitrosoguanidine or as spontaneously-occurring mutants. The levels of several developmental proteins have been assayed during sporu-l a t i o n time periods in these mutants. The spore germination c h a r a c t e r i s t i c s of two of the mutants are described. The r e l a t i o n -ship of a l k a l i n e protease accumulation to tyrosinase accumulation and spore germination is discussed. A large scale p u r i f i c a t i o n procedure for obtaining highly p u r i f i e d fungal chromatin is described. The isolated chromatin has normal r a t i o s of RNA:DNA and non-basic protein:DNA. However, the r a t i o of basic protein:DNA was extremely low. The p o s s i b i l i t y of p r o t e o l y t i c degradation of histdne during chromatin i s o l a t i o n was u n l i k e l y . Chromatin was kO - 50% as template active as DNA. It is concluded that basic proteins do not represent a major f r a c t i o n of M_. gypseum chromosomal proteins. V TABLE OF CONTENTS Page INTRODUCTION LITERATURE REVIEW 2 I. HEAT-INDUCED MACROCONIDIA GERMINATION 3 Materials and Methods '. 3 Organism, spore p u r i f i c a t i o n and conditions for spore germination 3 Results 5 Discussion 11 II. PHENYL METHYL SULFONYL FLUORIDE INHIBITION OF MACROCONIDIAL GERMINATION 13 Materials and Methods. 13 Germination media 13 Germination conditions 13 Spore coat and hyphal wall preparations . . . 13 High voltage electrophoresis \k Hydrolysis conditions ]k A n a l y t i c a l determinations 15 Protease p u r i f i c a t i o n 15 Disc gel electrophoresis 16 Preparation of c e l l - f r e e extract 16 Phenyl methyl sulfonyl f l u o r i d e 16 v i Page Results 17 Development of the germination system 17 Characterization of spore coats and hyphal eel 1 wal 1 s 17 Chemical analysis of germination supernatant f l u i d s 19 Analysis of germination supernatant f l u i d s for p r o t e o l y t i c a c t i v i t y and protease characterization 21 Effect of PMSF on p r o t e o l y s i s and germination . 26 Temperature response of protease a c t i v i t y and protease stimulation on germination . . . . . . 35 Discussion 40 III. THE RELATIONSHIP OF NUCLEAR SEGREGATION AND MACRO-CON I DIAL GERMINATION. 45 Materials and Methods 45 Nuclear D i s t r i b u t i o n Analysis 45 Results 46 Evaluation of nuclear staini n g procedure. . . . 46 Gross d i s t r i b u t i o n of nuclei in mycelia and spores germinated at 37 C 46 Nuclear d i s t r i b u t i o n analysis of spore germination at 37 C, 37 C in the presence of 10 - Z f M PMSF and at 25 C. 46 v i i Page Discussion 53 IV. THE BIOCHEMISTRY OF MACROCONIDIAL GERMINATION. . . 55 Materials and Methods 55 Sporulation and spore germination in the presence of mannitol, fructose and acetate . . 55 Preparation of c e l l - f r e e extracts. 55 A n a l y t i c a l determinations 55 Carbohydrate extraction and i d e n t i f i c a t i o n . . 55 o Enzyme assays 56 Results 58 Macroconidial u t i l i z a t i o n of a l c o h o l - and acid-soluble carbohydrates (saline-germination system) 58 Enzyme s p e c i f i c a c t i v i t y changes during periods of spore germination (glucose-germination system) 58 Mannitol metabolism in germinating macroconidia (mannitol, fructose or glucose-germinating system) 60 Evidence for the occurrence of a c o n s t i t u t i v e glyoxylate shunt (acetate or glucose-germination system) 6k Discussion 69 Page V. EARLY EVENTS IN MACROCON ID IAL GERMINATION 7k Materials and Methods: . . . , Ik Preparative procedures 7k Manometric techniques. 7k Uptake studies 7k Chemical f r a c t i o n a t i o n of macroconidia 75 C e l l - f r e e extract (CFX), preparation for DEAE chromatography 75 DEAE-Sephadex chromatography . . . 76 Results. 77 Stimulation of endogenous r e s p i r a t i o n 77 1 Zf Uptake of C glucose 77 Uptake of '^ C amino acids. 77 1 k Uptake of C u r a c i l 77 D i scuss ion 86 VI. ISOLATION AND PRELIMINARY CHARACTER IZATION OF DEVEL-OPMENTAL MUTANTS 88 Materials and Methods. 88 Mutagenesis and mutant screening conditions. . . 88 Growth and sampling conditions 88 Enzyme assays 89 Results 91 Visual c h a r a c t e r i z a t i o n 91 SP+, Pig+ 9 2 Page SP 2,Pig 92 SP|, Pig+O) and Sp|, Pig+(2) 92 S P ° P , Pig+- 92 SP~, Pig-orn 92 SP° P, Pig-yln 93 Spore germination mutants 93 NG sporulation mutants 100 Discussion 102 VII. ISOLATION AND PRELIMINARY CHARACTERIZATION OF CHROMATIN Materials and Methods 104 Extraction of hi stone 106 Chemical f r a c t i o n a t i o n of chromatin 106 Template a c t i v i t y determinations 106 Disc gel electrophoresis 106 Basic protease assay conditions 106 Chemical determinations 107 Results . 108 Chromatin i s o l a t i o n 108 Chemical ch a r a c t e r i z a t i o n of chromatin and nuclear f r a c t i o n s 108 Template a c t i v i t y 112 D i scuss ion . . 117 BIBLIOGRAPHY 119 X LIST OF TABLES Page T a b l e 1. E f f e c t o f i n c u b a t i o n t im e on p e r c e n t a g e o f g e r m i n a t i o n 7 T a b l e t l . E f f e c t o f I n c u b a t i o n t i m e a t 37 C and subsequen t r e t u r n t o 25 C on p e r c e n t a g e o f g e r m i n a t i o n 8 T a b l e I I I . E f f e c t o f I n c u b a t i o n medium on p e r c e n t a g e o f g e r m i n a t i o n i t ' 2k hr. o f i n c u b a t i o n 10 T a b l e IV. C h e m i c a l c o m p o s i t i o n o f s p o r e c o a t s and v e g e t a t i v e wal1 o f M i c r o s p o r u m gypseum 18 T a b l e V . Amino a c i d c o m p o s i t i o n o f 2-hr and 8-hr g e r m i n a t i o n medium s u p e r n a t a n t f l u i d 22 T a b l e V I . Amino a c i d c o m p o s i t i o n o f v e g e t a t i v e w a l l s , s p o r e c o a t s , and 7-hr s p o r e c o a t s o f M i c r o s p o r u m gypseum 28 T a b l e V I I . M e t a b o l i c enzyme l e v e l s d u r i n g s p o r e g e r m i n a t i o n 61 T a b l e V I I I . Hexose p h o s p h o e n o l p y r u v a t e t r a n s f e r a s e a c t i v i t i e s a f t e r 2k h r s s p o r e g e r m i n a t i o n 62 T a b l e IX. Hexose k i n a s e a c t i v i t i e s a f t e r 2k h r s s p o r e g e r m i n a t i o n 63 T a b l e X . A c t i v i t i e s o f m a n n i t o l m e t a b o l i s m enzymes f o l l o w i n g 2k h r g e r m i n a t i o n 65 T a b l e X I . G l y o x y l a t e and t r i c a r b o x y l i c a c i d c y c l e enzyme a c t i v i t i e s d u r i n g m a c r o c o n I d l a l g e r m i n a t i o n 66 T a b l e X I I . R e l a t i v e c o n c e n t r a t i o n s o f s p o r e r i b o n u c l e i c a c i d and p r o t e i n i n 0 t ime and 2k hr c e l l - f r e e e x t r a c t s 67 XI L i s t of Tables (Continued) Page 14 Table X U I . Chemical f r a c t i o n a t i o n of C glucose TCA-prectpitable material accumulated during spore germination. Counts are expressed as CPM/g mg spores f r a c t i o n -ated. 50 rag of spores were f r a c t i o n -ated at each sample time 83 Table XIV. DEAE-Sephadex chromatography of 2 hr c e l l - f r e e extract 84 Table XV. Spore germination at 37 C in sa l i n e germination system, 7 hrs incubation 94 Table XVI. Germination supernatant p r o t e o l y t i c a c t i v i t y . Saline germination system, 7 hrs Incubation at 37 C. 95 Table XVII. Free Amino nitrogen release Into the supernatant f r a c t i o n during spore germination. Saline germination system, 7 hrs incubation at 37 C. 96 Table XVIII. Germination supernatant a c t i v i t y against synthetic substrates. A c t i v i t y = AO.D./mg protein/min 97 Table XIX. Recovery of DNA during M_. gypseum chromatin i s o l a t i o n 109 Table XX. Chemical composition r a t i o s of M_. gypseum chromatin and nuclear f r a c t i o n s 110 Table XXI. Histone to DNA rat i o s of Stage V nucleohlstone (1.5 mg) before and a f t e r incorporation into the i s o l a t i o n p r o c edurelll Table XXII. Basic protease assay of M. gypseum nuclear lysate 114 X I I L IST OF FIGURES Page F i g . 1. E f f e c t o f i n c u b a t i o n t e m p e r a t u r e , f o r a 24-hr p e r i o d , on p e r c e n t a g e o f g e r m i n a t i o n . Each ba rogram t s t h e a v e r a g e o f a t r l p l i c a t e s a m p l i n g 6 F i g . 2 . R e l e a s e o f f r e e amino n i t r o g e n i n t o t h e s u p e r n a t a n t f r a c t i o n d u r i n g s p o r e g e r m i n a t i o n . G e r m i n a t i o n s y s t em was pH 6 . 5 p h y s i o l o g i c a l s a l i n e w i t h i n c u b a t i o n a t 37 C . M a c r o -con i d i a l c o n c e n t r a t i o n was 10 °/ml . 20 F i g . 3 . B a s i c d i s c g e l e l e c t r o p h o r e s i s o f d i a l y z e d , c o n c e n t r a t e d 8-hr s p o r e g e r m i n a t i o n s u p e r -n a t a n t f r a c t i o n . L e f t and c e n t e r p h o t o g r a p h s r e p r e s e n t e l e c t r o p h o r e s i s o f s u p e r n a t a n t f r a c t i o n f o r d i f f e r e n t t i m e p e r i o d s ; The o r i g i n i s a t t o p o f a l l p h o t o g r a p h s . The r i g h t p h o t o g r a p h shows l o c a l i z a t i o n o f p r o t e a s e a c t i v i t y i n an u n s t a i n e d d u p l i c a t e g e l o f t h e c e n t e r p h o t o g r a p h . The t r a n s p a r e n t a r e a , w h i c h a p p e a r s d a r k ow ing t o the b a c k g r o u n d , i s the zone o f c a s e i n h y d r o l y s i s . I n c u b a t i o n was f o r 1 h r a t 37 C , w h i c h caused some d i f f u s i o n o f t h e band 23 F i g . k. The pH p r o f i l e o f p r o t e a s e a c t i v i t y a g a i n s t c a s e i n . One u n f t e q u a l s t he amount o f enzyme w h i c h w i l l s o l u b i l i z e t h e e q u i v a l e n t o f 1 y g o f b o v i n e s e r u m , i n 1 m i n . I n c u b a t i o n was a t 37 C f o r 30 m i n . 2k F i g . 5- Therma l d e n a t u r a t i o n o f p r o t e a s e a c t i v i t y . P o r t i o n s (1 m l ) o f p u r i f i e d p r o t e a s e were p r e -i n c u b a t e d f o r 20 min a t t he t e m p e r a t u r e s i n d i c a t e d . P r o t e o l y t i c a c t i v i t y was then measured a g a i n s t pH 8 .0 c a s e i n , w i t h i n c u b a t i o n a t 37 C f o r 30 m i n . 25 X I I I L i s t of Figures (Continued) Page Fig . 6. Hydrolysis of purified spore coats by Microsporum protease. Reaction mixture was 50 jig of protease/ml in pH 6.5 s a l i n e , with 1 mg/ml of spore coats: Spore coats were pelleted at 600 x g_ for 1 min at the times indicated, and the op t i c a l density at 280 nm was determined on the supernatant f r a c t i o n s . 27 F i g . 7. Ef f e c t of PMSF on protease a c t i v i t y against pH 8.0 casein, with preincubation time of 30 min at 37 C. Assay conditions were 30 min incubation at 37 C. NPI, no preincubation with 10"^ M PMSF. 30 Fi g . 8. A. E f f e c t on free amino nitrogen release of zero time addition of 10"^ M PMSF to pH 6.5 sal i n e germination system, with incubation at 37 C. B. Re-plot of l e f t side data from the 1-hr sample point. 31 Fig . 9. A. L o c a l i z a t i o n of p r o t e o l y t i c a c t i v i t y in germination supernatant f r a c t i o n s , s o n i c a l l y disrupted p e l l e t s and t h e i r supernatant f r a c t i o n s . No 10" M PMSF added B. 10 _ 7 f M PMSF added at zero time 33 Fi g . 10. Ef f e c t of 10 M.PMSF addition on 2k hr to t a l c e l l u l a r protein values. Germination system; 1% (w/v) glucose, 0.1% (w/v) neopeptone, pH 6.5. Total incubation period was 2k hr at 37 C. 3k F i g . 11. E f f e c t of 10 _ Z f M PMSF addition on 24-hr germina-tion percentages. Germination system; \% (w/v) glucose, 0.1% (w/v) neopeptone, pH 6.5. 10"^ M PMSF was added to 10° spores/ml at times Indicated xiv L i s t o f F i g u r e s ( C o n t i n u e d ) Page F i g . 12 . T e m p e r a t u r e r e s p o n s e c u r v e f o r M i c r o s p o r u m p r o t e a s e . A s s a y c o n d i t i o n s : pH 8 .0 c a s e i n w i t h i n c u b a t i o n f o r 30 min a t t e m p e r a t u r e s I n d i c a t e d 37 F i g . 13 . E f f e c t o f p r o t e a s e a d d i t i o n t o 25 C g e r m i n a t i o n s y s t e m . G e r m i n a t i o n medium was pH 6 . 5 s a l i n e w i t h a t o t a l I n c u b a t i o n t ime o f 6 h r . P r o t e a s e , a t c o n c e n t r a t i o n s i n d i c a t e d , was added a t 0 t i m e . 63 38 F i g . 14 . D i s t r i b u t i o n o f n u c l e i pe r g e r m i n a t e d s p o r e c o m p a r t m e n t , a t 37 C . 48 F i g . 15- D i s t r i b u t i o n o f n u c l e i pe r g e r m i n a t e d s p o r e compar tment a t 25 C . 49 F i g . 16 . D i s t r i b u t i o n o f n u c l e i pe r g e r m i n a t e d s p o r e compar tment a t 37 C In the p r e s e n c e o f 10"*'* M PMSF. 50 F i g . 17 . U t i l i z a t i o n o f a l c o h o l and a c i d s o l u b l e c a r b o h y d r a t e f r a c t i o n s d u r i n g m a c r o c o n l d l a l g e r m i n a t i o n . 59 F i g . 18 . S t i m u l a t i o n o f endogenous r e s p i r a t i o n by g l u c o s e and n e o p e p t o n e . Spore c o n c e n t r a t i o n 10 mg/ml . S a l i n e g e r m i n a t i o n s y s t e m . 78 14 F i g . 1 9 . C g l u c o s e u p t a k e by g e r m i n a t i n g mac ro-c b n i d l a . S a l i n e g e r m i n a t i o n s y s t em c o n t a i n e d = s p o r e s a t 10 m g / m l ; 1 uc/m l g l u c o s e ; 0 . 33 mg/ml g l u c o s e , 0 .25 mg/ml neopep tone 79 14 F i g . 2 0 . C amino a c i d u p t a k e by g e r m i n a t i n g mac ro-c o n i d f a . S a l i n e g e r m i n a t i o n s y s t em c o n t a t n e d = s p o r e s a t 10 mg/ml ; 1 y c / m l ^ C p r o t e i n h y d r o -l y s a t e ; 0 . 33 mg/ml g l u c o s e , 0 . 2 5 mg/ml n e o -pep tone 80 XV L i s t of Figures (Continued) Page 14 Fig. 21. C u r a c i l uptake by germinating macro-conidia . Saltne germination system contained", spores at 10 mg/ml; 1 uc/ml ^ C u r a c i l ; 0.33 mg/ml glucose, 0.25 mg/ml neopeptone 81 Fig. 22. DEAE-Sephadex chromatography of a l k a l i n e phosphatase and mannitol dehydrogenase 85 F i g . 23. Enzyme accumulation during macroconidial development. Protease units: 1 unit = the amount of enzyme which w i l l hydrolyze the equivalent of 1 ug of bovine serum albumin. N-acetyl glucosammfdase and tyrosinase u n i t s . AO.D./mg protein/min. 98 F i g . 24. Enzyme accumulation during macroconidia1 development. Protease u n i t s : 1 unit = the amount of enzyme which w i l l hydrolyze the equivalent of 1 ug of bovine serum albumin. N-acetyl glucosaminidase and tyrosinase u n i t s . AO.D./mg protein/min. 99 F i g . 25. Enzyme accumulation during macroconidia1 development. Protease u n i t s : 1 untt =• the amount of enzyme which w i l l hydrolyze the equivalent of 1 pg of bovine serum albumin. N-acetyl glucosamtnidase and tyrosinase units = AO.D./mg protein/min. 101 Fig . 26. Polyacrylamide gel p r o f i l e of Stage V fish. nucleohistone a f t e r recovery from M_. gypseum i s o l a t i o n procedure. 20 ug of basic protein in 0.2 N HC1 containing 20% sucrose was applied to the gel. Electrophoresis time = 1 hr. 113 Fig. 27. RNA synthesis directed by M_. gypseum chromatin and DNA as a function of template concentrations. 114 ug of £_. col i RNA polymerase / 0.25 ml reaction mixture. Incorporation by the enzyme alone has been subtracted. 115 ACKNOWLEDGEMENTS I am deeply indebted to Dr. J . J . Stock for his continuing encouragement and advice which made this study possible. I am grateful to Dr. R. A. J . Warren for his helpful sug-gestions and advice. I thank Professor James Bonner and Dr. K e i j i Marushige for th e i r expert advice and stimulating discussions. I am indebted to Dr. D. J . Clark for his interest and con-s t r u c t i v e c r i t i c i s m throughout the course of t h i s work. I acknowledge a l s o , the assistance of Dr. T. H. Blackburn, Dr. J . J . R. Campbell, Dr. A. F. Gronlund and Dr. J u l i a Levy during c e r t a i n phases of t h i s i n v e s t i g a t i o n . INTRODUCTION Analysis of the molecular basis for c e l l u l a r development has resulted in tremendous advances in the understanding of c e l l d i f f e r e n t i a t i o n in such diverse organisms as sporulating bacteria, slime molds, sea urchins, salmon and peas. It was f e l t that fungi had been neglected within this context. These organisms o f f e r advantages a v a i l a b l e in few other developmental systems. Fungi represent t y p i c a l eucaryotic complexity with respect to c e l l u l a r organization and development while maintaining a reasonable generation time and concomitantly o f f e r i n g p o s s i b i l i t i e s for the genetic analysis of development not a v a i l a b l e in other higher organisms. Fungi are known to be amenable to many sophisticated experimental approaches applied to bacteria but which are extrapolated with great d i f f i c u l t y to organisms higher in the evolutionary sequence. Hence, i t seemed that an investigation into the molecular biology of fungal development ( i e . - asexual sporulation and spore germination) would be appropriate at th i s time. 2 LITERATURE REVIEW Since there have been no previous studies concerning the molecular biology of fungal development, i t is not possible to provide a pertinent h i s t o r i c a l review of other investigations in this context. There are several excellent reviews which contain most of the current knowledge avai1 able with respect to fungal sporulation and spore germination (Cochrane, 1966; G o t t l i e b , 1966 ; and Sussman and Halvorson, 1 9 6 6 ) . Schaeffer (1969) has published a review on B a c i l l u s development which contains several references concerning present knowledge about the association of e x t r a c e l l u l a r fungal enzymes with sporulation processes. It should be appreciated that none of the aforementioned reports concern work which has been done on Microsporum gypseum, and hence, may not be d i r e c t l y a pplicable to th i s organism. It would be an understatement to report that the knowledge of fungal biochemistry is meager, con-t r a d i c t o r y , and in the case of dermatophytes, such as M_. gypseum, of l i t t l e use in the analysis of c e l l d i f f e r e n t i a t i o n . Therefore the primary purpose of th i s investigation was to obtain some basic knowledge and to develop techniques from which a detai l e d study of M. gypseum development might proceed. 3 I. HEAT-INDUCED MACROCONIDIA GERMINATION Materials and Methods Organism, spore p u r i f i c a t i o n and condit ions, for spore germination. A s t r a i n of M_. gypseum (Bodin) Guiart and Grigorakis, 1928, o r i g i n a l l y obtained from F. Blank, Temple Uni v e r s i t y , Philadelphia, Pa., was used in a l l studies. The culture medium consisted of: glucose, \% (w/v); neopeptone (Difco), \% (w/v); agar, 2.5% (w/v); and d i s t i l l e d water (pH 6.4). The organism was grown for 7 days at 25 C in Roux fla s k s containing 150 ml of medium. Spores were harvested by f i l l i n g each f l a s k with 15 ml of s t e r i l e s a l i n e (0.85%, w/v), and vigorously shaking with glass beads (3.0 mm) to dislodge conidia from the surface of the mycelial mat. The r e s u l t i n g suspension was f i l t e r e d through two layers of s t e r i l e cheesecloth and subsequently through four layers of c l o t h . The spores were c o l l e c t e d from the f i l t r a t e s by cen t r i f u g a t i o n f or 120 sec at 450 x g_. The supernatant f l u i d was discarded and the pel let was. resuspended in one-half the o r i g i n a l volume of s a l i n e . The suspension was then dispersed by using a Vortex mixer for 30 sec and centrifuged at approximately 600 x cj_ for 25 sec. This resuspension and 25~sec centrifugation step was repeated two additional times. The r e s u l t i n g p e l l e t was re-suspended in culture medium to give 10 macroconidia per ml. 4 G e r m i n a t i o n e x p e r i m e n t s were c a r r i e d ou t by a d d i n g 1 ml o f s p o r e s u s p e n s i o n to 2 ml o f c u l t u r e medium in m e t a l - c a p p e d t e s t t u b e s (18 by 150 mm). The c u l t u r e t ubes were shaken a t 100 r e v / m i n i n a New B r u n s w i c k m e t a b o l y t e s h a k e r a t 2 3 , 2 5 , 27, 29, 3 1 , 3 3 , 3 5 , 3 7 , - 3 9 , 4 1 , 4 3 , 50 and 55 C . The a v e r a g e number o f c o n i d i a w i t h germ tubes was o b t a i n e d by c o u n t i n g a t o t a l o f 600 c o n i d i a f o r each i n c u b a t i o n t e m p e r a t u r e and t i m e . T r i p l i c a t e samp les were c o u n t e d and a v e r a g e d f o r each t ime and t e m p e r a t u r e . A P e t r o f f - H a u s e r c o u n t i n g chamber and phase c o n t r a s t o p t i c s a t 200 x were u s e d . 5 Res u11 s The procedure for the separation and concentration of macro-conidia was found to e f f e c t i v e l y remove a l l microconidia, mycelial fragments, and any macroconidia possessing germ tubes. Thus, i t was possible to obtain a suspension containing only ungerminated macroconidia. The data in Figure 1 demonstrate a basal l i n e of germination varying from 20 to 30%. A sharp increase in germination occurred at 35 C and was maximal at 37 C. The germination values decreased rapidly above 39 C. M_. gypseum macroconidia did not germinate at 50 C. After 24 hr these conidia were extremely vacuolated but would produce germ tubes i f returned to 25 C. A 15-min exposure to 55 C was l e t h a l . Prolonged incubation at t h i s temperature resulted in extreme vacuolation followed by fragmentation and l y s i s . Table I i l l u s t r a t e s the e f f e c t s of increased incubation times on germination values. There was no s i g n i f i c a n t increase in counts, from 2k to 48 hr. Table II shows the re s u l t s of incubation at 37 C for the times indicated, followed by incubation at 25 C for the remainder of the 24-hr period. This experiment was designed to determine whether a short exposure to 37 C was s u f f i c i e n t ' t o i n i t i a t e high levels of germination. There was no s i g n i f i c a n t increase in germination u n t i l 6 hr of exposure at 37 C. M_. gypseum macroconidia germinated with comparable percentage values in E f f e c t of incubation temperature, for a 24-hr period, on percentage of germination. Each barogram is the average of a t r i p l i c a t e sampling. 6 100 9 0 -8 0 Z o < rr LU CD 7 0 6 0 -5 0 Z LU 4 0 -O tr LU ° - 30 H 2 0 -10 0 2 3 2 5 2 7 2 9 31 3 3 3 5 3 7 3 9 41 4 3 5 0 T E M P E R A T U R E IN D E G R E E S (C) 7 Table I. E f f e c t of incubation time on percentage of germination. Temperature Germination Time C % hr 25 23 25 25 25 48 37 86 24 37 83 48 Table II. Effect of incubation time at 37 C and subsequent to 25 G on percentage of germination. Time at 37 C Time at 25 C Germination hr hr % 0.25 23.75 18 0.50 23.50 24 1 .00 23.00 21 2.00 22.00 20 3.00 21.00 26 6.00 18.00 38 9.00 15.00 83 24.00 0.00 87 d i s t i l l e d water, culture medium, and phosphate buffer, pH 6.5 (Table I I I ) . However, conidia germinated in d i s t i l l e d water and phosphate buffer exhausted t h e i r internal reserves soon a f t e r 2k hr and subsequently required glucose and neopeptone for further growth. It was not possible to increase basal germination values by exposure of these spores to leucine or o l e i c a c i d . Stimulation by these compounds was previously reported for another s t r a i n of t h i s organism under d i f f e r e n t experimental conditions (Barash et a l . 1967). T a b l e III. E f f e c t o f i n c u b a t i o n medium on p e r c e n t a g e o f g e r m i n a t i o n a t 2k h r o f i n c u b a t i o n . Medium T e m p e r a t u r e G e r m i n a t i o n C % C u l t u r e 25 22 C u l t u r e 37 91 D i s t i l l e d w a t e r 25 28 D i s t i l l e d w a t e r 37 89 O J H PO^ b u f f e r , pH 6 . 5 25 2k 0.1 M PO^ b u f f e r , pH 6 .5 37 89 11 D iscuss ion The optimal growth temperature for M_. gypseum was established as 27 C and the maximum at kk C (Paldrock. 1955). Hence the question a r i s e s as to why 37 C was found to be the optimum temp-erature for macroconidia1 germination and outgrowth. Further, the results (Table 11) indicated that an extended incubation period at 37 C was necessary for increased germination. There-fore, i t seems u n l i k e l y that the stimulation can be likened to the usual heat a c t i v a t i o n . The use of temperatures above 37 C, even for short periods of time does not appreciably increase the germination values, as exposure for 15 min at 55 C was found l e t h a l . The fact that these conidia would germinate in the absence of exogenous carbon and nitrogen sources suggested that endogenous re s p i r a t i o n may be the means by which outgrowth and synthesis occurred. The r e s u l t s show that increased germination values could be obtained by incubation of spores in d i s t i l l e d water at 37 C for 2k hr. The a c t i v a t i o n process may involve some type of heat-stimulated " t r i g g e r " , owing to the f a c t that spore germination counts did not increase, e i t h e r at 25 or 37 C, with extended incubation times. It seems that those spores germinated which were i n i t i a l l y a c t i v a t e d , whereas the ungerminated macroconidia continued to remain quiescent. Incubation at 37 C appears to have increased the i n i t i a l level of germinated spores. F u j i i (1957) showed that, in Trichophyton gypseum, the optimal growth tempera-ture is 30 C; however, the optimal temperature for endogenous re s p i r a t i o n is 37 C. Previous work in t h i s laboratory indicated a high level of endogenous r e s p i r a t i o n in M_. gypseum (McBride and Stock. 1966). M_. gypseum pools several carbohydrate reserve materials which decrease during early germination of spores (see section on Endogenous Reserves in Spores). Therefore, stimulation of endogenous metabolism at 37 C may p a r t i a l l y explain the increased germination values. II. PHENYL METHYL SULFONYL FLUORIDE INHIBITION OF MACROCONIDIAL GERMINATION Materials and Methods Germination media. Macroconidia (10° per ml) were germinated in e i t h e r pH 6.5 physiological s a l i n e or 0.1% (w/v) neopeptone (Difco) and 1.0% (w/v) glucose medium (pH 6 .5 ) . Germination conditions. Spores were germinated at 25 or 37 C in 50 ml Erlenmeyer f l a s k s containing 5 ml of medium. The f l a s k s were shaken at 125 rev/min in a R77 Metabolyte shaker water bath (New Brunswick S c i e n t i f i c Co., New Brunswick, N.J.). Spore coat and hyphal wall preparation. Macroconidia1 or hyphal suspensions in pH 6.5 physiological saline were mixed with 0.6 volume f i n e glass beads (100 urn, Sigma Chemical Co., St. Louis, Mo.) and blended at kC in a c o l l o i d m i l l ( G i f f o r d -Wood, Inc., Hudson, N.Y.), to 100% breakage as judged by micro-scopic examination. The glass beads were allowed to s e t t l e , and the supernatant f r a c t i o n was centrifuged at 600 x g_for 1 min. The p e l l e t obtained was resuspended in 10 volumes of cold physiolog-ic a l s a l i n e , pH 6 .5 , by mixing (Vortex J r . Mixer, S c i e n t i f i c Industries, Inc., Queen's V i l l a g e , N.Y.). Walls or spore coats were recovered by centr i f ugation at 600 x g_ f o r 1 min. The p e l l e t was washed eight times in physiological saline (pH 6.5) and eight times in d i s t i l l e d water by centrifugation at 600 x g_ for 1 min at k C. Vigorous dispersal of the p e l l e t was necessary a f t e r each washing. The f i n a l p e l l e t was resuspended in 10 volumes of 10% sodium lauryl s u l f a t e and extracted on a tube r o l l e r at 37 C for 3 days. The sodium lauryl s u l f a t e s o l u t i o n was changed once each day. The extracted p e l l e t was washed 10 times in d i s t i l l e d water at k C. P u r i f i e d spore coats and hyphal walls were then dried to a constant weightsHn vacuo at k C. High voltage electrophoresis. P u r i f i e d hyphal walls and spore coats were subjected to electrophoresis at 3000 v for kS min in a Gilson model D electrophorator by using a buffer (pH 6.5; a c e t i c a c i d , 4 ml; pyridine, 100 ml; and water, 900 ml). The paper s t r i p s were stained for free amino acids by the ninhydrin method of Smith (I960), f o r carbohydrate by the s i l v e r n i t r a t e method of Smith (I960) and for protein by nigrosiii (0.002% in 2% a c e t i c acid for 2k hr, decolorized in water). Hydrolysis conditions. Spore coats and hyphal walls at 1 mg/ml were hydrolyzed in 6 N HCl for 12 hr at 110 C p r i o r to amino acid a n a l y s i s . Germination medium supernatant f r a c t i o n s were mixed with an equal volume of 12 N HCl and hydrolyzed for 12 hr at 110 C pr i o r to amino acid a n a l y s i s . Glucosamine was liberated by hydro-lyzing coats or walls (1 mg/ml) in 6 N HCl for 6 hr at 110 C. Glucosamine standards were hydrolyzed under the same conditions. A n a l y t i c a l determinations. A l l protein estimations were made by the method of Lowry et_ a_l_. (1951). Prior to total c e l l u l a r protein estimation, the germinated spores were pelleted (4 , 0 0 0 x g_ for 1 min) three successive times through 10 ml of physiological s a l i n e (pH 6 . 5 ) . The r e s u l t i n g p e l l e t s were resuspended in equivalent volumes of s a l i n e (pH 6 . 5 ) . Total hexose was determined by the anthrone reaction (Morris. 1948). Free amino nitrogen analysis was c a r r i e d out by the method of Yemm and Cocking (1955). Ash was estimated by incinerating the walls or coats at 700 C for 6 hr. Glucosamine was determined by the method of Rondle and Morgan (1955). Amino acid residues were quantitated ih a Beckman model 120 B amino acid analyzer by the method of Spackman, Stein and Moore (1958). Protease a c t i v i t y was measured by the method of McDonald and Chen ( 1 9 6 5 ) . A l k a l i n e phosphatase a c t i v i t y was assayed as described by Garen and Levinthal ( i 9 6 0 ) . g Protease p u r i f i c a t i o n . Macroconidial suspension (50 ml, 10 conidia per ml), in physiological s a l i n e , (pH 6 . 5 ) , was placed in a 500 ml Erlenmeyer f l a s k , and the contents were shaken at 125 rev/min in a water bath at 37 C for 8 hr. The suspension was centrifuged at 2 0 , 0 0 0 x £ for 10 min at 4 C. The supernatant f l u i d was removed c a r e f u l l y and dialyzed against two changes of 200 volumes of physiological s a l i n e (pH 6 . 5 ) , during a period of 24 hr at 4 C. The d i a l y s a t e volume was reduced approximately 10-fold by d i a l y s i s against polyethylene glycol (20,000 molecular weight). The f i n a l y i e l d of enzyme was 3 to k mg of protease per 50 ml of o r i g i n a l spore suspension and was stored at -20 C u n t i l used. Disc gel electrophoresis. Disc gel electrophoresis was at pH 9.1, by using the procedure suggested for the Canalco (Rockville, Md.) model 6 system. P r o t e o l y t i c a c t i v i t y was located by placing the unstained gel on a s l i d e covered with a mixture of casein {]% w/v) and agar (1% w/v) at pH 8.0. The gel was removed a f t e r 15 min (visual l o c a l i z a t i o n of c l e a r i n g of area on s l i d e ) or a f t e r 1 hr (photographic l o c a l i z a t i o n ) at 37 C (see Figure 3). The s l i d e was flooded with a solution of mercuric chloride (1% w/v), pr i o r to f i n a l observation. Preparation of c e l l - f r e e extract. Samples (3 ml) of a g suspension of macroconidia (10 /ml) in 0.2 M tris-(hydrdxymethyl)-aminomethane ( T r i s ) , pH l . k , were s o n i c a l l y disrupted under nitrogen at a d i a l s e tting of 70 with a Biosonic model B (Bronwill S c i e n t i f i c Co., Rochester, N.Y.) f i t t e d with a microprobe attach-ment. The tube was held in ice during the treatment. The samples were given seven 30-sec bursts with a 30-sec interval for cooling between bursts. Debris was removed by cen t r i f u g a t i o n at 20,000 x g_ for 30 min at k C. Phenyl methyl sulfonyl f l u o r i d e . Solutions of 10 ' M PMSF .in t w i c e - d i s t i l l e d isopropanol were prepared immediately p r i o r to use. Results Development of the germination system. As a preliminary to this i n v e s t i g a t i o n , methods were developed for the rapid p u r i f i -cation of ungermtnated macroconidia and for the germination of 80 to 30% of a given spore suspension in physiological s a l i n e , pH 6 .5. It was also shown that the endogenous reserves of the macroconidia were s u f f i c i e n t for at least 8 hr of germination and outgrowth (see section on Endogenous Reserves in Spores). Hence, there existed a germination system which was independent of added nutrient materials. Characterization of spore coats and hyphal c e l l walls. Table IV l i s t s the gross chemical composition of the spore coats and hyphal c e l l walls. If the fungal spore has germinated as a r e s u l t of weakening the spore coat, as electron microscopic evidence c e r t a i n l y has indicated, (J. Das and S.H. Black. B a c t e r i o l . P r o c , p. 23, 1969), then presumably a protease, c h i t i n a s e , or e e l l u l a s e - 1 i k e enzyme could be involved. The question arose as to whether the protein values l i s t e d in Table IV were a c t u a l l y s t r u c t u r a l protein or adsorbed amino acids and peptides released during c e l l d i s i n t e g r a t i o n . Samples of p u r i f i e d spore coats and hyphal walls were placed on paper s t r i p s and examined for the presence of amino acids (ninhydrin), Table IV. Chemical composition of spore coats and vegetative wa11 of Microsporum gypseum. Spore Vegetative Component coats walls "/ °/ 'o 'a Hexose as glucose 31.2 40.4 Amino sugar as N-acetyl glucosamine 52.5 27.7 Protein 12.9 10.6 Ash 8.0 11.0 protein ( n i g r o s i n ) , and carbohydrate (AgNO^). Spore coats and hyphal walls gave no ninhydrin reaction, but were p o s i t i v e for carbohydrate and protein (possibly most of the free N-terminal amino acids in the c e l l wall protein were "blocked", i e . - nin-hydrin nonreactfve, owing to acetylation of the free amino groups). Ninhydrin staini n g a f t e r high-voltage electrophoresis did not reveal any reactive material and suggested that nonstructural amino acids were absent. Staining the paper s t r i p s for protein and carbohydrate revealed three glycoprotein bands in the spore coats and one glycoprotein band in the hyphal walls. Since carbohydrate and protein always migrated with each other, i t appears that the protein values t r u l y represent st r u c t u r a l protein. Chemical analysis of germination supernatant f l u i d s . If the spore coat was hydrolyzed during germination, the obvious place to look for degradation products would be in the germination super-natant f l u i d . Concentrated supernatant f l u i d s were assayed for Rondle-Morgan-positive material, anthrone-positive material, and ninhydrin-reactive material. Only the ninhydrin test gave a p o s i t i v e r e s u l t . Figure 2 shows the time course of free amino nitrogen release into the supernatant f l u i d . It is interesting to note that micro-scopic examination showed that the germination process was completed for a l l spores by 7 hr, i e . , those spores which were going to germinate, had done so by t h i s time. » Figure 2. Release of free amino nitrogen into the supernatant f r a c t i o n during spore germination. Germination system was pH 6.5 physiological s a l i n e with incubation at 37 C. Macroconidial concentration was 10^/ml. Therefore, i t was decided to characterize further the nature of t h i s ninhydrin-reactive material by amino acid a n a l y s i s . Table V l i s t s the amino acid composition of hydrolyzed 2- and 8-hour supernatants. The analyses were nearly i d e n t i c a l with respect to individual concentrations of the residues. This i n -formation suggested that the process producing t h i s material may be of a rather s p e c i f i c nature, as opposed to general i n t r a -c e l l u l a r turnover products d i f f u s i n g out into the supernatant f l u i d . Spore l y s i s was checked for during the course of the germination process by assaying 8-hr supernatant f r a c t i o n s f or a l k a l i n e phosphatase a c t i v i t y (a known c o n s t i t u t i v e i n t r a c e l l u l a r enzyme; R. Kelln, undergraduate thesis ( 1 9 6 9 ) ) . There was no detectable a l k a l i n e phosphatase a c t i v i t y in the supernatant f l u i d . Analysis of germination supernatant f l u i d s f o r p r o t e o l y t i c  a c t i v i t y and protease c h a r a c t e r i z a t i o n . Figure 3 presents the re s u l t s of basic d i s c gel electrophoresis on dialyzed, concentrated germination supernatant f l u i d . Only one protein band was detectable and t h i s band was shown to have p r o t e o l y t i c a c t i v i t y . Figure k i l l u s t r a t e s the pH p r o f i l e of the p u r i f i e d protease against casein; Figure 5 shows the thermal denaturation curve for p u r i f i e d protease. Both F i g . k and 5 suggested a single pro-t e o l y t i c a c t i v i t y and p-ovided corroborative evidence for the disc gel electrophoresis r e s u l t s . T a b l e V. Amino a c i d c o m p o s i t i o n o f 2-hr and 8-hr g e r m i n a t i o n medium s u p e r n a t a n t f l u i d 3 . . . . . , 2-Hr 8-Hr amino MCIO s u p e r n a t a n t s u p e r n a t a n t Lys i n e 3.75 3.80 H i s t i d i n e 1.99 1.95 A r g i n i n e 1.70 1.63 A s p a r t i c a c i d 6.97 6.93 T h r e o n i n e 2.40 2.10 S e r i n e 2.11 2.21 G l u t a m i c a c i d 10.3 11.9 P r o l i n e 1 .70 1.75 C y s t e i ne P r e s e n t P r e s e n t G l y c i n e 3.05 2.94 A l a n i n e 1.85 2.18 V a l i n e 1 .42 1 .37 Meth i o n i n e 0.21 0.29 1 s o l e u c i n e 2.36 2.35 Leuc i ne 1 .56 1.58 T y r o s i n e 0.67 0.65 P h e n y l a l a n i n e 0.46 0.52 E x p r e s s e d as m i c r o m o l e s o f amino a c i d p er 40 mg o f p r o t e i n i n t he s u p e r n a t a n t f r a c t i o n . Figure 3. Basic disc gel electrophoresis of dialyzed, concentrated 8-hr spore germination supernatant f r a c t i o n . Left and center photographs represent electrophoresis of super-natant f r a c t i o n for d i f f e r e n t time periods. The o r i g i n is at top of a l l photographs. The right photograph shows l o c a l i z a t i o n of protease a c t i v i t y in an unstained duplicate gel of the center photograph. The transparent area, which appears dark owing to the background, is the zone of casein hydrolysis. incubation was for 1 hr at 37 C, which caused some d i f f u s i o n of the band. 2k Ftgure W. The pH p r o f i l e o f protease a c t i v i t y a g a i n s t c a s e i n . One u n i t equals the amount of enzyme which w i l l s o l u b i l i z e the e q u i v a l e n t of 1 ug of bovine serum albumin In 1 min. Incubation was at 37 C f o r 30 min. Figure 5. The m i l denaturatton of protease acti v i t y . Portions (I ml) of purified protease were preIncubated for 20 min at the temperatures Indicated. Proteolytic activity was then measured against pH 8.0 casein, with Incubation at 37 C for 30 rain. 4 0 4 5 5 0 5 5 TEMPERATURE ( c ) 6 0 It was of interest to know whether the isolated protease would attack p u r i f i e d spore coats. The res u l t s of t h i s experiment are presented in Figure 6. Table VI l i s t s the amino acid composition of hyphal walls, ungerminated spore coats and 7 -hr spore coats. These analyses were repeated several times to check the r e p r o d u c i b i l i t y of the is o l a t i o n procedures. The individual residue values never varied more than 5%, and a l l analyses were q u a l i t a t i v e l y i d e n t i c a l . The hyphal wall and spore coat proteins appeared to be d i f f e r e n t types of protein q u a n t i t a t i v e l y . The residue per cent decrease during 7 hr of germination indicated a p r e f e r e n t i a l loss of aromatic residues. This observation was also supported by the fact that the protease was capable of attacking hippuryl phenylalanine but not benzoylarginine e t h y l e s t e r . These data suggested that the protease might be chymotrypsin-1ike. Effect of PMSF on proteo l y s i s and germination. The above information established that prot e o l y s i s occurred during spore germination, but, as yet, there was no conclusive evidence that t h i s r e l a t i o n s h i p was anything more than coincidence, to e s t a b l i s h a causal r e l a t i o n s h i p between prote o l y s i s and spore germination, a s e l e c t i v e protease i n h i b i t o r which would not disturb normal c e l l u l a r functions was required. Such an i n h i b i t o r was PMSF, a less toxic substitute for diiosopropylfluorophosphate (DFP). PMSF is p a r t i c u l a r l y e f f e c t i v e against chymotrypsin and to a lesser Figure 6. Hydrolysis of p u r i f i e d spore coats by Microsporum protease. Reaction mixture was 50 ug of protease/ml in pH 6.5 s a l i n e , with 1 mg/ml of spore coats. Spore coats were pelleted at 600 x £ for 1 min at the times indicated, and the o p t i c a l density at 280 nm was determined on the supernatant f r a c t i o n s . Symbols: 0 , reaction mixture, shaken at 150 rev/min in a New Brunswick rotary shaker at 37 C; • , substrate c o n t r o l , reaction mixture minus protease; 0 , enzyme co n t r o l , reaction mixture minus spore coats 27 Table VI. Amino acid composition of vegetative walls, spore coats, and 7 -hr spore coats of Microsporum gypseum 3. Vege- 7-Hr Amino acid t a t i v e Spore spore walls coats coats Lys i ne 2.37 9.50 8.10 14.7 H i s t i d i n e 1.17 4.88 3.78 22.5 Arg inine 1 .30 1.58 1.43 9.5 Aspartic acid 6.53 16.8 13.0 22.7 Threon i ne 6.29 8.30 6.30 24.1 Ser i ne 5.16 5.40 4.83 11.6 Glutamic acid 5.95 9.75 8.15 16.4 P r o l i ne 4.99 7.50 5.52 26.4 Cysteine Present Present Present Glycine 8.86 10.7 8.68 18.9 Alanine 5-75 4.65 4.33 6.9 V a l i ne 3.87 5.18 4.83 6.8 Meth i onine 0.467 0.250 0.180 28.0 Isoleucine 2.25 2.68 1.78 33.6 Leuci ne 2.92 2.72 2.30 16.4 Tyros ine 5.32 2.60 0.873 63.5 Phenylalanine 1 .00 2.35 0.973 58.7 Per cent decrease in residues duri ng 7 hr of germi na-t ion Expressed as micromoles of amino acid per 100 mg of walls or coats. degree, t r y p s i n . Figure 7 i l l u s t r a t e s the e f f e c t of various PMSF concentrations on protease a c t i v i t y against casein. Unfortunately, because of the limited s o l u b i l i t y of the i n h i b i t o r and the fac t that the solvent was fsopropanol, the highest usable concentration in our -k germination system was 10 M. Above th i s concentration, the isopropanol became s l i g h t l y i n h i b i t o r y to c e l l growth. The in h i b i t o r was e f f e c t i v e only when i t was preincubated with the enzyme. This was the expected r e s u l t , since PMSF is an i r r e v e r s i b l e type i n h i b i t o r . -4 The e f f e c t of 10 M PMSF addition on free amino nitrogen release into the germination supernatant f l u i d is depicted in Figure 8A. The i n h i b i t i o n of free amino nitrogen release became maximal a f t e r the f i r s t hour of incubation. To c l a r i f y the time course of PMSF i n h i b i t i o n of free amino nitrogen release, the data were replotted by using 1 hr as the point of o r i g i n (Figure 8B). The Inh i b i t i o n of free amino nitrogen release from 1 to 8 hr was approximately 70%. This value was in good agreement with the in v i t r o data (Figure 7). It was f e l t that the free amino nitrogen released during the f i r s t hour was probably from spores which were already committed to germinate at the time they were harvested and, therefore, were not susceptible to i n h i b i t i o n . An attempt was made to l o c a l i z e the p r o t e o l y t i c a c t i v i t y in the spore. S o n i c a l l y disrupted p e l l e t s , supernatant f l u i d s , and Figure 7. Ef f e c t of PMSF on protease a c t i v i t y against pH 8.0 casein, with preincubation time of 30 min at 37 C. Assay conditions were 30 min incubation at 37 C. NPI, no preincubation with 10 M PMSF. Effect on free amino nitrogen release of zero time addition of 10 M PMSF to pH 6.5 sa l i n e germination system, with incubation at 37 C. Re-plot of l e f t side data from the 1-hr sample point. Symbols: 0 , 10 M PMSF added; -k 0 , no 10 M PMSF addition 14 culture supernatant fluids were assayed for p r o t e o l y t i c a c t i v i t y . Most of the a c t i v i t y was l o c a l i z e d in the s o n i c a l l y disrupted p e l l e t and was released into the germination supernatant f l u i d with time (Figure 7A) . It seemed l i k e l y that the i n t r a c e l l u l a r a c t i v i t y was possibly just protease s o l u b i l i z e d from the spore coat by sonic di s r u p t i o n . A s i m i l a r experiment was done, but in the presence of PMSF. Due to i t s high negative charge, i t would seem u n l i k e l y that PMSF would cross the membrane, and hence, should act only on surface protease. Therefore, i f there were any true i n t r a c e l l u l a r a c t i v i t y , t h i s a c t i v i t y should increase r e l a t i v e to p e l l e t protease a c t i v i t y a f t e r washing and sonicating. This was not the case (Figure 9B), as i n t r a - and e x t r a c e l l u l a r levels of protease were s i m i l a r in the presence of PMSF. Hence, i t was concluded that the majority of the protease a c t i v i t y was l o c a l i z e d in the walls in the 20,000 x g_ p a r t i c u l a t e f r a c t i o n . Figure 10 i l l u s t r a t e s the e f f e c t of PMSF on germination by another approach. One must be cautious in extrapolating germination percentages to mean potential to outgrow and produce a hyphal network. Hence, a system was needed wherein one could c o r r e l a t e growth with germination percentage and i n h i b i t i o n e f f e c t . Here the germination medium was 1% (w/v) glucose and 0.1% (w/v) neo-peptone (pH 6.5). One-tenth per cent neopeptone, instead of the usual 1%, was used to minimize binding of PMSF by the medium. This medium was s u f f i c i e n t to support growth for several days. Figure 3. L o c a l i z a t i o n of p r o t e o l y t i c a c t i v i t y in germination supernatant f r a c t i o n s , s o n i c a l l y disrupted p e l l e t s and t h e i r supernatant f r a c t i o n s . A. No-10 M PMSF added; -k B. 10 M PMSF added at zero time. Symbols: 0 , germination supernatant f r a c t i o n (pH 6.5 saline) ; • , s o n i c a l l y disrupted 20,000 x £ p e l l e t ; A , s o n i c a l l y disrupted 20,000 x £ supernatant Figure 10. Effect of 10 M PMSF addition on 2k hr total c e l l u l a r protein values. Germination system; 1% (w/v) glucose, 0.1% (w/v) neopeptone, pH 6.5. Total incubation period was 2k hr at 37 C. -k 6 Symbols: 0 , 10 M PMSF added to 10 spores/ml at times indicated. • , no 10 _ Z t M PMSF added; 0.001% isopropanol (solvent for PMSF) added at zero time. 7 0 0 In t h i s medium, 100% m i c r o s c o p i c g e r m i n a t i o n was e f f e c t e d by 7 t o 8 h r . PMSF was added a t t he t i m e s i n d i c a t e d , and t h e s p o r e s were a l l o w e d t o g e r m i n a t e and grow f o r a t o t a l o f 2k h r . T o t a l c e l l u l a r p r o t e i n o f washed s p o r e s ( t h r e e - t i m e s washed i n pH 6 . 5 s a l i n e ) was measured and g e r m i n a t i o n p e r c e n t a g e s were a l s o d e t e r m i n e d a f t e r 2k h r ( F i g u r e 1 1 ) . Note t h a t the a d d i t i o n o f i n h i b i t o r a f t e r 7 h r had no e f f e c t on t o t a l p r o t e i n v a l u e s , i e . g r o w t h . -k T h i s i n f o r m a t i o n a l s o c l e a r l y d e m o n s t r a t e d t h a t 10 M PMSF had no e f f e c t on v e g e t a t i v e g r o w t h , s i n c e a d d i t i o n a f t e r 7 h r r e s u l t e d i n t h e same p r o t e i n v a l u e s as t h e c o n t r o l s y s t em w h i c h r e c e i v e d no PMSF. T e m p e r a t u r e r e s p o n s e o f p r o t e a s e a c t i v i t y and p r o t e a s e  s t i m u l a t i o n on g e r m i n a t i o n . The r e s u l t s o f F i g u r e 1 d e m o n s t r a t e d t h a t endogenous l e v e l s o f g e r m i n a t i o n (30% a t 25 C) c o u l d be s t i m u l a t e d t o 90% s p o r e g e r m i n a t i o n by r a i s i n g t h e i n c u b a t i o n t e m p e r a t u r e t o 37 C . F i g u r e 12 shows t h e t e m p e r a t u r e r e s p o n s e c u r v e o f t h e p r o t e a s e i n t h e c a s e i n a s s a y s y s t e m s . The enzyme and g e r m i n a t i o n t e m p e r a t u r e r e s p o n s e c u r v e s appea red t o be com-p a r a t i v e l y s i m i l a r up t o 37 C . Hence , one wou ld wonder whe the r i n c r e a s e d p r o t e a s e a c t i v i t y a t 37 C c o u l d e x p l a i n the i n c r e a s e d g e r m i n a t i o n . F i g u r e 13 i l l u s t r a t e s the e f f e c t o f a d d i t i o n s o f p u r i f i e d p r o t e a s e t o a s a l i n e g e r m i n a t i o n s y s t em a t 25 C . A t a p u r i f i e d p r o t e a s e c o n c e n t r a t i o n o f 63 u g / m l , t h e g e r m i n a t i o n Figure 11. Ef f e c t of 10 M PMSF addition on 24-hr germination percentages. Germination system; 1% (w/v) glucose, 0.1% (w/v) neopeptone, pH 6.5. IO - 2 - M PMSF was added to 10 spores/ml at times indicated. C, Control, no PMSF added; IPC, no PMSF, but 0.001% isopropanol added (solvent for PMSF). Temperature response curve for Microsporum protease. Assay conditions: pH 8.0 casein with incubation for 30 min at temperatures indicated. Figure 13- Effect of protease addition to 25 C germination system. Germination medium was pH 6.5 s a l i n e with a tota l incubation time of 6 hr. Protease, at concentrations indicated, was added at 0 time. 63 Control, 63 ug of protease per ml, heated at 100 C for 10 min p r i o r to addition. 38 J 8 NT •3 E el I 8 I 9 o o o o o 6s. . <g 6j 0> oo N o «n W CT NOiivNiwaao imSa§<i percentage at 25 C was equal to that obtained by incubating un-treated spores at 37 C. Heat i n a c t i v a t i o n of the protease (100 C for 10 min) completely destroyed i t s a b i l i t y to stimulate germination. D iscuss ion The concept that pro t e o l y s i s is an essential event in spore germination was suggested o r i g i r i a l l y more than 1 5 years ago (Powell and Strange. 1 9 5 3 ) . To test t h i s hypothesis c o n c l u s i v e l y , i t was necessary to have a method for s e l e c t i v e l y i n t e r f e r i n g with the p r o t e o l y t i c process. One approach would be the use of a s e l e c t i v e protease i n h i b i t o r , and PMSF was found to be such a compound. This reagent a c t i v e l y inhibited p r o t e o l y s i s in v i t r o and in vivo. This i n h i b i t o r also f u l f i l l e d the necessary require-ment of not i n h i b i t i n g vegetative growth. Sierra ( 1 9 6 7 ) was the f i r s t to attempt to i n h i b i t spore germination with a s e r i n e - a l k y l a t i n g agent. The germination of Baci1lus spores was inhibited completely by exposure to DFP. Unfortunately, 0 hr of exposure to the i n h i b i t o r was lethal to the c e l l s . The tolerance of fungal c e l l s to PMSF was possibly due to i t s high negative charge ( i e . - r e l a t i v e impermeability) and i t s reduced t o x i c i t y as compared to DFP. -k One question that arose was why 1 0 PMSF only inhibi t e d 70% of the p r o t e o l y t i c a c t i v i t y a f t e r 3 0 min of preincubation. Perhaps the i n h i b i t o r was not binding with high e f f i c i e n c y to the enzyme, either because of the conformation of the i n h i b i t o r or the enzyme acti v e s i t e . Hence, prolonged preincubation with PMSF would be necessary for complete i n h i b i t i o n . The prolonged preincubation of enzyme with i n h i b i t o r was not possible in t h i s in vivo system. Another p o s s i b i l i t y was that serine was not the a c t i v e - s i t e residue of t h i s protease but was very close to the a c t i v e s i t e . Hence, a l k y l a t i o n of the serine residue would only p a r t i a l l y inactivate the enzyme. This would be si m i l a r to the a l k y l a t i o n of methionine 192 (adjacent to a c t i v e serine 195) in chymotrypsin, which re-sulted in the loss of 80% of enzymatic a c t i v i t y (Lawson and Schramm. 1965). The r e l a t i o n s h i p between free amino nitrogen release (spore coat h y d r o l y s i s ) , the location and occurrence of the protease, and the PMSF i n h i b i t i o n data c e r t a i n l y support the contention that pro t e o l y s i s was es s e n t i a l for macroconidial germination. It was a singular f i n d i n g that the protease was the only large protein component recovered from the germination supernatant f l u i d . The thermal denaturation curve and the pH a c t i v i t y curve also supported the disc gel evidence that there was a single protease present in the supernatant f r a c t i o n . The Microsporum enzyme was found to be very s i m i l a r in pH optimum to Strange and Dark's enzyme (Strange and Dark. 1957), and also to the a l k a l i n e protease of B_. subt?1 is (Boyer and Carlton. 1968) . It is interesting that the B^. subt? 1 is al k a l ine protease was also susceptible to PMSF and had aromatic s p e c i f i c i t y . If a protease was p a r t i a l l y responsible for spore germination, one should be able to stimulate quiescent spores to germinate by a d d i n g the enzyme e x o g e n o u s l y under t h e p r o p e r c o n d i t i o n s . T h i s was the c a s e w i t h l y sozyme and the S t r a n g e and Dark enzyme (Gou ld and H f t c h i n s . 1 9 6 5 ) . T h i s p r e d i c t i o n was f ound a l s o t o be t r u e f o r t h i s s y s t e m . A d d i t i o n o f p r o t e a s e a t 63 j ig/ml r e s u l t e d i n g r e a t e r than a 2 . 5 - f o l d i n c r e a s e i n s p o r e g e r m i n a t i o n a t 25 C . The same e f f e c t can be a c h i e v e d by r a i s i n g t h e i n c u b a t i o n t e m p -e r a t u r e f r om 25 t o 37 C . Whether t h i s i n c r e a s e d s t i m u l a t i o n a t 37 C i n t h e absence o f added p r o t e a s e can be w h o l l y e x p l a i n e d by i n c r e a s e d p r o t e o l y t i c a c t i v i t y c a n n o t be c o n c l u s i v e l y a s c e r t a i n -ed a t t h i s t i m e . The f a c t t h a t exogenous p r o t e a s e c o u l d s t i m u l a t e q u i e s c e n t s p o r e s t o g e r m i n a t e i m p l i e d t h a t some s p o r e s may be p r o t e a s e r i c h w i t h r e s p e c t t o t h e p o p u l a t i o n . Tha t i s , abou t 30% o f t h e s p o r e s had enough p r o t e a s e t o g e r m i n a t e a t 25 C , whereas 60% o f t h e s p o r e s r e q u i r e d s u p p l e m e n t a t i o n under t h e s e e x p e r i m e n t a l c o n d i t i o n s . One e x p l a n a t i o n f o r t h i s o b s e r v a t i o n was s u g g e s t e d by t h e way i n w h i c h f u n g a l s p o r e s a r e f o r m e d . M a c r o c o n i d i a o f M_. gypseum a r e m u l t i - s e p t a t e s t r u c t u r e s c o n t a i n i n g a random d i s t r i b u t i o n o f n u c l e i per s e p t a t e d compartment ( E l - A n i . 1968). T h i s t y p e o f n u c l e a r d i s t r i b u t i o n i n s p o r e s has been o b s e r v e d a l s o i n Phycomyces ( H e i s e n b e r g and Cerda*-01medo. 1 9 6 8 ) . I f p r o t e a s e p r o d u c t i o n was a f u n c t i o n o f gene d o s a g e , t hen i t wou ld f o l l o w t h a t p o t e n t i a l i t y t o g e r m i n a t e a l s o s h o u l d f o l l o w the d i s t r i b u t i o n of nuclei per spore. Even under optimal conditions (37 C), one should see a gradient in the time required for the population of spores to germinate. Such a curve was observed when spores were germinated in the 0 .1% neopeptone, 1.0% glucose medium, and PMSF was added sequentially during the germination period. Sequential delays ih time of addition of i n h i b i t o r resulted in larger numbers of germinated spores at 37 C. This same type of time response was also seen in the s a l i n e germination system (no PMSF ad d i t i o n ) , in which the free amino nitrogen release ( i e . - amount of spore coat hydrolysis) increased with time. One additional fact common to these experiments was that there was a c e r t a i n percentage of spores (30%) which germinated with-out added protease at 25 C and were also not susceptible to i n h i b i t i o n . These spores presumably would have the largest amount of protease per spore in the population. If the protease concentration was s u f f i c i e n t l y high, the spore coats could be weakened enough so that j u s t t h e i r suspension in germination medium would be s u f f i c i e n t to i n i t i a t e germination. Hence, these spores would not be s e n s i t i v e to temperature s h i f t s or to i n h i b i t o r . If this was true, one should observe an i n i t i a l increase in free amino nitrogen release, re-s u l t i n g from the i n h i b i t o r - i n s e n s i t i v e germination of precommitted spores, followed by a plateau, r e s u l t i n g from i n h i b i t i o n of uncommitted spores. This is exactly the picture seen in the presence of 10 M PMSF. Also, fn the absence, of PMSF, one observes a fas t i n i t i a l r i s e in free amino nitrogen followed by a period of linear release. The results of t h i s study c l e a r l y support the concept that pro t e o l y s i s was an essential occurrence in dermatophyte germinati However, the question s t i l l remains as to whether the weakening of the spore coat per se or the appearance of protease hydrolysis products was the event essential to germination. I l l , THE RELATIONSHIP OF NUCLEAR SEGREGATION AND MACROCONIDIAL GERMINATION. Materials and Methods Nuclear D i s t r i b u t i o n Analysts Mycelial and spore nuclei were stained by the Giemsa method of Hejtmanek and Hejtma"nokova-Uhrova (1967), or the aceto-orceih method of El-AnI (1968) . Fourteen hundred germinated septated units of macroconidia were counted to determine the 37 C nuclear d i s t r i b u t i o n curve. Three hundred germinated septated units were counted to determine the 25 C and 37 C PMSF nuclear d i s t r i b u t i o n curves. Nuclear counts on germinated spores were made a f t e r 5 hrs (37 C incubation), or 8 hrs (25 C incubation). Chi-square and linear regression analyses were c a r r i e d out by standard s t a t i s t i c a l methods. Raw numbers were used in al1 cht-square c a l c u l a t i o n s . Linear regression analysis was performed . ^ r treated germination f r a c t i o n * ,^ , _ on a plot of — r - ^ 2 : — — : •?——-: versus the number of r control germination f r a c t i o n nuclei per germinated compartment. * Treated germination f r a c t i o n : - the f r a c t i o n of the to t a l popu-l a t i o n , including ungermtnated spores, which germinate. Results Evaluation of nuclear staini n g procedures. The aceto-orcein sta i n employedby E1-An i (1968), was useful in staining mycelial nuclei but did not give precise d e f i n i t i o n of germinating spore n u c l e i . The Giemsa nuclear staining method of Hejtmanek and Hejtma"riokova-Uhrova (1967) revealed well-defined spore and mycelial n u c l e i . It became evident that spore nuclei migrated r a p i d l y ' into the growing germ tube. Hence, i t was necessary to st a i n germinating spores at the e a r l i e s t possible times compatible with complete germination.. Optimal times for germination at 37 C were 5 hrs and at 25 C, 8 hrs. Gross d i s t r i b u t i o n of nuclei in mycelia and spores germinated at 37 C• Three day old mycelia were observed usually to possess two nuclei per hyphal septated u n i t . Four to eight nuclei were seen per hyphal growing t i p . One to eight nuclei were present per germinated spore septated unit at 37 C. Nuclear d i s t r i b u t i o n analysis of spore germination at 37 C, -L 37 C in the presence of 10 M PMSF and at 25 C. Under usual germination conditions 90 - 100% of the macroconidia present at 0 time, would germinate by 5 hrs at 37 C ( i t is not possible to detect i n i t i a l germ tube protrusion e a r l i e r than 3 h r s ) . Germ tube elongation continued u n t i l approximately 8 hrs at which time most of the spores endogenous reserves had been u t i l i z e d , (see s e c t i o n on Endogenous R e s e r v e s i n S p o r e s ) . However , i f s p o r e s -4 were g e r m i n a t e d a t 37 C i n t h e p r e s e n c e o f 10 M PMSF o r a t 25 C , o n l y 30% o f t h e m a c r o c o n i d i a w h i c h were p r e s e n t wou ld g e r m i n a t e (see s e c t i o n on PMSF I n h i b i t i o n o f M a c r o c o n i d i a l G e r m i n a t i o n ) . The 25 C g e r m i n a t i o n p e r c e n t a g e c o u l d be i n c r e a s e d t o 37 C v a l u e s by t h e a d d i t i o n o f p u r i f i e d g e r m i n a t i o n p r o t e a s e . The q u e s t i o n a r o s e as t o whe the r t h e r e were any s i m i l a r i t i e s between t h e 37 C , -4 10 M P M S F - i n h i b i t e d and 25 C p o p u l a t i o n s o f g e r m i n a t e d s p o r e s . I t was p r e v i o u s l y s u g g e s t e d t h a t t h o s e s e p t a t e d m a c r o c o n i d i a l u n i t s w h i c h c o n t a i n e d a h i g h enough number o f n u c l e i wou ld be p r e - c o m m i t t e d f o r g e r m i n a t i o n . Tha t i s , t h e s e p t a t e d compar tment wou ld have enough g e r m i n a t i o n p r o t e a s e p roduced d u r i n g s p o r e m a t u r a t i o n t o h y d r o l y z e t h e s p o r e c o a t s u f f i c i e n t l y so t h a t the s u b s e q u e n t s u s p e n s i o n o f t h e s p o r e i n g e r m i n a t i o n medium wou ld i n i t i a t e g e r m i n a t i o n . The s p o r e s wou ld then be i n s e n s i t i v e t o PMSF i n h i b i t i o n o r t e m p e r a t u r e s h i f t . One p o s s i b l e way t o v e r i f y t h i s h y p o t h e s i s f u r t h e r was by a n a l y z i n g n u c l e a r d i s t r i b u t i o n -4 c u r v e s o b t a i n e d f r om s p o r e s g e r m i n a t e d a t 37 C, 37 C i n 10 M PMSF and a t 25 C . F i g u r e s 1 4 , 15 and 16 i l l u s t r a t e t h e r e s u l t s o b t a i n e d f rom t h e s e a n a l y s e s . I n s p e c t i o n o f t h e d a t a i n d i c a t e d t h a t t h e n u c l e a r d i s t r i b u t i o n c u r v e s were s h i f t e d t o h i g h e r n u c l e a r numbers when -4 s p o r e s were g e r m i n a t e d a t 25 C o r i n the p r e s e n c e o f 10 M PMSF. Figure 14. D i s t r i b u t i o n of nuc le i per germinated spore com-partment, at 37 C. f(n) = the f r a c t i on of the tota l germinated compartments. D i s t r i b u t i o n of nuclei per germinated spore compartment at 25 C. f(n) = the f r a c t i o n of the total germinated compartments. 49 D i s t r i b u t i o n of nuclei per germinated spore -k compartment at 37 C in the presence of 10 M PMSF. f(n) = the f r a c t i o n of the t o t a l germinated compartments. 50 A l l of the experimental curves had good f i t to a chi-square d i s t r i b u t i o n . This suggested that the number of nuclei contained per spore septated unit was a random sample of the t o t a l nuclear population. S t a t i s t i c a l analysis of nuclear d i s t r i b u t i o n data. Prior to the a p p l i c a t i o n of s t a t i s t i c a l techniques to the d i s t r i b u t i o n data i t had been estimated that experimental counting error was no greater than 5% (a = 0.05). S t a t i s t i c a l comparison of the 37 C population to the 37 C, l o "^ M PMSF-inhibited and 25 C populations by chi-square analysis revealed that the control and PMSF-treated populations d i f f e r e d s i g n i f i c a n t l y even at the 0.001 l e v e l . A comparison of the 37 C, PMSF-treated and the 25 C popu-la t i o n indicated that the differences iii cumulative d i s t r i b u t i o n s were not s i g n i f i c a n t at the 0.05 l e v e l . Since the chi-square test is only s e n s i t i v e to differences in cumulative d i s t r i b u t i o n , the d i s t r i b u t i o n data was analyzed a l s o by linear regression analysis. This type of analysis (one-tailed), would indicate i f there were any s t a t i s t i c a l l y s i g n i f i c a n t differences throughout the populations in the d i r e c t i o n of higher nuclear numbers. It would indicate a l s o , i f one treatment was s i g n i f i c a n t l y more s e l e c t i v e for higher numbers of n u c l e i . Regression analysis suggested that both the 25 C and -k 37 C, 10 M PMSF-inhibited populations d i f f e r e d s i g n i f i c a n t l y (even at the 0.0005 l e v e l ) , from the 37 C population in t h e i r s e l e c t i o n of higher nuclear numbers. A comparison of slopes of the treated regression lines indicated that the 37 C, 10 M PMSF population s i g n i f i c a n t l y (at the 0.005 level) favored higher number of nuclei per septated unit for germination than that of the 25 C population. D i scuss ton Quantitative analysis of nuclear d i s t r i b u t i o n of M_. gypseum macroconidia indicated that the nuclear segregation mechanism was very s i m i l a r to that described in Phycomyces (Heisehberg and Cerda-Olmedo. T968). Nuclei are incorporated into the septated spore compartment by a random packaging of nuclei.from the av a i l a b l e mycelial nuclear population. This type of nuclear segregation during c o n i d i a t i o n has a concomitant e f f e c t on the p o t e n t i a l i t y for spore germination. If s e l e c t i v e pressures are applied to the germinating population, i e . - temperature s h i f t or germination protease i n h i b i t i o n , those spore compartments with higher nuclear numbers w i l l p r e f e r e n t i a l l y germinate. It seems l i k e l y that many of these spore compartments are pre-committed for germination. The f n f t f a l a c t i v a t i o n reactions have already occurred in the spore compartments due to the large number of nuclei present. The suspension of these spores in germination medium resulted in the immediate onset of germination processes which were not -k s e n s i t i v e to temperature s h i f t or the presence of 10 M PMSF. -k Comparative s t a t i s t i c a l analyses of 37 C, 37 C + 10 M PMSF -k and 25 C populations revealed that both the 37 C + 10 M PMSF and 25 C populations were s i g n i f i c a n t l y enriched in the number of nuclei possessed per germinated spore compartment. Linear regression analysis also suggested that use of PMSF was a more s e l e c t i v e treatment than was use of temperature s h i f t . The re-gression 1ine produced by PMSF addition s i g n i f i c a n t l y favored higher numbers of nuclei than does germination i n h i b i t i o n at 25 C. This is not an unexpected r e s u l t , since PMSF is an i r r e v e r s i b l e protease i n h i b i t o r . Once a protease molecule has bound a molecule of PMSF, i t is no longer a v a i l a b l e for further hydrolysis. How-ever,, at 25 C i t was possible that spores which were precommftted might release protease into the germination medium, which could subsequently a c t i v a t e spores containing lower numbers of n u c l e i . The enzymatic germination reaction can be slowed down by temperature s h i f t but the p o s s i b i l i t y of protease release and reabsorption cannot be eliminated. In view of the fact that M_. gypseum can be a human pathogen, the response of the germination system to temperature s h i f t may r e f l e c t a very useful adaptation to i t s p a r a s i t i c environment. Since the organism requires exogenous carbon and nitrogen sources for continued outgrowth processes a f t e r germination, i t would be unwise to germinate the total spore population at 25 C wherein conditions may not be s u f f i c i e n t l y adequate for the survival of the organism. However, at more elevated temperatures in a para-s i t i c environment, the mold wou.ld be better insured of a more suit a b l e environment for vegetative outgrowth and hence would germinate an optimal spore population. tV. THE BIOCHEMISTRY OF MACROCONIDIAL GERMINATION Materials and Methods Sporulation and spore germination in the presence of mannitol, fructose and acetate. Mannitol (1% w/v), fructose (1% w/v) or sodium acetate (3% w/v) was substituted f o r glucose (1% w/v) where indicated. Sporulation and spore germination always was carried out using the same carbon source. That i s , i f fructose was the carbon source used for spore germination studies, then these spores were harvested from a sporulation medium containing fructose as the single added carbohydrate. Preparation of c e l l - f r e e extracts. Extracts were obtained by the l i q u i d nitrogen method of'Bleyman and Woese (1969)• Debris was removed from the l i q u i d nitrogen grindate by centrifugation at 10,000 x g_ f o r 10 min at k C. In a l l cases, c e l l - f r e e extracts (CFX) were buffered in 0.2 M Trfs-HCl, pH l . k . A n a l y t i c a l determinations. Ribonucleic acid (RNA) was measured by the orcinol reaction (Schneider. 19^5) using yeast RNA standard. Carbohydrate extraction and i d e n t i f i c a t i o n . The 80% alcohol and s u l f u r i c acid soluble extracts were prepared as described by Lingappa and Sussman (1959). Alcohol soluble extracts were de-salted by passage through a mixed bed of Dowex-1-8 (Cl ) and Dowex 50W-X-4 (H ). Immediately a f t e r extraction and d e s a l t i n g , carbohydrates in the alcohol soluble pool were separated by de-scending paper.chromatography using a pyridine, ethyl acetate and water (50:120:40 v/v) solvent system. Sugars were detected by" developing the chromatograms with si1ver n i t r a t e (Trevelyan et a l . 1950), or benzidine (Harrocks. 1949), reagents. Unknown spots were i d e n t i f i e d by co-chromatography with authentic standards. Enzyme assays. Enzyme a c t i v i t i e s were determined by following the changes in o p t i c a l density of the reaction mixture at 25 C with a G i l f o r d Model 2400 automatic recording spectrophotometer (G i l f o r d Instrument Laboratories, Obelin, Ohio). Absorbance changes were measured with reference to reaction mixtures minus substrates. Measurements of reaction rates were made under conditions in which the i n i t i a l measured rate was linear with respect to time. S p e c i f i c a c t i v i t i e s are reported as the changes in op t i c a l density units per milligram of CFX protein per minute. Enzyme a c t i v i t i e s were measured as indicated below. A l k a l i n e phosphatase, glucose-6-PO^ dehydrogenase, aldolase, glyceraldehyde-3-PO^ dehydrogenase, enolase, fumarase and NADP-1 inked i s o c i t r i c dehydrogenase (as described in Biochimica Cata-logue, Boehringer Mannheim Corp., New York, N.Y.); mannitol-1-P0^ phosphatase (Strandberg. 1969); NADP-1inked mannitol dehydrogenase (Horikoshi e_t aj_. 1965); NAD-1 inked mannitol-1-P0^ dehydrogenase (Strandberg. 1969); fructokinase (Slei n et_ aj_. 1950); mannitol kinase (Sle in et_ a_l_. 1950); glucokinase (Slein et/a]_. 1950); transaldolase (De Vrfes e_t aj_. 1967); transketolase (De Vries et a l . 1967); i s o c i t r a t e lyase (Sjorgren et_ aj_. 1967); glucose phosphoenolpyruvate (PEP) transferase (Hanson and Anderson. I968); fructose PEP transferase (Hanson and Anderson. 1968); and mannitol PEP transferase (Hanson and Anderson. I968). Results Macrocoiiidlal ' u t i l i z a t i o n of alcohol - and acid-soluble carbo- hydrates (sal I ne-germination system). U t i l i z a t i o n of endogenous carbohydrate reserves is essential for the germination of conldia in many fungi. Since M_. gypseum macroconidia are capable of germinating in the absence of exogenous nutrients, i t was possible that endogenous carbohydrate reserves could provide part of the energy necessary for t h i s germination process. Chromatographic analysis of the 0 time 80% alcohol soluble pool, indicated the presence of glucose, fructose, mannitol, trehal and i n o s i t o l . The mannitol spot was no longer detectable 3 hr a f t e r i n i t i a t i o n of spore germination. Trehalose and i n o s i t o l , spots were no longer detectable a f t e r 8 hr. Figure 17 i l l u s t r a t e s the time course u t i l i z a t i o n of alcohol and acid-soluble carbo-hydrate f r a c t i o n s during germination. By 8 hr nearly a l l the a v a i l a b l e alcohol-soluble pool had been u t i l i z e d . No further germ tube elongation was observed a f t e r t h i s time. Enzyme s p e c i f i c a c t i v i t y changes during periods of spore  germination (glucose-germination system). 0.2 M Tris-HCl buffer, pH 7.4 was found to be superior to 0.2 M phosphate buffer, pH 7.4 for the preparation of c e l l - f r e e extracts. Phosphate at 0.2 M appeared to be an i n h i b i t o r to several of the enzymes assayed. Experimental variance in si n g l e enzyme s p e c i f i c a c t i v i t i e s between Ffgure 17. Ut 11 Ization of alcohol and acid soluble carbohydrate f r a c t i o n s during macroconidial germination. Symbols' f , Alcohol soluble f r a c t i o n 0 , Acid soluble f r a c t i o n subsequent experiments was never greater than 10%. The enzyme assays c i t e d were found to produce optimal reaction rates in this system. Table VII depicts the time course of the 12 metabolic enzymes assayed. Each enzyme also has been assigned an a c t i v i t y r a t i o (AR) , which is the 0 time s p e c i f i c a c t i v i t y divided by the 2k hr s p e c i f i c a c t i v i t y . The observed pattern does not appear to be an a r t e f a c t of c e l l d i s r u p t i o n . Spores disrupted by sonic o s c i l l a t i o n s or l i q u i d nitrogen gave ide n t i c a l AR level p r o f i l e s . Mannitol metabolism in germinating macroconidia (mannitol, fructose or glucose-germinating system). Sporulation on the various carbohydrates was completed by 7 days and the harvested spores from each carbohydrate source exhibited s i m i l a r germination time periods. That i s , spores harvested from the homologous carbo-hydrate source germinated to the 100% level by 8 hr in a l l cases. Macroconidia of M_. gypseum contained an NAD-1 inked mannltoI-J-PO^ dehydrogenase, a mannitol-1-P0^ phosphatase and an NADP-1inked mannitol dehydrogenase. Table VIII l i s t s the s p e c i f i c a c t i v i t i e s of glucose PEP transferase, fructose PEP transferase and mannitol PEP transferase a f t e r 2k hr germination in the presence of glucose, fructose or mannitol. Table IX l i s t s the s p e c i f i c a c t i v i t i e s of glucokinase, f r u c t o -kinase and mannitol kinase a f t e r 2k hr germination in the presence Table VII. Metabolic enzyme levels during spore germination. 24 Hrs a A c t i v i t y S p e c i f i c Ratio A c t i v i t y CAR) Fumarase 0, .100 0.072 0, .077 0 .030 3 .33 1socitrate lyase 0, .050 0.034 0, .021 0 .013 3 .85 Glyceraldehyde-3~phosphate .94 dehydrbgenase 0 .247 0.136 0 .124 0 .084 2 Transaldolase 0, .55 1.01 0 .90 0 .42 3 .69 Transketolase 0 .248 0.103 0 .068 0 .068 3 .65 Enolase 0 .310 0.126 0 .082 0 .084 3 .71 Aldolase I .05 0.908 0 .718 0 .500 2 . 10 Mannitol-1-phosphate dehydrogenase 1 .04 0.673 0 .416 0 .259 4 .02 Iso c i t r a t e dehydrogenase 0 .155 0.142 0 .158 0 .138 1 .12 G1ucose-6-phosphate dehydrogenase 0 .557 0.426 0 .408 0 .250 2 .23 Mannitol dehydrogenase 0 .620 0.668 0 .670 0 .555 1 .10 A l k a l i n e phosphatase 1 .12 1.27 1 .03 0 .233 4 .81 AO.D./min/mg c e l l - f r e e extract protein 0 time s p e c i f i c a c t i v i t y 24 hrs s p e c i f i c a c t i v i t y 0 Hrs 3 6 Hrs a 12 Hrs a S p e c i f i c S p e c i f i c S p e c i f i c t n z y m e s A c t i v i t y A c t i v i t y A c t i v i t y S p e c i f i c a c t i v i t y = A c t i v i t y Ratio (AR) = ON Table VIII. Hexose phosphoenolpyruvate transferase a c t i v i t i e s a f t e r 2k hrs spore germination. Carbohydrate source for sporulation and spore germination Enzymes Glucose Mannitol Fructose S p e c i f i c S p e c i f i c S p e c i f i c A c t i v i t y A c t i v i t y A c t i v i t y Glucose phosphoenolpyruvate transferase 0.022 0.026 0.028 Fructose phosphoenolpyruvate transferase 0.026 0.023 0.0^7 Mannitol phosphoenolpyruvate transferase 0.026 0.065 0.067 S p e c i f i c a c t i v i t y = AO.D./min/mg c e l l free extract protein Table IX. Hexose kinase a c t i v i t i e s a f t e r 2k hrs spore germination. Carbohydrate source f o r sporulation and spore germination Enzymes Glucose Mannitol Fructose S p e c i f i c S p e c i f i c S p e c i f i c A c t i v i t y A c t i v i t y A c t i v i t y Glucokinase 0.180 0.176 0.195 Fructokinase 0.0101 0.0102 0.0105 Mannitol kinase 0.0121 0.0110 0.0112 Sp e c i f i c a c t i v i t y = AO.D./min/mg c e l l free extract protein of glucose, fructose or mannitol. The above data suggested the presence of a " c o n s t i t u t i v e " glucokinase and that mannitol PEP transferase and fructose PEP transferase accumulated to higher s p e c i f i c a c t i v i t i e s when t h e i r respective substrates were added exogenously. It is of interest that both the fructose and mannitol PEP enzymes accumulated co-ordinately in the presence of fructose. Table X depicts the s p e c i f i c a c t i v i t y changes of mannitol dehydrogenase, mannitol-1-PO^ phosphatase and mannitol-1-P0^ dehydrogenase a f t e r 24 hr germination in the presence of glucose, mannitol or fructose. Identical enzyme accumulation data are obtained i f the spores are allowed to germinate for longer time periods, i . e . up to 120 hr. Evidence for the occurrence of a c o n s t i t u t i v e glybxylate  shunt (acetate or glucose-germination system). Table XI l i s t s the changes in s p e c i f i c a c t i v i t i e s of glyoxylate and TCA cycle enzymes during macroconidial germination. It is apparent that M_. gypseum possesses a c o n s t i t u t i v e glyoxylate shunt. It is also evident that germination in acetate resulted in increased levels of i s o c i t r a t e lyase and fumarase and a decrease in the level of i s o c i t r a t e dehydrogenase. RNA to c e l l - f r e e extract protein ratios during spore germination (glucose-germination system). Table XII l i s t s the RNA to CFX protein r a t i o s in 0 time and 24 hr spores. It is Table X. A c t i v i t i e s of mannitol 24 hrs germination. metabolism enzymes following Enzymes Carbohydrate source f o r sporulation and spore germination G1ucose S p e c i f i c Act i v i t y Mann i t ol S p e c i f i c Act i v i t y Fructose Spec i f ?c Act i v i ty Mannitol dehydrogenase Mann i tol-1-phosphate dehydrogenase 3 Mann i tol-1-phosphate phosphatase 0.5^0 0.300 9,5 0.995 0.320 16.0 1.07 0.568 27.0 a S p e c i f i c a c t i v i t y = AO.D./min/mg c e l l - f r e e extract protein. b S p e c i f i c a c t i v i t y = uM of inorganic phosphate released per 0.18 mg c e l l - f r e e extract protein per 75 min at 37 C. Table XI. Glyoxylate and t r i c a r b o x y l i c acid cycle enzyme a c t i v i t i e s during macroconidial germination. Carbohydrate source f o r sporulation and spore germination Enzymes Acetate Spec i f ic Act i v i t y c G1ucose Spec i f i c . A c t i v i t y 0 I s o c i t r a t e lyase Iso c i t r a t e dehydrogenase Fumarase 0.022 0.061 0.048 0.012 0.128 0.024 S p e c i f i c a c t i v i t y = AO.D./min/mg c e l l - f r e e extract protein Table XII. Relative concentrations of spore ribonucleic acid and protein in 0 time and 2k hr c e l l - f r e e extracts. j'm Ribonucleic acid Protein Ratio ribonucleic Cug/ml) Cug/ml) acid:protein 0 hours 2k hours 396 910 7,200 6,500 0.055 0.140 apparent that RNA accumulates to a greater extent than protei during spore germination. D iscuss ion Since there has been only one previous study of macroconidia1 germination in M_. gypseum . (Barash et^ aj_. 1967), t h i s preliminary investigation was undertaken to ascertain what basic processes occurred during spore germination, and hopefully to suggest possible germination s p e c i f i c proteins which might be examined subsequently in further d e t a i l . The composition of the alcohol-soluble pool was very s i m i l a r to that observed in Aspergi 1 lus (Horikoshi et_ aj_. 1965; Ikawa et a 1. 1968), and Neurospora (Ikawa et_ aj_. 1968; and Lingappa and Sussman. 1959). The ordered disappearance of mannitol and sub-sequently trehalose and i n o s i t o l was very s i m i l a r to the Aspergi11 us system. Horikoshi et_ aj_ (1965), have shown that mannitol is a repressor of trehalose and must be u t i l i z e d p r i o r to any trehalose degradat ion. The u t i 1 i z a t i o n pattern of the alcohol-soluble pool was found to be very s i m i l a r to that reported for Neurospora (Lingappa and Sussman. 1959). The biphasic nature of the u t i l i z a t i o n curve is probably the result of mannitol u t i l i z a t i o n p r i o r to the degrada-tion of trehalose and i n o s i t o l . It appears that M_. gypseum is unique in i t s a b i l i t y to u t i l i z e a considerable portion of the a c i d -soluble pool during spore germination. The pattern of enzyme s p e c i f i c a c t i v i t y changes observed during spore germination was somewhat unusual. A l l enzymic a c t i v i t i e s e i t h e r remained constant or decreased to some extent during the germination and hyphal outgrowth time period. This may be explained most e a s i l y by the f a c t that the vegetative nuclear-cytoplasmlc r a t i o was not preserved in the macroconidium. Upon germination, the concentrated spore nuclei migrate into the growing germ tube and r e e s t a b l i s h the lower mycelial nuclear cytoplasmic r a t i o (see section on Nuclear Segregation). Since germination enzymes of necessity would have to be synthesized p r i o r to and during germ tube elongation, i e . - "concentrated" p r i o r to rapid germ tube outgrowth, there could be a disproportionate amount of non enzymatic proteins synthesized during the outgrowth time period. This would give r i s e to an apparent decrease in enzyme s p e c i f i c a c t i v i t i e s during spore'germination. In t h i s type of a system, enzymes which maintain a constant s p e c i f i c a c t i v i t y during germination may in f a c t , represent a considerable amount of enzyme accumulation. It has been suggested by other workers that mannitol dehydro-genase and i s o c i t r a t e dehydrogenase are involved in fungal spore germination (Cal t r ider et^ a_l_. 1963 ; G o t t l i e b . 1966; Horikoshi e_^a_]_. 1965) (Proceeding of Soc. for General M i c r o b i o l . 1969 . The Glyoxylate Cycle as an Important Pathway in Fungal Morpho-genesis; by J.C. Galbraith and J.E. Smith; p. x i i ) . It is i n t e r e s t i n g t h a t t h e s e two enzymes have a low AR i n t h e M_. gypseum g e r m i n a t i o n system. I t i s p o s s i b l e t h a t t h e s e enzymes a r e i n v o l v e d i n g e r m i n a t i o n p r o c e s s e s . I n v e s t i g a t i o n s i n v o l v i n g o t h e r f u n g i and r e l a t e d o r g a n i s m s have s u g g e s t e d t h a t a I k a l f n e phosphatase ( G e z e l l u s and W r i g h t . 1965; Loom i s . 1969b; N a g a s a k i . 1968), and i s o c i t r a t e l y a s e ( C a l t r i d e r e t _ a j _ . 1963; G o t t l i e b . 1966) ( P r o c e e d i n g s o f Soc. f o r G e n e r a l M i c r o b i o l . 1969- The G l y o x y l a t e C y c l e as an Important Pathway i n Fungal M o r p h o g e n e s i s ; by J.C. G a l b r a i t h and J.E. S m i t h ; p. x i i ) , a r e p r e s e n t a t h i g h l e v e l s d u r i n g f u n g a l s p o r u l a t i o n . In a d d i t i o n , i t has been shown t h a t M_. gypseum a l k a l i n e phosphatase i s produced m a x i m a l l y d u r i n g p e r i o d s o f t e r m i n a l d i f f e r e n t i a t i o n (R. K e l I n , u n d e r g r a d u a t e t h e s i s . 1969). The s p e c i f i c i t y d a t a o b t a i n e d f o r a I k a l i n e p h o s p h a t a s e (R. K e l I n , u n d e r g r a d u a t e t h e s i s . 1969) have s u g g e s t e d t h a t i t may f u n c t i o n i h t h e s h u t - o f f o f i n t e r m e d i a r y m e t a b o l i s m and c a r b o -h y d r a t e r e s e r v e s y n t h e s i s . I t i s a l s o known t h a t t h e r e l e a s e o f i n o r g a n i c phosphates i s an e s s e n t i a l f e a t u r e o f m a c r o c o n i d i a 1 g e r m i n a t i o n i n M_. gypseum (W. Page, u n p u b l i s h e d d a t a ) . T h i s phosphate c o u l d be d e r i v e d e a s i l y from the a c t i o n o f a l k a l i n e p h o s p h a t a s e on f r u c t o s e - 6 - P O ^ and glucose-6-PO^. S i n c e t h e enzyme i s i n h i b i t e d by h i g h c o n c e n t r a t i o n s o f o r t h o p h o s p h a t e , (R. K e l I n , u n d e r g r a d u a t e t h e s i s ) , i t may r e g u l a t e i t s own s h u t - o f f by a t y p e o f e n d - p r o d u c t i n h i b i t i o n . The h i g h AR o f t h i s enzyme s u g g e s t s i t may not be accumulated during spore germination. Many fungi possess a c o n s t i t u t i v e glyoxalate shunt (Cochrane. 1966), and M_. gypseum appears to be no exception. When spores of th i s organism are germinated in acetate, i s o c i t r a t e lyase accumulated to a higher level than in glucose-germinated spores. In the presence of acetate the i s o c i t r i c dehydrogenase level decreases. This may be explained most rea d i l y by the observation of Osaki et^ aj_. (1968), which indicates that i s o c i t r a t e dehydrogenase is repressed by glyoxalate cycle products. Hence, an increase in glyoxalate cycle a c t i v i t y would be expected to result in a de-creased level of i s o c i t r a t e dehydrogenase a c t i v i t y . The f a c t that fumarase levels were high during acetate growth may be a result of increased succinate l e v e l s , and apparently a lack of i n h i b i t i o n of fumarase by glyoxylate cycle products. It is not s u r p r i s i n g that M_. gypseum macroconidia do not contain a high r a t i o of RNA to CFX protein. The terminal steps of sporulation occur under what are e s s e n t i a l l y starvation con-d i t i o n s (W. Page, unpublished data). This type of environment does not favor a high r a t i o of RNA to CFX protein. Fungal conidia cannot survive temperature extremes (Sussman and Halvorson. I966). In the case of £L gypseum, the macro-conidia are capable of surviving temperatures of 50 -55 C for only short time periods. However, macroconidia are capable of surviving for several months in the range k C to 25 C. The a b i l i t y of the macroconid turn to survive does not seem to reside in i t s possessing a protective physical environment, as is the case with fungal ascospores (Sussman and Halvorson. 1966). It is possible that the increased nuclear-cytoplasmic r a t i o in the macroconidium r e s u l t s in a large number of copies of essential information in the spore. This s e l e c t i v e concentration of nuclei increases the number of a v a i l a b l e targets and concomitantly the number of p o t e n t i a l l y lethal events that the spore is capable of s u r v i v i n g . V. EARLY EVENTS IN MACROCONIDIAL GERMINATION Materials and Methods Preparative procedures. For uptake studies, 7 day Roux f l a s k cultures were cooled to 4 C. A l l harvesting and p u r i f i c a t i o n procedures were c a r r i e d out at 4 C. P u r i f i e d spores were e q u i l i -brated at 37 C for 5 minutes prior to addition to the germination med ium. Manometric techniques. Oxygen consumption was measured by standard Warburg methods (Umbreit et a 1 . 1957), using si n g l e side arm f l a s k s . Glucose and neopeptone were contained in the side arm and added a f t e r 5 min preincubation at 37 C. 14 Uptake studies. The incorporation of C metabolites into whole macroconidia, t r i c h l o r a c e t i c acid (TCA) insoluble material and pools were determined by the f i l t r a t i o n method of Britten and McClure (1962). Whole c e l l counts were obtained by f i l t e r i n g macroconidia (5 mg) onto 1.2 y pore size f i l t e r s ( M i l l i p o r e Corp-ora t i o n , Bedford, Mass.) in a E8B p r e c i p i t a t i o n apparatus (Tracerlab, Waltham, Mass.). Conidia were washed with a 10X sample volume of germination medium at 37 C. A duplicate sample (5 mg) was combined with an equal volume of ice-cold 10% TCA and extracted at 0 C for 30 min. TCA-insoluble material was c o l l e c t e d by f i l -t r a t i o n (as above) and washed with a 10X sample volume of ice-cold 5% TCA. Dried f i l t e r s were placed in v i a l s containing 5 ml of s c i n t i l l a t i o n f l u i d ( L i q u i f l u o r , New England Nuclear Corp., Boston, Mass.) and counted in a model 725 l i q u i d s c i n t i l l a t i o n spectrometer (Nuclear Chicago Corp., Des Plaines, U l . ) . Chemical f r a c t i o n a t i o n of macroconidia. Macroconidial constituents (50 mg samples), were fractionated by the method of Roberts et_ aj_. (1955) as modified by C l i f t o n and Sobek (1961). Samples of the fractions, were plated onto s t a i n l e s s steel planchets, dried arid counted in a thin end-window Geiger tube attached to a Nuclear Chicago model 181A scaler equipped with an automatic gas-flow counter. Cel1-free extract (CFX) , preparation for DEAE chromatography. Macroconidia (375 mg) were germinated (2 hrs) in 25 ml of glucose-neopeptone (0.33 mg/ml and 0.25 mg/ml respectively) physiological s a l i n e containing 25 uC of a ' C protein hydrolysate. Total count incorporation irito TCA-precipitable material was approximately 1.0 x 10"7 CPM. Spores were broken by the l i q u i d nitrogen method of Bleyman and Woese (1969). Nucleic acids were pr e c i p i t a t e d with excess protamine s u l f a t e as suggested by Berlyn and Gile s (1969). The treated CFX was dialyzed (4 C, 2k hrs) against 2 changes (100 ~3 volumes)of 0.05 M Tris-HCl buffer (containing 10 M cysteine and 10 ^ M MgCl 2). The dialyzed CFX was chromatographed subsequently on DEAE-Sephadex. 76 DEAE-Sephadex chromatography. DEAE-Sephadex was prepared as an anionic exchanger, e q u i l i b r a t e d with 0.05 M Tris-HCl (con-taini n g 10 M cysteine and 10 M MgCl^) and packed in a 250 x 25 mm column. Dialyzed CFX was eluted with a 400 ml l i n e a r gradient of 0 to 1 M NaCl. Four ml f r a c t i o n s were c o l l e c t e d automatically and subsequently analyzed for a l k a l i n e phosphatase and mannitol dehydrogenase as described previously (see Biochemistry of Macro-coni d i a l Germination). One-tenth ml samples of the f r a c t i o n s containing enzymatic a c t i v i t y were assayed for r a d i o a c t i v i t y by mixing with 10 ml of s c i n t i 1 l a t i o n f l u i d (60 volumes toluene-L i q u i f l u o r + kO volumes absolute methanol) and counted in a l i q u i d s c i n t i l l a t i o n spectrometer. R e s u l t s S t i m u l a t i o n o f endogenous r e s p i r a t i o n . F i g u r e 18 d e p i c t s the e f f e c t o f a d d i t i o n s o f v a r y i n g amounts o f g l u c o s e and neopep tone on s p o r e r e s p i r a t i o n . I t was a p p a r e n t . t h a t 0.33 mg/ml g l u c o s e and 0.25 mg/ml neopep tone p r o v i d e d max ima l s t i m u l a t i o n . A d d i t i o n o f h i g h e r c o n c e n t r a t i o n s o f e i t h e r m e t a b o l i t e had no f u r t h e r s t i m u l a t o r y e f f e c t o n O ^ c o n s u m p t i o n . 14 Up take o f C g l u c o s e . F i g u r e 19 i l l u s t r a t e s the t i m e c o u r s e o f ' \ g l u c o s e u p t a k e d u r i n g e a r l y s p o r e g e r m i n a t i o n (germ t u b e s a r e not seen p r i o r t o 3 h r s ) . I t was e v i d e n t t h a t m a c r o c o n i d i a 14 were c a p a b l e o f immed ia te u p t a k e and c o n v e r s i o n o f C g l u c o s e i n t o T C A - i n s o l u b l e m a t e r i a l . 14 Up take o f C amino a c i d s . F i g u r e 20 p r e s e n t s t h e t i m e c o u r s e o f amino a c i d u p t a k e d u r i n g t h e f i r s t 2 h r s o f s p o r e g e r m i n a t i o n . I t was c l e a r t h a t t h e s p o r e was a b l e t o i m m e d i a t e l y a s s i m i l a t e and c o n v e r t a m i n o . a c i d s i n t o T C A - p r e c i p i t a b l e m a t e r i a l . 14 Up take o f C u r a c i 1 . F i g u r e 21 r e p r e s e n t s the t i m e c o u r s e 14 o f C u r a c i l u p t a k e d u r i n g e a r l y g e r m i n a t i o n t i m e p e r i o d s . A g a i n , i t was c e r t a i n t h a t m a c r o c o n i d i a were a b l e t o a c c u m u l a t e i m m e d i a t e l y and t o c o n v e r t u r a c i l i n t o T C A - p r e c i p i t a b l e m a t e r i a l . Owing t o t h e h i g h e x t e r n a l c o n c e n t r a t i o n s o f amino a c i d s , i t 14 wou ld seem u n l i k e l y t h a t much o f the C g l u c o s e wou ld be r e q u i r e d f o r p r o t e i n s y n t h e s i s . Hence , i t wou ld be o f i n t e r e s t t o know Figure 18. Stimulation of endogenous re s p i r a t i o n by glucose arid neopeptone. Spore concentration 10 mg/ml. Saline germination system. Symbols: § , endogenous; no glucose or neopeptone add i t ion. A , 0.33 mg/ml glucose, 0.05 mg/ml neopeptone 0 . 0 6 mg/ml glucose, 0 . 2 5 mg/ml neopeptone 0 0 . 3 3 mg/ml glucose, 0 . 2 5 mg/ml neopeptone or 0 . 6 6 mg/ml glucose, 0 . 5 0 mg/ml neopeptone Time (minutes) 14 Figure 19. C glucose uptake by germinating macroconidia. Saline germination system contained = spores at 14 10 mg/ml; 1 uc/ml C 0.25 mg/ml neopeptone. 10 mg/ml; 1 uc/ml C glucose; 0.33 mg/ml glucose, Symbols: © , Total counts A , TCA-precipitable counts 0 , Pool counts (Total counts = TCA pre-c i p i t a b l e counts) 79 50j 1 1 1 1 1 1 1 1 1 1 r 20 40 60 80 100 120 Time (minutes) 14 Figure 20. C amino acid uptake by germinating macroconidia, Saline germination system contained = spores at 14 10 mg/ml; 1 uc/ml C protein hydrolysate; 0.33 mg/ml glucose, 0.25 mg/ml neopeptone Symbols: 9 , Total counts A , TCA-precipitable counts 0 , Pool counts (Total counts = TCA-pr e c i p i t a b l e counts) 80 20 40 60 80 100 120 Time (minutes) 14 C u r a c i l uptake by germinating macroconidia. Saline germination system contained = spores at 14 10 mg/ml; 1 uc/ml C u r a c i l ; 0.33 mg/ml glucose 0.25 mg/ml neopeptone. Symbols: 8 , Total counts A , TCA-precipitable counts 0 , Pool counts (Total counts = TCA-pr e c i p i t a b l e counts) 81 Time (minutes) what type of macromolecules were synthesized from exogenous glucose. Table XIII l i s t s the composition of fractionated TCA-insoluble material which accumulated during early macroconidial germination. It was most interesting that the majority of the label was l o c a l i z e d In the acid alcohol-soluble f r a c t i o n . It had been suggested previously (The Biochemistry of Macro-coni d i a l Germination) that a l k a l i n e phosphatase and mannitol de-hydrogenase may represent sporulation and spore germination type proteins r e s p e c t i v e l y . If t h i s speculation was c o r r e c t , i t would be of interest to know i f the macroconidium d i f f e r e n t i a t e d synthe-t i c a l l y between these two proteins, one of which would be necessary for spore germination and the other not necessary and perhaps detrimental to germination processes. Table XIV l i s t s the p u r i f i -cation and recovery data from DEAE-Sephadex chromatography of the 14 two enzymes. Figure 22 i l l u s t r a t e s C amino acid incorporation into a l k a l i n e phosphatase and mannitol dehydrogenase peaks a f t e r 2 hrs of spore germination. It was apparent that mannitol de-hydrogenase was synthesized de novo during this time period.. It was u n l i k e l y that a l k a l i n e phosphatase was synthesized de novo to any s i g n i f i c a n t extent during early spore germination. Table XIII. Chemical f r a c t i o n a t i o n of C glucose TCA-precipitable material accumulated during spore germination. Counts are expressed as CPM/5 mg spores fr a c t i o n a t e d . 50 mg of spores were fractionated at each sample time. Time in Acid a l c o h o l - Hot TCA - NaOH-soluble Residue minutes soluble CPM soluble CPM CPM CPM 30 7,Hi 357 106 288 60 18,432 978 296 710 90 44,123 2,484 760 1,688 120 93,785 5,834 1,869 8,352 Table XIV. DEAE-Sephadex chromatography of 2 hr c e l l - f r e e extract. Enzyme Un i ts Units applted recovered Percent Specific., recovery a c t i v i t y applied Specific^. P u r i f i -a c t i v f t y cation recovered Alka1ine phosphatase 20 14.8 74 1.2 84.0 70X Mann i t o l dehydrogenase 17 10.1 60 0.57 48.5 85X Al k a l i n e phosphatase units = that amount of enzyme which w i l l cause a 1.0 O.D. (420 nm) change/min at 25 C Mannitol dehydrogenase units = that amount of enzyme which w i l l cause a 0.5 O.D. (340 nm) change/min at 25 C. S p e c i f i c a c t i v i t y = A0.D. /min/mg protein Figure 22. DEAE-Sephadex chromatography of a l k a l i n e phosphatase and mannitol dehydrogenase. Symbols: A , A l k a l i n e phosphatase units (1 unit =• the amount of enzyme which w i l l cause a 1.0 O.D. (420 nm) change/min at 2 5 C. 0 , Mannitol dehydrogenase units (1 unit = the amount of enzyme which w i l l cause a 0.5 O.D. (340 nm) change/min at 25 C. • , Rad i o a c t i v i t y . 1 4C CPM/ml/min x IO3 O O "7" "7" "7" *0 io o o cb ro t> n 1 1 1 1 1 1 1 1 1 1 1 r Discussion On the basis of the uptake studies presented, i t was clear that M_. gypseum macroconidia were able to assimilate immediately and convert glucose, amino acids and u r a c i l into macromolecules. This was a much more rapid response to germination conditions than was seen in A s p e r g i l l u s conidia (Horikoshi et_ aj_. 1965). The time course of glucose pool formation was very d i f f e r e n t from the pool accumulation seen in the case of amino acids and u r a c i l . The time course coordination of u r a c i l and amino acid pools seemed reasonable since the rate of protein synthesis was associated with RNA synthesis. The " s h i f t up" in the rate of u r a c i l conversion into TCA-insoluble material was in agreement with an e a r l i e r observation that the RNA to CFX protein r a t i o increased during spore germination (The Biochemistry of Maero-c o n i d i a l Germination). The glucose pool levels suggested a longer lag in incorporation into TCA-precipitable material. Since most of the exogenous glucose was incorporated into acid alcohol-soluble material ( i e . - l i p i d s , etc.) one may expect the synthetic rate to d i f f e r from those seen for RNA and protein. Lang and Lundgren (1970) have shown that the ba c t e r i a l spore l i p i d composition d i f f e r e d from vegetative 1ipid< material. One possible explanation for the large amount of glucose incorporated into the macroconidial l i p i d f r a c t i o n was that t h i s metabolite was being used to synthesize new mycelial type l i p i d s . The de novo synthesis experiment suggested that the spore was capable of ra p i d l y assessing the external environment arid d i f f e r e n t i a l l y synthesized a necessary germination protein. It seems that there was n e g l i g i b l e de novo synthesis of a l k a l i n e phosphatase (a sporulation type pr o t e i n ) , e s p e c i a l l y since i t has been demonstrated that a l k a l i n e phosphatase represented 1 -2% of the soluble protein in the organism (R. Kell n , unpublished data). If this enzyme was being synthesized de novo, one'would expect to see several times the number of counts a c t u a l l y accumu-lated under the enzyme peak.. In addition, M_. gypseum a Ikal ine phosphatase was capable of rapidly hydrolyzing fructose-6-PO^ and glucose-6-PO^ (R. Ke l l n , undergraduate t h e s i s ) . This f a c t and c e l l u l a r economy would make the synthesis of t h i s enzyme undesirable during a time when the organism was preparing for rapid macromolecular synthesis. VI. ISOLATION AND PRELIMINARY CHARACTERIZATION OF DEVELOPMENTAL MUTANTS Materials and Methods Mutagenesis and mutant screening conditions. Three hour germinating macroconidia (10 /ml, s a l i n e germination system), were mutagenized with N-methyl-N 1-nitro-N-nitrosuguanidine (NG) following the method of Yanagisawa et_ aj_. (1967). Survivors were plated on skim milk {2% w/v), glucose (1% w/v), neopeptone (Dlfco, \% w/v) agar plates (SGNP) , and incubated at 37 C for 5 to 7 days. Sus-pected mutants ( i e . - abnormal protein hydrolysis, unusual p i g -mentation or surface morphology), were rescreened at 37 C and subsequently maintained on glucose ( ] % w/v), neopeptone (Difco, \% w/v) agar slants (GNP) at 25 C. Selected mutants then were screened at 25 C on GNP plates to ascertain at what stage in development the mutation was expressed. Mutants which sporulated but exhibited abnormal hydrolysis (37 C, SGNP) were characterized further as to th e i r behavior in the saline germination system (37 C, 7 hrs), Growth and sampling conditions. A s u i t a b l e inoculum was taken from a 7 day GNP slant culture and inoculated Into glucose (\% w/v), neopeptone (Difco, \% w/v) broth medium, pH 6.5 and incubated on a Burrell wrist action shaker for k days at 25 C. The mycelium was c o l l e c t e d by aseptic f i l t r a t i o n and washed with ice-cold s t e r i l e physiological s a l i n e . The mycelial growth was resuspended in 100 ml of ice-cold s t e r i l e physiological s a l i n e and sheared at f u l l speed for 2 min in a V i r T i s ( V i r T i s Co., Gardiner, N.Y.) blender assembly (250 ml c a p a c i t y ) . The suspension was adjusted to 0.8 0-.D. at 660 nm (1 mg/ml) and inoculated onto Roux fla s k s in the usual manner. Since M_. gypseum does not sporulate when grown under shake-cultivation conditions, t h i s method is use-f u l in obtaining large amounts of non-sporulating mutant mycelia for inoculation purposes. At i n t e r v a l s , samples were scraped from the agar surface of Roux f l a s k cultures and washed with 300 ml of physiological s a l i n e and frozen (-20 C) u n t i l the developmental period was completed. C e l l - f r e e extracts (CFX) were prepared by the l i q u i d nitrogen method of Bleyman and Woese (1969) as described previously (see Biochemistry of Macroconidial Germination). Cultures also were observed by phase microscopy during the 7 day developmental period. Enzyme assays. A l k a l i n e protease a c t i v i t y was estimated as described previously (PMSF i n h i b i t i o n of Macroconidial Germination), except that 0.2 M barbital buffer (pH 9-0), was substituted for 0.2 M phosphate buffer (pH 8.0). N-acetyl glucosaminidase was assayed as suggested by Loomis (1969a). Tyrosinase was assayed spectrophotometrica1ly by the method of Horowitz and F l i n g (1953). The hydrolysis of hippuryl phenylalanine and hippuryl lysine was determined at 254 nm as described by Folk et_a_]_. (l960)(Folk and Schirmer. 1963). The aforementioned assay conditions provided maximal reaction rates. A l l i n i t i a l measured rates were linear with respect to time. Resu1ts The o r i g i n a l purpose of t h i s investigation was to obtain mutants d e f i c i e n t in a l k a l i n e protease, an enzyme which appeared to be necessary for spore germination (see section on PMSF Inhibition of Macroconidial Germination). Since i t has been suggested that the protease accumulated rather late in the developmental c y c l e , i t was thought such mutants might produce macroconidia which would not germinate. Such mutants would serve two purposes: corroboration of i n h i b i t i o n studies which suggested that the alka1ine protease was essential for spore germination; and possibly provide a means of assessing the role of the enzyme in other stages of development. Seventy mutant s t r a i n s have been isolated which were abnormal in e i t h e r sporulation or spore germination. Six of these mutants w i l l be described in t h i s section. Visual c h a r a c t e r i z a t i o n . Although M_. gypseum does not sporulate as e f f i c i e n t l y at 37 C as at 25 C, the higher temperature was chosen for screening because casein hydrolysis was much more pro-nounced under these conditions (10H5 mm zone of hydrolysis compared to 3-4 mm at 25 C). In the following designations, the SP subscript refers to the day at which a deviation from wild type development occurred; the SP superscript refers to the p r o t e o l y t i c a c t i v i t y of the mutant on SGNP medium.at 37 C, and Pig refers to the type of pigmentation produced on GNP medium a f t e r 7 days at 25 C. In a l l cases + re-presents the wild type character. SP+, Pig+. Brown surface pigmentation and reverse yellow pigmentation. Microscopic developmental sequence at 25 C on GNP; Day 1, germinated spores and mycelia; Day 2, conidiophores present; Day 3, con idiophores and aseptate conidia present; Day k, d i s t a l apical septation of the conidium began. Day 5, septation continued toward the proximal end of the conidophore. Day 6, septation of macroconidium completed. Day 7, mature macroconidia present ( i e . -capable of 100% germination), which could be dislodged from the con idiophore. Normal zone of casein hydrolysis. SP^jPig . A spontaneously-occurring mutant which had white f l u f f y surface growth. No con idiophores or conidia were produced. No zone of hydrolysis. SP|, Pig+(1) and SP^, Pig+(2). Normal pigmentation but grew as a " s w i r l " type of colony rather than r a d i a l l y . Macroconidia bulbous and larger than the wild type. Reduced zone of hydrolysis. SP°P, Pig-. S l i g h t l y reduced pigmentation. I n i t i a t i o n of sporulation delayed to Day k. Spores at 7 days small, non-septate with l i t t l e apparent i n t r a c e l l u l a r structure. Abnormally large zone of hydrolysis. 5?2> Pig-orn. Orange surface pigmentation. Aberrant coni-diophores at Day 2 but no spores v i s i b l e at Day 7. No zone of hy d r o l y s i s . SPj. , Pig-yln. Yellow surface pigmentation. Development normal, up to Day 5, at which time the macroconidia became aberrant and c o n s t r i c t e d . S l i g h t l y larger zone of hydrolysis. Spore germination mutants. Twenty mutants which produced macroconidia but had reduced zones of hydrolysis at 37 C were screened for t h e i r a b i l i t y to germinate in physiological s a l i n e at 37 C. Two of them (Table XV) had extremely low levels of spore germination, and were characterized further as to p r o t e o l y t i c a c t i v i t y per mg of conidia (Table XVI) and free amino nitrogen release per mg supernatant protein per mg of conidia (Table X V l l ) . It was evident that in these s t r a i n s , conidia were defective in the hydrolysis of both casein ( i e . - protease release into the germination supernatant) and spore coat material ( i e . - p r o t e o l y t i c attack of the spore coat substrate). Table XVI II l i s t s the a c t i v i t i e s of the wild type and the two mutants against hippuryl phenylalanine and hippuryl l y s i n e . The mutants did not hydrolyze either compound as e f f i c i e n t l y as the wild type. However, i t was interesting that mutant SPj,Pig+(2) only lost one-half of i t s hippuryl phenylalanine a c t i v i t y when assayed against hippuryl l y s i n e . Figures 23 and 2k i l l u s t r a t e the time course accumulation of a l k a l i n e protease, tyrosinase and N-acetyl glucosaminidase a c t i v i t y by SP~,Pig-, SP|,Ptg+(l) , SP|,Pig+(2) and SP +,Pig+(wi Id Sh Table XV. Spore germination at 37 C in sal i n e germination system, 7 hrs Incubation. Type Germination % SP + ,Pig+ 86 Sp|,Ptg+(l) 0 Sp|,Pig+(2) 5 Table XVI. Germination supernatant p r o t e o l y t i c a c t i v i t y . Saline germination system, 7 hrs incubation at 37 C. Type Protease units/mg protein/ml/min/mg spores SP + ,Pig+ 1.21 Sp|,PTg+(l) 0.09 SP|,P?g+(2) 0.10 Protease u n i t s . 1 unit = the amount of enzyme which w i l l hydrolyze the equivalent of 1 yg of bovine serum albumin. 96 Table XV i t . Free amino nitrogen release into the supernatant f r a c t i o n during spore germination. Saline germination system, 7 hrs incubation at 37 C. yg released/ml/mg " ^ p e protein/mg spores SP + ,Pig+ 0.256 Sp|,P?g+(l) 0.049 SP|,Pig+(2) 0.053 Table XVIII. Germination supernatant a c t i v i t y against synthetic substrates. A c t i v i t y = AO.D./mg protein/min. Type Hippuryl phenylalanine Hippuryl lysine SP + ,Pig+ 0.20 0.04 SP|,P?g+(l) 0.05 0.01 Sp|,P?g+(2) 0.04 0.02 Figure 23. Enzyme accumulation during macroconidial development. Protease units: 1 unit = the amount of enzyme which w i l l hydrolyze the equivalent of 1 ug of bovine serum albumin. N-acetyl g1ucosaminidase and tyrosinase units. AO.D./mg protein/min. Symbols: Protease 0 N-acetyl glucosaminidase Tyros i nase + + SP , Pig SO2,Pig-P r o t e a s e U n i t s / m g p r o t e i n / m l / m i n 3 C L Q ro O o o N - a c e t y l - g l u c o s a m i n i d a s e U n i t s T y r o s i n a s e 86 Figure 2k. Enzyme accumulation during macroconidial development. Protease units: 1 unit = the amount of enzyme which w i l l hydrolyze the equivalent of 1 yg of bovine serum albumin. N-acetyl glucosaminidase and tyrosinase units.= AO.D./mg protein/min. Symbols: A • Protease N-acetyl glucosaminidase Tyros i nase SP|,Pig+(D SP-,Pig+(2) P r o t e a s e U n i t s / m g p r o t e i n / m l / m i n CO h 3 CD 4^ "OL Q i n O CO O I I r N - a c e t y l - g l u c o s a m i n i d a s e U n i t s •fc-T y r o s i n a s e U n i t s 66 type). It is clear that none of the mutants accumulated a l k a l i n e protease or tyrosinase in the normal manner, however the two germination mutants (Sp|,Pig(l) and (2)) did accumulate normal levels of N-acetyl glucosaminida.se. It is most interesting that SP^jPig- did not accumulate any of,the enzymes to a s i g n i f i c a n t extent. NG sporulation mutants. During the se l e c t i o n of possible spore germination mutants, several unusual phenotypes were en-countered. A large class of abnormally pigmented mutants was isolated as well as a smaller class of near normally-pigmented mutants which produced very large zones of p r o t e o l y t i c c l e a r i n g at 37 C ( i e . - over-producers of protease). A l l of these mutants were found subsequently to be defective in the production of normal macroconidia. Figure 25 depicts the time course accumulation of a l k a l i n e protease, tyrosinase and N-acetyl glucosaminidase a c t i v i t y by SP°^ ,P i g- , SP2>Pig-orn, SP°'3 ,P i g-y 1 n mutants. 11 may be seen that a l l of them were affected in protease and tyrosinase accumula-t i o n . Both of the abnormally pigmented mutants did not accumulate N-acetyl glucosaminidase in the normal manner. Figure 25. Enzyme accumulation during macroconidial development. Protease u n i t s : 1 unit = the amount of enzyme which w i l l hydrolyze the equivalent of 1 ug of bovine serum albumin. N-acetyl glucosaminidase and tyrosinase units = AO.D./mg protein/min. Symbols: __, Protease , N-acetyl glucosaminidase , Tyrosinase • , SP°PfPtgi • , SP~,Pig-orn H , SP° P,Pig-yln P r o t e a s e U n i t s / m g p r o t e i n / m l / m i n 3 CD Q K3 O CO O N - a c e t y l - g l u c o s a m i n i d a s e U n i t s o T y r o s i n a s e U n i t s LOL D i scuss ion Mutant s t r a i n SP-,Pig+(l) appeared to be a " t y p i c a l 1 1 germina-ti o n protease-deficient mutant. It produced spores which did not germinate and, in addition, had the expected c h a r a c t e r i s t i c of not being able to accumulate normal levels of the a l k a l i n e protease during development. The absence of tyrosinase accumulation at Day 6 was expected, since the protease was thought to have aromatic s p e c i f i c i t y (see section on PMSF Inhibition of Macroconidial Germination) . SP^,Pig+(2) was a unique type of germination mutant. The accumulated level of protease a c t i v i t y was several times that of the wild type. In a d d i t i o n , a high level of a c t i v i t y persisted a f t e r Day 5- It was l i k e l y that t h i s protease molecule had suffered substrate s p e c i f i c i t y modification due to NG mutagenesis because tyrosinase did not accumulate in the usual manner, even though the protease s p e c i f i c a c t i v i t y was several times that of the wild type enzyme. The protease was proportionally twice as a c t i v e as would be expected against hippuryl l y s i n e , and the protease had an abnormal a c t i v i t y p r o f i l e against a number of n a t u r a l l y -occurring proteins (M. Dacy, undergraduate the s i s , 1970). It was possible that this s p e c i f i c i t y modification also allowed t h i s enzyme to escape the normal i n h i b i t i o n or degradation mechanism which caused a decrease in wild type a c t i v i t y a f t e r Day 5. It has been demonstrated by Pollock (1965) that point mutations in the peni-c i l l i n a s e molecule resulted in a concomitant a l t e r a t i o n in substrate s p e c i f i c i t y . The fact that both spore germination mutants produced bulbous macroconidia suggested that normal a l k a l i n e protease levels were also associated with spore coat i n t e g r i t y . SP° P,Pig-, SP^, Pig-orn and SP° P,Pig-yln were d i f f i c u l t mutants to c l a s s i f y . They appeared to be possible " c o n t r o l " type mutants ( i e . - t r a n s c r i p t i o n a l or t r a n s l a t i o n a l d e f e c t s ) , since several characters were affected ih each mutant. These mutants were not p a r t i c u l a r l y useful as far as providing information about the roles of s p e c i f i c developmental proteins. However, they did suggest that the M. gypseum developmental system may be very complex and highly branched. The absence of temporal u n i d i r e c t i o n a l pleiotrophy in these mutants ( i e . - abnormal early functions and some normal and some abnormal late f u n c t i o n s ) , suggested that several d i f f e r e n t developmental pathways may operate at the same time, each with i t s own s p e c i f i c regulatory system. SP2,Pig~ appeared to be a very useful mutant. This s t r a i n had a normal generation time and normal levels of several metabolic enzymes. However, i t did not sporulate or accumulate sporulation type proteins. Hence, t h i s mutant should prove most useful as a control for finding other sporulation s p e c i f i c proteins and as a means of separating metabolic and developmental processes.. VI I. ISOLATION AND PRELIMINARY CHARACTERIZATION OF CHROMATIN Materials and Methods Two l i t e r Erlenmeyer f l a s k s containing 600 ml of glucose, 1% (w/v) and neopeptone (Difco) , 1% (w/v), (pH 6.5), were inoculated with 10° conidia per ml and shaken at 250 RPM on a New Brunswick Controlled Environment Dry A i r Shaker (30 C) for k days. The mycelia were harvested by vacuum f i l t r a t i o n . The mycelial mat was washed k times with 1 l i t e r amounts of ice-cold physiological s a l i n e (pH 6.5). One hundred and f i f t y grams (wet weight) of mycelia were suspended in 350 ml of glycerol grinding solution (Stern. 1968), ( g l y c e r o l , 50% (w/v), 0.5 M sucrose, 0.001 M C a C l 2 and 0.05 M T r i s , pH 8.0). The re s u l t i n g s l u r r y was poured into an aluminum container and frozen by the addition of l i q u i d nitrogen. Several additions of l i q u i d nitrogen were necessary to completely freeze the material. The frozen s l u r r y was ground to a coarse powder and placed into a Waring Blender. When the temperature of the grinding solution reached -30 C, the blender was turned on to f u l l speed (110 v o l t s ) . Homogenization was continued u n t i l 0 C was reached. The homogehate again was poured into an aluminum container and p a r t i a l l y frozen ( i e . - to -20 C) by an addition of l i q u i d nitrogen. The p a r t i a l l y - f r o z e n s l u r r y subsequently was homogenized at f u l l speed u n t i l 0 C was reached. The freezing and homogenization steps were repeated u n t i l 70% microscopic breakage was obtained (usually 4 to 5 c y c l e s ) . The homogehate was f i l t e r e d two times through 1 layer of Miracloth and sub-sequently two times through three layers of Miracloth. The f i l t r a t e was centrffuged at 10,000 x £ for 10 min. The supernatant was removed with a large bore pipette and centrifuged at 30,000 x g_ (Sorvall SS-1, 16,000 RPM) for 1 hr. The 30,000 x £ pel let was resuspended (with the aid of a slowly rotating motorized homogenizer) into 90 ml of grinding s o l u t i o n . The resuspended nuclei were centrifuged again at 10,000 x £ for 10 min and the supernatant removed with a large bore pipette. The 10,000 x £ supernatant was centrifuged then at 30,000 x £ for 1 hr. The 30,000 x £ p e l l e t was resuspended in 10 ml of 0.01 M T r i s , pH 8.0, and s t i r r e d slowly overnight at 4 C. The nuclear lysate was centrifuged at 25,000 RPM (Spinco, 30 head), f o r 20 min. The 25,000 RPM supernatant was removed and layered onto a gradient con s i s t i n g of 1 ml of 50% sucrose (in 0.01 M T r i s , pH 8.0) and overlayed by 0.5 ml of 20% sucrose (In 0.01 M T r i s , pH 8.0). The chromatin was recovered by cent r i f u g a t i o n for 14 hr at 35,000 RPM (Spinco, SW39 ro t o r ) . The 35,000 RPM p e l l e t was resuspended in 0.01 M T r i s , pH 8.0, and dialyzed (4 hr) against 200 volumes of 0.01 M T r i s , pH 8.0. Aggregated material was removed by c e n t r i -fugation at f u l l speed in a c l i n i c a l centrifuge (approx. 3,000 x £) for 30 sec. The clear supernatant was used for a l l subsequent experiments. This material exhibited the typical u l t r a v i o l e t absorption spectra reported for isolated chromatins (Bonner et a l . 1968). Extraction of histone. Histone extraction using O.k N h^SO^ or 0.2 N HC1 was c a r r i e d out as suggested by Bonner et_ aj_. (I968). Histone extraction was also attempted by the 1 M C a C ^ method of Mohberg and Rusch (I969). Chemical f r a c t i o n a t i o n of chromatin. Chromosomal constituents were estimated as suggested by Marushige and Dixon (1969). Template a c t i v i t y determinations. DNA for template a c t i v i t y determinations was obtained by p e l l e t i n g chromatin from 3 M CSC1 (35,000 RPM for 2k hr, Spinco SW39 rotor) as described by Bonner et a 1. (1968). Template a c t i v i t y of DNA and chromatin was deter-mined as'suggested by Marushige and Dixon (1969). Disc gel electrophoresis. Chromosomal basic proteins were characterized by electrophoresis in polyacrylamide gels according to Bonner et al_. (1968) . Basic protease assay conditions. Nine-tenth ml of f i s h histone (1 mg/ml) was incubated with 0.1 ml of the nuclear lysate at 37 C for 1 hr. The reaction was terminated by the addition of 100% t r i c h l o r o a c e t i c acid (TCA), to give a f i n a l concentration of 25% (w/v). The control mixtures i n i t i a l l y contained 25% TCA,-and a.l 1 other constituents. TCA-insoluble material was pelleted at 107 12,000 x cj_ for 20 min. The protein concentration In the 12,000 x g_ supernatant was used as a measurement of p r o t e o l y t i c a c t i v i t y . Chemical determinations. DNA was quantItated by the d i -phenylanine method with c a l f thymus DNA as the standard (Dische. 1955). Results Chromatin i s o l a t i o n . Attempts to i s o l a t e fungal nuclei by the methods suggested f o r l i v e r or pea (Bonner et_ aJL 1 9 6 8 ) , were not successful. Nuclei always were sheared by these methods and speed s u f f i c i e n t to p e l l e t chromatin resulted in gross RNA contamination of the nuclear f r a c t i o n . It was f e l t that a more viscous grinding medium would afford greater protection to the nuclei during the c e l l breakage step. The glycerol grinding medium of Stern (1968) admirably s a t i s f i e d t h i s requirement. Table XIX l i s t s the recovery of DNA during the p u r i f i c a t i o n procedure. DNA recovery was quantitative in nuclear f r a c t i o n s 1 and 2 . A minimal amount of DNA was l o s t in the subsequent p u r i f i c a t i o n steps, and 150 - 200 ug of DNA as chromatin was recovered from 150 gm (wet weight) of mycelia. Chemical cha r a c t e r i z a t i o n of chromatin and nuclear f r a c t i o n s . Table XX l i s t s the DNA, RNA, total protein and basic protein composition of M_. gypseum chromatin and nuclear f r a c t i o n 2 . It was evident that there was an unusually low chromatin protein to DNA r a t i o and unusually low r a t i o s of basic protein to DNA in the chromatin and nuclear f r a c t i o n . The amount of chromosomal non-basic protein and RNA were close to the values reported f o r other eucaryotic c e l l s (Bonner et a 1 . 1 9 6 8 ) . Table XIX. Recovery of DNA during M_. gypseum chromatin i s o l a t i o n . Fraction Percent Recovery Homogenate 100% 1st 10,000 x g_ pel let 0% 1st 10,000 x £ supernatant 100% 1st 30,000 x £ supernatant 5% 1st 30,000 x £ p e l l e t (nuclear f r a c t i o n #1) 95% 2nd 10,000 x g_ pel let 0% 2nd 10,000 x £ supernatant 95% 2nd 30^000 x £ supernatant 5% 2nd 30,000 x £ p e l l e t (nuclear f r a c t i o n #2) 90% 25,000 RPM p e l l e t 0% 25,000 RPM supernatant 90% Sucrose supernatant 10% Sucrose pel let 80% 3,000 x £ pel l e t 10% 3,000 x £ supernatant 70% a The percentage values are averages from h separate experiments. Standard deviation is - 5%. Table XX. Chemical composition ratios of M_. gypseum chromatin and nuclear f r a c t i o n s Total DNA RNA O.k N h^SO^ 0.2 N 1.0 M Source protein . . HCl CaCl-y protein ^ . 2 r protein ^ .' r protein Nuclear f r a c t i o n 2 a 15(- 0.05) 1 5.001 (- 0.02) 0.05° 0.03° 0.05° Chromatin 1 3 1.05 (-0.05) 1 0.05 (-0.02).. 0.03° 0.05° — ° Values are the averages from six separate experiments. k Values are the averages from 2 separate experiments. Standard deviations are indicated in parentheses. C (-0.05) Table XXI. Histone to DNA ra t i o s of Stage V nucleohistone (1.5 mg) before and a f t e r incorporation irito the Isolation procedure. Source Historie DNAa Before addition 1.05(-0.05) 1 After i s o l a t i o n 1.00(-0.05) 1 DNA values corrected for the amount of fungal DNA present. Standard deviations are indicated in parentheses. The low h i s t o n e t o DNA r a t i o s s u g g e s t e d t h a t h i s t o n e - l i k e p r o t e i n s e i t h e r were not a ma jo r component o f t he chromosomal p r o t e i n s In t h i s o r g a n i s m o r were deg raded d u r i n g t h e i s o l a t i o n p r o c e d u r e . To a s s e s s t h e p o s s i b i l i t y o f t h e l a t t e r a l t e r n a t i v e s t a g e V f i s h n u c l e o h i s t o n e ( the gene rous g i f t o f K. M a r u s h i g e ) was added a t 1.5 mg c o n c e n t r a t i o n t o n u c l e a r f r a c t i o n II and t h i s m a t e r i a l was c a r r i e d t h r o u g h t h e s u b s e q u e n t i s o l a t i o n s t e p s . T a b l e XXI l i s t s the h i s t o n e t o DNA r a t i o s o f f i s h n u c l e o h i s t o n e b e f o r e and a f t e r r e c o v e r y f r o m the M_. gypseum c h r o m a t i n f r a c t i o n . I t was c l e a r t h a t t h e r e was no change i n t h e s e r a t i o s . F i g u r e 26 i l l u s t r a t e s t h e g e l p r o f i l e o f s t a g e V f i s h b a s i c p r o t e i n s a f t e r i s o l a t i o n f r om t h e M_. gypseum n u c l e a r f r a c t i o n . T h i s p r o f i l e was i d e n t i c a l t o t h a t r e p o r t e d f o r t h i s m a t e r i a l by M a r u s h i g e and D i x o n ( 1 9 6 9 ) . T a b l e XX I I l i s t s the r e s u l t s f rom a b a s i c p r o t e a s e a s s a y o f t he n u c l e a r l y s a t e m a t e r i a l . Here a g a i n , i t was e v i d e n t t h a t t h e r e was no d e t e c t a b l e h y d r o l y s i s o f f i s h b a s i c p r o t e i n s . I t a p p e a r e d u n l i k e l y t h a t p r o t e o l y t i c d e g r a d a t i o n o f h i s t o n e s d u r i n g c h r o m a t i n i s o l a t i o n was a r e a l p o s s i b i l i t y . P o l y a c r y l a m i d e g e l c h r o m a t o g r a p h y o f the s m a l l amount o f e x t r a c t a b l e b a s i c p r o t e i n ( e i t h e r f rom c h r o m a t i n o r n u c l e i ) d i d not i n d i c a t e any bands w h i c h m i g r a t e d i n t h e h i s t o n e a r e a . T e m p l a t e a c t i v i t y . F i g u r e 27 d e m o n s t r a t e s the t e m p l a t e a c t i v i t y o f DNA and c h r o m a t i n w i t h E s c h e r i c h i a c o l i RNA p o l y m e r a s e . Figure 26. Polyacrylamide gel p r o f i l e of Stage V f i s h nucleo-histone a f t e r recovery from M_. gypseum i s o l a t i o n procedure. 20 ug of basic protein in 0.2 N HC1 containing 20% sucrose was applied to the g e l . Electrophoresis time= 1 hr-Symbols: Fr, Front H , Histone P , Protamine e X X t l . Basic protease assay of M_. gypseum nuclear lysate ug/ml protein s o l u b i l i z e d pH 5.0 pH 8.0 Control 50 48 Reaction hi 52 Results obtained a f t e r incubation at 37 C for 1 hr. Histone concentration was 1 mg/ml. Reaction mixture was 0.9 ml histone +0.1 ml nuclear lysate. Figure 27. RNA synthesis directed by M_. gypseum chromat in and DNA as a function of template concentrations. 114 ug of E^ . col i RNA polymerase / 0.25 ml reaction mixture. Incorporation by the enzyme alone has been subtracted. 115 pg DNA as Chromatin or DNA/025ml It was apparent that M_. gypseum chromatin possessed kO - 50% of the template a c t i v i t y of DNA. There was no detectable hydrolysis of high s p e c i f i c a c t i v i t y H mouse RNA (the generous g i f t of1. Dr. J. Hudson) by e i t h e r the chromatin or DNA preparation. Hence, the decreased chromatin a c t i v i t y was not due to the presence of RNase. 117 D i scuss ion On the basis of the results reported, i t is concluded that basic proteins were not a major f r a c t i o n of M_. gypseum chromosomal proteins as obtained by the i s o l a t i o n and growth conditions. The p r o t e o l y t i c degradation of histones during chromatin i s o l a t i o n seemed a remote p o s s i b i l i t y . Since 70% of the DNA in the homogenate was recovered as chromatin, i t was l i k e l y that the material isolated was a representative sample of the chromatin in the fungal c e l l . It was found that RNA to DNA ratios greater than 0 .05:1.0 should raise suspicion of ribosomal contamination. Hence, d i r e c t comparison of these results with those obtained by other workers using Neurospora (Dwivedi et a 1. 1969), and yeast (Tonino and Rozijn. 1966), was not possible. However, in the case of Neurospora, i t appeared that this mold also possessed low "histone" to DNA r a t i o s . Stumm and van Went (1968) have reported that the water mold Allomyces does not contain histones. The a v a i l a b l e cytochemical evidence also suggests that histones are lacking in fungi (Bakerspiegel, 1959; Bloch. 1966). The high template a c t i v i t y of fungal chromatin perhaps was expected, since histones e s s e n t i a l l y were absent from this material. Van der V l i e t , Tonino and Rozijn (1969) have reported that yeast chromatin also exhibited high r e l a t i v e template a c t i v i t y . 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