UBC Theses and Dissertations

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UBC Theses and Dissertations

Localization and function of proteolytic enzymes in Bacteroides amylophilus H-18 Hullah, William Arthur 1969

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LOCALIZATION AND FUNCTION OF PROTEOLYTIC ENZYMES IN BACTEROIDES AMYLOPHILUS H-l8. by WILLIAM ARTHUR HULLAH B.Sc. (Agr.), U n i v e r s t i y of Guelph, ( I 9 6 7 ) . A THESIS SUBMITTED IN PARTIAL FULFILMENT-OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE DEPARTMENT . OF MICROBIOLOGY We.accept t h i s t h e s i s as conforming.to the required standard THE UNIVERSITY OF BRITISH COLUMBIA Ju l y , 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and S t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s thes. is f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada D e p a r t m e n t o f i . ABSTRACT B a c t e r o i d e s a m y l o p h i l u s p r o d u c e s a p r o t e o l y t i c enzyme o f w h i c h '20%. i s l i b e r a t e d i n t o t h e medium and 80%. i s bound.to t h e c e l l . Treatment .of t h e . c e l l s w i t h t o l u e n e o r m e c h a n i c a l d i s -i n t e r a t i o n does not i n c r e a s e t h e p r o t e o l y t i c a c t i v i t y , i n d i c a t i n g t h a t a l l t h e p r o t e a s e i s s u p e r f i c i a l l y l o c a t e d a t t h e b a c t e r i a l s u r f a c e . L e s s t h a n l%'.;of t h e t o t a l p r o t e a s e a c t i v i t y i s r e l e a s e d f r o m t h e c e l l s by o s m o t i c shock p r o c e d u r e s w h i c h i n d i c a t e d t h a t t h e p r o t e a s e i s n o t . f r e e i n t h e . p e r i p l a s m i c space. S p e r o p l a s t . f o r m a t i o n l i b e r a t e s 33% o f t h e . c e l l bound p r o t e a s e k0%.;of w h i c h i s s e d i m e n t a b l e by p r o l o n g e d h i g h speed c e n t r i f u g a t i o n . S o n i c d i s r u p t i o n o f s p h e r o p l a s t s r e l e a s e s 72% o f t h e p r o t e a s e . A f t e r g e n t l o s m o t i c r u p t u r e hh)% o f t h e enzyme a c t i v i t y , r e m a i n e d bound t o t h e s p h e r o p l a s t e n v e l o p e . P r o l o n g e d h i g h speed c e n t r i f u g a t i o n r e s u l t s i n t h e . s e d i m e n t a t i o n o f a l l b u t ±6% o f t h e t o t a l enzyme. T h e . r e s u l t s g i v e f u r t h e r e v i d e n c e t o t h e p a r t i c l e bound n a t u r e o f t h e p r o t e a s e o f B a c t e r o i d e s a m y l o p h i l u s . B a c t e r o i d e s a m y l o p h i l u s has a f a s t e r r a t e o f g r o w t h , w i t h a . r e d u c e d l a g phase and p r o d u c e s a g r e a t e r . c e l l y i e l d when t r y p t i c p e p t i d e s a r e i n c l u d e d i n t h e b a s a l medium. R a d i o a c t i v e amino a c i d s a r e i n c o r p o r a t e d i n t o . c e l l s i n s i g n i f i c a n t amounts, i n d i c a t i n g t h a t t h e y were n o t e x c l u d e d f r o m t h e c e l l by a p e r -m e a b i l i t y b a r r i e r . The amount o f i n c o r p o r a t i o n o f amino i i a c i d s i s f o u n d t o v a r y f o r d i f f e r e n t amino a c i d s and i s shown •Ik t o be c o n c e n t r a t i o n dependent. The exogenous C a m i n o . a c i d s were i n c o r p o r a t e d i n t o t h e c e l l p r o t e i n e i t h e r d i r e c t l y o r • a f t e r an i n t e r c o n v e r s i o n s t e p . The i n h i b i t i o n o f l ^ C amino a c i d s u p t a k e b y . p e p t i d e s and t h e d i r e c t u p t a k e o f -^C o l i g -p e p t i d e s d e m o n s t r a t e d p e p t i d e u p t a k e by B a c t e r o i d e s a m y l o p h i l u s . The r e s u l t s s u g g e s t t h a t o r g a n i c n i t r o g e n c o n t r i b u t e s t o t h e n u t r i t i o n o f B a c t e r o i d e s a m y l o p h i l u s . i i i T a b l e - o f . C o n t e n t s P a g e I N T R O D U C T I O N . . . . . . . . . . . . ; . . . . . . ; . . 1 L I T E R A T U R E . ' R E V I E W ; . 2 .1. L o c a l i z a t i o n o f P r o t e o l y t i c E n z y m e s . . . . . 3 I I . S p h e r o p l a s t F o r m a t i o n . . . . . . . . . . . . . . . . 3 I I I . E f f e c t o f A m i n o A c i d s a n d P e p t i d e s o n P r o t e a s e P r o d u c t i o n a n d B a c t e r i a l G r o w t h . . . . . . 5 I V . A m i n o A c i d U t i l i z a t i o n b y . R u m e n B a c t e r i a . . . : . 6 V . A m i n o A c i d I n c o r p o r a t i o n b y R u m e n B a c t e r i a . ; . 7 V I . P e p t i d e I n c o r p o r a t i o n b y R u m e n B a c t e r i a . . . 8 V I I . A m m o n i a I n c o r p o r a t i o n b y R u m e n B a c t e r i a . . . 9 V I I I . A m m o n i a I n c o r p o r a t i o n b y B . ' a m y l o p h i l u s . . . . 10 M A T E R I A L S AND METHODS . . . . . . . . . . . . . . . . . 12 . I . J M e d i a . . . . . . . . . . . • - . . . . : . . . . . . . . • 12 I I . B a c t e r i a a n d C u l t u r e C o n d i t i o n s . . . : . . . . lh I I I . . M e a s u r e m e n t . o f G r o w t h . . . . . , . . . . . . . . . . . . 15 I V . A m i n o A c i d I n c o r p o r a t i o n . . . 15 V . D r y . w e i g h t , p r o t e i n c o n t e n t a n d a m i n o a c i d . c o m p o s i t i o n o f C e l l s . . . • . . . . . . . . • • . • . 17 V I . P r o t e a s e D e t e r m i n a t i o n . . . , . , . . . . . . . . : . 18'. R E S U L T S A N D D I S C U S S I O N . . . . . . . . . . . . . . . 19-P A R T I : L o c a l i z a t i o n o f p r o t e o l y t i c e n z y m e s . . 19 I . O s m o t i c s h o c k p r o c e d u r e s 27 I I . O s m o t i c s h o c k . . . . . . . . . . : . : - 2 9 I I I . T h e E f f e c t o f a n a e r o b i c c o n d i t i o n s o n P r o t e a s e L i b e r a t i o n b y o s m o t i c s h o c k '. . . . . . . . . - 3 1 I V . T h e E f f e c t o f : s u c r o s e o n t h e P r o t e a s e A s s a y . . 3h' . V . M o d i f i e d o s m o t i c s h o c k p r o c e d u r e . . . . . . ; . . . . '38' V I . S p h e r o p l a s t f o r m a t i o n . . . . . . . . . . . . . . 38 i v P A E T . I I : Uptake ;of Amino A c i d s and P e p t i d e s . . • %6 .1. T h e . E f f e c t o f O r g a n i c N i t r o g e n on Growth. . . • 46 I I . Amino A c i d C o m p o s i t i o n o f ;B. a m y l o p h i l u s . . :-50 . I I I . The Uptake .of l l + c M i x e d Amino A c i d s . . .. . ; . ' 5 2 IV. The Uptake o f l ^ C Amino A c i d s i n t h e p r e s e n c e . o f T r y p t o s e . . . . . . . . . . . . . . . ; . . . 57 V. The'Uptake o f ' ^ C . p e p t i d e s .. .. .; . . . . . 62 V I . .1.. The Uptake a n d . C o n v e r t i o n .of Amino A c i d s . 62 . . 2 . . D e t e c t i o n o f l ^ C amino a c i d s on'. chromatograms66 ;3. R e c o v e r y o f l ^ C amino a c i d s f r o m c e l l u l o s e I . t h i n l a y e r chromatograms . . . ..... . . . . 68 : 4. I n t e r c o n v e r s i o n . o f amino a c i d s . . . . . 68 GENERAL DISCUSSION . . . . ; . . . . . . . ; . . . ; . . . . ' 72 . REFERENCES . . , . . . . . . . . . : . . . . . . . : . . . . . . 82 : 9 V T a b l e . I . L i s t .of T a b l e s P r o t e a s e a c t i v i t y ..after. o s m o t i c shock o f c u l t u r e s ; o f B . ' a m y l o p h i l u s . T a b l e . ' I I . The e f f e c t o f a n a e r o b i c c o n d i t i o n s on p r o t e a s e l i b e r a t i o n b y o s m o t i c shock. T a b l e . I I I . The r e l e a s e o f p r o t e a s e a c t i v i t y . f r o m B. a m y l o p h i l u s by o s m o t i c shock t r e a t m e n t under i p t i m u m . c o n d i t i o n s . T a b l e . :IV. The e f f e c t o f s p h e r o p l a s t f o r m a t i o n on p r o t e a s e l i b e r a t i o n . T a b l e . V. The l o c a t i o n o f p r o t e a s e i n s o n i c a l l y d i s r u p t e d . s p h e r o p l a s t s . T a b l e . V I . T h e . l o c a t i o n o f p r o t e a s e i n o s m o t i c a l l y : r u p t u r e d ' s p h e r o p l a s t s , Table.'. V I I . The e f f e c t o f o r g a n i c n i t r o g e n s h i f t s , on t h e growth r a t e . c o n s t a n t (k) and t h e g e n e r a t i o n t i m e o f B . a m y l o p h i l u s . T a b l e . . V I I I . Amino a c i d c o m p o s i t i o n o f h y d r o l y s a t e s .of B. ' a m y l o p h i l u s . T a b l e . I X . l ^ C amino a c i d u p t a k e i n t h e p r e s e n c e . o f h i g h l e v e l s o f c a s e i n h y d r o l y s a t e . T a b l e . X. The u p t a k e o f l ! +C amino . a c i d s i n t h e p r e s e n c e ! , o f t r y p t o s e . T a b l e . X I . The i n c o r p o r a t i o n o f " ^ C p r o t e i n i n t o . B . a m y l o p h i l u s , T a b l e . X I I . Uptake o f l ^ C amino a c i d s i n t h e p r e s e n c e o f an . i n d i v i d u a l s p e c i f i c amino a c i d c a r r i e r . T h i n l a y e r .chromatography .of amino a c i d s f r o m ;B_. a m y l o p h i l u s . C o n v e r s i o n o f '-^ C amino a c i d s i n B_. ' a m y l o p h i l u s . v i i L i s t ;of Figures F i g . . O p t i c a l dens i ty '(TOO) as a funct ion of BSA.concentrat ion . F i g . 2. O p t i c a l dens i ty (TOO), as a funct ion of protease concentrat ion . F i g . .3. Protease . a c t i v i t y , as a funct ion of length ..of incubat ion . F i g . .4. Rate of protease product ion by B. amylophi lus . F i g . .5 . Protease product ion as a func t ion;o f cu l ture temperature. F i g . .6. The temperature s t a b i l i t y of the protease . F i g . .7.' Protease a c t i v i t y .of B. 'amylophilus as a f fec ted by pH. F i g . .8. Osmotic shock procedure. F i g . - 9. Protease assays as a f fec ted by . sucrose . F i g . . 10'.. E l e c t r o n micrograph of B . amylophi lus . F ig . ..11.. Spheroplast formation procedure. . F i g . . ,12.', The e f fec t ;of amino a c i d concentrat ion on the growth..of B_. amylophi lus . F i g . . 13V The .effect of d i f f e r e n t sources .of organic n i t rogen .on the growth of B . ' a m y l o p h i l u s . F i g . . '14'.. S h i f t up and. s h i f t down • experiments. VX11 Fig. 15. The uptake of ^ C-mixed amino acids. Fig.. l6. Radioactive and ninhydrin detection of '^ G labelled amino acids. Acknowlegement I w o u l d l i k e t o e x t e n d my s i n c e r e g r a t i t u d e t o Dr. T.H. B l a c k b u r n f o r h i s c o n s t r u c t i v e c r i t i c i s m o f my r e s e a r c h and h i s e x h a u s t i v e e d i t i n g o f t h i s m a n u s c r i p t . I w o u l d l i k e t o expree my t h a n k s t o Dr. D.J. C l a r k f o r h i s e d i t i n g o f t h e t h e s i s . L a s t l y , I o f f e r my t h a n k s t o Mrs. R. Morgan f o r h e r t y p i n g o f t h e t h e s 1 INTRODUCTION B a c t e r o i d e s a m y l o p h i l u s s t r a i n H 18 i s a s t a r c h d i g e s t i n g rumen b a c t e r i u m w h i c h p r o d u c e s moderate amounts o f p r o t e o l y t i c enzyme. The s p e c i f i c f u n c t i o n o f t h e p r o t e o l y t i c enzyme i s unknown and i t s l o c a t i o n has no t b e e n c o n c l u s i v e l y d e f i n e d . The l o c a l -i z a t i o n o f t h e p r o t e a s e w o u l d a i d i n t h e e l u c i d a t i o n o f t h e f u n c t i o n o f t h e enzyme. B l a c k b u r n (1968a) d e m o n s t r a t e d t h a t t h e enzyme was n e i t h e r r e p r e s s e d n o r i n d u c e d b y amino a c i d s , p e p t i d e s o r p r o t e i n s . L i k e many o t h e r p r o t e o l y t i c m i c r o o r g a n i s m s i s o l a t e d f r o m t h e rumen, B. a m y l o p h i l u s u t i l i z e s ammonia i n p r e f e r e n c e t o o r g a n i c n i t r o g e n s o u r c e s (Abou Akkada and B l a c k b u r n , 1963). S t u d i e s on t h e n i t r o g e n m e t a b o l i s m o f B. a m y l o p h i l u s i n d i c a t e t h a t 93"^ o f t h e b a c t e r i a l p r o t e i n n i t r o g e n comes f r o m ammonia (Hobson, M c D o u g a l l and Summers, 1968). However, some rumen b a c t e r i a , e.g. Ruminococcus  f l a v e f a c i e n s , i n c o r p o r a t e exogenous amino a c i d s d u r i n g growth ( B r y a n t and R o b i n s o n , 1961). The p u r p o s e o f t h i s r e s e a r c h was t o i n v e s t i g a t e ; ( l ) t h e u t i l i z a t i o n o f exogenous amino a c i d s b y c u l t u r e s o f B. a m y l o p h i l u s ; and (2) t h e l o c a t i o n o f t h e p r o t e a s e . 2 L I T E R A T U R E R E V I E W M i c r o o r g a n i s m s h a v e b e e n s h o w n t o b e r e s p o n s i b l e f o r t h e b r e a k d o w n o f d i e t a r y p r o t e i n i n t h e r u m e n ( B l a c k b u r n a n d H o b s o n , i 9 6 0 ) . S o m e o f t h e r u m e n b a c t e r i a u s e t h e a m i n o a c i d s a n d p e p t i d e s p r o d u c e d b y p r o t e o l y s i s a s t h e i r m a i n s o u r c e o f n i t r o g e n w h i l e o t h e r s h a v e a r e q u i r e m e n t f o r a m m o n i a a s t h e i r m a i n s o u r c e o f n i t r o g e n ( B r y a n t a n d R o b i n s o n , 1962). M o s t B . a m y l o p h i l u s s t r a i n s p r o d u c e r e l a t i v e l y s m a l l q u a n t i t i e s o f f r e e a m i n o a c i d s f r o m p r o t e i n i n d i c a t i n g t h e a b s e n c e o f p e p t i d a s e a c t i v i t y . A b o u A k k a d a a n d B l a c k b u r n (1962) s t u d i e d t h e p r o d u c t s o f c a s e i n h y d r o -l y s i s o v e r a k d a y p e r i o d f o r s e v e r a l s t r a i n s o f B . a m y l o p h i l u s . T h e y f o u n d t h a t 86% o f t h e c a s e i n w a s h y d r o l y z e d , o f w h i c h 1% w a s r e c o v e r e d a s a m i n o a c i d n i t r o g e n ( p e r c e n t t o t a l c a s e i n n i t r o g e n ) , 64% p o l y p e p t i d e n i t r o g e n ( p e r c e n t t o t a l c a s e i n n i t r o g e n ) a n d 20% a m i n o a c i d n i t r o g e n ( p e r c e n t n o n p r o t e i n n i t r o g e n ) . N o .;dJl" '^epS^eds w e r e o b s e r v e d . E n d o p e p t i d a s e a c t i v i t y w i t h t r y p s i n - l i k e s p e c i f i c i t y w a s s h o w n t o b e p r e s e n t i n t h e e x t r a c t o f B . a m y l o p h i l u s ( B l a c k b u r n , 1968b). T h e r e w a s s o m e e v i d e n c e t h a t B . a m y l o p h i l u s c o n t a i n e d m o r e t h a n o n e p r o t e a s e . T h e p r o t e a s e a c t i v i t y w a s n o t i n h i b i t e d b y E D T A ( E t h y l e n e d i a m i n e t e t r a a c e t i c a c i d ) :.'or t h i o l r e a g e n t s , b u t w a s i n h i b i t e d b y d i i s o p r o p y l p h o s p h o f l u o r i d a t e (87%), ( B l a c k b u r n , 1968b). 3 ' I . L o c a l i z a t i o n o f P r o t e o l y t i c Enzymes Many enzymes a r e t h o u g h t t o be e x t e r n a l t o t h e c e l l membrane, i f a c c e s s i b l e t o impermeable s u b s t r a t e s o f i n h i b i t o r s (Demis e t a l . , 1954; M i t c h e l l , 1961), i f e l u t e d b y non-damaging s o l v e n t s (Weimberg and O r t o n , 1966), i f i n h i b i t e d b y s p e c i f i c a n t i b o d i e s (Kushner and P o l l o c k , 1961), i f r e l e a s e d b y o s m o t i c shock ( H e p p e l , 1967) o r ^ r e l e a s e d b y s p h e r o p l a s t p r o d u c t i o n (Malamy and H o r e c k e r , 1961). The l a t t e r two t e c h n i q u e s were employed t o d e t e r m i n e t h e l o c a t i o n o f t h e p r o t e a s e i n B. a m y l o p h i l u s . A number o f h y d r o l y t i c enzymes, a l k a l i n e p h o s p h a t a s e , c y c l i c p h o s p h o d i e s t e r a s e , 5 ' n u c l e o t i d a s e , a c i d p h o s p h a t a s e and t h e r i b o -n u c l e i c a c i d i n h i b i t e d - e n d o n u c l e a s e , were r e l e a s e d f r o m E. c o l i b y o s m o t i c shock ( W o s s a l and H e p p e l , I966). S p e c i e s o f S h i g e l l a ,  E n t e r o b a c t e r , C i t r o b a c t e r , E s c h e r i c h i a , S a l m o n e l l a and S e r r a t i a r e l e a s e d enzymes when o s m o t i c a l l y shocked. I n c o n t r a s t , P r o t e u s and P r o v i d e n c i a f a i l e d t o r e l e a s e t h e s e enzymes (Neu and Chou, I967). I I . S p h e r o p l a s t F o r m a t i o n Malamy and H o r e c k e r (1961) showed t h a t a l k a l i n e p h o s p h a t a s e was s i t u a t e d o u t s i d e t h e c e l l membrane, as i t was q u a n t i t a t i v e l y l i b e r a t e d i n t o t h e s u r r o u n d i n g medium when c e l l s were c o n v e r t e d t o s p h e r o p l a s t s w i t h lysozyme and EDTA ( e t h y l e n e d i a m i n e t e t r a a c e t i c ' a c i d ) . Malamy and H o r e c k e r (196U) d e m o n s t r a t e d t h a t t h e r e l e a s e " o f a l k a l i n e p h o s p h a t a s e r e q u i r e d t h e d i s s o l u t i o n o f t h e c e l l - w a l l , s i n c e r e l e a s e o f enzymes d i d n o t o c c u r when o s m o t i c a l l y s e n s i t i v e c e l l s were made w i t h p e n i c i l l i n o r c y c l o s e r i n e . They f e l t t h a t t h e enzyme was n o t bound t o e x i s t i n g c e l l - w a l l s t r u c t u r e s s i n c e i t was r e l e a s e d i n a s o l u b l e f o r m when p e n i c i l l i n o f c y c l o s e r i n e -s p h e r o p l a s t s were l y s e d b y t r e a t m e n t w i t h d i s t i l l e d w a t e r . T h e r e -f o r e , t h e enzyme was d e m o n s t r a t e d t o be l o c a t e d i n a compartment between t h e c e l l w a l l and t h e c e l l membrane termed t h e p e r i p l a s m i c space ( M i t c h e l l , I961). I n c o n t r a s t t o t h e 93% l i b e r a 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 , g l u c o s e - 6 - p h o s p h a t e dehydrogenase and g l u t a m i c dehydrogenase a c t i v i t i e s r e m a i n e d a l m o s t c o m p l e t e l y a s s o c i a t e d w i t h l y s o z y m e - s p h e r o p l a s t s . I n f a c t , t h e a c t i v i t y o f t h e l a t t e r enzyme o u t s i d e t h e s p h e r o p l a s t s e r v e d as an i n d e x o f t h e e x t e n t o f c e l l l y s i s d u r i n g s p h e r o p l a s t f o r m a t i o n (about 20%). When membrane i n t e g r i t y was d e s t r o y e d b y t o l u e n e t r e a t m e n t t h e r e was an i n c r e a s e i n t h e amount o f a l k a l i n e p h o s p h a t a s e a s s a y e d w h i c h f u r t h e r s u b s t a n t i a t e s t h e s u r f a c e bound l o c a 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 (Malamy and H o r e c k e r , 196k). Done e t a l . , (1965) i n d i c a t e d , b y c y t o c h e m i c a l t e c h n i q u e s , t h a t a l k a l i n e p h o s p h a t a s e was l o c a t e d i n t h e p e r i p l a s m . U n l i k e a l k a l i n e p h o s p h a t a s e , p e n i c i l l i n a s e i s membrane bound and r e m a i n s w i t h t h e membrane f r a c t i o n on l y s i s o f B a c i l l u s  l i c h e n i f o r m i s p r o t o p l a s t s (Kushner and P o l l o c k , 1961). 5 P e n i c i l l i n a s e was r e l e a s e d f r o m t h e p r o t o p l a s t b y t r e a t m e n t " w i t h t r y p s i n o r sodium d e o x y c h o l a t e . Lampen (1967a and 1967b) d e m o n s t r a t e d t h a t 60% o f t h e p e n i c i l l i n a s e o f B. l i c h e n i f o r m i s was r e l e a s e d when c e l l s were c o n v e r t e d t o p r o t o p l a s t s b u t was p a r t i c l e bound ( S a r g e n t e t a l . , 1968). C o l e s and Gross (1967) showed t h a t t h e l i b e r a t i o n o f p e n i c i l l i n a s e i n S t a p h y l o c o c c u s aureus d i d n o t i n v o l v e a c t i v e t r a n s p o r t a c r o s s t h e p l a s m a membrane s i n c e ATP was n o t r e q u i r e d . They c o n c l u d e d t h a t an e n z y m a t i c p r o c e s s was i n v o l v e d i n t h e l i b e r a t i o n o f p e n i c i l l i n a s e . I I I . E f f e c t o f Amino A c i d s and P e p t i d e s on P r o t e a s e P r o d u c t i o n  and B a c t e r i a l Growth P r o t e a s e s i n s e v e r a l s p e c i e s o f b a c t e r i a have been shown t o be i n d u c e d o r r e p r e s s e d b y amino a c i d s and r e p r e s s e d b y u t i l i z a b l e c a r b o h y d r a t e s . H o f s t e n and T j e d e r (1965), n o t e d t h a t t h e e x t r a -c e l l u l a r p r o t e a s e i n an A r t h r o b a c t e r sp. was i n d u c i b l e b y p r o t e i n h y d r o l y s a t e and peptone and r e p r e s s e d b y t h e c a r b o n s o u r c e s u t i l i z e d f o r g r o w th i . e . g l u c o s e and s u c c i n a t e , and by v a r i o u s f r e e amino a c i d s . I n B a c i l l u s c e r e u s , t h e e x t r a c e l l u l a r p r o t e a s e p r o d u c t i o n was r e p r e s s e d b y a m i x t u r e o f t h r e o n i n e and h i s t i d i n e (Newmark and C i t r i , 1962). I n B a c i l l u s m egaterium p r o t e a s e p r o d u c t i o n was r e p r e s s e d b y i n d i v i d u a l amino a c i d s , t h e most e f f e c t i v e b e i n g t h r e o n i n e , i s o l e u c i n e , l e u c i n e and v a l i n e ( C h a l o u p k a 6 'and K r e c h o v a , 1966). S p o r u l a t i o n and p r o t e a s e p r o d u c t i o n i n B a c i l l u s c e r e u s were i n h i b i t e d b y h i g h c o n c e n t r a t i o n s o f amino a c i d s ( L e v i s h o n and Ar o n s o n , 1967). Keen and W i l l i a m s (1967), d e m o n s t r a t e d t h a t i n Pseudomonas lach r y m a n s ^ p r o t e a s e p r o d u c t i o n was r e p r e s s e d b y o r g a n i c n i t r o g e n ( e . g . g l u t a m i c a c i d ) , i n t h e medium and was v i r t u a l l y i n d e p e n d e n t o f t h e s u c r o s e c o n c e n t r a t i o n . McDonald and Chambers (1966) p r o p o s e d t h a t t h e f u n c t i o n o f t h e e x t r a c e l l u l a r p r o t e a s e i n M i c r o c o c c u s sp. was t o ens u r e a s u p p l y o f c a r b o n f o r growth r a t h e r t h a n t o s u p p l y amino a c i d s f o r p r o t e i n s y n t h e s i s , s i n c e t h e p r o t e a s e f o r m a t i o n was i n d u c e d b y amino a c i d s and s u p p r e s s e d b y c a r b o n s o u r c e s such as m a l t o s e and m a l t o d e x t r a n s . I n t h e a n a e r o b i c o r g a n i s m C l o s t r i d i u m w e l c h i i , g l u c o s e i n h i b i t e d f o r m a t i o n o f t h e gamma p r o t e a s e ( B i d w e l l , 1950). B l a c k b u r n (1968a) d e m o n s t r a t e d t h a t t h e growth r a t e and p r o t e a s e p r o d u c t i o n i n B. a m y l o p h i l u s were n o t a l t e r e d b y t h e p r e s e n c e o f c a s e i n , t r y p t o s e , peptone, casamino a c i d s , g l u t a m i c a c i d , a s p a r t i c a c i d , l y s i n e o r a r g i n i n e . Growth and p r o t e a s e p r o d u c t i o n were shown t o be p r o p o r t i o n a l t o a l i m i t i n g ammonia c o n c e n t r a t i o n . Temperature a p p e a r e d t o be t h e o n l y o t h e r f a c t o r c o n t r o l l i n g p r o t e a s e p r o d u c t i o n ( B l a c k b u r n , p e r s o n a l c o m m u n i c a t i o n ) . IV. Amino A c i d U t i l i z a t i o n b y Rumen B a c t e r i a D i e t a r y p r o t e i n i s r a p i d l y h y d r o l y z e d i n t h e rumen t o amino a c i d s w h i c h a r e t h e n d e a m i n a t e d t o g i v e ammonia. Warner (1956), 7 W i l l i a m s e t a l . ( i 9 6 0 ) , and Le w i s and McgBoft"ald(l96~2), d e m o n s t r a t e d t h a t s o l u b l e p r o t e i n s y i e l d h i g h c o n c e n t r a t i o n s o f rumen ammonia. B l a c k b u r n and Hobson ( i 9 6 0 ) , showed t h a t t h e r a t e o f d e a m i n a t i o n was s l o w e r t h a n t h a t o f p r o t e o l y s i s s i n c e t h e r e was an i n c r e a s e i n amino a c i d s and p e p t i d e s i n t h e rumen a f t e r f e e d i n g . E v e n t u a l l y a l l amino a c i d s were deaminated and ammonia c o n c e n t r a t i o n was maximum t h r e e h o u r s a f t e r f e e d i n g . Amino a c i d s were degraded b y t h e rumen b a c t e r i a t o e q u l m o l a r q u a n t i t i e s o f ammonia, c a r b o n d i o x i d e , and v o l a t i l e f a t t y a c i d s . The h i g h e r t h e p r o t e i n c o n c e n t r a t i o n i n t h e d i e t t h e g r e a t e r was t h e m i c r o b i a l d e a m i n a t i n g power. There app e a r e d t o be some e v i d e n c e f o r a S t i c k l a n d - t y p e r e a c t i o n between amino a c i d s ^ s i n c e L - p r o l i n e was deaminated i n t h e p r e s e n c e o f o t h e r amino a c i d s ( L e w i s , 1955). Most amino a c i d s were p a r t i a l l y d egraded b y c o n c e n t r a t e d c e l l s u s p e n s i o n s and p r o l o n g e d i n c u b a t i o n ( L e w i s , 1962). B l a c k b u r n (1965) r e v i e w e d t h e breakdown o f d i e t a r y p r o t e i n s i n t h e rumen. V. Amino A c i d I n c o r p o r a t i o n b y Rumen B a c t e r i a Most m i c r o o r g a n i s m s i n c o r p o r a t e exogenous amino a c i d s ( S t r a s s m a n , 1955; R o b e r t s , 1957; R e i s s , 1955; F e r r o Luzzi-Ames, 196U; and Kay and G r o n l u n d , 1969). T h i s i n c o r p o r a t i o n o f amino a c i d s was accompanied b y r e p r e s s i o n and f e e d b a c k i n h i b i t i o n o f t h e amino a c i d b i o s y n t h e t i c enzymes. S i n c e t h e r e was no i n c o r p o r -8 ' a t i o n o f C - l e u c i n e t h e b i o s y n t h e s i s o f l e u c i n e was n o t i n h i b i t e d b y exogenous l e u c i n e i n Ruminococcus f l a v e f a c i e n s ( A l l i s o n e t a l . , 1962). The b i o s y n t h e s i s o f a s p a r t i c a c i d was n o t i n h i b i t e d i n S t r e p t o c o c c u s bo v i s b y h i g h c o n c e n t r a t i o n s o f exogenous a s p a r t i c a c i d , p r e s u m a b l y because o f l o w l e v e l s o f i n c o r p o r a t i o n . ( W r i g h t , i 9 6 0 ) . M i x e d rumen b a c t e r i a do n o t have an i m p a i r e d u p t a k e mechanism as t h e y a c t i v e l y m e t a b o l i z e , under a r t i f i c i a l rumen c o n d i t i o n s , "^C g l y c i n e ( W r i g h t and Hungate, 1967) a s p a r t i c and g l u t a m i c a c i d s ( P o r t u g a l and S u t h e r l a n d , 1963), w i t h t h e l i b e r a t i o n o f c a r b o n d i o x i d e , v o l a t i l e f a t t y a c i d s and ammonia. W r i g h t and Hungate (1967), d e m o n s t r a t e d t h a t "^C and "^N l a b e l l e d g l y c i n e was i n c o r p o r a t e d i n t o p r o t e i n w i t h o u t g o i n g t h r o u g h a d e a m i n a t i o n s t e p . V I . P e p t i d e I n c o r p o r a t i o n b y Rumen B a c t e r i a W r i g h t (1967) d e m o n s t r a t e d t h a t d i p e p t i d e s c o n t a i n i n g g l y c i n e were i n c o r p o r a t e d i n t o rumen b a c t e r i a more e f f i c i e n t l y t h a n g l y c i n e i t s e l f . S h e l t o n and N u t t e r (1964) showed a s i m i l a r e f f e c t w i t h d i p e p t i d e s i n t h e non-rumen m i c r o o r g a n i s m L e u c o n o s t o c m e s e n t e r o i d e s . P i t t m a n e t a l . (1967) f o u n d t h a t B a c t e r o i d e s r u m i n i c o l a i n c o r p o r a t e d Ill-l i t t l e l a b e l f r o m exogenous C l a b e l l e d L - p r o l i n e o r L - g l u t a m i c Ill-a c i d b u t i n c o r p o r a t e d p e p t i d e s l a b e l l e d w i t h C p r o l i n e . The Ik amount o f C i n c o r p o r a t e d i n t o p r o t e i n was d i r e c t l y p r o p o r t i o n a l t o t h e s i z e o f t h e p e p t i d e s up t o a m o l e c u l a r w e i g h t o f 2000. 9 V I I . Ammonia I n c o r p o r a t i o n b y Rumen B a c t e r i a Ammonia i s i n c o r p o r a t e d i n t o t h e p r o t e i n o f rumen b a c t e r i a even i n media c o n t a i n i n g amino a c i d s and p e p t i d e s (Warner, 1955; Abou Akkada and B l a c k b u r n , 1963). B r y a n t and R o b i n s o n (1962), d e m o n s t r a t e d t h a t some rumen b a c t e r i a r e q u i r e d an amount o f ammonia e q u a l t o o r g r e a t e r t h a n t h e amount o f c e l l u l a r n i t r o g e n p r o d u c e d d u r i n g g rowth, r e g a r d l e s s o f t h e amount o f amino a c i d and p e p t i d e n i t r o g e n p r e s e n t i n t h e medium. R. f l a v e f a c i e n s u s e d l a r g e amounts o f ammonia and v e r y l i t t l e C p r o t e i n h y d r o l y s a t e ( A l l i s o n , 1962) and L a c t o b a c i l l u s b i f i d u s i s o l a t e d f r o m t h e rumen, a c t i v e l y a s s i m i l a t e d ammonia ( P h i l l i p s o n e t a l . , 1962). Ruminococcus  f l a v e f a c i e n s , R. a l b u s and B a c t e r o i d e s s u c c i n o g e n e s , i m p o r t a n t c e l l u l o s e d i g e s t i n g b a c t e r i a , u t i l i z e ammonia f o r a l l p r o t e i n s y n t h e s i s , even i n t h e p r e s e n c e o f p r e f o r m e d amino a c i d s ( B r y a n t , 1961) . These s t r a i n s have an a b s o l u t e g r o w t h r e q u i r e m e n t f o r b r a n c h e d - c h a i n v o l a t i l e f a t t y a c i d s due t o an i m p a i r e d a b i l i t y t o s y n t h e s i z e t h e c a r b o n c h a i n o f t h e s e amino a c i d s and an i m p a i r e d amino a c i d u p t a k e s y s t e m ( B r y a n t and D o e t s c h , 1955; B r y a n t and Ro b i n s o n , I96I; B r y a n t and R o b i n s o n , 196*2). Thus a s t r a i n o f Ruminococcus f l a v e f a c i e n s was u n a b l e t o i n c o r p o r a t e more t h a n a Ik t r a c e o f C - l a b e l l e d l e u c i n e b u t i t was a b l e t o s y n t h e s i z e l e u c i n e f r o m "^C i s o v a l e r i c a c i d ( A l l i s o n e t a l . , 1959; A l l i s o n e t a l . , 1962) . B r y a n t and R o b i n s o n (1962) n o t e d t h a t a l l s t r a i n s o f 10 freshly, i s o l a t e d bacteria which required v o l a t i l e f a t t y acids, also required ammonia. Ammonia stimulated growth of Bacteroides ruminicola, B u t y r i v i b r i o fibrinosolvens and Succinovibrio dextrinosolvens. Ammonia was apparently incorporated by growing c e l l s of a l l these strains and by Ruminococcus albus, R. flavefaciens and Bacteroides amylophilus with no apparent u t i l i z a t i o n of amino acids (Bryant and Robinson, 1962). Lachnospira multiparus, Streptococcus bovis, Selenomonas ruminantium, Peptostreptococcus e l s d e n i i , some strains of But y r i v i b r i o f ibrinosolvens and S_. dextr inosolvens incorporated very l i t t l e ammonia but large amounts of amino acid carbon (Bryant et a l . , 1963). A l l strains of Bacteroides ruminicola appeared to u t i l i z e 14 very l i t t l e ammonia, yet f a i l e d to incorporate much C-labelled amino acid carbon (Bryant et a l . , 1963)• VIII. Ammonia Incorporation by Bacteroides amylophilus Blackburn (1968a) showed that protease production and growth of Bacteroides amylophilus were proportional to the ammonia concentration up to 3.5 umoles per ml medium. Tryptose, proteose peptone, casamino acids and casein did not replace the requirement for ammonia. Blackburn (1968a and 1968b) observed that the lag phase of b a c t e r i a l growth was reduced i n the presence of tryptose (0.1% v/v) and suggested that t h i s was due to the s t a b i l i z a t i o n of the EH i n the early stages .. of growth, rather than due to the u t i l i z a t i o n of amino acids and . 11. p e p t i d e s . Hobson, M c D o u g a l l and Summers (1968), u s e d N ammonia t o d e m o n s t r a t e t h a t 93% o f B. a m y l o p h i l u s c e l l n i t r o g e n was d e r i v e d f r o m ammonia r a t h e r t h a n f r o m o t h e r n i t r o g e n s o u r c e s . I n c o n t i n u o u s c u l t u r e s o f B a c t e r o i d e s a m y l o p h i l u s , Hobson and Summers (1967) showed t h a t ammonia a c c o u n t e d f o r 104% o f ' t h e c e l l n i t r o g e n . S i m i l a r n i t r o g e n u t i l i z a t i o n has been f o u n d i n o t h e r rumen b a c t e r i a ( r e v i e w b y Hungate et_ a l . , 1964). MATERIALS AMD METHODS I . M e d i a The m i n i m a l s a l t s medium d e v e l o p e d by Hungate (1950) c o n t a i n e d ( g / l ) K^RTO^, O .U5; K H ^ , 0 . L 5 ; ( N H ^ S O ^ , 0 . 9 ; N a C l , 0 . 9 ; MgSO^, 0.09; C a C l 2 , 0.09; r e s a z u r i n , 0 . 0 0 1 ; L-c y s t e i n e , ( H C l ) , 0 . 5 ; NaHCO^, 5 . 0 . The m i n e r a l and r e s a z u r i n s o l u t i o n s p l u s any o t h e r a d d i t i o n s t o t h e medium were p l a c e d i n a l i t r e b o t t l e and t h e volume made up t o 900 m l w i t h d i s t i l l e d w a t e r . The medium was a u t o c l a v e d a t 120 C f o r 15 m i n u t e s and t h e t o p s were t i g h t e n e d i m m e d i a t e l y upon r e m o v a l f r o m t h e a u t o -c l a v e , t o m a i n t a i n a n a e r o b i c c o n d i t i o n s . . L-cysteine»(HCl) 1.0% (w/v) was a u t o c l a v e d s e p a r a t e l y . Sodium b i c a r b o n a t e s o l u t i o n 10% (w/v) was s t e r i l i z e d b y h e a t i n g t o 100 C f o r 30 m i n o r by M i l l i p o r e f i l t r a t i o n w i t h an 0.45 l-mi p o r e s i z e f i l t e r . These two s o l u t i o n s were added t o t h e medium under a s t r e a m o f o x y g e n - f r e e 00^. The medium was d i s p e n s e d i n t o s m a l l e r t u b e s and u s e d f o r b a c t e r i a l growth. . A l l s t e p s were done under an atmosphere o f CO^. D i f f e r e n t media were named b y t h e i r a d d i t i o n a l c o n s t i t u e n t s , t h u s , m a l t o s e medium c o n t a i n e d b a s a l s a l t s medium p l u s 0.3% (w/v) m a l t o s e ; m a l t o s e and t r y p t o s e medium 0.1% (w/v) t r y p t o s e ( B a c t o L a b o r a t o r i e s , D e t r o i t ) , c o n t a i n e d 0.3% m a l t o s e p l u s 0.1% t r y p t o s e i n t h e b a s a l s a l t s Product Analysis Report for C-Protein Hydrolysate Amino acid Molecular weight Specific activity Percentage of mc/m mole total count Alanine 8 9 . 1 81 8 Arginine 174.2 195. 5 Aspartic acid 133.1 123 12.5 Glutamic acid 147.1 125 12.5 Isoleucine 131.2 175 10 Leucine 131.2. 175 5 Lysine 146.2 240 5 Phenylalanine 165.2 280 8 Proline 115.1 200 5 Serine 105.1 95 8 Threonine 119.1 130 8 Tyrosine 181.2 300 8 Valine 117.1 114 5 "medium. I n some e x p e r i m e n t s m a l t o s e was r e p l a c e d b y o t h e r c a r b o -h y d r a t e s a t t h e same c o n c e n t r a t i o n . I n many o f t h e amino a c i d u p t a k e e x p e r i m e n t s t h e L - c y s t e i n e - ( H C l ) was r e p l a c e d b y d i s o d i u m s u l f i d e and a s c o r b i c a c i d o r d i t h i o n i t e a l o n e a t f i n a l c o n c e n t r a -t i o n s o f 0.025%, 0.05% and 0.003% r e s p e c t i v e l y . Amino a c i d s were o b t a i n e d f r o m N u t r i t i o n a l B i o c h e m i c a l s Corp., C l e v e l a n d , Ohio. 14 C L - t r y p t o p h a n was o b t a i n e d f r o m N u c l e a r C h i c a g o Corp., w h i l e 14 Ik t h e o t h e r r a d i o a c t i v e amino a c i d s and C g l u c o s e and C m a l t o s e were p u r c h a s e d f r o m Schwartz B i o - R e s e a r c h I n c . , Orangeburg, N.Y. 14 3 as t h e u n i f o r m l y l a b e l l e d C p r o d u c t w i t h t h e e x c e p t i o n o f H L - h y d r o x y p r o l i n e . The r a d i o a c t i v e amino a c i d s were ch e c k e d f o r p u r i t y b y t h i n l a y e r chromatography (Jones and Heathcote,' 1966) and r a d i o a u t o g r a p h y . A l l p r e p a r a t i o n s c o n t a i n e d l e s s t h a n 5% i m p u r i t i e s (Kay and G r o n l u n d , 1969). I I . B a c t e r i a and C u l t u r e C o n d i t i o n s B a c t e r o i d e s a m y l o p h i l u s s t r a i n H - l 8 was i s o l a t e d f r o m a 10 d i l u t i o n o f rumen f l u i d ( B l a c k b u r n and Hobson, 1962). B. amy-l o p h i l u s i s a Gram n e g a t i v e p l e o m o r p h i c s m a l l c o c c o b a c i l l u s , a p p r o x i m a t e l y 0 . 9 t o 1.6 u b y 1.6 t o 4 . 0 u. and i t s c h a r a c t e r i s t i c s were c o n s i s t e n t w i t h t h o s e o f t h e t y p e s t r a i n ( H a m l i n and Hungate, 1956). T h i s o r g a n i s m f e r m e n t s o n l y s t a r c h o r s t a r c h h y d r o l y s i s p r o d u c t s such as m a l t o s e , t o a m i x t u r e o f a c e t i c , f o r m i c and s u c c i n i c a c i d s . S t o c k c u l t u r e s were m a i n t a i n e d i n s e m i s o l i d agar (0.7%) deeps c o n t a i n i n g b o t h m a l t o s e (0.3%) and t r y p t o s e ( 0.1%). These c u l t u r e s r e m a i n v i a b l e up t o 2 t o 3 weeks a t 4 C. I I I . Measurement o f Growth Two methods were u s e d t o measure growth. S e r i a l d e c i m a l d i l u t i o n o f t h e b a c t e r i a l c u l t u r e i n t o s e m i s o l i d agar c o n t a i n i n g 1 . 0 % a c i d washed c a s e i n ( B l a c k b u r n and Hobson, 1962), was u s e d t o d e t e r m i n e numbers o f v i a b l e c e l l s , and b a c t e r i a l d e n s i t y was measured a t 660 mu. i n a Bausch and Lomb S p e c t r o n i c 20 c o l o r i m e t e r . C e l l c o n c e n t r a t i o n as a f u n c t i o n o f Q.D.g^Q was l i n e a r t o an O.D.ggQ 0.5. No c o r r e c t i o n was a p p l i e d t o h i g h e r O.D. r e a d i n g s . IV. Amino A c i d I n c o r p o r a t i o n 14 The u p t a k e o f C amino a c i d s was s t u d i e d i n t h e p r e s e n c e o f i n d i v i d u a l amino a c i d s , c a s e i n h y d r o l y s a t e and p e p t i d e s i n 6 . 0 m l amounts o f m a l t o s e medium r e d u c e d b y a s c o r b i c a c i d and 14 sodium s u l f i d e . The p r o d u c t a n a l y s i s r e p o r t f o r t h e C p r o t e i n h y d r o l y s a t e i s p r e s e n t e d i n T a b l e I . One m l samples were f i l t e r e d and washed w i t h 2 .0 m l m a l t o s e medium on 0.45 P- p o r e s i z e f i l t e r s ( M i l l i p o r e Corp., B e d f o r d , M a s s . ) . R a d i o a c t i v i t y i n t h e c e l l s was c o u n t e d a f t e r p l a c i n g t h e d r i e d f i l t e r s i n s c i n t i l l a t i o n f l u i d (k2 m l L i q u i f l u o r p e r l i t e r o f t o l u e n e , New E n g l a n d N u c l e a r Corp., B o s t o n , M a s s . ) . The d i s t r i b u t i o n o f l a b e l i n t h e c e l l u l a r p r o t e i n was d e t e r m i n e d b y t h i n l a y e r chromatography. The c e l l s were h a r v e s t e d b y c e n t r i f u g a t i o n 9?000 x g_ f o r 10 m i n u t e s i n a S o r v a l R e f r i g e r a t e d a u t o m a t i c c e n t r i f u g e ( I v a n S o r v a l I n c . , Norwalk, C o n n e c t i c u t ) . They were washed once i n m i n e r a l media and f i n a l l y r e s u s p e n d e d i n 3 . 0 m l o f 6 N HC1. The p r o t e i n was h y d r o l y z e d f o r 18 h o u r s a t 110 C i n s e a l e d e v a c u a t e d g l a s s v i a l s . The a c i d was t h e n removed b y f l a s h e v a p o r a t i o n w i t h a Evapomix i n s t r u m e n t ( B u c h l e r I n s t r u m e n t s , F o r t Lee, N.J.) and t h e w a t e r -washed r e s i d u e was d r i e d and d i s s o l v e d i n a known q u a n t i t y o f d i s t i l l e d w a t e r . Samples were q u a n t i t a t i v e l y a p p l i e d t o c e l l u l o s e t h i n l a y e r p l a t e s ( C e l l u l o s e Powder MN 300, Macherey N a g e l and Co., Germany). The amino a c i d s were s e p a r a t e d two d i m e n s i o n a l l y b y th e method o f Jones and H e a t h c o t e (1966). The f i r s t d i m e n s i o n o f t h e chromatograms was r u n a l o n g t h e g r a i n o f t h e t h i n l a y e r p l a t e s i n a s o l v e n t c o n s i s t i n g o f p r o p a n o l - 2 : f o r m i c a c i d : w a t e r (40 : 2 0 : 1 0 v / v ) . The sec o n d d i m e n s i o n was r u n i n a s o l v e n t o f t - b u t a n o l : m e t h y l e t h y l ketone:ammonia:water (50: 30:10:10 v / v ) . The chromato-grams were exposed f o r 1 t o 2 weeks t o m e d i c a l X - r a y f i l m (Eastman Kodak Co., R o c h e s t e r , N.Y.). The f i l m s were d e v e l o p e d and t h e r a d i o a c t i v e a r e a s were drawn by vacuum i n t o s c i n t i l l a t i o n f l u i d and a s s a y e d f o r r a d i o a c t i v i t y i n a l i q u i d s c i n t i l l a t i o n s p e c t r o m e t e r . The a d d i t i o n o f f r o m 5 t o 60 mg o f c e l l u l o s e c a u s e d no i n c r e a s e i n q u e n c h i n g u n d e r t h e s e c o n d i t i o n s ( K a y a n d . G r o n l u n d , 1969). R e p l i c a t e t h i n l a y e r p l a t e s w e r e s p r a y e d w i t h n i n h y d r i n c o l l i d i n e c h r o m a g e n i c r e a g e n t t o l o c a t e a n d i d e n t i f y t h e a m i n o a c i d s . T h e n i n h y d r i n c o l l i d i n e c h r o m a g e n i c r e a g e n t c o n s i s t e d o f 0 . 3 g n i n -h y d r i n , 2 0 . 0 m l g l a c i a l a c e t i c a c i d a n d 5 .0 m l c o l l i d i n e ( 2 , 4 , 6 , t r i m e t h y l p y r i d i n e ) , i n e t h a n o l t o g i v e a f i n a l v o l u m e o f 100 m l . T e n p i o f S t a n d a r d s o l u t i o n s o f a m i n o a c i d s (2 u g / m l i n a q u e o u s p r o p a n - 2 - o l (10% v / v ) ) w e r e r u n t o v e r i f y t h e p o s i t i o n s o f t h e a m i n o a c i d s . V . D r y W e i g h t P r o t e i n C o n t e n t a n d A m i n o A c i d C o m p o s i t i o n o f C e l l s A 50 m l c u l t u r e O . D . 0 . 6 w a s h a r v e s t e d b y c e n t r i f u g a t i o n . T h e c e l l s w e r e w a s h e d t w i c e i n d i s t i l l e d w a t e r a n d r e s u s p e n d e d i n a v o l u m e o f 5-0 m l . Two m l s a m p l e s w e r e p l a c e d i n a l u m i n u m p a n s , d r i e d a t 88 C f o r 8 h o u r s a n d s u b s e q u e n t l y s t o r e d i n t h e d e s s i c a t o r f o r 48 h o u r s . T h e s a m p l e s w e r e t h e n h e a t e d t o 121 C f o r o n e h o u r , f o l l o w e d b y c o o l i n g i n t h e d e s s i c a t o r f o r 30 m i n u t e s p r i o r t o r e - w e i g h i n g . One m l w a s d i s s o l v e d i n 1 H NaOR", h e a t e d t o 80 C , c o o l e d , t h e n n e u t r a l i z e d w i t h I N H C 1 . P r o t e i n w a s d e t e r m i n e d b y t h e m e t h o d o f L o w r y e t a l . (1951) u s i n g b o v i n e s e r u m a l b u m i n ( B S A ) a s a s t a n d a r d . O n e m l w a s h y d r o l y z e d t o d e t e r m i n e t h e a m i n o a c i d c o m p o s i t i o n u s i n g a B e c k m a n M o d e l 120 C a m i n o a c i d a n a l y z e r , ( B e c k m a n I n s t r u m e n t s I n c . , P a l o A l t o , C a l i f o r n i a ) . V I . Protease Determination The assay procedure was a m o d i f i c a t i o n of the method used by McDonald and Chen ( I965) . Protease s o l u t i o n ( 0 . 2 ml) was added to 1.8 ml of 2% a c i d washed c a s e i n p l u s 0.01% m e r t h i o l a t e , d i s s o l v e d i n 0 . 1 M KH 2 P0^ at pH 6 . 7 . For other pH values KH^PO^ was omitted. The r e a c t i o n was stopped by the a d d i t i o n of 2 .0 ml of 0 .72 N t r i -c h l o r o a c e t i c a c i d (TCA) f o l l o w e d by r a p i d mixing and was kept at room temperature f o r 10 t o 15 minutes before sedimenting the TCA-i n s o l u b l e p r o t e i n at 1000 x g f o r 10 minutes. One ml of the p r o t e i n f r e e supernatant was added t o 5 .0 ml of a l k a l i n e copper sulphate s o l u t i o n ,(l.O ml of 0.5% CuSO^ i n 1.0% sodium c i t r a t e . 2H 2 0 per 50 ml of 0 . 2 N NaOH i n 2 .0 Na^O^). A f t e r a 15 minute i n c u b a t i o n at 38 C, 0.5 ml of 1 .0 H F o l i n Reagent was added and immediately mixed, and reincubated at 38 C f o r 20 minutes p r i o r to reading at 700 mu on a Bausch and Lomb Spectronic 20. The o p t i c a l d e n s i t i e s of unincubated TCA p r e c i p i t a t e d d i g e s t s c o n t a i n i n g the same amount of enzyme were subtracted from the o p t i c a l d e n s i t i e s of the t e s t s . The c o r r e c t e d readings are compared to a standard bovine serum albumen (BSA) curve. There was a l i n e a r r e l a t i o n s h i p between the c o n c e n t r a t i o n of BSA and O . D . ^ Q Q (Figure 1 ) . A u n i t of p r o t e o l y t i c a c t i v i t y was defined as the amount of enzyme which s o l u b l i z e the equivalent of 1.0 f ig BSA i n one minute. RESULTS AND DISCUSSION P a r t I : L o c a l i z a t i o n o f P r o t e o l y t i c Enzymes i n B. a m y l o p h i l u s P r e l i m i n a r y e x p e r i m e n t s d e f i n e d some c h a r a c t e r i s t i c s o f t h e p r o t e a s e a s s a y . The O . D . ^ Q Q p r o d u c e d b y BSA, w h i c h was u s e d as a s t a n d a r d i n t h e p r o t e a s e a s s a y , was p r o p o r t i o n a l t o BSA c o n c e n t r a -t i o n up t o 300 ug ( F i g u r e l ) . C h a r a c t e r i s t i c a l l y t h e c u r v e d i d n o t p a s s t h r o u g h t h e o r i g i n (Lowry e t a l . , 1951). A c u l t u r e o f B. a m y l o p h i l u s w h i c h c o n t a i n e d 16.6 p r o t e a s e u n i t s / m l was u s e d t o d e m o n s t r a t e t h a t t h e O . D . ^ Q Q o f t h e assay-was p r o p o r t i o n a l t o p r o t e a s e c o n c e n t r a t i o n ( F i g u r e 2) and p r o p o r t i o n a l t o t h e l e n g t h o f i n c u b a t i o n ( F i g u r e 3 ) . V a r y i n g l e n g t h s o f i n c u b a t i o n and c o n c e n t r a t i o n s o f enzyme c o u l d be u s e d f o r t h e d i g e s t s p r o v i d e d t h e O . D . ^ Q Q d i d n o t e x c e e d 0 . 7 0 . A t y p i c a l growth c u r v e and t h e p r o d u c t i o n o f t h e pH 6 .7 p r o t e a s e a c t i v i t y i s p r e s e n t e d i n F i g u r e 4. The p r o t e a s e was p r o d u c e d b y e a r l y e x p o n e n t i a l c e l l s and was p r o p o r t i o n a l t o t h e c e l l d e n s i t y . The r e l a t i o n s h i p between c e l l d e n s i t y and p r o t e a s e p r o d u c t i o n was f u r t h e r i n v e s t i g a t e d b y o b s e r v i n g t h e e f f e c t o f t e m p e r a t u r e on t h e r a t e o f g r o w th and p r o t e a s e p r o d u c t i o n . B a c t e r i a l c u l t u r e s were grown i n a t e m p e r a t u r e g r a d i e n t i n t h e range 36 C t o 42 C a t i n t e r v a l s t h e O . D . a n d p r o t e a s e c o n t e n t were d e t e r m i n e d . H a m l i n and Hungate (1956), n o t e d t h e B. a m y l o p h i l u s grew w e l l between 3? C and 45 C b u t n o t a t 30 C o r 55 C. The growth r a t e v a r i e d - w i t h t h e t e m p e r a t u r e o f i n c u b a t i o n and was maximum a t 42 C. _L _L _L _L J .25 .50 .75. 1-0 CULTURE VOLUME (ml) 0 50 100 150 200 250 300 BOVINE SERUM ALBUMIN(>ig) F i g . 1.. O p t i c a l d e n s i t y . ( -700. m y ) a s a f u n c t i o n o f B S A c o n c e n t r a t i o n . V a r i o u s c o n c e n t r a t i o n s o f B S A w e r e a d d e d t o . 5 . 0 m l o f . 0 .2 N N a O H i n . , 2 . 0 % N a 2 C 0 3 c o p p e r . s u l p h a t e s o l u t i o n ( .1 .0 m l o f .0.5% CuSOl^ i n .1.0%.. S o d i u m C i t r a t e p e r 50 m l ) c o n t a i n i n g ,0.5 ml. . ' .72 . N T C A , a n d t h e f i n a l v o l u m e s w e r e a d j u s t e d t o . 6 . 0 m l . A p r o t e i n d e t e r m i n a t i o n w a s c a r r i e d . . o u t o n t h e s e s o l u t i o n s a s d e s c r i b e d i n M e t h o d s . F i g . . 2 . . O p t i c a l . d e n s i t y (700mu) a s a f u n c t i o n o f p r o t e a s e , c o n c e n t r a t i o n . T h e d i g e s t c o n t a i n e d c a s e i n a t a f i n a l c o n c e n t r a t i o n of ,1 .8% p l u s , i n c r e a s i n g , v o l u m e s o f a c u l t u r e o f B . a m y l o p h i l u s , 0 . D . ^ n » .0.6 . T h e d i g e s t s w e r e i n c u b a t e d a t 38c f o r k h o u r s a n d t h e a c i d s o l u b l e p e p t i d e s w e r e a s s a y e d a s d e s c r i b e d i n M e t h o d s . o -20 c -10 -15 > < I U < O cc OL. 60 120 180 TIME (minutes) 240 3 4 6 8 10 TIME (hours) 12 F i g . 3 . F i g . h. P r o t e a s e a c t i v i t y as a f u n c t i o n o f l e n g t h o f i n c u b a t i o n . A c u l t u r e of. B. a m y l o p h i l u s was grown t o an O.D.ggrjs 0.62 and 0 .2 m l was p l a c e d i n a s e r i e s o f t u b e s c o n t a i n i n g 1.8 m l o f 2% c a s e i n d i s s o l v e d , i n 0 . 1 M. KR^POk a d j u s t e d t o pH 6.7 by NaOH. The d i g e s t s were i n c u b a t e d a t 38.C and t h e i r enzyme a c t i v i t y was s t i p p e d a t 30. m i n u t e i n t e r v a l s by t h e a d d i t i o n o f 0.72 N TCA. The p r o t e a s e a c t i v i t y o f each sample was a s s a y e d as d e s c r i b e d i n Methods. Rate o f p r o t e a s e p r o d u c t i o n by B. a m y l o p h i l u s . . • 200. m l o f m a l t o s e t r y p t o s e medium was i n o c u l a t e d w i t h 1.0 m l o f O.D.ggQj 0*5 c u l t u r e o f B. a m y l o p h i l u s . F i v e m l q u a n t i t i e s where removed a t t i m e i n t e r n a l s and the. c e l l d e n s i t i e s were r e a d O.D.ggo (o-p .). P r o t e o l y t i c a c t i v i t y o f t h e whole c u l t u r e ( ©-©) was d e t e r m i n e d as d e s c r i b e d i n Methods. ro J I I ! L_ 42 41 40 39 38 37 36 TEMPERATURE (C) F i g . 5. P r o t e a s e p r o d u c t i o n as a f u n c t i o n o f c u l t u r e t e m p e r a t u r e . One drop o f an O.B.^Q, 0.5 c u l t u r e o f B. a m y l o p h i l u s -was i n o c u l a t e d i n t o a s e r i e s o f t u b e s c o n t a i n i n g 8 .0 m l o f m a l t o s e t r y p t o s e medium p r e h e a t e d i n an Aluminum Thermal g r a d i e n t w i t h a'range f r o m h2C t o 36c. The s p e c i f i c a c t i v i t y ( p r o t e a s e u n i t s p e r O.D.ggg, 1 . 0 ) i n t h e range O.D.ggQ, 0.25 t o 0 .30 (o-o) and 0.60 t o 0.7^ '(o -o) were p l o t t e d a g a i n s t c u l t u r e t e m p e r a t u r e . The highest specific protease activity did not occur i n cultures grown at this temperature but was highest i n cultures grown at 38.5 C (Figure 5j curve A ) . The specific protease activities were calculated for cultures as they entered the early stationary phase (Figure 5; curve B) and were found to be considerably lower than those calculated for log phase cultures and there was no temperature at which peak protease production occurred. Continuous culture of B. amylophilus at dilution rates between 0.05 and 0.46 hr demonstrated a peak of protease activity at D 0.2 hr ^ (Hobson and Summers, 1967). These results indicated that there i s an optimum growth rate for protease production. Maximum enzyme production at a particular growth rate is not unique to B. amylophilus as Clarke et a l . (1968) showed a maximum rate for amidase production i n Pseudomonas aeruginosa 8602 at D 0.30-0.35 hr 1. Coleman (1967) observed that extracellular enzyme formation was greatest i n batch cultures of Bacillus subtilis during the slow growth following logarithmic growth and he suggested that this could be due to a slowing down of ribosome synthesis, leaving an increased nucleic acid precursor pool for synthesis of m-RNA concerned with extracellular enzyme formation. A similar mechanism could be involved in the increased enzyme synthesis of B. amylophilus at low growth rates, although i t was d i f f i c u l t to equate slow growth i n a continuous culture with slow growth at the end of a batch culture. J e r u s a l i m s k y (1958) o b s e r v e d t h a t m-KNA r e a c h e d a peak i n c o n t i n u o u s c u l t u r e s o f B. megateri u m a t d i l u t i o n r a t e s b e l o w t h e maximum w h i c h m i g h t c o r r e s p o n d t o a peak o f enzyme p r o d u c t i o n . The d e c r e a s e i n t h e s p e c i f i c p r o t e a s e a c t i v i t y , w h i c h was o b s e r v e d i n c u l t u r e s grown a t t e m p e r a t u r e s h i g h e r t h a n 38.5 C, c o u l d be e x p l a i n e d b y t h e t h e r m a l i n a c t i v a t i o n o f t h e p r o t e a s e a t t h e s e t e m p e r a t u r e s . A t h e r m a l i n a c t i v a t i o n c u r v e ( F i g u r e 6) d e m o n s t r a t e d a s m a l l l o s s i n s t a b i l i t y o v e r ko C and t h i s may have r e s u l t e d i n some p r o t e a s e i n a c t i v a t i o n a t t h e s e t e m p e r a t u r e s , b u t i t i s more l i k e l y t h a t t h e r e i s a p a r t i c u l a r g r o w t h r a t e d e t e r -mined b y t h e i n c u b a t i o n t e m p e r a t u r e , a t w h i c h maximum p r o t e a s e p r o d u c t i o n c a n o c c u r . P r o t e a s e a c t i v i t y was a s s a y e d a t pH 6 .7 b a s e d on t h e o b s e r v a t i o n b y B l a c k b u r n (1968), t h a t t h e r e was a p l a t e a u o f p r o t e a s e a c t i v i t y a t t h a t pH. There a r e , however, o t h e r pH o p t i m a ( B l a c k b u r n , 1968; Lesk, 1969), a t pH 5 . 0 , 6 . 7 , 8 . 0 and 11.5 w h i c h w o u l d s u g g e s t m u l t i p l e p r o t e a s e s b u t L e s k (1969) p u r i f i e d t h e pH 6 .7 p r o t e a s e 1 0 0 0 - f o l d w i t h o u t s e p a r a t i n g i t f r o m t h e p r o t e o l y t i c a c t i v i t y a t o t h e r pH v a l u e s . A t y p i c a l p H - a c t i v i t y p r o f i l e f o r t h e p r o t e o l y t i c a c t i v i t y i s p r e s e n t e d i n F i g u r e 7. The p r o t e o l y t i c a c t i v i t y o f a c r y s t a l l i n e p r o t e a s e o f B. s u b t i l i s s t r a i n N was shown ( M a t s a b a r a e t a l . , 1968), t o e x t e n d o v e r a wide pH range and t o have an i r r e g u l a r p l a t e a u , t h e r e f o r e i t cannot be c o n c l u d e d t h a t more t h a n one p r o t e a s e was 35 40 50 60 70 TEMPERATURE (c) F i g . 6. The t e m p e r a t u r e s t a b i l i t y o f t h e p r o t e a s e . .' E i g h t m l q u a n t i t i e s o f a : c u l t u r e o f B. a m y l o p h i l u s were added t o p r e h e a t e d t u b e s . o f d i f f e r e n t t e m p e r a t u r e s and.were i n c u b a t e d f o r 10. m i n u t e s . The t u b e s were c o o l e d and a s s a y e d . f o r r e s i d u a l p r o t e a s e a c t i v i t y . PROTEASE ACTIVITY (units/ml) ro ON p r o d u c e d b y B. a m y l o p h i l u s . I n some o f t h e subsequent e x p e r i -ments on p r o t e a s e l o c a l i z a t i o n b y o s m o t i c shock and s p h e r o p l a s t f o r m a t i o n , p r o t e o l y t i c a c t i v i t y a t pH 5-0, 6 .7 , 8 . 0 and 11.0 was examined t o d e t e r m i n e w h e t h e r • • q u a l i t a t i v e d i f f e r e n c e s c o u l d be d e t e c t e d between them. I . Osmotic Shock P r o c e d u r e s The p r o c e d u r e f o r o s m o t i c shock o f b a c t e r i a l c e l l s was e s s e n t i a l l y t h a t o u t l i n e d b y N o s s a l and H e p p e l (1966) ( F i g u r e 8) The c e l l s f r o m a c u l t u r e o f B. a m y l o p h i l u s O.D.g^Q, 0 . 4 t o 0 . 6 , were h a r v e s t e d b y c e n t r i f u g a t i o n a t 8 ,000 x g f o r 10 m i n u t e s . The c e l l s were washed t w i c e w i t h about t h e o r i g i n a l volume o f c o l d 0 . 0 1 M T r i s ( h y d r o x y m e t h y l ) amino methane ( T r i s ) and 0 .03 M N a C l a d j u s t e d t o pH 7 . 1 . One gram (wet w e i g h t ) o f c e l l s was suspended i n 40 m l o f 0.033 M T r i s pH 7-1 a t 24 C. A s m a l l sample was removed and p r o t e a s e a c t i v i t y was a s s a y e d b e f o r e an i n e q u a l q u a n t i t y o f 40% sucrose^O.0 3 3 M T r i s pH 7-1 was added. T h i s was f o l l o w e d b y t h e a d d i t i o n o f 0 .08 m l 0 . 1 M d i s o d i u m e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (EDTA) a t pH 7-1 t o g i v e a f i n a l -4 c o n c e n t r a t i o n o f 1.0 x 10 M. The m i x t u r e was s t i r r e d i n a M e t a b o l y t e w a t e r b a t h s h a k e r (New B r u n s w i c k S c i e n t i f i c Co., I n c . New B r u n s w i c k , New J e r s e y ) , f o r 10 m i n u t e s a t 180 rpm and c e n t r i f u g e d f o r 10 m i n u t e s a t 13,000 x g a t 4 C. The s u p e r -t F i g . 8. Osmotic Shock Procedure. 28 •200 ml cu l ture 9,000 x g_, 10 min. C e l l s Cul ture Supernatant C O ! . M T r i s . H C l • pH .7.1 0.03 M NaCl C e l l s Wash 1 C e l l s Wash 2 Washed c e l l s i n kO. ml 0.033' M-Tris .HCl, pH ;7.1 Add U C m l hot-sucrose i n 0.033 M T r i s . H C l pH 7 - 1 - l x l 0 _ 4 M EDTA.at 2kC Plasmolysed c e l l suspension 13,000 x £ , 15 min. P l a s m o l y s e d . c e l l . p e l l e t sucrose supernatant resuspend, i n hO ml 5 x l 0 _ i | - M MgCl2 centr i fuge 10,000 x £ , 15 min. h C Shocked.ce l l s ~1 S h o c k . f l u i d 7 n a t a n t f l u i d was removed. I n s t a g e 2, t h e p e l l e t was r a p i d l y d i s p e r s e d i n 80 m l o f i c e c o l d 5.0 x 10 ^ M M g C l ^ s o l u t i o n . The s u s p e n s i o n was g e n t l y s t i r r e d i n an i c e b a t h f o r 10 m i n u t e s , c e n t r i f u g e d a t 13,000 x g f o r 15 m i n u t e s i n a S o v a l r e f r i g e r a t e d a u t o m a t i c c e n t r i f u g e , and t h e s u p e r n a t a n t f l u i d was removed. P r o t e a s e a s s a y s were c o n d u c t e d a t pH 6.7, 5.0, 8.0 and 11.0 on t h e v a r i o u s f r a c t i o n s . I I . O smotic Shock P r e l i m i n a r y e x p e r i m e n t s were p e r f o r m e d b y t h e method o f N o s s a l and H e p p e l (1966). T h i s p r o c e d u r e was d e v e l o p e d f o r d e l i v e r i n g a m i l d o s m o t i c shock t o a e r o b i c s t r a i n s o f E n t e r o -b a c t e r i a c e a e , w h i c h r e s u l t e d i n t h e l i b e r a t i o n o f p e r i p l a s m i c enzymes b u t h a d no l e t h a l e f f e c t . ( A n r a k u and H e p p e l , 1967). I t was e x p e c t e d t h a t t h e a p p l i c a t i o n o f t h i s p r o c e d u r e t o a n a e r o b i c B. a m y l o p h i l u s c e l l s w o u l d r e s u l t i n d e a t h , b u t v i a b l e c e l l s were f o u n d a t t h e end o f t h e t r e a t m e n t . There was, however, a l o n g l a g b u t t h i s c o u l d have been due t o EDTA-treatment as n o t e d b y H e p p e l (1967) r a t h e r t h a n t o t h e o x i d i z i n g e n v i r o n m e n t . U l t r a s o n i c a t i o n o f whole c e l l s r e s u l t e d i n a d e c r e a s e i n p r o t e a s e a c t i v i t y a t pH 5.0, 6.7 and 8.0 o f 13%, 25% and 25% r e s p e c t i v e l y b u t i n an i n c r e a s e o f 14% i n p r o t e a s e a c t i v i t y measured a t pH 11.0 ( T a b l e I ) . T h i s c o n f i r m e d t h a t t h e p r o t e a s e , "Table I : P r o t e a s e a c t i v i t y a f t e r o s m o t i c shock o f c u l t u r e s o f B. a m y l o p h i l u s Sample P e r c e n t a g e o f o r i g i n a l c u l t u r e pH 5 .0 pH 6 .7 pH 8 . 0 pH 11.0 whole c u l t u r e 100 100 100 100 whole c u l t u r e s o n i c a t e d 87 75 75 114 c u l t u r e s u p e r n a t a n t 68 31 16 25 f i r s t T r i s - H C l wash 10 7 14 6 washed c e l l s b e f o r e s u c r o s e t r e a t m e n t 28 28 32 36 washed c e l l s a f t e r s u c r o s e t r e a t m e n t KR* KR KR KR s h o c k e d f l u i d 9 5 11 8 s h o c k e d c e l l s 1 5 4 7 * KR = no r e s u l t s P r o t e a s e was measured a t pH 5 . 0 , 6 .7 , 8 . 0 and 11 .0, and f o u n d t o be 1 8 . 2 , 8 . 9 , 5 . 6 , 9 . 1 u n i t s / m l r e s p e c t i v e l y . w i t h t h e p o s s i b l e e x c e p t i o n o f t h e p r o t e a s e a c t i v i t y a t pH 11.0, was l o c a t e d o u t s i d e t h e c e l l s ' p e r m e a b i l i t y b a r r i e r and t h i s s t e p was o m i t t e d i n subsequent e x p e r i m e n t s . The e x p e r i m e n t a l p r o c e d u r e i s o u t l i n e d i n F i g u r e 8 and t h e r e s u l t s a r e c o n t a i n e d i n T a b l e I . The washed c e l l s c o n t a i n e d a r e l a t i v e l y s m a l l p r o p o r t i o n o f p r o t e o l y t i c a c t i v i t y , a r e f l e c t i o n o f t h e age o f t h e c u l t u r e . There was a p p a r e n t l y a l a r g e i n c r e a s e i n t h e p r o t e o l y t i c a c t i v i t y due t o t h e a d d i t i o n o f sucrose-EDTA b u t t h i s a c t i v i t y c o u l d n o t be c a l c u l a t e d due t o t h e p r e s e n c e o f h i g h O . D . ^ Q Q r e a d i n g s i n t h e c o n t r o l s . T h i s was s u b s e q u e n t l y shown t o be c a u s e d b y t h e i n t e r a c t i o n o f TCA w i t h t h e s u c r o s e t o g i v e a p r o d u c t c a p a b l e o f r e a c t i n g w i t h t h e F o l i n Reagent. There was a p o o r r e c o v e r y o f t h e c e l l - b o u n d p r o t e a s e i n t h e sh o c k e d c e l l s and t h e shock f l u i d b u t p r o t e a s e a s s a y a t t h e f o u r pH v a l u e s showed a h i g h e r p r o p o r t i o n o f t h e enzyme i n t h e shock f l u i d . The a t y p i c a l l y h i g h p r o p o r t i o n o f p r o t e a s e i n t h e c u l t u r e s u p e r n a t a n t made i t d i f f i c u l t t o draw c o n c l u s i o n s f r o m t h i s e x p e r i m e n t b u t t h e r e was p r e s u m p t i v e e v i d e n c e t h a t p r o t e a s e a c t i v e a t a l l t h e pH v a l u e s was p e r i p l a s m i c . I I I . The E f f e c t o f A n a e r o b i c C o n d i t i o n s on P r o t e a s e L i b e r a t i o n  b y Osmotic Shock The c e l l s were k e p t u n d e r CO d u r i n g a l l p r o c e d u r e s . The e f f e c t o f a d d i n g c y s t e i n e -HC1 (0.1%) t o t h e w a s h a n d r e s u s p e n s i o n b u f f e r s t h r o u g h o u t t h e p r o c e d u r e r e s u l t e d i n i n c r e a s e d • v i a b i l i t y a n d s i n c e y o u n g e r - c u l t u r e s w e r e u s e d ^ a h i g h e r p r o p o r t i o n ( 8 0 % ) o f t h e p r o t e a s e - w a s f o u n d i n t h e w a s h e d c e l l s t h a n i n t h e s u p e r -n a t a n t . T h e r e c o v e r i e s . o f t h i s c e l l - b o u n d p r o t e a s e ( T a b l e I I ) i n t h e s h o c k e d c e l l s a n d s h o c k - f l u i d w e r e a p p r o x i m a t e l y 40% f o r a l l t h e t r e a t m e n t s . T h e r e w a s a g a i n a n i n t e r f e r e n c e b y s u c r o s e i n t h e a s s a y p r o c e d u r e s o i t w a s n o t k n o w n h o w m u c h p r o t e a s e w a s l i b e r a t e d d u r i n g p l a s m o l y s i s . M o r e p r o t e a s e , w i t h o r w i t h o u t c y s t e i n e i n ' t h e p r o c e d u r e , w a s r e t a i n e d b y t h e s h o c k e d c e l l s t h a n w a s l i b e r a t e d i n t o t h e s h o c k f l u i d . T h e p r o p o r t i o n , h o w e v e r , d i d v a r y a l i t t l e . A l a r g e a m o u n t , ( 3 ^ % ) o f t h e c e l l u l a r p r o t e i n w a s l i b e r a t e d d u r i n g t h e s h o c k p r o c e d u r e . T h i s w a s m u c h h i g h e r t h a n t h e 8% r e p o r t e d b y H e p p e l (I967) a n d w o u l d i n d i c a t e t h a t B . a m y l o p h i l u s c e l l s a r e m o r e o s m o t i c a l l y f r a g i l e t h a n m e m b e r s o f t h e E n t e r o b a c t e r i a c e a e . O n l y 20% o f t h e t o t a l p r o t e a s e o f B . a m y l o p h i l u s w a s r e l e a s e d i n t o t h e m e d i u m d u r i n g e x p o n e n t i a l g r o w t h o f t h e b a c t e r i a . U n l i k e t h e c e l l b o u n d p r o t e a s e o f a M i c r o c o c c u s s p . ( M c D o n a l d , 1961) o n l y 5-7% o f t h e p r o t e a s e o f B . a m y l o p h i l u s w a s l i b e r a t e d i n t o 2.0% R a C T , b u t M i c k l e d i s i n t e g r a t i o n o f l y o p h i l i z e d c e l l s s u s p e n d e d i n w a t e r g a v e g o o d y i e l d s o f p r o t e a s e . ( B l a c k b u r n ,1968a). •33 T a b l e I I : The e f f e c t o f a n a e r o b i c c o n d i t i o n s on p r o t e a s e l i b e r a t i o n b y o s m o t i c shock P r o t e a s e P r o t e i n minus c y s t e i n e c y s t e i n e c y s t e i n e + b i c a r b o n a t e c y s t e i n e b i c a r b o n a t e u n i t s u n i t s 1o u n i t s mg Washed c e l l s 100 4 . 4 100 6.8 100 4 .6 100 424 S u c r o s e s u p e r n a t a n t . KR* KR KR KR Shock f l u i d 16 0 . 7 2 0 . 1 4 0 . 2 3^ 142 Shocked c e l l s 23 1.0 3^ 2 .3 33 1.5 42 179 * = no r e s u l t s A c u l t u r e o f B. a m y l o p h i l u s , O.D.ggQ, 0 .43 was u s e d f o r t h e c y s t e i n e -minus and c y s t e i n e (0.05%) p l u s sodium b i c a r b o n a t e .(0.5%) e x p e r i m e n t s . A s e c o n d c u l t u r e , O.D.^Q, 0 .52 was u s e d f o r t h e c y s t e i n e (0.05%) e x p e r i m e n t . Normal o s m o t i c shock and p r o t e a s e a s s a y p r o c e d u r e s were c a r r i e d o u t as d e s c r i b e d i n t h e t e x t . A p r o t e i n d e t e r m i n a t i o n (Lowry e t a l . , 1951) was c o n d u c t e d on t h e c y s t e i n e f r a c t i o n s . IV. The E f f e c t o f S u c r o s e on t h e P r o t e a s e A s s a y Anomalous r e s u l t s were o b t a i n e d ( T a b l e s I and I I ) when p r o t e a s e was a s s a y e d i n t h e p r e s e n c e o f h i g h s u c r o s e c o n c e n t r a -t i o n s . S u c r o s e a p p e a r e d t o i n t e r f e r e b y r e a c t i n g w i t h TCA t o prod u c e a p r o d u c t w h i c h i n t u r n r e a c t e d w i t h t h e F o l i n Reagent. Enzyme b l a n k s were h i g h e r t h a n t h e enzyme t e s t s due t o t h e l o n g e r c o n t a c t between TCA and s u c r o s e i n t h e enzyme b l a n k s t h a n i n t h e t e s t s e r i e s . T h i s t i m e dependent i n c r e a s e i n b l a n k O.D.^^ i s i l l u s t r a t e d i n F i g u r e 9- EDTA was n o t f o u n d t o be a c o n t r i b u t a r y f a c t o r i n t h i s n o n - s p e c i f i c r e a c t i o n . I n o r d e r t o o b t a i n an a c c u r a t e a s s a y o f t h e p r o t e a s e a c t i v i t y i n t h e s u c r o s e s u p e r n a t a n t f r a c t i o n , samples were d i a l y z e d t o remove s u c r o s e b e f o r e p r o t e a s e a s s a y . V. M o d i f i e d Osmotic Shock P r o c e d u r e The f o l l o w i n g m o d i f i c a t i o n s were i n t r o d u c e d t o m i n i m i z e t h e s t r e s s on B. a m y l o p h i l u s c e l l s . EDTA c o n c e n t r a t i o n was r e d u c e d t o 1 x 10 M, t h e shoc k e d c e l l s were r e s u s p e n d e d m 40 m l 0.33 M T r i s ^ ^ H C l , c y s t e i n e - H C 1 was added t o a l l s o l u t i o n s t o g i v e a c o n c e n t r a t i o n o f 0.1% and e a r l y l o g phase c e l l s (O.D.gg l e s s t h a n 0.65) were u s e d t o m i n i m i z e l y s i s . Samples were d i a l y z e d a g a i n s t r e p e a t e d changes o f d i s t i l l e d w a t e r t o remove flu o 0.2 - J 1 - J l _ 10 20 30 40 TIME (minutes] 50 60 F i g . 9 . P r o t e a s e a s s a y s were p e r f o r m e d i n t h e absence o f any enzyme, by a d d i n g 0.2 ml t e s t s o l u t i o n to.1.8 m l 2.0% c a s e i n . The t e s t s o l u t i o n s c o n t a i n e d e i t h e r 2 . 5 x l 0 ~ % EDTA i n c u b a t e d w i t h TCA (O-O) and w i t h o u t TCA ($~# ), 20% s u c r o s e i n c u b a t e d w i t h TCA (&r&) and w i t h o u t TCA -At v a r i o u s t i m e s t u b e s were removed, TCA was added t o t h o s e t u b e s w h i c h d i d not a l r e a d y c o n t a i n TCA. The t u b e s were i m m e d i a t l y c e n t r i f u g e d and t h e O . D . J Q O was r e a d f o l l o w i n g t h e a d d i t i o n o f t h e T C A - s o l u b l e s u p e r n a t a n t t o t h e c o p p e r - b i c a r b o n a t e s o l u t i o n p l u s F o l i n r e a g e n t as d e s c r i b e d i n Methods. s u c r o s e and c y s t e i n e w h i c h i n t e r f e r r e d w i t h t h e p r o t e a s e and p r o t e i n d e t e r m i n a t i o n s . The r e s u l t s o f a t y p i c a l e x p e r i m e n t i n w h i c h t h i s m o d i f i e d p r o c e d u r e was u t i l i z e d , a r e p r e s e n t e d i n T a b l e I I I . The l o g phase c e l l s , p r o t e c t e d b y c y s t e i n e , r e m a i n e d v i a b l e t h r o u g h o u t t h e o s m o t i c shock t r e a t m e n t . The c u l t u r e s u p e r n a t a n t f r o m w h i c h t h e s e c e l l s were sedimented, c o n t a i n e d o n l y 24% o f t h e t o t a l p r o t e 3.SS ^  c h a r a c t e r i s t i c o f l o g phase c u l t u r e s ( B l a c k b u r n , 1968a). A d d i t i o n a l p r o t e a s e was l o s t i n t h e two wash t r e a t m e n t s b u t 65% o f t h e o r i g i n a l p r o t e a s e was r e t a i n e d b y t h e washed c e l l s . P l a s m o l y s i s . b y s u c r o s e a p p a r e n t l y l i b e r a t e d 18% o f t h i s bourid p r o t e a s e b u t o s m o t i c shock l i b e r a t e d o n l y l % ; a n d 80% was r e t a i n e d b y t h e s h o c k e d c e l l s . The p r o t e i n r e c o v e r y f i g u r e s a r e i n a c c u r a t e due t o t h e s m a l l amounts o f p r o t e i n i n s o l u t i o n b u t i t i s p r o b a b l e t h a t a t l e a s t "jk^fo o f t h e p r o t e i n was r e t a i n e d b y t h e whole c e l l s . T h i s was c o n s i s t e n t w i t h t h e i r good v i a b i l i t y . N o s s a l and H e p p e l (1966) f o u n d t h a t 4% o f t h e c e l l u l a r p r o t e i n was l i b e r a t e d b y o s m o t i c shock f r o m E. c o l i b u t t h i s p r o t e i n c o n t a i n e d t h e E. c o l i h y d r o l y t i c enzymes w h i l e t h e p r o t e a s e was f i r m l y r e t a i n e d b y t h e shocked c e l l s i n t h e s e e x p e r i m e n t s . The p r o t e a s e a p p a r e n t l y has a s u p e r f i c i a l l o c a t i o n , e x t e r n a l t o t h e p e r m e a b i l i t y b a r r i e r o f t h e c e l l , b u t i s n o t l o c a t e d f r e e i n t h e p e r i p l a s m o f t h e c e l l . T a b l e I I I : The r e l e a s e o f p r o t e a s e a c t i v i t y f r o m B. a m y l o p h i l u s " b y o s m o t i c shock t r e a t m e n t under optimum c o n d i t i o n s . * 1o p r o t e a s e p r o t e i n Washed c e l l s 100 100 S u c r o s e s u p e r n a t a n t 18 54 Shock f l u i d 1 59 Shocked c e l l s 80 74 The c e l l s f r o m a c u l t u r e o f B . a m y l o p h i l u s O . D . ^ Q , 0 . 5 , were o s m o t i c a l l y s h o c k e d under o p t i m a l c o n d i t i o n s . A l l s t e p s were k e p t a n a e r o b i c and a l l samples were d i a l y z e d f o r 4 8 h o u r s p r i o r t o p r o t e a s e a c t i v i t y and p r o t e i n d e t e r m i n a t i o n s . The washed c e l l s u s p e n s i o n c o n t a i n e d 11.6 p r o t e a s e u n i t s and 19 .3 M-g p r o t e i n p e r m l . V I . S p h e r o p l a s t F o r m a t i o n Under n o r m a l growth c o n d i t i o n s , B. a m y l o p h i l u s i s s u r r o u n d e d b y a t h i c k c e l l w a l l w h i c h d e f i n e s i t s shape as a s m a l l c o c c o -b a c i l l u s a p p r o x i m a t e l y 0.92 t o 1.2 n i n d i a m e t e r ( F i g u r e 10). The c e l l w a l l i s s e p a r a t e d f r o m t h e c y t o p l a s m i c membrane by a p e r i p l a s m i c space w h i c h was c a l c u l a t e d i n some B a c t e r o i d e s s p e c i e s t o be f r o m 150 t o 250 A ( B l a d e n and Waters, 1963). A c h a r a c t e r i s t i c o f p e r i p l a s m i c enzymes i s t h a t t h e y a r e l i b e r a t e d d u r i n g s p h e r o p l a s t f o r m a t i o n (Malamy and H o r e c k e r , 196I1-). S i n c e t h e l o c a t i o n o f t h e p r o t e a s e d i d n o t appear t o be p e r i p l a s m i c on t h e b a s i s o f o s m o t i c shock e x p e r i m e n t s , l i b e r a t i o n o f p r o t e a s e b y t h e lysozyme-EDTA t r e a t m e n t i n s p h e r o p l a s t f o r m a t i o n was examined. T h i s p r o c e d u r e has been u s e d i n d e t e r m i n i n g t h e l o c a t i o n o f i n v e r t a s e and m e l i b i o s e i n y e a s t ( S u t t o n and Lampen, 1962; I s l a m and Lampen, 1962) and o f p e n i c i l l i n a s e i n B a c i l l u s  l i c h e n i f o r m i s (Lampen, 1967a). The method o f Malamy and H o r e c k e r (196k) was f o l l o w e d w i t h o n l y a p p r o p r i a t e m o d i f i c a t i o n f o r a n a e r o b i c c e l l s o f B. a m y l o p h i l u s . C e l l s were grown i n m a l t o s e t r y p t o s e medium t o t h e d e s i r e d o p t i c a l d e n s i t y (0.D.gg Q, 0.5 t o 0 i 6 ) and 50 m l o f t h e c u l t u r e was h a r v e s t e d b y c e n t r i f u g a t i o n a t 9?000 x g f o r 10 m i n u t e s i n a S o r v a l r e f r i g -e r a t e d a u t o m a t i c c e n t r i f u g e . A f t e r a wash i n 50 m l o f 0.01 M T r i s b u f f e r , pH 7.k, t h e c e l l s were r e s u s p e n d e d i n 100 m l 0.033 M F i g . 10. A c u l t u r e o f B_. a m y l o p h i l u s O.D.ggg* 0*5 were f i x e d w i t h OsOij., d e h y d r a t e d w i t h e t h a n o l and a c e t o n e washes and embedded i n V e s t o p a l W ( M a r t i n and J a e g e r Co., Geneva S w i t z e r l a n d ) u s i n g t h e method o f K e l l e n b e r g e r and R y t e r , (1958). T h i n s e c t i o n s were c u t w i t h an LKB mic r o t o m e , s t a i n e d w i t h u r a n y l a c e t a t e and l e a d c i t r a t e and examined i n a P h i l i p s 100 e l e c t r o n m i c r o s c o p e . M a g n i f i c a t i o n X 25,000. The c e l l d i a m e t e r was 0.92u t o 1.2u. 39 a F i g . 11. S p h e r o p l a s t f o r m a t i o n p r o c e d u r e . 100 m l : c u l t u r e 9,000 x g_ 10 minutes'. C e l l s C u l t u r e : s u p e r n a t a n t 50 m l '.01. M - T r i s - H C l , pH 7.h C e l l s w a shings Resuspended i n 100 m l o f 20% s u c r o s e i n . 0 . 0 3 3 M T r i s - H C l pH .8.0 ,• UC 1 m l . o f 0.1 M EDTA and.0.1 m l lysozyme ( 5 mg/ml ) S p h e r o p l a s t s Osmotic f r a g i l i t y 1 m l sample p l u s 5 m l H2O iO'y'O'OO x g_ •15' m i n u t e s hC . p S j ^ r . o p l a s t s L y s o z y m e - s o l u b l i z e d f r a c t i o n wash 20%.sucrose i n .033 M T r i s - H C l pH 8.0 kC S p h e r o p l a s t s 1 Wash lOOml H 20 D i s r u p t e d s p h e r o p l a s t s Membranes s o l u b l e - c y t o p l a s m T r i s b u f f e r , pH 8 . 0 , c o n t a i n i n g 20% s u c r o s e . The s u s p e n s i o n was' k e p t a t 0 C i n an i c e b a t h and t o i t was added 1.0 m l o f 0 . 1 M EDTA, pH 8 . 0 ; 0 . 1 m l , 0. 5%'w/y,/lysozyme s o l u t i o n . The s u s p e n s i o n was r o t a t e d b y hand. One m l samples were t a k e n a t v a r i o u s t i m e i n t e r v a l s and d i l u t e d w i t h 5-0 m l o f d i s t i l l e d w a t e r . The o p t i c a l d e n s i t y o f t h e s e samples were m o n i t o r e d on a Bausch and Lomb S p e c t r o n i c 20 a t 660 mu. T h i s d e t e r m i n e d t h e o s m o t i c f r a g i l i t y o f t h e c e l l s . When t h e s p h e r o p l a s t f o r m a t i o n was complete as i n d i c a t e d b y t h e absence o f f u r t h e r d e c r e a s e i n t h e t u r b i d i t y o f t h e d i l u t e d sample, t h e s u s p e n s i o n was c e n t r i f u g e d a t 10,000 x g f o r 15 m i n u t e s a t k C. The s u p e r n a t a n t was removed and t h e p e l l e t was washed w i t h t h e s u c r o s e - T r i s b u f f e r s o l u t i o n . The p e l l e t was t h e n l y s e d b y t h e a d d i t i o n o f d i s t i l l e d w a t e r . A l l samples were d i a l y s e d p r i o r t o p r o t e i n and p r o t e a s e a s s a y t o remove t h e i n t e r -f e r e n c e c a u s e d b y s u c r o s e and c y s t e i n e . There was a 21% l o s s i n p r o t e a s e a c t i v i t y d u r i n g t h e s p h e r o -p l a s t i n g p r o c e d u r e , w h i c h cannot be e x p l a i n e d . The s p h e r o p l a s t s were a s s o c i a t e d w i t h 46% o f t h e a c t i v i t y b u t 21% was a p p a r e n t l y s o l u b l i s e d by l y s o z y m e t r e a t m e n t . C e n t r i f u g a t i o n o f t h i s f r a c t i o n a t 22,000 x g f o r 20 m i n u t e s r e s u l t e d i n t h e s e d i m e n t a t i o n o f more t h a n 50% o f t h e " s o l u b l e " enzyme and i t i s p r o b a b l e t h a t much o f t h e s o l u b l e enzyme was p a r t i c l e bound. T h i s c o u l d have o r i g i n a t e d f r o m l y s e d s p h e r o p l a s t s o r f r o m t h e breakdown o f s p e c i f i c membrane o r g a n e l l e s . There was t h u s good e v i d e n c e t h a t t h e major p o r t i o n T a b l e IV: The e f f e c t o f s p h e r o p l a s t f o r m a t i o n on p r o t e a s e l i b e r a t i o n % p r o t e a s e o f washed c e l l s Washed c e l l s u s p e n s i o n 100 S p h e r o p l a s t s + s o l u b l i z e d 79 f r a c t i o n S p h e r o p l a s t s k6 L y s o z y m e - s o l u b l i z e d 21 f r a c t i o n S p h e r o p l a s t s were f o r m e d as d e s c r i b e d i n t h e t e x t , f r o m c e l l s o f B. a m y l o p h i l u s grown i n a m a l t o s e - t r y p t o s e c u l t u r e O.D.ggQ' O'^L"* The washed c e l l s u s p e n s i o n c o n t a i n e d 12.7 p r o t e a s e u n i t s / m l . of the protease was bound to the c e l l w a l l cytoplasmic membrane f r a c t i o n and that l i t t l e , i f any, was located free i n the periplasm. I t i s not known how much disintegration of the c e l l w a l l of B. amylophilus occurred during the spheroplasting procedure and i t i s therefore not know whether the protease i s located i n the c e l l w a l l or on the cytoplasmic membrane. The 35 minutes required for complete osmotic f r a g i l i t y to develop due to lysozyme treatment was similar to that f o r E. c o l i ML 308 (30 min) and E. c o l i K12 (15 min) so i t i s probable that the same considerable quantities of muco-peptide were removed (Malamy and Horecker, 196L). High speed centrifugation (90,000 x g for 5 hours) of intact c e l l s of a B. amylophilus harvested from maltose tryptose medium O.D.ggQ 0.5 removed s l i g h t l y over 80% of the protease a c t i v i t y from the culture. This same culture subjected to sonic disruption sedimented approximately kcrfo of the enzyme a c t i v i t y under the same centrifugation procedure. Washed B. licheniformis c e l l s demonstrated that as much as 96% of the p e n i c i l l i n a s e a c t i v i t y sedimented, but that after disruption by sonication approximately 50% of the protease a c t i v i t y sedimented at these high speeds (Sargent, et a l . , 1968). • The results of another experiment involving spheroplasts are presented i n Table V. In t h i s there was no loss i n protease a c t i v i t y during spheroplast formation. Centrifugation at 22,000 x g sedimented 65% of the protease a c t i v i t y . Centrifugation at 160,000 x g, of. T a b l e V: T h e i l o c a t i o n .of p r o t e a s e i n . s o n i c a l l y d i s r u p t e d s p h e r o p l a s t s . p r o t e a s e % o f t o t a l . u n i t s ..... p r o t e a s e . A s p h e r o p l a s t s ik.5 63 non s e d i m e n t a b l e a t 22,000 x g_ 7.6 33' *non s e d i m e n t i b l e a t 160,000 x g_ - 20 B s p h e r o p l a s t e n v e l o p e s .5.1 22 s o l u b l e c y t o p l a s m . l 6 . 5 72 • * R e s u l t s o b t a i n e d f r o m a d i f f e r e n t e x p e r i m e n t . S p h e r o p l a s t s were formed, as d e s c r i b e d i n . t h e Methods, f r o m c e l l s o f ;B. a m y l o p h i l u s grown i n a m a l t o s e - t r y p t o s e . c u l t u r e , O.D.'66o>..0.7- The washed c e l l s c o n t a i n e d 22.9 p r o t e a s e u n i t s p e r m l . A p o r t i o n o f t h e s p h e r o p l a s t s (A) w e r e . c e n t r i f u g e d . a t .22,000 x g_ f o r 20 m i n u t e s t o sediment t h e e n v e l o p e s and s p h e r o p l a s t s . A n o t h e r p o r t i o n o f t h e s p h e r o p l a s t s (B) was d i s r u p t e d by s o n i c a t i o n and f r a c t i o n a t e d b y . c e n t r i f u g a t i o n a t 22,000 x g_ f o r 2 0 .minutes i n t o a c e l l - e n v e l o p e f r a c t i o n and a s o l u b l e c y t o p l a s m i c f r a c t i o n . a d i f f e r e n t p r e p a r a t i o n ( T a b l e V ) , s e d i m e n t e d a l l b u t 20% o f t h e p r o t e a s e . T h i s was f u r t h e r c o n f i r m a t i o n t h a t h i g h speed c e n t r i -f u g a t i o n s e d i m e n t e d t h e p r o t e a s e w h i c h was l i b e r a t e d b y lysozyme t r e a t m e n t and much o f t h i s p r o t e a s e may be assumed t o be p a r t i c l e bound. I n d i r e c t e v i d e n c e t h a t p a r t i c l e - b o u n d p r o t e a s e i s n o t c o m p l e t e l y s e d i m e n t e d i s p r e s e n t e d i n T a b l e V I where i t i s shown t h a t t h e p r o t e a s e f r o m s p h e r o p l a s t s , w h i c h a r e f r a g m e n t e d by u l t r a s o n i c a t i o n , does n o t sediment a t 22,000 x g. The d i s t r i b u t i o n o f p r o t e a s e i n f r a c t i o n s f r o m o s m o t i c a l l y r u p t u r e d s p h e r o p l a s t s a r e p r e s e n t e d i n T a b l e V I . There was a g a i n a 20% s o l u b l i z a t i o n o f p r o t e a s e b y lys o z y m e a c t i o n b u t 79$> r e m a i n e d bound t o t h e s p h e r o p l a s t s . T h i s p r o t e a s e was m o s t l y a s s o c i a t e d w i t h t h e c e l l e n v e l o p e s and o n l y l 6 % was n o n - s e d i m e n t i b l e a t l 6 o , 0 0 0 x g. The r e s u l t s o f e x p e r i m e n t s i n v o l v i n g s p h e r o p l a s t s were c o n s i s t e n t w i t h t h e e a r l i e r o b s e r v a t i o n s f r o m o s m o t i c shock e x p e r i m e n t s t h a t i n B. a m y l o p h i l u s t h e p r o t e a s e i s bound t o t h e o u t e r c e l l e n v e l o p e . Some l i b e r a t i o n o f t h i s p r o t e a s e o c c u r s d u r i n g t h e f o r m a t i o n o f s p h e r o p l a s t s b y lys o z y m e t r e a t m e n t b u t t h e r e was e v i d e n c e t h a t t h i s m i g h t be p a r t i c l e bound. L y s i s o f t h e p r o t o p l a s t and f r a c t i o n a t i o n o f t h e l y s a t e i n t o an e n v e l o p e f r a c t i o n and a s o l u b l e f r a c t i o n i n d i c a t e d t h a t t h e ma j o r p o r t i o n o f t h e p r o t e a s e was envelope-bound. T a b l e V I : The l o c a t i o n o f p r o t e a s e i n o s m o t i c a l l y r u p t u r e d s p h e r o p l a s t s P r o t e a s e P r o t e a s e F r a c t i o n u n i t s % washed c e l l s S p h e r o p l a s t 12.5 79 L y s o z y m e - s o l u b l i z e d .3.1 20 C e l l e n v e l o p e s 7.6 ^8 S o l u b l e c y t o p l a s m i c 2.5 l 6 S p h e r o p l a s t s were formed, as d e s c r i b e d i n t h e t e x t , f r o m c e l l s o f B. a m y l o p h i l u s grown i n m a l t o s e - t r y p t o s e c u l t u r e , O.D.g6o> 0 .L7 . The washed c e l l s u s p e n s i o n c o n t a i n e d 15.8 p r o t e a s e u n i t s p e r m l . The s p h e r o p l a s t s were s e d i m e n t e d a t 10,000 x g f o r 20 m i n u t e s . The s p h e r o p l a s t s were o s m o t i c a l l y r u p t u r e d and f r a c t i o n a t e d b y c e n t r i f u g a t i o n a t l6o,000 x g f o r k h o u r s i n t o a c e l l - e n v e l o p e f r a c t i o n and a s o l u b l e c y t o p l a s m i c f r a c t i o n . A l l samples were d i a l y z e d f o r 2k h o u r s , p r i o r t o p r o t e a s e a s s a y . "Part I I : uptake o f Amino A c i d s and P e p t i d e s b y B. a m y l o p h i l u s I . The E f f e c t o f O r g a n i c N i t r o g e n on Growth • B l a c k b u r n (1968) and Hobson e t a l . (1958) showed t h a t o r g a n i c n i t r o g e n was n o t i m p o r t a n t i n t h e n u t r i t i o n o f B. a m y l o p h i l u s . The e f f e c t o f . d i f f e r e n t o r g a n i c n i t r o g e n s o u r c e s and t h e e f f e c t o f s h i f t -up and s h i f t - d o w n c o n d i t i o n s on t h e growth o f B. a m y l o p h i l u s was examined t o c l a r i f y t h e s e o b s e r v a t i o n s . D i f f e r e n t c o n c e n t r a t i o n s o f c a s e i n h y d r o l y s a t e (O .65 mg/ml t o 3 . 3 mg/ml) d i d n o t a f f e c t t h e growth o f B. a m y l o p h i l u s ( F i g u r e 12) . There was no l a g phase b u t when B. a m y l o p h i l u s was grown w i t h s i n g l e amino a c i d s , c a s e i n h y d r o l y s a t e , o r t r y p t i c p e p t i d e s as o r g a n i c n i t r o g e n s o u r c e s , s l i g h t d i f f e r e n c e s d i d o c c u r ( F i g u r e 13) . There was a s l i g h t i n c r e a s e i n f i n a l c e l l d e n s i t y - w h e n B. a m y l o p h i l u s was grown i n t h e t r y p t o s e medium. R e s u l t s o f s h i f t e x p e r i m e n t s a r e g i v e n i n F i g u r e 13, and T a b l e V I I . Maximum growth r a t e s appear t o be t h e same f o r s h i f t - u p , s h i f t - d o w n and t h e t r y p t o s e c o n t r o l ( C u r v e s B, C, and A, r e s p e c t i v e l y ) . The maximum growth r a t e f o r t h e b a s a l c o n t r o l i s l o w e r t h a n f o r t h e o t h e r c o n d i t i o n s (0.3*+ h r 1 v s . a p p r o x i m a t e l y 0.5 h r 1 ) . A n o t h e r e f f e c t i s o b s e r v e d on t h e l a g a f t e r t h e s h i f t ( F i g u r e ik, i n s e r t ) . The s h i f t - u p ( c u r v e B) e x h i b i t s a g r e a t e r l a g t h a n e i t h e r t h e s h i f t - d o w n o r t r y p t o s e c o n t r o l ( c u r v e s C and A, r e s p e c t i v e l y ) . P r e - e x p o s u r e t o t r y p t o s e r e d u c e s t h e l a g , i n agreement w i t h r e s u l t s o b t a i n e d f r o m t h e g r o w t h r a t e e x p e r i m e n t s . The l a g i n t h e b a s a l yi«. 18*-';' fao offoet of axsiao aoifi eo»e«otratloB ©a too grwtb of B. ^lopti&litt. One drop of a 10 bow culture of 3* i ^ ^ f t U w T * ° ©Blto"* BoAiwa ime taooBlcteft iate ¥»0 m mXi&m awfilws mm>leEm&<£& srtth 0»6"s «g ^ ) » eg (a-®) aa& 3»3 ate (&-a) essoin fey&rolyioie po* ml of maltose a«fi4«o« "tofcorwrtlie** scacanfcratiOB* tooted gs*o stoils? sreotalta. She oel l density vis seoossed' afc 0*9«66Q fer Ml bowl* fig* 13# Sao effeot of differea* oeareooo^/orgastic nitrogen oa tfee grevta of &• oraloiy^ae* One too? of ft 13 - aoar oalttiro of fC i ^ ^ ^ ^ ^ V o w a la aoltoee aoalaa v e i l iaooalo&oA l i to « atrlee of tufces eoatoiatog 0#S trypffcoc* t ^ ) » 0*031 eaaoia ayttrelaraote (<».£>)* aeft 6.01$ ef oaob of toe aaturally occarrto^ aaloo *©ida (no), Th« co l l aeasity V M a«»»uy*d oft O.SUggg* •hi TIME (hours) 48 ' Table VII: The'.effect of organic nitrogen . s h i f t s . on. the growth rate constant (k) and the generation time of B.'amylophilus. Medium generation k % of max. . . time .' growth . . tryptose to tryptose 79 0.53 h r " 1 100 Basal to tryptose 84 .0.49 h r _ 1 92 tryptose to Basal •120.. 0.-34 h r _ 1 64 Basal to Basal 95 0.44 h r " 1 .83 C e l l s grown i n maltose and maltose plus tryptose (Q.1%) medium to an 0.D.g60> 0.6 were harvested by c e n t r i f u g a t i o n , washed, i n maltose medium and resuspended to.the o r i g i n a l volume ( 7 . 0 ml). From these 1.0 ml was added to each of three.4 . 0 ml amounts..of both, maltose and maltose plus tryptose medium. The o p t i c a l densities f o r a l l . c u l t u r e s were recorded. / f i g * ll. SStfft wp ma shift 6mm •xperiseats. Guitars* of B« reylottkilua wove grove ts *«ltc*e *ad maltose trygtoao ( o.UO atditsn to *n Q.&.gfo, 0 .6. ooll* war* cwrtrlfu«*4 at 9»^CKTX £ for 10 miautot, wwh«4 i a s&ltoso aeditm mSL y«»iM»j>onaod to t&o origiaal volusa ( 7.0 ml). Wtm tlteao, 1*0 tsl w u «$&«4 to «Mii of tfareo *»0 &l astowBto of both @altos« «a£ KBltoee tryptone acditus* 9ht ostic&l densities for •11 ettltufoo w e arocorded for ? feoisra OJS$ tfee a w N t g o of « M & «cri«» jetted* feyftooo "to t?yg*t©«« It A, fefttal to tryptose i t B, tryptoee te b*eal to 0 ea& toaaal to betel i t 1* The Insert dsaeastratea tfeo in Q*D»g&) for tfe* f i r s t tow after shift. c o n t r o l ( c u r v e D) was n o t e x p e c t e d . These o b s e r v a t i o n s s u g g e s t t h a t t r y p t o s e s u p p l i e s g r e a t e r p o o l s o r m a i n t a i n s b e n e f i c i a l i n t r a c e l l u l a r c o n d i t i o n s w h i c h a l l o w t h e c e l l s t o a d j u s t more r a p i d l y t o a new env i r o n m e n t . A g r e a t e r c e l l y i e l d i s o b s e r v e d i n t r y p t o s e medium ( c u r v e s A and B v s c u r v e s C and D). P r e -growth o f t h e c e l l s i n t r y p t o s e does n o t a f f e c t t h e y i e l d . I I . Amino A c i d C o m p o s i t i o n o f B. a m y l o p h i l u s The amino a c i d c o m p o s i t i o n o f B. a m y l o p h i l u s p r o t e i n and t h e p r o t e i n c o n t e n t o f t h e c e l l s was d e t e r m i n e d so t h a t t h e amount o f amino a c i d t a k e n up by. t h e c e l l s m i g h t be e x p r e s s e d as a f r a c t i o n o f t h e t o t a l amount o f t h a t amino a c i d i n t h e c e l l s . The d r y w e i g h t and t h e p r o t e i n c o n t e n t , were r e l a t e d t o t h e O.D.gg^ A s u s p e n s i o n o f ' B . a m y l o p h i l u s w i t h an O.D., 0 . 9 c o n t a i n e d 0.88 mg d r y w e i g h t and 0 .40 mg p r o t e i n p e r m l . The amino a c i d c o m p o s i t i o n o f B. a m y l o p h i l u s p r o t e i n i s p r e s e n t e d i n T a b l e V I I I . The r e s u l t s o b t a i n e d b y P u r s e r and B u e c h l e r (1966) f o r h y d r o l y s a t e s o f B. a m y l o p h i l u s p r o t e i n a r e p r e s e n t e d f o r comp a r i s o n . No c o r r e c t i o n was a p p l i e d t o t h e s e r e s u l t s t o ac c o u n t f o r t h e l o s s o f amino a c i d s d u r i n g h y d r o l y s i s . The c o m p o s i t i o n i s s i m i l a r t o t h a t f o u n d b y Purser, and B u e c h l e r (1966), w i t h t h e e x c e p t i o n o f v a l i n e . P u r s e r and B u e c h l e r (1966) r e p o r t e d a h i g h v a l i n e c o n c e n t r a t i o n f o r ' B . a m y l o p h i l u s i . e . 11.4 g/100 g t o t a l amino T a b l e V I I I : A m i n o a c i d c o m p o s i t i o n o f h y d r o l y s a t e s o f B . a m y l o p h i l u s A m i n o a c i d C o m p o s i t i o n C o m p o s i t i o n e x p e r i m e n t a l l y P u r s e r (1966) d e t e r m i n e d A l a n i n e 7.16 5 .3 A r g i n i n e 5.59 5.7 A s p a r t i c a c i d 10.5 9-9 C y s t e i n e 0 . 0 0 . 7 G l u t a m i c a c i d 12.59 10.3 G l y c i n e 7.4.3 5-9 H i s t i d i n e 2.19 2 .2 I s o l e u c i n e 6.46 6.3 L e u c i n e 8.25 7.7 L y s i n e 10.08 8 . 3 M e t h i o n i n e 2.58 2 .2 P h e n y l a l a n i n e 4 . 7 8 5 .2 P r o l i n e 4.02 3-9 S e r i n e 3.12 3.5 T h r e o n i n e 5.02 5-7 T y r o s i n e 4.27 4 . 2 V a l i n e 7.02 l l J -1 *> <H T h e c e l l s f r o m a 10 h o u r c u l t u r e o f B . a m y l o p h i l u s w e r e p r e p a r e d f o r a m i n o a c i d a n a l y s i s a s d e s c r i b e d i n t h e m e t h o d s ( M o o r e a n d S t e i n , I963). A l l a m i n o a c i d r e s u l t s w e r e e x p r e s s e d a s g r a m s p e r 100 g r a m s o f t o t a l a m i n o a c i d . T h e f i r s t c o l u m n w a s d e r i v e d f r o m t h e a v e r a g e v a l u e s f o r 2 a n a l y s e s e x p e r i m e n t a l l y d e t e r m i n e d f o r B . a m y l o p h i l u s H 18. T h e s e c o n d c o l u m n w a s t h e m e a n v a l u e o f t h e a m i n o a c i d c o m p o s i t i o n d e t e r m i n e d f o r a s t r a i n o f B . a m y l o p h i l u s b y P u r s e r a n d B u e c h l e r (1966). a c i d s , compared t o t h e amount p r e s e n t i n o t h e r rumen b a c t e r i a ( W e l l e r , 1957). I n c o n t r a s t , t h i s work i n d i c a t e s t h a t v a l i n e was p r e s e n t a t t h e same c o n c e n t r a t i o n f o u n d i n m i x e d p o p u l a t i o n s o f rumen b a c t e r i a . G l y c i n e and a l a n i n e c o n c e n t r a t i o n s i n t h e s e e x p e r i m e n t s , were s l i g h t l y h i g h e r - t h a n t h o s e f o u n d b y P u r s e r and B u e c h l e r (1966). The d i f f e r e n c e s i n t h e r e s u l t s c o u l d be a t t r i b u t e d t o e i t h e r s p e c i e s v a r i a t i o n or• v a r i a t i o n . i n t h e age o f t h e c u l t u r e s . P u r s e r and B u e c h l e r (1966) u s e d c u l t u r e s t h a t were o v e r 2k h o u r s o l d whereas 10 h o u r c u l t u r e s were u s e d i n t h e p r e s e n t work. P u r s e r and B u e c h l e r (1966) d i d n o t e e t h a t t h e r e was a v a r i a t i o n o f amino n i t r o g e n w i t h t h e age o f t h e c u l t u r e . B a c t e r o i d e s s p e c i e s p o s s e s s a t h i c k e r c e l l w a l l t h a n most gram n e g a t i v e b a c t e r i a ( B l a d e n and W a t e r s , 1963). T h i s m i g h t a c c o u n t f o r t h e h i g h v a l u e s f o u n d f o r a l a n i n e , g l y c i n e and g l u t a m i c a c i d , a l l o f w h i c h a r e known components o f t h e c e l l w a l l m ucopeptide. The amino a c i d chromatograms h a d a s m a l l peak w h i c h was t e n t a t i v e l y i d e n t i f i e d as d i a m i n o p i m e l i c a c i d , a c o n s t i t u e n t o f b a c t e r i a l m u c o peptide. 14 I I I . The-Uptake o f C M i x e d Amino A c i d s To f u r t h e r u n d e r s t a n d t h e r o l e o f amino a c i d s i n t h e n u t r i t i o n o f B. a m y l o p h i l u s t h e t o t a l amount o f i n c o r p o r a t i o n o f 14 C p r o t e i n h y d r o l y s a t e was i n v e s t i g a t e d . P r e l i m i n a r y e x p e r i m e n t s showed t h a t no amino a c i d s t a k e n i n t o t h e c e l l were i n c o r p o r a t e d i n t o p r o t e i n i n t h e p r e s e n c e o f oxygen, o r i n a sy s t e m t h a t l a c k e d a f e r m e n t a b l e c a r b o h y d r a t e s o u r c e . The main p u r p o s e o f t h e e x p e r i m e n t was t o p r o v i d e e v i d e n c e f o r t h e e n t r a n c e o f amino a c i d s i n t o t h e c e l l . The r e s u l t s ( F i g u r e 15) d e m o n s t r a t e d t h a t 3 m i n u t e s a f t e r t h e l a b e l l e d amino a c i d s were added t o t h e c u l t u r e 14% o r 8 . 8 x 10 ^ u moles o f t h e amino a c i d m i x t u r e was t a k e n up i n t o 1.0 m l o f c e l l s . B. a m y l o p h i l u s c e l l s make up 1.0% o f t h e volume o f t h e Q c u l t u r e when t h e c e l l s a r e a t a c o n c e n t r a t i o n o f 1 x 10 c e l l s p e r m l . Amino a c i d s were c o n c e n t r a t e d 14 t i m e s by. t h e c e l l s as c a l c u l a t e d f r o m t h e r a t i o o f t h e r a d i o a c t i v i t y i n t h e c e l l s t o t h e r a d i o a c t i v i t y i n t h e whole c u l t u r e . There was t h e p o s s i b i l i t y t h a t t h i s l o w l e v e l o f u p t a k e was due t o a b s o r p t i o n o f amino a c i d s t o t h e c e l l s u r f a c e . I t i s . n o t known whether i n d i v i d u a l amino a c i d s were t a k e n up p r e f e r e n t i a l l y . The u p t a k e i n t h i s e x p e r i m e n t w o u l d n o t be t h e maximum amount p o s s i b l e b y t h e c e l l because t h e c o n c e n t r a t i o n o f each amino a c i d p r e s e n t was e x t r e m e l y low. These r e s u l t s p r o v i d e p r e s u m p t i v e e v i d e n c e t h a t B. a m y l o p h i l u s does n o t have a p e r m e a b i l i t y b a r r i e r a g a i n s t t h e e n t r y o f amino a c i d s . The p e r m e a b i l i t y o f B. a m y l o p h i l u s t o amino a c i d s was f u r t h e r d e m o n s t r a t e d b y g r o w i n g t h e m i c r o o r g a n i s m s i n 1.0% "^C c a s e i n h y d r o l y s a t e , m a l t o s e medium. I t was c a l c u l a t e d t h a t t h e c a s e i n h y d r o l y s a t e c o n t r i b u t e d t o 20.4% o f t h e c e l l p r o t e i n . The • 5 4 5000 r TIME (minutes) F i g . 1 5 . The u p t a k e o f l ^ C - m i x e d a m i n o . a c i d s . ; B . a m y l o p h i l u s was. grown t o O.D.ggos i n m a l t o s e medium. T h e . c e l l s were r e s u s p e n d e d i n m a l t o s e medium w h i c h c o n t a i n e d 6 . 3 x l O ~ 5 moles mi x e d amino a c i d s ( C o m p o s i t i o n i n M a t e r i a l s ) and . 1 . 4 x l 0 ~ 3 y c l ^ C - p r o t e i n h y d r o l y s a t e . p e r m l . The u p t a k e o f '-^C-amino a c i d s was f o l l o w e d by t h e r a p i d f i l t r a t i o n method o f B r i t t e n and M c C l u r e , . ( 1 9 6 2 ) . A i n c o r p o r a t i o n o f C amino a c i d s f r o m media c o n t a i n i n g c a s e i n h y d r o l y s a t e a t two c o n c e n t r a t i o n s and t h e i r c o n t r i b u t i o n t o t h e c e l l p r o t e i n was examined and t h e r e s u l t s a r e i n T a b l e I X . A t w o - f o l d i n c r e a s e i n c a s e i n h y d r o l y s a t e i n t h e medium r e s u l t e d i n a t w o - f o l d i n c r e a s e i n t h e average c o n t r i b u t i o n o f t h e amino a c i d s t o t h e c e l l p r o t e i n . A t 2 .8 mg c a s e i n h y d r o l y s a t e / m l t h e ave r a g e c o n t r i b u t i o n was 43% and t h e range was 20% t o 76% f o r t h e i n d i v i d u a l amino a c i d s . A c u l t u r e O.D.gg^, 0 .6 w o u l d c o n t a i n 0.24 mg p r o t e i n / m l so i t was u n l i k e l y t h a t any amino a c i d was a t a l i m i t i n g c o n c e n t r a t i o n i n m e d i a c o n t a i n i n g 2 .8 mg c a s e i n h y d r o -l y s a t e / m l . T h i s was f u r t h e r e v i d e n c e t h a t t h e u p t a k e o f amino a c i d s was c o n c e n t r a t i o n dependent, t h a t s i g n i f i c a n t u p t a k e o c c u r r e d o n l y i n h i g h c o n c e n t r a t i o n o f amino a c i d and t h a t t h e mechanism o f u p t a k e was p r o b a b l y s i m p l e d i f f u s i o n . C a s e i n h y d r o l y s a t e , a t a c o n c e n t r a t i o n n o r m a l l y u s e d i n g r o w t h media ( .28%), c o n t r i b u t e d s i g n i f i c a n t l y t o t h e c e l l p r o t e i n . T h i s was n o t a n t i c i p a t e d as Hobson e t al. ( 1 9 6 8 ) showed t h a t ammonia was u s e d e x c l u s i v e l y b y B. a m y l o p h i l u s i n t h e p r e s e n c e o f o r g a n i c n i t r o g e n . Hobson e t a l . (1968) u s e d t r y p t o s e , a t r y p t i c h y d r o l y s a t e o f c a s e i n , r a t h e r t h a n f r e e amino a c i d s . 14 However, as no C t r y p t o s e was a v a i l a b l e , i t s d i r e c t u p t a k e c o u l d n o t be s t u d i e d . The c o m p e t i t i o n o f t r y p t o s e w i t h amino a c i d u p t a k e was t h e r e f o r e examined. T a b l e I X : X 4 C amino a c i d u p t a k e i n t h e p r e s e n c e o f h i g h l e v e l s o f c a s e i n h y d r o l y s a t e . Amino A c i d C a s e i n C o m p o s i t i o n 1.4 mg p e r m l 2 .8 mg p e r m l C o m p o s i t i o n o f C e l l % i n c o r p ug/ml % e x t e r n a l % i n c o r p ug/ml % e x t e r n a l ug/ml i n c o r p c o n t r i b u t i o n i n c o r p s c o n t r i b u t i o n A r g i n i n e 4.13. 11.2 8.1 4.7 42 4.3 5.0 45 A s p a r t i c a c i d 7.66 21.0 3.0 3.2 15 2.9 6.2 . 30 G l u t a m i c a c i d •23.55 25-2 2.0 6.6 26 1.3 8.6 34 H i s t i d i n e . 3.46 4.4 • 3.2 1.6 - 36 2.6 2.6 59 I s o l e u c i n e • 5.98 13.0 2.9 2.4 18 . 3.4 5.7 44 L e u c i n e 10.02 16.6 2.5 3.5 21 1.9 5.3 32 M e t h i o n i n e 5.2 1.9 0.9 17 '• 1.7 1.6 31 P h e n y l a l a n i n e 6.'o". 9.6 . 3.0 2.5 26 2.8 4.7 49 P r o l i n e 11.7 8.0 1.0 . 1.6 20 . 1.2 3.9 49 S e r i n e 5.9 6.2 3.7 3.1 50 2.9 4.8 76 T y r o s i n e 6.62 8.6 2.4 2.2 25 - 2.1 3.9 45 V a l i n e • 7.38 14.0 . 1.8 1.9 13 1.4 2.9 20 Average 26 43 An o v e r n i g h t c u l t u r e o f B_. a m y l o p h i l u s was i n o c u l a t e d i n t o t u b e s o f 6 m l m a l t o s e medium w h i c h c o n t a i n e d 0.018 uc o f i n d i v i d u a l T*C -amino a c i d s and e i t h e r 1.4 mg o r 2 .8 mg of. c a s e i n h y d r o l y s a t e p e r m l . The c u l t u r e s were grown t o an O.D.g6o» 0.5 and t h e r a d i o a c t i v i t y i n t h e c e l l s and t h e c e l l s p l u s t h e s u p e r n a t a n t was counted. The i n c o r p o r a t i o n o f l a b e l was. determined, and t h e c o n t r i b u t i o n of exogenous amino a c i d s t o b a c t e r i a l p r o t e i n was c a l c u l a t e d . The c a s e i n h y d r o l y s a t e and b a c t e r i a l c o m p o s i t i o n s were g i v e n as g/lOOg t o t a l amino a c i d , ( E l l i n g e r and Boyne, 1965). IV. The Uptake o f C.Amino A c i d s i n t h e P r e s e n c e o f T r y p t o s e C - l a b e l l e d amino a c i d s were added a t a h i g h s p e c i f i c a c t i v i t y and l o w c o n c e n t r a t i o n t o media w i t h and w i t h o u t t r y p t o s e . The p e r c e n t a g e i n c o r p o r a t i o n b y B. a m y l o p h i l u s o f t h e amino a c i d s was d e t e r m i n e d f o r b o t h s e r i e s and t h e r e s u l t s a r e p r e s e n t e d i n T a b l e X. The i n c o r p o r a t i o n o f each amino a c i d , w i t h t h e e x c e p t i o n o f g l y c i n e , was i n h i b i t e d b y t r y p t o s e . The average i n c o r p o r a t i o n was r e d u c e d f r o m 13.4% t o 4.8%. The c o n t r i b u t i o n o f .the amino a c i d s t o t h e b a c t e r i a l p r o t e i n , a t t h i s l o w c o n c e n t r a t i o n , was s m a l l and d i d n o t exce e d 1.0%. I t i s t h e r e f o r e i m p o s s i b l e t o deduce t o what e x t e n t t h e t r y p t o s e p e p t i d e s c o n t r i b u t e d t o t h e c e l l p r o t e i n b u t t h e ex p e r i m e n t c l e a r l y d e m o n s t r a t e d t h a t c o m p e t i t i o n between t h e p e p t i d e s and amino a c i d s o c c u r r e d and i t may be c o n c l u d e d t h a t some i n c o r p o r -a t i o n d i d o c c u r . The p r e f e r e n t i a l i n c o r p o r a t i o n o f p e p t i d e s i n t o B. r u m i n i c o l a was d e s c r i b e d b y P i t t m a n e t a l . (1967). 14 V. The Uptake o f C - p e p t i d e s The i n d i r e c t e v i d e n c e f o r t r y p t o s e i n c o r p o r a t i o n s u g g e s t 6.> -14 aetGrrox^e-.'t C - p e p t i d e s c o u l d be i n c o r p o r a t e d t o t h e c e l l p r o t e i n . The enzymes o f B. a m y l o p h i l u s h y d r o l y s e d p r o t e i n t o p e p t i d e w i t h l i t t l e f r e e amino a c i d (Abou Akkada and B l a c k b u r n , 1963). 14 To examine p e p t i d e u t l i z a t i o n b y B. a m y l o p h i l u s , C - p r o t e i n was T a b l e X: The u p t a k e o f C-amino a c i d s i n t h e p r e s e n c e o f t r y p t o s e . Amino a c i d E x t e r n a l % • "jo c o n c e n t r a t i o n i n c o r p o r a t e d i n c o r p o r a t e d i n mg/ml t r y p t o s e medium A l a n i n e .082 16.0 3.0 A r g i n i n e ./084 16.2 2.3 G l u t a m i c a c i d .074 16.7 KR G l y c i n e .090 10.0 14 . 5 H i s t i d i n e .042 9 . 2 3 .3 L e u c i n e .064 2 0 . 1 4 . 4 L y s i n e .061 3.3 1.4 M e t h i o n i n e .110 23 .4 11.4 P h e n y l a l a n i n e .038 17.6 8 . 3 P r o l i n e .048 2 0 . 0 3 . 3 S e r i n e .072 5-5 1.0 T h r e o n i n e .076 2.9 1.7 T y r o s i n e .040 5.3 KR V a l i n e .074 16.0 2.9* * KR = no r e s u l t s One drop o f an o v e r n i g h t c u l t u r e o f B. a m y l o p h i l u s was i n o c u l a t e d i n t o 6 . 0 m l amounts o f m a l t o s e medium c o n t a i n i n g O.O83 uc/ml o f i n d i v i d u a l amino a c i d a t t h e c o n c e n t r a t i o n s p e c i f i e d i n column one. T r y p t o s e (1.5 mg/ml) was added t o a d u p l i c a t e s e t o f c u l t u r e s . The c e l l s were grown t o O.D.^g , 0 . 6 . Samples o f t h e c e l l s and t h e c e l l s p l u s t h e s u p e r n a t a n t were d r i e d on M i l l i p o r e f i l t e r s and t h e r a d i o a c t i v i t y p r e s e n t was d e t e r m i n e d . i n c l u d e d i n t h e m e d i u m a s a s o u r c e o f p e p t i d e s . ' T h e l a b e l l e d p r o t e i n w a s i s o l a t e d f r o m B . a m y l o p h i l u s c e l l s a n d i t s r e i n c o r p o r a t i o n i n t o B . a m y l o p h i l u s w a s d e t e r m i n e d ( T a b l e X I ) . C o n s i d e r a b l e i n c o r p o r a t i o n o c c u r r e d e v e n t h o u g h t h e c o n d i t i o n s , w e r e p r o b a b l y n o t o p t i m u m f o r i n c o r p o r a t i o n a s t h e f i n a l p r o t e i n c o n t e n t i n t h e m e d i u m w a s l o w a n d p r e v i o u s r e s u l t s ( T a b l e I X ) s h o w e d t h a t a m i n o a c i d u p t a k e w a s c o n c e n t r a t i o n d e p e n d e n t . P r e v i o u s e x p e r i m e n t s h a v e b e e n c a r r i e d o u t u n d e r e i t h e r l i m i t i n g a m i n o a c i d c o n c e n t r a t i o n s o r u n d e r c o m p e t i t i v e p r e s s u r e o f e i t h e r a m i n o a c i d s o r p e p t i d e s . T h e i n c o r p o r a t i o n o f h i g h c o n c e n t r a t i o n s o f i n d i v i d u a l a m i n o a c i d s w a s e x a m i n e d , t o d e t e r m i n e t h e m a x i m u m a m o u n t o f i n c o r p o r a t i o n o f a n y o n e a m i n o a c i d i n t h e a b s e n c e o f o t h e r o r g a n i c n i t r o g e n s o u r c e s . T h e r e s u l t s a r e p r e s e n t e d i n T a b l e X I I . T h e a v e r a g e c o n t r i b u t i o n o f t h e l a b e l l e d a m i n o a c i d s t o t h e c o r r e s p o n d i n g a m i n o a c i d s i n t h e c e l l p r o t e i n w a s 54% w i t h a r a n g e o f 21% t o 88%. T h e s e f i g u r e s a r e c o m p a r a b l e t o t h o s e o b t a i n e d f o r t h e c o n t r i b u t i o n o f i n d i v i d u a l a m i n o a c i d s f r o m c a s e i n h y d r o l y s a t e ( T a b l e I X ) . T h e r e w a s n o t , h o w e v e r , g o o d c o r r e l a t i o n b e t w e e n t h o s e s h o w i n g a h i g h c o n t r i b u t i o n i n t h e t w o e x p e r i m e n t s . I n c a l c u l a t i o n , t h e c o n t r i b u t i o n o f i n d i v i d u a l a m i n o a c i d s , i t h a s 1 4 b e e n a s s u m e d t h a t t h e C a m i n o a c i d w a s n o t c o n v e r t e d t o o t h e r a m i n o a c i d s b e f o r e i n c o r p o r a t i o n i n t o p r o t e i n . T h e h i g h c o n t r i b u t i o n 14 o f s o m e C a m i n o a c i d s m a y b e d u e t o s u c h a c o n v e r t i o n . T h i s p o s s i b i l i t y w a s e x p l o r e d i n t h e f o l l o w i n g e x p e r i m e n t s . T a b l e X I : The i n c o r p o r a t i o n o f ' C - p r o t e i n i n t o E. 'amylophilus.. % i n c o r p o r a t e d . ug p r e s e n t p e r m l . ug p e r m l i n c o r p o r a t e d . . % c o n t r i b u t i o n t o t o t a l . c e l l p r o t e i n . -24 . 140. . 34 . 17 C e l l s . w e r e grown i n 100 m l o f m a l t o s e t r y p t o s e media i n . t h e p r e s e n c e o f 14C - n i a l t o s e ( 0.5 uc p e r m l o f media) t o an O.D.ggov'.0.52, h a r v e s t e d by . c e n t r i f u g a t i o n , e x t r a c t e d i n . hot. 10%'. TCA ( -80C. f o r 15 m i n u t e s ) , washed by 3 volumes o f a c e t o n e , r e d i s s o l v e d i n 2% Wa 2C0 3 i n . 0 . 2 N NaOH, f l a s h . e v a p o r a t e d t o .5.0 m l and n e u t r a l i z e d t o .pH .7.0 w i t h HC1. The r e s u l t i n g r a d i o a c t i v e p r o t e i n . c o n t a i n e d ,1.6 mg p r o t e i n . p e r m l and c o n t a i n e d 0.02 uc o f l a b e l p e r m l . One h a l f o f a m l o f t h e p r o t e i n s o l u t i o n was added t o ; 5 - 0 ml o f i n o c u l a t e d m a l t o s e .medium. The r a d i o a c t i v i t y w a s . d e t e r m i n e d by M i l l i p o r e f i l t r a t i o n , as d e s c r i b e d in. t h e Methods. Table X I I : Uptake of c amino acids i n the presence of an individual specific amino acid carrier Amino acid 1o incorporated Ug/ml incorporated 1o of the amino Alanine 6.7 100 47 Arginine 11.7 176 82 Histidine 4.9 74 34 Leucine 9.3 140 65 Proline 3.0 45 21 Serine 7.2 108 50 Tryptophan 12.6 I89 88 Valine 6.4 96 45 An overnight culture of B. amylophilus was inoculated into 6.0 ml of maltose medium containing 1.5 mg/ml of one specific amino acid plus .02 pc of the same ^ C amino acid, and grown to an O.D.gg , 0.5 V I . The Uptake and C o n v e r t i o n o f Amino A c i d s The f a t e o f C amino a c i d s was d e t e r m i n e d b y e x a m i n i n g t h e d i s t r i b u t i o n o f l a b e l i n t h e c e l l p r o t e i n . Some p r e l i m i n a r y e x p e r i m e n t s were p e r f o r m e d t o en s u r e t h a t adequate r e s o l u t i o n and r e c o v e r y o f amino a c i d s was p o s s i b l e b y t h e t h i n l a y e r c h r o m a t o g r a p h i c t e c h n i q u e . 1. Q u a l i t a t i v e amino a c i d a n a l y s i s b y t h i n l a y e r chromato-g r a p h y P r e l i m i n a r y work d e t e r m i n e d t h e c o n d i t i o n s r e q u i r e d t o o b t a i n a o p t i m u m d e t e c t i o n o f c e l l u l a r amino a c i d s on c e l l u l o s e t h i n l a y e r chromatograms. One d i m e n s i o n a l chromatography p r o v e d i n a d e q u a t e t o r e s o l v e a m i x t u r e o f amino a c i d s , c o n s e q u e n t l y two d i m e n s i o n a l chromatography b y t h e method o f Jones and H e a t h c o t e (1966) was u s e d i n a l l e x p e r i m e n t s . S t a n d a r d s o l u t i o n s o f amino a c i d s (2 mg/ml) were d i s s o l v e d i n aqueous p r o p a n - 2 - o l (10% v/v) were r u n t o v e r i f y t h e p o s i t i o n s o f t h e amino a c i d s . The R f v a l u e s r e m a i n e d r e l a t i v e l y c o n s t a n t when a l l t h e s o l v e n t f r o n t s were c a l c u l a t e d t o a common v a l u e o f 100 cm. L e s s t h a n 5 ug o f amino a c i d c o u l d n o t be d e t e c t e d on t h i n l a y e r chromatograms and s i n c e m e t h i o n i n e , c y s t e i n e , h i s t i d i n e and t r y p t o p h a n o c c u r r e d i n low c o n c e n t r a t i o n s i n B. a m y l o p h i l u s p r o t e i n , t h e y were d i f f i c u l t t o d e t e c t . Long s o l v e n t development.(18 cm) w e r e . r e q u i r e d . t o . s e p a r a t e v a l i n e f r o m m e t h i o n i n e , l e u c i n e f r o m i s o l e u c i n e , and g l y c i n e f r o m s e r i n e . C h a r a c t e r i s t i c . c o l o u r s f o r c e r t a i n a m i n o . a c i d s w e r e . f o u n d a f t e r s p r a y i n g w i t h t h e n i n h y d r i n - c o l l i d i n e s o l u t i o n : g r e e n . f o r a s p a r t i c a c i d , g r e y f o r c y s t i n e , brown f o r g l y c i n e , h i s t i d i n e , p h e n y l a l a n i n e and t y r o s i n e and y e l l o w f o r p r o l i n e and h y d r o x y -p r o l i n e . The R f v a l u e s f o r t h e amino a c i d s i n . t h e f i r s t d i m e n s i o n were more s i m i l a r . t o t h o s e o f Jones and H e a t h c o t e (I966), t h a n were the'.Rf v a l u e s . f o r t h e . s e c o n d d i m e n s i o n ( T a b l e X I I I ) ; r e p r o d u c i b i l i t y however was e x c e l l e n t . The p r o t e i n was h y d r o l y s e d f o r 12.,. 18. and 24. h o u r s t o d e t e r m i n e . t h e optimum t i m e f o r r e l e a s e o f t h e amino a c i d s f r o m t h e p r o t e i n . '• H y d r o l y s i s for"18 h o u r s p r o v e d t o be t h e most e f f i c i e n t and was u s e d i n a l l . s u b s e q u e n t e x p e r i m e n t s . H y d r o x y p r o l i n e was absent f r o m h y d r o l y s a t e s .of ;B_. a m y l o p h i l u s p r o t e i n , and t r y p t o p h a n was p r e s e n t o n l y when t h e p r o t e i n was h y d r o l y s e d . f o r 10^12 h o u r s . M e t h i o n i n e was n o t l i b e r a t e d by 12 hour h y d r o l y s i s b u t d i d appear a f t e r 18•hours. A l l o f t h e a s p a r a g i n e and g l u t a m i n e were h y d r o l y z e d t o g l u t a m i c a c i d and a s p a r t i c a c i d ; c y s t i n e was f o u n d o n l y i n t h e • o x i d i z e d f o r as c y s t e i c a c i d . " T a b l e X I I I : T h i n l a y e r chromatography o f amino a c i d s f r o m B. a m y l o p h i l u s . Amino a c i d F i r s t R f 100 d i m e n s i o n R f 100* Second Rf 100-d i m e n s i o n R f 100 : A l a n i n e 63 55 19 10 A r g i n i n e 32 24 13 3 A s p a r t i c a c i d 47 41 10 0 C y s t e i c a c i d 12 11 12 5 Diamino p i m e l i c a c i d 10 KR** 4 KR G l u t a m i c a c i d 57 52 10 1 G l y c i n e 43 36 14 7 H i s t i d i n e 24 15 12 9 I s o l e u c i n e 77 76 h9 ^5 L e u c i n e 80 78 52 52 L y s i n e 27 20 12 4 M e t h i o n i n e 7^ 66 4o 36 P h e n y l a l a n i n e 72 69 53 50 P r o l i n e 62 5^ 24 17 S e r i n e 43 36 19 13 T a u r i n e 3h 22 18 17 T h r e o n i n e 50 ^ 46 ^5 T y r o s i n e 69 60 40 21 V a l i n e 7h 69 40 30 S o l v e n t f r o n t 100 100 100 100 * = The r e s u l t s o f Jones and H e a t h c o t e (1966) p r e s e n t e d f o r c o m p a r a t i v e p u r p o s e s ** = Ko r e s u l t s 50 u l o f h y d r o l y s a t e (2 mg p r o t e i n / m l ) o f c e l l s f r o m m a l t o s e - t r y p t o s e medium was s p o t t e d o n t o t h i n l a y e r c e l l u l o s e MK 300 p l a t e s w h i c h were r u n two d i m e n s i o n a l l y (Jones and H e a t h c o t e , 1966) and were s p r a y e d w i t h n i n h y d r i n - c o H i d i n e , f o l l o w e d b y warm a i r d r y i n g . 2 . D e t e c t i o n o f C amino a c i d s on chromatograms B. a m y l o p h i l u s was grown on C-maltose w i t h o u t an o r g a n i c 14 n i t r o g e n s o u r c e t o c o n f i r m t h a t r a d i o a u t o g r a p h y w o u l d l o c a t e C-l a b e l l e d amino a c i d s i n p r o t e i n h y d r o l y s a t e s . The r e s u l t s o f t h i n l a y e r chromatography o f t h e p r o t e i n h y d r o l y s a t e i s g i v e n i n F i g u r e l6. A l l t h e common amino a c i d s , w i t h t h e e x c e p t i o n o f 14 c y s t e i n e , were f o u n d t o c o n t a i n C. C y s t e i n e , however, was l a b e l l e d i n a medium r e d u c e d by a s c o r b i c a c i d and sodium s u l f i d e i n s t e a d o f by c y s t e i n e - H C 1 . Two n i n h y d r i n s p o t s w i t h Rf v a l u e s s i m i l a r t o t h o s e o f h i s t i d i n e and d i a m i n o p i m e l i c a c i d c o n t a i n e d i n s u f f i c i e n t r a d i o a c t i v i t y t o expose X-ray f i l m . T r y p t o p h a n was f o u n d t o be n i n h y d r i n n e g a t i v e y e t r a d i o a c t i v i t y was p r e s e n t i n t h e a r e a where t r y p t o p h a n w o u l d be l o c a t e d on t h e c e l l u l o s e t h i n 14 ' / x l a y e r chromatogram. Two C s p o t s ( 2 2 and 23) appeared on t h e r a d i o a u t o g r a m s , w i t h Rf v a l u e s o f 17-5 and 54 i n t h e f i r s t d i m e n s i o n and 8 0 and 29 i n t h e second d i m e n s i o n . Spot 23 c o -chromatographed w i t h p u t r e s c i n e , a breakdown p r o d u c t o f a r g i n i n e , Spot 22 was n o t i d e n t i f i e d . Because t h e p r o t e i n was h y d r o l y z e d f o r o n l y 12 h o u r s t h e r e was t h e p o s s i b i l i t y o f i n c o m p l e t e h y d r o -l y s i s and s p o t 22 may have b e e n a p e p t i d e . ¥%C* 16. Bo4if*ottvt and Biafeydrin dottotioa of labelled catao aaida* ftta drop of a ealftart of B* aayloBailat vos iaooulatad lata 9 a l of aedltia eeat&ialas ©Vsf aaltoat aa& 9*1 ao/al **KJ aaltoaa* flao ealtara ttas grona to O.D.ggg, 0.6, etntylfstge4, tad ferdrolyMd for 12 aoara ana roeraipea&od i a S.O a l BgO. Fiftyvl of tsyfiyolysate tra* spotted oa cellalos© taia layer e!ureaato«r»paio plates, ran two dimeneicaaHy aad raatoatftotisas* @&do» aooordiag to taa procedure eatliaed in aetfcode. s$ot aaai&er asdao aoid OpOt BUS !ber oaiao aoid 1 leaoi&o 13 aoriao a i t tferooslao 3 Sfeaayl&laaia* IS argioiao ** valise 16 lyeioe ~ r i^etaioaiae 1? hiatl&iae tswaaftaa 18 dlesiaoplaelie T proline 1? eyeteio aoid 8 alaatae £0 eyeteiae 9 glataale aoid ai ornitaiae 19 asaartlo aeld aateaoira 11 mafeaova 23 aaSfiaoaa lit slyoiao 8$ tvyjy&oph&o iiaferdria toaitivo positive , ais&yflria «&d *%C pwitivo 1st DIMENSION 19 3. R e c o v e r y o f C amino a c i d s f r o m c e l l u l o s e t h i n l a y e r c h r o m a t o g r a p h i c p l a t e s C e l l s o f B. a m y l o p h i l u s were l a b e l l e d b y g r o w t h i n m a l t o s e medium w h i c h c o n t a i n e d 0 . 1 ug ~^C g l u t a m i c a c i d / m l . A p o r t i o n o f a h y d r o l y s a t e o f t h e s e c e l l s was d r i e d on a M i l l i p o r e f i l t e r and t h e r a d i o a c t i v i t y was c o u n t e d i n s c i n t i l l a t i o n f l u i d . I t was n e c e s s a r y t o d r y t h e sample b e f o r e c o u n t i n g as o n l y 17% o f t h e c o u n t s were r e c o r d e d when 50 u l o f t h e h y d r o l y s a t e was added d i r e c t l y t o t h e s c i n t i l l a t i o n f l u i d . The r e a d i o a c t i v e amino a c i d s were s e p a r a t e d on a c e l l u l o s e t h i n l a y e r chromatogram, l o c a t e d b y r a d i o a u t o g r a p h y and s c r a p e d i n t o s c i n t i l l a t i o n v i a l s . When a l l t h e s p o t s were c o u n t e d o n l y 82% o f t h e r a d i o a c t i v i t y was r e c o v e r e d . The e x p e r i m e n t was r e p e a t e d and a g a i n t h e r e were i n c o m p l e t e r e c o v e r i e s , p o s s i b l y due t o some l a b e l l i n g o f non-amino a c i d components i n t h e c e l l and a l s o t o q u e n c h i n g b y t h e c e l l u l o s e . The r e c o v e r i e s were q u i t e adequate t o a l l o w t h e f a t e o f t h e i n c o r p o r a t e d amino a c i d t o be f o l l o w e d . i Ik 4. I n t e r c o n v e r s i o n o f C amino a c i d s To d i f f e r e n t i a t e between t h o s e amino a c i d s w h i c h were d i r e c t l y i n c o r p o r a t e d i n t o p r o t e i n and t h o s e w h i c h were c o n v e r t e d i n t o d i f f e r e n t amino a c i d s b e f o r e t h e y were i n c o r p o r a t e d i n t o c e l l p r o t e i n , B. a m y l o p h i l u s was grown i n t h e p r e s e n c e o f each o f t h e common amino a c i d s , and t h e d i s t r i b u t i o n o f l a b e l i n t h e amino a c i d s o f t h e p r o t e i n was examined by t h i n l a y e r chromato-g r a p h y ( T a b l e XIV.). P l a t e s t r e a t e d w i t h n i n h y d r i n were n o t u s e d f o r r a d i o a c t i v e d e t e r m i n a t i o n s . L e u c i n e , p r o l i n e and h i s t i d i n e were i n c o r p o r a t e d i n t o p r o t e i n w i t h o u t any i n t e r c o n v e r s i o n o r r e s y n t h e s i s i n t o o t h e r amino a c i d s . The ma j o r p o r t i o n o f v a l i n e , t y r o s i n e , g l u t a m i n e and a r g i n i n e were i n c o r p o r a t e d d i r e c t l y i n t o p r o t e i n b u t some i n t e r c o n v e r s i o n d i d o c c u r . T r y p t o p h a n , t h r e o n i n e a l a n i n e and a s p a r t i c a c i d underwent m a j o r c o n v e r s i o n t o o t h e r amino a c i d s b u t i n t h e group o f amino a c i d s c o n s i s t i n g o f m e t h i o n i n e , g l y c i n e , s e r i n e and i s o l e u c i n e t r a c e amounts o f t h e amino a c i d were i n c o r p o r a t e d d i r e c t l y , most o f t h e r a d i o a c t i v i t y was f o u n d i n o t h e r amino a c i d s . The absence o f i s o l e u c i n e i s n o t r e a d i l y u n d e r s t o o d . The amino a c i d s t h a t have a l a r g e p r o p o r t i o n o f t h e t o t a l r a d i o a c t i v i t y c o n v e r t e d t o o t h e r amino a c i d s , h a d a g r e a t e r amount o f i n c o r p o r a t i o n t h a n t h e t o t a l c o n c e n t r a t i o n o f t h a t p a r t i c u l a r amino a c i d i n t h e c e l l . T here a p p e a r e d t o be s e v e r a l mechanisms o f r e a c t i o n r e s p o n s -i b l e f o r t h e i n t e r c o n v e r s i o n o f t h e amino a c i d s w h i c h were s u p p l i e d There i s some e v i d e n c e f o r b o t h d e - c a r b o x y l a t i o n and d e a m i n a t i o n r e a c t i o n s ; g l u t a m i c a c i d may have been d e c a r b o x y l a t e d t o b u t y r i c a c i d , o r n i t h i n e d e c a r b o x y l a t e d t o p u t r e s c e n e , and a r g i n i n e d e a m i n a t e d t o o r n i t h i n e . However, s e v e r a l o f t h e i n t e r c o n v e r s i o n s fable XX?t 8. eoy lo^ lg iB wa# i a o c « l a t « d iato 6.0 sal eBouota of maltose oedltsa* eaea ceataiol&i one of tee eossoa asdno aei&« st a eoacentration o f 0*035 -0 « l n e per a l . Calttarea were fcarvoated at O.D.^gs , 0 ,S oad uslae aolds l a tat aydroiy&ed "sectorial ce l l s vera *®par«ted by tain layer • e&remtegref&y. fhe ^reoeoee of radioactivity was located by darkened areas o n X -ray filsa asd t s © correepeadiBj* aroa o m tJae c e l l u l e s ® t M a -layer earoaatogrepaic plates were reached, collected i a r i a l © eeatalnl&g tsGiatilatiea f l u i d aad coasted. Sk© ossiao seida were tentatively identif ied aad re lat ive a&ouats of too t o t a l radioact ivity mre detersl&ed. * SBO ©ottota iaoemnred i n the mdae acid , ideat i f led by to l a layer eHroaato^rapb^ ae being t i e or ig ina l oso added to tae sodium, va* expressed at a poree&toge of too t o t a l coaats f&m& l a a l l tae radioaotlve spots. - T a b l e XIV: C o n v e r s i o n o f - L 4C-amino a c i d s i n B. ' a m y l o p h i l u s . Amino a c i d % d i r e c t l y i n c o r p o r a t e d Number o f 1 4 C amino a c i d s i n p r o t e i n T e n t a t i v e amino a c i d i d e n t i f i c a t i o n A l a n i n e 11. 2 .11%. a l a n i n e ; 89% unknown A r g i n i n e 45 5 92% g l u t a m i c a c i d and a r g i n i n e ; 4% o r n i t h i n e ; 6% unknowns A s p a r t i c A c i d • 7 2 7% a s p a r t i c a c i d ; 93% unknown C y s t e i n e NR 2 c y s t e i n e ; c y s t e i c a c i d G l u t a m i c A c i d 60 7 60% g l u t a m i c a c i d ; 15% p r o l i n e ; 15% a r g i n i n e ; 8 % unknown; and 1% l y s i n e . G l y c i n e 2 4 94% unknown; 2% g l y c i n e ; 2% g l u t a m i c a c i d ; 1% t h r e o n i n e 1% s e r i n e H i s t i d i n e 100 1 100% h i s t i d i n e I s o l e u c i n e 0 3 unknowns L e u c i n e 100 1 100%, l e u c i n e . . . . M e t h i o n i n e 2 • 7 1% i s o l e u c i n e and l e u c i n e ; 2% v a l i n e and m e t h i o n i n e 2% unknown P h e n y l a l a n i n e NR 4 p h e n y l a l a n i n e and unknowns P r o l i n e 100 1 100% p r o l i n e S e r i n e 1 5 s e r i n e and unknowns T h r e o n i n e 18 4 20%. i s o l e u c i n e ; 2 0 % t y r o s i n e ; 18% t h r e o n i n e ; 42% g l y c i n e T r y p t o p h a n 18 8 unknown,, a s p a r t i c a c i d and t r y p t o p h a n T y r o s i n e 76 3 t y r o s i n e and unknowns V a l i n e 85 3 v a l i n e , m e t h i o n i n e and l e u c i n e seemed - u n l i k e l y , i f t h e y o c c u r r e d b y t h e n o r m a l b i o s y n t h e t i c pathways. F o r example t h e r e i s no e x p l a n a t i o n f o r 20% o f t h e l a b e l l e d t h r e o n i n e a p p e a r i n g i n t y r o s i n e . The a u t o r a d i o g r a m s a l w a y s c o n t a i n e d some r a d i o a c t i v i t y a t t h e o r i g i n . T h i s r a d i o a c t i v i t y w o u l d have o r i g i n a t e d f r o m some o f t h e d e g r a d a t i o n p r o d u c t s t h a t w o u l d n o t r u n i n t h e s o l v e n t systems. GENERAL DISCUSSION I n r e c e n t y e a r s t h e s i t e o f s y n t h e s i s and t h e mechanism o f r e l e a s e o f b a c t e r i a l exo-enzymes have been e x t e n s i v e l y examined, b u t p r o t e a s e p r o d u c t i o n b y gram n e g a t i v e a n a e r o b i c b a c t e r i a has been n e g l e c t e d . The p r o t e a s e p r o d u c t i o n b y B. a m y l o p h i l u s was s t u d i e d b y B l a c k b u r n (1968a and 1968b), who d e m o n s t r a t e d t h a t t h e p r o t e a s e p r o d u c t i o n began i n t h e l o g a r i t h m i c phase o f growth and c o n t i n u e d u n t i l t h e b a c t e r i a c e a s e d t o d i v i d e . The c e l l - f r e e p r o t e a s e c o n s t i t u t e d 20% o f t h e t o t a l p r o t e a s e i n l a t e l o g a r i t h m i c phase. I n v e s t i g a t i o n s i n t o t h e l o c a t i o n o f t h e B. a m y l o p h i l u s p r o t e a s e was b y two methods. The f i r s t was s u g g e s t e d b y t h e work o f N o s s a l and H e p p e l , (1966), who u s e d m i l d o s m o t i c shock t o s e l e c t i v e l y r e l e a s e s u r f a c e enzymes w h i c h were n o t bound t o t h e c e l l w a l l s o r c e l l membranes. E a r l y l o g phase c e l l s o f B. a m y l o p h i l u s were sho c k e d under r e d u c e d c o n d i t i o n s t o m a i n t a i n v i a b i l i t y o f t h e a n a e r o b i c c e l l s . S u c r o s e was removed b y d i a l y s i s f r o m f r a c t i o n s , p r i o r t o p r o t e a s e a s s a y , as i t i n t e r f e r e d i n t h e a s s a y . L e s s t h a n 1% o f t h e t o t a l a c t i v i t y was r e l e a s e d t o t h e shock f l u i d , y e t o v e r 25% o f t h e t o t a l c e l l p r o t e i n was r e l e a s e d . The c e l l s were f o u n d t o be v i a b l e b u t o n l y a f t e r a l o n g l a g . Neu and Chou (1967) f o u n d t h a t a l a r g e number o f enzymes r e m a i n e d w i t h c e l l s w h i c h h a d l o s t 10% o f t h e c e l l p r o t e i n . The r e s u l t s i n d i c a t e d t h a t t h e p r o t e a s e was n o t f r e e i n t h e p e r i p l a s m i c space b u t t h a t t h e enzyme was a t t a c h e d t o a c e l l s t r u c t u r e w i t h i n t h e p e r i p l a s m i c space. S p h e r o p l a s t f o r m a t i o n was t h e second method u s e d f o r l o c a l -i z i n g t h e p r o t e a s e . I n t h e s p h e r o p l a s t ^ l a r g e a r e a s o f t h e c e l l w a l l a r e b r o k e n , e x p o s i n g t h e c y t o p l a s m i c membrane t o t h e medium ( B i r d s e l l and C o t a - R o b l e s , 1967), c o n s e q u e n t l y any enzyme f r e e i n t h e p e r i p l a s m w o u l d be r e l e a s e d i n t o t h e medium. Malamy and H o r e c k e r (1961), s t a t e d t h a t s e l e c t i v e r e l e a s e o f enzymes d u r i n g t h e f o r m a t i o n o f s p h e r o p l a s t s b y t r e a t m e n t w i t h EDTA and lyso z y m e , i m p l i e s a l o c a t i o n e x t e r n a l t o t h e c y t o p l a s m i c membrane. When t h e c e l l w a l l l a y e r s were p a r t i a l l y removed t h e s e e x t e r n a l enzymes w o u l d be r e l e a s e d , p r e s u m a b l y because t h e y e x i s t i n a f r e e s t a t e i n t h e p e r i p l a s m o r because t h e y c a n be e a s i l y d e t a c h e d f r o m t h e c e l l s u r f a c e . Lysozyme t r e a t m e n t l i b e r a t e d 33% o f t h e p r o t e a s e f r o m whole c e l l s b u t 4 0 % o f t h i s p r o t e a s e was se d i m e n t e d b y p r o l o n g e d c e n t r i -f u g a t i o n a t l 6 o , 000 x g. S o n i c d i s r u p t i o n o f t h e s p h e r o p l a s t s r e l e a s e d 72% o f t h e p r o t e a s e I n t o t h e s u p e r n a t a n t (22,000 g f o r 20 min) i n d i c a t i n g t h a t t h e enzyme was n o t l o c a t e d f r e e i n t h e p e r i p l a s m i c space b u t t h a t i t was bound t o c e l l s t r u c t u r e s . Other s p h e r o p l a s t e x p e r i m e n t s d e m o n s t r a t e d t h a t 79% o f t h e p r o t e a s e a c t i v i t y remained' w i t h t h e s p h e r o p l a s t and 20% was r e l e a s e d , t o t h e s u p e r n a t a n t (10,000 g f o r 20 m i n ) . Upon g e n t l e o s m o t i c , r u p t u r e , 48% o f t h e t o t a l p r o t e a s e a c t i v i t y r e m a i n e d w i t h t h e s p h e r o p l a s t e n v e l o p e ; t h e r e m a i n d e r was r e l e a s e d t o t h e s u p e r n a t a n t . P r o l o n g e d c e n t r i f u g a t i o n a t l6o,000 g o f t h e s u p e r n a t a n t f r a c t i o n r e s u l t e d i n t h e s e d i m e n t a t i o n o f a l l b u t l 6 % o f t h e enzyme a c t i v i t y . P e n i c i l l i n a s e i n B. l i c h e n i f o r m i s i s l o c a t e d i n a p e r i p l a s m i c s t r u c t u r e and i n t h e c y t o p l a s m i c membrane ( S a r g e n t e t a l . , 1968). T u b u l e s and v e s i c l e s w i t h s i n g l e l a y e r e d membranes and w i t h no v i s i b l e i n t e r n a l s u b s t r u c t u r e a r e seen i n t h e p e r i p l a s m and may r e p r e s e n t t h e p e n i c i l l i n a s e s e c r e t o r y a p p a r a t u s (Ghosh e t a l . , 1968). The m a j o r p a r t o f t h e c e l l - b o u n d p e n i c i l l i n a s e i s p r e s e n t i n p e r i p l a s m i c t u b u l e s w h i c h a r e r e l e a s e d on p r o t o p l a s t f o r m a t i o n ( S a r g e n t e t a l . , 1969). A r e a s o f a d h e s i o n e x i s t between t h e c e l l w a l l and t h e c y t o p l a s m i c membrane o f E. c o l i and t h e y p r o v i d e e v i d e n c e f o r t h e e x i s t e n c e o f p e r i p l a s m i c s t r u c t u r e s i n gram n e g a t i v e o r g a n i s m s ( B a y e r , I968). B l a d e n and Waters (1963), have i n d i c a t e d t h a t s i m i l a r p e r i -p l a s m i c s t r u c t u r e s a r e p r e s e n t i n a B a c t e r o i d e s sp. These f i n d i n g s may s u g g e s t t h a t t h e c e l l bound p r o t e a s e may go t h r o u g h a r e s i d e n t s t a g e on t h e c y t o p l a s m i c b r i d g e s p r i o r t o r e l e a s e . The p a r t i c l e bound n a t u r e o f B. a m y l o p h i l u s p r o t e a s e was s u g g e s t e d b y B l a c k b u r n (1968a) who showed t h a t p r o t e a s e r e l e a s e d f r o m d i s r u p t e d c e l l s , was e x c l u d e d f r o m Sephadex: G-200. S i m i l a r r e s u l t s were r e p o r t e d f o r t h e p e n i c i l l i n a s e o f B. l i c h e n i f o r m i s ; 60% o f t h e c e l l - b o u n d p e n i c i l l i n a s e was r e l e a s e d t o t h e s u p e r n a t a n t d u r i n g p r o t o p l a s t i n g , 50% o f t h i s p e n i c i l l i n a s e was se d i m e n t e d b y • c e n t r i f u g a t i o n a t 200,000 x g f o r 6 h o u r s . Most o f t h e p e n i c i l l i n -ase t h a t r e m a i n e d i n t h e s u p e r n a t a n t f l u i d a f t e r h i g h speed c e n t r i f u g a t i o n was e x c l u d e d b y Sephadex G--200, i n d i c a t i n g t h a t i t had a m o l e c u l a r w e i g h t g r e a t e r t h a n 200,000. By c o n t r a s t t h e exoenzyme h a d a m o l e c u l a r w e i g h t o f 2 8 , 0 0 0 . The c e l l - b o u n d p e n i c i l l i n a s e was c o v a l e n t l y l i n k e d t o t h e c e l l membrane t h r o u g h a p e p t i d e bond as t h e enzyme w h i c h was a t t a c h e d t o membrane f r a g m e n t s were n o t r e l e a s e d b y u l t r a s o u n d , u r e a , h y d r o x y l a m i n e o r d e o x y c h o l a t e , b u t was removed b y t r e a t m e n t w i t h t r y p s i n . I t i s p r o b a b l e t h a t t h e p r o t e a s e o f B. a m y l o p h i l u s i s f o u n d t e m p o r a r i l y i n a c e l l - b o u n d l o c a t i o n p r i o r t o i t s l i b e r a t i o n i n t o t h e g r owth medium. I n t h i s r e s p e c t i t w o u l d r e s e m b l e t h e p e n i c i l l i n a s e o f B. l i c h e n i f o r m i s (Lampen, 1965 and 1967b; C o l l i n s , 1964). P e r i p l a s m i c s t r u c t u r e s a c t as a p r i m e r o n t o w h i c h t h e p e n i c i l l i n a s e a t t a c h e s d u r i n g growth ( S a r g e n t e t a l . , 1969). The r e s i d e n t t i m e was f o u n d t o be s h o r t e r f o r l o g c e l l s (30 m i n u t e s ) t h a n f o r l a g c e l l s ( 3 . 5 h o u r s ) . T o l u e n e t r e a t m e n t , m e c h a n i c a l o r u l t r a s o n i c d i s i n t e g r a t i o n o f t h e i n t a c t c e l l s o f B. a m y l o p h i l u s d i d n o t i n c r e a s e t h e p r o t e a s e a c t i v i t y . T h i s i n d i c a t e d t h a t a l l t h e p r o t e a s e was s u p e r f i c i a l l y l o c a t e d a t t h e b a c t e r i a l s u r f a c e and was a c c e s s i b l e t o t h e c a s e i n s u b s t r a t e . P o l l o c k (1962), d e s c r i b e d t h e c h a r a c t e r i s t i c s o f a p a r t i a l l y c e l l - b o u n d enzyme. These a r e enzymes t h a t a r e c e l l - b o u n d b u t t h e y l e a k i n t o t h e g r o w t h medium. Because enzymes a r e l o c a t e d a t t h e s u r f a c e o f t h e c e l l , t h e a v a i l a b l e enzyme does n o t i n c r e a s e b y more t h a n 10% when t h e c e l l s a r e m e c h a n i c a l l y d i s r u p t e d . These enzymes a r e a b l e t o b r e a k down h i g h m o l e c u l a r w e i g h t s u b s t a n c e s and a r e o f t e n r e s p o n s i b l e f o r t h e i n i t i a l breakdown s t a g e s o f t h e s e m o l e c u l e s . The p r o t e a s e enzyme p r o d u c e d b y B. a m y l o p h i l u s appears t o be p a r t i a l l y c e l l bound. I t i s l o c a t e d i n s i d e t h e p e r i p l a s m i c space and p a r t i a l l y a t t a c h e d t o t h e c y t o p l a s m i c membrane. The e x p e r i m e n t s d e s i g n e d t o show t h e optimum pH a c t i v i t y o f t h e p r o t e a s e i n d i c a t e d t h a t e i t h e r one enzyme had s e v e r a l pH optimum o r t h a t t h e r e were s e v e r a l p r o t e a s e enzymes w i t h d i f f e r e n t pH optimum. There was no e v i d e n c e f o r t h e d i f f e r e n t i a l l i b e r a t i o n o f p r o t e a s e a c t i v i t i e s w i t h d i f f e r e n t pH o p t i m a . Other r e p o r t s e x i s t i n t h e l i t e r a t u r e d e m o n s t r a t i n g t h e p a r t i a l l y c e l l - b o u n d n a t u r e o f c e r t a i n enzymes. McDonald (1961) d e m o n s t r a t e d t h a t 66% o f t h e t o t a l p r o t e a s e i n M. f r e u d e n r e i c h i i was c e l l - b o u n d . Kushner and P o l l o c k (1961) n o t e d t h a t 85% o f t h e t o t a l p e n i c i l l i n a s e o f B. s u b t i l i s was c e l l bound. Amino A c i d U t i l i z a t i o n Few f r e e amino a c i d s o r p e p t i d e s a r e p r e s e n t i n t h e rumen o f a n i m a l s . R a p i d p r o t e o l y s i s b r e a k s down f o o d as i t e n t e r s t h e rumen •77 to amino acids and peptides, but these in turn are rapidly deaminated to volatile fatty acids and ammonia. Consequently, ammonia nitrogen is the largest source of soluble nitrogen available to the micro-organisms; the concentration varies from 6 to 130 mg HH^ W/lOO ml rumen contents (Hobson, McDougall and Summers, 1968). The nitrogen requirements for a number of anaerobic rumen bacteria have been tested in media containing different nitrogen sources as the criterion for the requirements. It has been demonstrated that bacterial isolates from the rumen have simple nitrogen requirements compared with bacteria from other sources. A rumen Streptococcus sp. had the least complex nitrogen requirements of any species of that organism studied (Niven et al., lykQ). Ammonium sulphate is always included as a nitrogen source, generally with^small concentration of protein hydrolysate, in culture media for B. amylophilus. It was demonstrated that cell growth is directly proportional to the' concentration of ammonia sulphate present in the medium (Blackburn, 1968a). However, B. amylophilus grows in medium containing only ammonia and cysteine, but with a longer lag phase than i f tryptose is present. No vitamins, volatile fatty acids, or other growth factors are needed (Hobson, McDougall and Summers, 1967). Preliminary experiments demonstrated a 15% increase in the final cell density, an increase in growth rate as well as a reduced lag phase in the presence of a supplementary organic nitrogen source. This change in growth characteristics indicated that amino acids play some role in the nutrition of B. amylophilus. Ik Initial uptake experiments, with C amino acids at high specific activity and low concentration, demonstrated that amino acids are not restricted in entry into the cell by a permeability barrier. Therefore once in the cell the labelled amino acid mix with the de novo synthesized amino acids and are incorporated into protein. Ik C amino acids were not incorporated into the cell in the presence of oxygen or in the leak of a fermentable carbohydrate source. The same observation was made by Hobson, McDougall and 15 Summers, (1967) w n o reported that some N B. amylophilus experi-ments showed resting suspensions did not take up amino acids, with or without maltose, but they also did not take up ammonia without maltose and growth. On the other hand the cells leaked cell N into the medium no matter what was outside. The entry of the amino acids into the cell is affected by the presence of peptides, supplied in the form of tryptose. The 50% reduction in amino acid uptake would suggest that peptides entered the cell in competition with the amino acids but in a much lesser quantity. Ik B. amylophilus protease hydrolyzed denatured C B. amy-lophilus protein into products that could be incorporated into B". amylophilus contributing 17% of the total cell protein. The 79 results suggested that the function of the protease was to hydrolyze protein for the purpose of supplying hydrolyzed products to the organism. 14 C-free amino acids were taken into the microorganism and incorporated into protein. The exogenous amino acids contributed 26% and 43% of the total cell protein in the presence of 1.4 mg and 2.8 mg/ml of casein hydrolysate. The two-fold increase in amino acid uptake corresponding with a two-fold increase in casein hydro-lysate concentrationjindicated that amino acids entered the micro-organism by simple diffusion. Most amino acids did not go directly into protein as they were interconverted to some extent to other 14 amino acids prior to incorporation; the addition of the C glutamic 14 14 acid, C methionine and C tryptophan to the growth medium labelled seven different amino acids in the bacteria. A proportion (20%) of the amino acid was not recovered as protein, suggesting that the amino acid was metabolized. Wright and Hungate (1967) noted that.rumen bacteria metabolized glycine to give carbon dioxide acetic acid and ammonia. Portugal and Sutherland (1966) reported the same type of degradation with glutamic and aspartic acid. Van Den Hende et al. (1963) had reported this phenomenon earlier but found that no CO^  was released and he con-cluded that the products were formed in a glycine reductase system. Stadtman (1966) showed that many anaerobic organisms, especially Clostridium sp. derived energy for growth from the fermentation of •'80 "amino acids. In some of these fermentations, 1 mole of amino acid (glycine) undergoes oxidative deamination whereas another undergoes reductive deamination. Further oxidation of the cc-keto acids was coupled with Co-ASH esterification and provides a potential source of energy for growth. Reductive deamination was also coupled with ATP formation i.e. one mole ATP formed per mole of glycine reduced (Stadtman, 1958). + -2 -3 + -k DPKH+H + glycine + HPO^  + ADP DPN + NH^  + A T P + acetate Stadtman (1966) showed that the amount of growth (dry weight of bacteria) obtained through anaerobic metabolism was directly propor-tional to the amount of substrate fermented and was absolutely related to the number of moles of ATP produced. It is not certain that amino acids could undergo a similar type of fermentation reaction in B. amylophilus. _ATP Hobson and Summers (1967) examined the 1 (g dry et. cell/ mole ATP) of B. amylophilus and found i t to be 20, as compared to 10-15 found for most other bacteria (Bauchop and Elsden, i 9 6 0 ) . They explained their results by suggesting that l i t t l e ATP energy was required for cell maintenance. This high ATP yield may be due partially to fermentation of amino acids ;yielding a quantity of ATP, and would aid in explaining the increased cell density and decreased log phase in medium containing a supplementary organic nitrogen source. Wright ( i 9 6 0 ) observed in Streptococcus bovis that the limited ability of amino acid utilization was due to the continued, non r e g u l a t e d s y n t h e s i s o f amino a c i d s . C ma l t o s e , w i t h and w i t h o u t a supplemented o r g a n i c n i t r o g e n s o u r c e p r e s e n t , l a b e l l e d a l l t h e common amino a c i d s . These r e s u l t s i n d i c a t e d t h a t t h e amino a c i d s were s y n t h e s i z e d c o n s t i t u t i v e l y and were n o t s u b j e c t t o end p r o d u c t r e p r e s s i o n ; amino a c i d u p t a k e was, t h e r e f o r e , l i m i t e d b y t h e c o n t i n u e d i n t r a c e l l u l a r s u p p l y o f amino a c i d s , even t h o u g h adequate l e v e l s were p r e s e n t i n t h e medium. McDonald and Chambers (1966) n o t e d t h a t ammonia was u t i l i z e d as a n i t r o g e n s o u r c e i n a M i c r o c o c c u s sp. and amino a c i d s and m a l t o s e were u t i l i z e d m a i n l y as a c a r b o n s o u r c e . Amino a c i d s cannot be u t i l i z e d b y B. a m y l o p h i l u s as a s o l e s o u r c e o f c a r b o n ( B l a c k b u r n , 1968a); t h e y may be u t i l i z e d i n a l i m i t e d c a p a c i t y o r a c t as a s t i m u l u s t o p r o t e i n s y n t h e s i s . I n c o n c l u s i o n , t h e s e e x p e r i m e n t s have d e m o n s t r a t e d t h a t amino a c i d s do p l a y a r o l e i n t h e n u t r i t i o n o f B. a m y l o p h i l u s b u t t h e p r e c i s e manner b y w h i c h t h e y f u n c t i o n i s n o t c l e a r l y u n d e r s t o o d . Amino a c i d s have been shown t o be i n c o r p o r a t e d d i r e c t l y and i n d i r e c t l y i n t o p r o t e i n , c o n t r i b u t i n g up t o 50% o f c e l l p r o t e i n . REFERENCES Abou Akkada, A.R., and Blackburn, T.H. (1963). Some observations on the nitrogen metabolism of rumen proteolytic bacteria. J. Gen. Microbiol. 31: 46l. Allison, M.J., Bryant, M.P., and Doetsch, R.N. (1959). 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