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Studies on the respiratory enzymes of the lactic acid and nitrogen-fixing bacteria Morgan, Joseph Francis 1942

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* STUDIES ON THE RESPIRATORY ENZYMES OF THE LACTIC ACID AND NITROGEN-FIXING BACTERIA  by Joseph F r a n c i s Morgan  A Thesis submitted•in P a r t i a l F u l f i l m e n t of The Requirements f o r the Degree o f MASTER OF SCIENCE IN AGRICULTURE  THE UNIVERSITY OF BRITISH COLUMBIA August, 1?42»  Contents  PART I  - FINAL REPORT TO THE NATIONAL RESEARCH  COUNCIL  PART I I - STUDIES ON RESPIRATORY ENZYMES 1. The Dehydrogenase Enzymes o f t h e R h i z o b i a . 2. V a r i a t i o n i n R e s p i r a t o r y Enzymes o f t h e Rhizobia. 3 . Rhizobium t r i f o l i i and Zoning Phenomena. 4• The Dehydrogenase Enzymes o f t h e L a c t i c Acid Bacteria. 5o O x i d a t i v e R e s p i r a t i o n o f S t r e p , l a c t i s . 6. R e s p i r a t i o n and F e r m e n t a t i o n by S t r e p * lactis. 7• I n f l u e n c e o f A d a p t a t i o n upon t h e R e s p i r a t i o n of Strep, l a c t i s . 8. I n f l u e n c e o f N i t r o g e n Source on F e r m e n t a t i o n and R e s p i r a t i o n *  STUDIES OH THE RESPIRATORY ENZYMES OF THE LACTIC ACID AND NITROGEN-FIXING BACTERIA  fines! B e p a r t o f . VJork c a r r i e s out tiader a .National R e s e a r c h C o u n c i l 'Bursarf i n A g r i c u l t u r a l B a c t e r i o l o g y -at '.til© . U a i v s r s i t y o f B r i t i s h , Columbia  Joseph f» Morgan  Sua.*! I f 4 2  gable: o f .Content's ' ''"*"- •  %,.. GENERAL SUI^&RY. » II. III.  2MTE0DU0TX0N  , .  1  .....................  4  .  .  r  8  EXPERIMENTAL' VTORK ' A. L a c t i c A c i d S t r e p t o c o c c i 1# -Aerobic O x i d a t i o n s ••»•«••••«•»•* 2, Fermentation 3,  anfi O x i d a t i o n ......  11  Peptone and A c i d P r o d u c t i o n •... .  12  4, Adaptiire*»Gonetitutive Bnaymeo B  8  8  16  Rhisobia !• Dehydrogenase Enzymes .« 2, A e r o b i c and Anaerobic  <  18  Oxidation »  20  3. Substrain Variation  22  4«*Zoning Phenomena •••*•••.........  28  If*  DISCUSSIOB OF RESULTS ,*.»..•,....».'».»...,  32  V.  CONCLUSION • ,,«*.,.•..•«.».•*•«....••.•*••.*  35  BIBLIOGRAPHY ,.. ....  • |6  I . GENERAL SUMMARY. 5?ho problem under i n v e s t . I g a t i o n has been "A Study o f t h e R e s p i r a t o r y Enzymes o f t h e L a c t i c A c i d and -Hitffogen-'Fixing Bacteria". T h i s study has e n t a i l e d a c o n s i d e r a t i o n o f both t h e a e r o b i c and a n a e r o b i c groupso  r e s p i r a t o r y mechanisms o f those two b a c t e r i a l  The dehydrogenase a c t i v i t y o f s e v e r a l s t r a i n s o f  R h i s o b i u n t r i f o l i i upon s i x t y o r g a n i c  compounds has been d e t e r -  mined by t h e methylene blue r e d u c t i o n technique The  o f Thunberg.  a e r o b i c o x i d i s i n g a b i l i t y o f both t h o L a c t i c A c i d S t r e p t o -  c o c c i and t h e N i t r o g e n - F i x i n g R h i z o b i a upon s e l e c t e d carbon sources has been determined q u a n t i t a t i v e l y by t h e B a r e r o f t manometric t e c h n i q u e .  The mechanism o f l a c t i c a e i d  production  by trashed b a c t e r i a l c e l l s has been i n v e s t i g a t e d , employing t h e method i n t r o d u c e d by H e g a r t y .  The a d a p t i v e  or c o n s t i t u t i v e '  c h a r a c t e r o f t h e b a c t e r i a l enzymes concerned has a l s o b©@a termined i n t h i s manner.  An e x t e n s i v e i n v e s t i g a t i o n has beon  conducted i n t o the problem o f v a r i a t i o n i s r e s p i r a t o r y a c t i v i t y  among s t r a i n s and. s u b s t r a i n s of Rhlzobiuia t r l f o l i l * s p e c i e s the i n f l u e n c e o f l a b o r a t o r y media upon growth  With t h i s char-  a c t o r s , r e s p i r a t o r y mechanism, and p h y s i o l o g i c a l n a t u r e hae a l s o boon studied,,  -  The e x p e r i m e n t a l r e s u l t s o b t a i n e d show d e f i n i t e l y t h a t the dehydrogenase a c t i v i t y o f Rh. t r i f o l i i  i s extremely  v a r i a b l e w i t h i n s t r a i n s o f the same s p e c i e c and even w i t h t h e same s t r a i n - at d i f f e r e n t t i m e s .  This v a r i a b i l i t y i n anaerobic  r e s p i r a t i o n r e n d e r s the methylene b l u e r e d u c t i o n t e s t o f l i t t l e value as a b a s i c - f o r c l a s s i f i c a t i o n o f these organisms*  The  oxygen uptake by trashed c e l l s of So, l a c t l s i n the presence o f v a r i o u s carbohydrates has been found t o be i r r e g u l a r i n manner and l i m i t e d i n degree.  No r e l a t i o n s h i p was found t o e x i s t  between the a n a e r o b i c o x i d a t i o n , the a e r o b i c o x i d a t i o n and the f e r m e n t a t i o n of c a r b o h y d r a t e s by t h i s organism. p r o d u c t i o n by washed b a c t e r i a l c e l l s was  Lactic acid  found t o be dependent  upon the presence of s m a l l amounts o f a n i t r o g e n sourc©tS'Qoa as peptone, a l o n g w i t h the c a r b o h y d r a t e , r a t h e r than upon t h e presence o r absence o f a v a i l a b l e oxygen.  '  By the a d a p t i v e -  c o n s t i t u t i v e enzyme method i t has been shown t h a t the L a c t i c A c i d S t r e p t o c o c c i u t i l i z e l a c t o s e as a carbohydrate source by f i r s t s p l i t t i n g t h e d i s a c c h a r i d e molecule i n t o i t s c o n s t i t u e n t monosaccharides, In  g l u c o s e and g a l a c t o s e .  i n v e s t i g a t i n g t h e r e s p i r a t i o n of t h e Rhisobium s p e c i e s  i t has been found p o s s i b l e to separate a s i n g l e s t r a i n o f t h i s  organism I n t o a l a r g e number o f s u b s t r a i n s which d i f f e r from one another and from the parent s t r a i n anaerobic r e s p i r a t o r y a c t i v i t y .  i n both a e r o b i c  and  With t h i s s p e c i e s i t has  also  been shown t h a t growth on l a b o r a t o r y media r e s u l t s i n a change i n the p h y s i o l o g y  of the organism,  T h i s change  Is.strikingly  i l l u s t r a t e d by the development of c l e a r e d sones about the c o l o n i e s when the c u l t u r e i s p l a t e d on Wilson's Agar. as c i r c u l a r  cleared 0ones  8  secondary soaes of h a a i i i e s s oj?  d e p o s i t i o n were a l s o d e t e c t e d from the c o l o n i e s .  The  As w e l l  i n the medium at some d i s t a n c e  appearance of t h e s e aoning pheomena i s  a s s o c i a t e d w i t h a change i n b o t h a e r o b l o  and  anaerObla  r e s p i r a t o r y a c t i v i t y o f the c u l t u r e and a l s o w i t h a change i n colonial  character.  Passage of t h e c u l t u r e through s o i l  e l i m i n a t e s the zoning' e f f e c t s and to i t s normal form.  causes the c u l t u r e to r e v e r t  II.  INTRODUCTION  '»  B i o l o g i c a l o x i d a t i o n o r r e s p i r a t i o n has been d e f i n e d (6) as t h e cum o f a l l those processes i n t h e l i v i n g  c e l l by which  oxygen i s i n t r o d u c e d i n t o t h e system and carbon d i o x i d e removed*  In i t s broadest eeneo„ r e s p i r a t i o n may be s a i d t o  i n v o l v e a l l chemical r e a c t i o n s i n l i v i n g energy.  c e l l o \~hich r e l e a s e  The study o f b a c t e r i a l r e s p i r a t i o n w i l l therefor©  i n v o l v e a c o n s i d e r a t i o n o f both a e r o b i c and a n a e r o b i c o x i d a t i v e processes* L i v i n g c e l l s o b t a i n t h e energy f o r t h e i r v i t a l processes. throvxgh t h e o x i d a t i o n o f compounds such as c a r b o h y d r a t e s , w i t h the o v o l u t i o n o f carbon d i o x i d e and t h e f o r m a t i o n o f -water and v a r i o u s end p r o d u c t s ,  The exact mechanism by which t h i s o x i d a -  t i o n i o c a r r i e d out has never been c o m p l e t e l y e s t a b l i s h e d .  It  i s b e l i e v e d t o c o n s i s t o f an enzymic c a t a l y s i s , t h e enzymes concerned b e i n g termed " r e s p i r a t o r y enzymes" •  I n ..this process  the f i r s t s t e p i s b e l i e v e d t o be t h e s p l i t t i n g  o f f of active  hydrogen from t h e o x i d i s a b l e compound, through t h e agency o f dehydrogenase enzymes.  T h i s hydrogon i s then passed over a  complex s e r i e s o f o x i d a t i o n - r e d u c t i o n systems w i t h l a t h e c o l l until  i t i s finally  converted i n t o water  0  During t h i s passage  the hydrogen g r a d u a l l y g i v e s up i t s energy t o t h e c e l l i n such a form t h a t i t can be immediately  utilised*.  The  problem o f b a c t e r i a l r e s p i r a t i o n has been  investigated University  extensively  by J . H. Q u a s t e l and h i s co-workers a t Cambridge ( 7 , 8, 9 ) .  Sine© t h e i r p i o n e e r trork, a good d e a l  of r e s e a r c h Into t h i s q u e s t i o n has been c a r r i e d , out „ m a i n l y i s England.  However, no s y s t e m a t i c i n v e s t i g a t i o n i n t o t h e  r e s p i r a t i o n o f t h e L a c t i c A c i d B a c t e r i a has y e t been r e c o r d e d . The  r e s p i r a t i o n o f t h e Rhlzobium s p e c i e s has been s t u d i e d  to a  c o n s i d e r a b l e extent by P. VJ. W i l s o n and t h e W i s c o n s i n r e s e a r c h workers ( l , 12, 14) • The  study o f b a c t e r i a l r e s p i r a t i o n has been g r e a t l y advanced  by t h e employment o f t h e " r e s t i n g c e l l " t e c h n i q u e .  In t h i s  method t h e organisms a r e employed as a suspension made up o f c e l l s which have been washed f r e e o f a l l t r a c e s o f c u l t u r e medium by repeated c e n t r i f u g i n g .  The use o f " r e s t i n g c e l l s " has  made i t -possible t o d i s s o c i a t e t h e phenomena o f growth and r e s p i r a t i o n and t o c a r r y out d e t a i l e d s t u d i e s on t h e r e s p i r a t o r y mechanism f r e e from t h e c o m p l i c a t i n g f a c t o r s o f growth and multipiicat1on•  I n t h e work r e p o r t e d upon h e r e i n t h e " r e s t i n g  c e l l " t e c h n i q u e has been employed almost The veniently  exclusively.  i n v e s t i g a t i o n o f anaerobic oxidations  I s most con-  c a r r i e d o u t by t h e method i n t r o d u c e d by Thunberg ( 1 3 ) .  In t h i s procedure t h e compound whose o x i d a t i o n  i s t o be t e s t e d  i s mixed w i t h a s u s p e n s i o n o f t h e organism b e i n g s t u d i e d d i l u t e s o l u t i o n o f methylene b l u e i s added.  A i r I s then  and a  removed from t h e tube by means o f a vacuum pump and o x i d a t i o n measured ae t h e time R e q u i r e d f o r d i s c o l o r i z a t i o n o f t h e methf len©.. .bltiei • • A e r o b i c o x i d a t i o n by t h e " r e s t i n g c o l l " t e c h n i q u e may be c a r r i e d out by s e v e r a l  raanomotric  procedures.  The apparatus  employed i n t h i s study has been t h e d i f f e r e n t i a l manometer i n t r o d u c e d by B a r e r o f t •  '•  For a d e t a i l e d d e s c r i p t i o n of t h i a  apparatus t h e monograph by Dixon (2) should be c o n s u l t e d . Considerable  c o n f u s i o n has been c r e a t e d i n t h e l i t e r a t u r e  by t h e use o f many d i f f e r e n t terms t o d e s c r i b e t h e processes of b l o l o g i o a l o x i d a t i o n .  I n t h i s r e p o r t t h e term "dehydrogenation"  w i l l be used t o d e s c r i b e anaerobic  o x i d a t i o n , w h i l e t h e term  " o x i d a t i o n " w i l l be c o n f i n e d t o t h e p u r e l y a e r o b i c mechanism. The  work h e r e i n r e p o r t e d on t h e R e s p i r a t o r y Ensymes o f t h e  L a c t i c A c i d and N i t r o g e n - F i x i n g B a c t e r i a r e p r e s e n t s an e x t e n s i o n of work p r e v i o u s l y c a r r i e d out on t h e Dehydrogenase Enzymes o f the L a c t i c A c i d B a c t e r i a (j>) and i n c l u d e s a study "of t h e a e r o b i c r e s p i r a t o r y a c t i v i t y o f these groups o f organisms.  The  dehydrogenase a c t i v i t y o f t w e l v e s p e c i e s o f L a c t i c A c i d S t r e p t o c o c c i on s i x t y t e a t compounds, i n c l u d i n g c a r b o h y d r a t e s ,  organic  a c i d s , amino a c i d s and a l c o h o l s , had a l r e a d y been determined. On account o f t h e marked v a r i a b i l i t y i n dehydrogenase a c t i v i t y , I t had been found I m p o s s i b l e t o use t h i s c h a r a c t e r i s t i c as a b a s i s f o r t h e c l a s s i f i c a t i o n o f t h i s group o f microorganisms®  I n an attempt t o e x p l a i n t h i s v a r i a b i l i t y i n o x i d a t i v e s t u d i e s have been c a r r i e d out on the enzyme q u e s t i o n  ability  adaptive-constitutive  and on the mechanism of l a c t i c a c i d  production,  employing the method e s t a b l i s h e d by H e g a r t y (4-3 f o r t h i s type of i n v e s t i g a t i o n .  In t h i s procedure two  per cent of  carbohydrate t o be t e s t e d i s added to a suspension of organism under study and  l a c t i c a c i d production  the the  i s then d e t e r -  mined by t i t r a t i n g samples o f the m i x t u r e at h a l f - h o u r l y i n t e r v a l s over an e i g h t - h o u r p e r i o d . The  determination  o f a e r o b i c and a n a e r o b i c r e s p i r a t o r y  enayrae c o n s t i t u t i o n has a l s o been a p p l i e d t o the  Nitrogen-  F i x i n g R h i z o b i a i n an attempt t o f i n d a p h y s i o l o g i c a l b a s i s f o r the c l a s s i f i c a t i o n o f t h i s group o f organisms.  T h i s study has  s u b s e q u e n t l y b©en extended to' an i n v e s t i g a t i o n of the  problems  o f s t r a i n v a r i a b i l i t y w i t h i n t h i s s p e c i e s and the e f f e c t of l a b o r a t o r y media upon t h e i r p h y s i o l o g y  and r e s p i r a t o r y a c t i v i t y .  T h i s work has been c a r r i e d out at The U n i v e r s i t y o f B r i t i s h Columbia i n the Departments o f D a i r y i n g and Agronomy, t o express my  s i n c e r e thanks t o Dr. B. A. E a g l e s and  L a i r d f o r t h e i r u n f a i l i n g i n t e r e s t and prosecution  of t h i s  research.  I wieh Dr. D.  encouragement i n the  G.  112,  EXPERIMENTAL WORK  A. THE LACTIC 'ACID STREPTOCOCCI Respiratory  s t u d i e s on t h e L a c t i c Aolfi S t r e p t o c o c c i have  heen c a r r i e d out, employing t h e "washed c e l l " t e c h n i q u e *  The  c u l t u r e medium used i n the p r e p a r a t i o n o f t h e b a c t e r i a l suspensions has c o n s i s t e d o f C a s e i n -Digest B r o t h , prepared' ' a f t e r the manner o f O r l a Jensen (3) , c o n t a i n i n g 0.5?» T o t a l N i t r o g e n and e n r i c h e d  w i t h 1 . 0 $ Dlfco "Zeaet E x t r a c t , 0.5f°  KgHPO^, and 0.5/° Glucose. have been:  The c u l t u r e s employed i n these s t u d i e s  Sc. l a c t i s . S.A, 3 0 , a t y p i c a l Sc. l a c t i s i s o l a t e d  from b u t t e r p o s s e s s i n g 374, o b t a i n e d  a caramel f l a v o u r (11) J Sc. l a c t i s A.T.C.  from the N a t i o n a l Type C u l t u r e C o l l e c t i o n a t  Washington, D.C.5  Sc. l a c t i s EHBg 1,  I s o l a t e d from a mature K i n g s t o n  a starch-fermenting  cheese.  strain  F o r comparative pur-  poses s t u d i e s have a l s o been c a r r i e d out employing B a c t • c o l l A.T.C. 4157 1.  o f tho American Type C u l t u r e C o l l e c t i o n .  Aerobic  Oxidations  The a e r o b i c r e s p i r a t o r y a c t i v i t y  o f the L a c t i c A c i d  S t r e p t o c o c c i has been s t u d i e d , employing the B a r c r o f t manometric technique.  The v a l u e s  expressed g r a p h i c a l l y i n F i g u r e s 1,  4 and 5 have been s e l e c t e d as t y p i c a l o f t h e r e s u l t s obtained.  2,  experimental  These graphs p o r t r a y the o x i d a t i v e a b i l i t y  o f Sc. l a c t i s A.T.C. j}74 upon c e r t a i n o f the carbohydrates  3,  tested*  Very s i m i l a r r e s u l t s have been obtained  employing  •So* l a e t i e S,A* ..JO-. In F i g u r e 1 and F i g u r e 3 oxygen uptake i n t h e presence o f v a r i o u s carbohydrates expressed  i s shown g r a p h i c a l l y , V a l u e s a r e  as c u b i c m i l l i m e t e r s o f oxygen u t i l i z e d per m i l l i g r a m  of dry c e l l weight over a one-hour p e r i o d * C o n t r o l d e t e r m i n a t i o n s have a l s o been c a r r i e d o u t , i n Triileh  oxygon uptake by the c e l l suspension  has been measured  i n the e n t i r e absence o f c x i d i z a b l e substance* o b t a i n e d i s known as the endogenous r e s p i r a t i o n .  The value so The mechanism  o f endogenous r e s p i r a t i o n has not y e t been e s t a b l i s h e d , but i t i s b e l i e v e d (14) t o c o n s i s t o f an o x i d a t i v e deamination o f c e l l u l a r amino a c i d s , u t i l i z i n g energy s o u r c e .  t h e c e l l p o l y s a c c h a r i d e as an  The s i g n i f i c a n c e of t h e a p p r e c i a b l e  aerobic  endogenous r e s p i r a t i o n w i t h t h e L a c t i c A c i d S t r e p t o c o c c i i s not c l e a r , s i n c e i t has a l r e a d y been shov;n (5) t h a t t h e s e organisms possess no d e t e c t a b l e anaerobic  endogenous r e s p i r a t i o n ,  Prom t h e curves shown i n F i g u r e s 1 and 3 i t i s apparent t h a t the oxygen uptake i s c h a r a c t e r i s t i c o f the substance b e i n g oxidised,.  Among the monosaccharides, g l u c o s e and f r u c t o s e are  s t r o n g l y o x i d i s e d , w h i l e g a l a c t o s e and arabinos© show an o x i d a t i o n almost i d e n t i c a l w i t h t h a t o f t h e endogenous. The r e s u l t s t a b u l a t e d i n f i g u r e 10 show t h a t t h i s v e r y  slight  10 o x i d a t i o n o f g a l a c t o s e i s due t o t h e presence o f an a d a p t I T © r a t h e r t h a n a c o n s t i t u t i v e easyme« ... Among t h e o t h e r c a r b o h y d r a t e s , d e x t r i n i s e s p e c i a l l y r a p i d l y o x i d i z e d , w h i l e l a c t o s e and r a f f i n o s e a r e o x i d i s e d t o a much l e s s e r e x t e n t .  With a d o n l t o l and m e l e a i t o s e , however,  the o x i d a t i o n a g a i n i s almost  ngliglble.  I n F i g u r e s ?. and 4 i s shown t h e carbon d i o x i d e product i o n from t h e carbohydrate  sources employed i n F i g u r e s 1 and 5*  In these graphs a l s o t h e r e appears a s i g n i f i c a n t endogenous respiration.  I n g e n e r a l t h e curves f o r carbon d i o x i d e produc-  t i o n correspond c l o s e l y t o t h e curves f o r oxygen uptake. i m p l i e s t h a t t h e o x i d a t i o n tends t o be c a r r i e d through  This  completely  to t h e carbon d i o x i d e s t a g e . In F i g u r e s £{a) and 5 ( b ) the . r e l a t i o n s h i p between acygen uptake and carbon d i o x i d e output i s shown by p l o t t i n g t h e R e s p i r a t o r y Q u o t i e n t s , which a r e o b t a i n e d by d i v i d i n g th© volume o f oxygen taken up by t h e volume o f carbon d i o x i d e g i v e n o f f . I n many cases t h e R e s p i r a t o r y Q u o t i e n t i s a s t r a i g h t l i n e o r tends toxvards a s t r a i g h t l i n e , as would n o r m a l l y be expected. o t h e r c a s e s , however, t h e R e s p i r a t o r y Q u o t i e n t s show a marked i r r e g u l a r i t y which can o n l y be e x p l a i n e d by assuming a sudden s h i f t i n c e l l metabolism i n s l o p e o f these ourvee«  t o correspond w i t h t h e sudden changes •  • ~  In  2, Fermentation  and O x i d a t i o n  I n Table 1 t h e comparative  dohydrosanations,  fermenta-  t i o n s , and a e r o b i c o x i d a t i o n s o f these same carbohydrates  by  Be, l a c t i s A.T.C. 374 have been summarized, A study o f t h i s t a b l e shows t h a t t h e r e i s no apparent i n t e r r e l a t i o n s h i p between t h e processes o f a e r o b i c o x i d a t i o n , anaerobic o x i d a t i o n and f e r m e n t a t i o n .  With, glucose „ f r u c t o s e  and l a c t o s e a l l t h r o e processes a r e c a r r i e d out s t r o n g l y .  With  arabinose and a d o n l t o l a l l t h r e e processes a r e e i t h e r very weak or e n t i r e l y negative.  With g a l a c t o s e and m e l e s l t o s e , however,  a e r o b i c and a n a e r o b i c o x i d a t i o n are weak, w h i l e a c i d is f a i r l y high.  production  With r a f f i n o S e and d e x t r i n a e r o b i c o x i d a t i o n  i s s t r o n g , w h i l e both a n a e r o b i c o x i d a t i o n and a c i d p r o d u c t i o n are very weak  0  From t h e r e s u l t s i n Table 1,  i t i s apparent t h a t an  organism may be a b l e t o o x i d i s e a compound which i t cannot dohydrogenate and cannot ferment.  I t may be able t o dohydro-  genate a compound which i t cannot o x i d i s e and cannot  ferment•  And i t may a l s o be a b l e t o ferment a compound which i t cannot o x i d i z e and cannot dehydrogenate.  These o b s e r v a t i o n s stand l a  d i r e c t c o n t r a d i c t i o n t o t h e g e n e r a l l y accepted  theories of  b i o l o g i c a l o x i d a t i o n , which h o l d t h a t a e r o b i c and anaerobic r e s p i r a t i o n are merely phases o f t h e same g e n e r a l mechanism o f which f e r m e n t a t i o n i s a measure o f t h e end-producto.  5» Peptone and A c i d P r o d u c t i o n The r e s u l t s r e p o r t e d I n Table 1 showed c l e a r l y t h a t l a c t i c a c i d p r o d u c t i o n was dependent upon some f a c t o r o t h e r t h a n a e r o b i c o r anaerobic o x i d a t i o n .  I t was therefor© d e t l d e d  t o i n v e s t i g a t e the a d a p t i v e - c o n s t i t u t i v e enzyme q u e s t i o n w i t h the L a c t i c A c i d S t r e p t o c o c c i i n an e f f o r t t o e x p l a i n t h e l a c k o f c o r r e l a t i o n between f e r m e n t a t i o n and o x i d a t i o n , and a l s o t o determine what s p e c i f i c I n f l u e n c e was e x e r t e d by t h e n i t r o g e n source, Hegarty ( 4 , 10) i n t r o d u c e d a method by which t h e a d a p t i v e or c o n s t i t u t i v e nature o f r e s p i r a t o r y enzymes c o u l d be determined by t i t r a t i n g t h e l a c t i c a c i d produced from carbohydrates washed suspension o f organisms.  by a  For a c i d p r o d u c t i o n t o take  p l a c e , he found i t necessary t o I n c l u d e O.J$ peptone i n t h e buffer mixture.  However, he d i d not i n v e s t i g a t e t h e p a r t p l a y e d  by peptone i n a c i d p r o d u c t i o n . The  procedure as d e t a i l e d by Hegarty (4) was t h e r e f o r e  c a r r i e d out and t^as extended to cover both a e r o b i c and anaerobic fermentation.  A b u f f e r m i x t u r e was prepared  o f peptone added.  and v a r y i n g amounts  To t h e m i x t u r e was added 2% g l u c o s e and  washed b a c t e r i a l c e l l s t o form a i f . s u s p e n s i o n .  For the aerobic  experiments samples were withdrawn at h a l f - h o u r l y i n t e r v a l s and t i t r a t e d w i t h 0»1 N sodium h y d r o x i d e .  The anaerobic  experiments  were c a r r i e d out i n a vacuum f l a s k under an atmosphere o f n i t r o g e n and* the apparatus so arranged  t h a t f r e s h methylene  blue c o u l d be added as r e d u c t i o n took place.,  Samples f o r  t i t r a t i o n were drawn o f f by s u c t i o n .  A l l samples were t i t r a t e d  t o pH 7 0 ,  The  o  u s i n g a Beckiaan pH meter.  summarised l a F i g u r e s 7  9  8* 9 and 1 0 ,  r e s u l t s obtained  l a a d d i t i o n t o pH  a c i d p r o d u c t i o n , the oxygen uptake by these v a r i o u s was a l s o determined.  These r e s u l t s are r e c o r d e d  are  and  mixtures  i n Figure  H,  F i g u r e 7 shows t h e change i n pH under a e r o b i c c o n d i t i o n s d u r i n g the course of t h i s experiment, w h i l e F i g u r e 8 shows the change i n pH under anaerobic  conditions.  The a d d i t i o n o f peptone  to the b u f f e r m i x t u r e has•caused a v e r y marked decrease i n pH under both a e r o b i c and anaerobic  conditions.  When more t h a n  0,25$ peptone has been added, however, the pH v a l u e s a l l tend to reach a constant  l e v e l at about pH  4,5*  F i g u r e 9 shows the i n f l u e n c e of peptone c o n c e n t r a t i o n upon l a c t i c a c i d p r o d u c t i o n under a e r o b i c c o n d i t i o n s , w h i l e F i g u r e 10 shows the c o r r e s p o n d i n g anaerobic  conditions•  r e s u l t s o b t a i n e d under  From the v a l u e s shown i n F i g u r e s 9 and  10  i t i s apparent t h a t l a c t i c a c i d p r o d u c t i o n has i n c r e a s e d w i t h i n c r e a s i n g peptone c o n c e n t r a t i o n . to r e a c h a constant  l e v e l , as was  There i s , however, no tendency found w i t h the pH v a l u e .  c o n t i n u e d i n c r e a s e i n t i t r a t a b i e a e l d l t y i s due to t h e h i g h  This  14 » b u f f e r i n g power of the phosphate mixture used, which has the pH at 4.5  held  w h i l e a l l o w i n g the t i t rat-able a c i d i t y t o • i n c r e a s e  regularly. I n F i g u r e 11 i s recorded the 'volume of oxygen taken by these v a r i o u s r e a c t i o n m i x t u r e s .  up  Under these c o n d i t i o n s ' -  the amount of oxygen absorbed has been found t o depend d i r e c t l y upon the cone eat r a t i o n of peptone.  Tinea 0*125$ peptone i s added  to the b u f f e r m i x t u r e , the oxygen uptake i s doubled! when i f . peptone i s added •  B  the oxygen uptake i s i n c r e a s e d s i x t i n e s ,  From F i g u r e s 7, 8, 9, 10 and 11 the marked i n f l u e n c e  of peptone i s apparent.  The  g r e a t e s t e f f e c t appears t o be  e x e r t e d i n the oxygen u p t a k e , where the s t i m u l a t i o n i s f a r g r e a t e r than c o u l d be e x p l a i n e d on t h e b a s i s of n i t r o g e n content The  alone,.  a d d i t i o n o f peptone has r e s u l t e d i n a marked decrease i s pH,  a n o t i c e a b l e r i e e i n titratabl© a c i d i t y , and a v e r y marked i n c r e a s e i n oxygen uptake,  A s i m i l a r stimulation"was  be e x e r t e d under both a e r o b i c and anaerobic  found to'  conditions.  Since  the change i n pH i s not p r o p o r t i o n a l t o peptone c o n c e n t r a t i o n . * but tends to reach a constant  l e v e l for a l l concentrations,  s i n c e both oxygen uptake and l a c t i c a c i d p r o d u c t i o n  and  a*©  p r o p o r t i o n a l t o peptone c o n c e n t r a t i o n , i t seems probable  that  t h e r e i s a v e r y c l o s e i n t e r r e l a t i o n s h i p bettreen ferment at l e a and oxygon u p t a k e .  T h i s c l o s e i n t e r r e l a t i o n s h i p , however, i s  not t h a t p o s t u l a t e d by t h e Glass t e a l Past©nj?-SIeywho£ t h e o r y i n ffnioh f e r m e n t a t i o n was s t i m u l a t e d by a decreased oxygen supply.  The r e s u l t s recorded  her© a r e r a t h e r t h e r e v e r s e o f  t h a t t h e o r y , s i n c e i t has been -shown that i n c r e a s e s oxygefc uptake i s a s s o c i a t e d w i t h i n c r e a s e d f e r m e n t a t i o n ,  These  experiments have i n d i c a t e d t h a t t h e mechanism o f f e r m e n t a t i o n does not depend upon t h e presence o r absence o f oxygen, but depends r a t h e r upon t h e presence o f a s u i t a b l e n i t r o g e n source in the reacting mixture.  4. Adapt j ye-Pon'st i t lit i v e • Snsyiae g. The a p p l i c a t i o n o f Hegarty*8 method t o the s t u d y .of A d a p t i v e - C o n e t i t u t i v e enzymes i s shown i n F i g u r e 1 2 .  The  washed ©ells were prepared from a g l u c o s e b r o t h c u l t u r e .  The  c o l l and b u f f e r m i x t u r e were made up as b e f o r e , and 0 . 5 $ peptone added.  To t h i s m i x t u r e was added 2$ o f t h e carbo-  hydrate t o be t e s t e d . a c i d i t y determined  The m i x t u r e was i n c u b a t e d , and t i t r a t a b l e  as p r e v i o u s l y d e s c r i b e d .  Where the organism  possesses a c o n s t i t u t i v e enzyme c o n t r o l l i n g dchydrogenation the c a r b o h y d r a t e , a c i d p r o d u c t i o n w i l l ocour a t once. where t h e organism possesses  of  However,  an a d a p t i v e enzyme f o r t h e carbo-  hydrate t e s t e d , a c o n s i d e r a b l e p e r i o d o f time w i l l e l a p s e b e f o r e a c i d I s produced i n any a p p r e c i a b l e q u a n t i t y .  This d i s t i n c t i o n  I D c l e a r l y shown i n F i g u r e 1 2 , where t h e organism  possesses  c o n s t i t u t i v e enzymes f o r g l u c o s e , mannose and f r u c t o s e , butpossesses an a d a p t i v e enzyme f o r g a l a c t o s e . I t i s b e l i e v e d t h a t a c o n s t i t u t i v e enzyme e x i s t s as an i n t e g r a l p a r t o f t h e c e l l s t r u c t u r e and I s t h e r e f o r e always present i n the c e l l i n an a c t i v e form.  An a d a p t i v e enzyme, on  the o t h e r hand, i s b e l i e v e d t o e x i s t i n t h e c e l l i n an i n a c t i v e s t a t e and t o r e q u i r e s t i m u l a t i o n or a d a p t a t i o n through  contact  w i t h t h e homologous substance b e f o r e enzymlc a c t i v i t y oan be demonstrated.  >  11 ~  In Figure. 1£ and F i g u r e 2.4- i t i s shown t h a t t h e organism possesses aa a d a p t I T ® essyme f o r l a c t o s e ,  I t i s alee  shown t h a t growth i n t h e presence o f l a c t o s e causes a d a p t a t i o n to b o t h l a c t o s e and g a l a c t o s e * w h i l e growth l a g a l a c t o s e b r o t h does not c a u s e a d a p t a t i o n ' t o l a c t o s e ,  This i n d i c a t e s that the  organism i s able t o u t i l i z e l a c t o s e o n l y t h r o u g h a p r e l i m i n a r y h y d r o l y s i s t o g a l a c t o s e and glucose«  '  I t i s a l s o a o t i e e a b l e t h a t a d a p t a t i o n t© g a l a c t o s e i s not achieved by growing t h e o r g a a i s a i& g a l a c t o s e t>'a*oth, but i s achieved by growing t h e organism i n l a c t o s e b r o t h .  I t has not  yet been p o s s i b l e to determine t h e cause o f t h i s p e c u l i a r adaptation  phettomeaoa*  B* 'BSfZOBlA. '  •  1. Dehydrogenase Enzymes The  dehydrogenase enzymes of the Rhlzobium s p e c i e s have  been i n v e s t i g a t e d ,  employing f i v e s t r a i n s of the Red  organism (Rhiao'biua t r i f o l l i ) and s t r a i n s employed were R.T. and  R.T.  39-1.  The  22B,  s i x t y ' t e s t compounds.  R.T,  224,  H,T,  226,  f i r s t f o u r s t r a i n s were s t o c k  39-1,  was  The  R.T.  231  cultures  o b t a i n e d from the U n i v e r s i t y of W i s c o n s i n i n 1930. s t r a i n , R.T.  clover  The  fifth  i s o l a t e d from red c l o v e r nodules at  U n i v e r s i t y of B r i t i s h Columbia i n 1939. proved by c r o s s - i n o c u l a t i o n  A l l s t r a i n s have been  experiments.  The  r e s u l t s obtained  i n the dohydrogenation t e s t s have been summarised i n Table A l l r e s u l t s have been c a l c u l a t e d The  f i v e s t r a i n s of Rh.  on the b a s i s Glucose  attempt at a r r a n g i n g the R h l z o b i a on the  W i l s o n (14)  and  Tarn and  W i l s o n (12)  the r e s p i r a t o r y enzymee of the R h l z o b i a a l s o v a r i a t i o n i n dehydrogenase a c t i v i t y . a t t a c h any  100.  This v a r i a t i o n i s  of t h e i r anaerobic r e s p i r a t i o n would appear to be of value.  s  2„  t r i f o l i i show a very marked  v a r i a t i o n i n t h e i r dehydrogenase a c t i v i t y . so great t h a t any  The  basis  little  i n t h e i r studies  on  encountered  However, they d i d  g r e a t s i g n i f i c a n c e t o t h i s v a r i a t i o n , but  not  considered  the dehydrogenase a c t i v i t y to bo c h a r a c t e r i s t i c of each Rhlzobium s p e c i e s .  The  r e s u l t s r e p o r t e d upon h e r e i n c o m p l e t e l y  d i s a g r e e w i t h those Of the Wisconsin•'workers,  The marked  v a r i a t i o n i n -anaerobic r e s p i r a t o r y a c t i v i t y w i t h i n f i v e s t r a i n s o f the R l i . t r i f o l l i  s p e c i e s makes I t evident t h a t t h e r e can be-  no s t a b l e s p e c i e s c h a r a c t e r i s t i c s ,  U n t i l such time as t h e  b a s i c f a c t o r s which govern r e s p i r a t o r y a c t i v i t y can be determined any attempt to e s t a b l i s h s p e c i e s c h a r a c t e r i s t i c s w i l l  /  be  futile.  - 20 2* A e r o b i c and Anaerobic  Oxidation.  A s y s t e m a t i c s t u d y o f the r e s p i r a t o r y enzymes o f the R h i z o b l u a s p e c i e s has been undertaken, the Red  employing a s t r a i n o f  c l o v e r organism, Rh. t r l f o l i i 224*  The  study o f the  dehydrogenase enzymes of the R h i z o b i a , r e p o r t e d i n Table 1, emphasized' the extreme v a r i a b i l i t y o f anaerobic within this species.  St was  had  respiration  t h e r e f o r e decided to conduct an  i n v e s t i g a t i o n i n t o the causes o f t h i s v a r i a b i l i t y by s t u d y i n g both the a e r o b i c and a n a e r o b i c r e s p i r a t o r y enzymes o f s t r a i n s and s u b s t r a i n s w i t h i n t h i s group o f organisms. The medium employed i n these e t u d i e s upon tho R h i z o b i a i s t h a t recommended by W i l s o n  (14)•  St c o n s i s t s o f a m i n e r a l  s a l t s base to which are added 1$ yeast e x t r a c t , 0.1$ excess c a l c i u m carbonate, suspension  and 1.5$  agar.  In preparing a  cell  f o r r e s p i r a t o r y s t u d i e s , the c u l t u r e i s grown upon  the s u r f a c e o f t h i s medium i n a l a r g e Roux f l a s k . hours' i n c u b a t i o n at 30° w i t h M/JO  glucose,  C. the growth l o washed from the s u r f a c e  phosphate b u f f e r o f pH 7 . 2 ,  resuspended i n b u f f e r .  A f t e r 48  The  c e n t r i f u g e s , washed, and  c e l l c o n c e n t r a t i o n i e made up t o  2$  by volume and r e s p i r a t i o n experiments ere then c a r r i e d out at 37°  C. Oxygen uptake and dehydrogenase a c t i v i t y were determined  i n the presence o f gluoose, m a n n l t o l , and sodium s u c c i n a t e , Oxygen uptake and methylene blue r e d u c t i o n i n t h e t o t a l absence  of carbon source were a l s o noted.  The  r e s u l t s of t y p i c a l  experiments ^ r e recorded i n F i g u r e s 15 and  16.  I n F i g u r e IS the -curves f o r oxygen uptake i n the presence o f g l u c o s e , m a n n i t o l , sodium succinate,, and water are reported.  With glucose and m a n n i t o l the oxygen Uptake i s  s i g n i f i c a n t l y h i g h , w h i l e w i t h sodium s u c c i n a t e I t i s v e r y  low.  I t i s a l s o n o t i c e a b l e t h a t t h e r e i s an a p p r e c i a b l e endogenous oxygen uptake* In F i g u r e 16 the comparative a e r o b i c and r e s p i r a t o r y c o e f f i c i e n t s are graphed.  In t h i s method o f i n t e r -  p r e t a t i o n , which i e used by the W i s c o n s i n s t u d i e s on the R h i a o b i a  anaerobic  workers i n t h e i r  ( l , 12, 1 4 ) , the r e d u c t i o n time w i t h  glucose and the volume of oxygen taken up i n the presence o f . glucose a r c taken as 100,  and c o r r e s p o n d i n g  values w i t h t h e  o t h e r compounds are reduced to percentage o f t h i s f i g u r e . c a l c u l a t i o n makes i t p o s s i b l e t o compare a e r o b i c and  This  anaerobic  r e s p i r a t i o n by r e d u c i n g both t o the same b a s i s . - - From t h e data i n F i g u r e 16 i t i s e v i d e n t t h a t the a e r o b i c and anaerobic  o x i d a t i o n o f m a n n i t o l and sodium s u c c i n a t e  occur x?ith v a r y i n g degrees o f i n t e n s i t y .  I t Is also noticeable  t h a t the endogenous r e s p i r a t i o n i s q u i t e pronounced under a e r o b i c c o n d i t i o n s , but i s e n t i r e l y absent under conditions.  anaerobic  S i n c e these r e s u l t s are c a l c u l a t e d on a comparative  b a s i s , i t would appear most probable t h a t d i f f e r e n t enzyme systems are I n v o l v e d i n a e r o b i c and anaerobie  respiration.,,  3. S u b s t r a i n V a r i a t i o n The  problem o f v a r i a t i o n i n r e s p i r a t o r y enzymes has  been estended t o a s t u d y o f the s u b s t r a i n s w i t h i n a g i v e n The  s t r a i n Rh. t r l f o l i i 224 was p l a t e d upon Wilson's  strain.  Agar and  14 separate c o l o n i e s p i c k e d t o form a s e r i e s o f s u b s t r a i n s . a e r o b i c and anaerobic  The  r e s p i r a t i o n o f these s u b s t r a i n s upon  g l u c o s e , m a n n i t o l , sodium s u c c i n a t e , and water has been d e t e r mined as p r e v i o u s l y d e s c r i b e d .  T y p i c a l r e s u l t s a r e summarized  In F i g u r e s 17 and 18. In F i g u r e 17 oxygen uptake i n t h e presence o f m a n n i t o l by t h e mother c u l t u r e , R.T. 224, and e l e v e n s u b s t r a i n s , i s shown g r a p h i c a l l y . There i s r e v e a l e d a tremendous v a r i a t i o n i n o x i d i s i n g a b i l i t y among those  s u b s t r a i n s , a v a r i a t i o n which  i n coma eases I s as great as s i x hundred per c e n t .  It is  apparent t h a t t h e mother c u l t u r e must have been extremely v a r i a b l e i n i t s r e s p i r a t o r y c h a r a c t e r and has been broken up i n t o subs t r a i n s p o s s e s s i n g a v e r y wide range o f o x i d a t i v e a b i l i t y . Although  p r e v i o u s xvorkers w i t h t h e R h i z o b i a have emphasized t h e  extreme v a r i a b i l i t y i n p h y s i o l o g i c a l c h a r a c t e r s e x h i b i t e d by t h i s s p e c i e s , t h i s i s t h e f i r s t demonstration  of i n s t a b i l i t y i n  r e s p i r a t o r y a c t i v i t y w i t h any group o f organisms. ' . I n F i g u r e 18 t h e comparative a e r o b i c and anaerobic r e s p i r a t o r y c o e f f i c i e n t s when sodium s u c c i n a t e i s employed as the s u b s t r a t e a r e r e p o r t e d f o r t h i s same group o f s u b s t r a i n s .  •  - 23. -  There i s a g a i n evident, an extreme v a r i a b i l i t y i n both a e r o b i c and anaerobic - o x i d a t i v e a b i l i t y .  Although both a e r o b i c and  anaerobic o x i d a t i o n a r e v a r i a b l e , they v a r y Independently of. each o t h e r ,  The data r e c o r d e d here,,, t h e r e f o r e \ f u r n i s h added  evidence t h a t t h e a e r o b i c and anaerobic- r e s p i r a t o r y ©nsyraee are d i s t i n c t l y d i f f e r e n t in c h a r a c t e r s as was i n d i c a t e d by the r e s u l t s d e t a i l e d i n figure-16»  - 24  -  In an attempt t o determine the cause of the of the Rh.  t r l f o l i l 224  culture i n respiratory a c t i v i t y ,  p r e v i o u s h i s t o r y of the c u l t u r e was employed i n these t e s t s was  investigated,  a stock l a b o r a t o r y  been c u l t u r e d upon l a b o r a t o r y  now  was  o b t a i n e d , l a b e l l e d R.T. isolated.  t h e n determined.  19 and  The  strain  s t r a i n which  c a r r i e d out  stock c u l t u r e , m a i n t a i n e d i n s t e r i l e s o i l .  Gxibstrains  The  the  media fox* more than a year*  f r e s h i s o l a t i o n of t h i s s t r a i n was  c u l t u r e was  Instability  had A  from the  A mass inoculum  2 24 E, p l a t e d and  six  r e s p i r a t i o n o f t h i s s e r i e s of  cultures  T y p i c a l r e s u l t s are r e c o r d e d i n F i g u r e s  20. In F i g u r e 19 the  comparative a e r o b i c and  anaerobic  r e s p i r a t o r y c o e f f i c i e n t s of these s t r a i n s i n the presence o f sodium s u c c i n a t e  are r e p o r t e d .  There i s e v i d e n t a marked  decrease i n v a r i a b i l i t y i n both a e r o b i c and ability.  A l t h o u g h t h i s v a r i a t i o n has  the a e r o b i c and  oxidative  been markedly decreased,  a n a e r o b i c r e s p i r a t o r y enzymes appear to r e t a i n  t h e i r i n d i v i d u a l character.  Variation in respiratory  appears t o have been d e p r e s s e d , but r e s p i r a t o r y enzymes are s t i l l of  anaerobic  the a e r o b i c and  activity  anaerobic  f u n c t i o n i n g w i t h d i f f e r e n t degrees  Intensity, In F i g u r e 20 the oxygen uptake by t h e s e f r e s h l y - I s o l a t e d  s t r a i n s i n the presence of glucose are r e p o r t e d .  I t i s apparent  t h a t the o x i d a t i v e a b i l i t y o f t h i s s e r i e s of c u l t u r e s i s v e r y  - 25 uniform*  T h i s u n i f o r m i t y I n o x i d a t i v e a b i l i t y among f r e s h l y -  i s o l a t e d s u b s t r a i n s stands i n marked c o n t r a s t t o t h e extreme v a r i a b i l i t y encountered among t h e s u b s t r a i n s :1Kelated from t h e old  laboratory s t r a i n .  I t i s t h e r e f o r e apparent t h a t o u l t u r l n g  on l a b o r a t o r y media f o r an extended p e r i o d has m o d i f i e d t h e p h y s i o l o g i c a l c h a r a c t e r o f t h e organism and has rendered i t extremely unstable  i s r e s p i r a t o r y activity®  -  The  26  -  change i n r e s p i r a t o r y a c t i v i t y undergone by  the  c u l t u r e as a r e s u l t o f prolonged c u l t i v a t i o n upon l a b o r a t o r y media i s c l e a r l y shown In F i g u r e s 21 and  22,  In these graphs  the r e s p i r a t o r y a c t i v i t y of t h e o l d l a b o r a t o r y s u b s t r a i n s i s compared w i t h t h a t o f the f r e s h l y i s o l a t e d  strains.  I n F i g u r e 21 t h e endogenous oxygen uptake o f a l l the s u b s t r a i n s i s shown g r a p h i c a l l y .  I t i s apparent t h a t i n the  f r e s h l y i s o l a t e d s t r a i n s on t h e l e f t of the graph the endogenous r e s p i r a t i o n i s uniform  i n c h a r a c t e r , w h i l e w i t h the o l d l a b o r a -  t o r y s u b s t r a i n s on the r i g h t the endogenous r e s p i r a t i o n i s extremely i r r e g u l a r . In F i g u r e 2 2 the glucose o x i d a t i v e a b i l i t y o f the f r e s h l y i s o l a t e d s t r a i n s i s uniform markedly w i t h the v a r i a b i l i t y and  i n c h a r a c t e r and  contrasts  i r r e g u l a r i t y e x h i b i t e d by  the  old laboratory substrains. A f u r t h e r important also noted.  The  change i n r e s p i r a t o r y a c t i v i t y  was  f r e s h l y i s o l a t e d s t r a i n s were found t o possess  a very marked r e d u c i n g a c t i v i t y upon methylene blue i n the sence of carbon source.  With the o l d l a b o r a t o r y s t r a i n s , on  the other hand, t h i s anaerobic e n t i r e l y disappeared. organism u p o n , l a b o r a t o r y d i m i n u t i o n i n anaerobic  ab-  endogenous r e s p i r a t i o n had  almost  I t would appear t h a t c u l t u r i n g the media has r e s u l t e d i n a endogenous r e s p i r a t i o n .  -  progressive  - 27  -  T h e r e f o r e , the endogenous r e s p i r a t i o n e x h i b i t e d by a Rhizobium euit.ure w i l l be dependent upon t h e r e s p i r a t o r y enzyme aake*up o f t h e c e l l as m o d i f i e d by the p r e v i o u s h i s t o r y of t h a t  culture*  - 28  -  4. Zoning Phenomena .' Xsveatigatlon Into the e f f e c t of c u l t u r i n g the Rhlaobia upon l a b o r a t o r y media has l e d t o t h e o b s e r v a t i o n o f a f u r t h e r change i n p h y s i o l o g i c a l and c u l t u r a l c h a r a c t e r i s t i c s . Xt wee observed t h a t p l a t i n g t h e o l d l a b o r a t o r y s t r a i n s o r s u b s t r a i n s upon W i l s o n ' s Agar l e d t o t h e development o f p e c u l i a r rough and f e a t h e r y c o l o n i e s o f a d i s s o c i a t e d appearance.  These c o l o n i e s  were surrounded by a c i r c u l a r zone i n which t h e suspended carbonate was completely transparent  removed from t h e medium.  zone t h e r e o c c u r r e d  medium remained unchanged. occurred  calcium  Outside t h i s  a much s m a l l e r zone i n w h i c h the  Beyond t h i s eeoond zone t h e r e  a further region of concentration  or p r e c i p i t a t i o n i n  which t h e c l o u d i n e s s and o p a c i t y o f t h e medium was n o t i c e a b l y increased.  I t was f u r t h e r e s t a b l i s h e d t h a t these  zoning  phenomena d i d n o t appear w i t h c u l t u r e s f r e s h l y i s o l a t e d from t h e soil.  Apparently,  t h e r e f o r e , c u l t u r i n g on l a b o r a t o r y media has  r e s u l t e d i n a change i n both c o l o n i a l type and c u l t u r a l  char-  acteristics. Experiments were c a r r i e d out t o determine t h e n a t u r e o f these phenomena and t h e p a r t played by t h e v a r i o u s present i n t h e medium.  constituents  Media were t h e r e f o r e prepared w i t h and  w i t h o u t y e a s t e x t r a c t , g l u c o s e , g e l a t i n e and c a l c i u m c a r b o n a t e .  Brom thymol b l u e i n d i c a t o r was added t o t r a c e t h e r e l a t i o n s h i p between a c i d p r o d u c t i o n and disappearance o f t h e c a l c i u m carbonate.  These v a r i o u s media were then employed t o prepare  p l a t e s from s u b s t r a i n s which showed v e r y a c t i v e sonine p r o p e r t i e s . • The r e s u l t s o b t a i n e d showed d e f i n i t e l y t h a t t h e primary zone o f complete c l e a r i n g i s dependent upon the presence o f glucose l a t h e medium.  • -.  E f f o r t s t o determine t h e cause o f t h e o u t e r r i n g o f d e p o s i t i o n i n t h e medium have so f a r proved f r u i t l e s s .  The  appearance o f t h i s phenomenon seems t o be dependent upon th© presence i n t h e medium o f t h e t h r e e f a c t o r s - g l u c o s e , y e a s t e x t r a c t and c a l c i u m c a r b o n a t e .  When any one o f these c o n s t i t u e n t s  was o m i t t e d t h e phenomenon f a i l e d t o d e v e l o p .  T h i s might  indicate  t h a t d e p o s i t i o n i s brought about through t h e p r e c i p i t a t i o n o f a e a l c i u m - p r o t e i n a t e complex from the y e a s t e x t r a c t by a change i n the i s o - e l e c t r i c p o i n t . The r e l a t i o n s h i p o f a c i d p r o d u c t i o n t o c l e a r i n g phenomenon appeared  rather uncertain.  The c a l c i u m carbonate  tended t o f i x a c i d p r o d u c t i o n w i t h i n t h e c o l o n y and prevent i t s d i s s o l u t i o n throughout t h e p l a t e .  When no carbonate was present  the a c i d d i f f u s e d r a p i d l y , but i n t h e absence o f carbonate no z o n i n g phenomena c o u l d be demonstrated.  » 30 * I t was a l s o observed t h a t g e l a t i n e e x e r t e d a profound e f f e c t upon 'Both colony type and c l e a r i n g phenomenon.  When  g e l a t i n e r e p l a c e d t h e y e a s t e x t r a c t i n the medium, complete c l e a r i n g zones developed but no d e p o s i t i o n took 'place and t h e c o l o n i e s a l l showed a t y p i c a l g r a n u l a t e d  appearance..  When  g e l a t i n e was added t o t h e medium c o n t a i n i n g y e a s t e x t r a c t , o n l y incomplete zoning was observed and t h e c o l o n i e s which developed were o f a f e a t h e r y d i s s o c i a t e d appearance. I t was f u r t h e r observed t h a t t h e z o n i n g  phenomenon w i l l  develop o n l y when t h e c o l o n i c s on a p l a t e a r e r e l a t i v e l y few i n number.  The presence o f a l a r g o number o f c o l o n i e s  represses  the zone f o r m a t i o n , w h i l e on a v e r y crowded p l a t e t h e phenomenon i s completely  inhibited.  I t has not y e t been p o s s i b l e t o  p o s t u l a t e an e x p l a n a t i o n f o r t h i s a n t a g o n i s t i c action,, From the r e s u l t B o b t a i n e d  i n t h i s study o f t h e  r e s p i r a t i o n o f t h e S h i z o b i a , i t appears p r o b a b l e t h a t  there  e x i s t s a d e f i n i t e c y c l e i n r e s p i r a t o r y a c t i v i t y which t h e c u l t u r e undergoes i n response t o c u l t i v a t i o n upon l a b o r a t o r y , media.  A c u l t u r e -when f r e s h l y i s o l a t e d from the s o i l  a watery t r a n s p a r e n t  possesses  growth o f c h a r a c t e r i s t i c appearance.  A f t e r prolonged c u l t i v a t i o n upon W i l s o n ' s Agar, t h e c u l t u r e l o s e s t h i s m o i s t , g l i s t e n i n g c h a r a c t e r i s t i c and becomes d r y and w r i n k l e d i n appearance.  The appearance o f t h i s  dry.  w r i n k l e d type o f growth c o i n c i d e s w i t h t h e development o f  z o n i n g phenomena about t h e c o l o n i e s on W i l s o n ' s Agar.  At t h e  same time a marked change i n both a e r o b i c and a n a e r o b i c r e s p i r a t i o n -takes p l a c e and t h e c u l t u r e becomes e x t r e m e l y unstable i n i t s oxidative a b i l i t i e s .  Passage o f t h e c u l t u r e -.  through s o i l n e u t r a l i z e s -these d i s s o c i a t i v e changes and restores the culture to i t s o r i g i n a l  condition*  * 31a  The a t t a c h e d photograph i l l u s t r a t e s the z o n i n g phenomena which develep when an o l d l a b o r a t o r y s t r a i n o f Rlu t r i f o l i i 2 2 4 I s p l a t e d upon W i l s o n ' s Agar. The comp l e t e zone o f c l e a r i n g which surrounds the c o l o n i e s p a r s b l a c k i n the p i c t u r e , w h i l e the- o u t e r zone o f d e p o s i t i o n appears as a h a z i n e s s i n the medium* Both, types of c o l o n i e s are a l s o . c l e a r l y shown, t h e f i r s t a f a i r l y r e g u l a r h a r d - c e n t r e d t y p e and the second a flat.,, s p r e a d i n g , v e i l - l i k e form. a B  e  - 12 IV. The e x p e r i m e n t a l  . DISCUSSION OF RF.STJLTS work r e p o r t e d h e r e i n has c o n s i s t e d o f  an i n v e s t i g a t i o n i n t o the R e s p i r a t o r y Enzymes o f t h e L a c t i c A c i d and N i t r o g e n - F i x i n g B a c t e r i a .  I n the. course o f t h i s  i n v e s t i g a t i o n , d e t a i l e d s t u d i e s i n t o t h e mechanisms o f l a e t i o a c i d f o r m a t i o n and o f s y m b i o t i c n i t r o g e n f i x a t i o n have been c a r r i e d out^ and i t i s f e l t that c o n s i d e r a b l e progress has been made towards an understanding of these fundamental  o f the p h y s i o l o g i c a l bases  processes.  The a e r o b i c and anaerobic r e s p i r a t i o n o f t h e L a c t i c A c i d S t r e p t o c o c c i have been e x t r e n s i v e l y s t u d i e d i n an e f f o r t t o e v o l v e a p h y s i o l o g i c a l method f o r t h e c l a s s i f i c a t i o n o f t h i s important  group o f micro-organisms.  Experimental  results  i n d i c a t e t h a t t h e l a c k of c o r r e l a t i o n between a e r o b i c o x i d a t i o n , anaerobic o x i d a t i o n and f e r m e n t a t i o n renders such a c l a s s i f i c a t i o n impracticable.  However, t h i s l a c k o f c o r r e l a t i o n  focuses a t t e n t i o n upon t h e mechanism of l a c t i c a c i d p r o d u c t i o n , a mechanism which a p p a r e n t l y i s a f u n c t i o n o f n e i t h e r t h e a e r o b i c nor t h e anaerobic  r e s p i r a t o r y processes.  I t has been  f u r t h e r shown t h a t l u e t i c a c i d f e r m e n t a t i o n i s governed not by the presence o r absence o f a v a i l a b l e oxygen, but by t h e presence o f an a v a i l a b l e n i t r o g e n  source.  These e x p e r i m e n t a l r e s u l t s stand i n d i r e c t c o n t r a d i c t i o n to t h e Pasteur t h e o r y t h a t f e r m e n t a t i o n c o n s i s t s I n t h e f o r m a t i o n  of by-products o f c e l l metabolism through the mechanism o f anaerobic o x i d a t i o n .  S i n c e the P a s t e u r t h e o r y i s the  basis  f o r many of the commercial f e r m e n t a t i o n processes c a r r i e d out by y e a s t s , moulds and r e s u l t s may  b a c t e r i a , i t i s p o s s i b l e t h a t these  prove t o have important i n d u s t r i a l a p p l i c a t i o n s *  S t u d i e s i n t o the a e r o b i c  and a n a e r o b i c r e s p i r a t o r y  mechanism o f the n i t r o g e n - f i x i n g b a c t e r i a have r e s u l t e d i n the demonstration of a d e f i n i t e c y c l e i n r e s p i r a t o r y a c t i v i t y . I t has been c o n c l u s i v e l y shown'that c u l t u r i n g the  organism  upon l a b o r a t o r y media over an extended p e r i o d causes the development of s t r i k i n g 'changes i n both p h y s i o l o g i c a l  character  and o x i d a t i v e a b i l i t y .  the  a e r o b i c and  I t i s therefore  apparent t h a t  a n a e r o b i c r e s p i r a t o r y a c t i v i t y o f an organism .are  l a r g e l y determined by the p r e v i o u s h i a t o r y o f the tested,  culture  This i s a fundamental p r i n c i p l e i n b a c t e r i a l r e s p i r a -  t i o n whieh appears t o have been c o m p l e t e l y o v e r l o o k e d i n previous  studies, Th©  demonstration of v a r i a t i o n i n r e s p i r a t o r y a c t i v i t y  i n response t o c u l t u r i n g Upon l a b o r a t o r y media i s of. . p r a c t i c a l importance i n the problem o f s y m b i o t i c  nitrogen f i x a t i o n , a  process which Is o f .great a g r i c u l t u r a l importance i n Relationt o the maintenance o f s o i l f e r t i l i t y . study of the r e s p i r a t o r y enzymes o f the  I t i s hoped t h a t t h i s fiMzobla  may  contribute  34' -  to a knowledge o f the p h y s i o l o g i c a l process by 'which t h e s e microorganisms convert the &its?ogen o f the atmosphere I n t o form a v a i l a b l e f o r p l a n t use* '  - 35 -  V,  CONCLUSION  The study o f t h e L a c t i c A c i d and  Nitrogen-Fixing  B a c t e r i a r e p o r t e d upon h e r e i n has opened up s e v e r a l f i e l d s f o r f u r t h e r study.  fertile  In p a r t i c u l a r the a p p l i c a t i o n o f  r e s p i r a t o r y onzyme s t u d i e s t o t h e  adaptive-constitutive  enzyme q u e s t i o n and t o the mechanism o f l a c t i c a c i d p r o d u c t i o n appears worthy o f f u r t h e r study.  Research i n t o t h e s e problems  may have important i n d u s t r i a l a p p l i c a t i o n s i n t h e  preparation  of s t a r t e r c u l t u r e s and i n t h e r e g u l a t i o n and e x t e n s i o n o f i n d u s t r i a l f e r m e n t a t i o n s c a r r i e d out .by microorganisms, 'The.' cause of v a r i a b i l i t y i n r e s p i r a t o r y enzymes and t h e i n f l u e n c e o f t h e p r e v i o u s h i s t o r y o f the c u l t u r e upon o x i d a t i v e  ability  w i t h t h e Rhizoblum s p e c i e s a r e fundamental q u e s t i o n s which should bo f u r t h e r i n v e s t i g a t e d .  The s o l u t i o n o f these problems  would be o f d i r e c t v a l u e t o t h e important a g r i c u l t u r a l process of symbiotic nitrogen  fixation.  - 36  -  BIBLIOGRAPHY .1.  B u r r i s , R»B ana W i l s o n , P»W.'- Gold S p r i n g Harbor Symposia on Quant i t at i ve B i o l o g y , 7* $49* 193?«  2,  Dixon, SI* - "Hanometrlo Methods  5.  Eagles,, B A, and S a d l e r , W. - Oan  4.  Hegarty, C.P. - J o u r . Baet. 37s  3.  Morgan, - B a c h e l o r s T h e s i s „ U n i v e r s i t y of B r i t i s h Columbia. ... ......  6.  Oppenhelsier, 0.. and .Stem, It. G« Sordemaan, 19.39. •  7.  Q u a s t e l , 3T.H. and Whetham., M.X5 - Bioehem. 3". 18: 519 0 1924.  8.  Q u a s t e l , J.Bt, and Whetham, M*D,  - Blochem. J . 19: 3 2 0  9.  Q u a s t e l , J.H. and Whet ham, M.D.  - Bioehem. .T. 19: 645, 1925*  10.  Rahn, 0., Hegarty, G . P . , D u e l l , 1471938,  R.E„ •« J o u r . Baet. 3jj; -  lie  S a d l e r , W, - Trans. Roy* See*. Can.  9  a  11  S  « Cambridge P r e s s s  t  1934.  3*. Research 7& 364., 1932.  145, 1939*  8  ~ "Biological Oxidation", • -  e  1 2 . ' Tam, .R.K. and W i l s o n , P..W.  - J o u r * .Baot, 42:  Thunberg «• Slcand® A r c t , P h y s i o l . 40: 1, 1920.  14*  W i l s o n * P.W=  your. Baot. $$t  1925®  Yolume 20, 1926«  13-  9  s  6 0 1 , 1938*  529, 1941  Abstract  The dehydrogenase enzyme a c t i v i t y of f i v e s t r a i n s of Rhizobium t r i f o l i i upon s i x t y t e s t compounds has been s t u d i e d , employing the Thunberg t e c h n i q u e . I t has been shown that the dehydrogenase a c t i v i t y of these s t r a i n s i s extremely v a r i a b l e , e s p e c i a l l y i n the a c t i v a t i o n of c a r b o h y d r a t e s . The dehydrogenase a c t i v i t y of Rhizobium t r i f o l i i  224  has been t e s t e d at i n t e r v a l s over a p e r i o d of one y e a r , and c o n s i d e r a b l e f l u c t u a t i o n i n r e s p i r a t o r y a c t i v i t y demonstrated. It  i s suggested t h a t t h i s marked s t r a i n v a r i a t i o n  may account f o r disagreement p r e v i o u s workers.  among the r e p o r t e d r e s u l t s o  I.  The  INTRODUCTION  mechanism o f r e s p i r a t o r y enzyme systems i n  symbiotic nitrogen investigators.  f i x a t i o n has been s t u d i e d  by s e v e r a l  W i l s o n (5) , working w i t h R. t r i f o l i i ,  r e p o r t e d a d e t a i l e d study o f t h e f a c t o r s i n f l u e n c i n g t h e preparation  of " r e s t i n g c e l l "  suspensions o f t h i s s p e c i e s  i n an e f f o r t t o develop a technique s u i t a b l e f o r t h e study of the a e r o b i c and anaerobic r e s p i r a t o r y a c t i v i t i e s o f t h i s organism.  Thome and Burr i s  (4) compared t h e  r e s p i r a t o r y systems o f "nodular" and " c u l t u r e d " and  found no s i g n i f i c a n t d i f f e r e n c e .  rhizobia  B u r r i s and W i l s o n ( l )  r e p o r t e d a survey o f t h e a e r o b i c r e s p i r a t o r y a c t i v i t y of ten s t r a i n s and f i v e s p e c i e s o f t h e root nodule bacteria,. Tarn and W i l s o n (3) s t u d i e d  t h e dehydrogenase systems o f  R. t r i f o l i i  and R. leguminosarum, employing some f o r t y t e s t  compounds.  The i n f l u e n c e o f i n h i b i t i n g agents upon t h e  r e s p i r a t o r y enzyme systems o f t h e R h i z o b i a has a l s o been studied  by B u r r i s and W i l s o n ( l ) and Tarn and W i l s o n ( 3 ) .  In the study o f r e s p i r a t o r y enzymes there has been a tendency to r e g a r d s u b s t r a t e a c t i v a t i o n as a constant species c h a r a c t e r i s t i c .  However, Tarn and Wilson  (3) found  a s t r a i n v a r i a t i o n , w h i c h was independent o f s p e c i e s , i n t h e a c t i v a t i o n of c e r t a i n s u b s t r a t e s .  The present  report  deals w i t h an i n v e s t i g a t i o n of the s t r a i n and s p e c i e s v a r i a t i o n among t h e r e d c l o v e r organisms.  II.  EXPERIMENTAL METHODS  The c u l t u r e s used i n these experiments c o n s i s t e d of f i v e s t r a i n s of Rhizobium t r i f o l i i .  Of these c u l t u r e s , f o u r ,  namely RT 22B, RT 224, RT 226 and RT 2 3 1 , were o b t a i n e d from the U n i v e r s i t y of W i s c o n s i n i n 1 9 3 0 , w h i l e the f i f t h , RT 3 9 - 1 , was i s o l a t e d from r e d c l o v e r nodules at The U n i v e r s i t y o f B r i t i s h Columbia i n 1 9 3 9 . The medium employed i n these s t u d i e s was t h a t recommended by W i l s o n ( 5 ) , c o n s i s t i n g o f t h e m i n e r a l s a l t s o f medium 79 (Fred, B a l d w i n , and McCoy, 1932) and e n r i c h e d w i t h 1.0$ D i f o C  yeast e x t r a c t and 0.1$ g l u c o s e * 1 . 5 $ agar was used as t h e s o l i d i f y i n g agent. The b a c t e r i a l suspensions r e q u i r e d f o r t h e " r e s t i n g  cell"  t e c h n i q u e were o b t a i n e d by growing the c u l t u r e s on the s u r f a c e of t h e medium i n l a r g e Roux f l a s k s .  A f t e r 48 hours*  i n c u b a t i o n a t 28° C. t h e growth was washed from t h e s u r f a c e o f the agar w i t h M/30 phosphate b u f f e r , c e n t r i f u g e d at 2,000 r.p.m., washed t w i c e and f i n a l l y re-suspended i n phosphate buffer.  A l l suspensions were a d j u s t e d t o a c e l l c o n c e n t r a t i o n  of 2$ by volume, employing t h e Hopkins v a c c i n e method ( 2 ) . Suspensions of t h i s c o n c e n t r a t i o n were found t o dehydrogenate glucose i n from 5 t o 10 m i n u t e s , and not t o reduce methylene blue endogenously i n l e s s t h a n two h o u r s .  Dehydrogenase technique  a c t i v i t y was determined by the  i n tubes c o n t a i n i n g 1 c.c. of " r e s t i n g  suspension,  Thunberg cell"  2 c.c. of phosphate b u f f e r , 1 c.c. of 1:7,000  methylene b l u e , and 1 c.c. of M/20  substrate.  The tubes  were-evacuated f o r two minutes on a water pump.  The  r e a c t i o n s were c a r r i e d out a t 40° C. and at a pH of 8.0, as recommended by Tarn and W i l s o n  (3).  Dehydrogenase  a c t i v i t y was measured as time r e q u i r e d to d e c o l o r i z e completely the methylene  blue.  The s u b s t r a t e s i n c l u d e d s i x t y t e s t compounds, e o n s i s t i n of c a r b o h y d r a t e s ,  o r g a n i c a c i d s , a l c o h o l s , and amines. A l l  o r g a n i c a c i d s were a d j u s t e d t o pH 7 . 0 w i t h N / i 0 hydroxide*  sodium  - 5 -  III.. A.  EXPERIMENTAL RESULTS  VARIATION AMONG STRAINS OF R. TRIFOLII The dehydrogenase a c t i v i t i e s of f i v e s t r a i n s o f Rhizobium m  t r i f o l i i are presented  i n Table 1.  From t h e v a l u e s  recorded  i n t h i s t a b l e i t i s apparent t h a t t h e r e i s an extreme v a r i a b i l i t y i n s u b s t r a t e a c t i v a t i o n among t h e v a r i o u s s t r a i n s tested.  This v a r i a b i l i t y i s most pronounced among t h e  carbohydrate  dehydrogenations, but i s a l s o encountered among  the a l c o h o l and o r g a n i c a c i d a c t i v a t i o n s . 1.  Carbohydrates Dehydrogenation of the hexoses i s c h a r a c t e r i z e d by  u n i f o r m i t y of a c t i v i t y .  With a l l f i v e s t r a i n s t e s t e d , g l u c o s e ,  mannose and f r u c t o s e are r a p i d l y a t t a c k e d , w h i l e g a l a c t o s e i s only slowly o x i d i z e d . one  With t h e p e n t o s e s , arabinose  and x y l o s e ,  s t r a i n , RT 224, o x i d i z e s both compounds r e a d i l y ; a second  s t r a i n , RT 251, a t t a c k s x y l o s e w i t h d i f f i c u l t y a n d a r a b i n o s e not at a l l ; the remaining "three s t r a i n s a c t i v a t e n e i t h e r compound.  Among t h e d i s a c c h a r i d e s , s u c r o s e , c e l l o b i o s e and  t r e h a l o s e are a t t a c k e d by a l l f i v e s t r a i n s , w h i l e maltose and m e l i b i o s e are a c t i v a t e d by f o u r s t r a i n s .  Lactose  by o n l y two s t r a i n s and those w i t h d i f f i c u l t y .  i s attacked  Among t h e  t r i s a c c h a r i d e s , r a f f i n o s e i s a t t a c k e d by a l l s t r a i n s and m e l e z i t o s e by t h r e e s t r a i n s .  Among t h e p o l y s a c c h a r i d e s ,  a c t i v a t i o n i s c a r r i e d out by a l l s t r a i n s *  The degree o f v a r i a t i o n i n dehydrogenase a c t i v i t y upon c e r t a i n carbohydrates  i s portrayed g r a p h i c a l l y i n Figure  1.  The u n i f o r m a c t i v a t i o n c h a r a c t e r i s t i c o f t h e hexoses i s c o n t r a s t e d w i t h the v a r i a b i l i t y o f t h a t o f t h e d i - , t r i - , and polysaccharides.  Among these l a t t e r s u b s t r a t e s , v a r i a t i o n s  up t o nine hundred p e r cent a r e encountered, w h i l e v a r i a t i o n s of f o u r and f i v e hundred per cent are common. 2.  Alcohols The dehydrogenase a c t i v i t y of R. t r i f o l i i s t r a i n s upon  the h i g h e r a l c o h o l s i s markedly i r r e g u l a r .  Of t h e compounds  t e s t e d , o n l y m a n n i t o l i s a c t i v a t e d by a l l s t r a i n s employed. G l y c e r o l and s o r b i t o l  are .attacked by t h r e e s t r a i n s , w h i l e  d u l c l t o l and i n o s i t o l a r e a t t a c k e d by one. E t h y l e n e  glycol  and e r y t h r i t o l a r e not a c t i v a t e d by any o f t h e s t r a i n s . S t r a i n v a r i a t i o n i n dehydrogenase a c t i v i t y upon these h i g h e r a l c o h o l s i s shown i n F i g u r e 1 .  There i s evident an  extreme v a r i a b i l i t y i n the a c t i v a t i o n o f m a n n i t o l and g l y c e r o l w i t h t h e s t r a i n s employed i n these s t u d i e s * 3•  Organic A c i d s Dehydrogenase a c t i v i t y upon o r g a n i c a c i d s was extremely  l i m i t e d w i t h t h e f i v e s t r a i n s o f R. t r i f o l i i employed i n these s t u d i e s ;  Sodium s u c c i n a t e was found t o be t h e o n l y  o r g a n i c a c i d a c t i v a t e d by a l l s t r a i n s t e s t e d .  Sodium malate  was a t t a c k e d by two b u t y r a t e , and  s t r a i n s , w h i l e sodium formate,  sodium l a c t a t e were a t t a c k e d by one  sodium strain*  A l l other organic a c i d s , i n c l u d i n g the m a j o r i t y of the monoand d i c a r b o x y l i c lower f a t t y a c i d s , were not a t t a c k e d any  strain,  B.  STRAIN VARIATION INFLUENCED BY TIME  by  The dehydrogenase a c t i v i t y of R, t r i f o l i i 224, t e s t e d at four i n t e r v a l s over a p e r i o d o f one y e a r , i s recorded Table 2,  in  The v a l u e s r e p o r t e d i n t h i s t a b l e show a marked  v a r i a b i l i t y i n s u b s t r a t e a c t i v a t i o n w i t h i n t h i s one at d i f f e r e n t t i m e s .  strain  T h i s v a r i a b i l i t y i s not l i m i t e d to  any  one group of s u b s t r a t e s , but i s found e q u a l l y among the carbohydrates,  a l c o h o l s , and o r g a n i c a c i d s .  Among the carbohydrates  the dehydrogenation of mannose  and f r u c t o s e remains f a i r l y c o n s t a n t .  That of g a l a c t o s e ,  however, shows extreme v a r i a t i o n , the R e s p i r a t o r y C o e f f i c i e n t i n c r e a s i n g from a v a l u e of 3 t o a value of 5 6 ,  Among the  pentoses the dehydrogenase a c t i v i t y upon x y l o s e decreases from a value of 133 to a v a l u e of 14,  Among the other  carbohydrates  a l s o t h e r e i s a c o n s i d e r a b l e f l u c t u a t i o n i n dehydrogenase v a l u e s , a f l u c t u a t i o n which appears t o be c h a r a c t e r i s t i c each i n d i v i d u a l carbohydrate  of  and does not appear t o show any  d e f i n i t e tendency towards i n c r e a s e d or decreased dehydrogenase a c t i v i t y on the part of the c u l t u r e i n g e n e r a l .  A s i m i l a r v a r i a t i o n i n dehydrogenase a c t i v i t y i s found when the a l c o h o l s are used as s u b s t r a t e s .  I t i s noticeable  t h a t i n t h e case of m a n n i t o l , v a l u e s of 84 o r 17 may be obtained f o r t h e R e s p i r a t o r y C o e f f i c i e n t , depending upon the time o f t e s t i n g ; and i n t h e ease o f g l y c e r o l i t i s evident t h a t a completely n e g a t i v e o r v e r y s t r o n g l y p o s i t i v e t e s t may be o b t a i n e d . With t h e o r g a n i c a c i d s t h i s f l u c t u a t i o n i s e q u a l l y pronounced. i n Table 1,  With t h e f i v e s t r a i n s of R. t r i f o l i i  reported  sodium s u c c i n a t e was found t o be t h e o n l y o r g a n i c  a c i d a t t a c k e d by a l l s t r a i n s .  However, the s i g n i f i c a n c e o f  such a f i n d i n g may be questioned when i t can be shown t h a t w i t h one o f these s t r a i n s t h e sodium s u c c i n a t e value may v a r y from 5 t o 8 4 , depending upon when t h e t e s t i s c a r r i e d o u t . This f l u c t u a t i o n i n dehydrogenase a c t i v i t y upon t h e carbohydrates, a l c o h o l s , and o r g a n i c a c i d s i s shown i n F i g u r e 2«  graphically  DISCUSSION The  d e t e r m i n a t i o n of dehydrogenase a c t i v i t y depends  upon the r a t e of r e d u c t i o n of methylene b l u e . of t h i s r e d u c t i o n may  Measurement  be c a r r i e d out by v i s u a l approxima-  t i o n s , u s i n g known c o n c e n t r a t i o n s of methylene b l u e . and Wilson (3)  Tarn  i n t h e i r s t u d i e s on the dehydrogenase systems  of R. t r i f o l i i and R. leguminosarum, employed a m o d i f i e d Thunberg method i n which the E v e l y n e l e c t r i c photometer and s p e c i a l l y designed Thunberg tubes were used.  T h i s method  was  found to g i v e more a c c u r a t e and c o n s i s t e n t r e s u l t s than the v i s u a l method g i v e s .  The v a l u e s r e p o r t e d i n the present paper  have been o b t a i n e d by measuring the time r e q u i r e d f o r complete d e c o l o r i z a t i o n of the methylene b l u e . converted to percentage  of glucose r e d u c t i o n time and  as r e s p i r a t o r y c o e f f i c i e n t s . Wilson (3)  A l l v a l u e s were then expressed  S i n c e the r e s u l t s of Tarn and  have a l s o been r e p o r t e d as r e s p i r a t o r y  coefficients,  c a l c u l a t e d from the s l o p e s of the l i n e s as determined  photo-  m e t r i c a l l y , the v a l u e s o b t a i n e d by these two methods should be comparable. I n v e s t i g a t i o n o f the dehydrogenase enzymes o f the R h i z o b i a i s c o m p l i c a t e d by the occurrence of endogenous  - 10 r e s p i r a t i o n , as shown by the a b i l i t y of " r e s t i n g  cell"  suspensions t o reduce methylene blue i n the absence of substrate.  T h i s endogenous r e s p i r a t i o n was a t t r i b u t e d by  W i l s o n (5) to the e l a b o r a t i o n and r e t e n t i o n of gummy m a t e r i a l which may reduction. after  then serve as a s u b s t r a t e f o r methylene blue The c u l t u r e s employed i n the present i n v e s t i g a t i o n ,  prolonged c u l t i v a t i o n on yeast e x t r a c t glucose agar,  were found t o possess a n e g l i g i b l e endogenous r e s p i r a t i o n , and proved unable t o reduce methylene b l u e without s u b s t r a t e i n under two hours.  I t was t h e r e f o r e p o s s i b l e to o b t a i n c l e a r - c u t  p o s i t i v e and n e g a t i v e dehydrogenase t e s t s from which the endogenous f a c t o r had been e l i m i n a t e d . These r e s u l t s are i n s t r i k i n g disagreement w i t h those r e p o r t e d by Tarn and W i l s o n (3) , who were unable t o reduce the endogenous r e s p i r a t i o n o f t h e i r s t r a i n s below a r e s p i r a t o r y c o e f f i c i e n t of 3 8 , Comparison o f the carbohydrate and a l c o h o l r e p o r t e d by W i l s o n (5)  dehydrogenations  and Tarn and W i l s o n (3) w i t h those  reported h e r e i n r e v e a l s a d e f i n i t e l a c k of c o r r e l a t i o n .  The  values o b t a i n e d by these i n v e s t i g a t o r s are markedly h i g h e r and more u n i f o r m than those o b t a i n e d w i t h the f i v e s t r a i n s cons i d e r e d i n the present r e p o r t .  It i s particularly noticeable  that x y l o s e , a r a b i n o s e , rhamnose, l a c t o s e , e t h y l e n e g l y c o l , e r y t h r i t o l , d u l c i t o l , i n o s i t o l and e t h y l a l c o h o l , which were found t o be r e a d i l y a t t a c k e d by the R, t r i f o l i i  202 and  209  s t r a i n s of Tarn and W i l s o n (3), were a t t a c k e d o n l y s l i g h t l y or not at a l l by the f i v e s t r a i n s r e p o r t e d h e r e i n .  - 11 There appears to be a f u r t h e r s i g n i f i c a n t d i f f e r e n c e between these*two groups of R. t r i f o l i i dehydrogenation of o r g a n i c a c i d s . Tarn and W i l s o n (3)  strains in their  Those s t r a i n s s t u d i e d  were found to a c t i v a t e n e a r l y a l l o r g a n i c  a c i d s t e s t e d , w h i l e the f i v e s t r a i n s r e p o r t e d i n the work were found to a t t a c k o n l y sodium s u c c i n a t e malate and  by  and  present sodium  to be c o m p l e t e l y i n a c t i v e upon a l l those r e p o r t e d  as p o s i t i v e by the o t h e r  investigators.  T h i s l a c k of agreement among v a r i o u s i n v e s t i g a t o r s the dehydrogenase enzyme systems of the R h i z o b i a may  of  possibly  be e x p l a i n e d by the occurrence of marked s t r a i n v a r i a t i o n within t h i s species.  I t has  been shown i n the present paper  t h a t f i v e s t r a i n s of R. t r i f o l i i  exhibit s u f f i c i e n t l y  d i s t i n c t i v e dehydrogenase a c t i v i t i e s t o be regarded almost as s e p a r a t e s p e c i e s .  I t has  dehydrogenase a c t i v i t y of any not  f u r t h e r been shown t h a t  the  s i n g l e s t r a i n of R. t r i f o l i i  a constant p h y s i o l o g i c a l c h a r a c t e r i s t i c but  s u b j e c t to extreme f l u c t u a t i o n .  is  is Itself  U n t i l such time as the  factors  which a f f e c t t h i s s t r a i n v a r i a t i o n i n dehydrogenase enzyme a c t i v i t y have been f u r t h e r c l a r i f i e d , any  attempt at a r r a n g i n g  or c l a s s i f y i n g the R h i z o b i a upon t h e i r r e s p i r a t o r y enzyme c h a r a c t e r would appear to be of l i t t l e  value.  - 12  -  REFERENCES B u r r i s , R. H. and W i l s o n , P. W. Cold S p r i n g Harbour Symposia on Q u a n t i t a t i v e B i o l o g y , V o l , V I I , 1939« Hopkins, D.  J o u r . Amer. Med. Ass'n. 6 0 : 1 6 1 5 , 1913.  Tarn, R. K. and W i l s o n , P.W. Thome, D.W.  and B u r r i s , R. H.  W i l s o n , P.W.  Jour. Bact. 35:  J o u r . B a c t . 41: 529, 1941. J o u r . B a c t . 36: 261, 1 9 3 8 . 601, 1938.  INTRODUCTION  The  f u n c t i o n of r e s p i r a t o r y enzyme systems i n the  process of s y m b i o t i c  nitrogen  several investigators.  fixation  has been s t u d i e d  by  Walker, Anderson and Brown (9).  employed the Warburg manometer to study oxygen uptake by R. leguminosarum and stimulated  found t h a t y e a s t e x t r a c t  respiration.  N e a l and Walker (6) s t u d i e d  oxygen uptake o f R. m e l i l o t i of v a r i o u s  greatly  carbon s o u r c e s .  and R.  j a p o n i cum  i n the  the presence  In both t h e s e i n v e s t i g a t i o n s ,  growing c u l t u r e s of the root nodule b a c t e r i a were employed. The activity  study of the mechanism of r e s p i r a t o r y enzyme among the R h i z o b i a  has been g r e a t l y f a c i l i t a t e d  the i n t r o d u c t i o n of the " r e s t i n g c e l l " t e c h n i q u e . reported  an e x t e n s i v e  the p r e p a r a t i o n aerobic  and  by  Wilson  (11)  i n v e s t i g a t i o n of the f a c t o r s i n f l u e n c i n g  of suspensions of R. t r i f o l i i s u i t a b l e f o r  anaerobic r e s p i r a t o r y s t u d i e s .  Thorne and  Burris  (8) compared the r e s p i r a t o r y enzyme systems of " c u l t u r e d "  and  "nodular" r h i z o b i a , and  found no s i g n i f i c a n t d i f f e r e n c e  r e s p i r a t o r y mechanism.  B u r r i s and W i l s o n (2)  survey of f i v e s p e c i e s and bacteria.  s t u d i e d the  systems of R. t r i f o l i i and R. some f o r t y t e s t compounds.  reported a  t e n s t r a i n s of the root  Tarn and W i l s o n (7)  nodule  dehydrogenase  leguminosarum, employing  The  s e l e c t i v e i n h i b i t i o n of  s p e c i f i c r e s p i r a t o r y enzyme systems of the R h i z o b i a been s t u d i e d  in  by B u r r i s and W i l s o n (2) and  by Tarn and  has Wilson  (7:).  A s i d e from the work of Thorne and has yet been made to study the  B u r r i s (8) no  s t a b i l i t y of  attempt  respiratory  enzyme systems of the R h i z o b i a i n response to changes i n environmental and The object  c u l t u r a l conditions.  work r e p o r t e d upon h e r e i n was  of d e t e r m i n i n g the  a c t i v i t y of s u b s t r a i n s  undertaken w i t h  the  comparative r e s p i r a t o r y enzyme  derived  from a s o i l stock  culture  and from a c u l t u r e c a r r i e d f o r a c o n s i d e r a b l e p e r i o d of time on l a b o r a t o r y  media.  EXPERIMENTAL METHODS  The organism used i n these experiments t r i f o l i i , W i s c o n s i n s t r a i n 224,  was Rhizobium  T h i s c u l t u r e had been  maintained c o n t i n u o u s l y upon l a b o r a t o r y media f o r a p e r i o d of eleven y e a r s *  At t h e end of t h i s time a d u p l i c a t e  t r a n s f e r had been i n o c u l a t e d i n t o s t e r i l e s o i l and maint a i n e d as a s t o c k c u l t u r e .  Both the o l d l a b o r a t o r y c u l t u r e  and the new s o i l s t o c k c u l t u r e (8 months i n s o i l ) were a v a i l a b l e f o r experimental  study.  S u b s t r a i n s were o b t a i n e d by p l a t i n g these two mother c u l t u r e s and p i c k i n g i s o l a t e d c o l o n i e s .  The s u b s t r a i n s  d e r i v e d from the l a b o r a t o r y s t r a i n have been l a b e l l e d  series  B and C; those f r e s h l y i s o l a t e d from the s o i l c u l t u r e have been l a b e l l e d s e r i e s E.  W i t h i n t h i s l a t t e r s e r i e s , RT 224 E  denotes t h e c u l t u r e r e - i s o l a t e d from the s o i l s t o c k c u l t u r e by a mass s u b - i n o c u l a t i o n , w h i l e the remaining s t r a i n s of t h e E s e r i e s r e p r e s e n t s u b s t r a i n s obtained by p l a t i n g t h i s mother c u l t u r e , RT 224 E. The c u l t u r e medium on which R« t r i f o l i i  224 had been  c a r r i e d c o n t i n u o u s l y was standard yeast water m a n n i t o l agar (4) .  For r e s p i r a t o r y enzyme s t u d i e s , however, the s p e c i a l  medium recommended by W i l s o n (11) was employed.  This  c o n s i s t e d of the m i n e r a l s a l t s o f Medium 79 (4)  enriched  w i t h ifo D i f c o yeast e x t r a c t and O.lf. g l u c o s e , and c o n t a i n e d 1*5% agar as the s o l i d i f y i n g agent© R e s t i n g c e l l suspensions f o r r e s p i r a t o r y s t u d i e s were o b t a i n e d by growing the organism on t h e s u r f a c e o f W i l s o n ' s medium (11) i n l a r g e Roux f l a s k s . t i o n at 28 the  A f t e r 48 hours' i n c u b a -  C. the growth was washed from the s u r f a c e of  agar w i t h M/30 phosphate b u f f e r o f pH 7 . 2 ,  c e n t r i f u g e d at  2000 r.p.m., washed t w i c e , and f i n a l l y resuspended i n phosphate b u f f e r .  A l l suspensions were a d j u s t e d t o a c e l l  c o n c e n t r a t i o n of 1% by volume, employing the Hopkins v a c c i n e tube method (5) • Dehydrogenase a c t i v i t y was determined by the Thunberg technique i n tubes c o n t a i n i n g 1 c . c . of r e s t i n g  cell  s u s p e n s i o n , 2 c . c . o f phosphate b u f f e r , 1 c.c. o f 1:7,000 methylene b l u e , and 1 c . c . o f M/20 s u b s t r a t e .  A l l tubes were  evacuated f o r 2 minutes on a water pump, and dehydrogenase a c t i v i t y measured as the time r e q u i r e d f o r complete t i o n o f t h e methylene b l u e .  decoloriza-  The t e s t s were c a r r i e d out at  40° C. and at pH 8 0 , as recommended by Tarn and W i l s o n ( 7 ) • o  A e r o b i c o x i d a t i v e a b i l i t y was measured m a n o m e t r i c a l l y by the B a r c r o f t a p p a r a t u s , as d e s c r i b e d by Dixon ( 3 ) .  The  cups used i n these experiments c o n t a i n e d 1 c c, of r e s t i n g 0  c e l l s u s p e n s i o n , 1 c . c . o f M/20 s u b s t r a t e , and 1 c . c . o f b u f f e r .  Carbon d i o x i d e was KOH  absorbed by f i l t e r paper soaked i n 20$  h e l d i n i n s e t tubes w i t h i n the cups.  c a r r i e d out at 37° The  C. and pH  A l l t e s t s were  7.2.  s u b s t r a t e s employed i n these experiments c o n s i s t e d  of g l u c o s e , m a n n i t o l and sodium s u c c i n a t e * A e r o b i c r e s p i r a t o r y a c t i v i t y has been expressed  as  cxibic m i l l i m e t e r s of oxygen taken up per m i l l i g r a m of dry weight per hour.  cell  This value i s known as the 0,0 .  Cell  2  dry weight was  determined by d r y i n g 2 c.c. of the  at 100°  deducting the weight of the s a l t s present i n  0. and  suspension  the b u f f e r s o l u t i o n . The  s u i t a b i l i t y of the Q,0  2  value f o r use i n r e s p i r a t o r y  s t u d i e s w i t h the R h i z o b i a has been questioned who  showed t h a t v a l u e s of the Q,0g  by W i l s o n  increased with increased  c o n c e n t r a t i o n of yeast e x t r a c t i n the growth medium. assumed these i n c r e a s e d Q-Og formation i n the c u l t u r e and standard, the q 0  2  (11),  He  v a l u e s to be due t o decreased proposed the use of a  gum  new  v a l u e , d e f i n e d as the oxygen uptake i n  cubic m i l l i m e t e r s per hour per m i l l i g r a m n i t r o g e n i n the  cells.  This method of c a l c u l a t i o n can be v a l i d only i f the assumption that oxygen uptake v a r i e s d i r e c t l y with the n i t r o g e n of the c e l l s can be proved.  content  Experiments c a r r i e d out upon  f o u r t e e n s u b s t r a i n s i s o l a t e d from the l a b o r a t o r y c u l t u r e R« t r i f o l i i 224 showed d e f i n i t e l y t h a t t h e r e i s c o n s i d e r a b l e •variation i n n i t r o g e n content The n i t r o g e n content  among s t r a i n s and s u b s t r a i n s .  o f t h i s group o f s u b s t r a i n s ranged  from 7,4?$ t o 12,93$,  F u r t h e r , oxygen uptake d i d not appear  to v a ry w i t h t h e n i t r o g e n content  o f the c e l l s , s i n c e those  s u b s t r a i n s w i t h h i g h n i t r o g e n contents  d i d not e x h i b i t  g r e a t e r r e s p i r a t o r y a c t i v i t y than those s u b s t r a i n s whose c e l l n i t r o g e n value was low. I t was a l s o noted t h a t oxygen uptakes on the b a s i s o f n i t r o g e n content  graphing  l e d to a  markedly g r e a t e r degree o f v a r i a t i o n than when t h e d r y weight b a s i s was used®  F o r these reasons i t was decided t o  c a l c u l a t e a l l r e s u l t s as QO2 v a l u e s , based on d r y weights®  EXPERIMENTAL RESULTS The a e r o b i c and anaerobic  r e s p i r a t o r y a c t i v i t i e s of the  s u b s t r a i n s d e r i v e d from t h e l a b o r a t o r y s t r a i n and from t h e s o i l s t o c k c u l t u r e o f R, t r i f o l i i Tables 1 and 2,  224 are presented i n  I n Table 1 are shown t h e comparative QOg  values o f these two groups o f s u b s t r a i n s when the g l u c o s e , m a n n i t o l , sodium s u c c i n a t e and endogenous o x i d a t i v e mechanisms  are c o n s i d e r e d *  In Table 2 the a e r o b i c and  anaerobic  r e s p i r a t o r y c o e f f i c i e n t s of the m a n n i t o l , s u c c i n a t e endogenous systems are d e t a i l e d .  and  A l l figures reported i n  t h i s l a t t e r t a b l e have been c a l c u l a t e d as percentage of the  glucose r e s p i r a t i o n , which i s given the value of  100,  R e s u l t s so o b t a i n e d are termed " r e s p i r a t o r y c o e f f i c i e n t s " . Substrain The  Variation  1  v a l u e s r e p o r t e d i n Tables 1 and  that t h e r e i s a fundamental d i f f e r e n c e s t r a i n s i s o l a t e d from the l a b o r a t o r y s t r a i n s i s o l a t e d from the of the B and "cultured"  2 show c l e a r l y  between the  s t r a i n and  s o i l stock c u l t u r e .  C s e r i e s , which have been d e r i v e d  s t r a i n , are  characterized  sub-  the  Substrains from the  by an extreme f l u c t u a -  t i o n i n respiratory a c t i v i t y ; substrains  of the E s e r i e s ,  which have been i s o l a t e d from the s o i l c u l t u r e , are a c t e r i z e d by a marked u n i f o r m i t y  in respiratory  This e s s e n t i a l d i f f e r e n c e between the two i s apparent upon a l l s u b s t r a t e s both a e r o b i c and  anaerobic  In f i g u r e s 1 and between these two  sub-  t e s t e d and  char-  activity.  groups of  substrains  h o l d s t r u e under  conditions.  2 the c h a r a c t e r i s t i c d i s t i n c t i o n  groups of s u b s t r a i n s  i s portrayed  graphically.  In F i g u r e 1 the anaerobic r e s p i r a t o r y c o e f f i c i e n t s  of s u b s t r a i n s  of the E s e r i e s are compared to those of the  and C s e r i e s , w i t h sodium s u c c i n a t e  as the s u b s t r a t e .  B  Within  the E s e r i e s these r e s u l t s show a marked u n i f o r m i t y , w i t h values r a n g i n g  from 62 to 100.  Among s u b s t r a i n s of the B  and C s e r i e s , on the other hand, there i s an  extreme  v a r i a t i o n i n r e s p i r a t o r y c o e f f i c i e n t s , and the values are found t o range between 14 and  118•  This u n i f o r m i t y among s u b s t r a i n s o f the E s e r i e s and v a r i a b i l i t y among s u b s t r a i n s of the B and C s e r i e s i s f u r t h e r emphasized by the r e s u l t s i l l u s t r a t e d  i n Figure  2•  In t h i s graph the endogenous 0,0 values of the two groups 2  of s u b s t r a i n s are shown.  Here a g a i n , the f r e s h l y - i s o l a t e d  s u b s t r a i n s show a r e g u l a r oxygen uptake which v a r i e s between 20 and 35 c u b i c m i l l i m e t e r s per hour.  With the " c u l t u r e d "  s u b s t r a i n s , however, the endogenous oxygen uptake f l u c t u a t e s between v a l u e s o f 13 and 64 c u b i c m i l l i m e t e r s per hour© From, the v a l u e s r e p o r t e d  i n Tables 1 and 2 and the  r e s u l t s graphed i n F i g u r e s 1 and 2, i t i s apparent t h a t c u l t u r i n g i n s o i l has e x e r t e d a profound i n f l u e n c e upon the physiology  o f the organism under s t u d y  0  Substrains i s o l a t e d  from the s o i l c u l t u r e e x h i b i t a u n i f o r m i t y i n r e s p i r a t o r y a c t i v i t y which i s i n marked c o n t r a s t t o the v a r i a b i l i t y of that of s u b s t r a i n s d e r i v e d from a " c u l t u r e d " s t r a i n .  It  would appear, t h e r e f o r e , t h a t c u l t u r i n g the organism i n s t e r i l e s o i l has s t a b i l i z e d the r e s p i r a t o r y enzyme mechanisms of R. t r i f o l i i  224*  Endogenous R e s p i r a t i o n The r e s u l t s r e p o r t e d  i n Tables 1 and 2 show d e f i n i t e l y  t h a t there i s a fundamental d i f f e r e n c e between the endogenous r e s p i r a t i o n o f f r e s h l y - i s o l a t e d s t r a i n s and t h a t of c u l t u r e d s t r a i n s .  This d i f f e r e n c e i s i l l u s t r a t e d i n  F i g u r e s 2 and 3 » In F i g u r e 2 the endogenous 0 , 0 of these two groups o f 2  substrains i s portrayed  graphically.  The endogenous  r e s p i r a t i o n o f the f r e s h l y - i s o l a t e d s u b s t r a i n s i s markedly u n i f o r m , w h i l e t h a t o f the c u l t u r e d s u b s t r a i n s i s extremely variable.  I n s p i t e of t h i s i r r e g u l a r i t y , however, the  average endogenous oxygen uptake o f the c u l t u r e d ( 3 1 « 3 c u . num.)  substrains  i s o n l y very s l i g h t l y g r e a t e r t h a n t h a t o f  the average o f the f r e s h l y - i s o l a t e d s u b s t r a i n s  ( 2 5 . 5 cu. num.)  I t would appear, t h e r e f o r e , t h a t , a l t h o u g h c u l t u r i n g i n s o i l has s t a b i l i z e d the a e r o b i c endogenous r e s p i r a t i o n , t h e r e has been e s s e n t i a l l y l i t t l e a l t e r a t i o n i n t h i s r e s p i r a t o r y mechanism. The anaerobic endogenous r e s p i r a t i o n o f these two groups of s u b s t r a i n s i s shown i n F i g u r e 3 »  This graph shows c l e a r l y  that a fundamental d i f f e r e n c e e x i s t s between the anaerobic endogenous.respiration cultured substrains.  o f t h e s o i l s u b s t r a i n s and t h a t o f the The r e - i s o l a t e d s u b s t r a i n s are  - 10 c h a r a c t e r i z e d by a very h i g h r a t e of endogenous r e s p i r a t i o n , w h i l e t h a t of' the c u l t u r e d s u b s t r a i n s i s almost n e g l i g i b l e . Among the f i f t e e n s u b s t r a i n s of t h i s l a t t e r group, o n l y shows a h i g h r a t e of endogenous r e s p i r a t i o n ; of the f o u r t e e n s u b s t r a i n s , e i g h t possess only very  one  remaining  slight  endogenous a c t i v i t y and f i v e have no endogenous r e s p i r a t i o n whatever.  T h i s would i n d i c a t e t h a t c u l t u r i n g the organism,  upon l a b o r a t o r y media has  caused the anaerobic  endogenous  r e s p i r a t i o n to decrease from a very h i g h v a l u e almost t o the vanishing point.  S i n c e dehydrogenase s t u d i e s on the  Rhizobia  are c o n d i t i o n e d by a low endogenous r e s p i r a t i o n , the . importance of t h i s f i n d i n g i s apparent. Glucose O x i d a t i o n The  o x i d i z i n g a b i l i t y upon glucose of these two  of s u b s t r a i n s i s p o r t r a y e d  !  i n F i g u r e 4«s  groups  Here a g a i n , t h e r e i s  apparent a s i g n i f i c a n t d i f f e r e n c e between the s u b s t r a i n s r e i s o l a t e d from s o i l and those i s o l a t e d from the o l d l a b o r a t o r y culture.  The  s o i l s u b s t r a i n s are c h a r a c t e r i z e d by a low  uniform r a t e of glucose  and  o x i d a t i o n , w h i l e the c u l t u r e d sub-  s t r a i n s e x h i b i t a g r e a t l y i n c r e a s e d and markedly i r r e g u l a r oxidizing Mannitol The  ability. Oxidation dehydrogenase a c t i v i t y of these two  s t r a i n s upon m a n n i t o l  i s shown i n F i g u r e 3•  groups o f subI t i s apparent  - 11 that t h e r e i s l i t t l e d i f f e r e n c e between the s o i l  strains  and the c u l t u r e d s t r a i n s i n r e g a r d to t h e i r dehydrogenation of t h i s hexahydric a l c o h o l . The a e r o b i c o x i d a t i o n of m a n n i t o l by these i s p o r t r a y e d i n F i g u r e 6,  substrains  I n marked c o n t r a s t to the  similarity  i n dehydrogenase a c t i v i t y shown i n F i g u r e 5» t h e r e i s r e v e a l e d here a very s i g n i f i c a n t d i f f e r e n c e i n the o x i d i z i n g of these two  groups of s u b s t r a i n s .  abilities  T]ie s t r a i n s r e - i s o l a t e d -  from s o i l possess a very low and u n i f o r m oxidase  activity,  w h i l e the s t r a i n s i s o l a t e d from the l a b o r a t o r y c u l t u r e show a g r e a t l y i n c r e a s e d and markedly i r r e g u l a r o x i d i z i n g ability© I t would seem, t h e r e f o r e , t h a t c u l t u r i n g on l a b o r a t o r y media has caused the m a n n i t o l o x i d i z i n g mechanism of t h i s organism t o become i n c r e a s e d i n s t r e n g t h and u n s t a b l e i n c h a r a c t e r . Succinate  Oxidation  The a e r o b i c and anaerobic  r e s p i r a t o r y a c t i v i t y upon  sodium s u c c i n a t e are graphed i n F i g u r e s I and  7»  I n F i g u r e 1 i s shown the dehydrogenating a c t i v i t y upon s u c c i n a t e of these two  s e r i e s of s u b s t r a i n s .  The  activating  power of the r e - i s o l a t e d s u b s t r a i n s i s very h i g h and r e g u l a r i n c h a r a c t e r , w h i l e t h a t of the l a b o r a t o r y s u b s t r a i n s i s very e r r a t i c i n behaviour.  Among t h i s l a t t e r group, v a l u e s  ranging from 14 to 1 1 8 have been r e c o r d e d .  There i s no  apparent tendency toward s t a b i l i z a t i o n w i t h i n t h i s group, and  - 12 the extreme f l u c t u a t i o n i n dehydrogenase a c t i v i t y can o n l y be e x p l a i n e d .as due t o the i n h e r e n t v a r i a b i l i t y  and  i n s t a b i l i t y i n r e s p i r a t o r y enzyme c h a r a c t e r of t h i s bacterial species. The  a e r o b i c r e s p i r a t o r y c o e f f i c i e n t s of these  substrains  w i t h s u c c i n a t e as the s u b s t r a t e are graphed i n F i g u r e 7© h i g h a c t i v i t y ' a n d s t a b l e c h a r a c t e r of the s u c c i n a t e  The  oxidase  mechanism among the s o i l s u b s t r a i n s are again emphasized. The  c u l t u r e d s t r a i n s , on the other hand, are c h a r a c t e r i z e d  by an extreme i r r e g u l a r i t y , t h e m a j o r i t y p o s s e s s i n g o n l y a very l i m i t e d a c t i v i t y upon s u c c i n a t e .  C u l t u r i n g on l a b o r a t o r y  media has r e s u l t e d i n a decrease i n the oxidase a c t i v i t y of t h i s c u l t u r e towards s u c c i n a t e s A e r o b i c and Anaerobic The  Mechanisms  a e r o b i c and anaerobic  r e s p i r a t o r y c o e f f i c i e n t s of  four s t r a i n s s e l e c t e d at random are i n d i c a t e d i n F i g u r e 8© The  values recorded  i n t h i s graph show d e f i n i t e l y t h a t  a c t i v a t i n g a b i l i t y under a e r o b i c and anaerobic does not m a i n t a i n a constant r e l a t i o n s h i p . of m a n n i t o l by s t r a i n RT 224 E the anaerobic  conditions  In the o x i d a t i o n activity is  a p p r e c i a b l y g r e a t e r than the a e r o b i c , w h i l e i n the o x i d a t i o n of s u c c i n a t e the r e v e r s e holds t r u e .  In the o x i d a t i o n of  mannitol by s t r a i n RT 224, on the other,hand, the  aerobic  a c t i v i t y i s much g r e a t e r than the a n a e r o b i c , and t h i s  order  - 13  i s a g a i n r e v e r s e d when s u c c i n a t e  -  acta as the s u b s t r a t e .  With the four s t r a i n s p o r t r a y e d i n F i g u r e 8 n e a r l y a l l poss i b l e i n t e r r e l a t i o n s h i p s between the a e r o b i c and r e s p i r a t o r y a c t i v i t i e s can be demonstrated.  anaerobic  T h i s l a c k of  d e f i n i t e r e l a t i o n s h i p can be shown i n the case of the mother c u l t u r e , s t r a i n RT RT  224 E, and  224,  the s t a b i l i z e d s o i l c u l t u r e , s t r a i n  any of the  parent cultures®  substrains  i s o l a t e d from these  A l l these r e s u l t s i n d i c a t e t h a t  and anaerobic r e s p i r a t o r y a c t i v i t y are c a r r i e d out separate and  two  aerobic by  d i s t i n c t enzyme systems•  DISCUSSION In s t u d i e s on the r e s p i r a t o r y enzyme systems of the assumption has  bacteria  been made t h a t the r e s p i r a t o r y a c t i v i t y  a c u l t u r e i s a constant and  s t a b l e c h a r a c t e r i s t i c of  s p e c i e s under investigation®  The  of  the  work r e p o r t e d upon h e r e i n ,  however, demonstrates that t h e r e e x i s t s a s t r i k i n g and fundamental d i f f e r e n c e  between the r e s p i r a t o r y enzyme systems  of a s t r a i n of R. t r i f o l i i media and sterile  the  c a r r i e d c o n t i n u o u s l y upon  laboratory  same s t r a i n m a i n t a i n e d as a s t o c k c u l t u r e  in  soils  I t has "cultured"  been shown t h a t s u b s t r a i n s  i s o l a t e d from a  s t r a i n e x h i b i t marked i r r e g u l a r i t i e s and  variability  - 14 in their respiratory a c t i v i t i e s .  This substrain v a r i a t i o n  shows that the r e s p i r a t o r y enzyme mechanisms of the s t r a i n c u l t u r e d upon l a b o r a t o r y media have become extremely u n s t a b l e i n character* reported  A somewhat s i m i l a r i n s t a b i l i t y has  by W i l s o n , Hopkins and  Fred (10)  i n r e g a r d to s t r a i n  v a r i a t i o n i n n i t r o g e n f i x a t i o n by the R h i z o b i a . f u r t h e r been shown by Almon and  been  Baldwin ( l ) t h a t  It  various  c u l t u r a l t y p e s d i s t i n c t from the t y p i c a l form of R. may  be i s o l a t e d .  has  trifolii  I t would appear, t h e r e f o r e , t h a t v a r i a t i o n  i n c u l t u r a l character  and n i t r o g e n - f i x i n g a b i l i t y of  R.  t r i f o l i i are accompanied by v a r i a t i o n i n r e s p i r a t o r y enzyme characters  as  wells  In c o n t r a s t to the marked i n s t a b i l i t y e x h i b i t e d substrains  from a " c u l t u r e d "  a c t i v i t i e s of s u b s t r a i n s  by  s t r a i n , the r e s p i r a t o r y enzyme  from a s o i l c u l t u r e have been shown  to be e x t r e m e l y u n i f o r m and  stable i n character.  i n s o i l f o r an eight-month p e r i o d has  Culturing  r e s u l t e d i n fundamental  changes i n r e s p i r a t o r y enzyme a c t i v i t y which are  characterized  by the development of marked s t a b i l i t y i n enzymic c o n s t i t u tion.  The  mechanism by which s o i l e x e r t s t h i s s t a b i l i z i n g  i n f l u e n c e has not as yet been determined© This demonstration of a r e s p i r a t o r y enzyme d i f f e r e n c e between " c u l t u r e d "  s t r a i n s and  " s o i l " s t r a i n s i s at .some  variance w i t h the r e s u l t s r e p o r t e d  by Thorne and  Burris  (8)  0  - 15 These workers i n v e s t i g a t e d the r e s p i r a t o r y enzyme mechanisms of "nodular"  and " c u l t u r e d " s t r a i n s of R h i z o b i a and  them to be e s s e n t i a l l y s i m i l a r * t h e i r " c u l t u r e d " s t r a i n s was may  found  The p r e v i o u s h i s t o r y of  not d e s c r i b e d , however, and  have been r e s p o n s i b l e f o r the r e s u l t s obtained by them, C u l t u r i n g upon l a b o r a t o r y media has r e s u l t e d i n profound  m o d i f i c a t i o n of the r e s p i r a t o r y enzyme systems of R, 224,  trifolii  Compared to t h a t of the s t a b i l i z e d s o i l s t r a i n s , the  r e s p i r a t o r y a c t i v i t y of the c u l t u r e d s t r a i n s i s c h a r a c t e r i z e d by a tremendously decreased anaerobic a g r e a t l y i n c r e a s e d oxidase  endogenous r e s p i r a t i o n ,  a c t i v i t y upon glucose  and  m a n n i t o l , and a decreased oxidase a c t i v i t y upon s u c c i n a t e s With the a e r o b i c endogenous r e s p i r a t i o n , and the dehydrogenase a c t i v i t y upon m a n n i t o l and s u c c i n a t e , t h e r e has  occurred  l i t t l e demonstrable change. I t has g e n e r a l l y been assumed t h a t the a e r o b i c anaerobic  and  r e s p i r a t o r y a c t i v i t i e s of an organism upon any  given s u b s t r a t e are c a r r i e d out through a common enzymic mechanism, i r r e s p e c t i v e of whether m o l e c u l a r  oxygen or  methylene blue f u n c t i o n s as the f i n a l Hydrogen Wilson  Acceptor,  ( 1 1 ) , working w i t h R, t r i f o l i i , found t h a t the  r e l a t i v e r a t e of r e d u c t i o n of methylene blue i n the presence of a given s u b s t r a t e was  g e n e r a l l y lower than the r a t e of  -  -  16  o x i d a t i o n of the same s u b s t r a t e .  The rank of the s u b s t r a t e s ,  however, as Hydrogen Donatorsy showed c l o s e agreement under a e r o b i c and anaerobic  conditions,.  The r e s u l t s shown g r a p h i c a l l y  i n F i g u r e 8 i n d i c a t e t h a t there i s no such c l o s e agreement among a e r o b i c and anaerobic mechanisms w i t h the s t r a i n s and s u b s t r a i n s s t u d i e d i n the present work, but r a t h e r a noticeable d i s s i m i l a r i t y  in activating ability*  This lack  of agreement i s f u r t h e r emphasized when the r e s u l t s o b t a i n e d from the v a r i o u s enzymic systems i n v e s t i g a t e d are compared* With the endogenous r e s p i r a t i o n i t has been shown t h a t c u l t u r i n g upon l a b o r a t o r y media causes almost complete disappearance of the anaerobic  reducing  a e r o b i c a c t i v i t y has not been a l t e r e d .  a c t i v i t y , while the With the m a n n i t o l  and s u c c i n a t e r e s p i r a t o r y mechanisms, c u l t u r i n g on l a b o r a t o r y media has caused an i n c r e a s e d o x i d a t i v b a b i l i t y upon m a n n i t o l and a decreased a c t i v i t y upon s u c c i n a t e , w h i l e i n n e i t h e r case has t h e dehydrogenating mechanism been a l t e r e d . r e s u l t s i n d i c a t e t h a t a e r o b i c and anaerobic  These  o x i d a t i o n of any  given s u b s t r a t e by an organism proceed through d i f f e r e n t enzymic mechanisms, o r t h a t , at any r a t e , these are independently  variable,  mechanisms <  The r e s u l t s r e p o r t e d h e r e i n f u r n i s h d e f i n i t e evidence that the r e s p i r a t o r y enzyme c h a r a c t e r o f the R h i z o b i a i s  -  17  -  extremely u n s t a b l e and i s markedly i n f l u e n c e d by environmental factors „  I t may be assumed, t h e r e f o r e , t h a t the r e s p i r a t o r y  a c t i v i t y e x h i b i t e d by a b a c t e r i a l c u l t u r e at any g i v e n time i s the r e s u l t a n t o f the i n f l u e n c e s of the environmental and c u l t u r a l c o n d i t i o n s t o which the organism has p r e v i o u s l y been exposed®  -  '  18  -  SUMMARY  The aerobic and anaerobic r e s p i r a t o r y a c t i v i t y of s t r a i n s i s o l a t e d from a , s o i l c u l t u r e and from a c u l t u r e of R. t r i f o l i i 224 have been compared. s t r a i n s and s u b s t r a i n s  laboratory These  have been s t u d i e d upon t h e i r  endogenous, g l u c o s e , m a n n i t o l , and sodium  succinate  r e s p i r a t o r y mechanisms. I t has been shown t h a t s u b s t r a i n s laboratory  i s o l a t e d from the  c u l t u r e e x h i b i t an extreme v a r i a b i l i t y i n  r e s p i r a t o r y a c t i v i t y upon a l l s u b s t r a t e s  tested.  Substrains  i s o l a t e d from the s o i l c u l t u r e , on the other hand, show a marked u n i f o r m i t y s o i l has t h e r e f o r e  i n respiratory a c t i v i t y .  Culturing i n  been shown t o s t a b i l i z e the r e s p i r a t o r y  enzyme systems o f R. t r i f o l i i 224© C u l t u r i n g on l a b o r a t o r y media has been proved t o modify the r e s p i r a t o r y enzyme mechanisms of t h i s s t r a i n .  The  anaerobic endogenous r e s p i r a t i o n has been decreased from a very h i g h v a l u e almost t o the v a n i s h i n g  p o i n t , oxidase  a c t i v i t y toward glucose and m a n n i t o l has been t h a t toward s u c c i n a t e  has been decreased.  increased,  A e r o b i c endogenous  r e s p i r a t i o n and dehydrogenase a c t i v i t y upon m a n n i t o l and succinate  have remained unchanged©  - 19 -  I t lias been shown t h a t a e r o b i c and anaerobic r e s p i r a t i o n upon any s u b s t r a t e are e i t h e r c a r r i e d  out by  separate enzyme systems or a r e independently variable©  \  -  20  -  REFERENCES ' Almon, L. and Baldwin, I . L . J o u r . Bact. 26: 229, 1933. B u r r i s , R.H and W i l s o n , P.W. Cold S p r i n g Harbor Symposia on Q u a n t i t a t i v e B i o l o g y , " V o l s 7 : 3 4 9 , 0  1939*  Dixon, M. "Manometric Methods", Cambrige U n i v e r s i t y P r e s s , 1934. F r e d , E.B. and Waksman, S„ "Laboratory Manual o f General M i c r o b i o l o g y " , 19 28. Hopkins, Do  J o u r . Amer. Med. Ass'n 6 0 : 1615, 1913. Jour. Bact. 3 2 : 1 8 3 , 1 9 3 6 .  N e a l , O.Ro and Walker, R.H. Tarn, R.K. and W i l s o n , P.W. Thorne, D.W.  J o u r . B a c t . 42: 3 2 9 , 1941.  and B u r r i s , R.H.  J o u r . B a c t . 3 9 : 1 8 7 , 1940.  Walker, R.H., Anderson, D.H., Brown, P.E, 38: 2 0 7 , 19 3 4 . W i l s o n , P.W., Hopkins, E.W., 32: 231, 1931. W i l s o n , P.W.  F r e d , E.B.  J o u r . B a c t . 3 3 : 6 0 1 , 1938  Soil  Soil  e  Science  Science  INTRODUCTION The  growth, p h y s i o l o g y and n i t r o g e n - f i x i n g a b i l i t y  of  the root nodule b a c t e r i a have been e x t e n s i v e l y s t u d i e d s i n c e t h e i r i s o l a t i o n i n 1886  by H e l l i e g e l and W i l l f a r t h .  Colonies  of these s p e c i e s possess a c h a r a c t e r i s t i c t r a n s p a r e n t , watery type of growth, which i s a t t r i b u t e d to the e l a b o r a t i o n of gummy m a t e r i a l of a p o l y s a c c h a r i d e n a t u r e .  While these  organisms have been shown to u t i l i z e carbohydrate growth medium f o r the s y n t h e s i s of c e l l u l a r  from t h e i r  polysaccharide  the changes o c c u r i n g w i t h i n the medium i t s e l f have r e c e i v e d scant attention© During  s t u d i e s upon the r e s p i r a t o r y enzymes of  Rhizobium t r i f o l i i ,  as r e p o r t e d p r e v i o u s l y ( 4 ) , i t was  observed t h a t zones of c l e a r i n g and p r e c i p i t a t i o n appeared i n the medium around the c o l o n i e s .  These zones appeared t o  be somewhat analogous to the Liesegang  phenomenon.  No  previous r e f e r e n c e to such growth c h a r a c t e r s has been r e p o r t e d w i t h the R h i z o b i a , although Niven, S m i l e y , and Sherman ( 7 ) reported the f o r m a t i o n of c l e a r e d zones around c o l o n i e s of S t r e p . s a l i v a r i u s i n a carbonate medium. The work r e p o r t e d upon h e r e i n c o n s i s t s of a d e s c r i p t i o n of t h i s zoning phenomenon and an i n v e s t i g a t i o n i n t o the v a r i o u s f a c t o r s which determine i t s occurrence.  EXPERIMENTAL METHODS The  s p e c i e s employed most e x t e n s i v e l y i n t h e t e s t s  r e p o r t e d h e r e i n was Rhizobium t r i f o l i i , W i s c o n s i n In  s t r a i n 224,  s t u d i e s on the d i s t r i b u t i o n of the zoning phenomenon among  s t r a i n s and s p e c i e s o f t h e R h i z o b i a a f u r t h e r group o f Wisconsin  s t r a i n s was employed,  R T 2 2 B , RT205, R T 2 2 6 , RT227,  R T 2 3 0 , RT231. A l l other s p e c i e s have  been  i s o l a t e d and  i d e n t i f i e d at t h e U n i v e r s i t y o f B r i t i s h Columbia and have been checked by c r o s s - i n o c u l a t i o n experiments. The medium employed was t h a t recommended by W i l s o n (8) and c o n s i s t s o f t h e m i n e r a l s a l t s o f M.79(3) , w i t h t h e a d d i t i o n of l.Of. D i f c o yeast e x t r a c t , 0.1/t g l u c o s e , O.J>% c a l c i u m c a r bonate , and 1.5f° agar. in  T h i s medium was prepared  f l a s k s containing approximately  200  p l a t e s poured i n a p p r o p r i a t e d i l u t i o n s .  and s t e r i l i z e d  c c . q u a n t i t i e s , and Before p o u r i n g , the  agar was cooled n e a r l y t o the s o l i d i f y i n g p o i n t and was s w i r l e d v i g o r o u s l y i n order t h a t the i n s o l u b l e c a l c i u m carbonate It  might be suspended u n i f o r m l y throughout t h e medium.  was noted t h a t p l a t e s prepared  i n t h i s manner j e l l e d  q u i c k l y and r e t a i n e d the c a l c i u m carbonate  u n i f o r m l y suspended 0  in  the medium.  A l l p l a t e s were incubated at 30 C.  The zones  of complete c l e a r i n g appeared w i t h i n t h r e e days and reached a maximum at about seven days; t h e zones o f d e p o s i t i o n appeared at about t e n days and reached f i f t e e n days.  a maximum at t e n t o  EXPERIMENTAL RESULTS A  e  D e s c r i p t i o n o f Zoning Phenomena The zones or areas o f complete c l e a r i n g and o f  d e p o s i t i o n which appeared when Rhizobium t r i f o l i i .  224 was  p l a t e d upon W i l s o n ' s Agar by t h e t e c h n i q u e a l r e a d y d e s c r i b e d a r e ' S h o w n i n p l a t e s 1 t o 6.  'In  P l a t e 1 the r e l a t i o n s h i p o f c o l o n i a l type to c l e a r i n g  of the medium i s emphasized. the  T h i s photograph was taken w i t h  source o f l i g h t thrown a g a i n s t the s u r f a c e o f t h e medium*  The s m a l l c o l o n i e s w i t h i r r e g u l a r shapes, seen toward the top,  c e n t r e and l e f t o f t h e p l a t e , a r e surrounded by c i r c u l a r  dark zones o f complete, c l e a r i n g .  These are s u b s u r f a c e  c o l o n i e s . The f l a t s p r e a d i n g c o l o n i e s w i t h t h e dark centres and w h i t e edges, seen toward the lower centre o f the p l a t e are  s u r f a c e c o l o n i e s and a p p a r e n t l y do not e x h i b i t  of t h e medium.  clearing  When t h e s u r f a c e growth i s scraped away,  however, the medium d i r e c t l y beneath .the c o l o n y i s seen to have been c l e a r e d .  T h i s p l a t e was photographed a t s i x days  p r i o r t o the appearance o f the secondary zones o f d e p o s i t i o n . A t h i r d c o l o n i a l type i l l u s t r a t e d on P l a t e s 2 and 3 appears as a v e r y t h i n , s p r e a d i n g , f e a t h e r y or v e i l - l i k e subsurface colony, which developed i n l a r g e numbers upon n e a r l y every plate*  This appeared t o be a d i s s o c i a t e d form which i s ,  however, s i m i l a r t o the more normal types i n zone  development.  The photograph as presented i n P l a t e 2 and a l l subsequent ones has been t a k e n w i t h the source o f l i g h t behind t h e p l a t e and s h i n i n g through the medium.  T h i s emphasizes the develop-  - 4 ment of r i n g f o r m a t i o n about the c o l o n i e s , r a t h e r than the colonies  themselves. P l a t e s 2 and 3 show c l e a r l y the i n n e r zones of  complete  c l e a r i n g and the outer zones of d e p o s i t i o n or  darkening i n the medium around the c o l o n i e s .  This e f f e c t i s  p a r t i c u l a r l y n o t i c e a b l e i n the areas between c o l o n i e s growing f a i r l y close together.  I t i s e v i d e n t t h a t surrounding the  c o l o n i e s t h e r e i s a c i r c u l a r r e g i o n i n which the opaque medium has been c o m p l e t e l y c l e a r e d , the area i n most cases appearing dark on the photographs.  Beyond t h i s dark zone  t h e r e occurs a r i n g i n which the opaque medium remains untouched.  Outside t h i s normal a r e a , a g a i n , t h e r e occurs  a t e r t i a r y zone  or r e g i o n  of d e p o s i t i o n i n which  the  o p a c i t y of the medium has been v e r y n o t i c e a b l y i n t e n s i f i e d . With some c o l o n i e s , towards the lower p a r t of the p l a t e s i n eaeh case, the p r i m a r y zones of complete l i g h t and the c o l o n i e s appear dark.  c l e a r i n g appear  -This i r r e g u l a r i t y i s  due to the i n h e r e n t d i f f i c u l t y of photographing  these  shadowy e f f e c t s . P l a t e 4 shows very c l e a r l y the r i n g f o r m a t i o n which develops around i s o l a t e d c o l o n i e s .  I n the examples shown  here the w i d t h of the v a r i o u s zones i s w e l l  illustrated.  In P l a t e s 3 and 6 unusual arrangements of these zones are shown. medium i s apparent colonies.  In P l a t e 5 a v e r y marked darkening of the i n the r e g i o n surrounding a group o f  The c o l o n i e s themselves  appear w h i t e , as do the  c l o s e l y a s s o c i a t e d zones of complete  clearing.  The d e p o s i t i o n  - 5 which, o c c u r s i s q u i t e pronounced and t a k e s p l a c e over a wide area at some d i s t a n c e from the c o l o n i e s .  In P l a t e 6 a f u r t h e r  example of t h i s heavy d e p o s i t i o n i s shown.  Growth  has  occurred i n a complete c i r c l e which i s surrounded by a v e r y wide a r e a of d e p o s i t i o n .  A f u r t h e r area o f p r e c i p i t a t i o n  then occurs i n the medium enclosed by t h i s r i n g of growth. The examples  shown i n P l a t e s 1 to 6 i l l u s t r a t e i n  every case the z o n i n g phenomena which develop when the l a b o r a t o r y s t r a i n of Rhizobium t r i f o l i i "Wilson' s Agar.  224 i s p l a t e d upon  When, however, s t r a i n s of t h i s c u l t u r e which  have been f r e s h l y i s o l a t e d from s o i l are employed, t h e r e i s no e f f e c t whatever upon the medium.  The organism develops as  s m a l l , t r a n s p a r e n t c o l o n i e s which resemble drops of water, and the medium remains untouched.  The t r a n s p a r e n c y of these  c o l o n i e s and the o p a c i t y of the medium made i t i m p o s s i b l e to secure a photograph which would i l l u s t r a t e t h i s normal type of growth.  However, the normal appearance of t h i s medium,  even i n the presence of non-zoning s t r a i n s , i s w e l l  illus-  t r a t e d i n the unchanged p o r t i o n s of P l a t e s 1 and 4. I t i s apparent from the photographs presented t h a t the type of growth d e v e l o p i n g on these p l a t e s i s not t y p i c a l of Rhizobium t r i f o l i i .  The s t r a i n employed, Rhizobium  trifolii  224, had been c u l t u r e d f o r over e l e v e n years upon L a b o r a t o r y media and had i n a d d i t i o n been c a r r i e d f o r s i x months on Wilson's ( 8 ) , medium which as a l r e a d y noted was employed i n these s t u d i e s .  T h i s s t r a i n on the u s u a l yeast water m a n n i t o l  agar (M.79), p o s s i b l y as a r e s u l t of prolonged l a b o r a t o r y  •- 6 c u l t i v a t i o n produced a dry, coarse and w r i n k l e d type of growth which would i n d i c a t e a profound change i n the hydrate a s s i m i l a t o r y process.  The  carbo-  i n f l u e n c e , which  c u l t u r i n g upon l a b o r a t o r y media e x e r t s upon r e s p i r a t o r y enzyme c h a r a c t e r , has a l r e a d y been r e p o r t e d ( 5 ) . B  *  FACTORS INFLUENCING ZONE DEVELOPMENT.. . Since p r e l i m i n a r y s t u d i e s i n d i c a t e t h a t development  of the zoning phenomenon i s a s s o c i a t e d w i t h the presence of c a l c i u m carbonate,  glucose and yeast e x t r a c t i n the  medium, an e x t e n s i v e i n v e s t i g a t i o n of the v a r i o u s f a c t o r s c o n t r i b u t i n g to the p r o d u c t i o n of these phenomena was undertaken.  The  i n f l u e n c e of v a r i o u s carbohydrates,  sources, carbonates,  nitrogen  and t r a c e elements, as w e l l as the  d i s t r i b u t i o n of t h i s phenomenon among s p e c i e s and s t r a i n s of the R h i z o b i a are r e p o r t e d h e r e i n . 1.  R e l a t i v e Number of  Colonies  I t has been observed t h a t w i t h r e l a t i v e l y few c o l o n i e s on a p l a t e the zoning phenomena developed completely f o r m a t i o n was  c l e a r , d i s t i n c t and e x t e n s i v e .  and  ring  However, as  the number of c o l o n i e s per p l a t e i n c r e a s e d the s i z e of the zones c o r r e s p o n d i n g l y d i m i n i s h e d , u n t i l i n very crowded p l a t e s the e f f e c t had  e n t i r e l y disappeared  and the  individual  c o l o n i e s were d i s t i n c t l y s m a l l . S i n c e these r e s u l t s may  suggest the e l a b o r a t i o n of  i n h i b i t o r y substances under crowded c o n d i t i o n s i t was to t e s t the p r o p e r t i e s of f i l t r a t e s of t h i s organism. f l a s k of the u s u a l medium was  prepared  without  decided A  agar, i n o c u l a t e d  w i t h Rh. t r i f o l i i  224, and incubated f o r t e n days at 30°  C.  The c u l t u r e was then passed through a S e i t z f i l t e r and a clear s t e r i l e f i l t r a t e obtained.  Due to the  possibility  t h a t an a c i d i n h i b i t o r y substance might be n e u t r a l i z e d by the excess c a l c i u m carbonate, d u p l i c a t e f i l t r a t e s were prepared from c u l t u r e s i n the b a s i c l i q u i d medium w i t h and w i t h o u t added carbonate. a s s o c i a t i o n w i t h growing  These f i l t r a t e s alone and i n c u l t u r e s were t e s t e d t o  determine  t h e i r i n f l u e n c e , i f any, upon the c l e a r i n g phenomenon. e f f e G t was  No  observed w i t h these  No  filtrates*  e x p l a n a t i o n f o r the i n h i b i t i o n of zoning on  densely seeded p l a t e s i s o f f e r e d at t h i s t i m e .  If clearing  phenomena are dependent upon the f o r m a t i o n of a c i d from the glucose i n the medium i t seems reasonable t o expect t h a t more a c i d and c o n s e q u e n t l y more c l e a r i n g would be produced i n a h e a v i l y - s e e d e d than i n a l i g h t l y - s e e d e d Experiments  plate.  i n which brom thymol blue- i n d i c a t o r was  to the medium t o determine  added  the r e l a t i o n s h i p of zone f o r m a t i o n  to a c i d p r o d u c t i o n gave i n c o n c l u s i v e r e s u l t s . 2*  Calcium Carbonate and Other S a l t s .  S i n c e p r e l i m i n a r y experiments the z o n i n g phenomenon was  had demonstrated  that  associated with three constituents  of the medium - c a l c i u m carbonate, glucose and yeast  extract,  i t appeared a d v i s a b l e t o t e s t the e f f e c t of v a r i o u s c a l c i u m carbonates and o t h e r r e l a t e d s a l t s .  Media were prepared  c o n s i s t i n g o f m i n e r a l s a l t s , agar, glucose and yeast e x t r a c t i n the u s u a l p r o p o r t i o n s and t o t h i s b a s i c medium were added  . - 8 0 . 3 $ c o n c e n t r a t i o n s of the f o l l o w i n g Calcium carbonate ( s i x samples),  salts:  calcium s u l f a t e , t r i  c a l c i c phosphate, and magnesium carbonate. With these media poured p l a t e s were prepared i n s u i t a b l e dilutions. The data r e p o r t e d i n Table 1 i n d i c a t e s t h a t the primary zone of complete c l e a r i n g develops i n the presence of a l l t h e v a r i o u s c a l c i u m carbonates t e s t e d .  The  zone of d e p o s i t i o n , however, occurs o n l y when one  secondary particular  carbonate i s used.  TABLE  1.  I n f l u e n c e of V a r i o u s Carbonates and R e l a t e d S a l t s upon the Zoning Phenomenon. S a l t Added  Growth C h a r a c t e r i s t i c s  CaCoj - c o n t r o l  Both c l e a r i n g and d e p o s i t i o n  CaCoj - S p e c i a l (Low i n A l k a l i s )  Growth, c l e a r i n g , no d e p o s i t i o n  CaCoj - (4 Samples)  C l e a r i n g , no d e p o s i t i o n  CaS0  No  4  -  CAj(P0 ) 4  MgCOj  2  growth  Normal growth, no c l e a r i n g Growth, v e r y s l i g h t  clearing  T h i s would i n d i c a t e t h a t the zone of primary c l e a r i n g i s a g e n e r a l phenomenon eaused by a c i d p r o d u c t i o n , w h i l e the secondary zone of d e p o s i t i o n i s a s p e c i f i c , phenomenon dependent upon the presence of some i m p u r i t y or group of i m p u r i t i e s o c e u r i n g i n the p a r t i c u l a r sample of c a l c i u m carbonate o r i g i n a l l y employed.  The r e s u l t presented  i n Table 1 suggest t h a t the primary c l e a r i n g i s p o s s i b l y more l i n k e d w i t h the carbonate r a d i c a l than w i t h the c a l c i u m i o n , s i n c e some s l i g h t c l e a r i n g developed i n the presence of magnesium carbonate but not i n the presence of calcium  phosphate,  3» E f f e c t of Trace Elements upon Zoning, In view of the r e s u l t s recorded i n Table 1 i t was decided to determine the e f f e c t of v a r i o u s t r a c e upon the development  elements  of the primary and secondary zones.  A c c o r d i n g l y the u s u a l b a s i c agar was prepared, c o n t a i n i n g 0. 3f« of a c a l c i u m carbonate which d i d not s t i m u l a t e the f o r m a t i o n o f secondary zones.  To 100 cc. q u a n t i t i e s o f t h i s  medium s a l t s o f v a r i o u s elements were added t o a c o n c e n t r a t i o n of 30 mgm.  per 100 cc.  These media were then p l a t e d i n  s u i t a b l e d i l u t i o n s w i t h Rh. t r i f o l i i  224.  The e x p e r i m e n t a l r e s u l t s r e c o r d e d i n Table 2 show t h a t s m a l l amounts of c e r t a i n t r a c e elements e x e r t a marked i n f l u e n c e upon the c l e a r i n g phenomenon.  The a d d i t i o n of  c a l c i u m c h l o r i d e f o r i n s t a n c e , r e s u l t e d i n a marked i n c r e a s e i n the s i z e of c l e a r e d a r e a s , w h i l e z i n c a c e t a t e e n t i r e l y  - 10 prevented t h e i r appearance.  .  Between these two extremes  aluminum n i t r a t e , b o r i c a c i d and l i t h i u m c h l o r i d e a r e observed to e x e r c i s e chloride of  no i n f l u e n c e ,  w h i l e ammonium s u l p h a t e , barium  and manganese s u l p h a t e markedly depressed the degree  clearing TABL1D  2  E f f e c t o f Trace Elements Upon C l e a r i n g Phenomenon - Rh. t r i f o l i i 224. Salt Added.  Grov/th  Characteristies  AC(Noj)^  P r i m a r y c l e a r i n g , no  (NH4)  P r i m a r y c l e a r i n g markedly depressed  2  SO^  deposition  BaCLg  P r i m a r y c l e a r i n g s l i g h t l y depressed  HjBOj  Very l i t t l e  CaClg  C l e a r i n g zones very g r e a t l y extended No d e p o s i t i o n . Medium g r a n u l a r Normal c l e a r i n g . C o l o n i e s have very  CuSO^  e f f e c t upon c l e a r i n g  noticeable dark, c o p p e r - c o l o r e d c e n t r e s . FeSO^  Restricted  c l e a r i n g . Only s u r f a c e  PbAc2  Normal c l e a r i n g . The c o l o n i e s are d i s t i n c t l y dark, denoting s u l f i d e f o r m a t i o n .  LiCl  Normal c l e a r i n g  MnS04  Very r e s t r i c t e d  ZnAC2  No c l e a r i n g at a l l  clearing  colonies.  - 11 Trace elements were a l s o found t o exert an i n f l u e n c e upon f a c t o r s other than t h e c l e a r i n g phenomenon* When copper - sulphate was added t o t h e medium normal c l e a r i n g developed but t h e c o l o n i e s possessed centres.  dark-colored  T h i s would i n d i c a t e t h a t the organisms possessed  the a b i l i t y t o f i x copper o r c o p p e r - p r o t e i n within their colonies.  complexes  When f e r r o u s sulphate was employed  i n t h e growth medium a reducing p o t e n t i a l was s e t up and t h e c o l o n i e s were able to develop o n l y on t h e s u r f a c e of the medium.  This o b s e r v a t i o n i s i n accord w i t h t h e p u b l i s h e d  r e s u l t s o f A l l y n and Baldwin  (1,2) r e l a t i v e t o t h e i n f l u e n c e  of o x i d a t i o n - r e d u c t i o n p o t e n t i a l s on t h e growth of Rhizobia.  When l e a d a c e t a t e was i n c o r p o r a t e d i n t h e medium  normal c l e a r i n g o c c u r r e d but t h e c o l o n i e s were v e r y n o t i c e a b l y darkened, denoting s u l f i d e f o r m a t i o n . t h i s darkening  I n t e r e s t i n g l y , however,  of the c o l o n i e s took p l a c e o n l y on l i g h t l y -  seeded p l a t e s ; when h e a v i l y seeded p l a t e s were examined the c o l o n i e s were of normal appearance.  There would, t h e r e f o r e ,  appear t o be a c l o s e r e l a t i o n s h i p between s u l f i d e  formation  and t h e a c i d c l e a r i n g phenomenon. 4.  I n f l u e n c e o f Carbohydrates upon Zoning. Since p r e l i m i n a r y experiments i n d i c a t e d t h a t no  zone f o r m a t i o n o f any s o r t developed i n t h e absence o f glucose i t appeared d e s i r a b l e t o t e s t the e f f e c t o f v a r i o u s other carbohydrates  sources  i n t h e growth medium.  s a l t s agar was t h e r e f o r e prepared  Mineral  as b e f o r e , and to t h i s  - 12 b a s i c medium v a r i o u s carbohydrates were added i n concentration.  0.1$  Poured p l a t e s were then prepared i n s u i t a b l e  dilutions. - , Reference t o the e x p e r i m e n t a l data as presented i n Table 3 d i s c l o s e s t h a t d i f f e r e n t carbohydrate sources exert a marked i n f l u e n c e upon the c l e a r i n g phenomenon. With the m a j o r i t y o f these c a r b o h y d r a t e s , c l e a r i n g develops t o an extent comparable t o that when glucose i s employed as the energy source.  This i s e s p e c i a l l y noticeable with  the monosaccharides,  a l t h o u g h w i t h g a l a c t o s e the c l e a r i n g  i s less extensive.  Of the pentoses, a r a b i n o s e i s observed  to produce complete  c l e a r i n g , w h i l e t h a t induced by x y l o s e  i s very uncomplete and r e s t r i c t e d .  T h i s r e s u l t i s the  exact r e v e r s e of the dehydrogenation r e a c t i o n s , as r e p o r t e d by Morgan, L a i r d and E a g l e s ( 4 ) , dehydrogenated all.  who  found t h a t x y l o s e was  r a p i d l y w h i l e a r a b i n o s e was not a t t a c k e d at  The d i s a c c h a r i d e s " e x c e p t l a c t o s e and m e l i b i o s e appear  to be q u i t e e f f e c t i v e ; l a c t o s e induces incomplete and r e s t r i c t e d c l e a r i n g w h i l e no zone f o r m a t i o n occurs w i t h melibiose.  TABLE - 3 E f f e c t of V a r i o u s Carbohydrate Sources upon C l e a r i n g Phenomenon Rh» t r i f o l i i 224. Carbon Source  Growth C h a r a c t e r i s t i c s .  Glucose  - 1.  Mannose  2*  Very good z o n i n g .  Galactose  3.  Good t o f a i r  Fructose  4.  Very good c l e a r i n g  Arabinose  3.  Very good c l e a r i n g .  Xylose  .6. •  Clearing  Rhamnose  7•  Very go od c l e a r i n g .  Methyl glucoside Sucrose  • 8. 9.  Very complete zone f o r m a t i o n .  No  clearing.  fair.  clearing.  Very good c l e a r i n g .  Cellobiose  10,  Good t o f a i r  Lactose  11.  Clearing  clearing.  f a i r t o poor.  Very good c l e a r i n g .  Maltose Trehalose  13.  Clearing  Melibiose  14.  No  clearing.  Raffinose  15.-  No  clearing.  Melizitose  16..  Clearing  d o u b t f u l , s l i g h t trace„  Dextrin  17.  Clearing  only f a i r .  Starch  18.  Clearing  good.  Salicin  19.  Very good c l e a r i n g .  Glycerol  20.  No  clearing.  Erythritol  21.  No  clearing.  Adonitol  22*  No  clearing.  Dulcitol  23.  No  clearing.  Mannitol  24.  Very good c l e a r i n g .  Sorbitol  23.  F a i r l y good c l e a r i n g .  Sod. s u c c i n a t e Sod* malate  26. 27.  No No  f a i r l y good.  clearing. clearing.  . - 14 A comparison of the r e s u l t s presented i n Table 3 w i t h those on dehydrogenase a c t i v i t y , u s i n g the same s t r a i n of R h i z o b i a and the same carbohydrates ( 4 ) r e v e a l s the i n t e r e s t i n g f a c t t h a t t h e r e i s l i t t l e c o r r e l a t i o n between these two processes.  I t i s p a r t i c u l a r l y n o t i c e a b l e t h a t t h e r e i s no  zone f o r m a t i o n when o r g a n i c a c i d s such as s u c c i n i c and m a l i c are i n c o r p o r a t e d i n the medium, a l t h o u g h these a c i d s are q u i t e readily j>.  dehydrogenated. I n f l u e n c e of N i t r o g e n Source upon Zoning. S i n c e p r e l i m i n a r y experiments i n d i c a t e d t h a t zone  f o r m a t i o n and d e p o s i t i o n are a s s o c i a t e d i n p a r t w i t h the yeast e x t r a c t c o n t a i n e d i n the medium i t was decided to t e s t the i n f l u e n c e of v a r i o u s o t h e r n i t r o g e n sources i n t h i s regards A c c o r d i n g l y m i n e r a l s a l t s , a g a r was prepared as u s u a l and c o n c e n t r a t i o n s of v a r i o u s n i t r o g e n sources added.  1.0$  The  c a l c i u m carbonate employed was the one which n o r m a l l y develops areas of complete c l e a r i n g but not areas of d e p o s i t i o n . The data recorded i n Table 4 shows t h a t the n i t r o g e n source present i n the medium e x e r t s a profound i n f l u e n c e upon the development of both c l e a r e d zones and r e g i o n s of d e p o s i t i o n .  The most important r e s u l t to be observed i n  t h i s c o n n e c t i o n i s the development of areas of d e p o s i t i o n i n a d d i t i o n t o c l e a r areas when e i t h e r beef e x t r a c t or alamine were employed as n i t r o g e n s o u r c e s .  S i n c e the yeast e x t r a c t  c o n t r o l showed o n l y areas o f primary c l e a r i n g the mechanism of the f o r m a t i o n of these secondary zones i s s t i l l  obscure•  - 15 TABLE - 4. I n f l u e n c e of V a r i o u s N i t r o g e n Sources upon C l e a r i n g and Zoning Phenomena Rh. t r i f o l i i N i t r o g e n Source  224,  Growth C h a r a c t e r i s t i c s . .  Edestin  Growth f a i r , c l e a r i n g hazy and incomplete.  Bacto beef  Growth good, o n l y s l i g h t  Gelatine  Good growth, e x t e n s i v e c l e a r i n g  Sod. C a s e i n a t e  Good growth w i t h  P r o t e o s e peptone  Clearing  Peptone  Good growth and c l e a r i n g  Difco  clearing  clearing  good, growth good*  Peptone. - W i t t e  P r i m a r y c l e a r i n g , good  Tryptone  Growth good, primary c l e a r i n g  Yeast e x t r a c t - D i f c o  Extremely good growth and c l e a r i n g  Yeast e x t r a c t - D i f c o (pantothenic a c i d fr ee)  Good growth, almost e n t i r e l y on the s u r f a c e , incomplete c l e a r i n g .  Yeast e x t r a c t  Very good growth, c l e a r i n g not extensive  -  -orla J ens-en.  growth  Yeast water  Very good growth, c l e a r i n g  Beef  C l e a r i n g w i t h areas of  extract  extensiv  deposition  Tyrosine  No growth, no c l e a r i n g  Asparagine  F a i r growth, good c l e a r i n g  Alamine  Good growth, w i t h c l e a r i n g and possibly deposition  Glycine  Very good growth, good  Urea  Growth f a i r , no c l e a r i n g at a l l  clearing  With the v a r i o u s n i t r o g e n sources  employed growth  occurred i n a l l cases, except w i t h t y r o s i n e , g l y c i n e and edestin.  I t i s to be observed a l s o t h a t primary c l e a r i n g was  obtained w i t h almost every n i t r o g e n source employed. are observed w i t h Bacto Beef and Orla-Jensen*s where growth was pantothenic  Exceptions  yeast e x t r a c t ,  good but c l e a r i n g very r e s t r i c t e d ; w i t h  a c i d - f r e e yeast e x t r a c t , where growth was  e n t i r e l y on the s u r f a c e and c l e a r i n g incomplete; urea, where growth was  almost  and  f a i r l y good but c l e a r i n g was  with entirely  absent. 6•  D i s t r i b u t i o n of Zoning Phenomenon.  In view of the c l e a r i n g and zoning phenomena w i t h trifolii  224,  i t was  Rh.  decided to determine the extent to which  these changes occur w i t h other s p e c i e s and s t r a i n s of R h i z o b i a . . A c c o r d i n g l y , a group of c u l t u r e s , r e p r e s e n t a t i v e of strains of Rh. t r i f o l i i , Rh. m e l i l o t i , Rh. p h a s e o l i , Rh. l u p i n i , Rh. leguminosarum, and the l o t u s and  coropea  organisms, were p l a t e d i n s u i t a b l e d i l u t i o n s upon the u s u a l medium. The  data as presented  i n Table 5 i n d i c a t e s t h a t the  a b i l i t y to cause c l e a r i n g of the medium i s q u i t e w i d e l y d i s t r i b u t e d among the v a r i o u s s p e c i e s of R h i z o b i a . phenomenon was  This  found to occur w i t h a l l s t r a i n s of Rh.  t e s t e d , but w i t h v a r y i n g degrees of completeness. observed to a marked degree w i t h c u l t u r e s coropea s t r a i n s .  trifolii  I t was  also  of the l o t u s and  Among s t r a i n s of Rh. leguminosarum, Rh.  l u p i n i and Rh. p h a s e o l i c l e a r i n g was  apparent, but to a very  l i m i t e d extent.  With Rh. m e l i l o t i , however, no c l e a r i n g was  observed w i t h any o f t h e f i v e s t r a i n s  tested.  TABLE 5. D i s t r i b u t i o n o f C l e a r i n g Phenomenon Among S t r a i n s and Species o f R h i z o b i a . Growth C h a r a c t e r i s t i c s .  Species o r S t r a i n R. t r i f o l i i 224 R.T.  227  R.T.  230  L i g h t growth, v e r y pronounced c l e a r i n g . Medium growth, complete c l e a r i n g . L i g h t growth, complete c l e a r i n g .  R.T.  231'  L i g h t growth, complete c l e a r i n g .  R.T.  205  Heavy gummy growth, o n l y p a r t i a l clearing.  R.T. 226 R.T. 40-1  Light  growth, c l e a r i n g not e x t e n s i v e .  Growth l i g h t , c l e a r i n g  extensive.  R.T, 22B  Growth medium, c l e a r i n g e x t e n s i v e .  R.T. 3 9 - 1  Growth l i g h t , complete c l e a r i n g •  R.T.  39-2  Growth l i g h t , complete c l e a r i n g .  Rh. m e l i l o t i 3 9 - 1  Gummy growth, some t r a c e s  R« mel. 4 0 - 1  Gummy growth, no c l e a r i n g .  R. mel. 4 0 - 2  Gummy growth, no c l e a r i n g *  R. mel. 41-2  Gummy growth, no c l e a r i n g .  R. m e l . 4 2 - 1  Gummy growth, no c l e a r i n g .  R. p h a s e o l i 4 2 - 1  Gummy growth, s l i g h t t r a c e of c l e a r i n g .  R. l u p i n i 3 9 - 1  L i g h t , watery growth, some c l e a r i n g *  R. l u p i n i 4 2 - 2  Medium growth, incomplete c l e a r i n g .  R. leguminosarum 4 1 - 1  Very gummy growth, p a r t i a l  R. legumin. 4 1 - 2  Very gummy growth, some t r a c e o f clearing.  R. l o t u s 42-2  Medium growth, v e r y complete c l e a r i n g .  R. coropea 42-1  V e r y gummj'- growth, complete c l e a r i n g .  of clearing.  clearing.  DISCUSSION. From the photographs presented ( P l a t e s 1 to 6 i n c l ) i t i s apparent t h a t the  c o l o n i a l c h a r a c t e r i s t i c s of  t r i f o l i i upon t h i s medium are  extremely a t y p i c a l  s u g g e s t i v e of a rough or d i s s o c i a t e d t h a t c u l t u r i n g upon the the p r o d u c t i o n of two extremely t h i n and  form.  and  I t would seem  p a r t i c u l a r medium employed has  types of c o l o n i e s ,  one  caused  of which i s  of a v e i l - l i k e or f e a t h e r y  These c o l o n i a l t y p e s , however, are not s i n c e i t has  Rh.  stable  structure. in character,  been proved to be i m p o s s i b l e to o b t a i n f i x e d  s u b s t r a i n s even by r e p e a t e d l y  p l a t i n g and  selectively  p i c k i n g f o r c o l o n i a l type over an extended p e r i o d .  I t would  appear t h a t some f a c t o r , p r o b a b l y the h i g h yeast content,  has  caused the p r o d u c t i o n of these a t y p i c a l c o l o n i a l forms, which are the r e s u l t of a d i r e c t s t i m u l a t i o n and  are  consequently not  t y p e , moreover, has of the 2 and  no  of a s t a b l e  by the medium i t s e l f  nature.  The  e f f e c t whatever upon the  colonial development  c l e a r i n g phenomenon, as i s shown c l e a r l y i n  Plates  3. The  l a c k of c o r r e l a t i o n between zone f o r m a t i o n  and  dehydrogenation w i t h v a r i o u s carbohydrates i n d i c a t e s t h a t zoning phenomenon i s i n t i m a t e l y p r o d u c t i o n mechanism of the  cell.  e x i s t s a l a c k of r e l a t i o n s h i p p r o d u c t i o n and  bound up w i t h the  r e s p i r a t i o n and  Laird  acid  I t would appear t h a t t h e r e  between the  processes of  acid  i n t h i s r e s p e c t i s somewhat  s i m i l a r to t h a t r e p o r t e d f o r the l a c t i c , a c i d by Morgan, E a g l e s and  (6).  the  The  streptococci  r e p r e s s i o n of zone  formation  and t h e r e f o r e a c i d p r o d u c t i o n upon  heavily-seeded  p l a t e s i n d i c a t e s t h a t the mechanism of a c i d p r o d u c t i o n extremely s e n s i t i v e to environmental changes.  is  F a i l u r e to  secure a c t i v e f i l t r a t e s from c u l t u r e s i m p l i e s t h a t  the  i n h i b i t o r y e f f e c t upon crowded p l a t e s i s caused by changes i n the p h y s i c a l - c h e m i c a l  nature of the medium i t s e l f r a t h e r than  by the e l a b o r a t i o n of t o x i c chemical substances,, From the r e s u l t s obtained  i n the study of the  influence  of v a r i o u s n i t r o g e n sources i n the medium upon the c l e a r i n g phenomenon i t would appear t h a t the R h i z o b i a are able to grow i n the presence of a wide range of n i t r o g e n o u s compounds, ranging  from simple amino a c i d s up t o complete p r o t e i n s .  It  would appear too t h a t c l e a r i n g of the medium i s a phenomenon independent of the growth requirements of the organism. Although c l e a r i n g does not appear to depend upon of any  s p e c i a l type of n i t r o g e n o u s organic  more complex than urea i s r e q u i r e d . a c u r i o u s l a c k of c o n s i s t e n c y preparations  the presence  compound, a source  There i s e v i d e n t ,  i n r e s u l t s when v a r i o u s yeast  are employed as the n i t r o g e n source.  i n d i c a t e t h a t the c l e a r i n g and r e l a t e d i n some way  too,  This  may  d e p o s i t i o n phenomena are  t o the a c c e s s o r y  f a c t o r s of the  Vitamin  B complex. The and  r e s u l t s obtained  s p e c i e s of R h i z o b i a  from a study of the v a r i o u s s t r a i n s  show t h a t the c l e a r i n g and  presumably  a l s o the d e p o s i t i o n phenomena are q u i t e w i d e l y d i s t r i b u t e d . I t i s n o t i c e a b l e , a l s o , t h a t these phenomena are most developed by the Rh.  trifolii  s t r a i n s , which are  completely  characterized  - 20 by l i g h t growth and absence o f gum formations,  The Rh.  m e l i l o t i s t r a i n s , which have a c h a r a c t e r i s t i c a l l y heavy and gummy growth, do not e x h i b i t t h i s c l e a r i n g o f the medium.  That t h e r e i s no c o n s i s t e n t r e l a t i o n s h i p between  gum p r o d u c t i o n and zone f o r m a t i o n i s shown by t h e s t r a i n s of the l o t u s and coropea organisms. developed  These c u l t u r e s  an extremely heavy gummy growth and a l s o e x h i b i t e d  markedly complete primary zones o f c l e a r i n g . The determined phenomena.  e x p e r i m e n t a l s t u d i e s r e p o r t e d h e r e i n have t h e i n f l u e n c e o f many f a c t o r s upon t h e zoning Hovsrever, t h e mechanism o f primary c l e a r i n g and,  more p a r t i c u l a r l y , t h e zone of d e p o s i t i o n , remains  obscure.  Whereas t h e development o f zones o f complete c l e a r i n g may be a t t r i b u t e d t o a c i d p r o d u c t i o n from the carbohydrate  i n the  medium w i t h the r e s u l t i n g d i s s o l u t i o n o f t h e suspended c a l c i u m carbonate the f o r m a t i o n of the r i n g s o f d e p o s i t i o n i s much more c o m p l i c a t e d .  T h i s d e p o s i t i o n i s not a t r u e darkening  of the medium but i s r a t h e r a g r e a t l y i n c r e a s e d o p a c i t y . I t i s demonstrable w i t h c o l o r l e s s media, such as those c o n t a i n i n g g e l a t i n e as the n i t r o g e n source.  I t has been d e f i n i t e l y shown  t h a t f o r d e p o s i t i o n t o take p l a c e , t h r e e f a c t o r s a r e necessary, a fermentable  carbohydrate  and c a l c i u m carbonate.  source, a s u i t a b l e n i t r o g e n source  I t i s important t o note, moreover,  t h a t o f a group o f s i x carbonates t e s t e d , o n l y one sample permitted true r i n g formation.  T h i s would i n d i c a t e t h e  n e c e s s i t y f o r one or more t r a c e elements o r a c c e s s o r y f a c t o r s present as i m p u r i t i e s adsorbed  on t h e c a l c i u m  carbonate.  - 21 This view i s strengthened  by the r e s u l t s obtained upon the  a d d i t i o n of v a r i o u s t r a c e elements to the medium as r e p o r t e d i n Table 2. . I t i s evident t h a t both c l e a r i n g and d e p o s i t i o n are markedly i n f l u e n c e d by the presence of s m a l l amounts of various s a l t s .  The most l o g i c a l e x p l a n a t i o n f o r the  formation  of the r i n g s of d e p o s i t i o n would be the d i f f u s i o n from the colony of some u n i d e n t i f i e d substance,  probably o r g a n i c i n  n a t u r e , which i s p r e c i p i t a t e d by the a c t i o n of c a l c i u m ions and c a t a l y s e d by the presence of t r a c e elements.  This  h y p o t h e s i s , however, does not account f o r the r i n g of unchanged medium which i n t e r v e n e s between the primary zone of complete c l e a r i n g and the o u t e r zone of d e p o s i t i o n . Although  the phenomenon of c l e a r i n g and  deposition  i n the media are i n t e r e s t i n g i n themselves, t h e r e i s the p o s s i b i l i t y t h a t they may c h a r a c t e r of the c e l l .  be r e l a t e d to the r e s p i r a t o r y enzyme  I t has been demonstrated, w i t h the  s t r a i n employed i n these t e s t s , Rh. t r i f o l i i 224, t h a t very e x t e n s i v e zones of c l e a r i n g and d e p o s i t i o n are formed by the " c u l t u r e d " or " l a b o r a t o r y " s t r a i n .  When, however, t h i s same  s t r a i n i s f r e s h l y r e - i s o l a t e d from s o i l i t causes no change whatever i n the medium. t r a n s p a r e n t watery forms.  The  c o l o n i e s now  appear as t y p i c a l  T h i s o b s e r v a t i o n i s i n accord w i t h  the p r e v i o u s r e p o r t of Morgan, L a i r d and Eagles " l a b o r a t o r y " s t r a i n s of Rh. t r i f o l i i  (5) t h a t  possess a s i g n i f i c a n t l y  d i f f e r e n t type of r e s p i r a t i o n from f r e s h l y - i s o l a t e d cultures.  "soil"  I t would appear, t h e r e f o r e , t h a t the development of  «. 22 these z o n i n g phenomena i s i n d i c a t i v e o f a d e f i n i t e i n s t a b i l i t y i n t h e r e s p i r a t o r y enzyme c h a r a c t e r of the c u l t u r e which i s a s s o c i a t e d w i t h a fundamental the carbohydrate mechanism of the c e l l .  change i n  - 2? SUMMARY 9  , -  The development of r i n g formations s i m i l a r to the Liesegang w i t h Rhizobium t r i f o l i i .  somewhat  phenomenon has been demonstrated T h i s zone f o r m a t i o n i s d e s c r i b e d  and i l l u s t r a t e d i n a s e r i e s o f p l a t e s . The i n f l u e n c e o f v a r i o u s f a c t o r s upon t h i s zone f o r m a t i o n has been s t u d i e d .  Carbohydrates, n i t r o g e n  source,  carbonates and t r a c e elements are a l l shown to exert an influence. The occurrence  of the zoning phenomenon has been  surveyed u s i n g a r e p r e s e n t a t i v e group of twenty-two s t r a i n s and s p e c i e s of R h i z o b i a . The mechanism o f zone f o r m a t i o n i s d i s c u s s e d and related to respiratory a c t i v i t y .  - 24 REFERENCES.  1.  A l l y n , W.P.. and B a l d w i n , I.L. - J o u r . B a c t . 20:417, 1930  2.  A l l y n , W.P. and B a l d w i n , I . L . - Jour. B a c t . 2 3 : 3 6 9 ,  3.  F r e d , E.B. and lakeman, S.- " L a b o r a t o r y Manual of General  1932  M i c r o b i o l o g y " 1928 4.  Morgan, J.F, L a i r d , D.G. E a g l e s , B.A.  3.  Morgan, J.F. L a i r d , ' D.G. E a g l e s , B.A.  6. 7.  Morgan, J.F. L a i r d , D.G, E a g l e s , B.A. N i v e n , C.F. S m i l e y , K.L. Sherman, J,M. - J o u r . B a c t . 41: . 4 7 9 , 1941.  8.  W i l s o n , P.W.  - J o u r . B a c t . 3 5 : 6 0 1 , 1938  ABSTRACT  Methods i n v o l v e d  i n the p r e p a r a t i o n  of r e s t i n g  cell  suspensions o f the l a c t i c a c i d b a c t e r i a s u i t a b l e f o r dehydrogenase s t u d i e s by the Thunberg technique have been investigated* The dehydrogenase a c t i v i t y  of f o u r t e e n s t r a i n s of l a c t i c  a c i d b a c t e r i a upon s i x t y t e s t compounds has been determined. V a r i a t i o n i n dehydrogenase a b i l i t y 'has been shown t o e x i s t w i t h i n s t r a i n s o f S t r e p , l a c t i s , S t r e p , cremoris and the B e t a c o c c i ,  and w i t h i n the same s t r a i n at d i f f e r e n t t i m e s .  I t has been found p o s s i b l e t o d i s t i n g u i s h the a e r o b i c pseudo l a c t i c a c i d b a c t e r i a from the t r u e l a c t i c a c i d c o c c i upon t h e i r r e s p i r a t o r y  strepto-  characters.  The a e r o b i c pseudo l a c t i c a c i d b a c t e r i a have been shown to possess an endogenous r e s p i r a t i o n which i s not e x h i b i t e d the l a c t i c a c i d s t r e p t o c i c e i .  by  The f e e b l e  dehydrogenation of l a c t o s e by the l a c t i c  s t r e p t o c o c c i , t h e r e l a t i o n s h i p of isomeric dehydrogenase a b i l i t y , the i n h i b i t o r y  alcohols to  e f f e c t of amines and the  methyl group upon dehydrogenation, and f a i l u r e t o dehydrogenate c i t r a t e have been d i s c u s s e d . The p o s s i b i l i t y of b a s i n g a c l a s s i f i c a t i o n o f the l a c t i c a c i d s t r e p t o c o c c i upon r e s p i r a t o r y enzyme c h a r a c t e r has been considered*  INTRODUCTION The f i r s t o b s e r v a t i o n s on the dehydrogenating  activities  of b a c t e r i a were recorded by Harden and Z i l v a ( 3 ) i n 1 9 1 5 , who n o t i c e d that washed suspensions o f B a c t . c o l i ability  a c q u i r e d the  to reduce methylene blue upon the a d d i t i o n o f v a r i o u s  i n a c t i v e reagents.  The s y s t e m a t i c i n v e s t i g a t i o n o f these  dehydrogenase enzymes was i n i t i a t e d by the i n t r o d u c t i o n of the a n a e r o b i c technique by Thunberg ( 1 8 ) i n 1 9 2 0 . The Thunberg method and " r e s t i n g c e l l " technique have been e x t e n s i v e l y a p p l i e d t o t h e study o f t h e r e s p i r a t o r y enzyme systems o f b a c t e r i a .  Q,uast e l and Whetham ( 9 ) s t u d i e d  the e q u i l i b r i a e x i s t i n g between s u c c i n i c , f u m a r i c , and m a l i c acids i n t h e presence o f " r e s t i n g " B a c t . c o l i , and found a c l o s e a s s o c i a t i o n between c h e m i c a l a c t i v i t y and p h y s i c a l s t r u c t u r e o f t h e organism.  Q,uastel and Whetham ( 1 0 ) continued  t h e i r study by t e s t i n g t h e dehydrogenase a c t i v i t y of B a c t . c o l i upon a l a r g e number o f s u b s t r a t e s , d i b a s i c a c i d s , hydroxy a c i d s , p o l y h y d r i c alcohols.  including fatty  acids,  and monohydric  In a f u r t h e r paper Q u a s t e l and Whetham ( l l ) r e p o r t e d  dehydrogenations by B a c t . c o l l i n the presence of v a r i o u s carbohydrates, and amino a c i d s .  K e n d a l l (6) i n v e s t i g a t e d the  dehydrogenase enzyme a c t i v i t i e s  of Bact. e o l i and other  r e l a t e d b a c t e r i a l s p e c i e s upon a v a r i e t y of s u b s t r a t e s . The dehydrogenase enzymes of t h e L a c t i c A c i d S t r e p t o cocci have not as y e t been e x t e n s i v e l y i n v e s t i g a t e d .  Farrell  (2) r e p o r t e d upon the r e s p i r a t o r y mechanism of 22 s t r a i n s of s t r e p t o c o c c i , s t r a i n s which c o n s i s t e d mainly of pathogenic types but which a l s o i n c l u d e d S t r e p , l a c t i s and S t r e p ,  fecalis  K a t a g i r i and K l t a h a r a (j>) showed the presence o f l a c t i c a c i d dehydrogenase among s e v e r a l s p e c i e s o f . l a c t i c  acid bacteria.  The work r e p o r t e d upon h e r e i n was undertaken w i t h the object o f o b t a i n i n g more d e t a i l e d i n f o r m a t i o n upon the dehydrogenase enzyme systems o f a l a r g e r number o f s p e c i e s of l a c t i c a c i d b a c t e r i a , and of determining  whether or not  t h i s i n f o r m a t i o n might prove v a l u a b l e i n t h e i r  classification.  EXPERIMENTAL METHODS 1•  Cultures Organisms r e p r e s e n t a t i v e o f c e r t a i n genera o f t h e t r u e  l a c t i c and pseudo l a c t i c a c i d b a c t e r i a were s e l e c t e d f o r study These i n c l u d e d :  S t r e p , l a c t i s S.A. 30, a t y p i c a l  i s o l a t e d from cream p o s s e s s i n g  Strep*  a caramel f l a v o r ( 1 2 ) ;  lacti  Strep.  l a c t i s A.T.C. 374, obtained from the N a t i o n a l Type C u l t u r e C o l l e c t i o n a t Washington, B.C.; S t r e p , l a c t i s EMB  2  1 (14);  Strep, cremoris HP (18); S t r e p , cremoris RW ( 1 8 ) ; S t r e p , cremoris EMB  X  1 9 5 (14); Betacoccus EMB  2  1 7 3 (14); S t r e p ,  c i t r o v o r u s A.T.C. 797; S t r e p , p a r a c i t r o v o r u s A.T.C. 7 9 8 ; 6058  S t r e p , b o v i s A.T.C.  ; T e t r a . c a s e i A.T.C. 391; T e t r a .  l i q u e f a c i e n s SM 3 ( 1 3 ) ; Bact. c o l i A.T.C. 4157, and B a c t . aerogenes A.T.C. 2 1 1 . 2.  Media Casein Digest B r o t h , prepared  a f t e r t h e manner o f O r l a -  Jensen ( l ) , c o n t a i n i n g 0 . 5 $ T o t a l N i t r o g e n , and e n r i c h e d w i t h 1.of. D i f c o yeast e x t r a c t , 0 . 5 $  K  H P 0 2  4  and 0 . 5 $ g l u c o s e ,  served  as the b a s i c medium. The  h i g h percentage o f yeast e x t r a c t i s a s t r i k i n g f e a t u r e  of t h i s medium.  The s t i m u l a t o r y e f f e c t o f s m a l l q u a n t i t i e s of  yeast e x t r a c t upon t h e l a c t i c a c i d b a c t e r i a was f i r s t by Orla-Jensen  reported  ( 8 ) , who l a t e r showed t h a t t h i s was due t o the  s u p p l y i n g o f c e r t a i n accessory growth f a c t o r s r e l a t e d to t h e v i t a m i n B complex.  Wilson  ( 1 9 ) , i n a study o f t h e r e s p i r a t o r y  enzymes o f t h e R h i z o b i a , found t h a t i n c r e a s i n g t h e yeast of the medium from  0.25$  to  1.0$  resulted i n c e l l  content  suspensions  which possessed markedly i n c r e a s e d dehydrogenase a c t i v i t y . Above loOf. t h e r e was no f u r t h e r s t i m u l a t i o n .  This e f f e c t was  a t t r i b u t e d t o a s t o r i n g - u p o f e s s e n t i a l coenzyme f a c t o r s w i t h i n ;  the c e l l s •  P r e p a r a t i o n o f Suspensions For the d e t e r m i n a t i o n  o f dehydrogenase a c t i v i t y , " r e s t i n g  c e l l " suspensions were prepared from young b r o t h cultures© A f t e r a s u i t a b l e i n c u b a t i o n p e r i o d at 30°. C. the c u l t u r e was c e n t r i f u g e d at 2,000 r.p.m. f o r 30 minutes i n f l a t - b o t t o m c e n t r i f u g e t u b e s , the supernatant l i q u i d poured from t h e sedimented c e l l s , and the c e l l s washed by m i x i n g and then r e c e n t r i f u g i n g w i t h M/30 phosphate b u f f e r o f pH 7,2.  A f t e r two  washings w i t h b u f f e r s o l u t i o n , the organisms were resuspended i n b u f f e r and employed as " r e s t i n g c e l l s " . 4•  S t a n d a r d i z a t i o n o f Suspensions I n dehydrogenation r e a c t i o n s w i t h b a c t e r i a l suspensions  the v e l o c i t y of o x i d a t i o n i s p r o p o r t i o n a l t o the c o n c e n t r a t i o n of organisms present. to s t a n d a r d i z e  I t t h e r e f o r e becomes extremely important  a l l suspensions before u s e .  Two methods o f s t a n d a r d i z a t i o n have been employed by v a r i o u s workers:  ( l ) the suspension i s d r i e d and weighed and  converted i n t o m i l l i g r a m s o f dry c e l l weight per cubic centimeter,  and (2) t h e n i t r o g e n content i s determined by the  m i c r o - K j e l d a h l method and suspensions compared on t h e b a s i s o f m i l l i g r a m s o f n i t r o g e n per cubic  centimeter.  Both these methods a f f o r d a b a s i s f o r comparison of r e s u l t s , but are open t o s e r i o u s o b j e c t i o n s .  I n the f i r s t  place,.both the moisture content and the n i t r o g e n content of the b a c t e r i a l c e l l are i n f l u e n c e d by c u l t u r a l c o n d i t i o n s , and s e c o n d l y , these procedures r e q u i r e c o n s i d e r a b l e time before r e s u l t s are o b t a i n e d . In order t o a v o i d these d i f f i c u l t i e s of  s t a n d a r d i z a t i o n has been adopted.  the f o l l o w i n g method  A 10-c.c. a l i q u o t p o r t i o n  of b r o t h c u l t u r e i s p l a c e d i n a Hopkins v a c c i n e tube (4) , c e n t r i f u g e d f o r 30 m i n u t e s , and t h e volume o f c e l l measured.  sediment  From t h i s d e t e r m i n a t i o n suspensions c o n t a i n i n g a  d e f i n i t e percentage by volume o f c e l l s are then p r e p a r e d . With the organisms employed i n the t e s t s r e p o r t e d h e r e i n a c o n c e n t r a t i o n o f i f . by volume was g e n e r a l l y found t o be most convenient.  P l a t e counts c a r r i e d out on suspensions of the  same organism prepared at d i f f e r e n t t i m e s showed a v a r i a t i o n of l e s s than 10f.. The procedure f o r s t a n d a r d i z i n g c e l l suspensions by t h i s method i s r a p i d , s i m p l e , and appears t o be q u i t e a c c u r a t e . Suspensions prepared by t h i s volume method may a l s o be conv e r t e d t o d r y weight and n i t r o g e n content bases w i t h l i t t l e difficulty.  S i n c e dehydrogenase a c t i v i t y i s l i n k e d up w i t h t h e s t r u c t u r e of t h e b a c t e r i a l c e l l , i t i s t o be expected t h a t the p e r i o d o f growth i n c u l t u r e before h a r v e s t i n g w i l l i n f l u e n c e t h e a c t i v i t y o f the b a c t e r i a l suspensions prepared therefrom. In order t o determine t h e optimum i n c u b a t i o n p e r i o d , b r o t h c u l t u r e s o f two s t r a i n s of S t r e p , l a c t i s and one s t r a i n o f S t r e p , cremoris were i n c u b a t e d at 30° C, and samples removed from t h e mother c u l t u r e s at r e g u l a r i n t e r v a l s .  These samples  were then plated, and the dehydrogenase a c t i v i t y upon glucose determined.  The r e s u l t s o b t a i n e d showed t h a t dehydrogenase  a c t i v i t y reached a maximum a t about 24 h o u r s , and then v e r y s l o w l y .declined, w h i l e maximum growth was not a t t a i n e d u n t i l 48 t o 72 h o u r s .  These f i n d i n g s i n d i c a t e d t h a t suspensions  prepared from c u l t u r e s i n t h e l o g a r i t h m i c growth phase were most a c t i v e .  A l l suspensions employed i n these t e s t s were  t h e r e f o r e prepared from c u l t u r e s grown from 18 t o 24 hours* These r e s u l t s are i n accord w i t h those o f Wooldridg©, Knox and. Glass ( 2 0 ) , who r e p o r t e d t h a t 24-hour c u l t u r e s o f B a c t , c o l l y i e l d e d t h e most a c t i v e suspensions f o r dehydrogenase s t u d i e s , 6,  Methylene Blue C o n c e n t r a t i o n The q u e s t i o n of c o n c e n t r a t i o n o f methylene  blue i s o f  importance not o n l y i n r e l a t i o n t o t h e r a t e o f r e d u c t i o n of t h e dye, but a l s o i n r e l a t i o n t o i t s t o x i c  action.  - 7 The t o x i c i t y of v a r y i n g c o n c e n t r a t i o n s of methylene blue upon B a c t , c o l i was  determined by exposing  suspensions  of  the organism i n Thunberg tubes f o r a one-hour p e r i o d to methylene blue c o n c e n t r a t i o n s ranging from 1:10,000 to 1:50,000. A f t e r one hour the suspensions  were p l a t e d and the number of  v i a b l e c e l l s determined, A c r i t i c a l t o x i c c o n c e n t r a t i o n was 1:15*000 and 1:20,000 methylene b l u e .  found to occur between Above 1:20,000 the  t o x i c e f f e c t i s very s l i g h t , w h i l e below 1:15,000 the rapidly lose t h e i r The blue.  viability.  c o n c e n t r a t i o n s e l e c t e d f o r use was T h i s was  cells  1:55,000 methylene  found to g i v e a c l e a r and d i s t i n c t  end-point  e i t h o u t e x e r t i n g any i n h i b i t o r y effects© The use of methylene blue i n dehydrogenase s t u d i e s w i t h the S t r e p t o c o c c i has been questioned  by F a r r e l l ( 2 ) , who  i n d i g o t e t r a s u l f o n a t e to be much l e s s t o x i c . and Wooldridge (15) and Wooldridge and Glass  found  However, S a n d i f o r d (21) showed  c l e a r l y t h a t dehydrogenase a c t i v i t y i s independent of v i a b i l i t y but i s a s s o c i a t e d e q u a l l y w i t h l i v i n g and dead c e l l s .  Parallel  experiments w i t h the l a c t i c a c i d s t r e p t o c o c c i , e m p l o y i n g methylene blue and  i n d i g o t e t r a s u l f o n a t e , showed no  differences.  appreciable  R e s t i n g c e l l suspensions of c e r t a i n b a c t e r i a l s p e c i e s possess the a b i l i t y t o reduce methylene blue i n the absence of substrate:,.  T h i s type o f r e d u c t i o n i s known as "endogenous  r e s p i r a t i o n " and has been a t t r i b u t e d t o an o x i d a t i v e deamination of c e l l u l a r amino a c i d s i n which the b a c t e r i a l c e l l s u t i l i z e t r a c e s of p o l y s a c c h a r i d e s o r c a p s u l a r m a t e r i a l as the energy source. Suspensions o f the l a c t i c a c i d s t r e p t o c o c c i showed no r e d u c i n g a c t i o n upon methylene blue i n the absence of s u b s t r a t e . When the more a e r o b i c organisms of the pseudo l a c t i c group, namely T e t r a . e a s e l , T e t r a . l i q u e f a c i e n s , Bact. c o l i and B a c t . aerogenes, were employed, the suspensions were observed t o reduce methylene b l u e i n the absence of s u b s t r a t e .  Repeated  washing by c e n t r i f u g i n g w i t h b u f f e r f a i l e d t o d e s t r o y the reducing a c t i v i t y , and i t was found necessary t o a e r a t e the suspensions, f o l l o w i n g the procedure of Q,uastel and Whetham (10).  A f t e r 60 minutes * a e r a t i o n the suspensions were found  to have l o s t t h e i r r e d u c i n g a c t i v i t y upon methylene b l u e . A l l suspensions of the a e r o b i c organisms were t h e r e f o r e s u b j e c t e d to 60 minutes' a e r a t i o n b e f o r e use. 8»  Thunberg Technique The dehydrogenase  a c t i v i t y of the l a c t i c a c i d b a c t e r i a  was  determined i n m o d i f i e d Thunberg tubes c o n t a i n i n g 1 c.c. of -  r e s t i n g c e l l suspension, pH 7 . 2 ,  phosphate b u f f e r of  c.c. of 1 : 7 , 0 0 0 methylene blue and  1  substrate. pump.  2 c.c. of M/30  A l l tubes were evacuated  1  c.c. of  f o r two minutes on a water  Tests were c a r r i e d out at 37,5° C. i n an  c o n t r o l l e d water b a t h .  M/20  electrically  Dehydrogenase a c t i v i t y was measured  as time r e q u i r e d f o r complete d e c o l o r i z a t i o n of the methylene blue.  A l l t e s t s which d i d not reduce w i t h i n two hours were  considered n e g a t i v e .  R e s u l t s o b t a i n e d have been expressed  as  percentage o f the r e d u c t i o n time of g l u c o s e , given the value of 1 0 0 .  T h i s value i s c a l l e d the r e s p i r a t o r y c o e f f i c i e n t .  EXPERIMENTAL RESULTS The  dehydrogenase a c t i v i t i e s of t h r e e s t r a i n s of S t r e p ,  l a c t i s and t h r e e s t r a i n s o f S t r e p , cremoris are recorded i n Table 1.  There i s evident a marked v a r i a t i o n i n dehydrogenase  a b i l i t y among s t r a i n s w i t h i n these two  species.  This v a r i a -  t i o n i s c l e a r l y shown i n the dehydrogenase r e a c t i o n s upon sucrose and t r e h a l o s e , r e a c t i o n s which y i e l d p o s i t i v e or negative v a l u e s , depending upon the s t r a i n employed. The dehydrogenase enzymes of S t r e p , l a c t i s and cremoris appear to be v e r y s i m i l a r i n c h a r a c t e r .  Strep,  Both s p e c i e s  are a b l e to dehydrogenate the four monosaccharides and m a j o r i t y o f the d i s a c c h a r i d e s and p o l y s a c c h a r i d e s .  the  They both  • - 10 f a i l , however, t o o x i d i z e x y l o s e , a r a b i n o s e , m e l i b i o s e , m e l e z i t o s e , rhamnose and methyl g l u c o s i d e .  Both s p e c i e s are  f u r t h e r c h a r a c t e r i z e d by a low r e a c t i v i t y upon the monohydric and p o l y h y d r i c a l c o h o l s and by a complete i n a b i l i t y t o a t t a c k the s a l t s o f o r g a n i c a c i d s .  An e x c e p t i o n t o t h e low r e a c t i v i t y  upon a l c o h o l s i s found i n t h e case o f S t r e p , cremoris RW, which has been found t o possess a r e s p i r a t o r y c o e f f i c i e n t o f 125 upon i s o p r o p y l a l c o h o l and o f 250 upon sec, b u t y l alcohol© T h i s c l o s e s i m i l a r i t y i n dehydrogenase enzyme a c t i v i t y makes i t extremely d i f f i c u l t t o attempt t o d i s t i n g u i s h these two s p e c i e s by t h e i r r e s p i r a t o r y enzyme c h a r a c t e r s .  The one  t e s t which might possess d i f f e r e n t i a l s i g n i f i c a n c e i s the dehydrogenation  of r a f f i n o s e .  A l l three s t r a i n s of Strep,  cremoris a t t a c k t h i s compound, w h i l e a l l t h r e e s t r a i n s o f S t r e p , l a c t i s proved unable t o do s o , The dehydrogenase a c t i v i t y o f S t r e p , b o v i s and t h r e e s t r a i n s o f B e t a c o c c i are t a b u l a t e d i n Table 2,  From the v a l u e s  recorded here t h e r e i s again e v i d e n t a marked v a r i a t i o n i n dehydrogenase a b i l i t y among t h e t h r e e s t r a i n s of B e t a c o c c i studied.  This v a r i a b i l i t y i s p a r t i c u l a r l y noticeable i n the  dehydrogenation  o f d e x t r i n , s a l i e i n and r a f f i n o s e .  Suspensions  of S t r e p , b o v i s appear t o possess v e r y s t r o n g o x i d i z i n g mechanisms upon r a f f i n o s e and s t a r c h ,  Dehydrogenation o f d e x t r i n , however,  i s a p p r e c i a b l y weaker, w h i l e t h a t o f maltose i s v e r y f e e b l e .  - 11  -  This would i n d i c a t e t h a t t h e organism u t i l i z e s s t a r c h d i r e c t l y , without  p r e l i m i n a r y h y d r o l y s i s to t h e d e x t r i n or maltose  stages.  The dehydrogenase a c t i v i t i e s of the pseudo l a c t i c a c i d "bacteria, namely, two s t r a i n s o f T e t r a c o c c i , Bact» c o l i , and Bact, aerogenes, are d e t a i l e d i n Table 3 ,  These s p e c i e s  e x h i b i t a very r a p i d o x i d i z i n g a b i l i t y upon n e a r l y a l l carbo- hydrates  tested,  T e t r a . c a s e i i s t h e o n l y organism s t u d i e d  which showed a s t r o n g dehydrogenase a c t i v i t y upon t h e pentoses, xylose and a r a b i n o s e .  These s p e c i e s a r e a l s o c h a r a c t e r i z e d  by o x i d a t i o n o f t h e hexahydric  a l c o h o l s , m a n n i t o l and s o r b i t o l ,  and by dehydrogenase a c t i v i t y upon t h e s a l t s o f c e r t a i n organic a c i d s , p a r t i c u l a r l y formate, l a c t a t e , s u c c i n a t e , and malate. O x i d a t i o n o f the monohydric a l c o h o l s i s a p p r e c i a b l e , e s p e c i a l l y i n the case o f T e t r a , l i q u e f a c i e n s , which shows a r e s p i r a t o r y c o e f f i c i e n t o f 200 i n t h e presence o f e t h y l , n p r o p y l and a l l y l alcohols.  The h y d r o g e n a t i o n o f formaldehyde and glutamine by  both Bact. c o l i and B a c t . aerogenes appears t o be o f some significance. The  dehydrogenase r e a c t i o n s o f S t r e p , l a c t i s S,A. 30 and  Strep, l a c t i s A.T.C. 374 were redetermined 18 months a f t e r the previous t e s t s had been c a r r i e d out. The r e s u l t s o f these two s e r i e s of t e s t s are recorded  i n Table 4,  - 12 The  dehydrogenase a c t i v i t i e s of these two  to be subject 'to c o n s i d e r a b l e the c u l t u r e i s t e s t e d . marked i n c r e a s e d substrates  s t r a i n s appear  v a r i a t i o n , depending upon when  With many s u b s t r a t e s  t h e r e has been a  i n dehydrogenase a b i l i t y , w h i l e w i t h other  t h e r e has been a marked decrease.  I t i s apparent,  t h e r e f o r e , t h a t t h i s v a r i a t i o n does not i n d i c a t e a d e f i n i t e tendency toward i n c r e a s e d of the  or decreased a c t i v i t y on the  c u l t u r e , but merely i l l u s t r a t e s the  part  essential  i n s t a b i l i t y of the r e s p i r a t o r y enzyme systems of t h i s b a c t e r i a l species*  DISCUSSION The  r e s u l t s o b t a i n e d from t h i s study of the  dehydrogenase  enzymes of the l a c t i c a c i d b a c t e r i a show c l e a r l y t h a t fundamental d i f f e r e n c e s  e x i s t between the r e s p i r a t o r y mechanisms  of the t r u e l a c t i c a c i d s t r e p t o c o c c i and the more pseudo l a c t i c a c i d c o c c i and  rod forms.  s t r e p t o c o c c i are c h a r a c t e r i z e d  The t r u e  aerobic lactic  by a dehydrogenase a c t i v i t y which  i s r e s t r i c t e d almost e n t i r e l y t o the carbohydrates and monohydric a l c o h o l s .  The  hydric and acids.  polyhydric  simpler  pseudo l a c t i c a c i d b a c t e r i a , on  other hand, possess much g r e a t e r the carbohydrates and,  acid  the  dehydrogenase a c t i v i t y upon  i n a d d i t i o n , are able t o a t t a c k monoa l c o h o l s as w e l l as the s a l t s of o r g a n i c  A f u r t h e r d i f f e r e n c e i n r e s p i r a t o r y character  between  - 13 these two groups i s shown by the endogenous r e s p i r a t i o n of the  aerobic species. The dehydrogenating a b i l i t y of the l a c t i c a c i d s t r e p t o c o c c i  upon carbohydrates i s q u i t e v a r i e d ,  a l l four  monosaccharides  and t h e ' m a j o r i t y of the d i - and p o l y s a c c h a r i d e s being a t t a c k e d . Among the monosaccharides, however, g a l a c t o s e i s o x i d i z e d much less r e a d i l y than g l u c o s e , f r u e t o s e or mannose, w h i l e among the  d i s a c c h a r i d e s the dehydrogenating a c t i o n upon l a c t o s e i s  very weako  T h i s low r e a c t i v i t y upon l a c t o s e i s r a t h e r s u r p r i s i n g ,  since the m a j o r i t y of these c u l t u r e s form s u f f i c i e n t l a c t i c  acid  to c l o t m i l k c u l t u r e s i n from 18 t o 48 hours. The a b i l i t y of organisms t o dehydrogenats o r g a n i c a c i d s appears t o be r e l a t e d to the a c i d s produced d u r i n g sugar fermentations.  The a c i d which accumulates i n the medium during  fermentations c a r r i e d out by the l a c t i c a c i d c o c c i i s almost entirely l a c t i c acid.  T h e r e f o r e these organisms should not be  expected t o possess an enzyme system capable of u t i l i z i n g lactate.  E x p e r i m e n t a l r e s u l t s have confirmed t h i s h y p o t h e s i s .  In the case of the t e t r a c o c c i , however, where l a c t i c a c i d i s not the o n l y a c i d accumulating i n the c u l t u r e medium, dehydrogenation o f l a c t a t e o c c u r s .  With B a c t • c o l i and B a c t .  aerogenes, where a gaseous m i x t u r e of carbon d i o x i d e and hydrogen i s produced, dehydrogenase enzymes f o r both l a c t a t e and formate are found.  - 14 The i n c l u s i o n of a l a r g e number of i s o m e r i c a l c o h o l s i n these tests- a f f o r d e d an o p p o r t u n i t y to study s p e c i f i c i t y of dehydrogenase a c t i o n .  stereochemical  With the organisms  tested here t h e r e appeared to be no such s p e c i f i c i t y ,  Tetra,  casei was  not  a b l e to dehydrogenate i s o p r o p y l a l c o h o l but  iso b u t y l a l c o h o l , n b u t y l a l c o h o l but not n p r o p y l or n amyl a l c o h o l s , sec, amyl a l c o h o l but not sec, b u t y l a l c o h o l * S i m i l a r e f f e c t s were observed  w i t h the other organisms.  most s t r i k i n g f e a t u r e o f the a l c o h o l dehydrogenation  was  The the  a b i l i t y shown by d i f f e r e n t s t r a i n s t o s e l e c t i v e l y a c t i v a t e one or two a l c o h o l s w i t h great r a p i d i t y .  This a c t i v a t i o n i s  apparently due e n t i r e l y to i n d i v i d u a l s t r a i n c h a r a c t e r . The m a j o r i t y of the organisms t e s t e d proved able to dehydrogenate e t h y l a l c o h o l but not one was e t h y l amine.  able to dehydrogenat  S u b s t i t u t i o n of an amino group f o r a h y d r o x y l  group has a p p a r e n t l y e l i m i n a t e d dehydrogenase a c t i v i t y .  This  o b s e r v a t i o n agrees w i t h the conception t h a t the amines are  final  and i n most cases t o x i c products of b a c t e r i a l a c t i o n . None of the organisms t e s t e d showed any dehydrogenase a c t i v i t y upon potassium  citrate.  T h i s was  t r u e of S t r e p ,  p a r a c i t r o v o r u s as w e l l as of B a c t , c o l l and B a c t , aerogenes• The a b i l i t y o f B a c t , aerogenes to grow w i t h c i t r a t e as the s o l e carbon source i s w i d e l y used to d i f f e r e n t i a t e these organisms.  two  U t i l i z a t i o n of c i t r a t e by B a c t , aerogenes must  - 15 t h e r e f o r e proceed i n some way other than by dehydrogenation. The ' i n a b i l i t y of S t r e p , p a r a c i t r o v o r u s t o dehydrogenate  citrate  i s of i n t e r e s t because o f the r e p o r t e d r e s u l t s of Slade.and Workman ( 1 6 ) , who showed t h a t c e l l s of S t r e p , p a r a c i t r o v o r u s grown i n c i t r a t e p l u s l a c t o s e were then able t o ferment c i t r a t e . A f u r t h e r p o i n t o f i n t e r e s t w i t h the carbohydrate denydrogenations i s the p o s s i b l e i n h i b i t o r y e f f e c t o f the methyl group.  A l l t h e organisms t e s t e d were able to dehydrogenate  glucose, w h i l e o n l y one was a b l e t o dehydrogenate alpha methyl glucoside.  Rhamnose, a methyl pentose, was not dehydrogenated  by organisms which a t t a c k e d x y l o s e and a r a b i n o s e .  Several  species were a b l e to dehydrogenate e t h y l a l c o h o l , but never methyl a l c o h o l .  These r e s u l t s a l l i n d i c a t e t h a t t h e methyl  group e x e r t s an i n h i b i t o r y e f f e c t upon dehydrogenase  activity.  This o b s e r v a t i o n agrees w i t h t h a t o f Koser and Saunders (7) » who showed t h a t m e t h y l d e r i v a t i v e s of s e v e r a l o f t h e commoner sugars were d i s t i n c t l y r e s i s t a n t t o b a c t e r i a l a t t a c k . The dehydrogenase  enzyme s t u d i e s r e p o r t e d h e r e i n were  c a r r i e d out i n an attempt t o e s t a b l i s h a b a s i s f o r t h e c l a s s i f i c a t i o n o f t h e l a c t i c a c i d s t r e p t o c o c c i . The r e s u l t s reported i n Tables 1 ,  2 and 3 show c l e a r l y t h a t no d e f i n i t e  species c h a r a c t e r i s t i c s e x i s t i n r e s p i r a t o r y enzyme  make-up.  There i s apparent a very marked s t r a i n v a r i a t i o n w i t h i n a l l  - 16 the s p e c i e s s t u d i e d *  -  This s t r a i n v a r i a t i o n i s further  emphasized by the r e s u l t s r e p o r t e d i n Table 4, which show that the  dehydrogenase a c t i v i t y of a s i n g l e s t r a i n v a r i e s  at  different times. From the r e s u l t s of these s t u d i e s  i t i s apparent t h a t  c l a s s i f i c a t i o n of the l a c t i c a c i d s t r e p t o c o c c i upon t h e i r r e s p i r a t o r y enzyme c h a r a c t e r i s not  feasible.  Such a  c l a s s i f i c a t i o n cannot be attempted u n t i l such time as  the  f a c t o r s i n f l u e n c i n g v a r i a t i o n i n dehydrogenase a c t i v i t y f u r t h e r clarified©  are  - 17, -  . ,  REFERENCES Can. Jour* Res. 1: 3 6 4 , 1 9 3 2 .  1.  E a g l e s , B A.,and S a d l e r , W,  2.  F a r r e l l , M.A.  3.  Harden, A. and Z i l v a , S.S.  4.  Hopkins, D.  5«  K a t a g i r i , H. and K i t a h a r a , K, 1938 •  6.  K e n d a l l , A, I . J o u r . I n f . D i s . 4 7 : 186, 1930.  7»  K o s e r , S.A. and Saunders, F.  8.  O r l a - J e n s e n , S.  9.  Q u a s t e l , J.H. and Whetham, M.D. 19 24.  Biochem. J o u r . 18: 5 1 9 ,  10.  Q u a s t e l , J.H. and Whetham, M.D.  Biochem. J o u r . 19: 520,  0  J o u r . B a c t . 2 9 : 411, 1 9 3 5 . Bioeliem. J o u r . 9 : 3 7 9 ,  1915.  J o u r . Amer. Med. A s *n. 6 0 : 1 6 1 5 , 1 9 1 3 . S  Bioeliem. J o u r . 3 2 : 1654,  J o u r . Bact. 26: 475, 1933*  J o u r . Bact. 12: 333, 1926.  1925.  11.  Q u a s t e l , J.H. and Whetham, M.D.  Biochem. J o u r . 19: 645,  1925.  12.  S a d l e r , W.  Trans. Roy, Soc. Can. V o l , 20, 1 9 2 6 .  13.  S a d l e r , W.  S c i e n t i f i c A g r i c u l t u r e , V o l , K, No, 2, 1927,  14.  S a d l e r , W,, E a g l e s , B.A. and Pendray, G. 370,  Can, J o u r . Res, 7  1932.  15.  S a n d i f o r d , B.R. and Wooldridge, W.R. 2 1 7 2 , 1931.  16.  S l a d e , H.D. and Workman, C.H.  17.  Thunberg  18.  Whitehead, H.R. and Cox, G.A.  19.  W i l s o n , P.W.  20.  Wooldridge, W.R., 30: 9 2 6 , 1936.  21.  Wooldridge, W.R.  Biochem. J o u r , 25•  J o u r . Bact. 41: 1 9 , A b  S c  1942.  Skand. A r c h . P h y s i o l . 40: 1, 1920. R  J o u r . D a i r y e s . 7: 556, 1936,  J o u r . B a c t . 35: 601, 1938, Knox, R., and C l a s s , V. and G l a s s , V.  Biochem.Jour•  Biochem, J o u r . 25: 2172, 1931,  ABSTRACT  The a e r o b i c r e s p i r a t o r y a c t i v i t y  of two s t r a i n s of  S t r e p , l a c t i s has been s t u d i e d upon t h i r t y  substrates,  c o n s i s t i n g o f c a r b o h y d r a t e s , a l c o h o l s and o r g a n i c acids© These organisms have been shown t o possess a v a r i e d o x i d a t i v e a b i l i t y upon the s u b s t r a t e s t e s t e d . I t has been shown t h a t t h e r e e x i s t s a c o n s i d e r a b l e v a r i a t i o n between t h e o x i d a t i v e systems o f t h e two strains studied* S t r e p , l a c t i s has been shown t o e x h i b i t a c o n s i d e r able endogenous oxygen uptake*  The r e l a t i o n s h i p o f t h i s  endogenous r e s p i r a t i o n t o the o x i d a t i v e systems and t o the mechanism o f r e s p i r a t i o n has been d i s c u s s e d .  INTRODUCTION A l t h o u g h t h e dehydrogenating a c t i v i t y o f many b a c t e r i a l species has been s t u d i e d , the a e r o b i c r e s p i r a t o r y mechanism has not y e t been e x t e n s i v e l y i n v e s t i g a t e d , , Stephenson ( 8 ) . have r e p o r t e d suspensions of B a c t • c o l i .  Cook and  upon t h e o x i d a t i v e a b i l i t y o f Lineweaver (15) s t u d i e d the  c h a r a c t e r i s t i c s o f o x i d a t i o n by A z o t o b a c t e r v i n e l a n d i i upon a v a r i e t y of s u b s t r a t e s  and found almost complete o x i d a t i o n  to carbon d i o x i d e and water.  W i l s o n ( 1 8 ) i n v e s t i g a t e d the  s u i t a b i l i t y o f t h e Rhizobium s p e c i e s f o r r e s p i r a t o r y s t u d i e s * Bernheim (4) determined t h e oxygen uptake o f the t u b e r c l e b a c i l l u s i n t h e presence of v a r i o u s s u b s t r a t e s  and demonstrated  a marked s t i m u l a t i o n i n the case o f a l l aldehydes t e s t e d . The o x i d a t i v e a c t i v i t y o f v a r i o u s s p e c i e s o f c o c c i has a l s o been i n v e s t i g a t e d .  Barron and H a s t i n g s ( l ) and Barron  (2) showed t h a t suspensions o f gonococci were able t o o x i d i z e a l p h a hydroxy and alpha k e t o n i c a c i d s t o carbon d i o x i d e and a c i d s c o n t a i n i n g one l e s s carbon atom, e.g.,  l a c t i c or p y r u v i c  a c i d t o a c e t i c a c i d and carbon  dioxide*  The r e s p i r a t o r y mechanism of the s t r e p t o c o c c i was s t u d i e d by F a r r e l l ( l l ) , employing twenty-two s t r a i n s , which mainly of pathogenic and h e m o l y t i c t y p e s .  consisted  Working w i t h the  hemolytic s t r e p t o c o c c i , Barron and Jacobs (3) o b t a i n e d a v a r i a t i o n both i n t h e number and r a t e s of o x i d a t i o n large number of  of a  substrates.  S t u d i e s upon the r e s p i r a t o r y mechanism o f the l a c t i c acid b a c t e r i a have been r e l a t i v e l y few.  Hunt (13) r e p o r t e d  upon the oxygen uptake by l i v i n g c u l t u r e s and washed suspensions o f t h e L a c t o b a c i l l i , emphasizing the e f f e c t o f v a r i o u s i n h i b i t i n g agents upon c e l l r e s p i r a t i o n .  Farrell ( l l )  i n h i s study o f t h e r e s p i r a t o r y mechanism, of the s t r e p t o c o c c i , included  t h r e e l a c t i c a c i d t y p e s among h i s v a r i o u s s t r a i n s .  Hansen (12) s t u d i e d t h e r e s p i r a t i o n of t w e l v e s p e c i e s of the rod-shaped l a c t i c a c i d b a c t e r i a and found great d i f f e r e n c e i n t h e i r r e s p i r a t o r y systems. The work r e p o r t e d upon h e r e i n was undertaken w i t h the object  of o b t a i n i n g  d e t a i l e d information  upon t h e a e r o b i c  r e s p i r a t o r y mechanism of the l a c t i c a c i d streptococci©  EXPERIMENTAL METHODS The c u l t u r e s employed i n t h i s study were S t r e p , l a c t i s S.A.  3 0 ( 1 0 ) and S t r e p , l a c t i s No. 374- o f t h e N a t i o n a l Type  Culture C o l l e c t i o n a t Washington, D.C.  Comparative  tests  were a l s o c a r r i e d out employing S t r e p , l a c t i s EMB 1 ( 1 7 ) , 2  and B a c t . c o l i A.T.C. 4137. Casein D i g e s t B r o t h , prepared a f t e r t h e manner o f O r l a Jensen ( 1 0 ) , c o n t a i n i n g 0 . 5 $ T o t a l N i t r o g e n , and e n r i c h e d with  1$  D i f c o yeast e x t r a c t ,  0.5$  K2HPO4,  and  0.5$  glucose,  served as the b a s i c medium. R e s p i r a t o r y a c t i v i t y was determined by the use of " r e s t i n g c e l l " s u s p e n s i o n s , prepared and s t a n d a r d i z e d as d e t a i l e d by Morgan, E a g l e s , and L a i r d  (16).  Oxygen uptake was measured m a n o m e t r i c a l l y i n the B a r c r o f t apparatus, as d e s c r i b e d by Dixon ( 9 ) •  The cups on t h i s  apparatus c o n t a i n e d a r e a c t i o n m i x t u r e c o n s i s t i n g of 1 c.c. o f r e s t i n g c e l l s u s p e n s i o n , 1 c.c. o f M / 2 0 s u b s t r a t e , and 1 c . c . of M / 3 0 phosphate b u f f e r .  Carbon d i o x i d e was absorbed i n  f i l t e r paper soaked i n 2 0 $ potassium hydroxide h e l d i n i n s e t tubes w i t h i n the cups. 37.5°  A l l experiments were c a r r i e d out a t  C. i n a t h e r m o s t a t i c a l l y c o n t r o l l e d water b a t h , and a t  a pH o f  7.2.  -  4'-  A l l r e s u l t s o b t a i n e d have been recorded  as Q0  and QC0  2  values* c a l c u l a t e d on the b a s i s of c e l l dry weight.  2  The  R e s p i r a t o r y Quotients were d e r i v e d by d i v i d i n g carbon d i o x i d e output by oxygen uptakes  EXPERIMENTAL RESULTS The  a e r o b i c r e s p i r a t o r y a c t i v i t y of two  s t r a i n s of  Strep, l a c t i s upon t h i r t y compounds i s recorded Values are expressed as Q0 , 2  at one  i n Table 1.  0,00 and R e s p i r a t o r y 2  Quotient  hour.  The  r e s u l t s r e p o r t e d i n t h i s t a b l e show t h a t these  s t r a i n s possess a v a r i e d o x i d a t i v e a c t i v i t y upon Determination  of carbon d i o x i d e p r o d u c t i o n and  the R e s p i r a t o r y Quotients  two  carbohydrates.  c a l c u l a t i o n of  have shown t h a t , i n most  cases,  o x i d a t i o n i s f a i r l y complete t o carbon d i o x i d e and water. The  rat© of oxygen uptake a g a i n s t time i n the ease of  Strep, l a c t i s S.A.  3 0 i s recorded  g r a p h i c a l l y i n F i g u r e 1©  These r e s u l t s have been s e l e c t e d as t y p i c a l of the type of curve obtained when the oxygen uptake i s p l o t t e d against time over a one-hour p e r i o d .  I t i s apparent from t h i s graph t h a t  the r a t e of oxygen uptake i s f a i r l y r e g u l a r and uniform i n characters  f  ~ 5 «  •jr..  The comparative oxygen uptake of S t r e p , l a c t i s S.A. 3 0 upon t e n s u b s t r a t e s i s shown g r a p h i c a l l y i n F i g u r e 2© •' O x i d a t i o n o f t h e monosaccharides, as e x e m p l i f i e d by f l u c o s e ' < and g a l a c t o s e , i s q u i t e h i g h i n v a l u e . f mannose and f r u c t o s e .  This i s also true of  With t h e pentoses, x y l o s e i s o x i d i z e d  I guite r e a d i l y , w h i l e a r a b i n o s e i s a t t a c k e d but s l i g h t l y . A l l ; the d i s a c c h a r i d e s t e s t e d were o x i d i z e d r e a d i l y , w i t h t h e f  •  •  J- exception o f m e l i b i o s e .  .  • •  •  Among t h e t r i s a c c h a r i d e s , both  i r a f f i n o s e and m e l e z i t o s e were a t t a c k e d w i t h ease. I  A l l the  p o l y s a c c h a r i d e s t e s t e d were o x i d i z e d , w i t h the exception o f i n u l i n , where oxygen uptake was very s l i g h t .  Among t h e  • a l c o h o l s , o x i d a t i v e a c t i v i t y was h i g h , except i n the case o f I  dulcitolo  When e t h y l a l c o h o l was the s u b s t r a t e , oxygen uptake  li was a p p r e c i a b l y g r e a t e r than t h a t o f g l u c o s e .  With e t h y l amine,  \ however, oxygen uptake was almost n e g l i g i b l e .  Tests on t h e  :  ' f ""• ••'  • • • •'  "  : a c t i v i t y o f t h i s organism toward t h e s a l t s o f o r g a n i c a c i d s { s h o w e d t h a t a l l t h e compounds s t u d i e d were r e a d i l y a t t a c k e d . :  fr '' I;  '• •' • The a e r o b i c r e s p i r a t o r y a c t i v i t y o f S t r e p , l a c t i s A.T.C.  374 upon t e n s u b s t r a t e s i s shown i n F i g u r e 3.  Oxidation of  the monosaccharides, g l u c o s e , mannose and f r u c t o s e , i s h i g h , 1  but t h a t o f g a l a c t o s e i s low. With t h e pentoses, a r a b i n o s e  i  i s o x i d i z e d r e a d i l y , but x y l o s e h a r d l y a t a l l .  •i  d i s a c c h a r i d e s , sucrose, c e l l o b i o s e and l a c t o s e are r e a d i l y  Among t h e  attacked, w h i l e m a l t o s e , t r e h a l o s e and m e l i b i o s e are a t t a c k e d :  I.  1  only weakly.  '  With t h e p o l y s a c c h a r i d e s t h e o x i d a t i o n o f s t a r c h  - 6 and d e x t r i n i s * seen to b© markedly g r e a t e r than even t h a t of glucose, w h i l e the o x i d a t i o n o f s a l i c i n and e s c u l i n i s low, and t h a t o f i n u l i n extremely s m a l l .  With t h i s  strain,activity  upon the a l c o h o l s i s very low except i n the case of g l y c e r o l , With ethylamine o x i d a t i o n i s low.  With o r g a n i c a c i d s the 0,0  2  i s very n e a r l y t h a t found i n the o x i d a t i o n of g l u c o s e . The comparative o x i d a t i v e a c t i v i t y o f S t r e p , l a c t i s •S,A, JO and S t r e p , l a c t i s A.T.C. 374 upon t e n s u b s t r a t e s i s shown g r a p h i c a l l y i n F i g u r e 4 ,  These r e s u l t s show t h a t , except  in t h e case o f the endogenous, glucose and l a c t o s e r e s p i r a t o r y mechanisms, t h e r e e x i s t s a marked v a r i a t i o n i n the a c t i v i t y o f these two s t r a i n s upon the m a j o r i t y o f the compounds t e s t e d . The e x i s t e n c e o f such a v a r i a t i o n i n o x i d a t i v e a c t i v i t y between two s t r a i n s o f the same s p e c i e s would imply t h a t  aerobic  r e s p i r a t o r y a c t i v i t y i s a s t r a i n , r a t h e r than a s p e c i e s , characteristic, The R e s p i r a t o r y Q u o t i e n t s  o f S t r e p , l a c t i s A.T.C. 374  upon f o u r s u b s t r a t e s are shown g r a p h i c a l l y i n F i g u r e 5,  The  curves shown i n t h i s graph a r e t y p i c a l of t h e r e s u l t s obtained upon v a r i o u s s u b s t r a t e s w i t h the two s t r a i n s employed.  The  R.Q.. i n the case o f f r u c t o s e i s shown t o be a s t r a i g h t l i n o which very n e a r l y approximates a value of l 0 . o  With s t a r c h  there i s demonstrated a decrease from a value o f  0<>7  to  0,6,  while w i t h l a c t o s e t h e R e s p i r a t o r y Quotient r i s e s from 0 6 O  to 0 , 8 . I n t h e case of i n o s i t o l a graph i s obtained which i s c h a r a c t e r i z e d by a v e r y sharp d i p i n the l i n e a t JO minutes.  T h i s same type o f curve has been noted w i t h a d o n i t o l  and d u l c i t o l w i t h S t r e p , l a c t i s A.T.C. 3 7 4 and w i t h arabinose in the case o f both s t r a i n s .  These r e s u l t s would i n d i c a t e ,  t h e r e f o r e , t h a t where such a sharp break i n the graph  of the  R e s p i r a t o r y Q u o t i e n t i s o b t a i n e d , t h e r e must e x i s t a d e f i n i t e and sharp break i n t h e metabolism o f the organism upon the substrate being t e s t e d . In F i g u r e 6 the glucose o x i d a t i o n o f S t r e p , l a c t i s S.A. 3 0 S t r e p , l a c t i s A.T.C. 374, and S t r e p , l a c t i s EMBg 1 i s compared with t h a t o f B a c t . c o l i A.T.C. 4157.  T h i s graph shows t h e  extreme d i f f e r e n c e i n o x i d a t i v e a c t i v i t y evidenced by a e r o b i c organisms, such as B a c t . c o l i , and t h e more anaerobic s p e c i e s , such as S t r e p , l a c t i s .  DISCUSSION An important c h a r a c t e r i s t i c o f t h e a e r o b i c r e s p i r a t i o n o f the l a c t i c a c i d s t r e p t o c o c c i i s the presence o f an a p p r e c i a b l e oxygen uptake i n t h e presence o f added o x i d i z a b l e s u b s t r a t e . This "endogenous r e s p i r a t i o n " i n the case o f t h e two s t r a i n s  - 8 studied h e r e i n amounts t o f i f t y or s i x t y per cent of the oxygen uptake i n the presence of g l u c o s e . as a c o m p l i c a t i n g f a c t o r which may  I t therefore acts  serve t o mask t r u e  o x i d i z i n g a b i l i t y i n the case of s u b s t r a t e s which are but weakly a t t a c k e d . The endogenous oxygen uptake of a v a r i e t y o f b a c t e r i a l species was  s t u d i e d by C a l l o w ( 5 ) , who  demonstrated a v e r y  a p p r e c i a b l e uptake i n the case of a e r o b i c organisms and a n e g l i g i b l e uptake i n the case of a n a e r o b i c s p e c i e s .  Strep,  l a c t i s was found t o resemble the a n a e r o b i c r a t h e r t h a n the aerobic s p e c i e s i n p o s s e s s i n g o n l y a v e r y s l i g h t endogenous oxygen uptake.  T h i s work was confirmed by F a r r e l l ( l l ) , who  found the endogenous oxygen uptake o f twenty-two  s t r a i n s of  s t r e p t o c o c c i t o be almost n e g l i g i b l e and c o r r e l a t e d t h i s f a c t with the absence of an indophenol-oxldase system i n t h e s e bacteria.  The two s t r a i n s o f S t r e p , l a c t i s r e p o r t e d h e r e i n  have been found t o possess an endogenous oxygen uptake which i s s i x or seven times the v a l u e r e p o r t e d by F a r r e l l . The mechanism of the endogenous r e s p i r a t i o n of b a c t e r i a i s s t i l l l a r g e l y unknown•  W i l s o n (18) , s t u d y i n g the r e s p i r a -  t i o n of the R h i z o b i a , found t h a t ammonia was produced  by  r e s t i n g c e l l suspensions,and p o s t u l a t e d endogenous r e s p i r a t i o n to be an o x i d a t i v e deamination of c e l l u l a r amino a c i d s i n  •.  - 9 -  which c e l l p l l y s a c e h a r i d e was u t i l i s e d as an energy source, Ingram ( 1 4 ) , s t u d y i n g the endogenous r e s p i r a t i o n o f B  0  cereus,  found t h a t the g r a i n - p o s i t i v e c h a r a c t e r and the high endogenous r e s p i r a t i o n were both a s s o c i a t e d w i t h a h i g h content o f f a t i n the c e l l .  The endogenous r e s p i r a t i o n o f  the l a c t i c a c i d s t r e p t o c o c c i , however, does not appear t o p a r a l l e l t h a t d e s c r i b e d by e i t h e r o f these two workers, s i n c e repeated t e s t s upon r e s t i n g c e l l suspensions demonstrate the p r o d u c t i o n of ammonia,  f a i l e d to  Furthermore, the  l a c t i c a c i d s t r e p t o c o c c i do not produce s i g n i f i c a n t amounts of c e l l p o l y s a c c h a r i d e and are not considered t o possess a high c e l l u l a r f a t content.  I t has f u r t h e r been shown t h a t oxygen  uptake by r e s t i n g c e l l s o f S t r e p , l a c t i s i s not c a r r i e d out through t h e f u n c t i o n i n g o f a g l u t a t h i o n e system, s i n c e repeated attempts f a i l e d t o secure a p o s i t i v e n i t r o p r u s s i d e r e a c t i o n with these s u s p e n s i o n s .  The endogenous mechanism of the l a c t i c  s t r e p t o c o c c i i s s t i l l f u r t h e r c o m p l i c a t e d by the o b s e r v a t i o n of Morgan, E a g l e s , and L a i r d ( l 6 ) t h a t , i n c o n t r a s t t o t h e i r a p p r e c i a b l e a e r o b i c endogenous oxygen uptake, these b a c t e r i a do not e x h i b i t any anaerobic endogenous r e s p i r a t i o n . The v a l u e s o f the R e s p i r a t o r y Q,uotents r e p o r t e d i n Table 1 are i n g e n e r a l v e r y c l o s e t o a f i g u r e o f u n i t y .  This i n d i c a t e s  that i n the m a j o r i t y of cases o x i d a t i o n o f these  carbohydrates  • - 10 proceeds completely through t o carbon d i o x i d e and water.  In  some cases, however, a p p r e c i a b l y lower values of the R e s p i r a t o r y Quotient have been r e c o r d e d , as i n the case of l a c t o s e .  This  observation suggests t h a t , w i t h these s u b s t r a t e s , o x i d a t i o n i s incomplete  and i n t e r m e d i a t e products may  accumulate©  a l t e r n a t i v e s u g g e s t i o n has been advanced by C l i f t o n  An (6,7),  who  showed t h a t w i t h washed, c e l l s of B a c t , c o l i the o x i d a t i o n of many s u b s t r a t e s was the s u b s t r a t e was  not c a r r i e d to c o m p l e t i o n , but a p o r t i o n of  a s s i m i l a t e d by the c e l l as  carbohydrate.  This a s s i m i l a t o r y process c o u l d be blocked by adding sodium ozide or d i n i t r o p h e n o l . The  values of the R e s p i r a t o r y Q u o t i e n t s r e p o r t e d i n Table 1  i n d i c a t e t h a t w i t h s e v e r a l s u b s t r a t e s c o n s i d e r a b l y more carbon dioxide i s evolved than i s oxygen u t i l i z e d . that i n such cases the f u r t h e r process some i n t e r m e d i a t e l y - f o r m e d  T h i s would suggest  of d e c a r b o x y l a t i o n of  compound may  be t a k i n g p l a c e .  The r e s u l t s r e p o r t e d h e r e i n show t h a t S t r e p , l a c t i s e x h i b i t s a s t r o n g o x i d i z i n g a b i l i t y upon a wide v a r i e t y of compounds, i n c l u d i n g c a r b o h y d r a t e s ,  a l c o h o l s and organic a c i d s .  The o x i d a t i o n of t h i s l a s t group appears to take p l a c e very s t r o n g l y w i t h a l l such compounds t e s t e d .  This i s r a t h e r  s u r p r i s i n g i n view of the r e p o r t by Morgan, E a g l e s , and (16) t h a t the true l a c t i c a c i d s t r e p t o c o c c i possess no dehydrogenating a c t i v i t y upon the o r g a n i c acids©  Laird  - 11 Comparison of t h e a e r o b i c r e s p i r a t o r y a c t i v i t y of S t r e p , l a c t i s S.A, 3 0 w i t h t h a t of S t r e p , l a c t i s A.T.C. 374 shows t h a t t h e r e i s a c o n s i d e r a b l e v a r i a t i o n i n t h e i r o x i d i z i n g a b i l i t y upon s e v e r a l s u b s t r a t e s .  This v a r i a t i o n  i n a c t i v i t y between two s t r a i n s o f t h e same s p e c i e s i m p l i e s t h a t such t e s t s w i l l have l i t t l e taxonomic s i g n i f i c a n c e .  -  12  REFERENCES 1.  B a r r o n , E.S.G. and H a s t i n g s , A.P. .- 91'. 7 3 , 1 9 3 2 ,  2,  B a r r o n , E.S.G.  3,  B a r r o n , E.S.G, and Jacobs, H.R, _ J o u r , Bact, 36: 433,  J u r . B i o l , Chem. 0  J o u r , B i o l , Chem. 113: 695 , 1936,  1938,  4,  Bernheim, F.  J o u r , B a c t , 41: 387, 1941,  5.  Callow, A,B,  Biochem, J o u r , 18: 507, 1924,  6. 7«  Clifton, Clifton,  8o  Cook, R.P. and Stephenson, M. 1928.  9•  Dixon, M. 1934,  10.  1939,  C.E. Enzymologia 4 : 246, 1 9 3 7 . C.E. and Logan, W.A. J o u r . B a c t . 37: 3 2 3 , Biochem. Jour. 2 2 : 1368  s  "Manoinetric Methods", Cambridge U n i v . P r e s s ,  E a g l e s , B.A. and S a d l e r , W.  Can* J o u r . Res. 7* 3 6 4 ,  1932.  11.  F a r r e l l , M.A.  J o u r , B a c t , 2 9 : 411, 1 9 3 5 .  12.  Hansen, P.A.  Sent, f u r B a c t . 9 8 : 2 8 9 , 1 9 3 8 .  13.  Hunt, G.A,  J o u r . B c t . 2 6 : 341, 1 9 3 3 ,  14.  Ingram, M,  Jour. Bact, 38: 599, 1939,  15.  Lineweaver, Hans  16.  Morgan, J . F . , E a g l e s , B.A,, and L a i r d ,  17.  S a d l e r , W., E a g l e s , B.A., and Pendray, G. Res, 7= 3 7 0 , 1 9 3 2 . W i l s o n , P.W. Jour. Bact. 35: 601, 1938,  18.  a  J o u r . B i o l , Chem. 99? 5 7 5 , 1 9 3 3 . D.G. Can. Jour.  ABSTRACT The a e r o b i c o x i d a t i o n , dehydrogenation and l a c t i c a c i d p r o d u c t i o n from t w e n t y - f i v e carbohydrates by two s t r a i n s o f S t r e p , l a c t i s have been s t u d i e d o I t has been shown t h a t t h e r e i s no c o r r e l a t i o n between the a e r o b i c and a n a e r o b i c r e s p i r a t i o n o f r e s t i n g c e l l suspensions and l a c t i c a c i d f e r m e n t a t i o n by growing c u l t u r e s of these  organisms.  I t has been suggested t h a t t h i s apparent  difference  between the mechanisms of r e s p i r a t i o n and f e r m e n t a t i o n may be due t o the i n f l u e n c e of n i t r o g e n sources i n the growth medium.  INTRODUCTION The r e s p i r a t o r y enzyme systems o f many b a c t e r i a l s p e c i e s have been i n v e s t i g a t e d , Q,uastel and Whetham ( l l , 12) s t u d i e d t h e dehydrogenase a c t i v i t y  o f Bact.  coli  upon o r g a n i c a c i d s , a l c o h o l s , amino a c i d s and carbohydrates.  K e n d a l l (7) r e p o r t e d upon t h e dehydrogenase  mechanisms o f Bact. c o l i and a group of other related species.  closely-  The a e r o b i c o x i d a t i v e mechanism of Bact.  c o l i has been s t u d i e d by Cook and Stephenson ( 2 ) •  Earrell  (5) r e p o r t e d upon the r e s p i r a t o r y enzyme systems o f t h e S t r e p t o c o c c i under a e r o b i c and anaerobic  conditions.  Morgan,  E a g l e s , and L a i r d (8) surveyed the dehydrogenase enzyme activity  of fourteen s t r a i n s of l a c t i c  a c i d b a c t e r i a , and  l a t e r (?) r e p o r t e d t h e a e r o b i c oxidase a b i l i t y o f S t r e p , lact i S. Although  a e r o b i c and anaerobic r e s p i r a t o r y enzymes  have been e x t e n s i v e l y i n v e s t i g a t e d , t h e r e has been attempt t o r e l a t e r e s p i r a t o r y a c t i v i t y ability.  K e n d a l l and Ishikawa  little  with fermentative  ( 6 ) , s t u d y i n g a l a r g e group  of b a c t e r i a l s p e c i e s , r e p o r t e d t h a t the r e d u c t i o n of methylene blue by r e s t i n g b a c t e r i a i n the presence o f c e r t a i n  carbo-  hydrates was p r e c i s e l y p a r a l l e l e d by the f e r m e n t a t i o n o f these same carbohydrates  i n c u l t u r e media,  Barron and  Eriedemann (1) s t u d i e d a group o f b a c t e r i a which were unable to ferment glucose and demonstrated an a p p r e c i a b l e a c t i v i t y upon t h i s  oxidase  carbohydrate.  The work r e p o r t e d upon h e r e i n r e p r e s e n t s a comparison of the a e r o b i c o x i d a t i o n , anaerobic o x i d a t i o n and l a c t i c f e r m e n t a t i o n of S t r e p , l a c t i s upon t w e n t y - f i v e  acid  carbohydrates,  EXPERIMENTAL METHODS The c u l t u r e s employed i n t h i s study were S t r e p , l a c t i s S.,A, JO ( 1 3 )  and S t r e p , l a c t i s A.T.C. 3 7 4 ,  the former  i s o l a t e d from b u t t e r p o s s e s s i n g a caramel f l a v o r , and the l a t t e r o b t a i n e d from t h e N a t i o n a l Type C u l t u r e C o l l e c t i o n at Washington,  D.C.  Casein D i g e s t B r o t h , prepared Jensen ( 4 ) ,  a f t e r the manner of Q r l a -  served as the b a s i c medium,  t i o n s , 2$ carbohydrate  Eor sugar  fermenta-  was i n c o r p o r a t e d , w h i l e f o r r e s p i r a t o r y  s t u d i e s the broth was e n r i c h e d w i t h 1 $ D i f c o yeast e x t r a c t , •0,5$  K 2 H P O 4 , and  0,5$  glucose©  R e s p i r a t o r y enzyme s t u d i e s were c a r r i e d out employing " r e s t i n g c e l l " suspensions prepared and s t a n d a r d i z e d as r e p o r t e d by Morgan, E a g l e s and L a i r d ( 8 ) . A e r o b i c o x i d a t i v e a b i l i t y was measured m a n o m e t r i c a l l y with, the B a r c r o f t apparatus, as d e t a i l e d by Dixon (3) .  Dehydrogenase  activity  was determined by t h e Thunberg t e c h n i q u e , f o l l o w i n g the procedure d e s c r i b e d by Morgan, E a g l e s and L a i r d (8) . Fermentation o f carbohydrates was measured by t i t r a t i n g fourteen-day c u l t u r e s i n sugar b r o t h s w i t h N/4 sodium h y d r o x i d e , u s i n g p h e n o l p h t h a l e i n as i n d i c a t o r .  After subtracting that  of the c o n t r o l , the remaining values were converted i n t o grams of l a c t i c a c i d per liter®  EXPERIMENTAL RESULTS The a e r o b i c oxygen uptake, dehydrogenase a c t i v i t y  and  l a c t i c a c i d p r o d u c t i o n by S t r e p * l a c t i s S A„ 30 and S t r e p , S  l a c t i s A.T.C. 374 upon t w e n t y - f i v e carbohydrates are presented i n Table 1.  Although these v a l u e s f u r n i s h d e t a i l e d  i n f o r m a t i o n about t h e r e s p i r a t o r y enzyme a c t i v i t y f e r m e n t a t i v e a b i l i t y of these two s t r a i n s ,  and  distinctly  d i f f e r e n t standards have been employed as the bases of these  t h r e e types of d e t e r m i n a t i o n ,  These r e s u l t s , t h e r e f o r e ,  cannot be s t u d i e d c o m p a r a t i v e l y u n t i l some common b a s i s of i n t e r p r e t a t i o n has been e v o l v e d .  The v a l u e s of the  Q,0  2  have a c c o r d i n g l y been r e c a l c u l a t e d as percentage of the glucose o x i d a t i o n v a l u e taken as 100, w h i l e the l a c t i c a c i d p r o d u c t i o n i n grams per l i t e r has l i k e w i s e been converted i n t o percentage of the a c i d produced from glucose. dehydrogenase a c t i v i t y had a l r e a d y been expressed  Since as  percentage of glucose r e d u c t i o n t i m e , a l l three  determina-  t i o n s are now based upon one g e n e r a l comparative  standard,  c a l l e d the R e s p i r a t o r y C o e f f i c i e n t s v a l u e s are presented  i n Table  The data recorded  These r e c a l c u l a t e d  2.  i n Table 2 i n d i c a t e d e f i n i t e l y t h a t  t h e r e i s no c o r r e l a t i o n between a e r o b i c r e s p i r a t i o n , anaerobic r e s p i r a t i o n and l a c t i c a c i d -production w i t h the two  s t r a i n s of S t r e p , l a c t i s s t u d i e d .  F u r t h e r , t h e r e does  not appear to be any r e l a t i o n s h i p between o x i d a t i v e a b i l i t y and f e r m e n t a t i o n , dehydrogenase a c t i v i t y and f e r m e n t a t i o n , o even between the a e r o b i c and anaerobic o x i d a t i v e mechanisms themselves. The l a c k of c o r r e l a t i o n between a e r o b i c and  anaerobic  r e s p i r a t o r y enzymes and f e r m e n t a t i o n i s f u r t h e r emphasized by the r e s u l t s shown i n F i g u r e s 1 and 2. the comparative  In these graphs  enzymic a c t i v i t i e s of each s t r a i n of S t r e p .  l a c t i s upon f i v e s e l e c t e d carbohydrates  are  portrayed.  In F i g u r e 1 the enzymic a c t i v i t y of S t r e p , l a c t i s S,A.30 upon g a l a c t o s e , s u c r o s e , l a c t o s e , t r e h a l o s e and m a n n i t o l i s shown.  The r e a c t i o n s upon g a l a c t o s e and l a c t o s e  are c h a r a c t e r i z e d by s t r o n g oxidase a c t i v i t y , weak dehydrogenase a c t i v i t y , and f a i r l y high l a c t i c a c i d production.  This r e l a t i o n s h i p between the three enzymic systems  i s the one most f r e q u e n t l y observed i n Table 2.  among the values r e p o r t e d  A r e v e r s a l of t h i s a c t i v i t y order i s observed  i n the case of t r e h a l o s e , where a c i d p r o d u c t i o n i s higher than oxidase a c t i v i t y , w h i l e dehydrogenase a c t i v i t y i s comparatively f e e b l e .  With sucrose as. the s u b s t r a t e , an  e n t i r e l y d i f f e r e n t p i c t u r e i s obtained.  Dehydrogenase a c t i v i t y  has become v e r y powerful and i s a p p r e c i a b l y g r e a t e r than oxidase a b i l i t y , w h i l e l a c t i c a c i d p r o d u c t i o n remains almost negligible,  Enzymic a c t i v i t y upon m a n n i t o l , on the other  hand, i s c h a r a c t e r i z e d by an extremely h i g h o x i d a t i v e a b i l i t y , w h i l e l a c t i c a c i d p r o d u c t i o n i s f e e b l e and dehydrogenase activity is entirely The  absent.  enzymic a c t i v i t y of S t r e p . l a c t i s A.T.C. 374 upon  g a l a c t o s e , s u c r o s e , r a f f i n o s e , d e x t r i n and i n u l i n i s shown i n F i g u r e 2,  With g a l a c t o s e t h e r e i s evident a moderate  oxidase a b i l i t y , low dehydrogenase a c t i v i t y , and very h i g h l a c t i c acid production.  With i n u l i n , on the other hand, the  exact r e v e r s e h o l d s t r u e , oxidase a b i l i t y being  low,  - 6 dehydrogenase a c t i v i t y high., and a c i d p r o d u c t i o n e n t i r e l y lacking.  When'sucrose serves as the s u b s t r a t e , dehydrogenase  a c t i v i t y i s very s t r o n g , oxidase a b i l i t y f a i r l y s t r o n g , and a c i d p r o d u c t i o n moderates a moderately  With r a f f i n o s e there i s demonstrable  s t r o n g oxidase mechanism, very s l i g h t  lactic  a c i d p r o d u c t i o n and no observable dehydrogenase a c t i v i t y .  A  somewhat s i m i l a r r e l a t i o n s h i p i s found i n the case of d e x t r i n , where t h e r e e x i s t s a tremendously  s t r o n g oxidase  activity,  t o g e t h e r w i t h o n l y f e e b l e dehydrogenase a c t i o n and l a c t i c acid formation. The r e s u l t s recorded i n Table 2 and shown g r a p h i c a l l y i n F i g u r e s 1 and 2 o f f e r d e f i n i t e proof t h a t no e x i s t s between the a e r o b i c and anaerobic  respiratory  processes and the mechanism o f l a c t i c a c i d However , the f e r m e n t a t i v e end-products may  be formed,there  must e v i d e n t l y  relationship  fermentations  of b a c t e r i a l  metabolism  e x i s t some d e t e r m i n i n g  mechanism apart from the enzymic processes of a e r o b i c and anaerobic  respiration.  V  DISCUSSION  •  The r e l a t i o n s h i p between o x i d a t i o n and  fermentation  i s d i s c u s s e d i n d e t a i l by Oppenheimer and S t e r n (10):  "The  time i s past when f e r m e n t a t i o n and o x i d a t i o n were  considered t q be two q u i t e d i s t i n c t types of b i o l o g i c a l processes and when the enzymes a c t i v e i n f e r m e n t a t i o n , the zymases, and those a c t i v e i n v i t a l o x i d a t i o n , were t r e a t e d as e n t i r e l y u n r e l a t e d *  Today we speak of one great system  of enzymes c a t a l y z i n g the o v e r - a l l phenomenon of energy p r o d u c t i o n i n the c e l l , termed desmolysis  I f the  a n o x y b i o n t i c metabolism does not pass over i n t o o x y b i o s i s , c e r t a i n r e a c t i o n s take p l a c e l e a d i n g to s t a b i l i z a t i o n of the a n a e r o b i c a l l y formed compounds, and l a c t i c a c i d or e t h y l a l c o h o l appear as the end-products  e  The  c o r r e l a t i o n between  f e r m e n t a t i o n and r e s p i r a t i o n , or r a t h e r between oxygen t e n s i o n and f e r m e n t a t i o n , i s maintained  by the s o - c a l l e d P a s t e u r -  Meyerhof r e a c t i o n , the mechanism of which i s s t i l l obscure.  largely  There i s one uniform mechanism o p e r a t i v e i n both  phases ( f e r m e n t a t i o n and r e s p i r a t i o n ) of desmolysis, namely, the t r a n s f e r of m e t a b o l i c hydrogen.  In a n a e r o b i o s i s i t  t e r m i n a t e s i n l a c t i c a c i d or a l c o h o l and i n a e r o b i o s i s i t t e r m i n a t e s i n water." T h i s view of the complete i d e n t i t y of the two of o x i d a t i o n and f e r m e n t a t i o n i s a l s o advanced by Gyorgyi  (14 ) , who  processes Szent-  p o i n t e d out t h a t both a e r o b i c and  breakdown o f sugars proceed through the same i n i t i a l namely, the s p l i t t i n g of hexose-phosphate i n t o t r i o s e  anaerobic steps,  - .8  phosphate  and the subsequent  three-carbon-compounds.  -  dehydrogenation of the r e s u l t i n g  The d i f f e r e n c e between these two  processes comes i n o n l y at the next.step.-. In o x i d a t i o n , m o l e c u l a r oxygen a c t s as the f i n a l Hydrogen Acceptor of e l e c t r o n s , w h i l e i n f e r m e n t a t i o n the hydrogen i s accepted through an i n t e r n a l rearrangement  of the molecules of the  o x i d i z a b l e substance. The r e s u l t s r e p o r t e d h e r e i n are d i f f i c u l t to r e c o n c i l e w i t h the accepted concept of the i d e n t i t y of the r e s p i r a t o r y and f e r m e n t a t i v e mechanisms.  With the l a c t i c a c i d  strepto-  c o c c i i t has been shown t h a t a p p r e c i a b l e f e r m e n t a t i o n of a carbohydrate may occur even when both a e r o b i c and anaerobic o x i d a t i v e mechanisms upon t h a t carbohydrate are very f e e b l e . I t has a l s o been shown t h a t l a r g e q u a n t i t i e s of l a c t i c a c i d may be formed from a carbohydrate a g a i a s t which the organism possesses a s t r o n g o x i d a t i v e but n e g l i g i b l e dehydrogenase activity.  I t has f u r t h e r been shown t h a t l i t t l e or no l a c t i c  a c i d may be formed when the organism e x h i b i t s a s t r o n g o x i d i z i n g and f e e b l e dehydrogenase a c t i v i t y , and even when the organism possesses both a s t r o n g o x i d i z i n g and a s t r o n g dehydrogenase a c t i v i t y upon a p a r t i c u l a r carbohydrate. These r e s u l t s suggest t h a t , w i t h the l a c t i c a c i d s t r e p t o c o c c i , f e r m e n t a t i o n by growing c u l t u r e s of these  -  9  -  organisms i s an enzymic process which appears t o be separate and d i s t i n c t " f r o m the a e r o b i c and anaerobic r e s p i r a t o r y mechanisms e x h i b i t e d by r e s t i n g c e l l suspensions. of  The l a c k  c o r r e l a t i o n between these two m e t a b o l i c processes may be  due t o the i n f l u e n c e o f n i t r o g e n source and a c c e s s o r y f a c t o r s c o n t a i n e d i n the growth medium. The presence of s m a l l amounts of a n i t r o g e n source has been found t o exert a pronounced i n f l u e n c e upon the r e s p i r a t o r y and f e r m e n t a t i v e mechanisms of r e s t i n g  cell  suspensions.  The r e s u l t s of t h i s study w i l l be presented i n  a subsequent  paper.  - 10 -  REFERENCES lo  Barron, E.S.G. and Friedemann, T.E. 137: 573, 1941.  J . B j o l . Chem,  2»  Cook, R.P. and Stephenson, M. 1928.  3.  Dixon, M. "Manometric Methods", Cambridge U n i v P r e s s , 1934  Biochem. Jour. 22: 1368  e  0  4.  E a g l e s , B.A., and S a d l e r , I ,  Can. J o u r . Res. 7: 3 6 4 ,  1932.  J o u r . B c t . 29: 411, 1935,  5*  E a r r e l l , M.A.  6.  K e n d a l l , A . I . and Ishikawa, M. 282,  a  J our. I n f Di s. 44: e  1929.  J o u r . I n f . D i s . 47: 1 8 6 , 1930  7.  Kendall, A.I.  8.  Morgan, J.F. , E a g l e s , B.A., L a i r d ,  D.G  9«  Morgan, J.F., E a g l e s , B.A., L a i r d ,  D.G.  10.  Oppenheimer, C. and S t e r n , K G. Nordemann, 19 39s  " B i o l o g i c a l Oxidation"  11.  Q u a s t e l , J.H. and Whetham, M.D. 5 2 0 , 1925.  Biochem. Jour. 19:  12.  Q u a s t e l , J.H. and Whetham, M.D. 645, 19 2 5 .  Biochem. J o u r . 1 9  13.  S a d l e r , W.  14.  S z e n t - G y o r g y i , A.Y. " O x i d a t i o n , Fermentation, V i t a m i n s , H e a l t h and D i s e a s e " , W i l l i a m s and W i l k i n s , 1939.  0  :  .Trans. Roy. Soc. Can. V o l . 20, 1926.  ABSTRACT  The i n f l u e n c e of carbohydrate  a d a p t a t i o n upon  the subsequent dehydrogenase a c t i v i t y and f e r m e n t a t i v e a b i l i t y of r e s t i n g c e l l suspensions  has been s t u d i e d  w i t h two s t r a i n s of S t r e p , l a c t i s . I t has been shown t h a t dehydrogenase a c t i v i t y v a r i e s markedly i n response to carbohydrate  changes i n  the growth medium. D i r e c t a d a p t a t i o n by dehydrogenase enzymes and a l s o more g e n e r a l eases of s t i m u l a t i o n and i n h i b i t i o n of dehydrogenation  have been shown to occur.  The a d a p t i v e and c o n s t i t u t i v e nature of the f e r m e n t a t i v e enzymes of S t r e p , l a c t i s has been determined. I t has been shown, by a d a p t i v e and c o n s t i t u t i v e enzymes, t h a t f e r m e n t a t i o n of l a c t o s e by S t r e p , l a c t i s must occur through a p r e l i m i n a r y h y d r o l y s i s to glucose and  galactose. The r e l a t i o n s h i p of these phenomena to the  mechanism of a d a p t a t i o n has been d i s c u s s e d .  INTRODUCTION The dehydrogenase a c t i v i t y of the L a c t i c A c i d S t r e p t o c o c c i has not as y e t been f u l l y F a r r e l l (5) r e p o r t e d  investigated.  upon the r e s p i r a t o r y mechanism  of 32 s t r a i n s of s t r e p t o c o c c i , i n c l u d i n g S t r e p , and  Strep, f e c a l i e .  K a t a g i r I and K i t a h a r a  lactis  (6) showed  the presence of l a c t i c a s i d dehydrogenase among s e v e r a l species  of l a c t i c a c i d b a c t e r i a .  Wood and Gunsalus (14)  studied  the v a r i o u s f a c t o r s i n f l u e n c i n g the p r o d u c t i o n  of  a c t i v e r e s t i n g c e i l suspensions of the Group B S t r e p t o c o c c i Morgan, E a g l e s and L a i r d (7) surveyed the dehydrogenase a c t i v i t y of a group of f o u r t e e n  s t r a i n s of l a c t i c a c i d  streptococci. Hegarty (5),and Rahn, Hegarty and D u e l l ( 9 ) , s t u d i e d l a c t i c ac id pr oduct i o n  by washed c e l l suspensions  of S t r e p , l a c t i s , T h e y demonstrated that the enzymes a t t a c k i n g glucose, while  mannose and f r u c t o s e were c o n s t i t u t i v e ,  those a t t a c k i n g a l l other sugars were The f i r s t o b s e r v a t i o n  adaptive.  of the phenomenon of  a d a p t i v e and c o n s t i t u t i v e enzymes was made by Wor tmann (15) i n the case of s t a r c h h y d r o l y s i s by an unknown b a c t e r i a l species.  The modern t e r m i n o l o g y of •'adaptive" and  " c o n s t i t u t i v e " enzymes was i n t r o d u c e d  by Karstrom,  V i r t a n e n and t h e i r a s s o c i a t e s ( 1 5 ) .  The i n f l u e n c e of  a d a p t a t i o n upon b a c t e r i a l enzymes was s t u d i e d b y  1  Stephenson and S t i c k l a n d (11) i n the case o f the hydrogenlyases,  Haines ( 4 ) w i t h b a c t e r i a l g e l a t i n a s e ,  Stephenson and Gale (12) w i t h Bact, c o l i ,  and Quaste 1  (8) w i t h M» l y s o d e i k t i c u s . The work r e p o r t e d upon h e r e i n was undertaken w i t h the o b j e c t of determining  the i n f l u e n c e exerted by a d a p t i v e  and c o n s t i t u t i v e enzymes upon the mechanisms of dehydrogena t i o n and l a c t i c a c i d f e r m e n t a t i o n by S t r e p . l a c t i s , , EXPERIMENTAL METHODS The c u l t u r e s employed i n t h i s study c o n s i s t e d of S t r e p , l a c t i s S.A. 30 ( 1 0 ) , i s o l a t e d from b u t t e r possess i n g a caramel f l a v o r , and S t r e p , l a c t i s A TO 374, obtained from the N a t i o n a l Type- C u l t u r e c o l l e c t i o n . C a s e i n D i g e s t B r o t h , prepared  a f t e r the manner of  Orla-Jensen  ( 2 ) , served as the b a s i c medium and was en-  riched with  0.5$ K 2 HPO4  and 1.0$ D i f c o y e a s t e x t r a c t .  To t h i s b a s i c medium v a r i o u s carbohydrates  were added to  a c o n c e n t r a t i o n of 0 . 5 $ . F o r the measurement of r e s p i r a t o r y enzyme  activity  and l a c t i c a c i d p r o d u c t i o n " r e s t i n g c e l l " suspensions S t r e p , l a c t i s were prepared  of  and s t a n d a r d i z e d as d e s c r i b e d  by Morgan, E a g l e s , and L a i r d ( 7 ) , L a c t i c a c i d p r o d u c t i o n was measured by t i t r a t i n g samples o f a r e s t i n g c e l l ,  carbo-  h y d r a t e , b u f f e r , peptone mixture a t r e g u l a r i n t e r v a l s over a s i x hour p e r i o d , f o l l o w i n g the method employed by Hegarty (5).  Dehydrogenase a c t i v i t y was determined by the Thunberg  technique, under the e x p e r i m e n t a l c o n d i t i o n s p r e v i o u s l y d e s c r i b e d by Morgan, E a g l e s and L a i r d ( 7 ) . EXPERIMENTAL RESULTS A. A d a p t a t i o n and Dehydrogenation The carbohydrate dehydrogenating a c t i v i t y of r e s t i n g c e l l suspensions o f S t r e p , l a c t i s SA s o prepared from c e l l s grown i n the presence o f v a r i o u s carbohydrates i s recorded i n Table 1.  The r e s u l t s r e p o r t e d i n t h i s t a b l e show t h a t  the presence o f s p e c i f i c c a i b o h y d r a t e s i n the growth medium has e x e r t e d a pronounced i n f l u e n c e upon the subsequent deh y d r o g e n a t i n g a b i l i t y o f r e s t i n g c e l l suspensions prepared therefrom.  There i s apparent a marked v a r i a t i o n i n carbo-  hydrate dehydrogenations  i n response  to change i n the carbon  source of the growth medium© T h i s v a r i a t i o n i s emphasized by the r e s u l t s shown i n F i g u r e 1.  I n t h i s graph the r e l a t i v e dehydrogenase a c t i v i -  t i e s upon a r a b i n o s e , l a c t o s e , maltose, r a f f i n o s e and s t a r c h are compared, u s i n g r e s t i n g c e l l s o b t a i n e d from g l u c o s e , l a c t o s e , s t a r c h and m a n n i t o l b r o t h s .  The s t i m u l a t o r y  e f f e c t of p r e v i o u s growth i n the homologous s u b s t r a t e i s  shown i n the case of l a c t o s e .  Growth i n l a c t o s e b r o t h  has i n c r e a s e d the dehydrogenase a c t i v i t y upon l a c t o s e f o u r t e e n time over the a c t i v i t y of c e l l s grown i n glucose broth.  I t i s a l s o n o t i c e a b l e that growth i n l a c t o s e b r o t h  has markedly lengthened the times r e q u i r e d f o r dehydrogena t i o n , as r e c o r d e d i n Table I , but a t the same time has i n c r e a s e d the s t r e n g t h of the r e l a t i v e r e d u c i n g c o e f f i c i e n t s . The i n f l u e n c e of the carbohydrate i n the growth medium upon subsequent  dehydrogenations i s very c l e a r l y  shown i n the case of arabinose and r a f f i n o s e .  With both  these sugars dehydrogenation f a i l s to occur when c e l l s are obtained from g l u c o s e b r o t h , but occurs very r e a d i l y when c e l l s are o b t a i n e d f r o m ' l a c t o s e b r o t h .  The r e v e r s e , of  t h i s phenomenon i s shown w i t h maltose and s t a r c h .  With  these carbohydrates dehydrogenation takes p l a c e vith.  cells  prepared from g l u c o s e l a c t o s e , or s t a r c h b r o t h , but does not take p l a c e w i t h c e l l s prepared from m a n n i t o l b r o t h . The carbohydrate dehydrogenating a c t i v i t y of suspensions of S t r e p , l a c t i s A TO 374 prepared from c e l l s grown i n g l u c o s e , l a c t o s e , and m a n n i t o l b r o t h s i s r e c o r d e d i n Table I I . The r e s u l t s shown i n t h i s table compare c l o s e l y w i t h those r e c o r d e d f o r S t r e p . l a c t i s SA 30 i n Table I . I t i s apparent w i t h both these s t r a i n s that the carbohydrate i n the growth  medium has markedly i n f l u e n c e d the dehydrogenase a c t i v i t y of the subsequent r e s t i n g c e l l suspensions. T h i s i n f l u e n c e i s f u r t h e r i l l u s t r a t e d i n F i g u r e 2, i n which the comparative dehydrogenations of mannose. g a l a c t o s e , l a c t o s e , m a n n i t o l and r a f f i n o s e a r e p o r t r a y e d . The c l e a r e s t example o f a d a p t a t i o n i s shown i n the ease of m a n n i t o l , suspensions o b t a i n e d from m a n n i t o l b r o t h dehydro g e n a t i n g m a n n i t o l twenty-seven  times as r a p i d l y as c e l l s  obtained from glucose b r o t h .  With the mannose dehydrogen  ase, t h e r e appears to be l i t t l e  e f f e c t e x e r t e d by the earb  h y d r a t e i n t h e growth medium.  In the case of g a l a c t o s e  dehydrogenase a c t i v i t y i s a p p a r e n t l y i n c r e a s e d by growing the c e l l s i n l a c t o s e or m a n n i t o l b r o t h s .  With r a f f i n o s e ,  dehydrogenation does n o t occur when the c e l l s have been prepared from g l u c o s e b r o t h , but does occur when the c e l l s have been prepared from l a c t o s e or m a n n i t o l b r o t h s , B. A dap t a t i on and Fer men t a t i o n L a c t i c a c i d p r o d u c t i o n from v a r i o u s carbohydrates by suspensions o f S t r e p , l a c t i s SA 30 i s shown i n Table 3. These r e s u l t s have been o b t a i n e d by t i t r a t i n g the a c i d produced by r e s t i n g c e l l suspensions i n phosphate  buffer  of pH. 7.2 c o n t a i n i n g 0.3$ peptone, to which has been added 2,0$ of the carbohydrate b e i n g t e s t e d ( 5 ) .  Where the  organism possesses a c o n s t i t u t i v e , enzyme c o n t r o l l i n g  f e r m e n t a t i o n of the s u b s t r a t e a c i d p r o d u c t i o n w i l l at once.  However, where the organism possesses  enzyme for" the carbohydrate  occur  an adaptive  under study, a c o n s i d e r a b l e  p e r i o d o f time w i l l elapse before a c i d i s produced i n any appreciable quantity. T h i s d i s t i n c t i o n between a d a p t i v e and c o n s t i t u t i v e enzymes i s c l e a r l y shown i n F i g u r e 3.  In t h i s graph  l a c t i c a c i d p r o d u c t i o n from the f o u r monosaccharides i s shown, employing r e s t i n g c e l l suspensions-obtained glucose b r o t h .  from  I t i s apparent from the graph that S t r e p ,  l a c t i s SA 30 possesses  c o n s t i t u t i v e enzymes f o r glucose,  f r u c t o s e and miannose, b u t an a d a p t i v e enzyme f o r g a l a c t o s e . The i n f l u e n c e of a d a p t i v e and c o n e t i t u t i v e enzymes upon the f e r m e n t a t i o n of l a c t o s e by suspensions l a c t i s SA 30 i s shown i n F i g u r e 4.  of S t r e p ,  C e l l s obtained from  glucose b r o t h or g a l a c t o s e b r o t h show p r a c t i c a l l y no f e r m e n t a t i v e a b i l i t y when p l a c e d i n the presence of lactose.  When c e l l s which have been grown i n l a c t o s e  b r o t h , however, a r e p l a c e d i n l a c t o s e there i s a r a p i d and heavy p r o d u c t i o n of l a c t i c a c i d .  This would i n d i c a t e  that growth i n l a c t o s e broth has s t i m u l a t e d the enzyme c o n t r o l l i n g l a c t o s e f e r m e n t a t i o n , which must t h e r e f o r e be an a d a p t i v e enzyme* The r e l a t i o n s h i p o f a d a p t a t i o n to the f e r m e n t a t i o n of g a l a c t o s e by suspensions  of S t r e p , l a c t i s 3A 30 i s shown  i n f i g u r e 5.  With, t h i s sugar i t would appear that growth  i n l a c t o s e b r o t h has s t i m u l a t e d the a d a p t i v e enzyme f e r menting g a l a c t o s e , although  growth i n g a l a c t o s e broth has  not .caused a corresponding s t i m u l a t i o n , , When S t r e p , l a c t i s ATC  374 was employed as the  test  organism, r a t h e r than S t r e p , l a c t i s SA30, e s s e n t i a l l y r e s u l t s were o b t a i n e d .  I t was  found t h a t , i n the  similar  fermentation  of the monosaccharides, t h i s organism a l s o possessed t i v e enzymes f o r glucose, mannose and f r u c t o s e , and  constituan  a d a p t i v e enzyme f o r g a l a c t o s e . However, an important d i f f e r e n c e between these s t r a i n s was  observed  two  i n the f e r m e n t a t i o n of l a c t o s e .  The  i n f l u e n c e of a d a p t a t i o n upon the f e r m e n t a t i o n of t h i s disacchar.de by suspensions shown i n F i g u r e 6.  of S t r e p , l a c t i s ATC  374 i s  I t i s apparent from t h i s graph t h a t  the organism possesses a c o n s t i t u t i v e enzyme f o r l a c t o s e f e r m e n t a t i o n , s i n c e c e l l s o b t a i n e d from glucose  broth  s t r o n g l y ferment l a c t o s e , and s i n c e growth i n l a c t o s e broth does not i n c r e a s e e i t h e r the r a t e or the amount of a c i d p r o d u c t i o n from l a c t o s e .  On the other hand, although  l a c t o s e i s fermented by cell's o b t a i n e d from glucose b r o t h there i s l i t t l e f e r m e n t a t i o n by c e l l s obtained from g a l a c t o s e br o th . • A f u r t h e r i n t e r e s t i n g r e l a t i o n s h i p i s found i n Figure 7 which shows the i n f l u e n c e of a d a p t a t i o n uponthe f e r m e n t a t i o n  of g a l a c t o s e by suspensions this  of Strep . l a c t i s ATC  374.  In  graph l a c t i c a c i d p r o d u c t i o n from g a l a c t o s e i s shown  to occur when c e l l s are prepared  from g a l a c t o s e b r o t h or  from l a c t o s e b r o t h , but not when the c e l l s are from glucose broth .  obtained  This i s f u r t h e r proof t h a t the  f e r m e n t a t i o n of g a l a c t o s e i s c a r r i e d out by an enzyme. . However, i t would appear that t h i s  adaptive  adaptive  enzyme f o r g a l a c t o s e f e r m e n t a t i o n has a l s o been s t i m u l a t e d by growth i n l a c t o s e b r o t h . DISCUSSION The r e s u l t s o b t a i n e d w i t h the two s t r a i n s of S t r e p , l a c t i s s t u d i e d show c l e a r l y that the carbohydrate  present  i n the growth medium e x e r t s a pronounced e f f e c t upon both the dehydrogenase a c t i v i t y of r e s t i n g c e l l The  and  the f e r m e n t a t i v e  ability  suspensions.  r e s u l t s recorded  i n Tables 1 and 3 and  portrayed  i n F i g u r e s 1 and 3 make i t evident that there i s an extreme v a r i a t i o n i n dehydrogenase a c t i v i t y carbohydrates  i n the growth medium.  growing these organisims  i n response to v a r i o u s I t i s apparent t h a t  i n l a c t o s e or m a n n i t o l broth very  g r e a t l y i n c r e a s e s the a b i l i t y of c e l l suspensions hydrogenase these s u b s t r a t e s . that these  to de-  I t would appear, t h e r e f o r e ,  dehydrogenase enzymes,are a d a p t i v e i n c h a r a c t e r .  A p a r t from these i n s t a n c e s of a d a p t a t i o n i n response to  the p r e s e n c e  of "She homologous c a r b o h y d r a t e  there  occur  other v a r i a t i o n s i n dehydrogenase a c t i v i t y which cannot e x p l a i n e d on  the b a s i s o f s i m p l e a d a p t a t i o n .  An  illustra-  t i o n o f such phenomena i s f o u n d i n the d e h y d r o g e n a t i o n raffinose.  With both  t i o n of r a f f i n o s e  s t r a i n s of Strep, l a c t i s  does n o t o c c u r i f the  been grown I n g l u c o s e b r o t h , but  be  of  dehydrogena-  c e l l s have p r e v i o u s l y  does o c c u r i f t h e c e l l s a r e  o b t a i n e d from media c o n t a i n i n g other  carbohydrates.  would appear t h a t g l u c o s e i n the growth  It  medium e x e r t s an  i n h i b i t o r y e f f e c t upon t h e r a f f i n o s e d e h y d r o g e n a s e enzyme of  subsequent suspensions.  F u r t h e r examples o f  phenomena are the i n a b i l i t y o f s u s p e n s i o n s SA 30  to dehydrogenat© m a l t o s e and  been grown i n m a n n i t o l b r o t h , and  similar  of S t r e p , l a c t i s  s t a r c h i f the c e l l s  the appearance of a s t r o n g  d e h y d r o g e n a s e a c t i v i t y upon arabinoe/e when the o r g a n i s m been p r e v i o u s l y grown i n l a c t o s e b r o t h *  These r e s u l t s  n o t a p p e a r to agree w i t h the o b s e r v a t i o n s o f Dubss ( 1 ) , has  e m p h a s i z e d the extreme s p e c i f i c i t y The  7 e m p h a s i z e the  has do who  o f a d a p t i v e enzymes.  r e s u l t s r e c o r d e d i n T a b l e 3 and  F i g u r e s 3,4,5,6 and  have  portrayed i n  i n f l u e n c e of  carbo-  h y d r a t e a d a p t a t i o n upon t h e f e r m e n t a t i v e a b i l i t y o f r e s t i n g cell  suspensions.  I t has been shown t h a t b o t h s t r a i n s o f  Strep, l a c t i s possess  c o n s t i t u t i v e .enzymes f o r t h e  t i o n o f g l u c o s e , mannose and  fermenta-  f r u c t o s e , but an a d a p t i v e  -10"enzyme f o r the f e r m e n t a t i o n o f g a l a c t o s e .  T h i s had p r e v -  i o u s l y been d e m o n s t r a t e d with S t r e p , l a c t i s by H e g a r t y ( 5 ) . In  c o n t r a d i c t i o n to h i s r e s u l t s , however', i t has been shown  t h a t one of the s t r a i n s , S t r e p , l a c t i s ATC 374. a constitutive With both  enzyme f o r t h e f e r m e n t a t i o n strains  of S t r e p , l a c t i s  possesses  of l a c t o s e .  i t has a l s o been  shown t h a t g r o w t h i n l a c t o s e b r o t h r e s u l t s ' i n t h e a d a p t a tion  to both  l a c t o s e and g a l a c t o s e , w h i l e  growth i n g a l a c -  t o s e b r o t h does n o t cause a d a p t a t i o n t o l a c t o s e . result one  This  has been o b t a i n e d w i t h two s t r a i n s of S t r e p ,  lactis,  of w h i c h h a s b e e n shown to c o n t a i n an a d a p t i v e and one  a c o n s t i t u t i v e enzyme f o r l a c t o s e .  T h i s would seem to  furnish  S t r e p , l a c t i s the  d e f i n i t e evidence  fermentation  that with  of l a c t o s e p r o c e e d s t h r o u g h  a preliminary  h y d r o l y s i s to i t s c o n s t i t u e n t m o n o s a c c h a r i d e s , and  glucose  galactose. With Strep, l a c t i s  adaptation  SA 30 i t was a l s o n o t i c e d  that  to g a l a c t o s e was o b t a i n e d by g r o w i n g t h e  o r g a n i s m i n the p r e s e n c e of l a c t o s e , but n o t i n t h e p r e s e n c e of g a l a c t o s e . formed t h r o u g h  This would i n d i c a t e t h a t  galactose  the h y d r o l y s i s of l a c t o s e i n t h e medium i s  more a c t i v e i n c a u s i n g  a d a p t a t i o n than  g a l a c t o s e added a s  s u c h to t h e medium. The r e l a t i o n s h i p o f t h e s e r e s u l t s  to the mechanism  o f a d a p t i v e and c o n s t i t u t i v e enzymes i s s t i l l It  obscure.  i s b e l i e v e d t h a t a c o n s t i t u t i v e enzyme e x i s t s a s a n  -11i n t e g r a l p a r t of the c e l l s t r u c t u r e and  i s t h e r e f o r e always  pre sent i n the c e l 1 i n an a c t i v e form.  An  adaptive  enzyme on the other hand, i s b e l i e v e d t o e x i s t i n the i n an i n a c t i v e s t a t e and  cell  to r e q u i r e s t i m u l a t i o n or adapta-  t i o n through c o n t a c t w i t h the homologous substance before enzymic a c t i v i t y , can be demonstrated.  This v i e w p o i n t ,  as  advanced by Y i r t a n e n (13), has been considered  inadequate  by Dubos ( 1 ) , Q u a s t e l  emphasized  ( 8 ) , and Haines ( 4 ) , who  the complex i n f l u e n c e s w h i c h enviromental  f a c t o r s may  e x e r t on the enzymic c o n s t i t u t i o n of the m i c r o b i a l c e l l . The r e s u l t s r e p o r t e d h e r e i n w i t h Stre p „ l a c t i s f u r n i s h f u r t h e r evidence of the important  i n f l u e n c e which the  c o n s t i t u t i o n of the c u l t u r e medium e x e r t s upon the enzymic s t r u c t u r e of the b a c t e r i a l  cell.  -12REFERENCES 1. Dubps, R . J . - B a c t e r . Rev. 4: 1, 19 40 2. E a g l e s , B. A. and S a d l e r , W. - Can. J o u r . R e s . 7:364 1933 3. F a r r e l l , M.A. - J o u r . B a c t . 29: 411, 1935. 4. Haines, R.B. - Biochem, J o u r . 37: 466, 1933. 5. Hegarty, C P . - Jour B a c t . 37: 145, 1939 . 6. K a t a g i r i , H. and K i t a h a r a , K. - Biochem, Jour* 32: 1654, 1938. 7. Morgan, J.F., E a g l e s , B.A., L a i r d , D.G. - I n publication. 8. Q u a s t e l , J.Ho - Enzymologia  2:37, 193 7.  9. Rahn, 0;, Hegarty, C.P. , D u e l l , R.E. - J o u r . ,Bact. 35: 547, 1938. 10. S a d l e r , W. - Trans. Roy. Soc. Canada, Vol.30, 1926. 11. Stephenson, M. and Stickla-nd, L.H. * Biochem, J o u r . 26 : 712, 1932. 13, Stephenson, M. and Gale, E.F. - Biochem, J o u r . 31: 1311, 1937. 13. Y i r t a n e n , A . I . - J o u r . B a c t . 28: 447, 1934, 14. Wood, A . J . and Gunsalus, I.C. - J o u r . B a c t . 44: 333 1942. 15. W ortmann, J . - Quoted from Dubos, R.J. - B a c t . Rev. 4:1, 1940 *  ABSTRACT  The s t i m u l a t i n g e f f e c t of f i f t e e n n i t r o g e n sources upon the f e r m e n t a t i o n and r e s p i r a t i o n of glucose by washed suspensions  cell  of S t r e p , l a c t i s has been s t u d i e d .  I t has been shown t h a t f e r m e n t a t i o n and r e s p i r a t i o n by c e l l s of S t r e p , l a c t i s are r e g u l a t e d , not by a e r o b i c or anaer o b i c c o n d i t i o n s , but by the presence i n the b u f f e r mixture of s m a l l amounts o f v a r i o u s nitrogenous  compounds.  The r e l a -  t i o n s h i p of t h i s phenomenon to the Pasteur e f f e c t i s d i s c u s s e d I t has been shown t h a t a e r o b i c and anaerobic l a c t i c f o r m a t i o n are s t i m u l a t e d t o a p p r o x i m a t e l y  acid  the same degree by  these d i f f e r e n t n i t r o g e n s o u r c e s , but t h a t the oxygen uptake i s stimulated i n a d i s t i n c t l y different  manner.  The r e l a t i v e s t i m u l a t o r y a c t i o n of d i f f e r e n t types of n i t r o g e n sources has been compared, and the q u e s t i o n of a c c e s s o r y f a c t o r s f o r f e r m e n t a t i o n and r e s p i r a t i o n d i s c u s s e d . Independent s t i m u l a t i o n o f the r e s p i r a t o r y and ferment a t i v e mechanisms has been demonstrated, and the p o s s i b i l i t y of separate enzymic systems p o s t u l a t e d .  INTRODUCTION  The  fermentation reactions  o f many s p e c i e s o f l a c t i c  s t r e p t o c o c c i have been i n v e s t i g a t e d . Orlai-Jensen and Hansen ( l ? ) s t u d i e d  acid  Orla-Jensen (18) and l a c t i c acid production  from carbohydrates by s p e c i e s and s t r a i n s of s t r e p t o c o c c i , including Strep, l a c t i s . such r e a c t i o n s this basis.  They emphasized the v a r i a b i l i t y o f  and suggested t h e f o r m a t i o n of sub-groups on  Such sub-group f o r m a t i o n , however, was opposed by  A y e r s , Johnson and Mudge ( 1 ) , by Hammer and Baker ( ? ) , and by Sherman (24), who c l a s s i f i e d  the true l a c t i c acid  strep-  t o c o c c i as S t r e p , l a c t i s and S t r e p , c r e m o r i s . The  r e s p i r a t o r y enzyme a c t i v i t y  t o c o c c i upon a v a r i e t y of s u b s t r a t e s Farrell  cremoris.  investigated.  i n c l u d i n g S t r e p . l a c t i s and S t r e p .  Morgan, E a g l e s and L a i r d (13) surveyed the dehyd-  rogenase a c t i v i t y also  has a l s o been  o f f o u r t e e n s t r a i n s of l a c t i c a c i d  (14) s t u d i e d  bacteria  t h e o x i d a t i v e a b i l i t y o f two s t r a i n s of  S t r e p , l a c t i s upon carbohydrate  substrates.  A l t h o u g h the l a c t i c a c i d p r o d u c t i o n by growing and  strep-  (8) r e p o r t e d upon t h e r e s p i r a t o r y mechanism of 22  s t r a i n s of s t r e p t o c o c c i  and  o f the l a c t i c a c i d  the r e s p i r a t o r y a c t i v i t y  cultures  of r e s t i n g c e l l suspensions o f  s t r e p t o c o c c i have both been s t u d i e d  i n t h e presence o f v a r i o u s  c a r b o h y d r a t e s , the r e l a t i o n s h i p o f r e s p i r a t i o n to f e r m e n t a t i o n i s s t i l l l a r g e l y obscure w i t h t h i s group o f microorganisms.  Rahn, Hegarty and D u e l l (20) and Hegarty (10) determined l a c t i a c i d p r o d u c t i o n by washed c e l l s of S t r e p , l a c t i s i n the presence of g l u c o s e .  They found l a c t i c a c i d p r o d u c t i o n to be  dependent upon the presence of s m a l l amounts of peptone i n the buffer mixture.  L a t e r , Rahn and Hegarty (21) showed that t h i s  was due t o the presence o f accessory f a c t o r s , o f which the most act i v e were a s c o r b i c a c i d and n i c o t i n i c a c i d .  Morgan,  E a g l e s and L a i r d (15) found no c o r r e l a t i o n between t h e amount of l a c t i c a c i d produced i n c u l t u r e and the a e r o b i c and anaerobic respiratory a c t i v i t y upon v a r i o u s carbohydrates.  of suspensions  of S t r e p ,  lactis  Smith and Sherman (25) s t u d i e d  the f e r m e n t a t i o n a b i l i t y of 151 c u l t u r e s of s t r e p t o c o c c i and determined the percentage of l a c t i c a c i d produced from They o b t a i n e d almost complete u t i l i z a t i o n of the even i n the absence of a n i t r o g e n  glucose  carbohydrate  source.  The work r e p o r t e d upon h e r e i n was undertaken w i t h the o b j e c t of d e t e r m i n i n g sources  the i n f l u e n c e which v a r i o u s n i t r o g e n  e x e r t upon t h e r e s p i r a t i o n and f e r m e n t a t i o n of r e s t i n g  c e l l suspensions  of Strep, l a c t i s .  I t was hoped t h a t such an  i n v e s t i g a t i o n would c l a r i f y the l a c k o f r e l a t i o n s h i p between r e s p i r a t i o n and f e r m e n t a t i o n w i t h t h i s group o f organisms. EXPERIMENTAL METHODS. The c u l t u r e s employed i n t h i s study were S t r e p ,  lactis  S.A. JO ( 2 2 ) , i s o l a t e d from b u t t e r p o s s e s s i n g a caramel f l a v o r and S t r e p , l a c t i s ATC 374, obtained from t h e N a t i o n a l Type C u l t u r e C o l l e c t i o n at Washington, D. C.  Casein D i g e s t B r o t h , prepared a f t e r the manner of O r l a (7),  Jensen  and e n r i c h e d w i t h 0.5%  K2HPO4,  0.5%  glucose and  1% D i f c o yeast e x t r a c t , served as the b a s i c growth medium. R e s t i n g c e l l suspensions of S t r e p , l a c t i s were prepared from . 18  - 2 0 hour c u l t u r e s i n t h i s medium by c e n t r i f u g i n g ,  t w i c e , and resuspending i n phosphate  washing  buffer to give a c e l l  c o n c e n t r a t i o n of 1% by volume, as measured by the Hopkins Vacc i n e Tube method ( 1 J ) . These suspensions were found t o contain  2,500,000,000  c e l l s per cu. ml. as measured by p l a t e  counts. L a c t i c a c i d p r o d u c t i o n under a e r o b i c c o n d i t i o n s  was  measured by the procedure d e s c r i b e d by Rahn, Hegarty and D u e l l (20).  For a n a e r o b i c s t u d i e s the procedure as o u t l i n e d by  these workers was m o d i f i e d .  The c e l l suspension,  suspended  i n a b u f f e r m i x t u r e c o n t a i n i n g 2% glucose and 0 . 5 o f the n i t r o g e n source being t e s t e d , was p l a c e d i n a 5 0 0 c c . s u c t i o n f l a s k and the whole evacuated.  A f t e r e v a c u a t i o n the contents  of the f l a s k were a d j u s t e d back to atmospheric pressure w i t h n i t r o g e n and the f l a s k clamped o f f . Methylene blue was  added  as n e c e s s a r y through a dropping f u n n e l and samples f o r t i t r a t i o n were removed by s u c t i o n through a c a p i l l a r y tube. A l l samples from both a e r o b i c and a n a e r o b i c experiments were t i t r a t e d t o pH 7 . 0 w i t h K / 1 0 sodium h y d r o x i d e , employing a Beckma nn pH.  meter. PH measurements were a l s o recorded on a l l samples fo  t i t r a t i o n and i t was found t h a t the pH decreased i n a u n i f o r m  manner as l a c t i c a c i d formation  proceeded.  In general,  there-  f o r e , these t e s t s have-been c a r r i e d out i n a h i g h l y - b u f f e r e d phosphate s o l u t i o n under a c i d c o n d i t i o n s .  That such c o n d i t i o n s  should be optimum was i n d i c a t e d by Niven and Smiley { 1 6 } , who showed t h a t under a c i d c o n d i t i o n s l a c t i c a c i d was the predomin a t i n g product of t h e f e r m e n t a t i o n  o f glucose by growing  c u l t u r e s of S t r e p , l i q u e f a e i e n s , w h i l e under a l k a l i n e c o n d i t i o n s formic a c i d and a c e t i c a c i d and e t h y l a l c o h o l would be produced i n large q u a n t i t i e s . R e s p i r a t i o n s t u d i e s were c a r r i e d out m a n o m e t r i c a l l y w i t h the B a r e r o f t r e s p i r o m e t e r , The  as d e s c r i b e d  by Dixon ( 6 ) .  n i t r o g e n sources employed i n t h i s study c o n s i s t e d o f  ammonium c h l o r i d e , ammonium s u l f a t e , sodium n i t r a t e ,  urea,  u r i c a c i d , 1 g l y c i n e , 1 c y s t i n e , 1 a s p a r a g i n e , t r y p t o n e , peptone D i f c o , peptone W l t t e , proteose peptone, sodium c a s e i n a t e , beef e x t r a c t and yeast e x t r a c t D i f c o . P r e l i m i n a r y s t u d i e s c a r r i e d out w i t h suspensions of Strep«. l a c t i s S.A. 3 0 i n d i c a t e d t h a t l a c t i c a c i d p r o d u c t i o n d i r e c t l y with increasing concentration  of peptone.  increased This r e s u l t  i s i n agreement w i t h t h e work of H i r s c h ( I I ) who showed t h a t acid production  by washed c e l l s o f E. c o l i i n t h e presence  of glucose i n c r e a s e d tration.  d i r e c t l y w i t h i n c r e a s i n g peptone concen-  On t h e b a s i s o f t h i s p r e l i m i n a r y study i t was  decided to employ  0.5°/.  under c o n s i d e r a t i o n .  concentrations Since  of a l l n i t r o g e n sources  i t was a l s o observed t h a t c e l l s o f  S t r e p , l a c t i s A.T.C. 3 7 4 gave a s l i g h t l y h i g h e r l a c t i c a c i d  -  5  ~  p r o d u c t i o n than those o f S t r e p , l a c t i s S.A. 3 0 , r e s t i n g suspensions quent  cell  of the former s t r a i n were employed i n the subse-  experiments. EXPERIMENTAL RESULTS.. The a e r o b i c and anaerobic l a c t i c a c i d p r o d u c t i o n and  the oxygen uptake a f t e r f i v e hours i n the presence of v a r i o u s n i t r o g e n sources are presented  i n Table 1.  A l l v a l u e s ex-  pressed i n t h i s t a b l e are c a l c u l a t e d on the common b a s i s o f 0.5$  nitrogenous  compound.  The r e s u l t s recorded  i n Table 1 show c o m p a r a t i v e l y the  degree o f s t i m u l a t i o n i n response t o the presence of these v a r i o u s n i t r o g e n sources i n the r e a c t i o n m i x t u r e .  It is  n o t i c e a b l e t h a t under a e r o b i c c o n d i t i o n s i n the absence of n i t r o g e n source no l a c t i c a c i d i s formed from g l u c o s e under anaerobic  f  whereas  c o n d i t i o n s an a p p r e c i a b l e amount i s e l a b o r a t e d .  T h i s format i o n of l a c t i c a c i d from glucose i n the absence of n i t r o g e n source  i s i n agreement w i t h the r e p o r t e d r e s u l t s of  Smith and Sherman ( 2 5 ) . i n the present  I n c o n t r a s t to t h e data  presented  paper, however, these workers o b t a i n e d a  s i m i l a r a c i d product i o n under a e r o b i c c o n d i t i o n s as w e l l . L a c t i c a c i d f o r m a t i o n under a e r o b i c and anaerobic d i t ions i n response t o the s t i m u l a t i o n of f i v e n i t r o g e n i s shown g r a p h i c a l l y i n F i g u r e s l a and l b .  consources  In F i g u r e l a i s  p o r t r a y e d the a c i d p r o d u c t i o n under a e r o b i c c o n d i t i o n s , w h i l e t h a t produced under anaerobic lb.  c o n d i t i o n s i s shown i n F i g u r e  From the r e s u l t s shown i n these graphs i t i s apparent t h a t l a c t i c a c i d p r o d u c t i o n i s very g e n e r a l l y s i m i l a r under a e r o b i c and anaerobic  conditions.  I t i s n o t i c e a b l e , however,  t h a t a c i d p r o d u c t i o n under a e r o b i c c o n d i t i o n s i s i n every case somewhat g r e a t e r than the a c i d produced under anaerobic ditions.  con-  I n g e n e r a l , the d i f f e r e n c e i s not very marked and  the curves f o r a c i d p r o d u c t i o n under these two c o n d i t i o n s are almost i d e n t i c a l . noted  The o n l y e x c e p t i o n t o t h i s r u l e i s t o be  i n t h e case of a s p a r a g i n e , where n e a r l y t w i c e as much  a c i d i s produced under anaerobic  as under a e r o b i c c o n d i t i o n s .  No e x p l a n a t i o n has been found t o account f o r t h i s  irregularity.  I t was a l s o n o t i c e d t h a t l a c t i c a c i d p r o d u c t i o n under anaerobic  c o n d i t i o n s was s t i m u l a t e d e q u a l l y w e l l whether or  not methylene blue was added t o t h e r e a c t i o n mixture t o serve as a Hydrogen A c c e p t o r .  This would i n d i c a t e e i t h e r t h a t  lactic  a c i d p r o d u c t i o n i s independent o f t h e dehydrogenase enzyme system o r t h a t t h e N i t r o g e n Source i t s e l f f u n c t i o n s as a Hydrogen Acceptor which i s a t l e a s t as e f f e c t i v e as the methylene blue. The  s t imulat i o n o f a e r o b i c l a c t i c a c i d product i o n from  glucose under the i n f l u e n c e of v a r i o u s n i t r o g e n sources i s f u r t h e r shown i n F i g u r e 2, a l l values being c a l c u l a t e d on the b a s i s of 0,51» n i t r o g e n o u s  compound.  The r e s u l t s p o r t r a y e d i n t h i s graph i n d i c a t e t h a t there i s an equal s t i m u l a t i o n o f f e r m e n t a t i o n whether urea, u r i c a c i d , ammonium c h l o r i d e , ammonium s u l f a t e , sodium n i t r a t e ,  g l y c i n e , a s p a r a g i n e , or beef e x t r a c t serves as the n i t r o g e n source.  However, when t r y p t o n e , peptone, or proteo.se peptone  serves as the n i t r o g e n o u s compound there i s a markedly g r e a t e r acid production. nitrogenous  T h i s s t i m u l a t i o n of f e r m e n t a t i o n by  simple  compounds i s i n some agreement w i t h the work of  Smythe ( 2 6 ) , who  r e p o r t e d t h a t v a r i o u s t i s s u e e x t r a c t s stimu-  l a t e the anaerobic  f e r m e n t a t i o n of glucose by Baker's y e a s t ,  but t h a t the a c t i v e agent i n these s t i m u l a t i o n s was ammonium chloride. The  s t i m u l a t o r y e f f e c t of n i t r o g e n o u s compounds upon the  r e s p i r a t i o n of washed c e l l suspensions shown i n F i g u r e 3•  of S t r e p , l a c t i s i s  Here a g a i n , i t i s apparent t h a t the more  complex o r g a n i c compounds, such as t r y p t o n e , beef e x t r a c t , and proteose peptone, have caused a f a r g r e a t e r s t i m u l a t i o n of r e s p i r a t i o n than have the s i m p l e r compounds such as u r e a , g l y c i n e , and sodium n i t r a t e . Comparison of the r e s u l t s shown i n F i g u r e 2 and 3 r e v e a l t h a t t h e r e i s a f a i r l y g e n e r a l agreement between the  extent  of a c i d p r o d u c t i o n and the s t i m u l a t i o n of oxygen uptake.  A  very n o t i c e a b l e e x c e p t i o n , however, i s found i n the case of beef e x t r a c t .  T h i s compound g i v e s o n l y a moderate s t i m u l a t i o n  of a c i d p r o d u c t i o n but a v e r y marked s t i m u l a t i o n of oxygen uptake. In F i g u r e 4 the r e l a t i v e s t i m u l a t i o n of a e r o b i c  and  anaerobic l a c t i c a c i d p r o d u c t i o n and r e s p i r a t i o n by v a r i o u s n i t r o g e n sources i s shown.  These r e s u l t s are based on the  common value of 0.5$ The  comparative  n i t r o g e n o u s compound. values shown i n F i g u r e 4 i n d i c a t e t h a t ,  i n most cases, a e r o b i c and anaerobic f e r m e n t a t i o n are stimul a t e d to the same extent by v a r i o u s n i t r o g e n sources.  Res-  p i r a t i o n , however, i s s t i m u l a t e d to an e n t i r e l y d i f f e r e n t degree by these n i t r o g e n o u s compounds.  With the m a j o r i t y of  the substances t e s t e d r e s p i r a t i o n i s s t i m u l a t e d t o a much l e s s e r extent than f e r m e n t a t i o n .  This appears to be t r u e when  the s i m p l e r chemical compounds are employed.  With the amino  a c i d s and v a r i o u s enzymic d i g e s t s other c o m p l i c a t i n g f a c t o r s are encountered.  In the case of c y s t i n e , r e s p i r a t i o n Is stimu-  l a t e d more than a c i d p r o d u c t i o n , w h i l e w i t h asparagine  anaerobic  l a c t i c a c i d f o r m a t i o n i s s t i m u l a t e d more than e i t h e r a e r o b i c acid, p r o d u c t i o n or r e s p i r a t i o n .  S i m i l a r cases of g r e a t e r  s t i m u l a t i o n of r e s p i r a t i o n than f e r m e n t a t i o n are found w i t h beef e x t r a c t , yeast e x t r a c t , and, to a l e s s e r e x t e n t , sodium caseinate.  The r e v e r s e o f t h i s e f f e c t , however, i s demonstrat-  ed w i t h W i t t e * s peptone.  With t h i s compound both a e r o b i c and  anaerobic l a c t i c acid' p r o d u c t i o n are h i g h , but r e s p i r a t i o n i s low.  I t would appear, t h e r e f o r e , on the bases of these  results,  t h a t the mechanism of r e s p i r a t i o n i s e i t h e r d i s t i n c t from the mechanism of f e r m e n t a t i o n o r , at l e a s t , i s capable of dent s t i m u l a t i o n by v a r i o u s a c c e s s o r y The  data presented  indepen-  factors.  i n Table 1 and shown g r a p h i c a l l y i n  F i g u r e s 1, 2, 3 and 4 have been c a l c u l a t e d on the b a s i s of 0.5$  n i t r o g e n source added to the r e a c t i o n m i x t u r e .  Since the  n i t r o g e n content of these compounds i s extremely v a r i a b l e i t  -  9  -  seemed, p o s s i b l e t h a t r e a r r a n g i n g the data on the b a s i s of a common n i t r o g e n content might give f u r t h e r i n f o r m a t i o n . A c c o r d i n g l y , the data as presented i n Table 1 have been r e c a l c u l a t e d t o the b a s i c value of 0.25 grams of n i t r o g e n .  These  r e c a l c u l a t e d v a l u e s are presented i n Table 2 . The r e s u l t s r e p o r t e d i n Table 2 are e n t i r e l y t h e o r e t i c a l and i n most cases are much h i g h e r than-could be obtained e x p e r i mentally.  Comparison of these r e s u l t s w i t h those presented i n  Table 1 r e v e a l s t h a t t h e r e has been a very marked change i n the r e l a t i o n s h i p o f the s t i m u l a t o r y power of these v a r i o u s compounds. I t i s n o t i c e a b l e from the n i t r o g e n contents recorded i n Table 2 and the r e l a t i v e s t i m u l a t i n g a c t i v i t y recorded i n Table 1 t h a t t h e l e a s t s t i m u l a t i o n has been obtained from the compound w i t h the h i g h e s t n i t r o g e n c o n t e n t , namely urea, w h i l e the g r e a t e s t s t i m u l a t o r y a c t i o n has been achieved by t h e compounds w i t h the lowest n i t r o g e n c o n t e n t s , namely, beef e x t r a c t and yeast e x t r a c t .  R e c a l c u l a t i n g these values on the b a s i s of a  common n i t r o g e n content w i l l t h e r e f o r e tend to emphasize the d i f f e r e n c e s between t h e s t i m u l a t o r y a c t i o n o f these v a r i o u s types o f n i t r o g e n o u s  compounds.  S t i m u l a t i o n of a e r o b i c l a c t i c a c i d p r o d u c t i o n by these v a r i o u s n i t r o g e n sources i s shown g r a p h i c a l l y i n F i g u r e 5 , on t h e b a s i s o f 0 . 2 5 gm. N i t r o g e n .  I t i s apparent t h a t t h r e e  l e v e l s of s t i m u l a t i o n e x i s t among these v a r i o u s compounds.  In,  the f i r s t l e v e l a c i d p r o d u c t i o n i s r e l a t i v e l y low, and the compounds f a l l i n g i n t o t h i s group are the simple ammonium s a l t s ,  - 10 the amino a c i d s and sodium c a s e i n a t e .  In the second  level  a c i d p r o d u c t i o n i s much h i g h e r , and the nitrogenous compounds i n v o l v e d c o n s i s t o f t h e products prepared by enzymic h y d r o l y s i s , the peptone group.  I n the t h i r d l e v e l a c i d p r o d u c t i o n i s very  great and the two s t i m u l a t i n g compounds are beef e x t r a c t and yeast e x t r a c t ,  There would appear, t h e r e f o r e , to be a f a i r l y  r e g u l a r grouping based on t h e chemical nature of these compounds and t h e i r probable a c t i v a t o r c o n t e n t , which i s i n f a i r l y c l o s e agreement w i t h t h e r e s u l t s shown i n F i g u r e 2 . S t i m u l a t i o n of r e s p i r a t i o n by these same nitrogenous compounds i s shown g r a p h i c a l l y , i n F i g u r e 6.  There i s apparent  a r e g u l a r i n c r e a s e from u r e a , w i t h the lowest v a l u e , t o beef e x t r a c t , with the highest value.  I n c o n t r a s t t o the r e s u l t s  shown i n F i g u r e j5 , t h e r e i s no grouping o f these compounds on the b a s i s of t h e i r c h e m i c a l n a t u r e .  I t i s a l s o apparent t h a t  t h e r e i s a g e n e r a l s i m i l a r i t y i n t he appearance of the two sets of curves shown i n F i g u r e s 5 and 6,  However, the s t i m u l a t i o n  of r e s p i r a t i o n i s much more u n i f o r m than t h e s t i m u l a t i o n o f a c i d product i o n . The s t i m u l a t o r y e f f e c t s when c y s t i n e i s employed as t h e n i t r o g e n source are r a t h e r i r r e g u l a r .  There i s a very marked  immediate s t i m u l a t i o n of oxygen uptake such t h a t a very high l e v e l i s reached w i t h i n two hours.  A f t e r two h o u r s , however,  the oxygen uptake appears t o reach a maximum value and the curve l e v e l s o f f . With l a c t i c a c i d p r o d u c t i o n , on t h e o t h e r hand, the s t i m u l a t i o n i s r e g u l a r and u n i f o r m and the curve r i s e s  ~ 11 s t e a d i l y t o a t t a i n a maximum value at f i v e hours. I t would appear, t h e r e f o r e , t h a t s t i m u l a t i o n of r e s p i r a b  tion y  c y s t i n e proceeds through a d i s t i n c t l y d i f f e r e n t mechan-  ism than s t i m u l a t i o n of f e r m e n t a t i o n .  These r e s u l t s are of  i n t e r e s t i n view of the r e p o r t e d work of Chaix and Fromageot (3), who  s t u d i e d the a c t i v a t i n g a b i l i t y of v a r i o u s compounds  upon the g l y c o l y t i c a c t i v i t y of Prop, pentosaceum, and found t h a t the g r e a t e s t s t i m u l a t o r y e f f e c t was  obtained with v a r i o u s  s u l f u r - c o n t a i n i n g compounds such as c y s t i n e , g l u t a t h i o n e and thiourea.  They concluded  e s s e n t i a l accessory  t h a t hydrogen s u l f i d e was  the  factor.  When W i t t e ' s peptone i s employed as the n i t r o g e n the s t i m u l a t o r y e f f e c t s are a g a i n i r r e g u l a r .  source  No l a c t i c a c i d  i s produced d u r i n g the f i r s t hour but d u r i n g t h i s p e r i o d there occurs an a p p r e c i a b l e s t i m u l a t i o n of oxygen uptake.  After this  one-hour p e r i o d , however, a c i d product i o n r i s e s w i t h extreme r a p i d i t y w h i l e the oxygen uptake remains at a f a i r l y low  level.  I t would appear, t h e r e f o r e , t h a t the two mechanisms of l a c t i c a c i d p r o d u c t i o n and of r e s p i r a t i o n are s t i m u l a t e d i n an l y d i f f e r e n t manner by the one n i t r o g e n source.  entire-  A somewhat  s i m i l a r e f f e c t i s encountered w i t h g l y c i n e and asparagine. two  These  compounds e x e r t an i d e n t i c a l s t i m u l a t i o n upon r e s p i r a t i o n  but a d i s t i n c t l y d i f f e r e n t s t i m u l a t o r y a c t i o n upon f e r m e n t a t i o n . The  slow i n i t i a l s t i m u l a t i o n of l a c t i c a c i d f o r m a t i o n  by W i t t e ' s peptone as c o n t r a s t e d t o t h a t of D i f c o peptone and proteose  peptone i s i n accord w i t h the work of S a d l e r , E a g l e s ,  and Pendray {23) who  found W i t t e - a peptone to be a t o t a l l y i n -  adequate n i t r o g e n source f o r the growth of the l a c t i c a c i d streptococci.  - 12 The t o t a l  s t i m u l a t i o n of a e r o b i c and anaerobic  fermenta-  t i o n and of r e s p i r a t i o n by these v a r i o u s n i t r o g e n sources i s shown c o m p a r a t i v e l y  i n F i g u r e 7.  a e r o b i c and anaerobic  These r e s u l t s emphasize t h a t  l a c t i c a c i d p r o d u c t i o n are g e n e r a l l y  s t i m u l a t e d to much the same degree by these d i f f e r e n t n i t r o g nous compounds, but t h a t r e s p i r a t i o n appears to be s t i m u l a t e d to an e n t i r e l y d i f f e r e n t degree.  In general the r e s u l t s  p o r t r a y e d i n F i g u r e 7 agree w i t h those shown i n F i g u r e 4. DISCUSSION The r e s u l t s presented  h e r e i n show t h a t  and r e s p i r a t i o n by washed c e l l suspensions are dependent not upon a e r o b i c or' anaerobic upon the presence of a n i t r o g e n source. lactis  fermentation  of S t r e p ,  lactis  c o n d i t i o n s , but  Washed c e l l s of S t r e p ,  suspended i n phosphate b u f f e r c o n t a i n i n g two  percent  g l u c o s e were found to form no l a c t i c a c i d under a e r o b i c cond i t i o n s , and o n l y a very s m a l l amount under anaerobic t i o n s , although The  condi-  a s l i g h t oxygen uptake could be demonstrated.  a d d i t i o n of a s m a l l amount (0»5$) of a group of n i t r o g e -  nous compounds r e s u l t e d , i n the e l a b o r a t i o n of very l a r g e amounts of l a c t i c a c i d under both a e r o b i c and anaerobic d i t i o n s , w h i l e at the same time ozygen uptake was  con-  very g r e a t l y  stimulated. I t was  a l s o observed t h a t , w h i l e a e r o b i c and  l a c t i c a c i d f o r m a t i o n were g e n e r a l l y s t i m u l a t e d to  anaerobic approximately  the same extent by i n d i v i d u a l n i t r o g e n sources, the s t i m u l a t i o n of r e s p i r a t i o n appeared to be d i s t i n c t l y n o t i c e d t h a t some n i t r o g e n o u s  different.  compounds possessed the  I t was ability  -'13  -  to s t i m u l a t e a c i d p r o d u c t i o n f a r more than r e s p i r a t i o n , w h i l e w i t h other compounds the r e v e r s e h e l d t r u e .  These r e s u l t s i n d i c a t e  t h a t r e s p i r a t i o n and f e r m e n t a t i o n are c a r r i e d out by d i f f e r e n t enzymic mechanisms, o r , at l e a s t , by mechanisms which are independently r e s p o n s i v e t o v a r i o u s s t i m u l a t i n g agents. These r e s u l t s are i n agreement w i t h the g e n e r a l l y accepted t h e o r i e s of the r e l a t i o n s h i p of r e s p i r a t i o n t o f e r m e n t a t i o n as d e t a i l e d by Oppenheimer and S t e r n ( 1 7 ) .  "There i s one  mechanism o p e r a t i v e i n both phases ( r e s p i r a t i o n and  uniform fermentation)  o f d e s m o l y s i s , namely, the t r a n s f e r o f metabolic hydrogen. In a n a e r o b i o s i s i t t e r m i n a t e s i n l a c t i c a c i d and i n a e r o b i o s i s i t t e r m i n a t e s i n water."  T h i s viewpoint i s r a t h e r d i f f i c u l t  to r e c o n c i l e w i t h the r e s u l t s r e p o r t e d i n the present work. I f the mechanism of both*processes  i s e s s e n t i a l l y the same  one would not expect to s t i m u l a t e both r e s p i r a t i o n and a e r o b i c f e r m e n t a t i o n at the same time nor would one expect to f i n d a e r o b i c l a c t i c a c i d f o r m a t i o n and anaerobic l a c t i c a c i d format i o n s t i m u l a t e d equally by v a r i o u s n i t r o g e n s o u r c e s . The r e s u l t s o b t a i n e d i n the present study a l s o f a i l to agree w i t h the c l a s s i c a l P a s t e u r and neo-Pasteur d i s c u s s e d by Burk and Lipman (2) and organisms possess  (12).  e f f e c t s as  "Most f a c u l t a t i v e  i n the P a s t e u r e f f e c t a r e g u l a t o r y device  t h a t enables them to use, as o c c a s i o n demands, e i t h e r t h e i r a e r o b i c or t h e i r anaerobic systems. e f f e c t t h e i r f e r m e n t a t i v e apparatus  By the o p e r a t i o n of t h i s i s blocked i n the  of s u f f i c i e n t oxygen, and energy i s f u r n i s h e d almost by the f a r more e f f i c i e n t and powerful r e s p i r a t o r y  presence exclusively  apparatus.  - 14 When oxygen i s l a c k i n g , however, the f e r m e n t a t i o n brought i n t o o p e r a t i o n . "  system i s  With the washed c e l l suspensions of  S t r e p , l a c t i s i t would appear that both r e s p i r a t i o n and  fermen-  t a t i o n are proceeding  and  t o g e t h e r under a e r o b i c c o n d i t i o n s  that- both processes can be independently nitrogenous  s t i m u l a t e d by  various  compounds.  I t i s p o s s i b l e t h a t much of t h i s l a c k of agreement a r i s e s from the use of r e s t i n g c e l l suspensions of a b a c t e r i a l which i s predominantly anaerobic  species  i n nature and which i s b e l i e v -  ed to possess a v e r y p r i m i t i v e type of m e t a b o l i c mechanism which i s probably  l a c k i n g i n the m a j o r i t y of the r e s p i r a t o r y en?yme  and hydrogen t r a n s p o r t systems possessed by the more a e r o b i c b a c t e r i a and The  yeast.  mechanism by which the nitrogenous  compounds s t u d i e d  e x e r t t h e i r s t i m u l a t o r y a c t i o n i s s t i l l obscure. tremely d i f f i c u l t  I t i s ex-  t o p o s t u l a t e a mechanism by which the r e s t i n g  c e l l s of S t r e p - l a c t i s are able to u t i l i z e  u r i c a c i d , which  c o n t a i n s i t s n i t r o g e n i n a r e l a t i v e l y s t a b l e and form, as an a c t i v a t o r f o r c e l l r e s p i r a t i o n and It i s also d i f f i c u l t  unavailable  cell  fermentation.  to v i s u a l i z e the manner i n which u r e a ,  ammonium c h l o r i d e or sodium n i t r a t e exert t h e i r s t i m u l a t i n g effects.  That some f a c t o r other than simply n i t r o g e n  content  of the a c t i v a t i n g substance must be i n v o l v e d i s apparent, s i n c e the compound w i t h the h i g h e s t n i t r o g e n c o n t e n t , namely u r e a , gave the l e a s t s t i m u l a t i o n , and the compounds w i t h the  lowest  n i t r o g e n v a l u e s , namely, beef and yeast e x t r a c t s , gave the greatest s t i m u l a t i o n .  ' -  15  -  From the r e s u l t s presented i n Table 1 i t i s n o t i c e a b l e that the f i g u r e s f o r anaerobic l a c t i c a c i d p r o d u c t i o n remain q u i t e c o n s t a n t , w i t h a value i n the neighborhood of 0 . 6 p e r c e n t , u n t i l t r y p t o n e and the v a r i o u s enzymic d i g e s t s are reached. These compounds then show a markedly g r e a t e r s t i m u l a t i o n which reaches a f a i r l y u n i f o r m l e v e l at about 1 . 0 percent.  T h i s may  i n d i c a t e t h a t substances of the peptone group possess a d d i t i o n a l a c t i v a t i n g compounds which have been e l a b o r a t e d d u r i n g the enzymic  processes e n t a i l e d i n t h e i r p r e p a r a t i o n . Such a d d i t i o n -  a l a c t i v a t o r s would not be encountered w i t h the pure s a l t s and amino a c i d s employed, and t h e i r s t i m u l a t o r y power would s e q u e n t l y be lower.  con-  Such a h y p o t h e s i s i s supported by the  r e s u l t s shown g r a p h i c a l l y i n F i g u r e s 2 and 5 , which  indicate  t h a t the s t i m u l a t o r y a c t i o n of these compounds may be arranged i n groups which c l o s e l y p a r a l l e l the s t a t e of chemical p u r i t y of the compounds s t u d i e d . The r e s u l t s r e p o r t e d h e r e i n are at marked v a r i a n c e w i t h the p u b l i s h e d r e s u l t s of Smith and Sherman ( 2 5 ) .  These workers  measured the percentage glucose converted t o l a c t i c a c i d by washed suspensions of the S t r e p t o c o c c i i n a b u f f e r s o l u t i o n without added n i t r o g e n source and o b t a i n e d almost utilization  o f the g l u c o s e i n twelve hours.  complete  I t i s pos s i b l e  t h a t the a b i l i t y of washed c e l l s t o u t i l i z e glucose i n the presence or absence of n i t r o g e n source may be governed by the f u n c t i o n i n g of t h e i r a s s i m i l a t o r y r a t h e r than t h e i r p r o c e s s e s , as demonstrated c o l i and Ps c a l c o - a c e t i c i .  respiratory  by C l i f t o n ( 4 , 5 ) i n the case of E.  -  16  -  The r e s u l t s o b t a i n e d i n t h i s study o f the i n f l u e n c e of v a r i o u s n i t r o g e n sources upon f e r m e n t a t i o n and r e s p i r a t i o n by washed c e l l s f u r n i s h an e x p l a n a t i o n f o r the r e p o r t e d observ a t i o n s of Morgan, E a g l e s , and L a i r d  (15 ) .  These workers  found t h a t no c o r r e l a t i o n e x i s t e d between the dehydrogenation and o x i d a t i o n of carbohydrates by washed suspensions of S t r e p , l a c t i s and the f e r m e n t a t i o n of these same carbohydrates by the organism i n c u l t u r e media.  I t i s apparent t h a t the fermen-  t a t i o n o f carbohydrates by growing c u l t u r e s i s governed by the i n f l u e n c e which a c t i v a t i n g substances i n the medium e x e r t upon the r e s p i r a t o r y and f e r m e n t a t i v e mechanisms of t h e c e l l ,  -  17  -  REFERENCES* 1.  A y e r s , S. H.,  and Mudge, C. S. - J . I n f .  3 4 : 2 9 . 1 9 24.  Dis.  2.  Johnson, W. T.,  Burk, Dean - Cold S p r i n g Harbour Symposia on Q u a n t i t a t i v e Biology, Vol. VII, 1 9 3 9 .  3.  Chaix, P. and Fromageot, C. - Enzymologia  4.  C l i f t o n , C. E. - Enzymologia  5.  C l i f t o n , C. E. and Logan, W. A. - Jour. B a c t .  6.,  Dixon, M. - "Manometric Methods", Cambridge Univ. P r e s s , 1 9 3 4 .  7.  Eages, B. A. and S a d l e r , W.  8.  E a r r e 1 1 , M.A.  - J o u r . Bact. 2 9 : 411,  9.  Hammer, B. W.  and Baker, M.P.  4: 24 6 ,  1:321,  1936.  1937.  - Can. J o u r . Res.  37:523,  7:364,  1939.  1932.  1935.  - Iowa Agr. Exp. S t a . , Tech.  Bull. 232, 1926. - J o u r . Bact. 3 7 : 145 , 1 9 39 .  10.  Hegarty,  11.  H i r s c h , J . - Enzymologia  12.  Lipman, F r i t z - "A Symposium on R e s p i r a t o r y Enzymes", U n i v e r s i t y of Wisconsin P r e s s , 1942. Morgan, J . F. , E a g l e s , B.A., and L a i r d , D. G. - In Publication. Morgan, J.F. , E a g l e s , B. A., and L a i r d , D. G. - In P u b l i c a t i on. Morgan, J . F., E a g l e s , B.A. , and L a i r d , D. G. - In  13. 14. 15.  CP.  4:94,  1939.  Publication. 16.  N i v e n , C.F.  17.  Oppenheimer,•C, and S t e r n , E.G.,  and S m i l e y , K.L.  - J o u r . Bact. 4 4 : 2 6 0 ,  1942.  "Biological Oxidation",  Nordemann, 1 9 3 9 . 18.  O r l a - J e n s e n , S. - " T h e . L a c t i c A c i d B a c t e r i a " , Copenhagen,1919.  19.  O r l a - J e n s e n , A.D.  20.  86:  6,  Rahn, 0 . , Hegarty, 547  ,  1938  and Hansen, P.A.  - Zent. Bact. I I Abt.  1932. .  C P . , and D u e l l , R.E.  - J o u r . Bact. 3 5 :  - 18  -  References. (Cont'd. 21.  Rahn, 0 . , and H e g a r t y , 38:  22. 23.  218,  S a d l e r , W.  CP.  - T r a n s . Roy. S o c . Can. V o l . 2 0 ,  7:  Med.  1938.  . S a d l e r , W. , E a g l e s , B.A., Res.  - P r o c . Soc. S x p l . B i o l .  1926.  and P e n d r a y , G. - Can. J o u r .  37 0, 19 3 2 .  24.  Sherman, J.M. - B a c t . R e v i e w s 1 : 3 ,  23.  Smith,  26.  Smythe, C.V.  1937.  P.A. , and Sherman, J.M. - J o u r . B a c t . - Enzymologia  6:9, 1939®  43:725,194-2.  Tabl© i CoBparative O x i d a t i o n , Dehydtogenation aad Fermentation o f Carbohydrates with S c . l a e t i s A.T.G, 374  COMPOUND  AEROBIC OXIDATIOH  Glucose  Strong  Fructose  Strong  Galactose  Weak  Arabinose  Very Weak  Lactose  F a i r l y Strotig  DEHYDROGENATIOK;  R.C.  GmoL,A« per l i t e r  Strong  Strong  75  6.5  100  Strong  Fair  Melezitose  Very Weak  Dextrin  Very Strong  Adonitol  Very Weak  7.4  Strong  8 Very Weak  4.5 Fairly  Negative  38 F a i r l y Strong  Negative  0 Negative  Strong  1.8  1  0 Haffinose  FERMENTATION  Very  Slight  5.4 Strong  1.1 Trace  , 4.1 Fairly  Strong  3 Trace  TracG  0  0.7  Negative  0.7  Trae©  2-  • T a b l e ^2'  ..  Dehydrogenase A c t i v i t y o f Rli, t r i f o l i l . Red.Time Glneose = B.C. Values ape Expressed as R e s p i r a t o r y C o e f f i c i e n t s ^^^^ lijae X Rh. t r i f o l i l SUBSTRATE  R.T, 231  100  Glucose  R.T, 224  R.T,39-1  R,T, 22B  100  100  100  45  100  75  Mannose  80  Galactose  20  0  5  6  5  Fructose  50  50  75  67  60  0  0  67  0  0  Xylose  16  0  133  0  0  Sucrose  55  100  57  100  20  Gelloblose  80  28  30  -  75  0  0  0  35  5  0  30  11  75  40  7  5  Arabinose  Lactose  •  R.T, 2 2 6  75  Maltose  56  Trehalose  10  20  Melibiose  34  0  57  4  5  Raffinose  25  7  18  67  35  0  67  30  50  Melezitos©  0 .  Mannitol  30  30  60  40  15  Sorbitol  50  0  0  7  16  1  3 Table 2  SUBSTRATE  R.T. 2 5 1  (cent.)  R.T. 2 2 6  R.T. 224  R.T.39-i  R.T. 22B  Salicin  9  20  80  67  50  Dextrin  18  14  56  50  50  18  55  80  67  42  Inulin  25  14  56  67  42  EsculiB  15  0  60  8  5  Rhamhose  0  0  0  0  0  Methyl g l u c o s i d e  0  0  0  0  0  Glycerol  0  0  75  8  6  Sod. format®  0  0  0  5  0  Sod. l a c t a t e  28  0  0  0  0  10  6  10  Starch  •  Sod. s u c c i n a t e  7  Sod. a c e t a t e  0  0  0  0  0  Sod. malate  7  0  0  0  27  A l l y l alcohol  0  0  0  0  7  -4-.  Table  1  R e l a t i v e Dehydrogenase A c t i v i t y o f R. t r i f o l i i S t r a i n s  RHIZOBIUM TRIFOLII STRAINS SUBSTRATES  RT 224  RT 2 2 6  RT 2 3 1  RT 3 9 - 1  Glucose  100  100  100  100  100  Mannose  75  45  75  80  100  Galactose  3  5  0  20  6  Fructose  60  75  50  30  67  Arabinose  0  67  0  0  0  Xylose  0  133  0  16  0  Rhamnose  0  0  0  0  0  Sucrose  20  37  100  55  100  Cellobiose  75  30  28  80  45  0  0  0  33  30  0  56  11  Lactose Maltose  5 75  Trehalose  5  40  20  10  7  Melibiose  5  37  0  34  4  Raffinose  35  18  7  25  67  Melezitose  50  67  0  0  50  Dextrin  50  56  14  18  50  Starch  42  84  33  18  67  Inulin  42  36  14  25  67  Esculin  5  60  0  13  Salicin  30  80  20  9  67  0  0  0  0  0  Methyl g l u c o s i d e  •  RT 2 2 B  8  5^  Table 1 (cont.)  RHIZOBITJM TRIFOLII STRAINS SUBSTRATES RT 2 2B  RT 224  RT 2 2 6  RT 2 3 1  RT 3 9 - 1  0  0  0  0  0  Grlycerol  6  75  0  0  8  Erythritol  0  0  0  0  0  Adonitol  0  28  0  0  0  Mannitol  15  60  30  30  20  Sorbitol  16  0  0  50  7  Dulcitol  0  3  0  0  0  Inositol  0  28  0  0  0  Sodium  formate  0  0  0  0  5  Sodium a c e t a t e  0  0  0  0  0  Sodium propionate  0  0  0  0  0  Sodium b u t y r a t e  0  0  0  0  5  Sodium l a c t a t e  0  0  0  28  0  10  10  4  7  6  0  0  0  0  0  Sodium malate  27  0  0  7  .0  Ethyl alcohol  0  0  0  0  0  n Propyl alcohol.  0  0  0  0  0  A l l y l alcohol  7  0  0  0  0  Ethylamihe - J;  0  •;0  0  0  7  n  0  6  0  0  0  Ethylene  glycol  Sodium s u c c i n a t e Sodium fumarate  •  Butylamine  *A11 values expressed  as percentage  .  .  of r e d u c t i o n time with glucose  Table 2 V a r i a t i o n i n Dehydrogenase A c t i v i t y with Time  RHIZOBIUM TRIFOLII 224 SUBTRATES  First  Tests  2 Months  8 Months  12 Months  100  100  Glucose  100  100  Mannose  45  100  46  63  5  33  25  56  Galactose Fructose  75  42  50  56  Arabinose  67  0  0  0  135  30  14  32  0  0  0  0  Sucrose  37  60  50  56  Cellobiose  30  75  46  63  Lactose  0  8  4  7  Maltose  30'  50  42  42  Trehalose  40  8  11  28  Melibiose  37  8  9  40  Raffinose  18  60  25  34  Melezitose  67  21  12  56  Dextrin  56  50  22  50  Starch  80 ,  42  30  30  Inulin  36  18  30  10  Esculin  60  60  45  32  Salicin  80  37  56  56  0  0  0  0  Xylose Rhamnose  •  Methyl g l u c o s i d e  7.  Table 2  (cont.)  RHIZOBIUM TRIFOLII 224 SUBSTRATES  Ethylene  glycol  Glycerol Erythritol  Tests  2 Months  8 Months  12 Months  0  0  0  0  75  0  9  63  0  0  0  0  Adonitol  28  0  0  0  Mannitol  60  18  17  84  16  20  21  Sorbitol  0  Dulcitol  3  0  0  0  Inositol  28  4  0  0  0  0  0  0  10  3  14  84  Sodium fumarate  0  6  0  9  Sodium male|iie  0  0  0  0  Sodium malate  0  38  0  14  Ethyl  0  0  20  10  0  0  0  11  0  0  15  0  0  0  0  20  0  0  0  10  i s o Butylamine  6  0  0  7  Formaldehyde  0  0  20  0  Sodium formate Sodium  succinate  alcohol  n Propyl n Butyl  alcohol alcohol  iso Butyl Allyl  t  First  alcohol  alcohol  Table 1 O2 Uptake  i n Cu.mra. per l i r . per mgm,  dry weight  w STRAIN  Endogenous  RT 224 E  34.7  93.6  ^1  28.3  54.2  46.7  62.6  ^2  24.7  65.5  35.3  56.6  ^5  27.7  57.8  29.7  83.5  ^6  22.4  50.6  13.9  E7  20.7  47.8  20.5  89.4  Es  20.2  57.3  25.0  53.2  27.2  58.9  68.6  19.6  %  42.3  80,3  76.2  33.5  ^2  17.1  65.0  58.9  19.4  28.8  126.0  43.7  42.3  °2  64.3  232.4  217.7  45.1  °3  58.2  128.2  115.5  93.2  °4  23.1  138.6  135.6  32.8  °5  19.6  129.4  97.4  28.8  C6  25.3  79.1  52.3  127.7  °7  29.9  80,6  83.2  35.1  13.0  30.8  31.7  22.1  30.8  179.1  132.8  47.6  18.6  60.3  60.0  15.3  21.0  68.3  54.5  20.2  41.9  167.8  RT 224  °1  ^  ^10  °12  Glucose  Mannitol  -  Sodium s u c c i n a t e  40.5  185.6  109.2  •  60.8  31.9  Table 2 Aerobic and Anaerobic R e s p i r a t o r y A a Anaerobic B = Aerobic Glucose r 1 0 0 Substrain  Endogenous  Mannitol  A RT 224 E ^^1  Coefficients  B  Sodium s u c c i n a t e  A  B  58  37.6  75.  44.2  82  ' 52.2  82.  86.1  A  B  62  118.4  62  115.4  80  86.4  88  144.4  •  70  37.7  80  88 •  47.9  75  H  61 .  44.2  80  27.4  80  120.1  ^7  60  43.3  66  42.8  66  187.0  ^8  90  35.2  100  92.8  116  33  RT 224  100  -  43.6  0  45  58  ^1  0  53  72  95  118  42  ^2  8  26  83  91  62  30  ^1  8  23  86  35  50  28  ^2  75  28  75  94  60  19  6  45  63  90  36  73  2  17  67  98  5  24  4  15  Al  75  14  22  20  32  86  66  30  161  , 0  37  57  44  22  103  °8  0  42  70  103  70  72  C9  2  17  42  74  13  27  °10  5  31  70  99  60  .25  4  31  60  80  60  30  0  25  80  110  100  19  °3 C4  H '1  C12  /  116  /o. Table 1 Dehydrogenase A c t i v i t i e s  SUBSTRATE  Sc. lactis S.A.30  of S t r e p , l a c t i s and S t r e p , c r e m o r i s *  Sc. lactis A.T.C.374  Sc. lactis EMB2 1  Sc. cremoris HP  Sc. cremoris EMBi 1 9 3  Sc. cremoris RW  d-Glucose  100  100  100  100  100  100  d-Mannose  30  75  70  80  50  7  d-Galactose  20  8  100  20  25  0  100  75  50  80  28  30  l-Arabinose  0  2  0  0  0  0  1-Xylose  0  2  0  0  0  0  0  0  0  0  0  0  Sucrose  30  100  0  0  50  0  Cellobiose  50  100  32  48  50  37  Lactose  6  38  100  33  50  11  Maltose  5  30  28  40  33  22  Trehalose  6  37  0  0  0  23  Melibiose  0  0  0  0  0  0  Raffinose  0  0  0  80'  40  11  Melezitose  0  0  0  0  0  0  Dextrin  0  3  6  9  23  Starch  80  50  22  80  50  37  Inulin  20  50  0  56  20  0  Esculin  0  3  0  12  0  0  Salicin  4  11  11  20  0  0  Methyl g l u c o s i d e  0  0  0  0  0  0  d-Fructose  Rhamnose  •  Sc. lactis A.T.G.574  Sc. lactis EMB2 1  0  0  0  0  0  0  Glycerol  0  3  0  0  0  0  Adonitol  0  0  0  0  0  0  d-Mannitol  0  3  0  0  0  0  d-Sorbitol  0  3  0  0  0  0  Dulcitol  0  0  0  0  0  0  Inositol  0  0  0  0  0  0  Sod,  formate  0  0  0  0  0  9  Sodo a c e t a t e  0  0  0  0  0  0  Sod,  0  0  0  0  0  0  0  0  0  0  0  10  5  0  20  7  0  n-Propyl a l c o h o l  8  0  0  0  0  10  n-Butyl a l c o h o l  4  0  0  0  0  0  A l l y l alcohol  15  17  0  0  0  20  SUBSTRATE  Ethylene  glycol  lactate  Sod, s u c c i n a t e Ethyl alcohol  Sc. lactis S.A.30  Sc. cremoris HP  Sc. cremoris EMBi 1 9 3  ^Recorded as percentage o f the r e d u c t i o n time shown by each organism i n the presence o f g l u c o s e .  .  Sc. cremoris RW  72  Table 2 Dehydrogenase A c t i v i t i e s  SUBSTRATE  Sc. bovis A.T.C. 6058  o f S t r e p , bovis and B e t a c o c c i  Betacoccus 17 3  EM®2  s  SC. citrovorus A.T.C. 7 9 7  Sc. paracitrovorus A.T.C.798  d-Glucose  100  100  100  100  d-Mannose  100  60  66  38  d-Galactose  12  0  10  25  d-Fructose  45  66  60  46  l-Arabinose  0  0  0  0  1-Xylose  0  0  0  0  0  0  0  0  Sucrose  89  72  50  16  Cellobiose  89  50  66  100  Lactose  55  6  10  12  Maltose  9  62  6  80  Trehalose  0  0  3  9  Melibiose  0  0  0  0  Raffinose  140  57  0  0  0  0  0  0  50  0  6  80.  Starch  140  4  50  50  Inulin  50  12  0  0  Esculin  78  0  0  0  ^Salicin  . 9  40  4  0  0  0  0  7  Rhamnose  Melezitose Dextrin  Methyl g l u c o s i d e  1^ • Table 2 (cont.)  SUBSTRATE  Sc. bovis A.T.C. 6 0 5 8  Betacoccus EMB2  175  Sc. citrovorus A.T.C.797  Sc. paracitrovorus A.T.C.798  Ethylene g l y c o l  0  0  0  0  Glycerol  0  0  7  0  Adonitol  0  0  0  0  Mannitol  0  0  0  22  d-Sorbitol  0  0  0  0  d-Dulcitol  0  0  0  0  Inositol  0  0  0  0  Sod. formate  0  0  0  0  Sod.  acetate  0  0  0  0  Sod. l a c t a t e  0  0  0  0  Sod.  succinate  0  0  0  0  Ethyl  alcohol  0  0  0  40  0  0  0  0  n-Propyl  alcohol  n-Butyl  alcohol  0  0  0  0  Allyl  alcohol  0  0  0  3  Recorded as percentage o f the r e d u c t i o n time shown by each organism i n the presence of g l u c o s e .  Table 3 Dehydrogenase A c t i v i t i e s  o f the Pseudo L a c t i c A c i d  Bacteria*  Tc. casei A.T.C. 3 9 1  Tc. liquefaciens  d-G-lucose  100  100  100  100  d-Mannose  20  65  80  75  d-Galactose  23  20  40  50  d-Fructose  33  100  67  44  l-Arabinose  25  0  12  0  1-Xylose  24  0  0  7  0  0  20  0  SUBSTRATE  Rhamnose  SM5  Bact. coli A.T.C. 4157  Bact. aerogenes A.T.C. 211  Sucrose  80  100  57  85  Cellobiose  30  90  25  67  Lactose  66  33  30  50  Maltose  66  75  45  60  Trehalose  25  16 '  67  75  Melibiose  45  . 0  10  9  Raffinose  80  90  45  50  Melezitose  12  16  11  0  Dextrin  50  89  30  80  Starch  40  0  36  68  Inulin  78  30  67  33  Esculin  0  0  -  -  Salicin  45  0  67  67  0  0  •  Methyl g l u c o s i d e  0  0  Table 3 (cont.)  SUBSTRATE  Tc. casei A.T.G. 391  Ethylene g l y c o l  0  Glycerol  0  Adonitol  0  Tc. liquefaciens SM5  Bacte coli A.T.C. 4157  Bact, aerogenes A.T.C. 211  0  0  20  25  0  0  0  16  36  100  0 30  d-Mannitol  80  d-Sorbitol  13  0  20  60  Dulcitol  0  0  0  0  Inositol  0  0  0  0  Sod. formate  0  7  40  57  Sod, a c e t a t e  0  0  0  0  11  32 27  Sod,  lactate  20  30  Sod, s u c c i n a t e  0  0  12  Sod, fumarate  0  0  10  0  17  0  14  12  0  200  2  67  0  200  3  24  Sod, malate Ethyl n-Propyl  alcohol alcohol  n-Butyl  alcohol  20  67  6  20  Allyl  alcohol  35  200  0  80  Formaldehyde  0  6  8  20  Glutamine  0  0  17  22  ^Recorded as percentage o f the r e d u c t i o n time shown by each organism i n the presence o f g l u c o s e .  16,. Table 4 Dehydrogenase Reactions o f S t r e p , l a c t i s at D i f f e r e n t . P e r i o d s o f Time  SUBSTRATE  Sc.  lactis  SA  30  Sc.  l a c t i s A.T.C. 374  Original Tests  18 mos. l a t e r  Original T e s t s  18 mos. l a t e r  d-Glucose  100  100  100  100  d-Mannose  30  100  75  100  d-Galactose  20  35  8  16  100  80  75  100  l-Arabinose  0  0  2  0  1-Xylose  0  0  2  0  0  0  0  0  Sucrose  30  4  100  8  Cellobiose  30  100  100  100  Lactose  6  20  38  37  Maltose  5  100  30  100  Trehalose  6  5  37  6  Melibiose  0  0  0  0  Raffinose  0  0  0  0  Melezitose  0  0  0  0  Dextrin  0  100  3  75  Starch  80  7  50  25  Inulin  20  100  50  42  Esculin  0  5  3  27  Salicin  4  12  11  37  0  0  0  0  d-Fructose  Rhamnose  •  Methyl g l u c o s i d e  Table  1  Aerobic Respiratory A c t i v i t y of Strep,  Strep. l a c t i s SUBSTRATE  S.A. 3 0  1  hour  1 hour  d-Glucose  26.6  26,6  d-Mannose  23.8  20.5  d-Galactose  21.7  d-Eructose 1-Xylose  S t r e p . l a c t i s A.T.C. 3 7 4  R.q.  Q.CO2  1  hour  lactis  QO2  1  hour  qc02  1  hour  R.Q. 1  hour  24.2  19.4  .80  ,86  30.8  25.0  ,81  21.0  .96  7.6  4.7  .73  27.9  22.8  .81  23.3  22.4  ,96  17.8  14.9  .83  7.5  5.5  .73  8.9  5.9  .66  10.2  9.2  .90  Sucrose  21.8  24.7  1.13  17.7  18.7  1.05  Cellobioae  23.8  16,8  .70  25.2  22.3  .88  Lactose  21.0  10.8  .51  21.4  17.5  .81  Maltose  22.4  22.4  13.0  11.0  .84  Trehalose  18.0  13.9  .77  14,9  14.0  .94  Melibiose  12,0  16.4  1.36  13.0  12.0  .92  Raffinose  18,7  20.2  1.08  14,0  13.1  .93  Melezitose  23.8  28.2  1.18  8,4  9.4  1.10  Dextrin  23.2  23.1  .91  37.1  30.4  .82  Starch  17.1  17.1  1.0  44.9  26,4  .59  Inulin  9.8  11.3  1.15  3.3  1.8  .54  Salicin  14.7  13.2  .89  12,1  10,2  .84  Esculin  19o6  19.6  14.9  10.0  .66  Alpha Methyl Glucoside  12.6  11.2  12.1  11,2  .92  l-Arabinose  1.0  1.0  1.0 .88  Table 1  Strep. l a c t i s SUBSTRATE  QO2  1 hour  QCO2  1 hour  Glycerol  18.2  21.2  Adonitol  7.0  5.3  d-Mannitol  24.9  d-Sorbitol  (cont.)  S.A. 3 0 R.q, . 1 hour 1.16  strep. QO2  1 hour  l a c t i s A.T.C. 374 qC0 2 1 hour  R.ft. 1 hour  27.3  21.5  .79  .78  10.2  10.2  1,00  20.3  .82  8,4  4.5  .53  21.0  16.6  .79  10.2  .91  Dulcitol  11,2  17.1  1.52  8.4  6.4  .76  Inositol  25.6  22.7  .88  9.3  8.3  .89  Ethyl  29.7  27.6  .92  -  -  -  8.7  3.8  .43  13.1  Sod. formate  26.6  25.2  .94  Sod.  19.2  19.2  1.00  24.4  29.1  1.19  alcohol  Ethylamine  lactate  Sod. s u c c i n a t e Sod, malate Sodi p o t i Endogenous  tartrate  -  -  -  23.8  18.7  .78  12.6  7.5  .59  11.2 ,  -  17.0  -  24.6  23.7  14,7  18,7  1.29  .96  1.27  Table  1  R e s p i r a t i o n and Fermentation vjitb S t r e p , Sc. l a c t i s SUBSTRATE  S.A, 3 0  IRespiratory  lactis  Sc. l a c t i s A.T.C. 374 ^Joefficient  Gm.L.A, per liter  8.1  24.2  100  7.4  30  7.7  30.8  75  7.4  21,7  .20  3.4  7.6  8  4.3  d-Fructose  27.9  100  7.4  23.3  75  6.3  1-Xylose  17.8  0  2.5  7.5  0  2.3  8,9  0  1.8  10.2  0  1.8  Sucrose  21.8  100  1.6  17.7  100  5.2  Cellobiose  23.8  50  2.7  25.2  100  6.3  Lactose  21.0  6  5.4  21.4  38  5.4  Maltose  22,4  5  3.6  13.0  30  5.0  Trehalose  18.0  6  7.0  14.9  37  4.7  Melibiose  12.0  0  1.6  13.0  0  0.9  Raffinose  18,7  0  2.0  14.0  0  1.1  Melezitose  23.8  0  2.0  8.4  0  4.1  Salicin  14.7  4;,  6.1  12.1  11  5.6  Dextrin  23.2  0  2.5  37.1  3  0.7  Starch  17.1  80  3.8  44,9  50  1.8  Inulin  9.8  20  0.9  3.3  50  0  19.6  0  2.7  14.9  3  2,0  12.6  0  1.4  12.1  0  0.9  Glycerol  18.2  0  1.4  27.3  0  1.8  Adonitol  7.0  0  0.7  10.2  0  0.7  d-Mannitol  24.9  0  0.7  8.4  3  2.7  ffllorbitol  21.0  0  1.1  11.2  3  0.7  Dulcitol  11.2  0  0.9  8.4  0  0.2  Endogenous  12.6  (3oef ficient  d-Glucose  26,6  100  d-Mannose  23.8  d-Galactose  l-Arabinose  Esculin Methyl  glucoside  Gm. L.A. per liter  Eiespiratory  q02  QO2  14.7  Table 2 Comparative R e s p i r a t o r y and Fermentative A c t i v i t y S t r e p , l a c t i s upon Carbohydrates w Sc. l a c t i s SUBSTRATE  of  Strep . l a c t i s A.T.C. 374  S.A. 3 0  Aerobic R.C.  Anaerobic R.C.  Acid R.C.  Aerobic R.C,  Anaerobic R.C.  Acid RiC.  d-Glucose  100  100  100  100  100  100  d-Mannose  89  30  95  127  75  100  d-Galactose  81  20  66  .31  8  60  104  100  91  96  75  87  1-Xylose  73  0  30  30  0  33  l-Arabinose  36  0  22  42  0  24  Sucrose  81  100  20  73  100  43  ,89  50  33  104  100  85  Lactose  78  6  66  88  38  72  Malto se  84  5  44  53  30  67  Trehalose  67  6  86  61  37  63  Melibiose  43  0  20  53  0  12  Raffinose  70  0  24  57  0  14  Melezitose  89  0  24  34  0  . 55  Salicin  53  4  75  50  11  75  Dextrin  94  . 0  30  153  3  9  Starch  64  80  46  185  50  24  Inulin  36  20  10  14  50  0  Esculin  73  0  33  61  3  27  Methyl g l u c o s i d e  47  0  17  50  0  12  Glycerol  68  0  17  0  24  Adonitol  26  0  8  42  0  9  d-Mannitol  93  0  8  34  3  (i^orbitol  78  0  13  46  3  9  Dulcitol  42  0  10  34  0  3  Endogenous  47  0  60  0  -  d-Fructose  Cellobiose  36  TABLE I ' Iffeot  of P r e v i o u s A d a p t a t l p n upon Carbohydrate Behydrogenations.^  Dehydrogenation Substrate  S t r e p , l a c t i s SA 30.  C e l l s Grown i n Presence Of Methyl Glucoae Lactose S t a r c h Manni to 1 G l u c o s i d e 6  d  Glucose  4  15  6  6  d  Mannose  13  21  7  6.  d  Galactose  20  28  16  d  Fruotoae  4  17  7  7  8  1  Arabinose  0  23  120  0  0  Sucrose  4  27  7  10  9  Cellobiose  8  26  7  9  10  Lactose  64  18  18  27  33  Maltose  89  69  11  0  0  Trehalose  62  18  ,29  16  22  Raffinose  0  12  9  13  13  Salicin  92  0  32  97  0  Dextrin  0  0  36  23  44  Starch  5  13  9  0  120  Inulin  21  37  25  6  9  0  0  0  0  0  d  Mannitol  B  12 20  A l l Values Expressed as R e d u c t i o n Time i n Minutes;  3i a .  •E&BLE I I E f f e c t of P r e v i o u s A d a p t a t i o n on Carbohydrate Dehydrogenation*Strep,  l a c t i s A.T.C. 374.  C e l l s Grown i n . Glucose  C e l l s Grown i n Lactose  d^ Glucose  3  2  8  d i Mannose.  4  3  10  38  6  12  4  5  10  Sucrose  3  4  11  Cellobiose  3  3  11  Lactose  8  5  12  Maltose  10  14  58  Trehalose  8  6  13  Raffinose  0  5  30  Sorbitol  90  56  31  Mannitol  90  21  10  Glycerol  110  8  36  88  20  64  Starch  6  4  9  Inulin  6  4  22  Dehydrogenation Substrate  d« Galactose d. F r u c t o s e  Salicin  C e l l s Grown in Mannitol  X A l l V a l u e s Expressed as Beduc t i o n Time i n Minutes^  Lactic Acid  TABLE I I I P r o d u c t i o n from Carbohydrates by Suspensions of S t r e p , l a c t i s SA 30 Adapted to Glucose. C e l l s Grown i n Presence  Time in Hoxrrs  \  Glucose Substrate  of Glucose  Mannose Substrate  Frutose Substrate  Galactose Substrate  Lactose Substrate.  Mannitol Substrate  0.5  • 306  • 216  i270  •018  0  0  1.0  ,432  • 414  • 342  • 018  0  0  1.5  .684  .432  • 468  • 056  0  0  2.0-  ,936  ^450  • 576  ;054  .036  0  2.5  .954  .540  .702  • 072  i054  0  3.0  .972  • 648  • 810  i072  i054  0  3.5  1.008  • 7ao  • 846  .090  • 072  0  4.0  1.026  .774  • 900  .108  .072  0  4.5  1.092  ,810  i900  .108  .090  0  5.0  1.116  .864  .900  .108  .108  0  5. 5  1.142  .900  • 936  .124 ,  .126  0  6,0  1.260  .918  • 972  .124  il26  0  TABLE I S t i m u l a t i o n of A c i d P r o d u c t i o n and Oxygen Uptake by V a r i o u s N i t r o g e n Sources upon R e s t i n g C e l l suspensions of S t r e p , l a c t i s A.T.C. 3 7 4 i n Presence of Glucose,  N i t r o g e n Source*  3 Hours Gm. L.A. per 1 0 0 ml. Aerobic Anaerobic  Q O2 3 hours  0  0.306  46.6  Ammonium C h l o r i d e  0.882  0.666  89.5  Ammonium S u l f a t e  1.008  0.666  64.3  Sodium N i t r a t e  0.846  0.630  60.6  Urea  0.774  0.594  52.1  0.828  0.666  -  Glycine  0.738  0.630  66.4  Cystine  0.882  0.684  159.5  Asparagine  0.396  0.630  66.4  Tryptone-DifCO  1.134  0.900  179.0  Peptone-DifCO  1.188  0.972  170.3  Peptone-Witte's  1,044  1.044  75.5  Proteose Peptone  1.170  0.918  242.6  Sodium Caseinate  0.630  0.576  101.0  Beef  0.864  0.666  207.9  1.206  0.828  187.4  C o n t r o l - No N i t r o g en  Uric  Acid  Extract  Yeast E x t r a c t - D i f C O  •*• A l l n i t r o g e n sources added to the r e s t i n g c e l l - glucose mixture i n 0.3fo c o n c e n t r a t i o n .  lis A  TABLE 2. Acid Production  and Oxygen Uptake R e c a l c u l a t e d  to Common Value o f 0,25 gm. N i t r o g e n .  Nitrogen  Source  Conversion % L a c t i c f. T.N. F a c t o r ' Acid Aerobic 3 Hrs.  % Lactic Oxygen Acid Uptake Anaerobic 5 Hrs. 3 Hrs.  Ammonium C h l o r i d e 2 6 . 2 0  1.9  1.675  1.265  170.0  Ammonium S u l f a t e  21.20  2.3  2.318  1.531  147.8  Sodium N i t r a t e  16.48  3.0  2.538  1-.890  181.8  Uric Acid  33.34  1.4  1.159  0.932  Urea  46.60  1.07  0.828  0.635  55.7  Glycine  18.66  2,6  1.918  1.638  172,6  Cystine  11.60  4.3  3.792  2,941  685.8  Asparagine  18.67  2.6  .1.029  1.638  172.6  Tryptone-DifCO  12,10+  4.1  4.649  3.690  733.9  Peptone-DifCO  15.40X  3.2  3.801  3.110  544.9  Peptone-Wltte  14,30*  3.5  3.654  3.654  264.2  Proteose Peptone  13.50^  3.7  4.329  3.396  897.6  Sodium  13.36  3.7  2.331  2.131  373.7  7.70  7.1  6,134  4,728  1476.0  5.9  7.115  4.883  1105.6  Caseinate  Beef E x t r a c t  Yeast E x t r a c t - D i f cSO 8 , 4 3  0.0  Factor Required t o Convert R e s u l t s t o Basic Value of 0 . 2 3 gm.N. B.A. and S a d l e r , VJ. - Can. J.Res.7: 364 , 1932 ® Value quoted from D i f c o Manual, 1 9 3 9 . ^ Values quoted from Eagles  Figure 1 Oiygen Uptake by Sc. l a c t i s A.T.C, 574 i n the Presence of Monosaccharides JO -I  Figure 2 Carbon D i o x i d e Production by Sc. l a c t i s A.T.C. 374 i n the Presence o f Uonosaccharides  Fructose Glucose  Endogenous Arabinose  Galactose  15  30  Time i n Minutes  45  60  2*.  Figure 3 Oxygen Uptake by Sc. L a c t i s i n the Presence o f Various Carbohydrates  Dextrin  Lactose  Raffinose  Adonitol Endogenous Melezitose  15  30 Time i n Minutes  45  60  Figure • Carbon Dioxide P r o d u c t i o n by Sc. l a e t i s A.T.C. 374. i n the Presence o f Various Carbohydrates  30;  Figure  5(a)  R e s p i r a t o r y Quotients - Sc. l a c t i s A.T.C. 374  .4 -  0  115  130  • 45  '60  Figure 6  Time i n Minutes In F i g u r e 6 i s shown the o x i d a t i o n o f glucose by s e v e r a l s p e c i e s of b a c t e r i a . T h i s graph emphasizes the comparatively s m a l l o x i d i z i n g a b i l i t y o f the L a c t i c A c i d S t r e p t o c o c c i i n comparison with more a e r o b i c species such as E , c o l i and R h i z o b i a ,  Figure 8 pH Ueasurementa  -  Anaerobic  Sc. l a c t i s S,A. 30  7.0-  6.5  No peptone  6.0 .  5.5  P4 5.0 4  4.5  4.0 .  3.5  0 . 1 2 5 ^ peptone 1 . 0 ^ peptone 0 . 2 5 > peptone 0 . 5 > peptone  Figure 9 I n f l u e n c e of Peptone Concentration upon Aerobic A c i d P r o d u c t i o n from Clueose by Sc. l a c t i s S.A, JO  1.0^  peptone  0,5^ peptone  0,251- pepton  0.125^ peptone  Figure  10  I n f l u e n c e o f Peptone Concentration upon Anaerobic A c i d P r o d u c t i o n from Glucose by Sc. l a c t i s S.A. 30  o o o  u  o  p.  1.0^  peptone  0.5^  peptone  0.25yL peptone 0 . 1 2 5 ^ peptone  •p o Hi  5  No  peptone  F i g u r e 11 I n f l u e n c e o f Peptone C o n c e n t r a t i o n upon Oxygen Uptake by Se* l a c t i s S.A. 30 i n Presence o f Glucose  200.  1,0^  peptone  0.5l^ peptone  o  a « M «  P4  0,231- peptone  <M O  0.125^  peptoK  — No peptone  2  3  Time i n Hours  31.  F i g u r e 12 L a c t i c A c i d P r o d u c t i o n from Monosaccharides - Sc. l a c t i s S.A, C e l l s Grown i n Glucose Broth  30 -  3«.  Figure  13  L a c t i c A c i d P r o d u c t i o n from Lactose - Sc. l a c t i s S.A,  30  4-0.  Figure  15  Oxygen Uptake by Washed C e l l s of Rh. t r i f o l i i (Cu. mgm,  of Og per mgm.  224  dry weight)  70 .  0  15  30 Time i n Minutes  45  60  Figure l6 Comparative Aerobic and Anaerobic R e s p i r a t o r y Rh. t r i f o l i i 224  Coefficients  Aerobic  Anaerobic 125-  100. n 13  m  75-  o  o  >. 1^  o' +> ed  U  50-  Pt at'  25  <D  o O P! o o  3  9  to  O  H O +>  a a as  ed C  •o to o CO  e +3  m  ¥>  a> m o o  o  bO •cs a w o  o -p  PI  a  CD  a a  •H  0  O CO  o o  F i g u r e 17 Oxygen Uptake from Mannitol by Rh. t r i f o l i i 224 and S u b s t r a i n s  210  180  150  .  120  90 .  60 .  30 .  R.T. 224  Bl  ^1  ^2  >3  °4  ^5  ^7  °11  ^12  18  Figure  Comparative Aerobic and Anaerobic R e s p i r a t o r y C o e f f i c i e n t s on Sodium succinate - R.T, 224 and S u b s t r a i n s  Anaerobic Aerobic  125  ra •p 0  o  100  o •H  u  Vi  (D O O  >> U  75  o  +> (d u  •H  <a 50  25  R.T.  B  1  C  1  c  2  c  3  C  4  C  5  c  7  224  Substrains  c„  8  c  9  C  11  C  12  ^4  Figure  19  ComparatlTe Aerobie and Anaerobic R e s p i r a t o r y C o e f f i c i e n t s on Sodium Succinate - R«T. Freshly Isolated Strains  Anaerobic Aerobic  175  150  m a  o o  o  125  100  p> u o •*»  m u  Pi  «  75  .  50  25  R.T,224  E  1  E  2  S  5  E,  6  S  7  ^8  4--5  Figure  20  Glucose O x i d a t i o n by F r e s h l y I s o l a t e d S t r a i n s of Rh. t r i f o l i i 224  100.  6  80-  60. M 0) 43  P, to  a  o to >.  40'  M O  20  E 2  E  8  v3  F i g u r e 21 Endogenous Oxygen Uptake with A n  S u b s t r a i n s o f Rh. t r i f o l i l  224  60.  50 «  o  40.  o M  od  p. p a  «  30.  >.  K O  20-  10-  ,E  E,  E.  E.  E.  E,  B RT 224  B,  '10 '11  Figure  22  Glucose O x i d a t i o n by A l l S t r a i n s of Rh,  trifolii  240  •  •  200  •• o  160 d> M 0  43  P< P 0  O tiD  120-  >» K  O  80  40  E  El  E.  E,  Er  Es  RT 2241  C6  224  Figure  1  S t r a i n V a r i a t i o n i n Dehydrogenase A c t i v i t y  1 2 3 4 3 Fructose  1 2 3 4 5 Salicin  1 2 5 4 3  1 23 4 5  1 23 4 5  Sucrose C e l l o b i o s e Raffinose  1 2 3 4 5 Mannitol  1  1 2 3 4 5  123 4 5  Xylose  Sod, malate  1. 2. 3. 4. 3.  RT 22B RT 224 RT 226 RT 231 RT 3 9 - 1  1 2 34 5 Starch  11 2 3 4 5 Glycerol  Figure  2  V a r i a t i o n i n Dehydrogenase A c t i v i t y with Time - R. t r i f o l i i 1 . F i r s t Tests 2 . A f t e r 2 months 3 . A f t e r 8 months 4 . A f t e r 1 2 months 100  75  50-  25  0 1 2 3 4  Mannose  1 2 3 4  Fructose  1 2 3 4  Xylose  1 2 3 4  1 2 3 4  Cellobiose  Raffinose  1 2 3 4  1 2 3 4  100  75 -  50  25 '  1 2 3 4  1 2 3 4  Glycerol  Sodium Mannitol succinate  1 2 3 4  Sodium malate  Ethyl alcohol  224  Figure 1 Dehydrogenase A c t i v i t y o f S t r a i n s and S u b s t r a i n s of R. t r i f o l i i upon Sodium s u c c i n a t e  100-  75-  50-  25-  E  E.  E.  E.  E.  E.  E,  Substrains  100  75 •  50 .  25  RT B. 224  B.  10 11 12 Substrains.  Figure 2 Endogenous 0,0^ of S t r a i n s and  S u b s t r a i n s of R. t r i f o l i i  60-  40 30 CO  20 10'  E  E  E.  E  E  E  E,  Substrains  6050 •  40 • 30o  CVJ  cf  20 • 10 •  RT  224  B,  1  B„ C, 2 1  C  2  C  3  c  5  c. 6  c  7  Substrains  8  c  9  c  10  c  11  12  224  Figure 3 Anaerobic Endogenous R e s p i r a t i o n with S t r a i n s and S u b s t r a i n s of R, t r i f o l i i 224  100 .  73 •  30  25  E  E.  E.  E.  E,  E.  E,  Substrains  100  75  •  50  25  RT 22  Li  '10 Substrains  '11  '12  Figure 4 A e r o b i c Glucose O x i d a t i o n of S t r a i n s and of R. t r i f o l i i 224  Substrains  240 •200 • 160 • 120 • 80 • 40 •  E  E,  E,  E.  E,  E<  Substrains  RT 224  B,  '11 Substrains  '12  Figure 5 Dehydrogenase A c t i v i t y upon M a n n i t o l of S t r a i n s and S u b s t r a i n s of R. t r i f o l i i 224  100  75  50  .  25 •  E  E  1  E  E  2  5  E  6  7  E,  Substrains  100 •  75  50 •  25 •  R 224  ^1 ^2 ^1  ^2 ^3 ^5  ^6 °7  Substrains  °8 °9  °10 °1]  Figure 6 Aerobic O x i d a t i o n of M a n n i t o l by S t r a i n s and of R. t r i f o l i i 224 150  Substrains  .T  100 .  O  CM  &  50  E  El  E,  E,  E,  1.  Ec  Substrains  150 .  CO  100  o  50  RT 224  "2 s  "7 Substrains  =8 =9  °10  °11  12  Figure 7 Aerobic R e s p i r a t o r y C o e f f i c i e n t s upon Sodium succinate of S t r a i n s and S u b s t r a i n s of R, t r i f o l i i 224  150-  100.  E  E.  E.  E  E,  E.  E,  8  Substrains  150-  100.  50.  RT 224  1  B„ C, 2 1  C^ C, 2 3  5  C/ 6  c„  Substrains  7  Cn 8  c  9  c  10  G  11  12  Figure 8 Comparative Aerobic and Anaerobic R e s p i r a t o r y A c t i v i t y of Four S t r a i n s o f R. t r i f o l i i 224 150  150.  100  100  50  50  End,  Mann.  AT-E  Sod. sueo,  End.  RT 224 E  RT 224  150  150-  100.  100.  50.  50,  A~B" End,  Mann,  RT 224 C,  B Sod. Mann. succ.  Sod. succ.  A B End.  A  Mann, RT 224 C  B_  Sod. succ. 12  Plate 1  i  5^ .  Plate 3  Plata  A.  P l a t e 5.  45.  Plate 6  Figure 1 Rate o f Oxygen Uptake - S t r e p , l a c t i s S.A,  Time i n Minutes  30  O  > a o o-  O  ft) CD 01 H-  ti  (B dO  O  p:  4 (D  ro CO  at)  o  CO CO  o  Figure 5 A e r o b i c R e s p i r a t o r y A c t i v i t y of S t r e p , l a c t i s A.T.C.  40 • 35 • 30 25 • 20 •  15 10 • 5 •  0  0  fl  ©  bD 0  0 0  a  w  CO  H  CO  0 +> 0  cd  <a  c»  a> m  0  1-4  >>  0)  0  0 42 0  +»  m  0)  iA  m  0  H  H  0  u  a  43  CO  ft  r-i H  0 4»  0 43  d  Pi  a  0  CO  H  374  Figure 4 Comparative O x i d a t i v e A c t i v i t y of S t r e p , l a c t i s and S t r e p , l a c t i s A.T.C. 3 7 4  S.A. JO  A. S t r e p , l a c t i s S.A. 30 B. S t r e p , l a c t i s A.T.C. 374 40 • 35 • 30 • 25 20 15 10  A B  A B  A B  AB  AB  AB  A B  A B  A B  o O  a  o o  XS  o m o o H  m o o  (0 1-4  (d  <D 09 O H  <D m o  43 o  (0  0)  m o  43  H  CO 1^  9  a  o 43  M a> H  AB  o  o ^l  aj  43  CO  43  ft  e  a  H  >. 43  Figure 5 R e s p i r a t o r y Quotients - S t r e p , l a c t i s A.T.C.  374  Figure 6 Comparative Glucose O x i d a t i o n s by S t r e p , and Bact. c o l i  0  15  30  45  Time i n Minutes  60  lactis  Figure  1  Comparative Enzymic A c t i v i t y upon Carbohydrates with S t r e p , S.A. 30 A. Aerobic R e s p i r a t o r y C o e f f i c i e n t s B. Anaerobic " " C. A c i d 100  lactis  .  GQ +>  a  © o  o  73  30  >1  u o  43  ^1 •H p  23 •  m  (D  A B C Galactose  A B C  A B C  Sucrose  Lactose  A  B C  A B C  Trehalose M a n n i t o l  Figure 2 Comparative Enzymic A c t i v i t y of S t r e p , l a c t i s A.T.C. 374 CQ  +3  fi <D  150 125  Q> O O >= fH  O  43  100 . 75 .  CO  u p4 ©  50 • 25  A B C Galactose  A  B .C  Sucrose  A B C Raffinose  A B C  A B C  Dextrin  Inulin  Figure  1  I n f l u e n c e of P r e v i o u s A d a p t a t i o n upon carbohydrate Dehydrogenation. S t r e p , l a c t i s SA. 30 A. B. C. D.  Cells Cells Cells Cells  grown grown grown grown  in in in in  Glucose b r o t h lactose broth s t a r c h broth mannitol broth  100.  75.  50.  25.  _L_ A B C D  A B C D  A B G D  Arab inose  Lactose  iltose  A B C D Baffinose  A B 0 D Starch  Subs trat.e  Expressed on Percentage of the Glucose Reduction Time.  Figure S I n f l u e n c e of Previous A d a p t a t i o n upon Carbohydrate Dehydrogenation. S t r e p i l a c t i s ATC 374 A. B. d.  C e l l s grown i n glucose broth C e l l s grown i n l a c t o s e b r o t h C e l l s grown i n mannitol broth  100  75  50 (D O  o  a o  25  3 a> A  B G  Mannose  A B C  A B C  Galac to se  Lac to se  A B C  Mannitol Raffinose  Substrate K  Expressed as Percentage S e d u c t i o n Time.  A B C  of the Glucose  Time In Houra  J^luene«  «  i  A d a p ^ t l e a upon Wwmim.1mlsi.9iB. « f  f'  i  t  4  ji  m  IS.  Figure 5 I n f l u e n c e of A d a p t a t i o n upon Fermentation of Galaotoee by Suspensions of S t r e p , l a o t i s SA. 30  Time i n Hours  Figure 6 I n f l u e n c e o f A d a p t a t i o n upon Fermentation o f Laotose t j Sttspensions o f S t r e p , l a c t i s ATC 374.  Time i n Hours  11.  Figure 7 I n f l u e n c e of A d a p t a t i o n upon Fermentation of Galactose by Suspensions of S t r e p , l a c t i a ATC.374  C e l l s from Galactose B r o t h  0  1  2  3 Time i n Hours  4  5  6  Figure 1  (a)  S t i m u l a t i o n of Aerobic P r o d u c t i o n of L a c t i c A c i d by R e s t i n g C e l l s of S t r e p , l a c t i s ATC 374 from Glucose  Figure  1  (b)  S t i m u l a t i o n of Anaerobic P r o d u c t i o n of L a c t i c A c i d by R e s t i n g C e l l s of S t r e p , l a c t i s ATC 374 from Glucose,  rH  1,03  o o u  Peptone-DifCO east  P  Extract  0,73 NH4  CI  Asparagine Sod,caseinate +3  0,45 d  C5  0,15 •  .h  FIGURE 2 S t i m u l a t i o n of A e r o b i o P r o d u c t i o n of L a c t i c A c i d by Suspensions of S t r e p , l a c t i s ATC 374 from G l u c o s e . E f f e c t o f R e p r e s e n t a t i v e N i t r o g e n Sources  ^ ^  ^  ^  / /  Beef E x t r a c t  /  ^  /  •1  Proteose peptone Tryptone  *2  '3 '4 Time i n Hours  Urea ^^^^ G l y c i n e  FIGURE 3 I n f l u e n c e of v a r i o u s N i t r o g e n Sources i n S t i m u l a t i n g R e s p i r a t i o n of Suspensions of S t r e p , l a c t i s ATC 314 with Glucose  ^Proteose  y  ^  /  //  // // 7  /  _  '  •2  Beef E x t r a c t  Tryptone  ^  .  •^  -3 Time i n Hours  peptone  . Glycine NaNO, 1 1 ^rea  Figure  4  B e l a t i v e S t i m u l a t i o n o f Aerobic L a c t i c A c i d P r o d u c t i o n , Anaerobic L a c t i c A c i d P r o d u c t i o n and H e a p i r a t i o n by VariouB N i t r o g e n Sources, A, B, 0,  Aerobic L a c t i s Acid Production Anaerobic L a c t i c A c i d Produc t i o n Oxygen Uptake  1.21,0 .6. .6. .4.2.  A B C  A B C  Amm* Amm. Chloride Sulfate  ABC Urea  ABC Cystine  ABO Asparagin  If %  Figure 4  (Continued)  R e l a t i v e S t i m u l a t i o n of Aerobic L a c t i c A c i d P r o d u c t i o n , Anaerobic L a c t i c A c i d P r o d u c t i o n and R e s p i r a t i o n by V a r i o u s N i t r o g e n Sources* A, B, C,  Aerobic L a c t i s A c i d Production Anaerobic L a c t i c A c i d Production Oxygen Uptake  1.2.  •180  w 1.0.  •150  (0  a  .8.  • 120  o  o OJ  .6.  . 90  .4.  . 60  .2.  • 30  o  A B C Peptone - Difco  ABC Peptone • Witte's  ABO  A B C  ABC  Sod» Casei nat  Beef Yeast Extrac t E x t r a c t  o  F i g u r e 5S t i m u l a t i o n of Aerobic L a c t i c A c i d P r o d u c t i o n by Various N i t r o g e n Sources. S t r e p , l a c t i s Suspension i n the Presence of Glucose. ^ Yeast  Extract  . Beef E x t r a c t  P r o t . peptone  O  o  Cystine - P e p t o n e - Witte  H P TJ •H  O  <  o ft •p  Sod.  o cd  caseinate  Glycine NH4CI  - Urea  '1  '2  '3 -4 Time i n Hours  A l l Values C a l c u l a t e d to the Common B a s i s 0 . 2 5  gm. Nitrogen,  FIGURE 6 S t i m u l a t i o n of R e s p i r a t i o n hj V a r i o u s N i t r o g e n Sources. S t r e p , l a c t i s ATC J74 Suspension i n . Presence of Glucose. ^ 1500.  Beef E x t r a c t  Time i n Hours X  A l l Values C a l c u l a t e d to the Common B a s i s 0 , 2 5 gm. N i t r o g e n  gIGUHE 7 R e l a t i v e S t i m - a l a t i o n of A e r d D i c L a c t i c A c i d P r o d u c t i o n , A n a e r o b i c L a c t i c A c i d P r o d u c t i o n and R e s p i r a t i o n by V a r i o u s N i t r o g e n Sources. A.  Aerobic  Lactic Acid  Production  B.  Anaerobic L a c t i c A c i d  C.  Oxygen Uptake  Production  1500 o  .1000  I CD  a t3 03 CD.  500 .  ABC Amm. chloride  AB  G  Amm. sulfate  iA BlC Urea  ABC  ABC  Glycine  Cystine  250  91.  gIGURE 7 _ ( c o n t r d j R e l a t i v e S t i m u l a t i o n of Aerobic L a c t i c I c i d P r o d u c t i o n , A n a e r o b i c L a c t i c A c i d P r o d u c t i o n and R e s p i r a t i o n by Various Hitrogen Sources. A.  Aerobic L a c t i c A c i d Production  B.  Anaerobic L a c t i c A c i d Production  G.  Oxygen Uptake  1500  a  o o .625H  ,1250 1000  CI nd •H O <^  .•37^-  7^0  .250-  500  .125-  250  CD  P. CD,  o •H  -P O 05  CfQ,  ABO Peptone L i fee  A B C  ABC  Sod. Pe pt one Wltte daseinate  ABC Beef Extract  ABC Yeast Extract  CD,  

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