* 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. 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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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UBC Theses and Dissertations
Studies on the respiratory enzymes of the lactic acid and nitrogen-fixing bacteria Morgan, Joseph Francis 1942
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