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Carbohydrate transport and metabolism in resting suspension of clostridium perfringens type A Groves, David John 1968

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CARBOHYDRATE TRANSPORT AND METABOLISM IN RESTING f  SUSPENSIONS OF CLOSTRIDIUM PERFRINGENS TYPE A  BY DAVID JOHN GROVES  B.Sc.  (Biochemistry), U n i v e r s i t y o f B r i t i s h Columbia,  1966  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 t o  required  standard  THE UNIVERSITY OF BRITISH COLUMBIA May,  1968  the  In p r e s e n t i n g  for  an  that  thesis  Library shall  i further  for  publication  w i t h o u t my  in p a r t i a l  the  make i t f r e e l y  agree that  by  written  Department of  be  this  thesis  Microbiology  6th September  Columbia  1968  the  British  for  g r a n t e d by  the  requirements  Columbia,  I agree  reference  and  for extensive  copying of  Head o f  It i s understood  for financial  permission.  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  of  available  permission  nils r e p r e s e n t a t i v e s .  of  f u l f i l m e n t of  University  s c h o l a r l y p u r p o s e s may  Department or  or  thesis  advanced degree at  the  Study.  this  gain  shall  my  that  not  this  be  copying  allowed  ABSTRACT  Suspensions  o f C.  p e r f r i n g e n s , when grown on a  peptone-  f r e e , s e m i - d e f i n e d medium, h a v e b e e n shown t o r e m a i n to autolysis  f o r extended  these suspensions on  has  The  stability  been compared w i t h t h a t o f c e l l s  found  grown on  this  semi-defined  t o c o n t a i n a l l o f t h e enzymes o f t h e  pathway o f g l y c o l y s i s ,  in addition  the p y r u v a t e - c l a s t i c system,  phosphate dehydrogenase a c t i v i t y Evidence  grown  has  been p r e s e n t e d  to l a c t i c acid  but  no e v i d e n c e  c o u l d be  f o r the  this  dehydrogenase of  glucose-6-  demonstrated.  i m p l i c a t i o n o f the  Embden-  degradation  organism.  Resting transport  suspensions  o f C.  p e r f r i n g e n s w e r e shown  r a d i o a c t i v e g l u c o s e and  carbohydrates  mannose, but  by a common m e c h a n i s m and  c o n c e n t r a t i o n s s e v e r a l hundred times external  medium w e r e  Embden-Meyerhof  M e y e r h o f p a t h w a y as t h e m a j o r p a t h w a y o f g l u c o s e by  of  complex media. Extracts of c e l l s  and  periods of time.  resistant  medium.  enzymatic,  The  accumulate  those  t r a n s p o r t system  not  was  to  other glucose  found  in the  found  t o be  an  e n e r g y - d e p e n d e n t , t e m p e r a t u r e - s e n s i t i v e , and  s p e c i f i c mechanism which  was  to  saturated at high substrate  highly  concentrations.  The carbohydrate was found to be accumulated  as an e q u i l i b r i u m mixture of phosphorylated hexoses. phosphorylation mechanism involved in accumulation was demonstrated to be other than the soluble hexo-kinase.  The  TABLE OF CONTENTS Page INTRODUCTION .  . .  2  I. Metabolism of Carbohydrates and Amino Acids .  . .  2  LITERATURE REVIEW  II.  1 . Metabolism of carbohydrates  2  2 . Metabolism of amino acids .  7  The Transport of Metabol i tes .  9  1 . Metabolite transport in mammals .  .  .  .  .  9  .  2 . Metabolite transport in b a c t e r i a  9 9  a. Amino acid transport  1 0  b. Carbohydrate transport i . Galactose and g a l a c t o s i d e s .  .  .  .  . 1 0  .  11  i i . Glucose i i i . Carbohydrate accumulation mechanisms  12  and components. 3 . Carbohydrate transport in yeast'and .fungi. MATERIALS AND METHODS . I.  .  .  . 1 3  . .  .  1  1 6  Organisms and Media 1 . Complex medium .  .  .  1  2 . Semi-defined medium  h. Growth of inoculum. Growth Conditions  6 16  3 . Stock cultures  II.  6  1 8 .  1 8  . .  1  9  Table of Contents  (Continued) Page  III.  Stab i 1 i ty of C e l 1 Suspens ions  2 0  IV. Enzyme Assays  .  1 . C e l l - f r e e extracts 2 . Enzyme assays  .  .  . .  .  . 2 1  .  .  .  .  2  1  . 2 3  . . . .  2 9  V. Carbohydrate Transport 1 . Preparation of anaerobic c e l l suspensions . \k  .  2 9  2 . Assays for  .  3 0  C-carbohydrate incorporation  .  3 . Determination of the nature of the accumulated  C-carbohydrate  V I . Assay of R a d i o a c t i v i t y  .  .  .  .  .  .  .  .  . .  .  . . .  1 . Mi 1 1 ipore f i Iters . 2 . Chromatograms and electrophoretograms  3 2  .  3k .  3k  .  .  .  3k  .  .  .  .  .  3 5  V I I I . A n a l y t i c a l and Preparative Techniques  .  .  .  .  3 6  .  3 6  VI I. Chromatography and Electrophoresis  1 . Dry weight 2 . Protein  . . . .  3 6  3 . Optical density measurements  3 6  .  k. Dephosphorylation of carbohydrates  .  .  .  .  3 7  5 . Glucose  .  .  .  .  .  .  3 7  IX. Chemicals, Enzymes and Substrates.  .  .  .  .  .  3 7  .  .  .  .  .  .  .  3 9  RESULTS AND DISCUSSION I. Growth and Resting C e l l Suspensions . 1 . Growth curves  .  3 9  . . . .  .  kO  vi  Table of Contents  (Continued) Page  2. Stab i 1 i ty of eel 1 suspens ions  43  I I. G l y c o l y t i c Enzyme Assays. -.III. Carbohydrate Transport  54  .  .  .  .  .  .  .  .  .  64  1. U t i l i z a t i o n of carbohydrates by C_. perf r i ngens  65  14 2. Transport of  C-carbohydrates.  .  .  .  .  .  (i) Transport of ^ C - g l u c o s e (ii)  Transport of  . 6 5  C-carbohydrates 6 7  other than glucose (iii)  Temperature dependence of glucose transport.  (iv)  .  .  C.  Energy requirement of the of glucose .  .  .  .  .  .  .  (vi)  C-mannose.  .  3. The pooling of  .  C-glucose .  (i) Pool capacity :  .  .  .  .  69  C.  .  .  lb  of  . . . .  71  . . .  .  .  . . . .  . . .  82  . . .  92  .  .  .  92  .  .  .  97  .  .  .  99  4. Mechanism of the accumulation of glucose  .  .  102  (ii)  .  .  . . .  S p e c i f i c i t y of the transport glucose and mannose .  .  transport  (v) K i n e t i c s of accumulation of glucose and  65  Internal concentration .  .  .  .  14  (iii)  Nature of the pooled  GENERAL DISCUSSION.  .  C  . . .  .104  Table of Contents (Continued) Page LITERATURE CITED .  .  .  .  .  .  .  .  .  .  .  .  .  108  vi i i  L I S T OF  TABLES  Page Table  Table  Table  Table  Table  I.  II.  The a u t o l y s i s o f e e l 1 s u s p e n s i o n s C. p e r f r i n g e n s u n d e r s t a r v a t i o n conditions  of  The s t a b i l i t y o f c e l l s u s p e n s i o n s o f £. p e r f r i n g e n s u n d e r s t a r v a t i o n c o n d i t i o n s when p r e v i o u s l y grown i n a c h e m i c a l l y d e f i n e d medium  49  50  I I I . S p e c i f i c a c t i v i t i e s o f g l y c o l y t i c enzymes i n e x t r a c t s o f C_. p e r f r i n g e n s . The e x t r a c t s w e r e p r e p a r e d f r o m c e l l s grown on s e m i d e f i n e d medium p l u s 0.6% glucose, except where i n d i c a t e d o t h e r w i s e 57 IV.  V.  G r o w t h o f £. p e r f r i n g e n s w i t h v a r i o u s c a r b o h y d r a t e s as t h e s o u r c e o f c a r b o n  66  S a t u r a t i o n k i n e t i c s o f the glucosemannose t r a n s p o r t s y s t e m . Estimated values of and V from t r i a l s i n F i g u r e s 15 and 17  81  A c c u m u l a t i o n o f c a r b o h y d r a t e s and c o m p e t i t i v e i n h i b i t i o n of the rate o f u p t a k e o f ' ^ C - g l u c o s e (8 x 10"'? mM, 1.4 u c / u m o l e ) and ^ C - m a n n o s e (1.6 x 10" mM, 1.4 u c / u m o l e ) , by a 100-fold e x c e s s o f t h e s e c a r b o hydrates  88  m  Table  VI.  2  ix  LIST OF FIGURES Page Fig.  1.  Growth of C. perfringens in complex medium plus 0.7% glucose.  41  Fig.  2.  Growth of £ . p e r f r i ngens i n complex medium plus 0.7% glucose  42  Fig.  3.  Growth of £ . p e r f r i ngens in secondary culture in semi-defined medium plus 0.7% glucose  44  Growth of C. perfringens in semi-defined medium plus 0.7% glucose  45  S t a b i l i t y of eel 1 suspensions of C. perfringens grown with 0.7% glucose  47  Fig. Fig.  4. 5.  Fig.  6.  The rate of l y s i s of suspensions <i>f c e l l s grown in complex medium  52  Fig.  7.  The a c t i v i t y of NADH^-oxidase in aerobic and anaerobic eel 1-free extracts of C. perfringens  58  The a c t i v i t y of 3-phospho-glyceraldehyde dehydrogenase in eel 1-free extracts of £ . perfr? ngens <  60  A c t i v i t y of the p y r u v a t e - c l a s t i c system in c e l l - f r e e extracts of C_. perf r i ngens  62  Fig.  Fig.  8.  9.  F i g . 10.  Early time course of uptake by whole ce 11 s of C_. perf r i ngens  F i g . 11.  Total uptake of r a d i o a c t i v i t y by c e l l s of C. p e r f r i ngens  70  F i g . 12.  Total incorporation of by c e l l suspensions  72  F i g . 13.  68  radioactivity  14 Saturation k i n e t i c s of C-glucose incorporation by C. p e r f r i ngens  75  X  Li st of Figures (Continued) Page 14 F i g . 14.. Saturation k i n e t i c s of C-glucose incorporation by C. p e r f r i hgens 14 F i g . 15- Saturation k i n e t i c s of C-glucose incorporation by C. p e r f r i hgens 14 F i g . 16. Saturation k i n e t i c s of C-mannose incorporation by C_. perf r i ngens 14 F i g . 17. Saturation k i n e t i c s of C-mannose incorporation by C_. perf r i ngens F i g . 18. Competition for glucose uptake in C_. p e r f r i hgens A and B  76 77 79 80 83  F i g . 18.  Competition for glucose uptake in C^_ perfringens C and D  84  F i g . 19.  Competition for mannose uptake in C_. p e r f r i ngens A and B  86  Competition for mannose uptake in C_. perf r i tigens C and D  87  Formation of a cold TCA s o l u b l e , radioactive p o o l , by c e l l suspensions  93  Formation of a cold TCA s o l u b l e , radioactive p o o l , by c e l l suspensions  94  Total incorporation of by c e l l suspensions  96  F i g . 19. F i g . 20. F i g . 21. F i g . 22.  radioactivity  ACKNOWLEDGEMENTS  My s i n c e r e g r a t i t u d e for her supervision for of  her help  i s extended  and encouragement o f t h e r e s e a r c h , and  and c o n s t r u c t i v e c r i t i c i s m  i n the preparation  the manuscript.  I would  like  t o thank Dr. J.J.R.  encouragement and f o r e d i t i n g  I would  also  consideration particularly of  t o Dr. A.F. Gronlund  Campbell  for his  the thesis.  l i k e t o t h a n k my w i f e  and s u p p o r t d u r i n g  H a l l i e f o r her  t h e r e s e a r c h , and  f o r h e r many h o u r s s p e n t  i n the preparation  the thesis.  Lastly, especially Mrs.  Rita  I o f f e r my t h a n k s t o my  D r . W.W. Rosbergen  Kay, f o r help  fellow-students,  i n my e x p e r i m e n t s , a n d t o  f o r the typing o f the thesis.  INTRODUCTION  The metabolism o f i n mamma 1 i a n of  obvious  by C . by not  interest.  While  the  technique,  become u n k n o w n . of £ .  perfringens  have m o t i v a t e d  physiology  and m e t a b o l i s m o f  characterization that  the  obligate  of  of  transport,  the  the enter  survival  of  the in  the  infection  recent  in  the  An  cell. resting  insight  the  organism  the  in  glucose  is of  great  the  metabolic  theoretical  a  intensive  a carbohydrate  is  the  semi-  However,  for  meaningful  suspensions of the  preparation  information the  the  austere  about  of  fate of  interest  the  such  the  environment  the s o i 1 . A knowledge o f  the  through  into  in  have  addition,  anaerobes warrants  carbohydrate  years  food  define  In  of  is  increasing  to  organism.  suspensions could a l s o provide  widespread  plus  degradation  uniform,  essential.  man,  o r g a n i s m as m i c r o - a e r o t o l e r a n t  part  organism are  of  this  integral  to  to  "gas-gangrene"  numerous a t t e m p t s  metabolism.  passage of  of  carbohydrate  as a c a u s a t i v e agent  it's  p e r m e a b l e membrane  stable  cases  of  initial  studies  this  contains only  investigation An  of  diminished  This danger,  poisoning,  genus  soluble  seriousness of  has been g r e a t l y  improved c l i n i c a l  recognition  major  s y s t e m s , b y an o r g a n i s m p a t h o g e n i c  perfringens  yet  g l u c o s e , the  transported  and p o s s i b l y o f  p r a c t i c a l value, due to the pathogenic nature of the organism. It was the object of t h i s i n v e s t i g a t i o n to devise a method of preparing resting c e l l suspensions of C. perfringens, an anaerobic s a c c h a r o l y t i c pathogen, to study the metabolism of glucose by t h i s organism, and to elucidate the system of entry of carbohydrates into the c e l l s .  2  LITERATURE  The gas  importance  gangrene  and  a Gram-positive studies  I.  of  the  in  f o o d p o i s o n i n g , and  by many w o r k e r s .  must  has  l o n g been  milk  that  is  from  glucose  Shaskan of Fe  (1944)  lactate,  as  extensive  p e r f r i hgens has been  to  of  the  toxin  the  and McCoy)  insure  the  gaseous  organism.  £.  1932).  fermentation  concentrations.  per  Fe  + +  stormy  p e r f r i ngens  Pappenheimer  Fe  acid  the  CO^ a n d  the  lactic  of  heterofermentative  medium a p p r o a c h e d z e r o ,  2 moles o f  that  into + +  much  production.  butyrate, e t h a n o l ,  Kmieciak,  of  for  the  studied  associated with  a saccharolytic,  acetate,  and  effects of  become a p p a r e n t ,  and  in order  l o o k e d more c l o s e l y  approached  importance  in  Acids  (Hastings  characteristic  ion c o n c e n t r a t i o n  p-roduction  growth 1932  medium  (Friedemann  g l u c o s e , and t h e + +  for  r e c o g n i z e d as b e i n g  organism producing  fairly  d i s s i m i l a t i o n was  h a s b e e n known s i n c e  fermentation  and A m i n o  reasons that w i l l  requirements  be a d d e d t o  general  both  organism.  g l u c o s e by C .  w o r k on c a r b o h y d r a t e  It  the  in  as a p a t h o g e n ,  carbohydrates  of  For  its  resulted  Carbohydrates  Metabolism of  metallic-ion  have  metabolism of  The d e g r a d a t i o n  this  Clostridium perfringens  anaerobe,  Metabolism of  1.  of  REVIEW  and  products As  lactic  mole o f  H^  the acid  glucose  3  fermented.  Thus low Fe  concentration s h i f t e d the normal hetero-  fermentation to a homofermentative production of l a c t i c a c i d . These workers also demonstrated the coincidence of optimum Fe  concentration for minimum l a c t a t e production, maximum growth, ++  and maximum t o x i n production.  Thus, Fe  is required for the  production of acetate and butyrate from pyruvate, the l a s t precursor common to both the v o l a t i l e f a t t y acids and to l a c t i c acid. Pappenheimer and Shaskan (I3kk) also demonstrated that while i r o n - d e f i c i e n t medium gave homofermentative g l y c o l y s i s , i r o n - f r e e medium produced by a a ' - d i p y r i d y l treatment, would not support growth.  On t h i s basis they assumed that iron played a r o l e in the  actual g l y c o l y t i c pathway to pyruvate as wel1 as in the heterofermentative steps beyond pyruvate. Bacon (19^9) indicated that carbon monoxide would a l s o cause a s h i f t from heterofermentation to homofermentation, and other workers (Lerner and P i c k e t t , 19^5; Kubowitz, 193^) showed that high cyanide concentrations caused an i n h i b i t i o n of gas production of other C l o s t r i d i a .  It  is known that a a ' - d i p y r i d y l a f f e c t s  inorganic  i r o n , while CO and CN bind heme i r o n . While i n v e s t i g a t i n g the dual function of i r o n , Bard and Gunsalus (1950) found that free i o n i c iron was required for phosphate a l d o l a s e .  Removal of F e  + +  fructose-1,6-di-  from eel 1 free extracts ++  completely i n h i b i t e d aldolase a c t i v i t y and addition of Fe reactivated the enzyme. activity  ++  or Co  They concluded that the presence of aldolase  indicated that the Embden-Meyerhof pathway of glucose  degradation was present.  In a d d i t i o n , they suggested that the  4  enzymes 3-phosphoglyceraIdehyde dehydrogenase, triose-phosphate isomerase, and ethanol dehydrogenase were operative In the organism; however, they did not present d e f i n i t i v e evidence. In a s e r i e s of p u b l i c a t i o n s , Shankar and Bard (1952, 1955a, 1955b) studied the e f f e c t s of Mg f r i ngens.  and Co  on the growth of £ . per-  By adding various ions to deionized medium (1952), they  demonstrated, as Webb (1948) had done, that Mg (1955a) produced filamentous c e l l s .  deficiency  They also showed t h a t , with  M g - d e f i c i e n t c e l l s , there was a considerable decrease in gas ++  evolution during growth.  In an unsuccessful attempt to demonstrate  a d e f i c i e n c y of Embden-Meyerhof pathway enzymes, they qua 1 i t a t i v e l y r  indicated the existence of hexokinase, phosphohexoseisomerase, phosphofructokinase, a l d o l a s e , and 3-phosphoglyceraldehyde dehydrogenase. In a d d i t i o n , they showed a s h i f t to homolactate fermentation from heterofermentation upon addition of excess C o , thus + +  that C o  + +  indicating  interfered with formation of the heterofermentative system  (1955b). Ivanov (1954) examined the hexokinase of C. perfringens c e l l - f r e e extracts and c e l l suspensions.  in  In e x t r a c t s , the pH optimum ++  was shown to range from 7.0 - 8.0, and Mg  ++  and Co  were found to  a c t i v a t e the enzyme. The a b i l i t y of £ . perfringens to grow under higher oxygen tensions than other C l o s t r i d i a is well documented (Fredette, P l a n t s , and Roy, 1967; Pr£vot, 1966).  Hi rano e_t al_. (1954) compared the aerobic  and anaerobic degradation of glucose by £ . perfringens and Escherichia co1i,  and found that while sodium azide stimulated aerobic degradation  of glucose by £ . c o 1 i , i t prevented the accumulation of pyruvate  5  from  g l u c o s e by £ .  respiration in  the  on  iron,  of  perfringens.  g l u c o s e and  presence of similar  Attempts metabolism,  carbon atoms,  that  define  using  found  t h a t whole  also  looked  cells  at  degraded  o x y g e n by a m e c h a n i s m s h o w i n g a d o u b l e  to  to  (1952)  Bard  of  the  the  major  radioactive  have  fermentation pathways  of  carbohydrates  been c a r r i e d  out.  of  glucose dependence  glucose to  h e x o s e and  labelled  at  Gibbs,  and  Paege,  the  acetate.  pentose specific  (1956)  Bard  14 used  C-glucose preparations  and C4 p o s i t i o n s glucose that  respectively  degradation  the  major  route  Embden-Meyerhof ethanol  to  at  the  and d e g r a d e d  show t h e  of  fate  of  C-1,  the  each  However,  lower  than  the  that  C-2,  C-6  was  They  indeed  acetate,  C-3  of concluded  via  the  of  the  specific activity  of  and  end p r o d u c t s  carbon.  glucose dissimilation  pathway.  p r o d u c e d was  labelled  indicating  that  12 C-ethanol C-1,  C-2  (1958) the  from  some o t h e r  a n d C-6  of  used c e l l s  ethanol  glucose.  Later,  grown o n p e n t o s e s ,  and a c e t a t e  activities.  s o u r c e had d i l u t e d  Either  products  the  had  however,  the  label  Embden-Meyerhof  the  C y n k i n and Gibbs  and w e r e a b l e nearly  from  to  identical  pathway  is  show  that  specific  the  major  pathway,  12 or  growth  on a p e n t o s e  "turns  off"  the  additional  source of  C-  ethanol.  (1958)  C y n k i n and D e l w i c h e dissimilation previously ferment those  of  in  been  ribose,  cell-free shown but  extracts  (Cynkin  not  the  existence  of  the  of £ .  and G i b b s ,  xylose or  glucose fermentation.  demonstrate  studied  enzymes  ribose  p e r f r i hgens. that  1957)  arabinose,  C y n k i n and ribokinase,  of  to  the  products  Delwiche  It  had  organism similar  were a b l e  phosphopentoisomerase  could to  to and  6  the  production  of  hexose-monophosphate  from  The  production  of  hexose-monophosphate  from  pentose and  indicated,  trans-ketolase  activity, NADP)  either  linked  indicating  In  a  and  the  In  in e i t h e r  glucose or  organism,  C.  enzymes  were  found  also  to  tetani, an  pathway. with  Oxidation  pyruvate  dehydrogenase or (Martinez  implicated  and  saccharolytic,  of  a pyruvate  thermophilic,  pathway,  pyruvate-clastic  as w e l l  as  demonstrated;(Lee  the  and O r d a l ,  The mechanism o f has been  Valentine,  been  studied  and J o h n s o n ,  1964).  pyruvate-clastic identified  the  or in  is  the  not  cells,  normally  fructose the  activity  1,6-diphosphate  presence of (NADh^)  suggested  the by  that  decarboxylase  was  1959). obi i gate  enzymes o f  a n a e r o b e , C_.  the  system,  Embden-Meyerhof have  been  1967).  heterofermentative extensively  19^3;  or  glucose  glucokinase  as s u b s t r a t e  Rittenberg,  of  (NAD  grown  of  r e d u c e d NAD  all  (Koepsell  pentose  inducible  thermo-saccharolyticum,  pyruvate  nucleotide  which  r e d u c e NAD w i t h  and t h i s  dehydrogenase  1961).  (Wood  be s a c c h a r o l y t i c ,  lactic  the  tri-phospho-pyridine  present  to  trans-aldolase  No g l u c o s e - 6 - p h o s p h a t e  not  extracts  operative  of  was  Embden-Meyerhof/  a  existence  h e x o s e - m o n o p h o s p h a t e pathway  substrate  either  the  phosphorylated  the  required  cell-free  or  found  related  considered  activity.  di-  was  claimed,  the  that  degradation  as  they  ribose-5-phosphate.  and  Mortlock,  T h e non-heme  iron  reviewed  Valentine,  containing  phosphoro-clastic system, and p u r i f i e d  from  breakdown  several  of  by s e v e r a l and W o l f e ,  component  ferredoxin, di f f e r e n t  of  workers 1959; the  has  Clostridia  7  and o t h e r  anaerobes which  has a l s o been  found  during  (Valentine,  growth  properties  The  i n C_.  of ferredoxin  excellently  reviewed  have  and to  The physical  perfringens independent catalyze  Although  Clostridia  electron have  NADr^-oxidase  2.  Metabolism  Most  oxidation-deamination (Nisman,  1954).  particularly (Nisman,  used  carbon  subsequently  1963).  and W o l f e ,  nor the presence o f  ferredoxin  and c h a r a c t e r i z e d  and f o u n d  t o be c y a n i d e  reduction  of  U  insensitive  T h e enzyme was shown  2~^2^  activity  from  u  s  '  n  9  NADr^.  cytochromes,  (Dolin,1959b)  in the  preparation.  acids  Clostridia reaction  However,  most  C. perfr? hgens,  1954).  being  n o t b e e n shown t o c o n t a i n  o f amino  proteolytic  1967).  (Valentine  isolated  showed c y t o c h r o m e c r e d u c t a s e  puri fied  been  i n C_. p e r f r i n g e n s .  1959a)  hydrogen  and c h e m i c a l  Tii ' C l o s t r i d i a - and p r o d u c e s  o f ^2^2 a s an i n t e r m e d i a t e .  a four  does n o t p r o d u c e  i s c o n s i d e r e d t o be t h e g e n e r a l  system,  has been  (Do!in,  Ferredoxin  e s t a b l i s h e d and have  and b u t y r a t e  the p y r u v a t e - c l a s t i c  been d e m o n s t r a t e d  Dolin  which  h y d r o g e n and a c e t y l - C o A , t h e l a t t e r  A NADH^ o x i d a s e C.  system  cleavage  synthesis of acetate  Neither  1964).  hydrogen.  ( M a l k i n and R a b i n o w i t z ,  mechanism o f p y r u v a t e  for  acidi-urici  are well  pyruvate-clastic  dioxide,  produce molecular  ferment  amino a c i d s  known a s t h e S t i c k l a n d  in a coupledreaction  s a c c h a r o l y t i c C l o s t r i d i a , and  do n o t u s e t h e S t i c k l a n d  mechanism  8  (19^2)  Woods and T r i m degrade  only  threonine arginine the  5 amino a c i d s .  were m e t a b o l i z e d p r o d u c e d NH^ and  degradation  of  characterized  of  (Hughes  glutarate  acid,  of  the  oxaloacetate,  to  keep  The with to  be  C.  inhibited and  Cysteine,  nitrate  or  by  decar-  and  oxaloacetate  resulted This  as w e l l  found  that  by w a s h e d c e l l  has  b e e n shown t o  and u r e a  the  (Hicks,  from  and de-  provided as  the  neither  organism's  d e p e n d i n g on t h e cystine  organisms  to  suspensions, occur  sulphate,  sulphur  strain  produce H S.  of  sulphite  or  requirements.  used,  and g l u t a t h i o n e  and  1965).  sulphur metabolism  h o m o c y s t e i n e were demonstrated  cysteine,  by t h e  nitrite  studied  c o u l d supply the  requirements,  degraded  to  as s u b s t r a t e ,  (1957)  and h a v e  cystine  thiosulphate,  in o p e r a t i o n ,  aspartate  •y-ami n o b u t y r a t e  aspartate  of  transaminase  that  followed  oxaloacetate.  by K C N , i o d o a c e t a m i d e , Bonde  perfringens  these  of  glucose or ethanol  thiosulphate  amounts  no  organism.  alanine.  reduction  Fuchs  system  found  and C O ^ , b u t  and p y r u v a t e  transaminated the  who  demonstrated  and  Evidence for  concluded that  aspartate  Tytel1  has b e e n p u r i f i e d  1952).  catalytic  while  a n d NH^ by t h e  CO^  to  and  1952,  In  transamination,  was  was a b l e  cysteine  2>  (195*0,  by H i c k s  It  C0  no  orthinine,  a simple  formed o n l y and t h a t  product,  but  and W i l l i a m s o n ,  if  carboxylation  major  to  perfringens  C C ^ , NH^ and H ^ ,  C_. p e r f r i h g e n s  had o c c u r r e d .  a-ketoglutarate  produce  H^,  C.  cystine,  produced a l a n i n e  be e x p e c t e d  boxylation  glutamic  to  has b e e n p r e s e n t e d  and c t - k e t o as w o u l d  Serine,  arginine  The g l u t a m i n a s e  activity  showed t h a t  to  while  satisfy sulphate,  w e r e shown t o  be  9  I I.  The T r a n s p o r t o f  The  importance o f  metabolites  through  t h e s e membranes met w i t h  which  are  times  that  1965;  are  highly  to  concentrate  the  external  cells  reviewed  ions,  (Wilbrandt  transport  Amino a c i d  studied  s p e c i f i c and  levels  several  thousand  of  membranes o f  and c a r b o h y d r a t e s  and  various have  R o s e n b e r g , 1961;  been  Quastel,  paralleled  of  of  and  investigations  bacteria  a m i n o a c i d s by m i c r o o r g a n i s m s has reviewed  the  their  in  transport  and M c C l u r e ,  The workers  with  years.  1967).  Metabolite  Britten  to  fifteen  has  environment.  by a m i n o a c i d s ,  The t r a n s p o r t  1956)  metabolites  permeation  extensively  environment,  have been d e s c r i b e d w h i c h  numerous s t u d i e s o f  Albers,  internal last  i n mammals  a.  1962;  cell's  p r o c e s s e s by w h i c h  the  adequately  2.  the  and t h e  transport  mammalian most  membranes,  can m a i n t a i n  Metabolite  The  p r o c e s s by w h i c h m i c r o o r g a n i s m s p a s s  recognition within  able of  the  their  increasing  Systems  1.  Metabolites  (Holden,  Kepes and  Cohen,  1962).  Pasteur  Institute  s t u d i e d on t h e  into  1962;  been  the  nature  of  (Cohen and R i c k e n b e r g ,  transport  of  g-galactosides  amino a c i d t r a n s p o r t .  They  developed in  that  accounted  a s p e c i f i c , energy-dependent  defined E.:  a model  several  coli.  specific  Recently,  the  1968). was  specific  As w e l l ,  shown  on the  to  for  the  for  transport  of  the  structural  amino a c i d s  is  while  found  to  of  amino  by  several  acids  acids  across the  (Kay,  cell  accumulation  be an  by  of  amino  acids  (1962)  concentration  of  the  amino  and M c C l u r e  be m e d i a t e d  "families"  of  membrane  Britten  amino a c i d  shown t o  movement  concentration  and a c c u m u l a t i o n  be e n e r g y - i n d e p e n d e n t ,  inside  the  process.  systems  by Pseudomonas a e r u g i n o s a was permeases  for  membrane  process  energy-dependent  process.  b.  Carbohydrate  i.  transport  G a l a c t o s e and  galactosides  The g a l a c t o s i d e of  the  most w e l l  mechanism. Buttin  defined,  The workers  and M o n o d ,  both  of  1956)  the  permeation as  to  specificity,  Pasteur  showed  the  system o f  Institute  existence  of  E_. c o l i  control,  is and  (Rickenberg, an  one  inducible  Cohen, system  14 that  selectively  transported  C-thiomethyl-g-D-galactopyranoside  14 (  C-TMG)  kinetics  into  and was  enzyme-like the  the  labile  properties,  transport  dependence o f on t h e - e x i t ' : filtration  cell,  in to  enzyme  they  function. the  a process that  technique.  In  t h e word  (1957)  p r o c e s s and d i d  and e n t r a n c e  inhibitors.  coined  Kepes  kinetic  w o r k by  "permease"  studies the  Koch  saturation  Because o f  demonstrated  processes, using later  followed  to  these describe  theienergy(Kepes,  rapid  (1964),  1960)  Millipore cells  preloaded  w i t h TMG to and  a t a low t e m p e r a t u r e ,  c o n t a i n an e x i t  t o a l l o w e x i t , were  p r o c e s s t h a t was f a c i l i t a t e d by e n e r g y  an e n e r g y - i n d e p e n d e n t  s t u d i e s demonstrated constant of exit  t h e n warmed  entrance process.  Further  t h a t e x i t was  thus a l l o w i n g c o n c e n t r a t i o n o f the s u b s t r a t e w i t h i n ( W i n k l e r and W i l s o n ,  the rate  prevented, the c e l l s  1966).  The s p e c i f i c i t y o f t h e v a r i o u s systems g a l a c t o s i d e s have been s t u d i e d Rotman  inhibition,  kinetic  t h a t e n e r g y was u t i l i z e d t o a l t e r  (K^) i n a manner s u c h  shown  fairly  f o r g a l a c t o s e and  extensively.  Ganeson and  (I966) r e v i e w e d t h e e v i d e n c e f o r two TMG p e r m e a s e s i n  E. c o l i ,  as w e l l  as a permease f o r a m e t h y l - g - D - g a l a c t o s e .  permease  I, the o r i g i n a l  permease s t u d i e d  by t h e P a s t e u r  ( R i c k e n b e r g , C o h e n , B u t t i n and Monod, 1956) D - g a l a c t o p y r a n o s i d e s and i t was  TMGgroup  t r a n s p o r t e d a - and 3-  i n d u c e d by compounds c o n t a i n i n g  unsubstituted  galacto-pyranose ring.  by g a l a c t i n o l  a n d t r a n s p o r t e d TMG  The second  s y s t e m was  but not l a c t o s e .  The  an  induced  third  g a l a c t o s i d e permease t r a n s p o r t e d m e t h y l - 3 - D - g a l a c t o p y r a n o s i d e b u t n o t TMG. has  also  A system  f o r t h e t r a n s p o r t o f D - g a l a c t o s e by E_. c o l i  r e c e i v e d much a t t e n t i o n  ( H o r e c k e r , Thomas, a n d Monod,  1960a;  1960b; O s b o r n , M c L e l l a n a n d H o r e c k e r , 1961) and f o u n d t o e x h i b i t kinetics  of exit  ii.  and u p t a k e  very s i m i l a r  t o t h o s e o f t h e TMG  Glucose  In c o n t r a s t t o t h e g a l a c t o s i d e p e r m e a s e s y s t e m , p e r m e a s e f o r g l u c o s e i n E_. c o l i has b e e n d e s c r i b e d w h e r e of  the energy  permease  s u p p l y was shown t o c a u s e  a  removal  an a c c u m u l a t i o n o f a - m e t h y l -  glucoside (otMG), a non-metabolizable analogue of glucose (Hoffee, Englesberg and Lamy, 1964). The glucose transport system in E. c o l i had e a r l i e r been shown, by competition and k i n e t i c studies (Cohen and Monod, 1957; Kessler and Rickenberg, 1963), to accumulate aMG. H a g i h i r a , Wilson and Lin (1963) have shown further that mutants defective in glucose uptake cannot concentrate aMG.  In  competition studies with substituted d e r i v a t i v e s of aMG, the same workers showed the s p e c i f i c i t y of the system depended upon the substituents on C-2, C-3, and C-6 of the aMG. Addition of an exogenous energy source by Hoffee et a l . (1964) resulted in a depression of accumulation.  These r e s u l t s were presented as  evidence for the existence of an energy-requiring e x i t in glucose transport.  reaction  The s p e c i f i c i t y of aMG uptake has also  been studied by these workers and i t was shown that a t e n - f o l d excess in concentration of gMG or glucose competed very strongly with aMG uptake, while maltose allowed 68% of normal accumulation and mannose, deoxy-glucose, and sucrose each allowed 80% of normal activity.  Other carbohydrates had e i t h e r 1 i t t l e e f f e c t or  stimulated aMG uptake.  These r e s u l t s have been confirmed by Halpern  and Lupo (1966). Studies of a galactose-negative s t r a i n of E. c o l i showed that galactose was entering v i a the glucose permeation system (Rogers and Yu, 1962). Although the s t r a i n lacked galactokinase, as much as50% of the galactose was phosphorylated in the p o o l , suggesting an accumulation mechanism based on phosphorylation during transport.  III.  Carbohydrate accumulation mechanisms and components  The study of carbohydrate transport in Gram-positive  organisms  has b e e n  limited  1966).  (Egan and M o r s e ,  functions, while  evidence  indicated with  transport  maltose, was  counterflow  S.  aureus  carbohydrates  1965).  (Egan a n d M o r s e ,  It  protein  transport  Wang a n d M o r s e  (1968)  carrier  mutants  coli  mutants  have  for  E.  the  1967;  Fraenkel  Tanaka,  Attempts transport  found  and A e r o b a c t e r  to  lack  isolate  has  long been  recognized  inhibit  Rothstein .  for  recently  (1967)  transport  carbohydrate  ability  to  mutation  lactose,  carbohydrates  Egan and M o r s e ,  of  1967;  pleiotropic  aerogenes. two  (Tanaka  These protein  and  e_t a j _ . ,  Lin,  1-9'67).  components o f  successful  in yeast  received that  a great  carbohydrate  (Fox and  Kennedy,  the  fungi  transport  carbohydrates It  Cobaltous, Nickelous  and U r a n y l  ions  metabolism  deal  of  attention.  implicated  transport  and  of  Van S t e v e n i n c k  have  carbohydrates.  1966).  carbohydrate  (1-951).;  the  similar  Simoni  relatively  T h e m e c h a n i s m and s p e c i f i c i t y o f by y e a s t  the  either  and c h a r a c t e r i z e  been  and S t e i n ,  Carbohydrate  1967;  and L i n ,  number o f  that with  characterized  to  a  defined  genetic  by a s i n g l e  p h o s p h o t r a n s f e r a s e system  s y s t e m s have  Kolber  Dawson  lost  and  that  (Hengstenberg,  been d e m o n s t r a t e d  components o f  could  for  indicated  also  experiments  experiments  c o u l d be  was  1968).  3.  Staphylococcus aureus  s u c r o s e and aMG, p h o s p h o r y l a t i o n o f  coincident with  1965;  coccus  a common c a r r i e r  a mutant o f  eight  the  Extensive competition  permease  Studies  to  by y e a s t .  (1966)  and  and Van S t e v e n i n c k  p o l y p h o s p h a t e s as  in y e a s t s ,  Hurwitz  the  as N i c k e l o u s  energy ions  has  and source  interfered  with polyphosphate s t r u c t u r e , and i n h i b i t e d transport.  In a d d i t i o n ,  these workers have claimed the existence of an a c t i v e , induced, energy-dependent transport system for galactose, using the same c a r r i e r mechanism as that used in non-induced,  facilitated  d i f f u s ion. The s p e c i f i c i t y of the carbohydrate transport system in yeast has been studied to a considerable extent 1962; C i r i l l o , 1962; C i r i l l o , 1968).  (Scharff and Kraemer,  From competition studies  on the uptake of L-sorbose and D-xylose using 25 d i f f e r e n t carbohydrates, C i r i l l o (1968) has defined the s p e c i f i c s t r u c t u r a l requirements of the c o n s t i t u t i v e yeast monosaccharide transport system.  He demonstrated that the conformation and substituents  of every carbon except C-2 contribute to the carbohydrates' a b i l i t y to compete with the transport of the non-metabolized sugars, sorbose and x y l o s e .  In a d d i t i o n , C i r i l l o (1968)  demonstrated that the sugar must be in the pyranose form and that while removal of the anomeric l-OH has l i t t l e e f f e c t on t r a n s p o r t , s u b s t i t u t i o n at t h i s p o s i t i o n completely destroyed competitive a c t i v i t y .  M u l t i p l e a l t e r a t i o n s at carbon atoms  other than C-2 had an e f f e c t greater than the sum of individual  their  effects.  A study of the mechanism of transport of carbohydrate by yeast c e l l s under anaerobic conditions, by k i n e t i c a n a l y s i s , and a t e n t a t i v e model, based on c a r r i e r mediated d i f f u s i o n , anaerobic carbohydrate transport (Scharff and Kraemer, 1962).  for  in g e n e r a l , has been published  From a comparison of rate constants  for transport and for p u r i f i e d hexokinase, these workers concluded  that hexokinase did not play an important, d i r e c t part in the anaerobic transport of sugars by yeast.  They have suggested that  metabolism plays e s s e n t i a l l y no part in anaerobic sugar  transport.  Further, c a r r i e r f a c i l i t a t e d d i f f u s i o n , being the mechanism of transport  in erythrocytes and anaerobic yeast, is the method of  transport  for a l l c e l l s capable of anaerobic metabolism.  MATERIALS AND METHODS  1. 0 rgah ? sms arid Med i a  C l o s t r id i urn perf r i ngens (BP6K) was used throughout t h i s study.  1. Complex mediurn  The composition of the complex medium was as f o l l o w s : yeast e x t r a c t , 2 gm; proteose peptone, 5 gm; sodium ascorbate, 0.2 gm; and FeSO^.yH^ (0.5% s o l u t i o n ) , 1.0 ml per l i t e r .  These  were dissolved in d i s t i l l e d water, the s o l u t i o n was neutralized with 1N NaOH and 160 ml of 0.5 M phosphate buffer added.  (pH 7-2) were  The volume was adjusted to 1 l i t e r , and the medium was  autoclaved.  S t e r i l e solutions of glucose and MgSO^^r^O were  added to 0.7% and 0.02% r e s p e c t i v e l y .  2. Semi-defined medium  This medium was a modification of that described by Boyd, Logan and T y t e l l  (1948). Stock solutions were prepared as follows  2.0 mg of b i o t i n were dissolved in 100 ml of d i s t i l l e d water, 10 mg of r i b o f l a v i n and 20 mg of Ca-d-pantothenate were dissolved in 100 ml of d i s t i l l e d water, and 50 mg of u r a c i l and 87 mg of  adenine  sulfate  20 mg o f of  of  water,  gm e a c h o f  at  To  FeS0^.7H20, was  replace  the  0.83  gm o f  L-tryptophan  with  0.25  gm o f  by u s i n g  adjusted Five  adenine  ml  and  added t o  medium,  low h e a t  j  All  with  gm o f  acid  used per  of  the  salts ml  5.0  ml o f  the  ml  MgS0^.7H20 100  ml  were  solution, of  the  mixed,  neutralized  to  11.25  diluted  7.2  with  amino a c i d  decanted  15 m i n ,  Before  for  into  then  use,  filter  out  the  distilled  cooling,  one  frozen  12 h r s liter  the  pH  to  solution,  4 C.  Nalgene  thawed,  and d i s p e n s e d i n t o  uracil  and  ml  of  100 ml w i t h . d i s t i 1 l e d  precipitate at  the  pantothenate  1N NaOH.  and s t o r e d a t  medium was  and  a d d e d , and the  D u r i n g a u t o c l a v i n g , a heavy settle  Logan  together  s o l u t i o n , and 0.25  distilled-water.  to  These,  ml o f  riboflavin  pyridoxine  pH a d j u s t e d  1000  Boyd,  h y d r o l y z e d c a s e i n and  After  ml w i t h  Millipore  100  per  d i s s o l v e d in  adjusted  to  the  liter.  were  N^ f o r  10 g m o f  addition,  1N.NaOH.  s o l u t i o n were  the  then  d i s s o l v e d in  amino a c i d s o f  (pH 7 . 2 )  was  In  stock solutions  phosphate b u f f e r  allowed  HG1.  MnSO^H^,  and a g i t a t i o n .  7.2  the  16.7  were  to  2.5  biotin  water  a n r  sodium a s c o r b a t e , were  solution,  solution, the  NaCl  prepared.  individual  (1948)  was  0.2N  4 C.  and T y t e l 1  water  50 ml o f  and a s o l u t i o n c o n t a i n i n g  d i s t i l l e d water,  stored  d i s s o l v e d in  p y r i d o x i n e m o n o h y d r o c h l o r i d e were  distilled  and 0 . 5  were  This mixture 100 ml o f total  formed  0.5  -20  and t h i s  bubbled  was  fluid with  C.  filtered  sterile,  M  v o l u m e was  The s u p e r n a t a n t bottles,  was  through  a 0.3  water-jacketed  u  reaction v e s s e l s .  S t e r i l e solutions of glucose or other substrates  were added a s e p t i c a l l y .  3. Stock cultures  Cultures were grown in the complex medium supplemented with 0.5% acid hydrolyzed c a s e i n , u n t i l an o p t i c a l density (O.D.) at 660 my of 1.2 was reached, then s t e r i 1 e glycerol was added to 15%.  The mixture was dispensed a s e p t i c a l l y in 2.0 ml  amounts into s t e r i l e tubes, frozen and held for as long as several months at -20 C (Pivnick et aj_., 1964). Cultures were routinely checked for p u r i t y by streaking onto blood agar plates which had been previously spread with gas-gangrene antitoxin  (Connaught Medical Research L a b . , Toronto).  Control  p l a t e s , without a n t i t o x i n , were also streaked with the c u l t u r e s . Plates were incubated for 2k hrs at 37 C, under aerobically. and p a r t i a l  and a l s o  The plates were then examined for colony morphology i n h i b i t i o n of haemolysis in the presence of a n t i t o x i n .  k. Growth of inoculum  A stock culture was thawed by slowly warming i t and 0.5 ml was added to a tube of Roger's meat medium with the desired carbohydrate substrate, and incubated at 37 C.  The c u l t u r e was  used as an inoculum when i t was a c t i v e l y gassing (2.5 hrs incubation) .  II.  Growth Conditions  Cultures were usually grown in 100 or 500 ml water-jacketed reaction vessels heated to 37 C by a temperature c o n t r o l l e d c i r c u l a t i n g waterpump.  S t e r i l e nitrogen was passed over the  surface of the medium at 50 ml/min to keep the cultures anaerobic.  Sterile  was bubbled through the medium for 20 min  p r i o r to the a d d i t i o n of a 5% inoculum from an a c t i v e l y gassing meat tube c u l t u r e .  With the semi-defined medium, to ensure a  r a p i d l y growing c u l t u r e with a minimum of carry over from the meat medium, the cultures were grown, with s t i r r i n g , to an O.D. of 1.0 at 660 mu (approximately 3 h r s ) .  A 5% inoculum from t h i s  c u l t u r e was transferred to a second f l a s k of semi-defined medium prepared as the f i r s t , and grown to the desired O.D. at 660 my. On occasion, the inoculated meat tube was held in ice in an i n s u l a t e d , water-jacketed reaction vessel u n t i l a t i m e r - c o n t r o l l e d c i r c u l a t i n g waterpump warmed the reaction vessel to 37 C and i n i t i a t e d a c t i v e growth of the c u l t u r e .  A f t e r 2.5 hrs growth,  a t i m e r - c o n t r o l l e d air-pump a s e p t i c a l l y forced approximately 2 ml of the supernatant f l u i d of the a c t i v e l y gassing meat tube into 100 ml of the semi-defined medium in a water-jacketed reaction v e s s e l .  The medium was maintained under anaerobic  conditions by bubbling s t e r i l e  through i t .  Simultaneous  w i t h . i n o c u l a t i o n , the medium was warmed to 37 C by a timer-  c o n t r o l l e d c i r c u l a t i n g waterpump.  When the growing c u l t u r e reached  an O.D. of 1.0 at 660 mu, i t was used to inoculate a second reaction vessel of the semi-defined medium.  No s i g n i f i c a n t  d i f f e r e n c e was observed between the c e l l s r e s u l t i n g from manual or automatic subculture. During growth of the second culture in the semi-defined medium or the c u l t u r e in complex medium, a l i q u o t s were removed a s e p t i c a l l y and the O.D. at 660 my was measured.  In a d d i t i o n , the pH of  the medium was determined and an a l i q u o t was frozen for the q u a n t i t a t i v e determination of glucose.  III.  Stabi1i ty of Cel1 Suspens ions  Cultures were grown to an O.D. at 660 my of 1.75 in the complex medium and to 0.75 in the semi-defined medium and harvested by c e n t r i f u g a t i o n at room temperature at 5,000 x £ . The c e l l s were washed by resuspending them in 0.85% NaCl (pH 7.2) and c e n t r i f u g i n g again at 5,000 x £ . This washing procedure was repeated and then the c e l l s were resuspended to the desired O.D. at 660 my in 0.05 M tris(hydroxymethyl)amino methane-HCl(tris-HCl) buffer  (pH 7.2) and held under s t e r i l e  N^ in a water-jacketed reaction vessel at 37 C. the suspension was flushed with s t e r i l e  The surface of  at a rate of 50 ml  per min and samples of the c e l l suspension were removed at vari>oustime i n t e r v a l s . A l i q u o t s of the samples were d i l u t e d with t r i s - H C l buffer  (pH 7.2) for the determination of O.D. at 660 my.  Other  a l i q u o t s were centrifuged at k C for 10 min at 10,000 x £ and ultra-violet  (UV) adsorption spectra from 200 to 300 my were  determined on the r e s u l t i n g supernatant f l u i d with a Beckmann model DBG recording spectrophotometer.  IV. Enzyme Assays  1. C e l l - f r e e extracts  Cultures were grown to an O.D. at 660 my of 0.6 in the semidefined medium with 0.7% glucose.  The cultures were then trans-  ferred to a screw-capped 300 ml p l a s t i c centrifuge b o t t l e . surface of the c u l t u r e was flushed with  The  for 5 min and the  b o t t l e was sealed and centrifuged at room temperature at 5,000 x £ for 7 min.  The p e l l e t was resuspended in the following  s o l u t i o n , which had previously been bubbled with  to remove  oxygen: 0.2 M t r i s - H C l (pH 7 . 5 ) , 10~ M MgCl and 6 x 10"^ M 3  cysteine-HC1.  The suspension was t r a n s f e r r e d , with a 5.0 m l ,  l i g h t l y greased, glass syringe f i t t e d with a 3 i n c h #21 needle, _  to a t h i c k - w a l l e d , 16 mm centrifuge tube with a rubber stopper. The tube had previously been made anaerobic by f l u s h i n g i t with N^,, using two #20 needles inserted in the rubber stopper (anaerobic centrifuge tube).  The suspension was centrifuged at  room temperature, at 5,000 x £ for 7 min, the supernatant  fluid  was removed, and the pel l e t was resuspended in the anaerobic buf fer, M g , c y s t e i ne s o l u t i o n . ++  A l i q u o t s were transferred by a  syringe and needle, as described p r e v i o u s l y , to several anaerobic centrifuge tubes.  A f t e r c e n t r i f u g a t i o n for 7 min at 5000 x g_,  the supernatant f l u i d s were removed and the pel 1ets were flushed with N for 5 min. 2  The needles were removed from the stopper  and the tubes were stored at -70 C. C e l l - f r e e extracts were prepared from these c e l l s w i t h i n a maximum time i n t e r v a l of one week.  There was no apparent loss  of enzyme a c t i v i t y during t h i s storage period at -70 C.  As  required, the c e l l p e l l e t s were thawed and resuspended to 50 times the growth concentration (approximately 12 mg dry weight/ml) by the a d d i t i o n of the anaerobic buffer-Mg  -cysteine solution.  The  suspension was subjected to 2.5 min of sonic o s c i l l a t i o n at 88 watts (Bronwill B P - l i , 1/8 i n . d i a . probe, Bronwill Industries, Rochester, N.Y.).  Sonication was performed in anaerobic centrifuge tubes  which were maintained in an ice-water bath.  Nitrogen was  flushed over the surface of the suspensions at 50 ml per min, and sonication was applied for 1/2 min i n t e r v a l s , with a l t e r n a t e 1/2 min i n t e r v a l s for c o o l i n g .  The sonicate was  centrifuged in the anaerobic centrifuge tube for 7 min at 10,000 x g_ and the supernatant anaerobic extract was transferred by means of a 5.0 ml l i g h t l y greased, glass syringe f i t t e d with a 3 inch #21 needle to a second anaerobic centrifuge tube, where i t was kept under  at O X .  A sample of each eel 1-free extract  was frozen and subsequently assayed for protein content.  2.  Enzyme assays  In the following s e r i e s of assays, with the exception of enolase and the p y r u v a t e - c l a s t i c system, the reduction of NADP or NAD, or the o x i d a t i o n of NADH^ were followed spectrophotom e t r i c a l l y by observing the change in O.D. at 3^0 my.  All  assays, except that for the p y r u v a t e - c l a s t i c system, were c a r r i e d out at 37 C. Anaerobic assays were prepared in 2.5 or 1.5 ml t o t a l volumes in 3.0 ml, 1 cm l i g h t - p a t h , quartz cuvettes (Beckmann Instruments  I n c . , F u l l e r t o n , C a l i f o r n i a ) with c i r c u l a r openings  sealed with a rubber serum stopper.  To remove oxygen, the  reaction mixtures were e q u i l i b r a t e d with nitrogen by f l u s h i n g the surface of the solutions with  at the rate of 20 ml per min,  using two #21 hypodermic needles inserted through the rubber serum stoppers.  A l l s o l u t i o n s , which were added a n a e r o b i c a l l y , were  e q u i l i b r a t e d under N^ at 0 C and added through the serum stopper v i a a l i g h t l y greased, 1.0 ml glass syringe f i t t e d with a 3 i n c h #21 needle. _  Aerobic assays were set up in 1.0 or 0.5 ml t o t a l volume systems in quartz cuvettes with 1 cm l i g h t - p a t h of the appropriate s i z e .  Anaerobic c e l l - f r e e extracts were used for  a l l assays in order to l i m i t enzyme degradation by protein oxidation.  The buffer used in a l l assays, except the p y r u v a t e - c l a s t i c system, was t r i s - H C l (pH  7.5).  Control reaction mixtures were prepared for each enzyme assay to ensure that the observed reactions were dependent upon the presence of the various substrates employed.  Enzyme  a c t i v i t i e s were corrected for the control values and expressed 2 as ymoles of substrate u t i l i z e d x 10 /min/mg of p r o t e i n .  a. Hexokinase was assayed in an aerobic system of 1.0 ml t o t a l volume containing: b u f f e r , 80 ymoles; glucose (fructose, mannose or g a l a c t o s e ) , 5 umoles; adenosine .5 triphosphate (ATP)[, k ymoles; NADP, 0.1 ymoles ; and commercial glucose-6-phosphate dehydrogenase, 5-35 yg.  The reaction was  i n i t i a t e d by the addition of the anaerobic e x t r a c t .  b. Glucose-6-phosphate dehydrogenase was assayed in an anaerobic, 2.5 ml system containing: b u f f e r , 2k0 ymoles; glucose-6-phosphate, 1.0 ymoles; and MgCl^, 20 ymoles. A f t e r e q u i l i b r a t i o n at 37 C with  for 7 min,anaerobic extract was  added and the reaction started by the addition of anaerobic NADP or NAD.  c. Phosphohexose isomerase was assayed in an anaerobic system of 1.0 ml t o t a l volume,containing: b u f f e r , 60 ymoles; fructose-6-phosphate,  5 ymoles; glucose-6-phosphate dehydrogenase  0.3 y g ; NADP, 0.125 ymoles; and anaerobic e x t r a c t .  d. Phospho-fructokinase was measured in a 2.5 ml t o t a l volume under anaerobic conditions c o n t a i n i n g : b u f f e r , 200 ymoles; ATP, 10 ymoles; a - g l y c e r o l phosphate dehydrogenase, 20 y g ; a l d o l a s e , 20 yg; M g C l , 30 ymoles; and NAD.H , 0.25 ymoles. 2  After  equilibrat-  ion at room temperature under N for 7 min, the reaction was 2  started by the addition of anaerobic e x t r a c t .  e. Fructose-1,6-diphosphate aldolase was assayed by a modified procedure of Groves e_t aj_. (1966) in an anaerobic system of 2.5 ml t o t a l volume containing: b u f f e r , 120 ymoles; potassium a c e t a t e , 240 ymoles; cobalt c h l o r i d e , 1.68 ymoles; c y s t e i n e - H C l , 0.24 ymoles; a-glycerophosphate dehydrogenase, 20 yg; and t r i o s e phosphate isomerase, 20 yg. under  After equilibration  for 7 min at 37 C,  anaerobic extract and 0.25 ymoles of NADH^ were added  and the reaction was started by the addition of 10 ymoles of fructose-1,6-phosphate.  f. Triose-phosphate isomerase was measured in an anaerobic system of 2.5 ml t o t a l volume c o n t a i n i n g : b u f f e r , 240 ymoles; iodoacetamide, 10 ymoles; and 20 yg of a - g l y c e r o l phosphate dehydrogenase. under  The mixture was e q u i l i b r a t e d at room temperature  for 7 min, then anaerobic extract and 0.25 ymoles of  NADH^ were added.  The reaction was started by the addition of  7.5 ymoles of anaerobic 3~phospho-glyceraldehyde.  g. Three-phosphoglyceraldehyde dehydrogenase was measured by a modification of Krebs' (1955) procedure in an anaerobic, 2.5 ml system containing: b u f f e r , 240 ymoles; c y s t e i n e - H C l , 1.5 ymoles; sodium arsenate, 30 ymoles; sodium f l u o r i d e , 30 ymoles; and 3~phosphoglyceraldehyde, 7.5 ymoles. A f t e r e q u i l i b r a t i o n at room temperature for 7 min with N^, anaerobic extract was added and the reaction mixture  equilibrated  under  for a f u r t h e r 5 min at room temperature and 2 min at  37 C.  The reaction was i n i t i a t e d by the addition of 0.25 ymoles  of NAD.  In a p a r a l l e l experiment, 10 ymoles of  iodoacetamide  was added to the reaction mixture p r i o r to the f i r s t e q u i l i b r a t i o n with N  2  (Krebs, 1955).  h. Three-phospho-glyceric acid kinase was measured in a 1.5 ml t o t a l volume system under anaerobic conditions. reaction mixture contained: b u f f e r , 60 ymoles;  The  3-phosphoglycer-  aldehyde dehydrogenase, 50 yg; ATP, 1.5 ymoles; M g C ^ , 12.5 ymoles; cysteine-HCl , 9 ymoles; and 7.5 ymoles of 3 phospho-  glycerate.  A f t e r e q u i l i b r a t i o n at room temperature  under  for 7 min, 0.15 ymoles of anaerobic NADH^ were added and the assay started by the addition of the anaerobic e x t r a c t .  i . Mutase.  The a c t i v i t y of t h i s enzyme was assayed in  an anaerobic system with 1.5 ml t o t a l volume containing:  b u f f e r , 60 ymoles; MgCl^, 15 ymoles; ATP, 0.75 ymoles; 2,3  _  diphosphoglyceric a c i d , 0.625 ymoles; 2-phosphoglyceric a c i d , 7.5 ymoles; c y s t e i n e - H C l , 9 ymoles; 3-phosphoglyceraldehyde dehydrogenase, 20 yg; and 3-phosphoglycerate kinase, 20 yg. A f t e r 7 min under  at room temperature, 0.15 ymoles of anaerobic  NADH^ were added, and the reaction i n i t i a t e d by the addition of anaerobic e x t r a c t .  j.  Pyruvate kinase was assayed in an anaerobic 2.5 ml  system by a modification of the procedure of BUcher and P f l e i d e r e r (1955).  The reaction mixture contained the f o l l o w i n g :  b u f f e r , 100 ymoles; KC1 , 187 ymoles; M g C ^ , 20 ymoles; l a c t i c dehydrogenase, 1 yg; and ADP, 0.125 ymoles.  After  equilibrating  with N2 for 7 min at room temperature, anaerobic extract and 0.5 ymoles of NADH were added and the reaction started by the 2  addition of 2.5  moles of anaerobic phosphoenol pyruvate.  k. L a c t i c dehydrogenase was assayed in a 1.5 ml system under U^, that contained the f o l l o w i n g : b u f f e r , 40 ymoles; NADh^, 0.15 ymoles; and sodium pyruvate, 5 ymoles. A f t e r 7 min at room temperature, the reaction was started by the a d d i t i o n of anaerobic e x t r a c t .  1.  NADH2  oxidase was measured under both aerobic and  anaerobic conditions.  28  (i) The aerobic a c t i v i t y of NADH^ oxidase was measured in a 2.5 ml system containing 30 ymoles of buffer and anaerobic extract.  The reaction mixture was mixed in a i r and warmed to  37 C, a f t e r which the reaction was started by the addition of 0.45 ymoles of aerobic NADH^.  (ii)  The a c t i v i t y of the oxidase was measured in the  absence of molecular oxygen by using a 2.5 ml system containing 250 ymoles of buffer e q u i l i b r a t e d under  for 7 min at 37 C.  Anaerobe extract was added, and the reaction was started by the addition of 0.45 ymoles of anaerobic NADH . 2  m. Enolase was measured by following the change in O.D. at 230 my, according to the method of Westhead (1966).  An  aerobic system of 0.5 ml t o t a l volume was used and i t contained the f o l l o w i n g : b u f f e r , 20 ymoles; MgCl^, 1 ymole; and 2-phospho.1  g l y c e r a t e , 2.5 ymoles.  1 .  A f t e r warming to 37 C, the assay was  i n i t i a t e d by the addition of d i l u t e d anaerobic e x t r a c t . n. P y r u v a t e - c l a s t i c system.  This enzyme system was assayed  by a modification of the method of Lovenberg e_t a_l_. (I963), which was based on the assay of Lipmann and T u t t l e (19^5) for acetyl phosphate.  A 10 ml reaction mixture containing the f o l l o w i n g :  phosphate buffer  (pH 6 . 5 ) , 250 ymoles; sodium pyruvate, 100 ymoles;  and coenzyme A, 0.35 mg; was bubbled with temperature, and then for 2 min at 30 C.  for 5 min at room The reaction was  started by the a d d i t i o n of the anaerobic extract and  was  flushed over the surface of the reaction mixture during the experiment.  One ml samples were removed at various time i n t e r v a l s  and added to a mixture of 100 ymoles of acetate buffer  (pH 5.4)  and-14 mg of neutralized hydroxy lamine-HC1 in a t o t a l volume of 2.0 ml. A f t e r mixing, the suspension was allowed to stand at room temperature f o r 10 min, a f t e r which 1.0 ml of 3N HCI, 1.0 ml of 10% t r i c h l o r o a c e t i c acid (TCA) and 1.0 ml of 5% FeCl^.SH^O in 0.1N HCI were added with vigorous mixing. The mixtures were centrifuged f o r 5 min at 12,000 x £ and the supernatant f l u i d was examined for O.D. at 540 my. A molar e x t i n c t i o n c o e f f i c i e n t was calculated from a standard curve obtained by measuring the O.D. at 540 my of solutions of the complex of f e r r i c ions and the hydroxamate of s u c c i n i c anhydride (Lipmann and T u t t l e , 1945).  V.  Carbohydrate Transport  .• 1.'; Preparation :of anaerobic c e l l suspensions  Cultures were grown in the semi-defined medium with 0.6% sodiurn pyruvate and 0.1% glucose unless otherwise i n d i c a t e d . The c e l l s were harvested and washed twice with semi-defined medium plus 0.6% pyruvate under N^, as described f o r the preparation of eel 1-free e x t r a c t s .  A f t e r the f i n a l washing, the  c e l l s were resuspended to an O.D. at 660 my of 0.28 (0.078 mg dry weight/ml)  in the semi-defined medium plus 0.6% sodium pyruvate,  held under N and at 18 C in a water-jacketed reaction v e s s e l . 2  The O.D. at 660 my was monitored during the course of the experiment to ensure that the c e l l density remained constant.  2. Assays for  14 C-carbohydrate incorporation  A small reaction vessel c o n s i s t i n g of a shortened 18 mm test-tube was f i t t e d with a rubber stopper, a #18 hypodermic needle for  i n l e t , and an open port for sampling, a d d i t i o n of  substrates and c e l l s , and  exit.  The reaction vessel contained  a small magnetic s t i r r i n g bar driven by an underwater magneticstirrer  (Bronwill  Industries, Rochester, N . Y . ) , was flushed  with  at 50 ml per min, and was warmed to 30 C in a water-  bath. 14 C - l a b e l l e d carbohydrate substrates were prepared in 1.0 ml volumes of the semi-defined medium and placed in a small tube which was f i t t e d with a rubber stopper, with a needle and an open port.  inlet  The substrate s o l u t i o n was held in  t h i s tube on i c e , under nitrogen, for 5 min and then warmed to 30 C for 3 min.  Four ml of the anaerobic c e l l suspension was  transferred with a 5.0 ml syringe f i t t e d with a 3-inch #21 hypodermic needle, to the anaerobic, 30 C reaction v e s s e l .  The  c e l l suspension was warmed to 30 C, s t i r r e d for 3 min and the reaction was started by the a d d i t i o n of the 30 C, anaerobic substrate s o l u t i o n , using a 1.0 ml syringe f i t t e d with a 3 i n c h -  #21 needle.  At time i n t e r v a l s , 1.0 ml a l i q u o t s of the reaction  mixture were removed with a s i m i l a r 1.0 ml syringe and needle.  The samples were analyzed for incorporation of  C-  l a b e l l e d carbohydrates into the whole eel Is by the method of B r i t t e n and McClure (1962).  C e l l s were fi1tered onto a  Tracer lab E8B p r e c i p i t a t i o n apparatus (Tracer l a b , Waltham, M a s s . ) , containing a 0.45 y M i l l i p o r e f i l t e r , and q u i c k l y washed with 2.0 ml of semi-defined medium containing 0.6% sodium pyruvate. Samples were a 1 so added to test-tubes containing equal volumes of i c e - c o l d 5% TCA, held at 0 C for 15 min and f i l t e r e d on the p r e c i p i t a t i o n apparatus containing a 0.45 y M i l l i p o r e as above.  The tubes were rinsed twice with i c e - c o l d 5% TCA and  the rinse solutions were added to the f i 1 t e r ( B r i t t e n and McClure, 1962). of the  This procedure was used to determine the 14  incorporation  C-label into the cold TCA insoluble proteins and nucleic  acids (Roberts et_ a)_.,  1955).  To study the competitive i n h i b i t i o n of carbohydrate t r a n s p o r t , 12 a 100-fold excess of the  C possible competitor was added to the  14 C - l a b e l l e d carbohydrate s o l u t i o n and the rate of t o t a l 14 incorporation of C into the whole eel Is was compared to 14 12 the rate of incorporation of C in the absence of the Ccarbohydrate. 14 In cases where the  C-carbohydrate did not accumulate in  t h e . c e l l s against a concentration gradient, extreme caution in f i I t e r i n g was necessary in order to prevent the retention of high levels of background r a d i o a c t i v i t y  in the fi1tered samples,  which would obscure the r e l a t i v e l y smal1 changes in  radioactivity  b e i n g measured.  The M i l l i p o r e  f i l t e r s were pre-washed w i t h  2.0 ml o f s e m i - d e f i n e d medium c o n t a i n i n g t h e  12  C-substrate at  14 t h e same c o n c e n t r a t i o n a s t h e r e a c t i o n m i x t u r e t o reduce The in  danger o f r e t a i n i n g  of  s p e c i f i c adsorption to the f i l t e r .  cells  suction  3 t i m e s w i t h 1.0 ml v o l u m e s  i n s t e a d o f o n c e w i t h 2.0 m l , a s i n n o r m a l  incorporation experiments. on  mixture  by t h e u s e o f h i g h e r t h a n n o r m a l  the f i l t e r e d  t h e wash s o l u t i o n ,  substrate i n the  s m a l l volumes o f t h e r e a c t i o n  t h e f i l t e r was r e d u c e d  r a t e s , and washing  C-labelled  C a r e was t a k e n  the p r e c i p i t a t i o n apparatus  f o r equal  t o keep t h e f i l t e r s  lengths of time  t h e y w e r e removed f o r d r y i n g a n d c o u n t i n g .  before  Control values f o r  non-specific retention of label  by t h e f i l t e r s w e r e d e t e r m i n e d  performing  for total  parallel  experiments  incorporation  5.0 ml o f s e m i - d e f i n e d medium c o n t a i n i n g 0.6% s o d i u m r a d i o a c t i v e s u b s t r a t e , a n d no c e l l c a r e f u l l y washed and d r i e d  suspension.  by  using  pyruvate,  The f i l t e r s  were  as above, and t h e v a l u e s t h a t were  o b t a i n e d were s u b t r a c t e d from  the values f o r total  incorporation  into eelIs. 14 C-labelled  c a r b o h y d r a t e s were added a t t h e v a r i o u s c o n c e n t r a t -  i o n s and s p e c i f i c a c t i v i t i e s  indicated  i n t h e R e s u l t s and  Discussion. 14 3. D e t e r m i n a t i o n o f t h e n a t u r e o f t h e a c c u m u l a t e d  C-  carbohydrate A r e a c t i o n m i x t u r e was p r e p a r e d  as d e s c r i b e d f o r t h e study  of t o t a l incorporation of  C into c e l l s .  The e n t i r e  reaction  mixture was removed with a 5.0 ml syringe f i t t e d with a #21 hypodermic needle at 0.5 min a f t e r addition of the substrate.  C-labelled  The mixture was injected onto a 0.45 y M i l l i p o r e  f i l t e r , and washed once with 2.0 ml of semi-defined medium containing 0.6% sodium pyruvate.  The f i l t r a t e from the reaction  mixture was recovered and held at 0 C for a n a l y s i s .  The f i l t e r  was q u i c k l y removed and immersed in a beaker containing 3.0 ml of i c e - c o l d 5% TCA. The f i l t e r and TCA s o l u t i o n were placed in an 18 mm t e s t - t u b e , the beaker was rinsed with 2.0 ml of i c e - c o l d 5% TCA, and the rinse s o l u t i o n was added to the t e s t tube.  A f t e r vigorous a g i t a t i o n of the f i l t e r in the TCA s o l u t i o n ,  the s o l u t i o n was removed and the f i l t e r was further extracted with two washings of 2.0 ml of i c e - c o l d 5% TCA, which were added to the o r i g i n a l s o l u t i o n .  The combined solutions were c e n t r i -  fuged at 12,000 x £ for 15 min at 4 C, to remove the p r e c i p i t a t e d material and the supernatant f l u i d was extracted four times with equal volumes of cold ethyl ether to remove the TCA. The 14 remaining aqueous s o l u t i o n , containing the pooled  C-carbohydrate,  was concentrated to a small volume by warming to 30 C in a f l a s h evaporator (Laboratory Glass and Instruments C o r p . , New York, N.Y.) with the condenser cooled in an ice-water bath.  The  concentrated s o l u t i o n was subjected to a n a l y s i s by electrophoresis and paper chromatography.  An a l i q u o t of the material was  fractionated by preparative paper electrophoresis and the separated f r a c t i o n s were eluted with d i s t i l l e d water and  de-phosphorylated with b a c t e r i a l a l k a l i n e phosphatase.  The  de-phosphorylated preparations were concentrated and subjected to analyses by paper electrophoresis and paper chromatography. The f i l t r a t e of the o r i g i n a l  reaction mixture was assayed  for t o t a l r a d i o a c t i v i t y and then concentrated to a small volume and the nature of the radioactive compounds was determined by the same procedures as used for the accumulated radioactive compounds. The recovery of the r a d i o a c t i v i t y  in a l l of the samples was  monitored during the preparative and a n a l y t i c a l procedures, and n e g l i g i b l e losses occurred.  V I . Assay of R a d i o a c t i V i t y  1.  Millipore fiIters  Dried M i l l i p o r e f i l t e r s  were placed in the v i a l s containing  5 ml of s c i n t i 1 l a t i o n f l u i d ( L i q u i f l u o r , New England Nuclear Corporation) and the v i a l s were assayed for r a d i o a c t i v i t y  in  a Nuclear Chicago 1iquid s c i n t i 1 l a t i o n spectrometer model 725.  2.  Chromatograms and electrophoretograms  Radioactive chromatograms were scanned by running 1-inch s t r i p s through a Nuclear Chicago model C 100 B Actigraph II with a gas flow counter and a model 1620 B A n a l y t i c a l Count ratemeter equipped with a chart recorder.  The r a d i o a c t i v i t y of compounds from electrophoretograms and chromatograms were q u a n t i t a t i v e l y determined by c u t t i n g out the areas where label had been detected, drying under an i n f r a red lamp, and counting in the 1iquid s c i n t i 1 l a t i o n counter.  V I I . Chromatography and Electrophores?s  Paper chromatography was routinely performed on substrate solutions and extracted pool materials by spotting samples on Whatman No. k paper, and running them by the descending technique with the solvent system of Grado and Ballou (1961) as f o l l o w s : e t h y l - a c e t a t e / p y r i d i n e / s a t u r a t e d aqueous b o r i c acid (60/25/20). Electrophoresis was c a r r i e d out with a water-cooled apparatus s i m i l a r to a Resco model E-800-2B equipped with a Resco model 1911 power supply.  The buffer system routinely employed was  0.1 M ammonium carbonate (NH^HCO^.NH^COONH^-Analar) (pH 8.6) and samples were spotted onto e i t h e r Whatman No. k paper or Whatman No. 3 paper for preparative e l e c t r o p h o r e s i s .  The  maximum voltage of 750 v o l t s was applied for 1.5 to 2.0 h r s . A f t e r drying chromatograms overnight at kO C and e l e c t r o phoretograms for 20 min at 100 C, they were developed in the following manner.  Carbohydrates were detected by dipping  chromatograms in 0.12% m-periodate in acetone and spraying with 0.18% benzidine in acetone ( C i f o n e l l i , 195*0. Reducing sugars were detected by dipping chromatograms in 0.5% AgNO^ in acetone and developing in 0.5N NaOH in 70% N a ^ O ^ S H ^  (Smith, I960).  36  VIII.  Ahalytical  1.  Dry  arid P r e p a r a t i v e  weight  The dry w e i g h t centrifuging  of  cell  pellet  was  resuspended  in d i s t i l l e d  water  in o n e - t e n t h  water.  s u s p e n s i o n was  placed  the  original  temperature.  final  This  s a m p l e s was  of  at  placed  under  p r o c e d u r e was  pellet  i_n  concentrated  pre-weighed  95 C f o r  The  volume,  this  by  resuspending  re-centrifuging.  aluminum  2.5 h r s ,  room t e m p e r a t u r e  and a g a i n  for  then  2k h r s .  vacuum a t  repeated  until  dish.  placed  The  room the  weight  constant.  Protein  Protein method o f  content  Lowry  egg a l b u m i n  3.  of  dried  pans were w e i g h e d ,  2.  and  determined  suspension,  in a p r e - d r i e d ,  i n a vacuum d e s s i c a t o r a t  the  the  A measured a l i q u o t  T h e d i s h and s a m p l e w e r e  of  s u s p e n s i o n s was  a known v o l u m e o f  the  distilled  Techniques  five  Optical  Optical  of  s a m p l e s was d e t e r m i n e d  e_t aj_. . ( 1 9 5 1 ) . times  density  densities  The p r o t e i n  according to  standard  the  u s e d was  recrystal1ized.  measurements  in  the  visible  range were d e t e r m i n e d  using  a Beckman model B spectrophotometer. d e n s i t i e s in the u l t r a - v i o l e t  Values for o p t i c a l  range were taken from spectra  obtained using a Spectronic 505 or Spectronic 600 spectrophotometer (Bausch and Lomb).  To record the change in O.D. with time,  for enzyme assays, a G i l f o r d recording spectrophotometer, model 2000, was used.  4. Dephosphorylation of carbohydrates  The removal of phosphate from phosphorylated intermediates was c a r r i e d out by incubation with a commercial b a c t e r i a l a l k a l i n e phosphatase in 0.1 M t r i s - H C l buffer  (pH 8.0) at 37 C.  5. Glucose  Samples were analyzed for glucose content with the enzymatic Glucostat procedure (Worthington Biochemical C o r p . , Freehold, N.J.).  IX. Chemicals, Enzymes and Substrates  A l l chemicals, enzymes and substrates were purchased from commercial sources. with NaOH.  Where necessary the substrates were neutralized  Barium was removed from substrates by treatment with  Dowex-50 H and the r e s u l t i n g free acids were neutralized with +  NaOH.  C-U-maltose, 1-lactose and  C-U-D-ribose,  C-1-D-galactose,  C-  14 C-U-aMG were obtained from the Nuclear 14  Chicago Corp. (Des P l a i n e s , 111.); Me-  C-thio-D-galactoside  14 and  C-lhglucose were purchased from Schwarz Bioresearch Corp.  (Orangeburg, N . Y . ) . A l l l a b e l l e d and unlabelled carbohydrates were found to be chromatographically homogeneous by the ethyl saturated aqueous boric acid solvent system.  acetate/pyridine/  RESULTS AND DISCUSSION.  I. Growth and Resting Cell Suspensions  The tendency of Gram-positive organisms, both anaerobic and f a c u l t a t i v e l y anaerobic, to undergo a u t o l y s i s has long been recognized (Jones, Stacey, and Webb, 1 9 4 9 ) .  This rapid  a u t o l y s i s occurs in both stationary phase cultures and resting c e l l suspensions and has prevented extensive metabolic studies from being c a r r i e d out with C_. perfr?ngens and s i m i l a r microorganisms.  Jones, Stacey, and Webb ( 1 9 4 9 )  defined the system of  a u t o l y t i c enzymes in C. p e r f r i hgens and in Staphylococcus c i t r e u s , as  c o n s i s t i n g of a ribonuclease and two p r o t e o l y t i c enzymes. It has been noted t h a t , when in a magnesium-deficient,  enriched medium, C_. perfringens formed filamentous c e l l s which were r e s i s t a n t to a u t o l y s i s (Webb, 1 9 4 8 ) . (1948)  Boyd, Logan, and T y t e l l  demonstrated that the production of toxins by C_. perf r i ngens  could be repressed, by growing the organism in a defined medium. It was assumed that possibly a c o r r e l a t i o n existed between the production of exotoxins and a u t o l y t i c enzymes.  A modification of  Boyd, Logan, and T y t e l l ' s medium was employed and the s t a b i l i t y c e l l s grown in t h i s semi-defined medium was compared with the s t a b i l i t y of c e l l s grown in the complex medium.  To prevent  of  loss by p r e c i p i t a t i o n , magnesium was added to the complex medium a f t e r autoclav? ng.  1. Growth curves  Cultures grown in the enriched and in the semi-defined media, with glucose as the energy source, produced somewhat d i f f e r e n t growth curves. c e l l density  A f t e r a considerable l a g , rapid growth to a high  (O.D.^Q,  approximately 4.5) was evident in the  enriched medium ( F i g . 1).  The hydrogen ion concentration rose  very rapidly during logarithmic growth ( F i g . 1) and, as there was excess glucose present during the stationary phase, low pH was most l i k e l y the factor which limited growth.  C a l c u l a t i o n s from a  logarithmic plot of the c e l l density (optical density at 660- my) versus time indicated that the generation time was approximately 48 min ( F i g . 2 ) . Microscopic observations of Gram-stained preparations of the c e l l s from the complex medium demonstrated the t y p i c a l square-ended, r e l a t i v e l y short t h i c k , Gram-positive b a c i l l i , : described by other workers (Breed, Murray, and Smith, 1957).  During log phase growth,  the cultures produced considerable q u a n t i t i e s of gas and gave o f f the rancid odor c h a r a c t e r i s t i c of a mixture of a c e t i c and b u t y r i c acids. Neither high c e l l d e n s i t y , high acid concentration, nor low glucose concentration limited growth in the semi-defined medium  41  Fig.  1.  G r o w t h . o f C. p e r f r i n g e n s i n c o m p l e x medium 0.7% g l u c o s e . o o, O.D. a t 660m u ; A —A, pH.  plus  5.0  HOURS Fig.  2.  G r o w t h . o f C. p e r f r i n g e n s i n c o m p l e x medium p l u s 0.7% g l u c o s e . L o g a r i t h m i c p l o t o f O.D.,, v e r s u s time.  ( F i g . 3).  Rather, depletion of some required growth factor from  the medium was more l i k e l y .  When compared with the 2-3 hrs lag  in growth, routinely observed in the complex medium, the lag in both the primary and secondary culture in the semi-defined medium was n e g l i g i b l e .  The generation time of the secondary culture  in  the semi-defined medium was Ml min, which was;, s i g n i f i c a n t l y shorter than the 51 min generation time of the primary  culture  ( F i g . k). Microscopic observation of logarithmic phase c e l l s from the second c u l t u r e in semi-defined medium revealed d r a s t i c a l l y altered c e l l u l a r morphology, although the Gram-reaction of the c e l l s was s t i l l  positive.  The majority of the c e l l s were filamentous  and much narrower than the c e l l s from the complex medium. lengths of the filaments varied considerably.  The  In a d d i t i o n ,  the gas produced by cultures grown in the semi-defined medium was greatly reduced, and the rancid odor was not detectable.  2. S t a b i l i t y of c e l l suspensions  Resting c e l l suspensions prepared from c e l l s grown in the semi-defined medium, and suspended in 0.05 M t r i s - H C l (pH 7.4) _o  and 10  _|__|_  M Mg  were stable to a u t o l y s i s as determined by measuring  the O.D. of the suspensions at 660 mu.  C e l l s which had been grown  in the enriched medium lysed r a p i d l y and a 60% decrease in O.D. 660 occurred during the course of a 3 hrs experiment ( F i g . 5 ) . In preliminary experiments, harvesting and resuspending of the  kk  0 Fig. 3 .  2  4 HOURS  6  8  Growth.of £ . perfringens in secondary c u l t u r e in semidefined medium.plus 0 . 7 % glucose, o o, O.D.^^ ; A- —A, pH; Q '•—Q , glucose concentration. Q  k. ' G r o w t h  o f C.~:perfringens  0.7%glucose. o-  o,  i n semi-defined  Logarithmic  primary  culture;  plot A  -  medium  o f O.D.g,  0  plus  versus, time,  - A ,secondary  culture.  c e l l s from the complex medium were c a r r i e d out under various conditions of anaerobiosis, a g i t a t i o n , and temperature.  In  a d d i t i o n , the c e l l s were resuspended in various concentrations of phosphate and t r i s - H C l b u f f e r s , as well as in 0.05 (pH 7.4). with 10 sucrose.  -2  M tris-HCl  M M g , 5% mannitol, 0.5% s o r b i t o l , or 3.5% + +  The rate of c e l l l y s i s was not affected by any of these  attempts to produce stable c e l l suspensions.  Suspensions of  these c e l l s in 0.05 M t r i s - H C l (pH 7.**) containing 5% galactose or 0.05% glucose remained r e l a t i v e l y s t a b l e , however the pH of the medium decreased in both cases, demonstrating that galactose and glucose were being metabolized.  In the absence of galactose or  glucose, c e l l l y s i s commenced immediately, and t h i s indicated that the a c t i v i t y of the a u t o l y t i c enzymes were, e s s e n t i a l l y , completely repressed by the presence of an adequate energy source. The concentration of casein hydrolysate in the semi-defined medium was the l i m i t i n g factor of growth, i f the primary  culture  of the semi-defined medium was allowed to reach the stationary phase. A tendency to form carbonaceous storage products has been observed in other organisms (Dawes and Ribbons, 1962; Doudoroff and S t a n i e r , 1959), when grown under conditions of carbon excess and nitrogen limitation.  No d i r e c t evidence of such a product has been demonstrated  in C. p e r f r i hgens.  U t i l i z a t i o n of a carbonaceous storage product  could have served the same purpose in repressing the a c t i v i t y  of  a u t o l y t i c enzymes as the addition of low l e v e l s of glucose did in suspensions of c e l l s grown on complex medium.  2  3  F i g . 5. .Stabi1j ty of eel 1 suspens ions of C_." perfringens grown wi th 0.7% gl ucose i n : o o, complex medium; ©———©, semi-defined medium.  As the c e l l s in the suspensions lysed and released t h e i r i n t r a c e l l u l a r m a t e r i a l , the u l t r a v i o l e t absorption of the suspending f l u i d increased s i g n i f i c a n t l y .  The O.D. at 260 my  in the supernatant f l u i d s , increased by a factor of 4 . 6 8 , over the zero-hour value with the c e l l s grown in the complex medium (Table I ) , and by a f a c t o r of 4.5 for the c e l l s grown in the semidefined medium during a 3 hrs period (Table I I ) .  However, the zero-  time r a t i o s of the concentration of u l t r a v i o l e t absorbing material (O.D. at 260 my) to the c e l l d e n s i t i e s (O.D. at 660 my) were found to be 0.0652 for the c e l l s grown on semi-defined medium (Table  I),  and 0.314 for the suspensions of c e l l s from the complex medium. This high i n i t i a l concentration of u l t r a v i o l e t absorbing material in the suspensions of c e l l s from the complex medium demonstrated that a s i g n i f i c a n t amount of l y s i s had occurred during the washing and resuspension procedures.  This tendency to lyse immediately  upon resuspension indicated that the c e l l s were induced to synthesize the a u t o l y t i c enzymes during growth on the complex medium. The f a c t that the suspension of c e l l s grown in the semi-defined medium did not have t h i s high i n i t i a l O . D . ^ ^ Q suggested that the synthesis of the a u t o l y t i c enzymes had been repressed, by some f a c t o r , during growth in t h i s medium. The i n i t i a l concentration of the c e l l suspensions could not have conferred s t a b i l i t y on the c e l l s grown in the semi-defined medium  49  Table  I.  The a u t o l y s i s of under s t a r v a t i o n  c e l l suspensions of conditions.  OPTICAL  INCUBATION  C.  p e r f r i hgens  DENSITY  TIME (HR)  660 my cell  suspension  260 my supernatant  5.10  1.60  1  5.10  2.60  1  4 ..85  5.40  2  3.85  5.70  3  2.30  7.50  0.45  4.68  3 hr/0  hr  fluid  Table II.  The s t a b i l i t y of eel 1 suspensions of C. perfringens under starvation conditions when previously grown in a chemically defined medium.  STARVATION MEDIUM  3  NO ADDITION  1 0  INCUBATION TIME  ~  3  M MgCl  10~ M MgCl 2  2  2  OPTICAL DENSITY  660 my  b  260 my  C  660 my  260 my  660 my  220 my  0  2.91  0.19.  2.70  0.20  2.76  0.16  1  2.85  0.82  2.79  0.59  2.80  0.49  2  2.67  1.80  2.87  0.71  2.90  0.66  3  2.53  2.70  2.88  0.90  2.90  0.80  6  2.28  4.20  2.96  1.40  2.90  0.95  18  2.16  9.10  3.34  3.05  3.12  1.95  3 hr/0 hr  0.87  14.2  1,07  4.50  1.05  5.0  18 hr/0 hr  0.74  83.5  1.24  1.13  12.2  a  in T r i s  - 0.05  buffer (pH 7 . 4 ) ;  ^ - O.D.ggg.of c e l l suspension; - O.D gQ of supernatant <2  fluid.  15.2  as the rate of decrease in O . D . ^ Q , with time, for c e l l suspensions grown on complex medium, increased l i n e a r l y with increasing i n i t i a l c e l l density ( O . D . ^ )  ( F i g . 6).  One of the major differences between the complex and the semidefined media was the s u b s t i t u t i o n of a c i d - c a s e i n hydrolysate, for the proteose peptone as the source of amino a c i d s .  It has  been demonstrated that exotoxins were not produced when the C_. perfri hgens was grown on the semi-defined medium (Boyd, Logan, and T y t e l l , 1948) and the importance of peptides f o r toxinogenesis by C. perfrihgens type D has been demonstrated (HauschiId, 1965)If a c o r r e l a t i o n existed between the production of the exotoxins, e x t r a c e l l u l a r p r o t e o l y t i c enzymes, and a u t o l y t i c enzymes (Jones, Stacey, and Webb, 1949), a l l three classes of enzymes may have been coordinately induced by the presence of the peptides in the complex medium.  The absence of these substrates of p r o t e o l y t i c  enzymes from the semi-defined medium, could have repressed the synthesis of the three classes of enzymes, and thus produced stable eel 1 suspensions. Attempts were made to improve the s t a b i 1 i t y of suspensions of c e l l s grown in the semi-defined medium by the addition of various concentrations of Mg the c e l l s .  to the buffer used to wash and resuspend  S i g n i f i c a n t changes in the rate of decrease in O . D . ^ ^ Q  and increase in 0- ' 60 u  w e r e  2  observed (Table I I ) .  SIight a u t o l y s i s  of the suspension in 0.05 M t r i s - H C l (pH 7.2).was demonstrated with  I of the i n i t i a l  O.D.^Q  remaining,after 3 h r s .  During the same  6.  The rate of l y s i s of suspensions of eel Is grown in complex medium, as a function of the i n i t i a l c e l l concentration of the suspensions.  p e r i o d , the O . D . ^ g was increased to 14.2 times the -2 value.  The addition of 10  initial  ++ M Mg  to the r e s t i n g suspension  resulted in 105% of the i n i t i a l O.D.^ at the end of 3 h r s , Q  with only a f i v e - f o l d  increase in 260 my absorbing m a t e r i a l .  -3  The a d d i t i o n of 10  ++  M Mg  gave r e s u l t s nearly i d e n t i c a l  those found with the addition of 10  M Mg  (Table  to  II).  ,j |  The addition of the Mg  ions may have served to s t a b i l i z e  the eel 1 suspensions in e i t h e r of two ways, for which no evidence is presented.  The existence of low l e v e l s of  autolytic  enzymes in the suspensions of c e l l s grown in the semi-defined medium was evident from the slow, but d e f i n i t e , rate of l y s i s of the.eel 1 .suspensions.' Magnesium ions, in the concentrations used, may have d i r e c t l y i n h i b i t e d the a c t i v i t y of the a u t o l y t i c enzymes.  However, M g  ++  is a l s o known to s t a b i l i z e the structure of ribosomes in v i t r o and in vivo (McCarthy, 1962; T i s s i e r e s e_t aj_., 1959)  and therefore the  magnesium may have prevented the d i s s o c i a t i o n of the ribosomes and the release of r i b o n u c l e i c acid and proteins which would have served as substrates for the ribonuclease and p r o t e o l y t i c enzymes which have been demonstrated to be involved in a u t o l y s i s (Jones, Stacey, and Webb, 19^9). The u l t r a v i o l e t  absorbing material released by the stable  suspensions of C. p e r f r i hgens (Table II)  may have been the  degradation products of the endogenous metabolism of the organism,  as has been observed in other microorganisms (Campbell, Gronlund, and Duncan, 1963; Strange e_ta_l_., 1963). The gradual increase in c e l l numbers (O.D.ggg) '  n  the stable suspensions ( F i g . 2)  may have resulted from t h i s u t i l i z a t i o n of an endogenous storage product. The lower concentration of Mg , 10  M_, conferred adequate  s t a b i l i t y on the eel 1 suspensions, and was considered to be more s u i t a b l e for use in routine studies of resting suspensions.  II. G l y c o l y t i c Enzyme Assays  The Embden-Meyerhof  pathway has often been implicated as  being the major pathway of g l y c o l y s i s in the C l o s t r i d i a (Bard and Gunsalus, 1950; Shankar and Bard, 1955a), however the enzymes of t h i s pathway have been demonstrated d e f i n i t e l y in only one C l o s t r i d i urn, C. thermosaccharo1yticum (Lee and Ordal , 1967).  One major reason  for the lack of a successful demonstration of the complete pathway in C_. perfringens has undoubtedly been the existence of a strong NADH -oxidase ( D o l i n , 1959a, 1959b), which interferes with the 2  most e f f i c i e n t procedure f o r assaying g l y c o l y t i c enzymes, that of following the oxidation or reduction of pyridine nucleotides spectrophotometrically. The NADH ~oxidase a c t i v i t y 2  in c e l l - f r e e extracts was  considerably greater than the a c t i v i t y of phosphohexose isomerase, phosphofructokinase, and triosephosphate isomerase and more than  t e n - f o l d greater than hexokinase, 3-phosphoglyceraldehyde dehydrogenase, pyruvate kinase, and l a c t i c dehydrogenase.  In  preliminary experiments, oxidase a c t i v i t y prevented the detection of a l l enzymes except those which could be linked to NADP reduction v i a a commercial source of glucose-6-phosphate dehydrogenase. Attempts were made to decrease the oxidase a c t i v i t y by various procedures.  Extended periods of i r r a d i a t i o n of c e l l -  free extracts with l i g h t at 365 my ( D o l i n , 1959b), using the monochrometer of a Bausch and Lomb Spectronic 20 spectrophotometer, f a i l e d to reduce the oxidase a c t i v i t y A combination of 10  -3  -h  M atabrine and 10  sufficiently.  M H2O2 reduced the  NADh^-oxidase a c t i v i t y to a low l e v e l , however, the r i s k of destroying other enzymes by oxidation with 1^02 and a l s o the very high absorption at 3^0 my of a t a b r i n e , combined to severely l i m i t the use of these compounds. Centrifugation at 25,000 x g_ for 30 min caused p r a c t i c a l l y no reduction in the oxidase a c t i v i t y and confirmed the reported soluble nature of the enzyme ( D o l i n , 1959a).  Because of the enzyme's dependence on molecular  oxygen as an electron acceptor for the oxidation of NADh^ ( D o l i n , 1959a), the possible usefulness of the exclusion of oxygen by preparing c e l l - f r e e extracts under . l ^ , and of assaying the enzymes anaerobically was examined.  This technique reduced the NADr^  -  oxidase a c t i v i t y of the extracts to less than \% of the a c t i v i t y  56  evident under aerobic conditions (Table III,  Fig. 7).  That molecular oxygen was not the p h y s i o l o g i c a l electron acceptor for the NADr^-oxidase has been demonstrated by the lack of involvement of H^O^ as an intermediate in the r e a c t i o n , and by the f a c t that other enzymes of the organism produced t o x i c q u a n t i t i e s of r^C^ when exposed to oxygen ( D o l i n , 1961). With the exception of 3-phosphoglyceraldehyde dehydrogenase, each of the enzymes of the Embden-Meyerhof pathway was measured spectrophotometrically by using the product of each r e a c t i o n , as a substrate for one of the f o l lowing>. commercial ly prepared enzymes-, glucose-6-phosphate dehydrogenase, a-glycerophosphate dehydrogenase, or l a c t i c acid dehydrogenase.  When required,  the product of the enzyme being assayed was converted to the appropriate substrate by the use of a second commercial enzyme preparation.  The commercial enzyme preparations were always  added in excess, t h e r e f o r e , the enzyme in the c e l l - f r e e was r a t e - l i m i t i n g .  extract  A l l of the enzymes of the Embden-Meyerhof  pathway were independently shown to be present in the extracts 2  and s p e c i f i c ' . ^ a c t i v i t i e s , expressed as umoles x 10  of product  formed per min per mg of p r o t e i n , were calculated (Table  III).  Preliminary growth studies demonstrated that the organism would grow r e a d i l y on mannose, fructose and glucose. Kinase a c t i v i t y with mannose could not be demonstrated, however, the phosphohexose isomerase was a c t i v e with mannose6-phosphate.(Table I 11).  The f a i l u r e to detect the mannose  kinase even in the presence of high concentrations of the substrate  Table III.  S p e c i f i c a c t i v i t i e s of g l y c o l y t i c enzymes in extracts of C. p e r f r i hgens. The extracts were prepared from eel 1s grown on semi-defi ned medi urn pi us 0.6% glucose, except where indicated otherwise. ORGANISM  ENZYME  C_. perf r i hgens  Hexokinase glucose fructose mannose Phospho-hexose isomerase fructose-6-phosphate mannose-6-phosphate Glucose-6-phosphate dehydrogenase in extracts from semi-defined medium, log phase semi-defined medium, s t a t . phase complex medium, log phase Phospho-fructo kinase Fructo 1,6-diphosphate aldolase Triose-phosphate isomerase  C_. thermosaccharolyticum  specific activity 5.87 ' 0.210 0 21.4 13.95  8.69  13.4  0 0 0 72.5 3,940  9.6 1,84  37.7  192  3-phospho-g1ycera1dehyde dehydrogenase  7.9  3-phospho-glycerate kinase  726  Mutase  210  Enolase  20.8 840  1 ,416  Pyruvate, kinase  5.85  L a c t i c acid dehydrogenase P y r u v a t e - c l a s t i c system in extracts from enriched medium semi-defined medium semi-defined medium plus 5 mg Fe /ml  8.5  Lee and Ordal (1967);  1.6  16.3 4.1  56,000 1,28  88  6.5 b  2 -.(TO.) umoles/min/mg p r o t e i n .  a  58  F i g . 7.  The a c t i v i t y of NADH -oxidase in aerobic and anaerobic c e l l - f r e e extracts or C. p e r f r i hgens.  and e x t r a c t ,  indicated that e i t h e r the assay system was  inadequate or a d i f f e r e n t system f o r a c t i v a t i n g t h i s hexose was operative in the organism.  A phosphoenol-pyruvate  dependent  phospho-transferase system has been demonstrated to phosphorylate several carbohydrates in E_. col ? and Aerobacter aerogenes (Kundig and Roseman, 1966).  The r e s u l t s from mannose transport  studies  suggest that a phospho-transferase system o r , at least a very s i m i l a r system, is probably the agent for a c t i v a t i n g mannose f o r metaboli sm in C. perfri hgens. Three-phosphoglyceraldehyde dehydrogenase was measured d i r e c t l y by recording the reduction of NAD in the presence of excess 3-phosphoglyceraldehyde.  The enzyme was s e n s i t i v e to  iodoacetamide (Krebs, 1955) and k um/ml of the i n h i b i t o r caused 100% i n h i b i t i o n of a c t i v i t y .  It was necessary to add 12 umoles/ml  of sodium arsenate and 30 umoles/ml of sodium f l u o r i d e to the assay mixture to obtain measurable a c t i v i t y of the enzyme ( F i g . 8). The arsenate ion rendered the reaction i r r e v e r s i b l e by subs t i t u t i n g f o r phosphate and subsequently forming the unstable product, 1-arseno-3-phosphoglycerate.  This product decomposed  to 3-phosphoglyceric acid and therefore, prevented the accumulation of the i n h i b i t o r y reaction (Krebs, 1955).  end products of the dehydrogenase  Fluoride and arsenate ions  enolase a c t i v i t y by forming a complex wi th Mg  inhibited  (BUcher, 1955),  and consequently, t h i s prevented the oxidation of the accumulated  60  0.5 TEST  3  E  O  CONTROL <  o  0.2  a.  - FLUORIDE  O  _  0.1  ARSENATE  + I0D0ACETAMIDE  0 2 S C U T E S Fig.  8.  The a c t i v i t y of 3-phospho-glyceraldehyde dehydrogenase in c e l l - f r e e extracts of C. perfringens. Control minus 3-phospho-glyceraldehyde. lodoacetamide (k mM) or the omission of f l u o r i d e and arsenate from the reaction mixture i n h i b i t s a c t i v i t y completely.  NADH2  by the subsequent action of l a c t i c acid dehydrogenase.  The p o s s i b i l i t y of interference by 3-phosphoglyceraldehyde dehydrogenase a c t i v i t y  in other spectrophotometric assays was  n e g l i g i b l e when f l u o r i d e and arsenate were not present in the reaction mixture ( F i g . 8). A comparison was made of the s p e c i f i c a c t i v i t i e s of the g l y c o l y t i c enzymes in C. perfringens with those values determined by Lee and Ordal (1967) f o r glucose-grown cultures of C_. thermosacchafblyti cum (Table I I I ) . In a d d i t i o n to the g l y c o l y t i c enzymes from glucose to pyruvate, l a c t i c acid dehydrogenase and the p y r u v a t e - c l a s t i c system were present in the organism.  The method of assaying  the p y r u v a t e - c l a s t i c system had some undesirable c h a r a c t e r i s t i c s which should be considered when i n t e r p r e t i n g the r e s u l t s . The rate of acetyl-phosphate production in the reaction mixture decreased over the duration of the experiment ( F i g . 9).  This  decrease in the rate of product accumulation may have been due to e i t h e r the conversion of acetyl phosphate to a c e t i c and b u t y r i c a c i d s , two of the normal end-products of the metabolism of pyruvate in C. p e r f r i ngens or to the end-product  inhibition.  Recently, Biggins and Ditworth (1968) demonstrated a control of the p y r u v a t e - c l a s t i c system in C.pasteurianum extracts by ADP and acetyl phosphate concentrations.  Acetyl phosphate was found  to.be a " p r o d u c t : i n h i b i t o r " of the c l a s t i c r e a c t i o n .  High ADP  62  5  1 0  MINUTES  ' F i g . 9.  A c t i v i t y of the p y r u v a t e - c l a s t i c system iri c e l l - f r e e extracts of C_. perf r i hgens. The role of accumulation of acetyl-phosphate is extrapolated from the i n i t i a l rate.  15  63  concentrations stimulated t h e a c t i v i t y of the c l a s t i c system, probably by lowering the acetyl-phosphate concentration  through  stimulation of acetate kinase. The non-1inear accumulation of acetyl-phosphate w i t h time precluded exact measurements of the a c t i v i t y of the pyruvatec l a s t i c system, however, 1inear extrapolations from the i n i t i a l rates of product accumulat ion gave comparable estimates of the a c t i v i t y of the system under various conditions ( F i g . 9, Table I II). The p y r u v a t e - c l a s t i c system was more a c t i v e in c e l l s grown in the enriched medium than in the.semi-defined medium (Table Ml).  Attempts to stimulate the a c t i v i t y of this system by the  addition of excess ferrous iron to the semi-defined medium were only p a r t i a l l y successful and resulted in an increase in a c t i v i t y of 60%.  This increased a c t i v i t y however, was only k0%  of the value obtained with eel Is grown in the enriched medium. This indicated that some factor other than F e  + +  (required  for  the synthesis of ferredoxin) was lacking in the semi-defined medium.  The presence of both l a c t i c acid dehydrogenase and the  p y r u v a t e - e l a s t i c system explains the heterofermentative  production  of l a c t i c , a c e t i c , and b u t y r i c a c i d s , as we11 as the production of carbon dioxide and hydrogen from glucose by cultures of £ . perfri hgens. The f a i l u r e to demonstrate the existence of glucose"-6phosphate dehydrogenase a c t i v i t y , e i t h e r NAD- or NADP-1inked, in the e x t r a c t s , argued against the hexose-monophosphate pathway  as a major route of g l y c o l y s i s in t h i s organism.  The f a i l u r e  of glucose-grown c e l l s to accumulate acetate under conditions of low F e  + +  concentration, probably indicated the absence of  the phosphoketolase system of g l y c o l y s i s (De V r i e s , Gerbrandy, and Stouthamer, 1967).  Five-carbon s k e l e t o n s , for the  synthesis of nucleic a c i d s , are probably generated by a reversal of the transketolase-transaldolase system which was demonstrated in C_. perfringens by Cynkin and Delwiche (1958).  III.  Carbohydrate Transport  An integral step in the u t i l i z a t i o n of carbohydrates by any c e l l is the transporting of the carbohydrate across the semipermeable membrane into the i n t e r i o r of the c e l l .  The a b i l i t y  to transport carbohydrates s p e c i f i c a l l y and rapidly across the membrane and, to a greater extent, the a b i l i t y to accumulate metabolites w i t h i n the c e l l at concentrations many times above that of the external concentration, confer a d e f i n i t e  biological  advantage upon a microorganism. 14  The mechanism of transport of  C-glucose by C. perfringens  was studied with regard to i t s k i n e t i c s , s p e c i f i c i t y and energy dependence.  The a b i l i t y to accumulate radioactive carbohydrates  and the nature of the accumulated product were also investigated. As a r e s u l t of the v a r i e t y of techniques used for  studying  metabolite uptake and for segregating the component functions of "membrane passage and metabolite accumulation", the term " t r a n s p o r t " has come to have several meanings (Hengstenberg,  Egan, and Morse, 1968).  In these s t u d i e s , „the term transport  has been considered to encompass the two f u n c t i o n s , although, as w i l l be discussed, attempts were made to separate them in t h i s organism.  1. U t i 1 i z a t i o n of carbohydrates by C. perfringens  Growth of C. perfringens with various carbohydrates as the source of carbon was studied.  Flasks with anaerobic semi-  defined medium containing 0.5% of the appropriate carbohydrate were inoculated as for growth s t u d i e s , and the increase in O.D. at 660 my was observed over 5.5 h r s , two hours beyond the beginning of stationary phase of glucose-grown cultures (Table IV). Carbohydrates which f a i l e d to support growth were added to 3 sequential subcultures of organisms growing on glucose, but no inducible u t i l i z a t i o n of these carbohydrates was observed.  14  2. Transport of  C-carbohydrates  (i) Transport of ^ C - g l u c o s e . When suspensions of eel Is grown with 0.5% pyruvate plus 0.1% glucose as the energy sources (pyruvate-grown eel 1 s ) , -2 and resuspended to 7.8 x 10. mg dry weight/ml (1.4 y c u r i e / ymole) 14 in the presence of pyruvate and the t o t a l C incorporated into 14 the c e l l s was determined, the C entered the c e l l s at a l i n e a r  66  Table IV.  Growth of C. perfringens with various carbohydrates as the source of carbon. Data obtained from two separate t r i a l s .  CARBOHYDRATE  INITIAL  FINAL  Control  0.17  0.36  Sorbose  0.165  0.31  Mannose  0.2  1.11  Control  0.06  0.137  1-Arabinose  0.125  0.16  Xylose  0.125  0.44  Ribose  0.04  0.952  Trehalose  0.125  0.44  Sucrose  0.09  0.45  Lactose  0.12  0.465  Maltose  0.05  0.45  Fructose  0.105  0.435  Galactose  0.08  0.22  Glucose  0.12  1.38  rate for 1.5 min, and then the rate decreased during the remainder of the experiment ( F i g . 10).  As w i l l be demonstrated,  12,  the concentration of  C in the c e l l s at 1.5 min was several  hundred times greater than in the external environment. (ii)  Transport of ^C-carbohydrates other than glucose.  Of the seven radioactive compounds examined, only glucose and mannose were concentrated in detectable amounts (Table V I ) .  Ribose, maltose, aMG, lactose and galactose  were not concentrated in the c e l l s during the i n t e r v a l s studied. Growth of the organism on lactose did not induce an accumulation mechanism for t h i s carbohydrate.  Attempts to study the process  of e q u i l i b r a t i o n of these sugars across the c e l l membrane, or to detect concentrations only s l i g h t l y above the e x t r a c e l l u l a r l e v e l , were interfered with by the n o n - s p e c i f i c retention of r a d i o a c t i v i t y by the membrane f i l t e r s even in the absence of 14 cells.  The amount of  C retained was of the same order of  magnitude as one would expect to enter the c e l l i f a concentration gradient was not e s t a b l i s h e d .  The amount of the  retained r a d i o a c t i v i t y was s u f f i c i e n t l y e r r a t i c to obscure changes in the pool s i z e .  This retained r a d i o a c t i v i t y could  not be removed by washing the f i l t e r s twice with medium, by p r e - t r e a t i n g the f i l t e r with unlabel led substrate at the same 14 concentration as the C-substrate, or by varying the  I  2  3  4  5  MINUTES  1.0.  -6 14 Early time course of uptake of 8 x 10 M C-lhglucose (1.4 yc/ymole) by whole c e l l s of C. perfringens.  temperatures of the f i l t e r holder and wash medium.  In  preliminary experiments, the organism was shown to grow r e a d i l y with any of these carbohydrates as the carbon source, except aMG, which was not tested.  Therefore, i t  is  likely  that these compounds entered the c e l l by f a c i l i t a t e d  diffusion.  However, the l i m i t a t i o n s discussed above make studies o f . t h i s type of transport process a technical i m p o s s i b i l i t y at present.  (iii)  Temperature dependence of  1 /, C-glucose transport.  The rate of transport was dependent on the temperature of the reaction mixture as determined by observing the t o t a l 14 accumulation of  C at 37 C and 30 C.  While the maximum s i z e  of the pool remained nearly constant, the i n i t i a l rate of uptake was found to be considerably less at the lower ( F i g . 11).  temperature  Although the c e l l s were grown at 37 C, i t was  found necessary to routinely observe the uptake of carbohydrates at 30 C to obtain adequate periods of l i n e a r uptake.  The  i n i t i a l rate of uptake was found to increase by 81% over the seven degree change in temperature ( F i g . 11).  The O^Q of the  transport system was estimated from these data to be 2.54. This value compared well with the t h e o r e t i c a l value of 2.0 for chemical r e a c t i o n s , and with the value of 2.2 found for components of the glucose transport system'in E. c o l i et a l . , 1963).  (Hoffee  TOTAL UPTAKE  11. Total uptake of r a d i o a c t i v i t y by cells of X . 1 with 1.25 x 10-5 M ^C-U-glucose (1.4 yc/ymoleT oo, at 37 C; A— 4 , at 30 C.  Ingens  (iv) Energy requirement of the transport of glucose.  The processes of accumulation have been shown to be energy dependent for $-galactosides (Kepes, 1957), galactose (Horecker, Thomas, and Monod, 1-9'60a), and amino acids (Kay, 1968).  Hoffee, Englesberg and Lamy . (1-963)' demonstrated that  a lack of energy caused an increase in accumulation by the glucose transport system in suspensions of E. c o 1 i . Attempts to demonstrate energy dependence of  transport  in £ . perfringens were hampered by the fact that the normal uncouplers of energy for transport systems, such as 2 , 4 - d i n i trophenol  (Winkler and Wilson, 1966; Horecker, Thomas, and  Monod, 1960a), are of l i t t l e use in l i m i t i n g the energy y i e l d i n g reactions of an o b l i g a t e anaerobe.  This could be due to the  fact that t h i s organism undoubtedly r e l i e s l a r g e l y on substrate level phosphorylation. The a d d i t i o n of pyruvate to the c e l l suspension was demonstrated to be necessary for the achievement of reproducible rates of accumulation ( F i g . 12).  However, i t s omission from  the resuspension medium did not eliminate accumulation, but did reduce the i n i t i a l rate of uptake by approximately 50%. A f t e r the f i r s t 0.5 min of the experiment, the rate of accumulation  Ik of  C was demonstrated to increase r a p i d l y .  Addition of 1.5 mM  iodoacetamide to the s o l u t i o n without pyruvate during resuspension  72  +PYRUVATE  -PYRUVATE  -PYRUVATE + IODOACETAMIDE  .5  0.5 MINUTES F i g . 12.  Total incorporation of r a d i o a c t i v i t y by c e l l suspension with 8 x 10"^ M.glucose . (1.4 yc/ymole). o o, with 0.6% pyruvate; A — A». minus pyruvate, Q rj, minus pyruvate, plus 1,5 x 10 3 M iodoacetamide. :  _  of the c e l l s did not affect the i n i t i a l rate of glucose accumulation without pyruvate, but prevented the increase in the rate observed when iodoacetamide was not present ( F i g . 12). The a b i l i t y of t h i s organism to metabolize f i v e of the amino acids present in the medium (Woods and Trim, 19^2), probably accounted for the base level of uptake observed in the absence of added pyruvate.  Attempts were made to s i m p l i f y  the resuspension medium in order to define more c l o s e l y the source of energy for accumulation.  Various buffer solutions  and combinations of the components of the medium with and without added pyruvate, were used to wash and resuspend the c e l l s but only the complete semi-defined medium with 0.6% pyruvate was found to y i e l d reproducible rates of accumulation. The increase in the rate of transport with time in the pyruvate-less c e l l s was probably due to the supply of energy from metabolism of the glucose which had entered the c e l l s during the e a r l y time course of the experiment.  Iodoacetamide  has been shown to i n h i b i t the production of energy by g l y c o l y s i s , through i n h i b i t i n g 3~phosphoglyceraldehyde dehydrogenase activity  (Krebs, 1955).  A c t i v e transport  in c e r t a i n systems has been shown to occur  by the counter-transport of a s i m i l a r compound using the same membrane c a r r i e r (Wilbrandt, and Rosenberg, 1961).  It was  d i f f i c u l t to demonstrate that pyruvate or some metabolic product of pyruvate was not involved in counter-transport with glucose, thus providing the energy required for accumulation.  However,  i t is u n l i k e l y that a system capable of d i f f e r e n t i a t i n g  between  sugars epimeric in one hydroxyl , as w i l l be shown, would a l s o recognize a low molecular weight a c i d .  Therefore, i t would  appear that the accumulation of glucose was dependent on energy gained, at least in p a r t , by the metabolism of exogenously added pyruvate.  (v) K i n e t i c s of accumulation of ^ C - g l u c o s e and 14_ C-mannose. 14 Exposure of pyruvate-grown c e l l s to  C-U-glucose  resulted in t y p i c a l saturation k i n e t i c s for t o t a l uptake.  The  rate of uptake of glucose was found to increase 1inearly with concentration of substrate u n t i l 0.01 mM, then the rate of change in v e l o c i t y with increasing substrate concentration decreased as the v e l o c i t y approached a maximum ( F i g . 13). When the rates were plotted in the Lineweaver-Burke f a s h i o n , a l i n e a r r e l a t i o n s h i p between the reciprocal of the v e l o c i t y of uptake and the reciprocal of the substrate concentration was' found ( F i g . 14). Three separate t r i a l s were performed for qlucose ( F i g . 15) and the values of K and V for the three t max y  t r i a l s were estimated and averaged (Table V ) .  A s i m i l a r response  of the rate of uptake of mannose to increasing external concent r a t i o n of mannose was observed with saturation of the uptake mechanism becoming apparent a f t e r 0.016 mM mannose was present ( F i g . 16). Values of K and V were estimated from the t max reciprocal plot of these data ( F i g . 17), and compared to those values for glucose uptake (Table V ) .  0  F i g . 13.  0.01 GLUCOSE  0.02 C O N C mM  0.03  Saturation k i n e t i c s . o f C-glucose (1.4 yc/ymole) incorporation by £ . perfringens. C e l l s at 30 C were exposed for 1 min t o ' i n c r e a s i n g concentrations of C - g l ucose. The rate of t o t a l uptake is plotted against concentration of glucose. l 4  2 w  G L U C O S E C O M C x IO"° M F i g . 14.  Saturation k i n e t i c s . o f O g l u c o s e (1.4 yc/ymole) incorporation by C. perfringens.• Liheweaver-Burke plot of rate of uptake against glucose concentration from t r i a l in Figure 13.  4LCELLS)  -  -  v.  V.'  ^  1  o *  '  0  •  I  i  2  '  3  1  4  §  G L U C O S E C O N C x 10" F i g . 15.  U  Saturation k i n e t i c s of ^C-glucose (1.4 yc/ymole) incorporation by C . perfringens. Lineweaver-Burke plot of rate of uptake against glucose concentration for three d i f f e r e n t t r i a l s performed as in Figure 13. 1  The change from l i n e a r response of the rate of uptake to substrate concentration was demonstrated to occur over a very narrow range of substrate concentration, a phenomenon which has been shown to be t y p i c a l of carbohydrate transport systems (Hoffee, Englesberg, and Lamy, 1964; Egan and Morse, 1966; Horecker, Thomas, and Monod, 1960).  This abrupt saturation of  the transport system caused technical d i f f i c u l t i e s  in accurately  determining the changes in the rate of uptake as the transport system approached s a t u r a t i o n .  The s l i g h t v a r i a t i o n in the slope  of the three reciprocal plots of v e l o c i t y versus substrate concentration for glucose and the subsequent v a r i a t i o n in the estimates of the K and V values resulted from t h i s t max uncertainty. While the K value for mannose transport was over three times as large as the average value for glucose transport, the V  •  ft  for  m a x  mannose was only 33% larger than the average value for glucose (Table V ) .  The  v a l u e s , while not absolute measures of  a f f i n i t y for the substrate, served to indicate that the accumulation mechanism had a much higher a f f i n i t y for glucose than for mannose. The difference between the V values for the transport of the max r  sugars was small enough to indicate that both carbohydrates were accumulated at approximately the same maximum r a t e .  It has been  demonstrated that glucose and mannose each i n h i b i t e d the accumulation of the other, and thus were both accumulated by the same mechanism, probably at the same maximum v e l o c i t y . Values of K of the order of 10 ^ M have been demonstrated to be t y p i c a l of microbial carbohydrate transport mechanisms (Egan,  79  o.oso F i g . .'16.  0.020  0.025  Saturation k i n e t i c s of ' 'C-mannose (1.4 uc/umole) incorporation by C . p e r f r i n g e n s . C e l l s at 30 C were exposed for 1 min to increasing concentrations of C-mannose. The rate of t o t a l uptake is plotted against the concentration df mannose. :  i.O  o  0  0.2  0.4  0.6  0.8  I  MAM.MOSE CONG % 8 0 - % . 17.  Saturation k i n e t i c s of C-mannose (1.4 yc/ymole) incorporation by C. perfringens. Lineweaver-Burke plot of the rate of uptake against mannose concentration from Figure 16.  81  Table V.  Saturation k i n e t i c s of the glucose-mannose transport system. Estimated values of K and ^ ' f r o m t r i a l s in Figures 15 and 17. t  TRIAL  K (M) t  x  V a max  Glucose A  2.0  x 10~  38.9  B  1.67 x 10~  31.3  C  2.38 x 10~  29.5  Average  2.02 x 10~  33.2  Mannose  7.70 x 10~  kk.k  5  5  5  5  5  - ymoles/min/mg dry weight.  82  and Morse, 1966; Hoffee, Englesberg, and Lamy, 1964; and Horecker, Thomas, and Monod, 1960).  It should be noted that  t h i s e x h i b i t i o n of saturation k i n e t i c s did not d i f f e r e n t i a t e between enzymatic and adsorptive processes of accumulation (Egan, and Morse, 1966).  (vi) S p e c i f i c i t y of the transport of glucose and mannose.  As previously mentioned, the demonstration of 14 saturation k i n e t i c s f o r the accumulation of  C-carbohydrates  did not d i f f e r e n t i a t e between adsorptive and enzymatic mechanisms. In order to demonstrate that the concentrative mechanism was enzymatic, i t was necessary to a s c e r t a i n that the accumulation of carbohydrates was s p e c i f i c , a property that adsorption would not have e x h i b i t e d . 14 14 When the eel 1s were exposed to C-U-glucose, or to C-U14 mannose, and the t o t a l uptake of C observed over 1.5 min, a 14 l i n e a r concentration of C with time occurred for both sugars ( F i g . 18a, 19a).  In p a r a l l e l experiments, unlabel led carbo-  hydrates were added to 100 times the concentration of the  14 C-  s u b s t r a t e , and the rate of t o t a l incorporation was compared to 12 the rate observed in the absence of added  C-carbohydrate. The  demonstration that f o r both glucose ( F i g . 18) and mannose ( F i g . 19) most carbohydrates did not competitively i n h i b i t transport  indicated  that not al1 carbohydrates metabolized by the c e l l s were transported  0  60  BO  90  0  10  SO  90  SECONDS F i g . 18.  Competition for glucose uptake in C. perfr?hgens. The rate of incorporation o f . 8 x 10~ C - g l ucose (1.4 yc/urnole) in the presence or absence of 8 x 10~ M C-carbohydrates. A. o — , C-glucose o n l y ; © — , '^C-pentoses added ( r i b o s e , x y l o s e , arabinose). B . 0 — , C - d i s a c c h a r i d e s except maltose added ( l a c t o s e , sucrose, t r e h a l o s e ) ; A — , C-maltose. b  , H  2  ±  so  0  30  SECONDS Competition for glucose uptake in C. perfringens. The rate of incorporation of 8 x 1 0 " ^ C - g l u c o s e Cl. 4'uc/ymol e) in the presence or absence.of C-carbohydrates. C. AC-fructose; gCgalactose; 0 — , ^C-mannose; ®—, glucpse control D. C-glucose o n l y : A-—. ' T - m a H n i fc —, C-mannose; glucose control . 6  1  into the c e l l by a common mechanism, but rather that glucose and mannose were accumulated by mechanisms of a highly s p e c i f i c nature.  The mutual i n h i b i t i o n of mannose and glucose concentration  by each other (Table VI) demonstrated that they were accumulated or transported by the same mechanism (Wilbrandt, and Rosenberg, 1961). It was demonstrated that f r u c t o s e , r i b o s e , and sucrose, a l l r e a d i l y metabolized by t h i s organism for growth, did not i n h i b i t uptake of e i t h e r mannose or glucose, while the disaccharide maltose did reduce the rate of accumulation (Table V I ) .  Thus,  the s p e c i f i c i t y of the accumulation mechanism was not determined s o l e l y by the s i z e or number of r i n g s , but was probably determined by the configuration of the substituents on the carbohydrate r i n g . At t h i s point i t is necessary to consider the d i s t i n c t i o n between transport and accumulation.  These functions have been  separated in several metabolite transport systems (Winkler, and Wilson, 1966; Osborn, McLel l a n , and Horecker, 1-961; and Kay, 1968) by observing the s p e c i f i c i t y of displacement of preloaded 14 pools of one  C-carbohydrate when a second, unlabel led carbo-  hydrate was added.  Competitive i n h i b i t i o n of i n i t i a l uptake was  considered to act on e i t h e r the transport mechanism or the accumulation mechanism.  However, the displacement of an  accumulated pool was only possible i f the two compounds shared the same accumulation mechanism. Stable pools of metabolites were found necessary f o r t h i s type of study, and techniques for generating these pools are  0  20  40  SO  SO  0  20  40  60  SO  SECONDS Fig.  19.  C o m p e t i t i o n f o r mannose u p t a k e i n £ . p e r f r i n g e n s . The r a t e o f incorporation o f 1.5 x 10"5 M ^ C - m a n n o s e (1.4 yc/ymole) in the p r e s e n c e o r a b s e n c e o f 1.6 x 10"3 M C-carbohydrates. A . 0—, '^C-mannose a l o n e ; A — , C-sucrose; trehalose, 1 a c t o s e ; iH -, C-ma1tose a d d e d ; 0 — , C - g l u c o s e added; © — , C-mannose c o n t r o l . B.Q— C - f r u c t o s e ; /S—, C-aMG added. 1  1 2  1 2  1 2  1 2  l 2  1  2  1 2  oo  A  SECONDS 19-  Competition for mannose uptake in C. perfringens. The rate of incorporation of 1.5 x 10"-^ M ^C^mannose (1.4 yc/ymole) in the presence or absence of 1.6 x 10 C-carbohydrates. C, A - , C - x y l o s e added;D—j S c arab inose added; f — , C-ribose added. 12  12  88  Table VI.  Accumulation of carbohydrates and competitive i n h i b i t i o n of the rate of u p t a k e ^ f C-glucose (8 x 10"? mM, 1.k uc/umole) and C-mannose (1.6 x 1 0 " mM, 1 A uc/umole), by a 100-fold excess of these carbohydrates. 2  12  C-carbohydrate added in 100-fold excess  Accumulation by C_. perfringens  % I n h i b i t i o n of , 14  1  C  ,, '14, f -uptake  1 ^ r - n I n r n c p  C-glucose ^C-mannose 0 0  Lactose  0  Trehalose  -  0  0  Sucrose  - .  0  0  Maitose  0  33  52  Ribose  0  0  0  Xylose  -  0  0  Arabinose  -  0  0  aMG  0  0  0  Galactose  0  0  0  Fructose  0  0  0  Glucose  +  100  100  Mannose  +  79  100  known for other organisms (Kay, 1968; Hoffee, and Englesberg, 1962).  The techniques used depended upon the use of non- or  slowly metabolizable substrates to form the pools, or the use of mutant s t r a i n s of the organism that did not immediately degrade the pooled m a t e r i a l .  Neither non-metabolizable analogues which  could be accumulated, nor adequate mutant s e l e c t i o n techniques, were a v a i l a b l e for the study of transport  in C. perfringens.  Studies of the transport mechanism alone are possible i f analogues of carbohydrates that are transported, but not accumulated, are a v a i l a b l e .  The competitive i n h i b i t i o n of the  '•14' transport of C-U-aMG was attempted, but, as mentioned p r e v i o u s l y , n o n - s p e c i f i c retention of the r a d i o a c t i v i t y on the membrane f i l t e r obscured the r e l a t i v e l y small changes in 14 incorporated  C levels.  The f i n a l possible method f o r segre-  gating transport from accumulation was the use of metabolic poisons to remove the energy supply f o r those accumulation mechanisms which are energy dependent (Wilbrandt, and Rosenberg, 1961; Winkler, and Wilson, 1966; Hoffee et a_j_., 1963). As w i l l be discussed, i t was not possible to completely destroy accumulation through energy l i m i t a t i o n .  Had i t been possible  to remove accumulation by t h i s technique, the technical difficulties  mentioned previously in observing simple  e q u i l i b r a t i o n of substrates across the membrane would have interfered. Therefore, as i t proved impossible to d i f f e r e n t i a t e  between  the functions of trans-membrane passage and accumulation of carbohydrates in C. p e r f r i hgens, the terms transport and accumulation w i l l be used interchangeably.  Based on the s p e c i f i c i t y of i n h i b i t i o n of the uptake of mannose and glucose, the s t r u c t u r a l  requirements of a substrate  of the accumulation process were e l u c i d a t e d .  The transport  mechanism was shown to have the higher a f f i n i t y for glucose (Table V ) , with substituents or with any modification in structure reducing i t s a c t i v i t y .  In compounds with any changes other than  at the C-2 p o s i t i o n , accumulation was completely eliminated. Epimerization of the -OH at the C-2, to produce mannose, raised the K value by a factor of three, but s t i l l  allowed appreciable  accumulation and approximately the same V  as for glucose  accumulation (Table V ) .  m a x  As a r e s u l t of t h i s lowered a f f i n i t y ,  the competitive a c t i v i t y of mannose was found to be only 79% that of glucose, for glucose uptake (Table V I ) .  Replacement of  the C-1 hydroxyl with any s u b s t i t u e n t s , as in aMG, t r e h a l o s e , or sucrose, completely destroyed the transport a c t i v i t y .  The  sugar a l l o s e , which is the epimer of glucose at the C-3 hydroxyl, was not a v a i l a b l e for study of i t ' s  inhibitory  activity.  Epimerization of the C-k hydroxyl group to form galactose, completely eliminated a c t i v i t y as a competitor for the glucosemannose accumulation system, and was not i t s e l f accumulated by the system.  However, s u b s t i t u t i o n of the 4-hydroxyl in the  (3-conf i g u r a t i o n , by the formation of maltose, a 1 lowed 33% of the i n h i b i t o r y a c t i v i t y to remain, while accumulation was found to be-eliminated.  The s i m i l a r s u b s t i t u t ion with galactose at  the k carbon of glucose in the B-configuration to form l a c t o s e , completely eliminated both forms of a c t i v i t y system.  in the accumulation  Therefore, on the basis of these r e s u l t s , the s p e c i f i c i t y of the glucose-mannose accumulation system was found to be only p a r t i a l l y dependent upon the configuration about C-2, and very s e n s i t i v e to changes in the configuration about C-k. The system was a l s o found to be very s e n s i t i v e to substituents at C-1, and s u b s t i t u t i o n s at C-k resulted in varying degrees of reduction of competitive  activity.  Studies by H a g i h i r a , Wilson, and L i n (1963) on the s p e c i f i c i t y of glucose uptake by E. cbl i , using d e r i v a t i v e s of aMG, demonstrated that substituents on C-2 affected the a c t i v i t y of the carbohydrates.  Other workers have demonstrated  30% i n h i b i t i o n of aMG concentration by £ . cbl ? by the addition of maltose, while mannose and sucrose each caused a 20% i n h i b i t i o n of a c t i v i t y  (Hoffee, Englesberg, and Lamy, 1963).  The s p e c i f i c i t y of the glucose concentration system in E. c b l i appeared therefore to d i f f e r greatly from that demonstrated in s us pens'? ons of C. perf r i hgens, 1 n that the configuration about the C-2 in the substrate or competitor of the C. perfringens mechanism was not found to be c r i t i c a l , while changes at t h i s p o s i t i o n reduced the rate of accumulation in suspensions of E. c b 1 i . The " a n a e r o b i c transport mechanism" f o r carbohydrates in yeast, as described by Scharff and Kramer (1962), d i d not appear in C. perf r l hgens  as fructose was shown to be transported by the  yeast system, while i t had no a c t i v i t y accumulation system studied.  in the glucose-mannose  The " c o n s t i t u t i v e monosaccharide  transport system" of yeast ( C i r i l l o , 1968) is more s i m i l a r to  the system described for C. perfringens.  S u b s t i t u t i o n of the  anomeric.hydroxyl completely destroyed a c t i v i t y , and, substituents or configurational changes at a l l other carbons except C-2 decreased the a c t i v i t y of the glucose ring in both systems. However, the carbohydrates were compared as competitive i n h i b i t o r s of the transport of d - x y l o s e , or of ^-sorbose, which are not metabolized by yeast.  D-xylose was found to be inactive  in the system for the accumulation of glucose by C. perfringens l i m i t i n g the value of comparisons between the two systems. Studies of the s p e c i f i c i t y of the carbohydrate accumulation system of the Gram-positive S. aureus, have indicated that carbohydrate accumulation is very s p e c i f i c , with very competition for entry between the carbohydrates.  little  Maltose had  no detectable e f f e c t on the rate of concentration of glucose, and mannose was not tested for a c t i v i t y  in the system (Egans, and  Morse, 1966), making comparisons with the s p e c i f i c i t y of C_. perfr?hgens uptake d i f f i c u l t .  14  3. The pooling of  C-glucose  (i) Pool capacity. In experiments in which both the t o t a l  incorporation  and the Jncorporation into the cold TCA insoluble materia 1 were measured over 40 min, the amount of  C in the soluble pool rapidly  reached a maximum value ( F i g . 20, 21), a f t e r which the s i z e of 14 the pool remained r e l a t i v e l y constant as the C of both the t o t a l  93  suspensions with 8 x 10" .M C-U-glucose (1.4 uc/umole) 0-0, t o t a l incorporation; A—A, soluble radioactive p o o l ; El—Q, cold TCA p r e c i p i t a t e . . 1  94  TOTAL  0  10  20  30  40  iyiiNUT.es 21. Formation of cold TCA s o l u b l e , radioactive pool by eel 1 suspensions in the 1.82 x l O " ' ' M ^ C - U - g l ucose (1.4 yc/ymole). 0-0, t o t a l incorporation; A—A, cold TCA p r e c i p i t a t e ; D—0, soluble radioactive pool. 1  95  and cold TCA insoluble f r a c t i o n s  increased at the same r a t e .  The pool s i z e decreased towards the end of the experiment, presumably due to the metabolism of glucose ( F i g . 21). The decrease in the rate of uptake a f t e r the i n i t i a l min, may have been the r e s u l t of any of several events.  1.5 The  transport system may have become limited by the depletion of exogenous glucose, by the rate of energy production or by the 14 e q u i l i b r i u m constant of the transport process. may have been rapidly metabolized by the c e l l to  The  C-glucose  intermediates  or end-products which were released to the medium, making the system g l u c o s e - l i m i t i n g . When the pool s i z e was maximal ( F i g . 10), the f i l t r a t e of the suspension was concentrated and passed through a combined column of Dowex-50-H and Dowex-1-formate ion-exchange r e s i n s . +  E s s e n t i a l l y a l l of the r a d i o a c t i v i t y was eluted f r o n t a l l y with d i s t i l l e d water.  When the eluate was chromatographed, i t was  found to co-chromatograph with the glucose standard.  Thus,  glucose had not been converted to a degradation product and the system had not become g l u c o s e - l i m i t i n g .  When the density of  the c e l l suspension was reduced by one-half in an attempt to prolong the time required to form the maximum pool s i z e , the 14 rate of uptake and the level of the by one-half  ( F i g . 22).  C-pool were also decreased  This demonstrated that the rate of  accumulation, as well as the t o t a l accumulation, was proportional to c e l l mass and that the transport enzymes were saturated under these experimental  conditions.  The decrease in maximum pool s i z e , proportional  to the decrease  96  TOTAL  20  40  60  80  SECONDS FIG. 22.  Total incorporation of r a d i o a c t i v i t y by c e l l suspensions with2.5 x 10" M ^C-U-glucose.(1.4 yc/ymole).A-A, 7.8 x 10" mg dry weight of c e l l s / m l ; 0-0, 3.9 x 10 dry weight of c e l l s / m l . 5  2  1  in c e l l mass, indicated that some property of the c e l l the s i z e of the pool of accumulated carbohydrate.  limited  Assuming that  the concentration of the carbohydrate was f a c i l i t a t e d by an energy-requiring system, then the s i z e of the pool may have been determined by the energy supply or by the rate of energy production. As the c e l l s were resuspended in 0.6% pyruvate at the beginning of the experiments, and as the rate of accumulation of  C  remained constant over several hours of sequential experiments, the rate of energy production, rather than the amount of substrate a v a i l a b l e , may have been the l i m i t i n g  factor.  An a l t e r n a t i v e explanation is that the equi1ibrium constant of the transport process, a measure of the a f f i n i t y of the transport mechanism for the carbohydrate, may have l i m i t e d the pool s i z e by defining the maximum r a t i o of internal to external concentrations of the carbohydrate.  As w i l l be shown l a t e r ,  the  C inside the c e l l was in the form of a charged d e r i v a t i v e of glucose, and therefore was not in a form d i r e c t l y in e q u i l i b r i u m across the membrane with the glucose.  However, the r a t i o of  the concentration of the d e r i v a t i v e to the concentration of free exogenous glucose determined by the rate constant of the accumulation mechanism, could have defined the maximum pool size possible.  (ii)  Internal concentration.  During the e a r l y time course of transport the  C  entered the c e l l s at a l i n e a r r a t e , and a f t e r 1 min, 32% of  the t o t a l label was present in the soluble pool ( F i g . 20). 14 There was a 15 to 20 sec lag in the incorporation of the  C  into the cold TCA insoluble f r a c t i o n and t h i s was followed by increasing rate of incorporation for the remainder of the experiment. 14 The s i z e of the soluble pool of C was estimated from the 14 level of soluble  C at 1.5 min ( F i g . 20), a f t e r which time the  rate of incorporation decreased as the pool capacity was reached ( F i g . 10).  Based on the assumption that 80% of the c e l l weight  was water ( L u r i a , 1966), and that 10% of t h i s c e l l was i n t e r 14 c e l l u l a r , the concentration of the C in the i n t r a c e l l u l a r water at 1.5 min was c a l c u l a t e d to be 396 times the concentration of 14 14 14 the e x t r a c e l l u l a r C. Losses of C as C 0^ from the system over the course of the experiments were n e g l i g i b l e . A comparison of t h i s concentration r a t i o with values obtained with other microorganisms was not p a r t i c u l a r l y valuable because of the w i l d range of published r a t i o s . 4 Galactose was concentrated by 10 - f o l d in E. c b l i  (Horecker, 3  Thomas, and Monod, 1960a).  Lactose was accumulated 7.2 x 10  times above the external concentration in S. aureus while the same organism was found to concentrate ma 1tose by a f a c t o r of 700, sucrose by a factor of 520, and aMG by a f a c t o r of 370 over the e x t r a c e l 1 u l a r concentration (Egan, and Morse, 1966).  These  l a t t e r values are of a magnitude comparable with the level of accumulation demonstrated in C. p e r f r i ngens.  Higher concentration  factors have been demonstrated f o r the accumulation of amino acids by microorganisms, with values running into the tens of thousands ( B r i t t e n and McClure, 1962).  (iii)  Nature of the pooled  C.  The accumulation of pools of soluble  C which occurred 14  when suspens ions of C. perfr? hgens were exposed to  C-U-glucose  has been described as the accumulation of glucose, even though the nature of the pooled material was not known.  In S . aureus,  however, i t has been demonstrated that maltose, sucrose, aMG, l a c t o s e , and i s o p r o p y l - t h i o galactosides were accumulated as phosphorykated d e r i v a t i v e s (Hengstenberg, Egan, and Morse, 1968). In a d d i t i o n , the i m p l i c a t i o n of the phosphoenolpyruvate-dependent phospho-transferase system for the phosphorylation of carbohydrates, in the accumulation of carbohydrates by E. col? and S. typhimuriurn has been demonstrated (Tanaka and L i n , 1967; Tanaka, Fraenkel, and L i n , 1967;  Simoni et_ aj_., 1967).  An attempt to e l u c i d a t e the  14  nature of the  C material accumulated w i t h i n the c e l l  indicated  14  that in £ . perfringens, the  C from glucose and mannose was  concentrated as a d e r i v a t i v e . 14  When the C material present in the cold TCA soluble pool at 0.5 min was isolated and concentrated, chromatography of 14  the pool demonstrated no detectable  C which chromatographed  as free glucose or as free mannose.  Rather, in both cases two  peaks containing a l l of the r a d i o a c t i v i t y were shown to remain near the o r i g i n .  When a l i q u o t s of the pool were separated by  e l e c t r o p h o r e s i s , some free carbohydrate was present near the  o r i g i n of the electrophoretogram, probably as a r e s u l t of hydrolysis during preparation or during the actual e l e c t r o phoresis.  The two charged peaks were observed to move the  same distance as glucoses-phosphate and fructose 1 , 6 - d i phosphate standards with approximately 65% of the label in the singly-charged f r a c t i o n .  The remainders of the concentrated  pools were separated by preparative e l e c t r o p h o r e s i s , and the f r a c t i o n s were recovered and concentrated. f r a c t i o n s from both  14  C-glucose and  14  Aliquots of these  C-mannose pools were  dephosphorylated with commercial b a c t e r i a l a l k a l i n e phosphatase. The dephosphorylated f r a c t i o n s were concentrated, and then cochromatographed with f r u c t o s e , glucose and mannose standards. Upon dephosphorylation, a l l of the pooled materials chromatographed as uncharged hexoses.  The pool of  14 C-glucose was then found to  contain monophosphorylated glucose, monophosphorylated fructose 14 and diphosphorylated f r u c t o s e .  The accumulated pool of  C from  14 C-mannose contained monophosphorylated d e r i v a t i v e s of mannose, glucose and f r u c t o s e , in addition to a diphosphorylated d e r i v a t i v e of f r u c t o s e .  The pooled material  i s o l a t e d a f t e r 4 min exposure  14 to  C-glucose was found by electrophoresis to be l a r g e l y in the  highly charged f r a c t i o n , probably as a diphosphorylated  derivative  of f r u c t o s e . It has been demonstrated that c e l l membranes which are relatively  impermeable to charged d e r i v a t i v e s of carbohydrates,  allow the transport of the free carbohydrates (Hengstenberg, Egan, and Morse, 1968).  Thus, an e f f i c i e n t method of  concentrating  carbohydrates in the c e l l would be to phosphorylate them on the  inside of the membrane.  The uncharged carbohydrate would be  transported in by f a c i l i t a t e d d i f f u s i o n , and the charged product of phosphorylation would be unable to leave the c e l l , forming a concentration gradient of carbohydrate.  thus  Although an  a c t i v e soluble hexokinase could have phosphorylated the glucose and mannose during accumulation by t h i s organism, evidence has been accumulated for a phospho-transferase or kinase, of the membrane-bound type described for E. c o l i Ghosh, and Roseman, 1964).  (Kundig,  Comparisons were made of the  s p e c i f i c i t y of the accumulation mechanism with that determined for the hexokinase a c t i v i t y  in c e l l - f r e e e x t r a c t s .  The hexo-  kinase was found to phosphorylate glucose and f r u c t o s e , with fructose being phosphorylated at a rate 10% that of glucose (Table I I I ) .  No a c t i v i t y of hexokinase for mannose was observed,  even when excess q u a n t i t i e s of mannose and extract were added. While the f a i l u r e to detect t h i s enzyme did not completely disprove i t ' s e x i s t e n c e , there remained 1 i t t l e probabi1ity that mannose-kinase was involved in the accumulation of mannose. 14 Studies of the s p e c i f i c i t y of  C-carbohydrate accumulation  mechanism have demonstrated the opposite e f f e c t . neither accumulated nor has any a c t i v i t y  Fructose i f  in the i n h i b i t i o n of  glucose accumulation, while mannose is accumulated and i n h i b i t s the rate of glucose uptake by 70%.  It therefore appeared that  the phosphorylation of the carbohydrates during accumulation was performed by a mechanism which d i f f e r e d from that of the soluble hexoki nase. The absence of detectable free glucose or mannose in the  pooled material  indicated that these carbohydrates were  phosphorylated upon transport,  implicating a phosphotrans-  ferase or kinase associated with the membrane. This mechanism of accumulation t e c h n i c a l l y cannot be c o r r e c t l y described as a glucose-concentration mechanism, because a d e r i v a t i v e of glucose and not free glucose was demonstrated to be accumulated.  k. Mechanism of the accumulation of glucose  The accumulation of glucose and mannose in suspensions of C. perfr?ngens has been demonstrated to occur by a system d i s p l a y i n g saturation k i n e t i c s , temperature s e n s i t i v i t y , and energy dependence.  In a d d i t i o n , by i t s high degree of  s p e c i f i c i t y the reaction appeared to be enzymatic in nature. The mechanism was demonstrated to accumulated glucose to concentrations several hundred times the external  concentration,  and t h i s pooled material was shown to be in the form of phosphorylated d e r i v a t i v e s .  The method of phosphorylation was not  the soluble hexokinase, but rather was probably due to a membranebound phosphotransferase or kinase. Metabolism of exogenously-added pyruvate or previously accumulated glucose probably supplied the high energy i n t e r mediates necessary for phosphorylation.  The l i m i t e d soluble  pool s i z e might well have resulted from e i t h e r the depletion of the reserve'of the high energy intermediate, or from the attainment of an equi1ibrium across the phosphorylating mechanism.  This mechanism as proposed bears more than s u p e r f i c i a l resemblance to the phosphorylation mechanisms of accumulation by :S. aureus (Egan, and Morse, 1966) and the phosphoenolpyruvate dependent phospho-transferase system of accumulation of carbohydrates in E. col? (Simoni et a l . , 1967). The advantages of possession of such an accumulation mechanism to the c e l l are s e v e r a l .  The carbohydrate which served as the  most common energy source in the organism's normal environment was e f f i c i e n t l y scavenged from the external medium and the energy expended by the accumulation mechanism simultaneously served t o . a c t i v a t e the carbohydrate for metabolism by glycolysis.  GENERAL DISCUSSION  Necrotic mammalian t i s s u e during pathological  conditions,  the lower region of the mammalian d i g e s t i v e system under normal c o n d i t i o n s , and s o i l , are known to be the three major e c o l o g i c a l systems of C. perfr?ngens (Breed, Murray, and Smith, 1957). There are two major mechanisms by which t h i s organism may survive extended periods of exposure to the d e f i c i e n c y of nutrients  in s o i l .  C_. perfringens is known to form spores,  and as c e r t a i n s t r a i n s are important causative agents of food poisoning, extensive studies of the formation, s u r v i v a l , and germination of the organism's spores have been published ( C o l l e e , Knowlden, and Hobbs, 1961; Weiss, and Strong, 1967). Studies in c u l t u r a l conditions have indicated that only under a l k a l i n e c o n d i t i o n s , with a source of carbohydrate present, w i l l spores be formed (Wi11 i s , 1964).  These conditions  occur in the lower region of the mammalian d i g e s t i v e system, so considerable numbers of spores may be excreted in wastes, to u l t i m a t e l y be d i s t r i b u t e d  in the s o i l .  The maintenance of the i n t e g r i t y , over 18 h r s , of the vegetative c e l l s grown on the peptone-less, semi-defined medium, would seem to provide an a l t e r n a t i v e explanation for the s u r v i v a l of £ . perfringens in the austere conditions of  the s o i l .  In the absence of substrates for the  proteolytic  exo-enzymes, the formation of the a u t o l y t i c enzyme system would be repressed, and the magnesium present in the s o i l d would repress the small ^amounts of a u t o l y t i c present.  activity  The c e l l s could u t i l i z e any carbonaceous or  nitrogenous endogenous supply of energy that they may possess, and the v i a b i l i t y and i n t e g r i t y of the vegetative c e l l s could be maintained for extended periods of time. Any glucose or mannose present in the s o i l , could be immediately accumulated to l e v e l s far above the external concentration.  Other carbohydrates which would support growth  could not be accumulated, but rather could be rapidly transported into the c e l l by f a c i l i t a t e d  diffusion.  During growth in necrotic mammalian t i s s u e s , the supply of glucose and nutrients would allow rapid growth.  Peptides  would be present, and the production of the toxins upon which the pathogenicity of £ . perfringens depends, would be induced. Induction of the a u t o l y t i c enzymes by the presence of peptides would not destroy the c u l t u r e , as the presence of an energy source has been shown to repress the a c t i v i t y of the a u t o l y t i c enzymes. The accumulated glucose would be metabolized v i a the Embden-Meyerhof pathway, with the heterofermentative  production  of l a c t i c , a c e t i c , and b u t y r i c a c i d s , in addition to the carbon-dioxide and molecular hydrogen which produce the gaseous d i s r u p t i o n of t i s s u e in "gas-gangrene".  Five-carbon sugars  for the production of n u c l e i c acids may be produced by the t r a n s k e t o l a s e / t r a n s a l d o l a s e system, from fructose-6-phosphate and from 3-phospho-glyceraldehyde, in the absence of a hexose-monophosphate pathway or complete pentose c y c l e . To provide the energy required f o r the rapid growth, the maintenance of a low oxidation p o t e n t i a l , and the extensive production of e x o - t o x i n s , required f o r the pathogenic s t a t e , the organism could concentrate the a v a i l a b l e glucose from the t i s s u e by the economical means of  activating  the glucose, through phosphorylation, for g l y c o l y s i s .  Thus,  in one step the organism would simultaneously r e t a i n the carbohydrate molecule w i t h i n the c e l l , and prepare i t f u r t h e r metabolism.  for  This system is twice as e f f i c i e n t as  that which has been proposed for the accumulation of g galactosides in E_. co 1 i , by the use of one mole of ATP to concentrate one mole of carbohydrate through a l t e r i n g a f f i n i t y of the transport mechanism (Kepes,  the  1960).  An organism which used t h i s system, would then be required to expend another mole of ATP to phosphorylate the carbohydrate during g l y c o l y s i s . The other Gram-positive organism which has been studied f o r carbohydrate t r a n s p o r t , S. aureus, accumulated eight d i f f e r e n t sugars v i a permeases highly s p e c i f i c f o r each carbohydrate, coupled to a general membrane c a r r i e r mechanism (Egan, and Morse,  1966).  The evolution of C. perfringens  "'into a form which, in nature, grows rapidly only in mammalian  systems, where the major soluble carbohydrate is glucose, may have been related to the loss of the accumulation mechanisms for carbohydrates other than glucose.  A study  of the s p e c i f i c i t y of the transport mechanism for nonaccumulated carbohydrates in C. perfringens would serve to c l a r i f y t h i s concept, but has proven to be t e c h n i c a l l y difficult.  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