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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.  ( A g r . ) , U n i v e r s t i y o f Guelph,  (I967).  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 t h e required  standard  THE UNIVERSITY OF BRITISH COLUMBIA J u l y , 1969  In p r e s e n t i n g an a d v a n c e d the  degree  Library shall  I further for  this  agree  scholarly  by  his  of  this  written  thesis  in p a r t i a l  fulfilment  of  at  University  of  Columbia,  the  make  that  it  permission  purposes  may be  representatives. thes.is  for  freely  It  financial  available for  permission.  Department  of  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  by  the  understood  gain  Columbia  for  extensive  granted  is  British  shall  Head o f  be  requirements  reference copying  that  not  the  of  I agree and this  or  allowed  without  that  Study. thesis  my D e p a r t m e n t  copying  for  or  publication my  i  . ABSTRACT  Bacteroides  amylophilus  produces 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 a n d  80%. i s b o u n d . t o  cell.  or mechanical  T r e a t m e n t .of t h e . c e l l s w i t h t o l u e n e  i n t e r a t i o n does not  increase the p r o t e o l y t i c  i n d i c a t i n g that a l l the protease at the b a c t e r i a l surface. protease  activity  L e s s t h a n l%'.;of t h e  located  total  c e l l s by  osmotic  shock procedures which i n d i c a t e d t h a t the protease f r e e i n t h e . p e r i p l a s m i c space. l i b e r a t e s 33% sedimentable  k0%.;of  of the  r u p t u r e hh)% o f t h e enzyme  remained bound t o the  which i s  h i g h speed c e n t r i f u g a t i o n .  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% A f t e r g e n t l osmotic  i s not .  Speroplast.formation  o f t h e . c e l l bound p r o t e a s e by p r o l o n g e d  spheroplast  envelope.  ±6%  o f t h e t o t a l enzyme.  f u r t h e r evidence protease  activity, Prolonged  Bacteroides  The.results  t o t h e p a r t i c l e bound n a t u r e  of Bacteroides  Sonic  protease.  speed c e n t r i f u g a t i o n r e s u l t s i n the . sedimentation a l l but  dis-  activity,  is superficially  i s r e l e a s e d from the  the  high  of  give of  the  amylophilus.  amylophilus  has  a f a s t e r r a t e of growth, w i t h  a . r e d u c e d l a g p h a s e a n d 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 peptides  a r e i n c l u d e d i n t h e b a s a l medium.  acids are i n c o r p o r a t e d i n t o . c e l l s i n d i c a t i n g t h a t t h e y were not meability barrier.  The  Radioactive  i n significant  excluded  amounts,  from t h e c e l l by  amount o f i n c o r p o r a t i o n o f  amino  a  per-  amino  ii  acids i s found t o vary f o r d i f f e r e n t  a m i n o a c i d s a n d i s shown  •Ik t o be c o n c e n t r a t i o n d e p e n d e n t .  The e x o g e n o u 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 • 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 .  C amino.acids d i r e c t l y or  The i n h i b i t i o n o f l ^ C a m i n o  a c i d s u p t a k e b y . p e p t i d e s a n d t h e d i r e c t u p t a k e o f -^C peptides  demonstrated p e p t i d e uptake by B a c t e r o i d e s  The r e s u l t s  olig-  amylophilus.  suggest t h a t organic n i t r o g e n c o n t r i b u t e s t o the  n u t r i t i o n of Bacteroides  amylophilus.  i i i  Table-of.  Contents  Page  INTRODUCTION  . .  . .  .  .  . .  . .  .  . ;  .  . .  .  .  . ;  LITERATURE .'REVIEW; .1.  Localization  II.  of  Spheroplast  III.  Effect  of  Proteolytic  Formation  . . .  Amino A c i d s  and B a c t e r i a l  IV.  Amino A c i d  Utilization  V.  Amino A c i d  Incorporation  Peptide  .  .  .  .1  .  2  .  3  .  . . . . . . . . . . . . .  and Peptides  Production  VI.  Enzymes  .  Growth.  by.Rumen by  on .  3  Protease .  .  .  .  Bacteria. .  Rumen B a c t e r i a  5  . :  .  6  .;  .  7  Incorporation  by  Rumen B a c t e r i a  .  .  .  8  VII.  Ammonia I n c o r p o r a t i o n  by  Rumen B a c t e r i a  .  .  .  9  VIII.  Ammonia I n c o r p o r a t i o n  by  B. 'amylophilus  . .  M A T E R I A L S A N D METHODS .I . J M e d i a . II.  . .  Bacteria  . .  IV.  Amino A c i d  V.  Dry.weight, . composition  VI.  Protease  . .  and  I I I . .Measurement  . . . . .  I:  .  . , .  content  Cells .  . .  . .  •  12  .:  .  .  .  .  lh  . .  . .  . .  . .  ..  15  .  15  and amino  acid  . .  . .  •  • .  • .  17  Determination. .  . ,  . ,  . .  .  . .  . .  . :  .  18'.  .  .  Localization  shock  .  .  of  .  .  .  .  .  .  . . . . .  enzymes  .  19.  Liberation  by  osmotic  The  of:sucrose  osmotic  shock  formation  19 27  ..  anaerobic  Spheroplast  .  procedures  of  Effect  .  proteolytic  Effect  Modified  . .  . .  .  Osmotic  VI.  . .  . .  . .  II.  . V.  . : . .  12  • .  shock  IV.  . .  10  . .  Osmotic  The  - .  Conditions..  protein  I.  III.  •  Growth  RESULTS AND D I S C U S S I O N PART  . .  Incorporation  of  .  . . . . . . . . . . . . . .  Culture  .of  .  .  .  .  .  conditions  shock on the  '. .  on . .  Protease  procedure .  .  .  . . .  . : . :  - 2 9  Protease .  . .  .  Assay. . .  -31 .  3h'  . ; . .  . . '38'  . . . . . . . . . . . . .  38  iv  PAET.II: .1.  U p t a k e ;of A m i n o A c i d s a n d P e p t i d e s .  The.Effect  II.  o f Organic  N i t r o g e n on Growth.  .  . • 46  o f ;B. a m y l o p h i l u s  .  . :-50 .  Amino A c i d C o m p o s i t i o n  III.  T h e U p t a k e .of  l l + c  . • %6  M i x e d Amino A c i d s  '52  . . .. . ; .  I V . 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 of Tryptose  .  .. .  .  .. .  .2.. ;3.  .. .. .. .; .. . .  62  T h e U p t a k e a n d . C o n v e r t i o n .of A m i n o A c i d s  .  62 .  .. .. .; .  D e t e c t i o n o f l ^ C a m i n o a c i d s on'. chromatograms66 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 .  68  .....  68  . t h i n l a y e r c h r o m a t o g r a m s . . . ..... . . . :  4.  Interconversion.of  amino a c i d s  . .. .; .. .. .. .; .. .; .. .  .  ' 72 .  REFERENCES . . , . . . . . . . . . : . . . . . . . : . . . . .  .  82 :  GENERAL D I S C U S S I O N  9  57  . ..  V. T h e ' U p t a k e o f ' ^ C . p e p t i d e s V I . .1..  .  V  L i s t .of T a b l e s Table. I.  P r o t e a s e a c t i v i t y ..after. o s m o t i c cultures;of  Table.'II.  B.'amylophilus.  The e f f e c t o f a n a e r o b i c l i b e r a t i o n by osmotic  Table. I I I .  osmotic  c o n d i t i o n s on p r o t e a s e  shock.  The r e l e a s e o f p r o t e a s e by  shock o f  a c t i v i t y . f r o m B.  shock treatment  amylophilus  under iptimum.conditions.  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  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 .  Table. V I .  The.location of protease  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 , o n t h e g r o w t h rate.constant  liberation.  i n osmotically:ruptured' spheroplasts,  (k) and t h e g e n e r a t i o n t i m e o f  B.amylophilus.  Table..VIII.  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 .of B. ' a m y l o p h i l u s .  Table. IX.  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  high  levels  of casein hydrolysate.  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  tryptose.  Table. XI.  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 ,  Table. X I I .  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 .individual specific  amino a c i d  carrier.  o f an  T h i n l a y e r . c h r o m a t o g r a p h y .of a m i n o 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 a m i n o a c i d s i n B_. ' a m y l o p h i l u s .  vii  L i s t ;of F i g u r e s  Fig.  . O p t i c a l d e n s i t y '(TOO) as a f u n c t i o n of B S A . c o n c e n t r a t i o n .  Fig.  2.  Fig.  .3.  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 ..of i n c u b a t i o n .  Fig.  .4.  Rate of p r o t e a s e  Fig.  .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  Fig.  .6.  The temperature s t a b i l i t y of the  Fig.  .7.'  Protease  Fig.  .8.  Osmotic shock p r o c e d u r e .  Fig.-  9.  Fig..  10'..  Optical density  Protease  (TOO), as a f u n c t i o n o f p r o t e a s e  concentration.  p r o d u c t i o n by B . a m y l o p h i l u s .  c u l t u r e temperature.  protease.  a c t i v i t y .of B . 'amylophilus as a f f e c t e d by p H .  assays as a f f e c t e d  by.sucrose.  E l e c t r o n micrograph of B . amylophilus.  Fig...11..  Spheroplast formation procedure..  F i g . . ,12.',  The e f f e c t ;of amino a c i d c o n c e n t r a t i o n on the  growth..of  B_. a m y l o p h i l u s . Fig..  13V  The . e f f e c t  of different  sources .of o r g a n i c  the growth o f 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.  nitrogen.on  VX11  Fig.  15.  Fig.. l 6 .  The uptake of ^C-mixed amino acids. Radioactive and ninhydrin detection of '^G labelled amino acids.  Acknowlegement I would l i k e his  t o e x t e n d my  constructive criticism  of t h i s  s i n c e r e g r a t i t u d e t o D r . T.H. o f my  Blackburn f o r  r e s e a r c h and h i s e x h a u s t i v e  editing  manuscript.  I w o u l d l i k e t o e x p r e e my t h a n k s t o D r . D . J . C l a r k f o r h i s e d i t i n g the  of  thesis.  Lastly,  I o f f e r my t h a n k s t o M r s . R. M o r g a n f o r h e r t y p i n g o f t h e t h e s  1  INTRODUCTION  Bacteroides amylophilus  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 m o d e r a t e amounts o f p r o t e o l y t i c enzyme. and  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 u n k n o w n  i t s l o c a t i o n has not been 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 the protease would a i d i n the e l u c i d a t i o n o f the f u n c t i o n o f t h e enzyme. was  Blackburn  (1968a) d e m o n s t r a t e d  that the  enzyme  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  proteins.  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  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 organic n i t r o g e n sources  isolated  ammonia i n p r e f e r e n c e t o  (Abou A k k a d a a n d B l a c k b u r n , 1963).  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  Studies  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 ( H o b s o n , M c D o u g a l l and  Summers, 1 9 6 8 ) .  flavefaciens,  e.g. Ruminococcus  i n c o r p o r a t e exogenous amino a c i d s d u r i n g g r o w t h  (Bryant and Robinson, to  H o w e v e r , some r u m e n b a c t e r i a ,  investigate;  1961).  The p u r p o s e o f t h i s r e s e a r c h was  ( 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 ;  a n d (2) t h e l o c a t i o n o f t h e  protease.  2  L I T E R A T U R E  M i c r o o r g a n i s m s  b r e a k d o w n  o f  d i e t a r y  i960).  S o m e  p e p t i d e s  p r o d u c e d  w h i l e  o f  o t h e r s  n i t r o g e n  s t r a i n s  f r o m  l y s i s  T h e y  o f  a n d  f o u n d  k  t h a t  r e c o v e r e d  64%  p o l y p e p t i d e  w e r e  a c i d  p e r i o d  86%  a s  o f  a m i n o  n i t r o g e n  s p e c i f i c i t y  was  ( B l a c k b u r n ,  1968b).  n o t  m o r e  i n h i b i t e d  r e a g e n t s ,  b u t  ( B l a c k b u r n ,  s h o w n  t h a n  b y  was  t o  t h e  s e v e r a l  T h e r e  n o n  o n e  E D T A  o f  ( p e r c e n t  i n  t h e  T h e  m a i n  s o u r c e  a c i d s  A b o u  c a s e i n  B.  h y d r o -  a m y l o p h i l u s .  o f  w h i c h  1%  c a s e i n  n i t r o g e n )  n i t r o g e n ) ,  20%  a n d  .;dJl"^'epS^eds  No  t r y p s i n - l i k e  t h a t  p r o t e a s e  ( 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  b y  o f  e x t r a c t  e v i d e n c e  n i t r o g e n  a m i n o  n i t r o g e n ) .  w i t h  o f  a c t i v i t y .  t o t a l  c a s e i n  a n d  a m y l o p h i l u s  f r e e  h y d r o l y z e d ,  a c t i v i t y  s o m e  o f  B.  p r o d u c t s  p r o t e i n  p r o t e a s e .  i n h i b i t e d  1968b).  M o s t  t h e  H o b s o n ,  a c i d s  t h e i r  p e p t i d a s e  t o t a l  p r e s e n t  was  a s  f o r  a n d  s o u r c e  s t r a i n s  was  n i t r o g e n  ( p e r c e n t  b e  m a i n  1962).  s t u d i e d  E n d o p e p t i d a s e  a m i n o  a m m o n i a  o f  ( p e r c e n t  t h e  q u a n t i t i e s  c a s e i n  a c i d  r e s p o n s i b l e  ( B l a c k b u r n  t h e i r  a b s e n c e  f o r  t h e  n i t r o g e n  o b s e r v e d .  c o n t a i n e d  (1962)  u s e  f o r  s m a l l  b e  r u m e n  a s  R o b i n s o n ,  t h e  t o  t h e  b a c t e r i a  r e l a t i v e l y  d a y  was  a m i n o  a n d  i n d i c a t i n g  a  i n  p r o t e o l y s i s  B l a c k b u r n  o v e r  s h o w n  r e q u i r e m e n t  ( B r y a n t  p r o d u c e  r u m e n  b y  a  b e e n  p r o t e i n  t h e  h a v e  p r o t e i n  A k k a d a  h a v e  R E V I E W  o f  B.  B.  a m y l o p h i l u s  a m y l o p h i l u s  a c t i v i t y  a c i d )  was  :.'or  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  t h i o l  (87%),  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  M a n y enzymes a r e t h o u g h t t o b e e x t e r n a l t o t h e c e l l membrane, if  a c c e s s i b l e t o impermeable s u b s t r a t e s  1954; and  1961),  Mitchell,  O r t o n , 1966),  Pollock,  1961),  antibodies  i f r e l e a s e d by osmotic shock (Heppel,  i n B.  (Kushner and 1967)  5'nucleotidase,  osmotic shock (Wossal and Heppel,  II.  ^  cyclic  a c i d phosphatase and t h e r i b o w e r e r e l e a s e d f r o m E. c o l i b y  I966).  Species  of Shigella,  Citrobacter, Escherichia, Salmonella  and S e r r a t i a  r e l e a s e d e n z y m e s when o s m o t i c a l l y s h o c k e d . and  r  amylophilus.  nucleic acid inhibited-endonuclease,  Enterobacter,  o  were employed t o determine t h e l o c a t i o n  A number o f h y d r o l y t i c e n z y m e s , a l k a l i n e p h o s p h a t a s e , phosphodiesterase,  (Weimberg  ( M a l a m y a n d H o r e c k e r , 1961).  production  two t e c h n i q u e s  of the protease  (Demis e t a l . ,  i f e l u t e d b y non-damaging s o l v e n t s  i f i n h i b i t e d by s p e c i f i c  released by spheroplast The l a t t e r  of inhibitors  In contrast,  Proteus  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 ( N e u a n d C h o u , I967).  Spheroplast  Formation  Malamy and H o r e c k e r  (1961) s h o w e d 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, a s 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 the surrounding to spheroplasts  medium w h e n c e l l s w e r e  w i t h l y s o z y m e a n d EDTA  converted  (ethylenediaminetetraacetic  M a l a m y a n d 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 "  'acid).  of a l k a l i n e phosphatase 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 w e r e 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  that  the  enzyme was n o t b o u n d 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 -  spheroplasts  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 water.  There-  f o r e , t h e enzyme was d e m o n s t r a t e d t o b e l o c a t e d i n a c o m p a r t m e n t b e t w e e n t h e c e l l w a l l a n d t h e c e l l membrane t e r m e d t h e p e r i p l a s m i c space  (Mitchell,  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  I961).  a l k a l i n e phosphatase, glucose-6-phosphate glutamic  dehydrogenase  a c t i v i t i e s remained almost  associated with lysozyme-spheroplasts.  I nfact,  the  latter  enzyme o u t s i d e t h e s p h e r o p l a s t  the  extent  of cell lysis  during  an increase  served  spheroplast  When membrane i n t e g r i t y was d e s t r o y e d was  dehydrogenase a n d completely the activity of as a n index o f  formation  by toluene  (about  20%).  treatment there  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  assayed  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 phosphatase  ( M a l a m y a n d H o r e c k e r , 196k).  indicated, by cytochemical was  techniques,  Done e t a l . , (1965)  that a l k a l i n e phosphatase  located i n the periplasm. 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  b o u n d a n d r e m a i n s w i t h t h e membrane f r a c t i o n o n l y s i s o f B a c i l l u s licheniformis protoplasts  ( K u s h n e r a n d 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 " Lampen (1967a a n d 1967b)  w i t h t r y p s i n o r sodium deoxycholate. demonstrated  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 w h e n c e l l s w e r e 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 (Sargent e t a l . ,  1968).  Coles and Gross  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  (1967)  i nStaphylococcus  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 .  was  involved i n the 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 .  III.  They c o n c l u d e d t h a t a n e n z y m a t i c  process  E f f e c t o f Amino 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 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 h a v e b e e n 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 a n d r e p r e s s e d b y u t i l i z a b l e  carbohydrates.  Hofsten and Tjeder  (1965),  noted t h a t the  extra-  c e l l u l a r p r o t e a s e i n a n A r t h r o b a c t e r s p . 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 andpeptone and repressed b y the carbon  sources  u t i l i z e d f o r growth i . e . glucose 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 .  InBacillus  cereus, t h ee x t r a c e l l u l a r  protease  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 a n d h i s t i d i n e (Newmark a n d C i t r i ,  1962).  I n B a c i l l u s megaterium protease  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 effective being threonine, isoleucine,  l e u c i n e and v a l i n e  (Chaloupka  6  1966).  'and K r e c h o v a , Bacillus acids  Sporulation andprotease production i n  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  (Levishon and Aronson,  demonstrated  1967).  t h a t i n Pseudomonas  Keen and W i l l i a m s  lachrymans ^protease 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 ) , medium a n d was v i r t u a l l y M c D o n a l d a n d Chambers e x t r a c e l l u l a r protease of  (1967),  independent  i n the  o f t h e sucrose concentration.  (1966) p r o p o s e d  that the f u n c t i o n o f t h e  i n Micrococcus  s p . was t o e n s u r e  a supply  c a r b o n f o r g r o w t h 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 a n d s u p p r e s s e d maltodextrans.  b y carbon  sources  I n the anaerobic organism  such as maltose and  Clostridium welchii,  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 Blackburn  (1968a) d e m o n s t r a t e d  1950).  (Bidwell,  t h a t the growth r a t e and protease  p r o d u c t i o n i n B. a m y l o p h i l u s w e r e 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 , acid,  lysine or arginine.  Growth a n d p r o t e a s e p r o d u c t i o n were  shown t o b e 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 appeared t o be t h e o n l y other f a c t o r protease production (Blackburn, personal  IV.  aspartic  controlling  communication).  A m i n o 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 acids which  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  Williams  e t a l . (i960),  a n d L e w i s a n d McgBoft"ald(l96~2), d e m o n s t r a t e d  that soluble proteins y i e l d high Blackburn was s l o w e r  and Hobson (i960),  concentrations  o f rumen  ammonia.  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  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 a n i n c r e a s e i n  amino a c i d s a n d 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 .  Eventually  a l l a m i n o a c i d s w e r e d e a m i n a t e d a n d ammonia c o n c e n t r a t i o n was maximum three hours a f t e r  feeding.  Amino a c i d s were d e g r a d e d 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, The h i g h e r  carbon d i o x i d e , and v o l a t i l e f a t t y  the protein concentration  t h e m i c r o b i a l d e a m i n a t i n g power. for  a Stickland-type  i n t h e d i e t t h e g r e a t e r was  T h e r e a p p e a r e d t o b e some e v i d e n c e  r e a c t i o n b e t w e e n amino a c i d s ^ s i n c e  was d e a m i n a t e d 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-proline  (Lewis,  M o s t amino a c i d s were p a r t i a l l y d e g r a d e d b y c o n c e n t r a t e d suspensions and prolonged incubation (1965)  V.  acids.  (Lewis,  1962).  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  1955).  cell  Blackburn  i n t h e rumen.  A m i n o 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 microorganisms i n c o r p o r a t e ( S t r a s s m a n , 1955; 196U;  Roberts,  1957;  exogenous amino  Reiss,  a n d K a y a n d G r o n l u n d , 1969).  This  1955;  Ferro  acids Luzzi-Ames,  i n c o r p o r a t i o n o f amino  a c i d s was a c c o m p a n i e d b y r e p r e s s i o n a n d f e e d b a c k i n h i b i t i o n o f the  a m i n o a c i d b i o s y n t h e t i c enzymes.  Since  t h e r e was no i n c o r p o r -  8  'ation o f by  C-leucine  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  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  Streptococcus  bo v i s  by high  concentrations  o f exogenous a s p a r t i c  a c i d , presumably 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 , i960).  M i x e d r u m e n b a c t e r i a do n o t h a v e a n i m p a i r e d  mechanism as t h e y conditions, and  a c t i v e l y metabolize,  "^C g l y c i n e ( W r i g h t  glutamic  acids  uptake  under a r t i f i c i a l  rumen  a n d H u n g a t e , 1967)  aspartic  1963), w i t h t h e  ( P o r t u g a l and Sutherland,  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 a n d ammonia. W r i g h t a n d Hungate (1967),  d e m o n s t r a t e d t h a t "^C a n d "^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 deamination  VI.  going  through a  step.  Peptide  Wright  I n c o r p o r a t i o n b y Rumen B a c t e r i a  (1967)  demonstrated that dipeptides containing 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 itself.  Shelton and Nutter  dipeptides  little  found that Bacteroides  l a b e l f r o m exogenous  a c i d but  effect  with  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 .  e t a l . (1967)  Pittman  (1964) s h o w e d a s i m i l a r  Ill-  incorporated peptides  ruminicola  incorporated  C l a b e l l e d L-proline o r L-glutamic labelled with  Ill-  C proline.  The  Ik amount o f to  the  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  s i z e o f the peptides  up t o a molecular  w e i g h t o f 2000.  9  VII.  A m m o n i a 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 r u m e n b a c t e r i a 1955;  e v e n i n m e d i a c o n t a i n i n g amino a c i d s a n d p e p t i d e s (Warner, Abou A k k a d a a n d B l a c k b u r n , 1963).  (1962),  Bryant and Robinson  d e m o n s t r a t e d t h a t some r u m e n b a c t e r i a r e q u i r e d a n 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  produced  d u r i n g g r o w t h , r e g a r d l e s s o f t h e amount o f a m i n o a c i d a n d 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  a m o u n t s o f ammonia a n d v e r y l i t t l e 1962)  and L a c t o b a c i l l u s b i f i d u s  C protein hydrolysate  i s o l a t e d f r o m t h e rumen,  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 . , flavefaciens,  1961) .  1962).  (Allison,  actively  Ruminococcus  R. a l b u s a n d 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  cellulose digesting bacteria, u t i l i z e synthesis,  large  ammonia f o r a l l  protein  e v e n 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  (Bryant,  These s t r a i n s have a n a b s o l u t e growth 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 a n 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 a n d a n i m p a i r e d a m i n o a c i d u p t a k e s y s t e m ( B r y a n t a n d D o e t s c h , 1955; Robinson, I96I; Ruminococcus  B r y a n t a n d R o b i n s o n , 196*2).  Bryant and  Thus a s t r a i n o f  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 trace of  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  f r o m "^C i s o v a l e r i c 1962) .  acid  (Allison et al.,  Bryant and Robinson  leucine  1959; A l l i s o n e t a l . ,  (1962) n o t e d t h a t a l l  strains of  10  freshly, i s o l a t e d b a c t e r i a 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 f i b r i n o s o l v e n s and Succinovibrio dextrinosolvens. Ammonia was apparently incorporated by growing c e l l s of a l l these s t r a i n s and by Ruminococcus albus, R. f l a v e f a c i e n s and Bacteroides amylophilus w i t h 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 s t r a i n s of  B u t 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 a c i d carbon (Bryant et a l . , 1963).  A l l s t r a i n s of Bacteroides ruminicola appeared t o u t i l i z e 14  very l i t t l e ammonia, yet f a i l e d t o incorporate much  C - l a b e l l e d amino  a c i d carbon (Bryant et a l . , 1963)• V I I I . Ammonia Incorporation by Bacteroides  amylophilus  Blackburn (1968a) showed that protease production and growth of Bacteroides amylophilus were p r o p o r t i o n a l to the ammonia concentration up to 3.5 umoles per ml medium.  Tryptose, proteose peptone, casamino  acids and casein d i d not replace the requirement f o r ammonia. Blackburn (1968a and 1968b) observed that the l a g 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 t o the s t a b i l i z a t i o n of the EH i n the e a r l y stages .. of growth, rather than due t o the u t i l i z a t i o n of amino acids and .  11.  peptides. to  H o b s o n , M c D o u g a l l a n d Summers  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  (1968), u s e d  N ammonia  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 continuous  cultures o f Bacteroides  (1967)  amylophilus,  H o b s o n a n d Summers  t h a t ammonia a c c o u n t e d f o r 1 0 4 % o f ' t h e  c e l l nitrogen.  n i t r o g e n u t i l i z a t i o n has been found i n other b y H u n g a t e et_ a l . ,  1964).  showed  Similar  rumen b a c t e r i a  (review  M A T E R I A L S AMD METHODS  I.  Media  The  m i n i m a l s a l t s medium d e v e l o p e d b y H u n g a t e  contained  ( g / l ) K^RTO^, O.U5;  0.9;  NaCl,  MgSO^, 0.09;  c y s t e i n e ,(HCl), 0.5;  in  0.09;  CaCl , 2  NaHCO^, 5 . 0 .  s o l u t i o n s p l u s any other  0.L  K H ^ ,  (1950) 0.9;  (NH^SO^,  5 ;  r e s a z u r i n , 0.001;  The m i n e r a l a n d r e s a z u r i n  a d d i t i o n s t o t h e medium were  placed  a l i t r e b o t t l e a n d t h e v o l u m e made u p t o 900 m l w i t h  water.  a t 120  The m e d i u m was a u t o c l a v e d  the tops were t i g h t e n e d i m m e d i a t e l y clave, to maintain 1.0%  anaerobic  ( w / v ) was a u t o c l a v e d  L-  distilled  C f o r 15 m i n u t e s a n d  upon removal from t h e auto-  conditions.  separately.  . L-cysteine»(HCl) Sodium  bicarbonate  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 b y M i l l i p o r e f i l t r a t i o n w i t h a n 0.45  l-mi p o r e s i z e  filter.  These two s o l u t i o n s were added t o t h e medium u n d e r a s t r e a m o f o x y g e n - f r e e 00^.  The m e d i u m was d i s p e n s e d  and u s e d f o r b a c t e r i a l growth. a t m o s p h e r e o f CO^.  into  smaller  . A l l s t e p s w e r e done u n d e r a n  D i f f e r e n t m e d i a w e r e 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 s a l t s m e d i u m p l u s 0.3% ( w / v ) m a l t o s e ; m e d i u m 0.1% contained  tubes  (w/v) t r y p t o s e  (Bacto  0.3% m a l t o s e p l u s 0.1%  basal  maltose and t r y p t o s e  Laboratories,  Detroit),  tryptose i n the basal  salts  Product Analysis Report for  Amino acid  Molecular weight  C-Protein Hydrolysate  Specific activity mc/m mole  Percentage of total count  89.1  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  Alanine  "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 a m i n o  acid  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 alone a t f i n a l concentrat i o n s o f 0.025%,  0.05% a n d 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 C o r p . , w h i l e the  o t h e r r a d i o a c t i v e amino a c i d s a n d  14  C glucose and  Ik  C maltose  w e r e p u r c h a s e d f r o m S c h w a r t z B i o - R e s e a r c h I n c . , O r a n g e b u r g , N.Y. as t h e u n i f o r m l y l a b e l l e d L-hydroxyproline.  14  C product with the exception o f  3 H  The r a d i o a c t i v e a m i n o a c i d s w e r e c h 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 c h r o m a t o g r a p h y ( J o n e s a n d H e a t h c o t e , ' 1966) and r a d i o a u t o g r a p h y . impurities  II.  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%  (Kay and Gronlund,  1969).  B a c t e r i a and Culture 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 a n d H o b s o n , 1962).  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 a p p r o x i m a t e l y 0 . 9 t o 1.6  u b y 1.6  B. amy-  coccobacillus,  t o 4 . 0 u. a n d 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 those o f t h et y p e s t r a i n (Hamlin a n d Hungate, 1956).  This organism ferments only starch o r starch  p r o d u c t s such as maltose, t o a m i x t u r e o f a c e t i c , succinic  acids.  hydrolysis  f o r m i c and  (0.7%)  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 deeps c o n t a i n i n g b o t h m a l t o s e  (0.3%) a n d 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 u p t o 2 t o 3 w e e k s a t 4 C.  III.  Measurement o f Growth  Two m e t h o d s w e r e u s e d t o m e a s u r e g r o w t h .  S e r i a l decimal  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 1.0%  a c i d washed c a s e i n  (Blackburn  d e t e r m i n e numbers o f v i a b l e c e l l s ,  a n d H o b s o n , 1 9 6 2 ) , was u s e d t o a n d b a c t e r i a l d e n s i t y was  m e a s u r e d a t 660 mu. i n a B a u s c h a n d Lomb S p e c t r o n i c C e l l concentration O.D.ggQ 0.5.  IV.  20 c o l o r i m e t e r .  a s a f u n c t i o n o f Q.D.g^Q was l i n e a r t o a n  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  Amino A c i d  containing  O.D.  readings.  Incorporation  14 The u p t a k e o f  C a m i n o 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 peptides 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 a n d 14 sodium s u l f i d e . hydrolysate  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  i s presented i nTable I .  C protein  One m l s a m p l e s w e r e  f i l t e r e d a n d w a s h e d w i t h 2 . 0 m l m a l t o s e medium o n 0.45 P- p o r e size f i l t e r s  ( M i l l i p o r e Corp., Bedford, Mass.).  Radioactivity  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  scintillation fluid  (k2 m l L i q u i f l u o r p e r l i t e r  E n g l a n d N u c l e a r Corp., i n the cellular The c e l l s  Boston, Mass.).  The d i s t r i b u t i o n o f l a b e l  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.  w e r e 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 Sorval Refrigerated automatic centrifuge Norwalk,  o f t o l u e n e , New  Connecticut).  and f i n a l l y r e s u s p e n d e d  (Ivan Sorval Inc.,  They were washed once i n m i n e r a l m e d i a 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  vials.  The a c i d was t h e n r e m o v e d b y f l a s h e v a p o r a t i o n w i t h a E v a p o m i x instrument ( B u c h l e r Instruments, F o r t Lee, N.J.)and t h e waterw a s h e d r e s i d u e was d r i e d a n d d i s s o l v e d i n a k n o w n q u a n t i t y o f d i s t i l l e d water. thin layer plates Germany).  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 ( C e l l u l o s e P o w d e r MN 300, M a c h e r e y N a g e l a n d Co.,  The a m i n o a c i d s w e r e s e p a r a t e d t w o d i m e n s i o n a l l y b y (1966).  the  method o f Jones a n d Heathcote  The f i r s t  dimension o f  the  c h r o m a t o g r a m s 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  plates  i n a solvent 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 acid:water (40:20:10 v/v).  T h e s e c 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 k e t o n e : a m m o n i a : w a t e r (50: 3 0 : 1 0 : 1 0 v / v ) .  The c h r o m a t o -  grams w e r e e x p o s e d f o r 1 t o 2 w e e k s t o m e d i c a l X - r a y f i l m K o d a k Co., R o c h e s t e r , N . Y . ) .  (Eastman  The f i l m s w e r e d e v e l o p e d a n d t h e  r a d i o a c t i v e a r e a s were drawn b y vacuum i n t o s c i n t i l l a t i o n f l u i d a n d assayed f o r r a d i o a c t i v i t y i na l i q u i d  scintillation  spectrometer.  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  quenching  under  Replicate  thin  chromagenic ninhydrin hydrin,  these layer  reagent  collidine  20.0  Standard  propan-2-ol amino  (10%  were  locate  sprayed with ninhydrin c o l l i d i n e  and i d e n t i f y  chromagenic acetic  i n ethanol solutions  v/v))  Dry Weight  A  were  reagent  the  to  of  give  amino  run to  amino  consisted  a c i d a n d 5.0  P r o t e i n Content  50 m l c u l t u r e  cells  were  washed  a volume  of  5-0  dried  88  C for  for  to  1969).  ( K a y and. G r o n l u n d ,  acids.  of  0.3  acids  verify  g  ml collidine  a f i n a l volume (2  the  nin-  (2,4,6,  of  100  ug/ml i n positions  The  ml.  aqueous of  the  acids.  V.  The  plates  ml glacial  trimethylpyridine), Ten p i o f  conditions  at  48  hours.  followed  by  ml.  O . D . 0 . 6 twice  cooling  was  i n the  harvested  were  by  were  dessicator  for  centrifugation.  to  in  i n aluminum pans,  stored  then heated  Cells  and resuspended  placed  and subsequently  samples  Composition of  i n d i s t i l l e d water  Two m l s a m p l e s  8 hours  The  and Amino A c i d  i n the  121  dessicator  C for  30 m i n u t e s  one  prior  hour,  to  re-weighing. One m l w a s then of  dissolved  i n 1  n e u t r a l i z e d w i t h I N HC1.  Lowry et  al.  One m l was  H NaOR", h e a t e d P r o t e i n was  (1951) u s i n g b o v i n e hydrolyzed to  using  a B e c k m a n M o d e l 120  Inc.,  Palo Alto,  to  C,  serum a l b u m i n (BSA)  determine  the  cooled,  determined by  amino  C amino a c i d a n a l y z e r ,  California).  80  acid  as  the a  method standard.  composition  (Beckman  Instruments  VI.  Protease Determination  The a s s a y p r o c e d u r e was  a m o d i f i c a t i o n o f t h e method used  Protease s o l u t i o n (0.2  McDonald and Chen ( I 9 6 5 ) . t o 1.8  m l o f 2% a c i d washed c a s e i n p l u s 0.01%  i n 0.1  M K H P 0 ^ a t pH 6.7.  added  merthiolate,  dissolved  F o r o t h e r pH v a l u e s KH^PO^ was  2  The r e a c t i o n was  ml) was  by  omitted.  stopped by t h e a d d i t i o n o f 2.0 m l o f 0.72  N  tri-  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 m i x i n g and was k e p t a t room temperature f o r 10 t o 15 minutes b e f o r e s e d i m e n t i n g t h e i n s o l u b l e p r o t e i n a t 1000 f r e e s u p e r n a t a n t was  x g f o r 10 m i n u t e s .  One m l o f t h e p r o t e i n  added t o 5.0 ml o f a l k a l i n e copper s u l p h a t e  s o l u t i o n ,(l.O m l o f 0.5%  CuSO^ i n 1.0%  o f 0 . 2 N NaOH i n 2.0 N a ^ O ^ ) . 0.5 m l o f 1.0  TCA-  sodium c i t r a t e . 2H 0 p e r 50 m l 2  A f t e r a 15 minute i n c u b a t i o n a t 38  H F o l i n Reagent was  added and i m m e d i a t e l y mixed,  C,  and  r e i n c u b a t e d a t 38 C f o r 20 minutes p r i o r t o r e a d i n g a t 700 mu on a Bausch and Lomb S p e c t r o n i c 20.  The o p t i c a l d e n s i t i e s o f u n i n c u b a t e d  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 t h e same amount o f enzyme were s u b t r a c t e d 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  r e a d i n g s are compared t o a s t a n d a r d b o v i n e serum albumen curve.  There was  o f BSA and  O.D.^QQ  (BSA)  a l i n e a r r e l a t i o n s h i p between t h e c o n c e n t r a t i o n (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  d e f i n e d as t h e amount o f enzyme w h i c h s o l u b l i z e t h e e q u i v a l e n t o f 1.0  fig BSA  i n one  minute.  was  RESULTS AND  Part 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 i n B.  P r e l i m i n a r y experiments protease  DISCUSSION  assay.  The  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  O.D.^QQ  produced  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 t i o n up  t o 300  ug  not pass through B.  (Figure l ) . the o r i g i n  amylophilus which  demonstrate t h a t the protease of  w h i c h was  p r o p o r t i o n a l t o BSA  1951).  the  used  as  concentra-  C h a r a c t e r i s t i c a l l y the curve  c o n t a i n e d 16.6  did  A culture  p r o t e a s e u n i t s / m l was  of  used to  o f t h e assay-was p r o p o r t i o n a l t o  c o n c e n t r a t i o n ( F i g u r e 2)  i n c u b a t i o n ( F i g u r e 3).  b y BSA,  (Lowry et a l . ,  O.D.^QQ  amylophilus  and p r o p o r t i o n a l t o t h e  length  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 b e 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 the  O.D.^QQ  p r o d u c t i o n o f t h e pH The  0.70.  d i d not exceed  p r o t e a s e was  6.7  produced  A t y p i c a l g r o w t h c u r v e and  protease a c t i v i t y i s presented by e a r l y e x p o n e n t i a l c e l l s and  the  i n Figure was  p r o p o r t i o n a l to the c e l l density. The was  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  f u r t h e r i n v e s t i g a t e d by observing the e f f e c t of temperature  t h e r a t e o f g r o w t h and p r o t e a s e p r o d u c t i o n . were grown i n a temperature at  production  i n t e r v a l s the O . D . a n d  g r a d i e n t i n t h e r a n g e 36 protease  c o n t e n t were  H a m l i n a n d H u n g a t e ( 1 9 5 6 ) , n o t e d t h e B. b e t w e e n 3? C a n d  45  C b u t n o t a t 30  v a r i e d - w i t h the temperature  Bacterial  on  cultures  C t o 42  C  determined.  a m y l o p h i l u s grew w e l l  C o r 55  C.  o f i n c u b a t i o n a n d was  The  growth r a t e  maximum a t 42  C.  4.  0 Fig.  1..  F i g . .2..  J _L _L _L _L 50 100 150 200 250 300 BOVINE SERUM ALBUMIN(>ig)  .25 .50 .75. CULTURE VOLUME (ml)  1-0  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 concentrations 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 NaOH 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 TCA, a n d t h e f i n a l volumes were a d j u s t e d to.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 was 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 . 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 . The digest 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 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 c i d s o l u b l e p e p t i d e s were assayed as d e s c r i b e d i n Methods.  contained of and the o  c  -20 -15  >  -10  IU  < <  O cc  OL.  60 120 180 TIME (minutes) Fig.3.  Fig.  h.  240  3  4  6  8 10 TIME (hours)  12  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 g r o w n t o a n O.D.ggrjs 0 . 6 2 a n d 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 b y NaOH. 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 3 8 . C a n d 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 b y 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 e a c h s a m p l e was a s s a y e d a s d e s c r i b e d i n Methods. R a t e o f 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 . . • 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 w h e r e r e m o v e d a t t i m e i n t e r n a l s a n d the. c e l l d e n s i t i e s w e r e 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 w h o l e c u l t u r e ( ©-©) was d e t e r m i n e d a s d e s c r i b e d i n Methods.  ro  42  41  J 40  I 39  TEMPERATURE Fig.  5.  I 38  ! 37  L_ 36  (C)  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 d r o p o f a n 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 a n A l u m i n u m T h e r m a l g r a d i e n t w i t h a ' r a n g e f r o m h2C t o 3 6 c . The s p e c i f i c activity ( 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 r a n g e O.D.ggQ, 0.25 t o 0 . 3 0 (o-o) a n d 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 a c t i v i t y 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 a c t i v i t i e s  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 D 0.2 hr  demonstrated a peak of protease a c t i v i t y at  ^ (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 i s 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 s u b t i l i s 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 i n 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 p e a k i n c o n t i n u o u s c u l t u r e s o f B. m e g a t e r i u m maximum w h i c h m i g h t The  a t d i l u t i o n r a t e s below t h e  c o r r e s p o n d t o a p e a k o f enzyme p r o d u c t i o n .  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 g r o w n a t t e m p e r a t u r e s h i g h e r t h a n 3 8 . 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 at these temperatures. demonstrated  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)  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 a n d t h i s may h a v e  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 it  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 -  m i n e d 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 production can occur. 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 p H 6 . 7 b a s e d o n 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. o p t i m a ( B l a c k b u r n , 1968; 11.5  which would  There a r e , however, o t h e r pH  L e s k , 1969),  a t pH 5 . 0 , 6.7,  suggest m u l t i p l e proteases b u t Lesk  p u r i f i e d t h e pH 6.7 p r o t e a s e 1000-fold  8.0 and (1969)  without separating 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 typical  pH-activity p r o f i l e f o r the proteolytic a c t i v i t y i s presented 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 . ,  e x t e n d over a wide pH range a n d t o have a n i r r e g u l a r  1968), t o plateau,  t h e r e f o r e i t c a n n o t b e c o n c l u d e d t h a t m o r e t h a n o n e p r o t e a s e was  35  40  50 TEMPERATURE  Fig.  6.  60 (c)  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 a n d . w e r e 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 w e r e c o o l e d a n d a s s a y e d . f o r r e s i d u a l protease a c t i v i t y .  70  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 s u b s e q u e n t  experi-  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 a n d 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 a n d 11.0  was  examined t o determine whether••qualitative 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 b e t w e e n 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 s h o c k 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 Heppel The  (1966) ( F i g u r e 8)  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 ,  w e r e 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 , 0 0 0 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 0.01 M T r i s  ( h y d r o x y m e t h y l ) amino methane ( T r i s ) a n d 0.03 M  N a C l a d j u s t e d t o pH 7.1. suspended  One g r a m ( w e t w e i g h t ) o f c e l l s was  i n 4 0 m l o f 0 . 0 3 3 M T r i s p H 7-1  a t 24 C.  A  small  s a m p l e was r e m o v e d a n d 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 a n in e q u a l q u a n t i t y o f 40% s u c r o s e ^ O . 0 3 3 M T r i s p H 7-1  was a d d e d .  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 . 0 8 m l 0 . 1 M d i s o d i u m ethylenediaminetetraacetic acid  (EDTA) a t p H 7-1  t o give 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.  Metabolyte water bath shaker  The m i x t u r e was s t i r r e d (New B r u n s w i c k S c i e n t i f i 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 c e n t r i f u g e d f o r 10 m i n u t e s  i na Co., I n c .  a t 180 r p m a n d  a t 1 3 , 0 0 0 x g a t 4 C.  The s u p e r -  28  t  F i g . 8.  Osmotic Shock P r o c e d u r e .  •200 ml c u l t u r e 9,000 x g_, 10 min. Culture Supernatant  Cells  CO!. M Tris.HCl • pH .7.1 0.03 M NaCl  Cells  Wash 1  Cells  Wash 2  Washed c e l l s i n kO. ml 0.033' M - T r i s . H C l , 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 M EDTA.at 2kC _ 4  P l a s m o l y s e d c e l l suspension 13,000 x £ , 15 m i n .  Plasmolysed.cell.pellet  sucrose  supernatant  resuspend, i n hO ml 5 x l 0 - M MgCl2 c e n t r i f u g e 10,000 x £ , 15 m i n . h C _i|  Shocked.cells  ~1  Shock.fluid  7  I n s t a g e 2,  n a t a n t f l u i d was r e m o v e d .  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  t h e p e l l e t was  x 10 ^ M M g C l ^ s o l u t i o n .  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 a n i c e b a t h f o r 10 centrifuged at automatic  II.  The  minutes,  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  centrifuge,  a n d t h e s u p e r n a t a n t f l u i d was r e m o v e d . a t p H 6.7,  P r o t e a s e assays were conducted various  rapidly  5.0,  8.0  a n d 11.0  on the  fractions.  Osmotic  Shock  P r e l i m i n a r y experiments N o s s a l and Heppel  (1966).  d e l i v e r i n g a m i l d osmotic  were performed  b y t h e method o f  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 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 -  bacteriaceae, which r e s u l t e d i n the 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 n o l e t h a l e f f e c t . ( A n r a k u a n d H e p p e l , 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 B.  amylophilus c e l l s would r e s u l t  noted b y Heppel  I t  t o anaerobic  i n death, but v i a b l e  were found a t the end o f the t r e a t m e n t . a long l a g but t h i s  1967).  cells  T h e r e was, h o w e v e r ,  c o u l d have b e e n due t o EDTA-treatment as  (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  environment.  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 decrease i n p r o t e a s e a c t i v i t y a t p H 5.0,  6.7  a n d 8.0  o f 13%,  25% a n d 25%  r e s p e c t i v e l y but i n an increase o f 14% i n protease m e a s u r e d a t p H 11.0  (Table I ) .  activity  This confirmed that the protease,  "Table I :  Protease a c t i v i t y B. a m y l o p h i l u s  a f t e r osmotic  Sample  Percentage  whole  culture  whole c u l t u r e culture  sonicated  supernatant  Tris-HCl  wash  5.0  of o r i g i n a l  of  culture  11.0  pH 6.7  pH 8 . 0  100  100  100  100  87  75  75  114  68  31  16  25  10  7  14  6  pH  first  shock o f c u l t u r e s  pH  washed c e l l s b e f o r e treatment  sucrose  28  28  32  36  washed c e l l s treatment  sucrose  KR*  KR  KR  KR  11  8  after  shocked  fluid  9  5  shocked  cells  1  5  *  KR  =  no  8.9,  7  results  P r o t e a s e was m e a s u r e d a t pH 5 . 0 , be 18.2,  4  5.6,  9.1  units/ml  6.7,  8.0  a n d 11.0,  respectively.  and f o u n d t o  with the p o s s i b l e exception of the protease  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 a n d t h i s was o m i t t e d i n s u b s e q u e n t e x p e r i m e n t s .  experimental  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  procedure i n Table  I.  The w a s h e d c e l l s  contained a r e l a t i v e l y  proportion of proteolytic activity, the  The  step  culture.  small  a r e f l e c t i o n o f t h e age o f  T h e r e 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 s u c r o s e - E D T A 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 readings  O.D.^QQ  to  i n the controls.  T h i s was  of high  subsequently  shown  b e 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 product  capable  o f r e a c t i n g w i t h t h e F o l i n Reagent.  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 cells values  and t h e shock f l u i d b u t p r o t e a s e  i n the shocked  assay a t the f o u r  s h o w e d a h i g h e r p r o p o r t i o n o f t h e enzyme  fluid.  experiment  made i t d i f f i c u l t  i n the culture  t o draw c o n c l u s i o n s f r o m  b u t t h e r e was p r e s u m p t i v e  evidence  pH  i n t h e shock  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  supernatant  There  that  this  protease  a c t i v e a t a l l t h e p H v a l u e s was p e r i p l a s m i c .  III.  The E f f e c t o f A n a e r o b i c by  Osmotic  C o n d i t i o n s on Protease  Liberation  Shock  The c e l l s w e r e k e p t u n d e r CO  during a l lprocedures.  The  effect  of  buffers and of  throughout  since the  in  protease-was  the  a l l in  The  procedure  There  assay procedure  cysteine  so  liberated  did  vary  was  l i b e r a t e d during the the  a  of  the  shock  A large  cells  are  p r o p o r t i o n (80%)  cells  than  again not  super-  (Table  II)  a p p r o x i m a t e l y 40%  for  an interference  by  sucrose  known how much p r o t e a s e  retained by  fluid.  the  without  shocked  cells  the  shock procedure.  was  with or  The p r o p o r t i o n ,  amount,(3^%)of  more  i n the  however,  cellular  T h i s was  protein  much  (I967) a n d w o u l d i n d i c a t e osmotically fragile  than  than  higher that  members  Enterobacteriaceae. Only  released  into  the  resuspension  i n increased •viability  More protease,  was  and  cell-bound protease  was  8% r e p o r t e d b y H e p p e l  amylophilus  1961)  into  l i t t l e .  B.  Unlike  i t  procedure,  was  than  was  during plasmolysis. in'the  washed  this  wash  higher  and s h o c k - f l u i d were  treatments.  liberated  the  resulted  found i n the  cells  to  were used^a  recoveries.of  shocked  the the  the  younger - cultures  natant.  (0.1%)  cysteine-HC1  adding  into  the only  2.0%  suspended  of  20%  the  the  t o t a l protease  RaCT,  of but  i n water  B.  amylophilus  medium d u r i n g e x p o n e n t i a l growth  c e l l bound protease 5-7%  of  the  protease  Mickle gave  of  a Micrococcus  of  B.  of  the  liberated  lyophilized  protease.  bacteria.  (McDonald,  a m y l o p h i l u s was  disintegration of  good y i e l d s  sp.  of  was  cells  (Blackburn,1968a).  •33  Table  II:  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 o n p r o t e a s e l i b e r a t i o n b y osmotic shock  Protease minus cysteine  cysteine  units cells  100  Sucrose supernatant.  KR*  Washed  Shock  fluid  Shocked  *  =  cells  4.4  units  100  6.8  KR  Protein cysteine + bicarbonate  1o 100  cysteine bicarbonate  units  4.6  KR  mg  100  424  KR  16  0.7  2  0.1  4  0.2  3^  142  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 . 4 3 was u s e d f o r t h e c y s t e i n e m i n u s a n d c y s t e i n e (0.05%) p l u s s o d i u m 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 . 5 2 was u s e d f o r t h e c y s t e i n e (0.05%) experiment. Normal osmotic shock and p r o t e a s e assay procedures were c a r r i e d o u t as d e s c r i b e d i nt 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) w a s c o n d u c t e d o n 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 Protease  Assay  Anomalous r e s u l t s were o b t a i n e d ( T a b l e s I a n d I I ) when p r o t e a s e was a s s a y e d tions.  Sucrose  i n t h e presence  o f high sucrose  concentra-  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  produce a product which  i n turn reacted with the Folin  Reagent.  Enzyme b l a n k s w e r e 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 b e t w e e n TCA a n d s u c r o s e test is  series.  illustrated  This time  dependent i n c r e a s e i n b l a n k  i n F i g u r e 9-  contributary factor  i n t h e enzyme b l a n k s t h a n i n t h e  EDTA was n o t f o u n d t o b e a  i n this non-specific reaction.  to o b t a i n an accurate assay o f the protease sucrose  supernatant  fraction,  sucrose before protease  V.  a c t i v i t y i n the  samples were d i a l y z e d t o remove  Procedure  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 o n B. a m y l o p h i l u s reduced  I n order  assay.  M o d i f i e d Osmotic Shock  The  O.D.^^  t o 1 x 10  cells.  M, t h e s h o c k e d  40 m l 0.33 M T r i s ^ ^ H C l ,  EDTA c o n c e n t r a t i o n was c e l l s were resuspended  cysteine-HC1  was a d d e d 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 l e s s t h a n 0.65) w e r e u s e d t o m i n i m i z e  m  lysis.  (O.D.gg  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 water  t o remove  flu  o  0.2  -J  10  F i g . 9.  1 20 TIME  - J  l  _  30 40 (minutes]  50  60  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 e n z y m e , b y a d d i n g 0.2 m l 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 . T h e 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) a n d 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&) a n d 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 w e r e r e m o v e d , TCA was a d d e d t o t h o s e t u b e s w h i c h d i d n o t a l r e a d y c o n t a i n TCA. T h e t u b e s w e r e i m m e d i a t l y c e n t r i f u g e d a n d 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 TCA-soluble supernatant t o t h e copper-bicarbonate s o l u t i o n plus F o l i n reagent as d e s c r i b e d i n M e t h o d s .  sucrose  and  protein  determinations.  The procedure  cysteine which i n t e r f e r r e d with the protease  r e s u l t s o f a t y p i c a l experiment was  phase c e l l s ,  utilized,  are presented  p r o t e c t e d by  i n which t h i s  i n Table  III.  c y s t e i n e , remained v i a b l e The  and  the osmotic  shock treatment.  which these  c e l l s w e r e s e d i m e n t e d , c o n t a i n e d o n l y 24%  modified  The  log  throughout  c u l t u r e supernatant  from of  the  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 of l o g phase c u l t u r e s (Blackburn, 1968a). but  65%  A d d i t i o n a l p r o t e a s e was  o f t h e o r i g i n a l p r o t e a s e was  Plasmolysis.by sucrose protease but by the  osmotic  shocked c e l l s .  The  that at l e a s t  viability.  "jk^fo  t h e p r o t e a s e was  consistent with their  l i b e r a t e d by osmotic  protease  but  retained good  t h a t 4% o f  the  s h o c k f r o m E.  coli  c o l i h y d r o l y t i c enzymes w h i l e  f i r m l y r e t a i n e d by the  The  retained  are  o f t h e p r o t e i n was  N o s s a l a n d H e p p e l (1966) f o u n d  cells.  o f t h i s bourid  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  t h i s p r o t e i n c o n t a i n e d t h e E.  experiments.  r e t a i n e d by t h e washed  p r o t e i n recovery figures  T h i s was  c e l l u l a r p r o t e i n was  treatments  ;  it  by the whole c e l l s .  wash  s h o c k l i b e r a t e d o n l y l % a n d 80% was  to the  i s probable  i n t h e two  a p p a r e n t l y l i b e r a t e d 18%  i n a c c u r a t e due  but  lost  shocked c e l l s  a p p a r e n t l y has  a superficial  e x t e r n a l to the p e r m e a b i l i t y b a r r i e r of the c e l l , l o c a t e d f r e e i n the periplasm of the  cell.  i n these  but  location,  i s not  Table 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 u n d e r optimum c o n d i t i o n s .  *  Washed c e l l s Sucrose  supernatant  Shock f l u i d Shocked c e l l s  1o  protease  protein  100  100  18  54  1  59  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 , w e r e o s m o t i c a l l y shocked under o p t i m a l c o n d i t i o n s . A l l steps 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 hours 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 w a s h e d 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 a n d 1 9 . 3 M-g p r o t e i n per ml.  VI.  Spheroplast  Formation  U n d e r n o r m a l g r o w t h c o n d i t i o n s , B. a m y l o p h i l u s by a t h i c k c e l l w a l l which  d e f i n e s i t s shape a s a s m a l l  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  bacillus  is  surrounded cocco-  ( 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 b y a p e r i p l a s m i c s p a c e w h i c h w a s 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 b e f r o m  150 t o 250 A  ( B l a d e n a n d W a t e r s , 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 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 , Since the l o c a t i o n o f the protease on t h e b a s i s o f osmotic  examined.  This procedure  l i b e r a t i o n o f protease  i n s p h e r o p l a s t f o r m a t i o n was  hasbeen used i n determining  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 1962;  I s l a m a n d Lampen, 1962)  licheniformis The  196I1-).  d i d not appear t o be p e r i p l a s m i c  shock experiments,  by t h e lysozyme-EDTA treatment  are  the  ( S u t t o n a n d Lampen,  ando f p e n i c i l l i n a s e  i n Bacillus  (Lampen, 1967a).  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 density by  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  (0.D.gg , 0.5 t o 0 i 6 ) a n d 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 Q  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 -  erated automatic  centrifuge.  T r i s b u f f e r , p H 7.k,  A f t e r a w a s h i n 50 m l o f 0.01 M  t h e c e l l s were resuspended  i n 100 m l 0.033 M  Fig.  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 w e r e 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 a n d a c e t o n e w a s h e s a n d embedded i n V e s t o p a l W ( M a r t i n a n d J a e g e r C o . , 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 a n d 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 m i c 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 a n d l e a d c i t r a t e a n d e x a m i n e d i n a P h i l i p s 100 e l e c t r o n microscope. 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.  Spheroplast  formation procedure . 100 m l : c u l t u r e 9,000 x g_ 10 minutes'.  Culture : supernatant  Cells  50 m l '.01. M - T r i s - H C l , pH 7.h  washings  Cells  R e s u s p e n d e d 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 a n d . 0 . 1 m l lysozyme ( 5 mg/ml )  Osmotic f r a g i l i t y 1 m l sample p l u s 5 m l  Spheroplasts  iO'y'O'OO x g_ •15' m i n u t e s  .p  Lysozyme-solublized fraction  Sj^r.oplasts wash 20%.sucrose pH 8.0 kC  i n .033 M T r i s - H C l 1  Spheroplasts lOOml Disrupted Membranes  hC  Wash  H 0 2  spheroplasts soluble-cytoplasm  H2O  T r i s b u f f e r , pH  8.0,  c o n t a i n i n g 20%  sucrose.  k e p t a t 0 C i n a n i c e b a t h a n d t o i t was EDTA, pH was  8.0;  0.1ml,  The  a d d e d 1.0  suspension m l o f 0.1  0. 5%'w/y,/lysozyme s o l u t i o n .  r o t a t e d b y hand.  One  ml  The  was' M  suspension  samples were t a k e n a t v a r i o u s time  i n t e r v a l s a n d d i l u t e d w i t h 5-0  ml of d i s t i l l e d water.  The  optical  d e n s i t y o f t h e s e s a m p l e s w e r e m o n i t o r e d o n a B a u s c h a n d Lomb S p e c t r o n i c 20 of  a t 660  the c e l l s .  mu.  This determined the osmotic  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  i n d i c a t e d by the absence o f f u r t h e r decrease 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  for  15  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  a t k C.  minutes  The  fragility  complete  i n the t u r b i d i t y  s u p e r n a t a n t was  removed and t h e  solution.  a 21%  w e r e a s s o c i a t e d w i t h 46% s o l u b l i s e d by lysozyme at  t h a n 50%  t h e s o l u b l e enzyme was  explained.  o f t h e a c t i v i t y b u t 21%  treatment.  of the "soluble"  inter-  cysteine.  cannot be  2 2 , 0 0 0 x g f o r 20 m i n u t e s  was  A l l samples were  loss i n protease a c t i v i t y during the  p l a s t i n g procedure, which  g  pellet  The p e l l e t  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  T h e r e was  of  c e n t r i f u g e d a t 10,000 x  then l y s e d by the a d d i t i o n of d i s t i l l e d water.  f e r e n c e caused b y s u c r o s e and  as  sphero-  The s p h e r o p l a s t s was  apparently  Centrifugation of t h i s  fraction  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  enzyme a n d i t i s p r o b a b l e t h a t much o f  p a r t i c l e bound.  This c o u l d have  originated  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 b r e a k d o w n o f s p e c i f i c membrane organelles.  T h e r e was  thus good evidence t h a t the major  portion  Table 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 o n p r o t e a s e liberation  % protease  Washed c e l l Spheroplasts fraction  suspension + solublized  o f washed  cells  100 79  Spheroplasts  k6  Lysozyme-solublized fraction  21  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 g r o w n 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 protease units/ml.  of the protease was bound t o 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 f r e e i n the periplasm. I t i s not known how much d i s i n t e g r a t i o n 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 f o r  complete osmotic f r a g i l i t y t o develop due t o lysozyme treatment was s i m i l a r t o 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 q u a n t i t i e s of mucopeptide were removed (Malamy and Horecker, 196L). High speed c e n t r i f u g a t i o n (90,000 x g f o r 5 hours) o f i n t a c t 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 c u l t u r e subjected t o sonic d i s r u p t i o n  sedimented approximately kcrfo of the enzyme a c t i v i t y under the same c e n t r i f u g a t i o n procedure. Washed B. l i c h e n i f o r m i s 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 a f t e r d i s r u p t i o n by s o n i c a t i o n approximately 50% of the protease a c t i v i t y sedimented at these high speeds (Sargent, et a l . , 1968). • The r e s u l t s o f another experiment i n v o l v i n g spheroplasts are presented i n Table V.  I n t h i s there was no l o s s i n protease a c t i v i t y  during spheroplast formation.  C e n t r i f u g a t i o n a t 22,000 x g sedimented  65% of the protease a c t i v i t y .  C e n t r i f u g a t i o n at 160,000 x g, o f .  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 spheroplasts.  in.sonically disrupted  protease % o f total . u n i t s ..... p r o t e a s e .  A  B  spheroplasts n o n s e d i m e n t a b l e a t 22,000 x g_ * n o n s e d i m e n t i b l e a t 1 6 0 , 0 0 0 x g_ spheroplast envelopes soluble cytoplasm  ik.5 7.6 -  63 33' 20  .5.1 . l6.5  22 72  • * Results obtained from a d i f f e r e n t  experiment.  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, from c e l l s o f ;B. a m y l o p h i l u s g r o w n 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>..0.7The 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 per 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) were.centrifuged. a t .22,000 x g_ f o r 20 m i n u t e s t o s e d i m e n t t h e e n v e l o p e s a n d spheroplasts. 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 . m i n u t e 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 a n d a soluble cytoplasmic 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 (Table V), protease.  bound.  a n d much o f t h i s p r o t e a s e may b e a s s u m e d t o b e p a r t i c l e I n d i r e c t evidence  that the protease from ultrasonication, protease  that particle-bound protease  i s not  i s p r e s e n t e d i n T a b l e V I w h e r e i t i s shown spheroplasts, which  a r e fragmented  by  d o e s n o t s e d i m e n t a t 2 2 , 0 0 0 x g. The d i s t r i b u t i o n  i n f r a c t i o n s from o s m o t i c a l l y ruptured spheroplasts  are presented of  centri-  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 l y s o z y m e  c o m p l e t e l y sedimented  of  a l l b u t 20% o f t h 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 s p e e d  f u g a t i o n sedimented treatment  sedimented  i n Table V I .  T h e r e was a g a i n a 20% s o l u b l i z a t i o n  p r o t e a s e b y l y s o z y m e a c t i o n b u t 79$> r e m a i n e d b o u n d t o t h e  spheroplasts. envelopes  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  a n d o n l y l 6 % was n o n - s e d i m e n t i b l e  r e s u l t s o f experiments  a t l 6 o , 0 0 0 x g.  The  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 observations from osmotic 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  shock experiments  i s bound t o t h e outer c e l l  that  envelope.  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 spheroplasts b y lysozyme treatment t h i s m i g h t be p a r t i c l e bound. fractionation of the lysate into  b u t t h e r e was e v i d e n c e  that  L y s i s o f t h e p r o t o p l a s t and an envelope  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 major p o r t i o n o f t h e protease was  envelope-bound.  Table VI:  The l o c a t i o n o f p r o t e a s e spheroplasts  Fraction  i n osmotically ruptured  Protease units  Protease % washed c e l l s  12.5  79  Lysozyme-solublized  .3.1  20  C e l l envelopes  7.6  ^8  Soluble cytoplasmic  2.5  l6  Spheroplast  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 g r o w n 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 . L 7 . The w a s h e d 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 w e r e 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 w e r e o s m o t i c a l l y r u p t u r e d a n d 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 :  I.  uptake  o f A m i n o A c i d s a n d P e p t i d e s b y B.  amylophilus  The E f f e c t o f O r g a n i c N i t r o g e n o n G r o w t h  • Blackburn  (1968) a n d H o b s o n e t a l . (1958) s h o w e d 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 organic n i t r o g e n sources and t h e e f f e c t o f s h i f t up  a n d s h i f t - d o w n c o n d i t i o n s o n t h e g r o w t h 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 these 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 . 6 5 mg/ml t o 3 . 3 mg/ml) d i d n o t a f f e c t t h e g r o w t h o f B. a m y l o p h i l u s  ( F i g u r e 12).  T h e r e was no l a g p h a s e b u t when B. a m y l o p h i l u s was g r o w n w i t h  single  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 a s o r g a n i c nitrogen sources,  slight  d i f f e r e n c e s d i doccur  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 g r o w n i n t h e t r y p t o s e medium. g i v e n i n F i g u r e 13,  (Curves  There  B. a m y l o p h i l u s was  R e s u l t s o f s h i f t experiments a r e  and Table V I I .  t o b e t h e same f o r s h i f t - u p ,  ( F i g u r e 13).  Maximum g r o w t h r a t e s a p p e a r  shift-down and t h e t r y p t o s e c o n t r o l  B, C, a n d A, r e s p e c t i v e l y ) .  T h e maximum g r o w t h 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 a p p r o x i m a t e l y 0.5 h r Another insert).  1  vs.  ) .  e f f e c t i s observed  The s h i f t - u p  1  on the l a g after the s h i f t  (Figure  ( 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  either  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 a n d A, r e s p e c t i v e l y ) . Pre-exposure  t o t r y p t o s e reduces  t h e l a g , i n agreement w i t h  obtained from t h e growth r a t e experiments.  results  The l a g i n t h e b a s a l  ik,  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 o e l l density v i s 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? offt13 - aoar oalttiro of f C i ^ ^ ^ ^ ^ V o w a l a aoltoee aoalaa v e i l iaooalo&oA l i t o « 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« c o l l aeasity V M a«»»uy*d oft O.SUggg*  •hi  TIME  (hours)  48  ' Table VII:  The'.effect of o r g a n i c n i t r o g e n . s h i f t s . on. the growth r a t e constant (k) and the g e n e r a t i o n time o f B.'amylophilus.  Medium ..  generation time .'  k  % of max. growth . .  100  tryptose to tryptose  79  0.53  hr"  Basal to tryptose  84  .0.49  hr  _ 1  92  tryptose to Basal  •120..  0.-34  hr  _ 1  64  95  0.44  hr"  Basal to Basal  1  1  .83  C e l l s grown i n maltose and maltose p l u s t r y p t o s e (Q.1%) medium t o an 0.D.g60> 0.6 were h a r v e s t e d by c e n t r i f u g a t i o n , washed, i n maltose medium and resuspended t o . t h e o r i g i n a l volume ( 7 . 0 m l ) . From t h e s e 1.0 ml was added t o each o f t h r e e . 4 . 0 ml amounts..of both, maltose and maltose p l u s t r y p t o s e medium. The o p t i c a l d e n s i t i e s 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 . o o l l * 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 «$&«4 to «Mii of tfareo *»0 &l astowBto of both @altos« «a£ KBltoee tryptone acditus* 9ht ostic&l densities for •11 ettltufoo w e arocorded for ?feoisraOJS$ tfee a w N t g o of « M & «cri«» j e t t e d * feyftooo "to t?yg*t©«« I t 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) w a s n o t e x p e c t e d .  These o b s e r v a t i o n s  that tryptose supplies greater pools o r maintains intracellular  beneficial  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 e n v i r o n m e n t .  A greater c e l l yield  i s observed  i n t r y p t o s e medium ( c u r v e s A a n d B v s c u r v e s C a n d D ) . growth o f t h e c e l l s  II.  suggest  Pre-  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 .  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 a m i n o 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 a n d 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 of  s o t h a t t h e amount  a m i n o a c i d t a k e n u p by. t h e c e l l s m i g h t b e 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 a m i n o a c i d i n t h e c e l l s . The d r y w e i g h t  a n d t h e p r o t e i n c o n t e n t , w e r e 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 a n 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  a n d 0 . 4 0 mg p r o t e i n p e r m l .  The a m i n o  acid  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 The r e s u l t s o b t a i n e d b y P u r s e r a n d B u e c h l e r of  (1966) f o r h y d r o l y s a t e s  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 c o m p a r i s o n .  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 a c c o u n t of  amino a c i d s d u r i n g h y d r o l y s i s .  to  t h a t f o u n d b y Purser, and B u e c h l e r  of  valine.  Purser and Buechler  concentration for'B. amylophilus  VIII.  No  f o r the loss  The c o m p o s i t i o n i s s i m i l a r (1966), w i t h t h e e x c e p t i o n  (1966) r e p o r t e d a h i g h i . e . 11.4  g/100 g t o t a l  valine amino  T a b l e  V I I I :  A m i n o B.  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  a m y l o p h i l u s  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  (1966)  P u r s e r  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  0.0  0.7  C y s t e i n e  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  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  4.78  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 -  G l u t a m i c  a c i d  L y s i n e  1 *> <  H  10  T h e  c e l l s  f r o m  a  f o r  a m i n o  a c i d  a n a l y s i s  S t e i n , 100  p e r f r o m f o r  I963). g r a m s  t h e B .  A o f  a v e r a g e  l  t h e  a m i n o  a c i d  B.  a m y l o p h i l u s  b y  c u l t u r e a s  l a m i n o  t o t a l  H  18.  a c i d  B.  a n d  s e c o n d  t h e  e x p r e s s e d  f i r s t  c o l u m n f o r  c o l u m n  (1966).  w a s a  p r e p a r e d  ( M o o r e  e x p e r i m e n t a l l y  d e t e r m i n e d B u e c h l e r  w e r e  m e t h o d s  w e r e  T h e  a n a l y s e s  T h e  a m y l o p h i l u s  i n  r e s u l t s  a c i d . 2  f o r  c o m p o s i t i o n P u r s e r  o f  d e s c r i b e d  a m i n o  v a l u e s  a m y l o p h i l u s  o f  h o u r  t h e  s t r a i n  a s w a s  a n d g r a m s d e r i v e d  d e t e r m i n e d m e a n o f  v a l u e  acids,  c o m p a r e d t o t h e amount p r e s e n t  (Weller,  1957).  was p r e s e n t  i n o t h e r rumen b a c t e r i a  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  a t t h e same c o n c e n t r a t i o n f o u n d  o f rumen b a c t e r i a .  i n mixed populations  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 these  experiments,  were s l i g h t l y higher- 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).  attributed t o either of the cultures. were over  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  Purser and Buechler  Purser and Buechler  (1966) u s e d c u l t u r e s t h a t  (1966) d i d n o t e e t h a t  there  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 .  Bacteroides species possess  a t h i c k e r c e l l w a l l t h a n most  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). for  c o u l d be  2k h o u r s o l d w h e r e a s 10 h o u r c u l t u r e s w e r e u s e d i n t h e  p r e s e n t work. was  The d i f f e r e n c e s i n t h e r e s u l t s  the h i g h values found  gram  This might  account  f o r alanine, g l y c i n e and glutamic  acid,  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 u c o p e p t i d e . The a m i n o a c i d c h r o m a t o g r a m s h a d a s m a l l p e a k 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 diaminopimelic a c i d ,  a constituent of bacterial  mucopeptide.  III.  The-Uptake o f  14  C M i x e d Amino  To f u r t h e r u n d e r s t a n d n u t r i t i o n o f B. a m y l o p h i l u s  Acids  t h e r o l e o f amino a c i d s i n t h e 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 .  Preliminary  experiments  s h o w e d t h a t no a m i n o a c i d s t a k e n i n t o t h e c e l l w e r e  incorporated  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 system t h a t a fermentable carbohydrate source. e x p e r i m e n t was t o p r o v i d e acids  into the c e l l .  lacked  The m a i n p u r p o s e o f t h 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  The r e s u l t s ( F i g u r e 15)  demonstrated  that  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  i n t o 1.0  x 10  ml of  ^ u m o l e s o f t h e amino a c i d m i x t u r e  was t a k e n u p  cells.  B. a m y l o p h i l u s  c e l l s make u p 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 per ml.  Amino a c i d s were c o n c e n t r a t e d  calculated from the r a t i o  t i m e s by. t h e c e l l s  surface.  as  T h e r e was t h e p o s s i b i l i t y  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  to the c e l l  cells  of the r a d i o a c t i v i t y i n the c e l l s to the  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 . that this  14  o f 1 x 10  o f amino  acids  I t i s . n o t known w h e t h e r 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  experiment  w o u l d n o t b e t h e maximum amount p o s s i b l e b y t h e c e l l b e c a u s e t h e concentration  o f e a c h amino a c i d p r e s e n t  results provide  was e x t r e m e l y l o w .  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  These  does n o t  h a v e 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 a m i n o 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% hydrolysate, hydrolysate  m a l t o s e medium.  I t was  c o n t r i b u t e d t o 20.4%  "^C  casein  calculated that the casein  of the c e l l protein.  The  • 5 4  5000 r  TIME (minutes) Fig.  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. g r o w n 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 m i 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 b y 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  incorporation of hydrolysate  C amino a c i d s f r o m m e d i a c o n t a i n i n g  a t two c o n c e n t r a t i o n s  casein  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 e x a m i n e d a n d t h e r e s u l t s a r e i n T a b l e I X . two-fold increase i n casein hydrolysate  A  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 a v e r a g e c o n t r i b u t i o n o f t h e amino A t 2.8  acids to the c e l l protein.  mg c a s e i n h y d r o l y s a t e / m l  the  a v e r a g e c o n t r i b u t i o n was 43% a n d t h e r a n g e was 20% t o 76% f o r the  i n d i v i d u a l amino a c i d s .  0.24 mg p r o t e i n / m l a limiting lysate/ml.  A c u l t u r e O.D.gg^, 0 . 6  would  contain  s o i t was u n l i k e l y t h a t a n y amino a c i d was a t  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  casein hydro-  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 d e p e n d e n t ,  t h a t s i g n i f i c a n t uptake  occurred  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 Casein hydrolysate, g r o w t h m e d i a (.28%),  diffusion.  at a concentration normally  contributed s i g n i f i c a n t l y t o the c e l l protein.  T h i s was n o t a n t i c i p a t e d a s H o b s o n e t a l . ( 1 9 6 8 ) ammonia was u s e d e x c l u s i v e l y b y B. of organic  nitrogen.  t r y p t i c hydrolysate  used i n  showed t h a t  amylophilus i n the presence  H o b s o n e t a l . (1968) u s e d t r y p t o s e ,  a  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 H o w e v e r , a s no not be s t u d i e d .  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 The c o m p e t i t i o n  u p t a k e was t h e r e f o r e  examined.  o f t r y p t o s e w i t h amino a c i d  could  Table IX:  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  Amino A c i d  Arginine Aspartic acid Glutamic a c i d Histidine Isoleucine Leucine Methionine Phenylalanine Proline Serine Tyrosine Valine Average  levels of casein  Casein Composition 1.4 mg p e r m l Composition o f C e l l % incorp ug/ml % external ug/ml incorp contribution  4.13. 7.66 •23.55 . 3.46 • 5.98 10.02 6.'o". 11.7 5.9 6.62 • 7.38  11.2 21.0 25-2 4.4 13.0 16.6 5.2 9.6 8.0 6.2 8.6 14.0 .  8.1 3.0 2.0 • 3.2 2.9 2.5 1.9 . 3.0 1.0 . 3.7 2.4 1.8  4.7 3.2 6.6 1.6 2.4 3.5 0.9 2.5 1.6 3.1 2.2 1.9  42 15 26  - 36 18 21 17 •' 26  20 . 50 25  13 26  hydrolysate.  2 . 8 mg p e r m l % incorp ug/ml % e x t e r n a l incorpscontribution  4.3 2.9 1.3 2.6 . 3.4 1.9 1.7 2.8 1.2 2.9 - 2.1 1.4  5.0 6.2 8.6 2.6 5.7 5.3 1.6 4.7 3.9 4.8 3.9 2.9  45 . 30 34 59 44 32 31 49 49 76 45 20 43  A n 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 u c o f i n d i v i d u a l T*C - a m i n o a c i d s a n d 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 w e r e g r o w n t o a n O.D.g6o» 0 . 5 a n d 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 a n d 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. T h e i n c o r p o r a t i o n o f l a b e l was. d e t e r m i n e d , a n d t h e c o n t r i b u t i o n of exogenous a m i n o 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 . T h e c a s e i n h y d r o l y s a t e a n d b a c t e r i a l c o m p o s i t i o n s w e r e g i v e n a s g/lOOg t o t a l a m i n o a c i d , ( E l l i n g e r a n d B o y n e , 1 9 6 5 ) .  IV.  The U p t a k e o f  C.Amino A c i d s  i n the Presence o f Tryptose  C - l a b e l l e d amino a c i d s w e r e a d d e d a t a h i g h 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 without  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  specific  tryptose.  activity The  o f t h e a m i n o a c i d s was  determined f o r both s e r i e s and the r e s u l t s are presented  i n T a b l e X.  The i n c o r p o r a t i o n o f e a c h a m i n o 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  inhibited by tryptose.  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 a m i n o a c i d s t o t h e  bacterial protein, at this e x c e e d 1.0%.  The a v e r a g e i n c o r p o r a t i o n was r e d u c e d  l o w c o n c e n t r a t i o n , was s m a l l a n d d i d n o t  I t i s therefore impossible  the t r y p t o s e peptides  t o deduce t o what  extent  contributed t o t h e c e l l p r o t e i n butt h e  experiment c l e a r l y demonstrated t h a t c o m p e t i t i o n between t h e peptides and  amino a c i d s o c c u r r e d  a t i o n d i d occur.  a n d i t may b e c o n c l u d e d  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  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  V.  t h a t some i n c o r p o r -  The U p t a k e o f  14  into  e t a l . (1967).  C-peptides  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 protein.  C-peptides 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  The enzymes o f B. a m y l o p h i l u s  peptide with l i t t l e  hydrolysed  protein to  f r e e amino a c i d (Abou A k k a d a a n d B l a c k b u r n ,  To e x a m i n e 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 ,  14  C - p r o t e i n was  1963).  T a b l e X:  The u p t a k e o f tryptose.  Amino a c i d  C-amino a c i d s i n t h e p r e s e n c e o f  External concentration mg/ml  % • incorporated  "jo incorporated i n t r y p t o s e medium  Alanine  .082  16.0  3.0  Arginine  ./084  16.2  2.3  .074  16.7  KR  Glycine  .090  10.0  14.5  Histidine  .042  9.2  3.3  Leucine  .064  20.1  4.4  Lysine  .061  3.3  1.4  Methionine  .110  23.4  11.4  Phenylalanine  .038  17.6  8.3  Proline  .048  20.0  3.3  Serine  .072  5-5  1.0  Threonine  .076  2.9  1.7  Tyrosine  .040  5.3  KR  Valine  .074  16.0  Glutamic  *  KR  acid  =  2.9*  no r e s u l t s  One d r o p o f a n 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 a m o u n t s o f m a l t o s e m e d i u m c o n t a i n i n g O.O83 u c / m l 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 a d d e d t o a d u p l i c a t e s e t o f cultures. The c e l l s w e r e g r o w n t o O.D.^g , 0 . 6 . Samples o f the c e l l s and t h e c e l l s p l u s t h e supernatant were d r i e d on M i l l i p o r e f i l t e r s a n d 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 .  included  i n the  p r o t e i n was into  B.  isolated  amylophilus  incorporation not  medium as  medium was  was  even  Previous amino  acid  concentrations  amino  acids  or peptides.  i n d i v i d u a l amino  amount  of  other  organic  Table  XII.  to  the  a  range  The  of  21%  for  hydrolysate between  to  the  88%.  those  the  (Table  protein IX)  carried  reincorporation  probably  content  showed  out  competitive  examined,  any  one  to  amino The  acids  There  a high  its  Considerable  in  that  figures  high  determine  acid  i n the  results  i n the  under  the  amino  the  are  the  are  not,  absence  presented  acids  i n d i v i d u a l amino  with  those from  good two  acids  54%  to  of i n  amino  comparable  however,  either  maximum  c e l l p r o t e i n was  contribution i n the  contribution of  of  limiting  concentrations  labelled  i n d i v i d u a l amino was  either  pressure  incorporation of  These  IX).  showing  calculation,  under  contribution of  (Table  labelled  c o n d i t i o n s , were  contribution of  amino  and  XI).  final  been  sources.  average  corresponding  obtained  In  nitrogen  have  was  incorporation of  the  The  dependent.  The  acids  the  results  or  cells  (Table  though  concentration experiments  peptides.'  amylophilus  i n c o r p o r a t i o n as  was  of  determined  low and p r e v i o u s  a c i d uptake  of  source  from B.  occurred  optimum f o r  a  casein  correlation experiments.  acids,  i t  has  14  been amino  assumed  that  acids  before  the  C amino  a c i d was  not  converted  incorporation into protein.  to  The h i g h  other contribution  14 of  some  C amino  possibility  was  acids  explored  may be i n the  due  to  such  following  a  convertion.  experiments.  This  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  %  incorporated . ug  . -24 .  present per ml  140.  i n t o E. ' a m y l o p h i l u s . .  . ug p e r ml % contribution incorporated.. t o t o t a l . c e l l p r o t e i n  . 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 m e d i a i n . t h e p r e s e n c e o f 1 4 C - n i a l t o s e ( 0.5 u c p e r m l o f m e d i a ) t o a n O.D.ggov'.0.52, 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 , e x t r a c t e d i n . h o t . 10%'. TCA ( -80C. f o r 15 m i n u t e s ) , w a s h e d b y 3 v o l u m e s o f a c e t o n e , r e d i s s o l v e d i n 2% W a C 0 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 a n d 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 a n d c o n t a i n e d 0.02 u c 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 a d d e d t o ; 5 - 0 m l 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 b y 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. 2  3  Table X I I :  Amino acid  Uptake of c amino acids i n the presence of an individual specific amino acid carrier  Ug/ml  1o of the amino  1o incorporated  incorporated  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  12.6  I89  88  Tryptophan 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  VI.  The U p t a k e a n d C o n v e r t i o n  The f a t e o f the  o f Amino  Acids  C a m i n o 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  distribution of label i nthe c e l l protein.  Some p r e l i m i n a r y  experiments were performed t o ensure t h a t adequate r e s o l u t i o n and  recovery  o f a m i n o 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  chromatographic  1.  technique.  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-  graphy  P r e l i m i n a r y work determined t h e c o n d i t i o n s r e q u i r e d t o obtainaoptimum detection o f c e l l u l a r l a y e r chromatograms. inadequate t o resolve  amino a c i d s o n c e l l u l o s e t h i n  One d i m e n s i o n a l c h r o m a t o g r a p h y p r o v e d 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 . acids  S t a n d a r d s o l u t i o n s o f amino  (2 mg/ml) w e r e d i s s o l v e d i n a q u e o u s 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 . values  The R f  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  w e r e c a l c u l a t e d t o a common v a l u e amino a c i d c o u l d n o t be d e t e c t e d  o f 100 cm.  fronts  L e s s t h a n 5 ug o f  on t h i n l a y e r chromatograms and  since methionine, c y s t e i n e , h i s t i d i n e and tryptophan occurred i n low to  concentrations detect.  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 w e r e  difficult  Long s o l v e n t  d e v e l o p m e n t . ( 1 8 cm)  were.required.to.separate  v a l i n e from methionine, leucine  from i s o l e u c i n e , and g l y c i n e  serine.  f o r c e r t a i n amino.acids were.found  Characteristic.colours  after spraying aspartic  with  the ninhydrin-collidine solution:  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 ,  p h e n y l a l a n i n e and t y r o s i n e  from  green.for  histidine,  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 -  proline . The R f v a l u e s f o r t h e a m i n o a c i d s  in.the  first  w e r e more s i m i l a r . t o t h o s e o f J o n e s a n d H e a t h c o t e the'.Rf v a l u e s . f o r  dimension  (I966),  the.second dimension (Table X I I I ) ; r e p r o d u c i b i l i t y  h o w e v e r 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. a n d 24. h o u r s t o d e t e r m i n e . t h e o p t i m u m t i m e f o r r e l e a s 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  be t h e most e f f i c i e n t  a n d was u s e d i n a l l . s u b s e q u e n t  experiments. amylophilus  p r o t e i n , a n d 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 h y d r o l y s e d . f o r 10^12  hours.  b u t d i d appear a f t e r 18•hours.  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  f o r as c y s t e i c  acid.  was  M e t h i o n i n e was n o t l i b e r a t e d b y A l l of the  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 and  of the  for"18 hours proved t o  H y d r o x y p r o l i n e was a b s e n t f r o m h y d r o l y s a t e s .of ;B_.  12 h o u r h y d r o l y s i s  t h a n were  in  acid  the•oxidized  " Table  XIII:  Amino  T h i n l a y e r c h r o m a t o g r a p h y o f amino a c i d s B. a m y l o p h i l u s .  acid  First  100  Rf  dimension R f 100*  from  Second dimension R f 100-  R f 100  Alanine  63  55  19  10  Arginine  32  24  13  3  47  41  10  0  12  11  12  5  10  KR**  4  KR  57  52  10  1  Glycine  43  36  14  7  Histidine  24  15  12  9  Isoleucine  77  76  h9  ^5  Leucine  80  78  52  52  Lysine  27  20  12  4  Methionine  7^  66  4o  36  Phenylalanine  72  69  53  50  Proline  62  5^  24  17  Serine  43  36  19  13  Taurine  3h  22  18  17  Threonine  50  ^  46  ^5  Tyrosine  69  60  40  21  Valine  7h  69  40  30  100  100  100  Aspartic Cysteic  acid acid  Diamino p i m e l i c Glutamic  acid  Solvent f r o n t *  =  ** =  acid  100  The r e s u l t s o f J o n e s a n d H e a t h c o t e comparative purposes Ko  :  (1966) p r e s e n t e d f o r  results  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 m e d i u m 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 w e r e r u n t w o d i m e n s i o n a l l y ( J o n e s a n d H e a t h c o t e , 1966) a n d 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 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.  B.  Detection of  amylophilus  C amino a c i d s on  was  grown on  chromatograms  C-maltose w i t h o u t  an  organic 14  n i t r o g e n source labelled  to confirm that radioautography  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 .  would l o c a t e The  results  t h i n l a y e r chromatography of the p r o t e i n h y d r o l y s a t e F i g u r e l6.  A l l the  Cof  i s given i n  common a m i n o a c i d s , w i t h t h e e x c e p t i o n  of  14 c y s t e i n e , were f o u n d t o c o n t a i n labelled  i n a medium r e d u c e d by  i n s t e a d of by  cysteine-HC1.  s i m i l a r to those  of h i s t i d i n e  C.  C y s t e i n e , however,  a s c o r b i c a c i d and  Two  n i n h y d r i n spots w i t h Rf  and  diaminopimelic  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 . f o u n d t o be  sodium  acid  was sulfide values  contained  Tryptophan  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  present  t h e a r e a w h e r e t r y p t o p h a n w o u l d be l o c a t e d o n t h e c e l l u l o s e 14 ' / x l a y e r chromatogram. Two C s p o t s (22 a n d 23) a p p e a r e d o n radioautograms, d i m e n s i o n and  w i t h Rf v a l u e s 80 a n d  29  i n the  o f 17-5  and  54  i n the  second dimension.  S p o t 22 was for  o n l y 12  lysis  and  not  identified.  h o u r s t h e r e was  spot  22 may  B e c a u s e t h e p r o t e i n was  peptide.  thin the  co-  of arginine, hydrolyzed  the p o s s i b i l i t y of incomplete  have been a  in  first  S p o t 23  chromatographed w i t h p u t r e s c i n e , a breakdown product  was  hydro-  ¥%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. F i f t y v l 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$ot aaai&er asdao aoid 1 leaoi&o  a3 **  r T 8 9  19 11  lit  Sfeaayl&laaia* valise i^etaioaiae tswaaftaa proline alaatae glataale aoid asaartlo aeld mafeaova slyoiao  iiaferdria toaitivo positive , ais&yflria «&d * C pwitivo %  OpOt BUS!ber 13 it IS  16  1? 18 1? £0 ai  23 8$  oaiao aoid aoriao tferooslao argioiao lyeioe ~ hiatl&iae dlesiaoplaelie eyeteio aoid eyeteiae ornitaiae aateaoira aaSfiaoaa tvyjy&oph&o  1st DIMENSION  19  3.  Recovery 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  chromatographic 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 medium w h i c h c o n t a i n e d 0.1 of a hydrolysate o f these and  u g ~^C g l u t a m i c  acid/ml.  A portion  c e l l s was d r i e d o n a M i l l i p o r e  filter  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 .  was n e c e s s a r y the  were l a b e l l e d b y growth i n m a l t o s e  counts  t o d r y t h e sample b e f o r e  counting  I t  a s o n l y 17% o f  w e r e 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 a d d e d  directly to the s c i n t i l l a t i o n f l u i d . a c i d s were s e p a r a t e d  The r e a d i o a c t i v e a m i n o  on a c e l l u l o s e t h i n l a y e r chromatogram,  located by radioautography  and scraped  into  scintillation  vials.  When a l l t h e s p o t s w e r e 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 recovered. incomplete  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  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 n o n -  amino a c i d components i n t h e c e l l a n d a l s o t o q u e n c h i n g b y t h e cellulose.  The r e c o v e r i e s w e r e q u i t e a d e q u a t e 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  4.  Interconversion of  Ik  C amino a c i d s  To d i f f e r e n t i a t e b e t w e e n 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 those w h i c h were 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  converted into  c e l l p r o t e i n , B. a m y l o p h i l u s was g r o w n i n t h e p r e s e n c e o f e a c h o f t h e common a m i n o a c i d s , a n d 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 a m i n o a c i d s o f t h e p r o t e i n was e x a m i n e d b y t h i n l a y e r graphy (Table  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 radioactive determinations. were i n c o r p o r a t e d resynthesis tyrosine,  Leucine,  p r o l i n e and h i s t i d i n e  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  i n t o other  amino a c i d s .  The m a j o r p o r t i o n o f v a l 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  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 . alanine  Tryptophan,  threonine  t o other  amino  c o n s i s t i n g of methionine,  d i r e c t l y , m o s t o f t h e r a d i o a c t i v i t y was f o u n d  amino a c i d s .  understood.  The a b s e n c e o f i s o l e u c i n e i s n o t r e a d i l y  The a m i n o a c i d s t h a t h a v e a l a r g e p r o p o r t i o n  t o t a l r a d i o a c t i v i t y converted t o other  amino a c i d s , h a d a  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 particular  greater  amino a c i d i n t h e c e l l .  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 T h e r e 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 reactions;  of the  of that  There appeared t o be s e v e r a l mechanisms o f r e a c t i o n  acid,  into  s e r i n e a n d 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 incorporated i n other  directly  and a s p a r t i c a c i d underwent major conversion  a c i d s b u t i n t h e g r o u p o f amino a c i d s glycine,  chromato-  glutamic  deaminated t o o r n i t h i n e .  supplied  and deamination  a c i d may h a v e b e e n d e c a r b o x y l a t e d  o r n i t h i n e decarboxylated  respons-  to butyric  t o putrescene, and a r g i n i n e  However,  several of the interconversions  fable XX?t  8. e o y l o ^ l g i B wa# i a o c « l a t « d i a t o 6.0 sal eBouota of maltose oedltsa* eaea ceataiol&i one o f tee eossoa asdno aei&« st a eoacentration o f 0*035 0«lne per a l . Calttarea were fcarvoated at O . D . ^ g s , 0 , S oad uslae aolds l a t a t aydroiy&ed "sectorial c e l l s vera *®par«ted by t a i n l a y e r • e&remtegref&y. fhe ^reoeoee of r a d i o a c t i v i t y was located by darkened areas o n X - r a y 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, c o l l e c t e d i a r i a l © eeatalnl&g tsGiatilatiea f l u i d aad coasted. Sk© ossiao seida were t e n t a t i v e l y i d e n t i f i e d aad r e l a t i v e a&ouats o f too t o t a l r a d i o a c t i v i t y mre detersl&ed. * SBO ©ottota iaoemnred i n the mdae a c i d , i d e a t i f l e d by t o l a l a y e r eHroaato^rapb^ ae being t i e o r i g i n a l oso added t o tae sodium, va* expressed at a poree&toge o f too t o t a l coaats f&m& l a a l l tae radioaotlve spots.  - Table XIV:  Amino  C o n v e r s i o n o f - C-amino a c i d s  acid  L4  % directly incorporated  11. 45  Alanine Arginine  Number o f C amino a c i d s i n protein 1 4  2 5  60  2 2 7  Glycine  2  4  Histidine Isoleucine Leucine Methionine  100 0 100 2  Phenylalanine Proline Serine Threonine  NR  100 1 18  4 1 5 4  Tryptophan Tyrosine Valine  18 76 85  8 3 3  Aspartic Cysteine Glutamic  Acid  • 7 NR  Acid  i n B. ' a m y l o p h i l u s .  1 3 1 • 7  Tentative  amino a c i d  identification  .11%. a l a n i n e ; 89% unknown 92% g l u t a m i c a c i d a n d a r g i n i n e ; 4% o r n i t h i n e ; 6% unknowns 7% a s p a r t i c a c i d ; 93% unknown cysteine; cysteic acid 60% g l u t a m i c a c i d ; 15% p r o l i n e ; 15% arginine;8% u n k n o w n ; a n d 1% l y s i n e . 94% u n k n o w n ; 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 100% h i s t i d i n e unknowns 100%, l e u c i n e . . . . 1% i s o l e u c i n e a n d l e u c i n e ; 2% v a l i n e a n d 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 a n d unknowns 100% p r o l i n e s e r i n e a n d unknowns 20%. i s o l e u c i n e ; 2 0 % t y r o s i n e ; 18% threonine; 42% g l y c i n e unknown,, a s p a r t i c a c i d a n d t r y p t o p h a n t y r o s i n e a n d unknowns v a l i n e , methionine and l e u c i n e  seemed - u n l i k e l y , pathways. labelled  i f they occurred by the normal b i o s y n t h e t i c  F o r e x a m p l e 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 threonine appearing  i n tyrosine.  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  The  autoradiograms  at the origin.  This  r a d i o a c t i v i t y w o u l d h a v e 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 products  t h a t would not r u n i n t h e s o l v e n t systems.  GENERAL DISCUSSION  In  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 the mechanism o f  r e l e a s e o f b a c t e r i a l exo-enzymes h a v e b e e n e x t e n s i v e l y examined, but 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 h a s been neglected.  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 w a s  s t u d i e d b y B l a c k b u r n (1968a a n d 1968b), who d e m o n s t r a t e d  thatt h e  protease p r o d u c t i o n began i n the l o g a r i t h m i c phase o f growth a n d continued u n t i l the b a c t e r i a ceased t o d i v i d e . protease  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  The c e l l - f r e e i nlate logarithmic  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. work o f N o s s a l andHeppel, to  The f i r s t was s u g g e s t e d b y t h e  ( 1 9 6 6 ) , who u s e d m i l d o s m o t i c  shock  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 w e r e n o t b o u n d 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 p h a s e c e l l s o f B. a m y l o p h i l u s w e r e s h o c k e d reduced  conditions t omaintain v i a b i l i t y o f t h eanaerobic  Sucrose  was r e m o v e d b y d i a l y s i s  assay, total  under cells.  from f r a c t i o n s , p r i o r t o protease  as i t i n t e r f e r e d i n the assay.  L e s s t h a n 1% o f t h e  a c t i v i t y was r e l e a s e d t o t h e s h o c k f l u i d , y e t o v e r 25%  of  thet o t a l  c e l l p r o t e i n was r e l e a s e d .  to  be v i a b l e b u t o n l y a f t e r a long l a g .  The c e l l s w e r e  found  N e u a n d C h o u (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  which  had  lost  protease  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  was n o t f r e e i n t h e p e r i p l a s m i c  was a t t a c h e d  to a cell  Spheroplast  structure within the periplasmic  formation  i z i n g the protease.  s p a c e b u t t h a t t h e enzyme  was t h e s e c o n d m e t h o d u s e d f o r l o c a l -  I n the spheroplast^large  w a l l a r e broken, exposing the cytoplasmic ( B i r d s e l l and Cota-Robles,  1967),  areas o f the c e l l  membrane t o t h e m e d i u m  c o n s e q u e n t l y a n y enzyme f r e e i n  t h e p e r i p l a s m w o u l d b e r e l e a s e d i n t o t h e medium. Horecker  (1961),  the formation  space.  Malamy a n d  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  o f spheroplasts  during  b y t r e a t m e n t w i t h EDTA a n d l y s o z y m e ,  implies a l o c a t i o n external t o the cytoplasmic  membrane.  When t h e  c e l l w a l l l a y e r s w e r e p a r t i a l l y r e m o v e d 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 , presumably because they e x i s t i n a f r e e s t a t e i n t h e  periplasm  o r because they can be e a s i l y detached from t h e c e l l  surface. L y s o z y m e 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 c e l l s but40%o f t h i s protease f u g a t i o n a t l 6 o , 000 x g .  was s e d i m e n t e d b y p r o l o n g e d  Sonic  r e l e a s e d 72% o f t h e p r o t e a s e  from whole  disruption of the  centri-  spheroplasts  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 m i n ) 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 periplasmic  s p a c e b u t t h a t i t was b o u n d t o c e l l  Other spheroplast protease  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  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  ,to the supernatant  structures.  ( 1 0 , 0 0 0 g f o r 20 m i n ) .  a n d 20% was r e l e a s e d  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 spheroplast envelope;  t h e remainder  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  fraction  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 Penicillinase  activity.  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 1968).  s t r u c t u r e a n d 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 . , Tubules  a n d 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 a n d 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 s e e n i n t h e p e r i p l a s m a n d may r e p r e s e n t the p e n i c i l l i n a s e  s e c r e t o r y apparatus  (Ghosh e t a l . ,  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 p e r i p l a s m i c tubules which (Sargent e t a l . ,  1969).  1968). i s present i n  a r er e l e a s e d on p r o t o p l a s t formation Areas  o f adhesion e x i s t between the  w a l l a n d t h e c y t o p l a s m i c membrane o f E. c o l i a n d t h e y p r o v i d e  cell evidence  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 organisms (Bayer,  I968). B l a d e n a n d W a t e r s (1963), h a v e i n d i c a t e d t h a t s i m i l a r  p l a s m i c s t r u c t u r e s are p r e s e n t i n a B a c t e r o i d e s s p . These may  peri-  findings  s u g g e s t t h a t t h e c e l l b o u n d 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  stage on the cytoplasmic bridges p r i o r t o release. The  p a r t i c l e b o u n d 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 w a s  s u g g e s t e d b y B l a c k b u r n (1968a) who s h o w e d 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 - 2 0 0 .  Similar  r e s u l t s w e r e 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 ; 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  60%  during protoplasting,  50% o f t h i s p e n i c i l l i n a s e was s e 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 . ase t h a t remained  i n the supernatant f l u i d  Most o f t h e p e n i c i l l i n after high  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 S e p h a d e x 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.  exoenzyme h a d a m o l e c u l a r w e i g h t  o f 28,000.  By c o n t r a s t t h e 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  through  a p e p t i d e b o n d a s t h e enzyme w h i c h was a t t a c h e d t o membrane 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 b u t was removed b y t r e a t m e n t w i t h It  fragments  o r deoxycholate,  trypsin.  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  temporarily i n a cell-bound l o c a t i o n p r i o r t o i t sl i b e r a t i o n t h e g r o w t h medium.  into  I n t h i s r e s p e c t i t would resemble 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 a n d 1967b;  Collins,  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 primer onto which 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  (Sargent e t a l . ,  r e s i d e n t t i m e was f o u n d t o b e s h o r t e r f o r l o g c e l l s for  1969).  The  (30 m i n u t e s )  than  (3.5 h o u r s ) .  lagcells Toluene  treatment, mechanical o r u l t r a s o n i c  disintegration  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 activity.  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 a n d was a c c e s s i b l e t o t h e c a s e i n substrate.  P o l l o c k (1962),  described the characteristics of a  partially  c e l l - b o u n d enzyme.  T h e s e 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. at  the surface of the c e l l ,  t h e a v a i l a b l e enzyme d o e s n o t i n c r e a s e  b y m o r e t h a n 10% w h e n t h e c e l l s enzymes a r e a b l e t o b r e a k  B e c a u s e enzymes a r e l o c a t e d  are mechanically disrupted.  down h i g h m o l e c u l a r w e i g h t  These  substances and  are 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 stages o f these molecules. to  T h e p r o t e a s e enzyme p r o d u c e d  be p a r t i a l l y  c e l l bound.  space and p a r t i a l l y  b y B. a m y l o p h i l u s  appears  I t i s located inside the periplasmic  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 o p t i m u m p H 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 o n e enzyme h a d s e v e r a l p H o p t i m u m or  t h a t t h e r e w e r e 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 p H o p t i m u m .  T h e r e 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 protease  a c t i v i t i e s w i t h d i f f e r e n t pH optima.  Other r e p o r t s e x i s t i n t h e  literature  demonstrating the p a r t i a l l y  cell-bound nature o f c e r t a i n  enzymes.  M c D o n a l d (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 w a s 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 w a s cell  bound.  Amino A c i d  Utilization  Few f r e e a m i n o 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 r u m e n o f animals.  Rapid p r o t e o l y s i s breaks  down f o o d a s i t e n t e r s t h e r u m e n  •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 microorganisms;  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 c e l l 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 f i n a l c e l l density, an increase in growth rate as well as a reduced lag phase i n the presence of a supplementary organic nitrogen source.  This change i n growth characteristics indicated that amino acids play some role i n the nutrition of B. amylophilus. Ik  I n i t i a l uptake experiments, with  C amino acids at high  specific activity and low concentration, demonstrated that amino acids are not restricted i n entry into the c e l l by a permeability barrier.  Therefore once i n the c e l l 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 c e l l in the presence of oxygen or i n 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 c e l l N  into the medium no matter what was outside. The entry of the amino acids into the c e l l is affected by the presence of peptides, supplied i n the form of tryptose.  The  50% reduction i n amino acid uptake would suggest that peptides entered the c e l l i n competition with the amino acids but i n 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 c e l l 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 c e l l protein i n the presence of 1.4 mg and 2 . 8 mg/ml of casein hydrolysate.  The two-fold increase i n amino  acid uptake corresponding with a two-fold increase i n casein hydrolysate concentrationjindicated that amino acids entered the microorganism 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 i n 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 a l . (1963) had reported this  phenomenon earlier but found that no CO^ was released and he concluded that the products were formed i n 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  DPKH+H + glycine + HPO^  -3  + ADP  +  DPN  -k  + NH^  +  A T P  +  acetate  Stadtman (1966) showed that the amount of growth (dry weight of bacteria) obtained through anaerobic metabolism was directly proportional 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. c e l l /  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 c e l l 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 c e l l density and decreased log phase i n medium containing a supplementary organic nitrogen source. Wright ( i 9 6 0 ) observed i n 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 without  s y n t h e s i s o f amino a c i d s .  a supplemented organic  C maltose, w i t h  n i t r o g e n source present,  and  labelled  a l l t h e common a m i n o 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  t o end p r o d u c t r e p r e s s i o n ; l i m i t e d by the continued  a m i n o a c i d u p t a k e was,  intracellular  supply  even though adequate l e v e l s were p r e s e n t M c D o n a l d a n d Chambers as  (1966)  subject  therefore,  o f amino a c i d s ,  i n t h e medium.  n o t e d t h a t ammonia was  utilized  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 .  u t i l i z e d b y B. a m y l o p h i l u s  Amino a c i d s c a n n o t be  as a s o l e s o u r c e o f c a r b o n  1968a);  t h e y may b e u t i l i z e d  stimulus  to protein  (Blackburn,  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  synthesis.  In conclusion,  these experiments have demonstrated  that  a m i n o 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 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 A m i n o a c i d s h a v e b e e n shown t o b e i n c o r p o r a t e d  but  understood.  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 u p 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). Conversion of isovalerate to leucine by Ruminococcus flavefaciens. Arch. Biochem. Biophys. 851 2 4 5 . Allison, M.J., Bryant, M.P., and Doetsch, R.N. (1962). Studies on the metabolic function of branched-chain volatile fatty acids, growth factors for ruminococci. I. Incorporation of isovalerate into leucine. 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