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Regulation of sucrose metabolism and constitutive nitrogen fixation in Azotobacter vinelandii Jacobson, Marty 1983

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REGULATION OF SUCROSE METABOLISM AND CONSTITUTIVE NITROGEN FIXATION IN AZOTOBACTER VINELANDII by MARTY JACOBSON A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Microbiology) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1983 (c) Marty Jacobson, 1983 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a i f u l f i l l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s m a y b e g r a n t e d b y t h e H e a d o f my D e p a r t m e n t o r b y h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f M i c r o b i o l o g y T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 6 1 7 4 U n i v e r s i t y B l v d . V a n c o u v e r , B . C . , C a n a d a V 6 T 1W5 DATE i i ABSTRACT Azotobacter v i n e l a n d i i was found to exhibit diauxie growth i n sucrose containing Burk's medium supplemented with 29 mM ammonium ace-tate as nitrogen source. The diauxie growth curve was determined to be a re s u l t of p r e f e r e n t i a l u t i l i z a t i o n of acetate as carbon source during the f i r s t part of the growth curve. No detectable disappearance r 14 I of LU- CJ sucrose was noted i n samples taken before the lag separating the two portions of the diauxie curve. Repression of both sucrose transport and metabolizing a c t i v i t y was observed i n pre-lag, acetate-u t i l i z i n g c e l l s when the medium contained a high (58 mM) sucrose con-centration. When a lower i n i t i a l sucrose concentration (7 mM) was used, both sucrose transport and glucose-6-phosphate dehydrogenase a c t i v i t y were induced but acetate was s t i l l used p r e f e r e n t i a l l y and diauxie growth ob-served. Mutant s t r a i n s which f i x N. i n the presence of NH,+ exhibit 2. 4 diauxie growth curves as well and are severely reduced i n t h e i r a b i l i t y to reduce C^H^ ( f i x N ) while u t i l i z i n g acetate i n the pre-lag growth phase. In vivo nitrogenase a c t i v i t y i n the presence of NH^+ increases dramatically a f t e r the lag. The low nitrogenase a c t i v i t y i n pre-lag cultures i s not due to a reduced supply of energy or reductant because measurement of nitrogenase i n c e l l free extracts provided with ATP and d i t h i o n i t e y i e l d s the same r e s u l t s . The amount of nitrogenase protein detectable as a n t i g e n i c a l l y cross reactive materials varies with the density of the culture. We conclude that the presence or u t i l i z a t i o n of acetate i n some way i n t e r f e r e s with the synthesis or s t a b i l i t y of nitrogenase. A derepressed mutant Azotobacter v i n e l a n d i i (UW59) was i s o l a t e d as a revertant of a Nif mutant (UW2. Proc. Natl. Acad. S c i . , USA 6jh 3501-3503). A deri v a t i v e of UW59, UW590, expresses approximately twice the a c t i v i t y of UW 59 when grown with e i t h e r ^ or NH^+ as n i t r o -gen source. Biochemical and genetic c h a r a c t e r i z a t i o n was undertaken to determine the nature of the mutation(s) r e s u l t i n g i n the co n s t i t u t i v e phenotype. Strains UW59 and UW590 were used as donors i n genetic trans-59 formation of UW2. Results indicate that the n i f mutation .is responsi-c 590 ble for the co n s t i t u t i v e (Nif ) phenotype whereas the n i f mutation permits good growth on N^ but not c o n s t i t u t i v i t y . The rate of NH^+ uptake by UW590 was lower than that of the wild type at pH 5.9. The decreased NH^+ uptake a b i l i t y was correlated with an increased c o n s t i t u -t i v e synthesis of nitrogenase at low pH . A defect i n CH^NH^+ uptake was detected i n UW59 and UW590 at pH 7.3 and pH 5.9. A k i n e t i c analysis of the derepressed mutant UW59 revealed that at high CH^ NH^ "1" concentrations the mutant i s s i g n i f i c a n t l y d i f f e r e n t from the wild type. The r e s u l t s suggest that the c o n s t i t u t i v e nitrogenase synthesis might be due to an a l t e r a t i o n i n NH,+ (CH„NH„ +) a s s i m i l a t i o n . 4 3 3 TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS v i i i INTRODUCTION 1 I. Regulation of Sucrose U t i l i z a t i o n by Acetate 1 I I . Regulation of Constitutive Nitrogenase 2 A. Regulation of Nitrogenase i n K l e b s i e l l a pneumoniae . . 3 B. Regulation of Nitrogenase i n Azotobacter v i n e l a n d i i . . 5 MATERIALS AND METHODS 13 I. B a c t e r i a l Strains 13 I I . Media and Culture Conditions 13 II I . Chemicals 14 IV. Genetic Transformation 14 V. Determination of Labelled Substrate U t i l i z a t i o n . . . . 15 VI. Measurement of Sucrose Uptake 15 VII. Metabolic Enzyme Assays 16 VIII. Nitrogenase Assays 18 IX. Immunoelectrophoresis 20 X. Ammonium Disappearance from Medium 21 XI. Measurement of NH4+ Uptake Rates 22 XII. Methylamine (CH3NH3+) Uptake 22 XIII. Protein Determination 24 RESULTS 25 I. Regulation of Sucrose U t i l i z a t i o n by Acetate 25 A. Diauxic Growth 25 B. P r e f e r e n t i a l U t i l i z a t i o n of Acetate 25 C. Sucrose Transport A c t i v i t y i n Acetate U t i l i z i n g C e l l s . 30 D. Metabolic Enzyme A c t i v i t i e s i n Acetate U t i l i z i n g C e l l s . 32 E. E f f e c t of a Lower Sucrose Concentration i n the Growth Medium 35 F. Regulation of Sucrose U t i l i z a t i o n 37 II . Regulation of Constitutive Nitrogenase 42 A. Nitrogenase A c t i v i t y 42 B. E f f e c t of pH on Derepressed Nitrogenase Synthesis . . 43 C. NH44" Uptake 49 D. CH3NH3+ Transport 50 E. CH„NH~ + Uptake i.Under a Defined Atmosphere . . . . 57 F. Ki n e t i c s of CH3NH3+ Transport 59 II I . E f f e c t of Acetate on Constitutive Nitrogenase A c t i v i t y . . 65 A. I s o l a t i o n and Properties of Methylalanine Resistant Mutants 65 B. E f f e c t of Acetate on Constitutive Nitrogenase . . . 68 C. E f f e c t of Acetate on Constitutive In V i t r o Nitrogenase A c t i v i t y 72 D. E f f e c t of Acetate on Constitutive Synthesis of Nitrogenase Protein 76 V Page DISCUSSION 78 I. Regulation of Sucrose Transport and Metabolism by Acetate 78 I I . E f f e c t of NH4+/CH3NH3+ Uptake on Constitutive Nitrogenase A c t i v i t y 83 I I I . E f f e c t of Acetate on Constitutive Nitrogenase A c t i v i t y 95 REFERENCES 100 v i LIST OF TABLES Table Page c 1 Derivation and r e l a t i v e phenotypes of Nif mutants derived from s t r a i n UW2 7 2 Genetic transformation 10 59 590 3 Separation of n i f and n i f by transformation 11 r 14 i 4 I n i t i a l rates of LU- CJ sucrose uptake 31 5 Metabolic a c t i v i t i e s of Azotobacter v i n e l a n d i i during pre- and post-lag growth 34 6 Comparison of uptake and metabolic a c t i v i t i e s i n pre-lag c e l l s grown i n medium containing 28 mM acetate plus a high (58 mM) or low (7 mM) sucrose concentration 36 7 E f f e c t of pH on i n vivo nitrogenase a c t i v i t y 46 8 Comparison of nitrogenase a c t i v i t y and proteins at d i f f e r e n t c u l -ture densities for c e l l s grown using ammonium s u l f a t e as N source. 49 9 Rate of NH^+ uptake at pH 5.9 53 10 CH^ NH^ "1" (1 mM) uptake rates of the wild type (JK) and mutant s t r a i n s of A. v i n e l a n d i i 55 11 CH^NH^+ (1 mM) uptake under defined atmospheres 58 12 K i n e t i c s of CH^NH^+ transport i n A. v i n e l a n d i i s t r a i n UW59 as determined from several d i f f e r e n t graphing techniques 64 13 Levels of nitrogenase i n w i l d type and methylalanine r e s i s t a n t (Mal r) mutants 67 14 Constitutive nitrogenase a c t i v i t y of methylalanine r e s i s t a n t mutants grown with various concentrations of acetate 71 15 E f f e c t of acetate on c o n s t i t u t i v e synthesis of nitrogenase protein 77 v i i LIST OF FIGURES Figure Page 1 Diauxie growth of Azotobacter v i n e l a n d i i 27 2 U t i l i z a t i o n of [U-^C ]acetate and [ U-"^C ]sucrose during diauxie growth of Azotobacter v i n e l a n d i i 29 r 14 i 3 E f f e c t of acetate on the uptake and incorporation of LU- CJ su-crose i n cultures of Azotobacter v i n e l a n d i i induced f or sucrose u t i l i z a t i o n 40 4 The ef f e c t of pH on i n vivo nitrogenase a c t i v i t y i n s t r a i n UW590 grown on medium containing ammonium s u l f a t e as N-source. . . . 45 5 Ammonium remaining i n the culture medium of st r a i n s JK and UW590 during growth 52 6 A plo t of v e l o c i t y (v) versus substrate concentration ( s ) for methylammonium (CH^NH^+) uptake 61 7 A Lineweaver-Burk plot for CH^ NH^ "1" uptake rates. 63 8 Growth and c o n s t i t u t i v e nitrogenase a c t i v i t y i n the methylalanine r e s i s t a n t (Mai ) mutant s t r a i n JK1 70 9 Growth and c o n s t i t u t i v e nitrogenase a c t i v i t y i n the mutant s t r a i n UW590 74 14 + 10 K i n e t i c s of c a r r i e r transport of CH NH i n Azotobacter v i n e l a n d i i (JK) 91 11 A Lineweaver-Burk plot f o r 1 4CH 3NH 3 + i n Azotobacter v i n e l a n d i i ( J K ) , 93 v i i i ACKNOWLEDGEMENTS I w o u l d l i k e t o t h a n k D r . J . K. Gordon w i t h o u t whose p a t i e n c e , s u p p o r t and g u i d a n c e t h i s p r o j e c t w o u l d n o t have been p o s s i b l e . My t h a n k s a l s o go t o R. A . Moore f o r h i s many h e l p f u l d i s c u s s i o n s and t o Ms . E . L e a f f o r h e r t e c h n i c a l a s s i s t a n c e on p a r t o f t h i s p r o j e c t . 1 INTRODUCTION I. Regulation of Sucrose U t i l i z a t i o n by Acetate Diauxic growth, or diauxie, the occurrence of two successive growth curves, separated by a period of lag, was demonstrated by Monod (52). He found that when Escherichia c o l i was provided with both glucose and lactose, growth stopped when the preferred carbon source, glucose, was exhausted from the medium and resumed a f t e r a period corresponding to the time required for the induction of lactose transport and degrading enzymes. He also noted sequential substrate u t i l i z a t i o n when other combinations of sugars and sugar alcohols were provided (52). Diauxie has been observed i n a number of other bacteria as w e l l . In some cases, p r e f e r e n t i a l u t i l i z a t i o n of sugars i s noted (12,52,65), whereas, i n other cases, compounds such as organic acids are preferred (33,40,55,88). In general, i t appears that the preferred carbon source a f f e c t s u t i l i z a t i o n of the second carbon source by regula-t i o n of transport and/or metabolism. It was observed i n t h i s study that AzotObacter v i r i e l a r i d i i e x h i bits diauxie when grown i n Burk's medium (sucrose as carbon source) supplemented with ammonium acetate as nitrogen source. Other investigators have mentioned s i m i l a r observations (87, T. Melton, Abst. Annu. Meet. Am. Soc. M i c r o b i o l . 1980, 124, p. 88). This i n v e s t i g a t i o n into the cause of diauxie revealed that repression of both sucrose transport and metabolizing a c t i v i t y i s effected by acetate which i s used p r e f e r e n t i a l l y by A. v i n e l a n d i i . This i s of p a r t i c u l a r i n t e r e s t i n that u t i l i z a t i o n of organic acids a f f e c t s N_-fixing a c t i v i t y (refer to section II.B.). 2 I I . Regulation of Constitutive Nitrogenase Fixed nitrogen from sources such as l i g h t n i n g and combustion, b i o l o g i c a l ^ f i x a t i o n , and i n d u s t r i a l ammonia synthesis i s required to support the needs of the en t i r e b i o l o g i c a l community. B i o l o g i c a l N^ f i x a t i o n i s thought to account for approximately 70% of the t o t a l ^ f i x e d (10). Thus, the economic importance of b i o l o g i c a l m f i x a t i o n i s evident. Our understanding of the process of 1$ f i x a t i o n , although advancing, i s s t i l l somewhat l i m i t e d due to the complexity of the biology, biochemistry, regulation and genetics of the process. Therefore, to r e a l i z e any p r a c t i c a l applications involving improvements i n N^ f i x a t i o n or the creation of novel associations, a large and accurate data base must be a v a i l a b l e . Bacteria capable of N f i x a t i o n are found i n a v a r i e t y of genera. F r e e - l i v i n g N^-fixing b a c t e r i a include members of K l e b s i e l l a , Azotobacter, Clostridium, Rhodospirillum, Azospirillum, and various cyanobacteria. Those ba c t e r i a which f i x N only when symbiotically associated with a plant include Rhizobium spp. (which nodulate legumes [22, 94]), c e r t a i n actinomycetes (which nodulate Comptinia and alder [89]) and Anabaena  azola (which fi x e s N^ within the l e a f pores of the water fern A z o l l a [59]). Nitrogenases from the d i f f e r e n t b a c t e r i a have some very s i m i l a r properties (17). The enzyme consists of two oxygen l a b i l e proteins. Component I, or nitrogenase, has two copies of two d i f f e r e n t subunits (o^,^) a n ^ contains both i r o n and molybdenum atoms (17,18,32,46,86,90). Component I I , or nitrogenase reductase, has two copies of a single subunit 3 and c o n t a i n s i r o n atoms (5,17,18,32,68,90). Component I c o n t a i n s t h e s i t e o f s u b s t r a t e b i n d i n g and s u b s t r a t e r e d u c t i o n . The a c t i v e s i t e o f t h e n i t r o g e n a s e comp lex r e s i d e s i n an i r o n - m o l y b d e n u m c o f a c t o r ( F e M o - c o ) t h a t i s a s s o c i a t e d w i t h component I (5,75,76), y e t c a n be d i s a s s o c i a t e d by a c i d t r e a t m e n t o f component I (75,76). The r o l e o f component I I i s t o r e d u c e component I (5,32,68). A . R e g u l a t i o n o f N i t r o g e n a s e i n K l e b s i e l l a pneumoniae The m a j o r i t y o f i n f o r m a t i o n about t h e g e n e t i c s and r e g u l a t i o n o f N2 f i x a t i o n has been o b t a i n e d f rom s t u d i e s i n K l e b s i e l l a pneumon iae . T h e r e have b e e n between 15-20 n i f genes i d e n t i f i e d f o r K. pneumoniae a r r a n g e d i n 7 o p e r o n s (14,37,68). The known gene f u n c t i o n s have been r e c e n t l y r e -v i e w e d by Kennedy e t a l . (37) and R o b e r t s and B r i l l (67,68). The s t r u c t u r a l genes f o r t h e o<(ni fp) and QjnlfK) s u b u n i t s o f component I and component I I ( n i f H ) have b e e n a s s i g n e d by i d e n t i f i c a t i o n o f e l e c t r o p h o r e t i c a l l y a l t e r e d p r o t e i n s (69). T r a n s c r i p t i o n a l r e g u l a t i o n o f t h e K. pneumoniae n i f r e g u l o n a p p e a r s t o i n v o l v e a t l e a s t t h r e e m a j o r s y s t e m s (1,16,68,84,91): r e g u l a t i o n i n b o t h a p o s i t i v e and n e g a t i v e f a s h i o n by e l e m e n t s o f t h e g e n e r a l n i t r o g e n m e t a b o l i s m s y s t e m a t n i f R';' p o s i t i v e r e g u l a t i o n by t h e n i f A p r o d u c t a t t h e o p e r a t o r r e g i o n o f e a c h o f t h e n i f t r a n s c r i p t s ; and n e g a t i v e r e g u l a t i o n i n t h e p r e s e n c e o f o x y g e n . The nifA._ gene p r o d u c t h a s b e e n shown t o be n e c e s s a r y (6,37,50,67) and s u f f i c i e n t (19,47) as a p o s i t i v e r e g u l a t o r f o r a l l n i f o p e r o n s e x c e p t i t s own, n i fRLA (48,67), and i s r e q u i r e d c o n t i n u o u s l y f o r t h e e x p r e s s i o n o f t h e o t h e r t r a n s c r i p t s (37). I n a d d i t i o n , t h e q u a n t i t y o f t h e n i f A p r o t e i n a v a i l a b l e t o o t h e r n i f p r o m o t e r s may be a f a c t o r i n v o l v e d i n t h e l e v e l o f t r a n s c r i p t i o n a c h i e v e d f rom t h e s e p r o m o t e r s (6,37). However , 4 t h e n i f A p r o d u c t does n o t a p p e a r t o i n t e r a c t d i r e c t l y w i t h any m e t a b o l i c s i g n a l s o r f a c t o r s t o m o d u l a t e n i f e x p r e s s i o n (47). M u t a t i o n s i n n i f L a l t h o u g h r e s p o n s i v e t o ammonium c o n c e n t r a t i o n s , were no l o n g e r n o r m a l l y o x y g e n r e p r e s s e d (34). T h u s , t h e n i f L . gene p r o -d u c t h a s been shown t o be n o n - e s s e n t i a l f o r n i f e x p r e s s i o n and p o s s i b l y i n v o l v e d as a n e g a t i v e e f f e c t o r i n t h e 0^ r e p r e s s i o n o f n i t r o g e n a s e (34,68). T h e r e i s e v i d e n c e t o s u g g e s t t h a t t h e n i f L, p r o d u c t may a l s o be i n v o l v e d i n r e p r e s s i o n o f n i f genes a t l o w b u t n o t h i g h l e v e l s o f ammonium (7,37). R e g u l a t i o n by f i x e d n i t r o g e n compounds a p p e a r s t o o c c u r by a g e n e r a l n i t r o g e n m e t a b o l i s m r e g u l a t o r y s y s t e m (1,16,68,84,91). The m a j o r i t y o f t h e l o c i i n v o l v e d i n t h i s s y s t e m h a v e t h e d e s i g n a t i o n g i n and u n t i l r e c e n t l y t h e g i n A g e n e , w h i c h c o d e s f o r g l u t a m i n e s y n t h e t a s e , was t h o u g h t t o be t h e m a j o r n i t r o g e n r e g u l a t o r y gene i n t h e c e l l (92). However , b e c a u s e o f r e c e n t work done i n K. pneumoniae (44) and by a n a l o g y w i t h work i n o t h e r s y s t e m s s u c h as S a l m o n e l l a (42,66) and E. c o l i (92), i t a p p e a r s t h a t r e g u l a t i o n by t h i s s y s t e m i s much more c o m p l e x . The t r a n s c r i p t i o n a l e f f e c t o f t h i s r e g u l a t o r y s y s t e m seems t o be g e n e r a t e d by i n t e r a c t i o n i n a s i n g l e r e g i o n o f t h e n i f o p e r o n , a t n i f R s i n c e m u t a t i o n s i n t h i s r e g i o n r e n d e r e x p r e s s i o n o f n i t r o g e n a s e i n d e p e n d e n t o f t h e g e n e r a l n i t r o g e n r e g u l a t o r y s y s t e m (67,68). A t h i g h c o n c e n t r a t i o n s o f ammonium, i n w i l d t y p e K. pneumon ia , t h e n i f A and n i f L gene p r o d u c t s a r e n o t s y n t h e s i z e d (67,68). C o n t r o l o f t h e i r e x p r e s s i o n by f i x e d n i t r o g e n compounds o c c u r s by means o f a r e g u l a t o r y s i g n a l a c t i n g a t t h e u n i q u e r e g u l a t o r y s i t e , n i f R (67), p r o b a b l y a t o r n e a r t h e p r o m o t e r f o r t h e n i f R L A o p e r o n . I n a r e c e n t r e v i e w a r t i c l e by R o b e r t s and B r i l l (68), i t i s s u g g e s t e d t h a t K l e b s i e l l a may a l s o have a fo rm o f p o s t - t r a n s c r i p t i o n a l c o n t r o l . 5 They indicate findings that nif-coded mRNAs of K l e b s i e l l a have unusually long h a l f - l i v e s of approximately 30 min. They suggest that NH^+ and 0^ might also act to d e s t a b i l i z e these nif-coded mRNAs as well as being involved i n t r a n s c r i p t i o n a l regulation. However, no data have yet been published. B. Regulation of Nitrogenase i n Azotobacter v i n e l a n d i i Although advances have been made i n our understanding of nitrogenase regulation in.K. pneumoniae, very l i t t l e i s known about the process i n A. v i n e l a n d i i . Some information has been obtained from biochemical ch a r a c t e r i z a t i o n of Nif mutants (21,54,75,78). Even though such mutants are valuable for understanding the nature of the active s i t e of nitrogenase, i t i s d i f f i c u l t to determine genetic linkages of the mutations. Genetic analysis has been hampered i n A. v i n e l a n d i i due to the lack of markers close to n i f mutations. However, a crude genetic map of n i f mutations with respect to each other has been deduced from r a t i o test crosses r e l a t i v e to transformation of a r i f marker (3). It i s i n t e r e s t i n g to note that a mutation causing a defect i n nitrogenase component I i s r e l a t i v e l y c l o s e l y linked to a mutation producing an i n a c t i v e component II (.3), which might indicate close association between the nitrogenase s t r u c t u r a l genes i n A. v i n e l a n d i i as i s seen i n K. pneumoniae (14,37,68). In agreement with t h i s i s the find i n g that an EcoRI r e s t r i c t i o n fragment of A. v i n e l a n d i i chromosomal DNA hybridizes with probes containing e i t h e r n i f H or n i f D from K. pneumoniae (71). As previously mentioned, both components of the nitrogenase complex are extremely oxygen l a b i l e (9,17). Azotobacter i s a s t r i c t aerobe and therefore must protect i t s nitrogenase from being inactivated. It i s believed that t h i s i s accomplished by the tremendous resp i r a t o r y a c t i v i t y of t h i s organism (60). This form of oxygen protection has been named 6 r e s p i r a t o r y protection (61,62,97). Respiratory protection simply means that the rate of reduction i s s u f f i c i e n t l y fast to maintain the 0^ con-centration at the c e l l surface at zero and the environment of nitrogenase anaerobic (96). Oxygen i s r a p i d l y reduced by means of the electron transport system and the i n t e r n a l oxygen concentration probably remains very low. To maintain t h i s high r e s p i r a t o r y a c t i v i t y , a large amount of carbon substrate i s u t i l i z e d (15). It has been shown that when the flow of electrons i s s u f f i c i e n t l y lowered, components I and II of nitrogenase are complexed with another i r o n - s u l f u r protein c a l l e d the Shethna protein or Fe-S protein II (31,70,93). These proteins are found i n t h i s oxygen stable complex i n a 1:1:1 r a t i o . The complex i s apparently not a c t i v e , but a c t i v i t y can be restored when the electron flow (release from carbon l i m i t a t i o n ) i s increased so that the organism i s capable of keeping the i n t e r n a l 0^ tension s u f f i c i e n t l y low. The presence of ammonium i n the culture medium of A. v i n e l a n d i i r e s u l t s i n strong repression of nitrogenase synthesis such that no a c t i v i t y nor a n t i g e n i c a l l y detectable cross reactive material i s detectable (77,82). Some findings indicate that glutamine synthetase may play a r o l e i n t h i s regulation since addition of methionine s u l f o x i -mine (MSX), or methionine sulfone (MSF), i n h i b i t o r s of glutamine synthetase (26), derepresses nitrogenase synthesis i n A. v i n e l a n d i i i n the presence of excess NH^+ as i t does i n K. pneumoniae (26) and A. c y l i n d r i c a <80) . and others. Because ^ reduction requires the expenditure of a large amount of energy and reductant (8), t i g h t c o n t r o l of t h i s system would be expected to be b e n e f i c i a l to the organism. Therefore, a mechanism of feedback i n h i b i t i o n would seem to be an advantage to the N 9 - f i x i n g 7 bacterium. However, recent work by Gordon et^ a l . (28) indicates that nitrogenase i s not subject to feedback i n h i b i t i o n upon addition of NH^+ to derepressed cultures of A. v i n e l a n d i i . Similar r e s u l t s were found f o r K. pneumoniae and C^ . pasteurianum (28). I n s e n s i t i v i t y of nitrogenase synthesis to repression by ammonium i s a property not normally found i n asymbiotic N^-fixing b a c t e r i a . However, s e l e c t i o n f o r mutant s t r a i n s with t h i s property can be accomplished i n a var i e t y of asymbiotic ^ - f i x i n g b a c t e r i a (24,25,47,79,84,87,95). I s o l a -t i o n of such derepressed mutants which synthesize nitrogenase i n the presence of ammonium would be useful i n the determination of factors responsible f o r repression. Some N i f + revertants of the A. v i n e l a n d i i Nif mutant UW2, which i s unable to synthesize e i t h e r component of nitrogenase, were found to be derepressed f o r nitrogenase synthesis (25) . Coordinate synthesis of nitrogenase components I and II i n the wild type (77), the simultaneous loss of both components by a single mutation i n a putative regulatory gene, and simultaneous appearance of both components i n derepressed revertants (25) indic a t e that a common regulatory gene i s required f o r the repression of synthesis of the two components of nitrogenase. Also, the fa c t that only about 30% of the revertants of the Nif mutant UW2 were derepressed f o r n i f expression and that the derepressed l e v e l of nitrogenase a c t i v i t y and synthesis i s less than that l e v e l found i n f u l l derepressed wild type (25) are indi c a t i o n s that control may occur through a p o s i t i v e e f f e c t o r or ac t i v a t o r protein, perhaps analogous to the n i f A product of K. pneumoniae. c The derivation as well as the relevant phenotypes of the Nif st r a i n s derived from UW2 are presented i n Table 1. A genetic analysis 8 Table 1. Derivation and r e l a t i v e phenotypes of Nif s t r a i n s derived from Azotobacter v i n e l a n d i i (UW2). St r a i n UW UW2 & UW1 UW59 UW590 Selection Procedure Phenotype Nif N i f " N i f + Nif° In vivo Igitrogenase A c t i v i t y (using as a N-source) *2 0.35 0 0.18 NH.+CHo00" -4 3 p e n i c i l l i n s e l e c t i o n f o r mutants unable to f i x ^ se l e c t i o n f o r revertant which i s able to grow with N^ as sole nitrogen source s e l e c t i o n f o r f a s t e r growth Nif Nif 0.35 with N^ as sole nitrogen source 0 0 0.07 0.12 \Table complements of Dr. J . K. Gordon. Abstracts of the 2nd Steenbock-Kettering International Symposium on Nitrogen F i x a t i o n . nmoles C„H formed/min ml K l e t t unit 9 c of the mutations i n these Nif s t r a i n s was ca r r i e d out by Dr. J . Gordon (Dr. J . K. Gordon. Abstracts of the 2nd Steenbock—Kettering International Symposium on Nitrogen F i x a t i o n ) . A summary of t h i s analysis follows (data compliments of Dr. Gordon). Transformation was used to map the mutations i n UW59 and UW590. When UW59 DNA was used as donor, a l l of the + c Ni f transformants of the parent s t r a i n UW2 were Nif (Table 2), i n d i c a t i n g that one (or two c l o s e l y linked) mutation i s responsible for both the N i f + c and N i f phenotypes of UW59. When UW590 DNA was used as donor, only some (the small colony type) of the transformants of UW2 (or UW1) were Nif 59 590 (Table 3), i n d i c a t i n g that the n i f and n i f are not c l o s e l y linked 590 because they are e a s i l y separated. Furthermore, the n i f mutation does c 590 not cause the Nif phenotype, however, the n i f mutation does compensate 2 1 + for the n i f and n i f mutations, restoring the Nif phenotype to both UW2 and UW1 (large colony type transformants). Crosses between UW59 and str a i n s which have mutations i n the s t r u c t u r a l genes f o r nitrogenase, UW10 10 38 59 (ni f ) and UW38 (nif ), indicate that the n i f mutation i s unlinked to n i f ^ and n i f ( T a b l e 2). However, i t i s also possible that expression c 2 of the Nif phenotype of UW59 requires the n i f mutation. A biochemical analysis of these derepressed mutant s t r a i n s , UW59 and UW590, was undertaken to further our understanding of the mechanism(s) which c o n t r o l nitrogenase synthesis. The r e s u l t s indicate that a defect i n NH^+ uptake or a s s i m i l a t i o n e x i s t s i n these s t r a i n s and may be responsi-b l e f o r the observed phenotype. Other Nif mutants of A. v i n e l a n d i i , i s o l a t e d as methylalanine r e s i s t a n t (Mai ) mutants, were also studied i n hopes of understanding the mechanism(s) c o n t r o l l i n g nitrogenase synthesis. An unexpected 10 Table 2. Genetic transformation. Donor Recipient % of N i f+ Transformants that are N i f c UW59 UW2 100 UW59 UW1 100 UW UW2 0 UW59 UW38 0 UW59 UW10 2 b Table complements of Dr. J . K. Gordon. Abstracts of the 2nd Steenbock-Kettering International Symposium on Nitrogen F i x a t i o n . The transformants which simultaneously become Nif and Nif are probably a r e s u l t of incorporation of more than one piece of donor DNA. 11 Table 3. Separation of n i f a n d n i f by transformation. 3 Donor UW590 Recipient UW2 Appearance of Transformation Colony small large % Nif 100 6 UW590 UW1 small large 100 0 Small Nif transformant from the cross UW590 X TJW1 UW1 small 100 Table complements of Dr. J . K. Gordon. Abstracts of the 2nd Steenbock-Kettering International Symposium on Nitrogen F i x a t i o n . 12 o b s e r v a t i o n made w i t h t h e s e N i f m u t a n t s was t h a t t h e mutant s t r a i n s e x h i b i t e d d i f f e r e n t l e v e l s o f n i t r o g e n a s e s p e c i f i c a c t i v i t y as t h e d e n s i t y o f t h e c u l t u r e c h a n g e d . A n a l y s i s o f t h i s phenomenon r e v e a l e d t h a t d u r i n g t y p i c a l d i a u x i e g r o w t h , c o n s t i t u t i v e n i t r o g e n a s e a c t i v i t y i n t h e s e s t r a i n s i s dependent on t h e c a r b o n s o u r c e t h e c u l t u r e i s u t i l i z i n g . T h i s may d e -f i n e a r o l e f o r c a r b o n m e t a b o l i s m i n t h e r e g u l a t i o n o f n i t r o g e n a s e . T h i s r e p o r t d e s c r i b e s e x p e r i m e n t s d e s i g n e d t o d e t e r m i n e t h e e f f e c t s o f N H ^ + u p t a k e and u t i l i z a t i o n o f a c e t a t e as c a r b o n s o u r c e on c o n s t i t u t i v e n i t r o g e n a s e s y n t h e s i s and a c t i v i t y . 13 MATERIALS AND METHODS I. B a c t e r i a l Strains The wild type organism used was Azotobacter v i n e l a n d i i OP (11) obtained from the American Type Culture C o l l e c t i o n and i s referred to as s t r a i n JK. S t r a i n UW2 i s a Nif (non-N^-fixing) mutant derived from a d i f f e r e n t wild type s t r a i n designated as UW (25). It was noted that s t r a i n UW subsequently had undergone some modification r e s u l t i n g i n a decreased y i e l d of nitrogenase a c t i v i t y i n fermenter cultures (V.K.Shah, personal communication). Therefore, s t r a i n JK was used i n these studies as the w i l d type i n an attempt to most c l o s e l y mimic s t r a i n UW. St r a i n UW59 i s a mutant derepressed for nitrogenase synthesis i n the presence of NH^+ which was derived from s t r a i n UW2 (25). S t r a i n UW590, another derepressed mutant, was i s o l a t e d as a fast growing d e r i v a t i v e of s t r a i n UW59 (29). Strains JK1, JK2, JK3, and JK4 are another class of derepressed mutants which were i s o l a t e d as methylalanine r e s i s t a n t (Mai ) mutants i n t h i s laboratory by Ms. L i s a Thorson. D e t a i l s of the i s o l a t i o n procedure r and properties of these Mai mutants are presented i n the re s u l t s section. I I . Media and Culture Conditions A l l cultures were grown on a modified Burk's medium (82) with sucrose and/or acetate at the concentrations indicated. Ammonium grown cultures were provided with 400 jag N/ml as ei t h e r ammonium acetate or ammonium s u l f a t e as indicated unless otherwise noted. A l l cultures were grown at 30°C on a rotary shaker i n b a f f l e d f l a s k s , shaking at 260 rpm. Growth was measured using a Klett-Summerson ph o t o e l e c t r i c colorimeter with a number 640 f i l t e r . One K l e t t unit i s equivalent to 5.3x10 c e l l s / m l as determined with a Petroff-Hauser counting chamber (attached c e l l s which appeared to have completed c e l l d i v i s i o n were counted as two c e l l s ) . 14 I I I . Chemicals [U-^C] sodium acetate, [U-^C]sucrose and Aquasol were purchased from New England Nuclear (Lachine, Quebec). [u-^c]methylamine-HCl was obtained both from New England Nuclear (Lachine, Quebec) and Amersham Corp. (Oakville, Ontario). The 2,5-diphenyloxazole (PPO) used for a toluene-based s c i n t i l l a t i o n c o c k t a i l was obtained from Mallinckrodt, Inc. (Paris, Ky.). A l l other chemicals used were reagent grade, a v a i l -able commercially. IV. Genetic Transformation Genetic transformation was performed according to Page and Sadoff (57). Competent r e c i p i e n t c e l l s were grown as described by Page and von Tigerstrom (58) i n Burk's iro n - f r e e (OFe) medium supple-mented with 200 jig N/ml as ammonium acetate for 20-22 hours at 30°C ( u n t i l t u r b i d with a b r i l l i a n t green c o l o r ) . Strains used as donors were grown on agar slants of Burk's medium containing 400 pg N/ml as ammonium acetate at 30°C for 2-3 days. DNA was prepared by l y s i s of the donor c e l l s i n 5 ml of 15 mM saline-15 mM sodium c i t r a t e buffer, pH 7.0, containing 0.05% sodium dodecyl s u l f a t e at 60°C for 60 min. The lysate was then allowed to cool at room temperature. This crude DNA was e i t h e r used d i r e c t l y i n transformation studies or stored f or future use at -20°C. Transformation was c a r r i e d out i n a s t e r i l e test tube containing 0.3 ml of transformation buffer (20 mM MOPS, 16 mM MgCl 2 > pH 7.2), 50 u l of compe-tent r e c i p i e n t c e l l s and 50 yd. of the crude donor DNA for 20-30 min at 30°C Transformation was terminated by the addition of 0.1 ml of DNase I (25 Jig/m to the mixture with gentle mixing. The c e l l suspension was then d i l u t e d as necessary and s e l e c t i o n of N i f + transformants was accomplished using Burk's N-free medium s o l i d i f i e d with Difco p u r i f i e d agar. 15 V. Determination of Labelled Substrate U t i l i z a t i o n To determine which carbon source i s u t i l i z e d p r i m a r i l y by pre-lag c e l l s , p a r a l l e l cultures were grown i n 500 ml flasks containing 100 ml of Burk's medium supplemented with 29 mM ammonium, 14 mM acetate plus 2% 14 (58 mM) sucrose or 7 mM sucrose. To one culture C-labelled acetate (1.5jiCi) was added at the time of i n o c u l a t i o n . To the second f l a s k 14 C-labelled sucrose (0.75jiCi) was added at the time of ino c u l a t i o n . Growth of the cultures and disappearance of the l a b e l from the medium were followed. At the times indicated, 0.5 ml samples of the b a c t e r i a l cultures were subjected to ce n t r i f u g a t i o n for 2 min i n an Eppendorf microfuge (Brinkman Instruments Inc., Westbury, N.Y.). The supernatant so l u t i o n was saved and the r a d i o a c t i v i t y i n duplicate 0.1 ml samples determined by counting with 10 ml Aquasol as s c i n t i l l a t i o n f l u i d i n eit h e r a Nuclear Chicago Isocap 300 l i q u i d s c i n t i l l a t i o n counter or a Beckman LS7500 l i q u i d s c i n t i l l a t i o n counter. VI. Measurement of Sucrose Uptake Cultures were grown to an appropriate c e l l density as indicated. B a c t e r i a l c e l l s were c o l l e c t e d from 40 ml of a 25 K l e t t unit culture or 20 ml of a 75 K l e t t unit culture using vacuum f i l t r a t i o n (0.45 Jim pore s i z e f i l t e r s ) . E x t r a c e l l u l a r sucrose was removed by washing the f i l t e r s containing the b a c t e r i a l c e l l s twice with 10 ml of sucrose-free Burk's medium. The c e l l s were resuspended i n 10 ml sucrose-free Burk's medium and kept at 0-5°C u n t i l assayed. A 10 min aerobic incubation at 30°C preceded ..- 14 ' the addition of IU- CJ sucrose to i n i t i a t e the uptake assay. For assay of sucrose uptake i n the presence of acetate, 7 or 20 mM sodium acetate was added p r i o r to the 10 min pre-incubation. The concentration of sucrose i n the uptake assay was 1.5 mM (0.05%) with s p e c i f i c a c t i v i t y equal to 16 0.57 j i C i / mol. Assays were performed on a rotary shaker at 30°C i n 20 ml glass s c i n t i l l a t i o n v i a l s containing .1.'2 ml medium. At various times within a 2% min period, 5 samples of 200yl volume were removed and the c e l l s c o l l e c t e d by vacuum f i l t r a t i o n on a 0.45 jam pore s i z e f i l t e r (25 mm diame-t e r ) . The wash buffer, Burk's medium containing unlabelled sucrose at 0.05%, was maintained at 30°C and added i n at least 20-fold excess of the sample volume. The f i l t e r s were dried and the r a d i o a c t i v i t y deter-mined using as s c i n t i l l a t i o n f l u i d 10 gm 2,5-diphenyloxazol (PPO) per l i t e r toluene. A l l data were f i t t e d to s t r a i g h t l i n e s using l i n e a r regression a n a l y s i s . VII. Metabolic Enzyme Assays Harvesting c e l l s and preparation of crude extracts. B a c t e r i a l extracts were prepared by the procedure of Senior and Dawes (74). Cul-tures were grown i n Burk's medium containing sucrose and/or acetate as carbon source at the concentrations indicated supplemented with 29 mM NH. as e i t h e r ammonium acetate or ammonium su l f a t e . The c e l l s were 4 harvested at the culture densities indicated by centrifugation at 9,000 rpm for 10 min, washed twice and resuspended i n approximately 5 volumes; of the b u f f e r appropriate to the p a r t i c u l a r enzyme being studied. These buffers were as follows: for glucose-6-phosphate dehydrogenase, 120 mM imidizole-HCl (pH 7.3); 6-phosphogluconate dehydratase plus 2-keto-3-deoxy-6-phosphogluconate aldolase, 100 mM triethanolamine-HCl (pH 7.7); pyruvate dehydrogenase, 200 mM T r i s - H C l (pH 7.9) containing 1 mM d i t h i o -t h r e i t o l . U l t r a s o n i c a t i o n with a Bronwill Biosink (eight 5 sec bursts) was used to disrupt the c e l l s . The sonicated suspension was then sub-jected to c e n t r i f u g a t i o n at 35,000xg for 20 min. Protein was determined by the procedure of Bradford (4). A standard curve was prepared using bovine gamma glo b u l i n . 17 Glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Reaction tubes contained the following i n a 2.5 ml volume: 120 mM imidizole-HCl (pH 7.3), 1.5 ml; 10 mM glucose-6-phosphate, 0.25 ml; 5 mM NAD+, 0.5 ml; c e l l extract, 0.1 ml. The reactants were pre-incubated at 30°C and the reac-t i o n was i n i t i a t e d with the addition of NAD+. Enzyme a c t i v i t y at 30°C was determined by measuring the increase i n absorbance at 340 nm using a G i l f o r d Stasar III spectrophotometer. Values were corrected for any endogenous NADH oxidation occurring. 6-phosphogluconate dehydratase (EC 4.2.1.12) plus 2-keto-3-deoxy- 6 — phosphogluconate (KDPG) aldolase (EC 4.1.2.14). A combined assay for these enzymes measured pyruvate production from 6-phosphogluconate using l a c t a t e dehydrogenase and NADH. Reaction tubes contained the following i n a 3.2 ml volume: 80 mM MnCl 2, 0.01 ml; 80 mM d i t h i o t h r e i t o l , 0.2 ml; la c t a t e dehydrogenase (0.5 mg/ml), 0.01 ml; 10 mM NADH, 0.2 ml; c e l l extract, 0.1 ml; 50 mM 6-phosphogluconate, 0.2 ml; 100 mM triethanolamine-HC1 (pH 7.7), 2.48 ml. The reactants were preincubated at 30°C. The rate of any endogeneous NADH oxidation was measured before the reaction was i n i t i a t e d by addition of 6-phosphogluconate. Enzyme a c t i v i t y was determined at 30°C by measuring the decrease i n absorbance at 340 nm. Pyruvate dehydrogenase (EC 1.2.4.1). Due to the presence of NADH oxidase i n crude extracts the reaction was ca r r i e d out i n the presence of 10 mM KCN which has been shown to i n a c t i v a t e NADH oxidase a c t i v i t y (56). Reaction tubes contained the following i n a volume of 3.0 ml: 200 mM Tris-HCL (pH 7.9), containing d i t h i o t h r e i t o l , 1.85 ml; 166 mM L-cysteine, 99 p i ; 6.2 mM Coenzyme-A (CoASH), 60 jil; 15 mM NAD+, 0.3 ml; 10 mM cocarboxylase, 0.15 ml; 0.25 M MgCl 2, 0.12 ml; 0.25 M sodium pyruvate, 0.12 ml; c e l l free extract, 0.3 ml; 300 mM KCN, 0.1 ml. A l l 18 reactants were preincubated at 30 UC and the reaction was i n i t i a t e d by the addition of sodium pyruvate. Enzyme a c t i v i t y was measured as the increase i n absorbance at 340 nm. mM The m i l l i m o l a r e x t i n c t i o n c o e f f i c i e n t for NADH i s E„._ = 6.2. 340 VIII. Nitrogenase Assays In vivo nitrogenase assays were c a r r i e d out at 30°C with shaking i n 8.5 ml serum v i a l s to which 1.0 ml culture samples were added and stoppered with rubber serum stoppers. Acetylene (0.5ml) was added to st a r t the reaction which was terminated a f t e r 15 min by the addition of 0.1 ml of 30% t r i c h l o r o a c e t i c a c i d (TCA). Production of ethylene (CyRj) was monitored by i n j e c t i o n of 0.3 ml of the gas phase into a Perkin-Elmer 3920 gas chromatograph equipped with a Porapak N column. A c t i v i t y i s reported as nmoles C^H^ formed/min per ml per K l e t t u n i t . The overnight acetylene reduction assay was performed i n the f o l -lowing manner. Strains to be tested were streaked into patches on ammon-ium containing agar plates of Burk's medium and allowed to incubate at 30°C f o r 24 hours. S t e r i l e a p p l i c a t o r s t i c k s were used to transfer the c e l l s from 24 hour patches to tubes containing 0.5 ml Burk's medium supplemented with ammonium acetate. The tubes were incubated with shaking at 30°C for approximately 8 hours ( u n t i l f a i n t l y t u r b i d ) . The contents of the tubes were transferred to 8.5 ml serum v i a l s , stoppered and the reaction i n i t i a t e d by i n j e c t i o n of 0.5 ml acetylene. Overnight incubation with shaking at 30°C was followed by quantitation of C^H^ formed as before, with the exception that the reaction was not stopped by the addition of t r i c h l o r o a c e t i c acid. Extracts f o r measurement of i n vitro nitrogenase a c t i v i t y were prepared as described by Shah et^ a l . (77), with the su b s t i t u t i o n of 19 d i t h i o n i t e f o r d i t h i o t h r e i t o l i n the l y s i n g buffer. A l l buffers were sparged with N 2 gas made oxygen free by passage over a heated copper c o i l . The b a c t e r i a l culture was harvested under an N 2 atmosphere by centrifugation at 10,000xg for 10 min. The p e l l e t was resuspended i n N^-sparged 0.025 M Tris-HCl (pH 7.4) buffer and transferred to a 15 ml corex centrifuge tube, stoppered and flushed several times with N 2 gas. B a c t e r i a l c e l l s were p e l l e t e d by centrifugation as before. The supernatant was removed with a flushed syringe and the tube immediately flushed with N 2 gas. The p e l l e t was resuspended i n 9-10 volumes of sparged 0.025 M Tris - H C l (pH 7.4) buffer containing 4 M g l y c e r o l by vortexing, immediately followed by flu s h i n g with N 2 gas. The resuspended c e l l s were incubated 30 min at room temperature followed by cen t r i f u g a t i o n at 10,000xg for 10 min to p e l l e t the c e l l s . The supernatant was anaerobically removed as before and the tube immediately flushed with N 2 gas. Ly s i s was accomplished by the addition of 3 volumes of l y s i n g buffer (0.3 mg/ml d i t h i o n i t e , 5-10 ^ug/ml DNase I, and sparged 0.025 M T r i s - H c l , pH 7.4 buffer) which had been flushed several times with N 2 gas. Lysed c e l l s were immediately flushed with N 2 upon resuspension i n l y s i s buffer to remove any trace oxygen contamination. C e l l debris and unlysed c e l l s were removed by ce n t r i f u g a t i o n at 10,000xg for 30 min at 0-4°C. The supernatant was anaerobically re-moved and placed i n a sealed serum v i a l which had been previously made anaerobic by flu s h i n g several times with N 2. Upon addition of the extract the serum v i a l was again flushed several times with N 2 > Assay v i a l s f o r the jLn v i t r o reaction were prepared as follows. An ATP generating system containing creatine phosphate (17.2 mg/ml), ATP (2.62 mg/ml), creatine kinase (0.33 mg/ml), 8.2 mM MgCl 2, and N 2 sparged 0.025 M Tris - H C l (pH 7.4) to 55 ml was prepared and flushed several 20 times with N . To stoppered anaerobic 8.5 ml serum v i a l s 0.6 ml of the ATP generating system was added. The v i a l was again flushed with N a n d brought to atmospheric pressure. Removal of 0.9 ml of gas with a flushed syringe was performed to accomodate the following additions: 0.2 ml d i t h i o n i t e s o l u t i o n (105 mg d i t h i o n i t e i n 7 ml of 0.013 N NaOH flushed with N ) and 0.5 ml acetylene. The reaction was i n i t i a t e d by the addi-t i o n of 0.2 ml of c e l l extract and incubated at 30°C for 15 min with shaking. The reaction was terminated by the addition of 0.3 ml of 30% t r i c h l o r o a c e t i c acid. Ethylene (C^H^) produced was quantitated as previously described. The s p e c i f i c a c t i v i t y i s calculated as nmoles ^2^^ f o r m e d / m i n per mg protein. IX. Immunoelectrophoresis Quantitation of nitrogenase component I and component II proteins was accomplished using a modification (23) of the rocket Immunoelectro-phoresis technique described by L a u r e l l (43). Antisera to components I and II were prepared using p u r i f i e d components kind l y provided by Drs. W. J . B r i l l and V. K. Shah, University of Wisconsin. Cultures were harvested at densities indicated by c e n t r i f u g a t i o n and washed twice i n 0.025 M Tr i s - H C l (ph 7.4) buffer. Extracts were pre-pared i n 1.0 ml of the same buffer using a French pressure c e l l , then centrifuged at lOOOxg to remove c e l l debris. The supernatant was removed and assayed for t o t a l p rotein by e i t h e r the procedure described by Bradford (4) or Gornall et a l . (30). Rocket Immunoelectrophoresis was performed i n b a r b i t a l buffer with an i o n i c strength (JJ) of 0.10 (0.12 M b a r b i t a l , 0.1 N NaOH). A 1% agarose gel was prepared i n 0.05 b a r b i t a l buffer, cooled to approx-imately 45°C and a n t i s e r a against e i t h e r component I or II of nitrogenase 21 was added at a r a t i o of 1:8 (one part antisera:8 parts agarose) and mixed we l l . The s o l i d i f i e d gel was pre-electrqphoresed for 20 min at 25 mA (constant current) using a Hoefer PS1200 power supply (Hoefer S c i e n t i f i c Instruments, San Francisco). Ten m i c r o l i t e r samples were loaded into 1 cm diameter wells and the gels run for 12 hours at 100 v o l t s (constant voltage). Peak heights were resolved by soaking the gel i n 0.5 M NaCl overnight. To enhance r e s o l u t i o n the peaks could be stained for less than one minute i n coomassie blue R and quickly destained. Samples were t i t r a t e d i n order to obtain optimal peak heights for measurement. X. Ammonium Disappearance From Medium The amount of ammonium remaining i n the medium was determined using an NH^+ d i s t i l l a t i o n procedure described by Strandberg and Wilson (82). Cultures were grown i n 200 ml of Burk's medium containing 100 yg N/ml as ammonium s u l f a t e . At the times indicated during growth, 3 ml samples were removed in t o two 1.5 ml Eppendorf tubes and centrifuged f o r 2 min i n an Eppendorf microfuge. The supernatant was removed and stored at -20°C u n t i l assayed f o r NH^+ content. The amount of NH^+ i n the supernatant of each sample taken was examined by the d i s t i l l a t i o n of 1 ml samples i n 8.5 ml serum v i a l s . Care-f u l addition of 1 ml of a saturated K^CO^ s ° l u t : ' - o n t o t n e supernatant so as not to mix was followed by stoppering the v i a l with a rubber stopper containing an acid etched glass rod previously coated with 4 N H^SO^. Upon stoppering the v i a l the contents were gently mixed so as not to allow the supernatant-K^CO^ mixture to come into contact with the etched glass rod. The v i a l s were allowed to d i s t i l l overnight at room tempera-ture. The stoppers were then c a r e f u l l y removed without touching the glass rod to the sides of the v i a l and.the d i s t i l l e d NH.+ was rinsed 22 from the glass rod with 2.5 ml of d i s t i l l e d water into a test tube. The amount of NH^+ present was quantitated using Nesslers reagent. XI. Measurement of NH^+ Uptake Rates NH^+ uptake rates were determined by measuring the rate of disap-pearance of NH^+ from the medium as described by Gordon and Moore (27). C e l l s were harvested by centrifugation, washed twice, and suspended to the o r i g i n a l density i n Burk's N-free medium. The washed c e l l suspension was Incubated a e r o b i c a l l y at 30°C f o r 10 min to allow for depletion of remaining e x t r a c e l l u l a r NH^+. At that time, NH^+ (as ammonium sulfate) was added to a f i n a l concentration of 0.8 mM. At 1 min i n t e r v a l s , 1 ml samples were removed and f i l t e r e d through a 0.45 jam pore-size f i l t e r (Gelman, Inc., Ann Arbor, Mich.). The f i l t r a t e c o l l e c t e d was assayed f o r NH^+ content with a glutamate dehydrogenase assay (83). XII. Methylamine (CH 3NH 3 +) Uptake 14 i Uptake of LU- CJ methylammonium was quantitated using the procedure of Gordon and Moore (27). Unless otherwise noted, cultures were grown with NH^+ as nitrogen source to a density of 80 K l e t t u n i t s . For assay of CH 3NH 3 + uptake, a l l traces of e x t r a c e l l u l a r NH^+ were removed p r i o r to i n i t i a t i o n of the assay. This was done by c o l l e c t i n g the c e l l s on a 0.45 um pore-size f i l t e r (Gelman, Inc., Ann Arbor, Mich, or Amicon Canada Ltd., O a k v i l l e , Ontario) and washing twice with 10 ml Burk's N-free medium at a pH corresponding to assay conditions. The f i l t e r was removed, and the c e l l s were suspended i n N-free medium at indicated pH, concentrated two-fold the o r i g i n a l density. C e l l suspensions were kept at 0-5°C u n t i l assayed. A 10 min aerobic incubation at 30°C 14 + preceded the addition of CH 3NH 3 to i n i t i a t e the reaction. Assays 23 were performed i n 20 ml s c i n t i l l a t i o n v i a l s i n volumes ranging from 1.2 to 14 + 1.4 ml. The s p e c i f i c a c t i v i t y of the CH^NH^ was adjusted to permit accurate quantitation of the uptake rates and ranged from 0.2 to 0.4 pCi/ mole. At various times within a 2.5 min period, f i v e samles of 200 ^ i l volume were removed and c e l l s were c o l l e c t e d by vacuum f i l t r a t i o n on a 0.45 jam pore-sized f i l t e r (25 mm diameter). The wash; buffer used was the same as that used i n the uptake assay and contained a concentration of unlabelled CH^ NH^ "1" equivalent to that i n the assay. Wash buffers, maintained at 30°C were added i n at least a 20-fold excess of the sample volume. The f i l t e r s containing b a c t e r i a l c e l l s were dried at 55°C for 60 min and the r a d i o a c t i v i t y was determined by counting i n a Nuclear Chicago Isocap 300 or a Beckman LS7500 l i q u i d s c i n t i l l a t i o n counter. A l l data were f i t t e d to st r a i g h t l i n e s by l i n e a r regression a n a l y s i s . CH^NH^+ uptake under defined atmosphere was performed i n an 8.5 ml serum v i a l containing 1.2 ml of culture washed and concentrated two-fold as previously described. The v i a l s were stoppered and flushed with helium (He) and then brought to one atmosphere. Oxygen was added by i n j e c t i o n to 20% the gas volume, the f i n a l volume of gas being 9.0 ml (under p o s i t i v e pressure). A 10 min incubation at 30°C preceded the 14 + addition of CH^NH^ to i n i t i a t e the reaction. The f i n a l concentration 14 + of CH^NH^ i n the assay was 80pm (0.96 juCi). At various times within 3 min, f i v e 100 p i volume samples were removed with a 10 0 y l Hamilton syringe (Hamilton Co., Reno, Nev.) and assayed as before. At approximately 3.5 min, the 9 ml gas volume was removed with a syringe and 9 ml of a premixed gas containing e i t h e r He/0 2 (80%/20%) or N 2/0 2 (80%/20%) was added. At various times within the next 3 min, f i v e additional 100 jil samples were removed and f i l t e r e d as previously described. The f i l t e r s were dried and 24 the amount of radioactive methylammonium incorporated was determined as previously described. XIII. Protein Determination A modification (73) of the Lowry procedure (45) was used to measure whole c e l l protein f or sucrose and methylamine uptake assays and i n v i t r o nitrogenase assays. The c e l l suspension was centrifuged and the p e l l e t washed with Burk's buffer before being resuspended to the o r i g i n a l density i n d i s t i l l e d water. A 1.0 ml sample of the washed c e l l suspension was mixed with 1.0 ml of 1 N NaOH and immersed i n a b o i l i n g water bath for 5 min. A f t e r cooling, a sample was removed and the pro-t e i n content determined using bovine serum albumin as a standard. The protein concentrations of the c e l l - f r e e extracts used for the metabolic enzyme assays were determined by the method of Bradford (4) using reagents supplied by Bio-Rad (Missasauga, Ontario). Bovine gamma glo b u l i n was used as a standard. 25 RESULTS I. Regulation of Sucrose U t i l i z a t i o n by Acetate A. Diauxie Growth Growth of Azotobacter v i n e l a n d i i on a modified Burk's medium (32) supplemented with 400 jig N/ml as ammonium acetate (29 mM) re s u l t s i n a diauxie growth curve as seen i n Figure 1. Exponential growth to a density of 45 K l e t t units i s characterized by a generation time of 1 hour 40 min. A period of severely reduced growth rate, referred to as a lag, p e r s i s t s f o r approximately 20 min. This lag i s followed by a period of growth characterized by a generation time of 1 hour 50 min. An i d e n t i -c a l growth curve i s observed when a culture i s grown i n medium supplemented with 29 mM NH^+ as ammonium s u l f a t e plus 29 mM sodium acetate. Reduction of the acetate concentration to 14 mM re s u l t s i n onset of the lag at a lower c e l l density but does not a f f e c t the pre- or post-lag generation times. These r e s u l t s suggest that the lag i s due to the depletion of acetate from the medium and that acetate i s , i n fa c t , the preferred carbon source. B. P r e f e r e n t i a l U t i l i z a t i o n of Acetate Acetate can be used as sole carbon source by A. v i n e l a n d i i (20). To determine i f pre-lag cultures u t i l i z e acetate as sole carbon source or use i t i n conjunction with sucrose, duplicate cultures were grown with 28 mM NH.+, 14 mM sodium acetate and 58 mM sucrose. To one culture, 4 r 14 i LU- Cjacetate was added at the time of inoc u l a t i o n . The other culture 14 received [U- C]sucrose. Growth of both cultures was followed together with the number of cpm i n the culture medium. As seen i n Figure 2A, no 14 loss of cpm from the medium containing C-labelled sucrose could be detected i n samples taken before the lag, whereas there was s i g n i f i c a n t 26 Figure 1. Growth curve of Azotobacter v i n e l a n d i i OP on a modi-f i e d Burk's medium containing 400 pg N/ml as ammonium acetate (28 mM) . < Klett units co ui (D ui o o o o ho 28 Figure 2. U t i l i z a t i o n of [U- C]acetate and [ U- C]sucrose during diauxic growth of Azotobacter v i n e l a n d i i . Duplicate cultures were grown i n 100 ml Burk's medium containing 14 mM sodium acetate, 28 mM NH + as ammonium s u l f a t e and 58 mM sucrose (panel A) or 7 mM sucrose (panel B). Labelled acetate (13.3 pCi) was added to one f l a s k (0 0) and l a b e l l e d sucrose (1.7 uCi) added to a duplicate f l a s k (X X) at the time of in o c u l a t i o n . Samples were c o l l e c t e d at the times indicated and pro-cessed as described i n the text. B a c t e r i a l culture density was also followed (• 1) . 30 reduction i n cpm i n samples taken from the culture which had received 14 C-labelled acetate. To enhance the s e n s i t i v i t y of the assay, a lower i n i t i a l concentration of sucrose (7 mM, 0.025%) was used f o r growth of the culture. As seen i n Figure 2B, no disappearance of cpm from the medium of pre-lag cultures could be detected under these conditions e i t h e r . I f sucrose were being u t i l i z e d by the pre-lag culture, one might expect the rate of loss of cpm from the medium (% cpm/ml super-natant per hour) to be approximately threefold lower than that seen for acetate u t i l i z a t i o n i n Figure 2A and, therefore, detectable. This p r e d i c t i o n i s based on the fact that the concentration of sucrose carbon (.84 mM) i s thr e e f o l d greater than that of acetate carbon (28 mM), thus lowering the s e n s i t i v i t y of detection approximately threefold. Loss of cpm from the medium of [JJ-^^C ]sucrose grown cultures i s detectable i n samples taken a f t e r the lag as shown i n Figures 2A and 2B. The r e s u l t s i n d i c a t e that i n medium containing both acetate and sucrose as p o t e n t i a l carbon sources, acetate i s used f i r s t i n preference 14 to sucrose. Similar data was obtained i n experiments using C-labelled 14 glucose i n place of C-labelled sucrose (data not shown). Presumably, acetate u t i l i z a t i o n e f f e c t s regulation of sucrose/glucose u t i l i z a t i o n by interference with transport and/or metabolism of the sugar. C. Sucrose Transport A c t i v i t y i n Acetate U t i l i z i n g C e l l s Organic acids have been shown to a f f e c t regulation of sugar transport systems i n Pseudomonas aeruginosa (51,53) and Arthrobacter  c r y s t a l l o p o i e t e s (40). To determine i f acetate i n t e r f e r e s with sucrose u t i l i z a t i o n i n A. v i n e l a n d i i by a l t e r i n g the sucrose transport a c t i v i t y , the rates of sucrose transport i n acetate u t i l i z i n g pre-lag and sucrose u t i l i z i n g post-lag c e l l s were compared. The r e s u l t s are shown i n Table 4. 31 r— 14—| Table 4. I n i t i a l rates of (TJ- CJ sucrose uptake. UPTAKE RATE CULTURE DENSITY Kl e t t U n i t s 3 CARBON SOURCES PRESENT Sucrose (mM) Acetate (mM) nmol sucrose/min per mg protein .,„, p l u s No Addition ."" ,20 mM Acetate 75 25 25 58 58 0 28 28 60 5.09 0.25 0.00 5.75 0.60 c 75 K l e t t unit cultures are s u c r o s e - u t i l i z i n g post-lag cultures 25 K l e t t unit cultures are a c e t a t e - u t i l i z i n g pre-lag cultures ' i n i t i a l concentrations present i n Burk's medium supplemented with 29 mM NH,+ 4 not tested 32 The derepressed rate of sucrose uptake i n c e l l s taken from post-lag (75 K l e t t unit) cultures grown i n medium containing f u l l sucrose (58 mM) and f u l l acetate (28 mM) was 5.09 nmoles sucrose/min per mg protein, as shown. The uptake a c t i v i t y of c e l l s taken from acetate u t i l i z i n g pre-lag (25 K l e t t unit) cultures was only 5% that rate (0.25 nmoles/min per mg protein). Assays using c e l l s from a culture grown i n medium containing 60 mM acetate but no sucrose revealed no uptake a c t i v i t y . The r e s u l t s i n d i c a t e that acetate (or acetate u t i l i z a t i o n ) does a f f e c t sucrose uptake and might, i n that way, i n t e r f e r e with sucrose u t i l i z a t i o n . To determine i f acetate i n h i b i t s sucrose uptake a c t i v i t y , the e f f e c t of including acetate i n the sucrose uptake assay was examined. The r e s u l t s are shown i n Table 4. No i n h i b i t i o n of sucrose uptake a c t i v i t y resulted from the addition of 20 mM acetate (or 7 mM acetate, data not shown) when e i t h e r pre-lag or post-lag c e l l s from f u l l sucrose plus f u l l acetate medium were used. In f a c t , acetate addition was stimulatory to the sucrose uptake rate of c e l l s taken from pre-lag (25 K l e t t unit) cultures. The r e s u l t s i n d i c a t e that sucrose uptake a c t i v i t y i s not i n h i b i t e d by the presence of acetate and, therefore, that the low sucrose uptake rate i n pre-lag c e l l s grown with f u l l (58 mM) sucrose may be a r e s u l t of repression of the synthesis of a sucrose uptake p r o t e i n (or pro t e i n s ) , or i n h i b i t i o n of sucrose metabolism. D. Metabolic Enzyme A c t i v i t i e s i n Acetate U t i l i z i n g C e l l s Because a reduced rate of sucrose transport would not preclude sucrose metabolism, I sought to determine i f the a c t i v i t i e s of some sucrose metabolizing enzymes might also be affected by acetate. 33 Because A. v i n e l a n d i i metabolizes glucose p r i m a r i l y by the Entner-Doudoroff pathway (81), the a c t i v i t y of glucose-6-phosphate dehydrogenase, the f i r s t oxidative enzyme of the pathway was examined. A comparison of the enzyme a c t i v i t y i n pre-lag and post-lag cultures can be found i n Table 5. Acetate u t i l i z i n g , pre-lag (25 K l e t t unit) cultures, grown i n medium containing f u l l acetate (29 mM) and f u l l sucrose (58 mM) were found to have a maximum glucose-6-phosphate dehydrogenase a c t i v i t y only 44% that found i n sucrose u t i l i z i n g , post-lag (75 K l e t t unit) cultures grown i n the same medium. Cultures grown with 60 mM acetate as sole carbon source, as expected, had severely reduced glucose-6-phosphate dehydrogenase a c t i v i t y as noted i n Table 5. The next metabolic enzyme of the Entner-Doudoroff pathway, 6-phospho-gluconate dehydratase, which catalyzes the conversion of 6-phosphogluconate to 2-keto-3-deoxy-6-phosphogluconate (KDPG) could not be assayed d i r e c t l y . The a c t i v i t y of the enzyme KDPG aldolase, which catalyzes the conversion of KDPG into pyruvate and glyceraldehyde-3-phosphate, also could not be d i r e c t l y measured due to lack of substrate. Therefore, a combined reaction fo r these two sequential enzymes was used. Measurement of pyruvate pro-duced from 6-phosphogluconate was accomplished by addition of la c t a t e dehydrogenase to the reaction mixture and measuring the rate of NADH oxidation as described i n the Materials and Methods. A comparison of a c t i v i t i e s of pre- and post-lag cultures i s presented i n Table 5. Acetate u t i l i z i n g , pre-lag (25 K l e t t u n i t ) , cultures were found to have only 15% of the a c t i v i t y seen i n sucrose u t i l i z i n g , post-lag (75 K l e t t unit) cultures. The a c t i v i t y of pyruvate dehydrogenase, the l a s t enzyme of the Entner-Doudoroff pathway, i s also presented i n Table 5. Acetate u t i l i z i n g , 34 Table 5. Metabolic a c t i v i t i e s of A. v i n e l a n d i i during pre- and post-lag growth. 6-Phospho-gluconate Dehydratase Glucose-6- + Culture Carbon Source Present phosphate KDPG Pyruvate Density Sucrose Acetate Dehydrogenase Aldolase ^  Dehydrogenase (Klett units) 3 (mM) (mM) (Sp. act.) (Sp. act.) (Sp. act.) 75 58 28 46.8 29.0 2.5 25 58 28 23.9 4.3 0.8 25 0 60 2.2 NDS ND 75 K l e t t unit cultures are post-lag, sucrose u t i l i z i n g cultures 25 K l e t t unit cultures are pre-lag, acetate u t i l i z i n g cultures I n i t i a l concentrations present i n Burk's medium supplemented with 29 mM NH, + . 4 S p e c i f i c a c t i v i t y = nmoles NAD+ reduced/min per mg protein S p e c i f i c a c t i v i t y = nmoles NADH oxidized/min per mg protein Not done 35 pre-lag (25 K l e t t unit) cultures were found to have 32% of the pyruvate dehydrogenase a c t i v i t y found i n sucrose u t i l i z i n g , post-lag (75 K l e t t unit) cultures. These r e s u l t s indicate that the presence or u t i l i z a t i o n of acetate does a f f e c t sucrose metabolic enzymes, however, i t appears that the extent of t h i s e f f e c t may vary from enzyme to enzyme. It i s expected that t h i s e f f e c t of acetate on the uptake and metabolism of sucrose i s s u f f i c i e n t to explain the observed p r e f e r e n t i a l u t i l i z a t i o n of acetate during pre-lag growth. E. E f f e c t of a Lower Sucrose Concentration i n the Growth Medium Growth of A. v i n e l a n d i i i n medium containing 14 mM acetate plus only 7 mM sucrose r e s u l t s i n diauxie as previously discussed (Figure 2B). It was expected that both sucrose transport and metabolic a c t i v i t i e s would be repressed i n pre-lag c e l l s grown under these conditions, as they are i n pre-lag c e l l s grown i n medium containing 28 mM acetate and 58 mM sucrose. Data for sucrose uptake and metabolic a c t i v i t i e s observed i n pre-lag c e l l s grown i n medium containing an 8-fold lower i n i t i a l sucrose concentration (7 mM) i s presented i n Table 6. The a c t i v i t y of the com-bined reaction f or the conversion of 6-phosphogluconate into pyruvate and glyceraldehyde-3-phosphate was found to be s i m i l a r to that observed i n pre-lag c e l l s grown i n medium containing f u l l (58 mM) sucrose. Under both culture conditions the a c t i v i t y of t h i s combined reaction involving the enzymes 6-phosphogluconate dehydratase and KDPG aldolase was repressed to about 15-17% of that l e v e l observed i n sucrose u t i l i z i n g post-lag c e l l s (compare Tables 5 and 6). Likewise, the a c t i v i t y of pyruvate dehydrogenase i n pre-lag c e l l s grown i n medium containing 7 mM sucrose was found to be s i m i l a r to that observed i n pre-lag c e l l s grown i n medium containing 58 mM sucrose (Table 6). 36 Table 6. Comparison of uptake and metabolic a c t i v i t i e s i n pre-lag c e l l s grown i n medium containing 28 mM acetate plus a high (58 mM) or low (7 mM) sucrose concentration. I n i t i a l Sucrose Concentration Sucrose Uptake Rate 3 Glucose -6-phosphate 6-Phospho-gluconate Dehydratase + KDPG Pyruvate Dehydrogenase Aldolase Dehydrogenase 7 mM 58 mM 6.71 0.25 55.2 23.9 4.9 4.3C 1.0 0.8' nmoles sucrose/min per mg protein nmoles NAD+ reduced/min per mg protein nmoles NADH oxidized/min per mg protein 'data from Table 4 data from Table 5 37 No s i m i l a r i t y between pre-lag c e l l s grown i n medium containing 7 mM sucrose compared to pre-lag c e l l s grown i n medium containing 58 mM sucrose was observed f o r sucrose uptake nor for glucose-6-phosphate dehydrogenase a c t i v i t y (Table 6). Both of these a c t i v i t i e s were derepressed i n pre-lag c e l l s taken from medium containing 7 mM sucrose. In f a c t , the l e v e l s of derepression observed were higher than those l e v e l s observed i n sucrose u t i l i z i n g , post-lag c e l l s taken from cultures grown i n medium containing 29 mM acetate plus 58 mM sucrose (compare Tables 4, 5 and 6). The data indicate that sucrose uptake and the enzyme glucose-6-phosphate dehydrogenase are regulated d i s s i m i l a r l y from the remainder of the Entner-Doudoroff pathway enzymes. It appears that the enzymes early i n the sucrose metabolic pathway are repressed by acetate when the growth medium contains high l e v e l s of sucrose (58 mM). However, they are also s e n s i t i v e to low l e v e l s of sucrose i n the growth medium. Under such con-d i t i o n s (low l e v e l s of sucrose i n the- growth medium) the a c t i v i t y of sucrose uptake and glucose-6-phosphate dehydrogenase i s derepressed to a maximal or greater than maximal l e v e l . The remainder of the Entner-Doudoroff pathway enzymes however, appear not to be s e n s i t i v e to low l e v e l s of sucrose i n the growth medium and remain repressed under such conditions i n the presence of acetate. It i s predicted that t h i s repres-sion of the l a t e r enzymes of the Entner-Doudoroff pathway i s responsible f o r the diauxic growth pattern observed i n Figure 2B. F. Regulation of Sucrose U t i l i z a t i o n Derepression of glucose transport and metabolic a c t i v i t i e s have been noted f o r Pseudomonas aeruginosa (13) and Arthrobacter c r y s t a l l o p o i e t e s (40) when these organisms were grown with an organic acid and a low i n i t i a l concentration of glucose (7-15 mM) as carbon sources. However, i t 38 was not stated whether or not. p r e f e r e n t i a l u t i l i z a t i o n of the organic acid s t i l l occurred. A. v i n e l a n d i i , when grown i n medium containing 14 mM acetate and a sucrose concentration of 7 mM, . e x h i b i t s , diauxie growth (Figure 2B). Although both sucrose uptake and glucose-6-phosphate dehydrogenase a c t i v i t i e s are f u l l y derepressed (compare Tables 4, 5 and 6), pyruvate dehydrogenase and the a c t i v i t y of the combined reaction i n v o l v i n g the enzymes 6-phosphogluconate dehydratase and KDPG aldolase are not derepressed at low sucrose concentration. To determine i f acetate a f f e c t s sucrose u t i l i z a t i o n by i n h i b i t i n g these sucrose metabolic enzymes, duplicate cultures were grown i n medium containing 28 mM NH^+, 14 mM acetate and 7 mM sucrose. To one culture [U-^C ]sucrose (9 pCl) and 2.9 mmoles of sodium acetate were added when the culture reached a post-lag (sucrose induced) density of 40 K l e t t u n i t s . The other culture received only[U-^C]sucrose (9 jaCi) at the same culture density. Both disappearance of cpm from the medium and incorpora-t i o n of cpm into c e l l u l a r material were followed. The r e s u l t s seen i n Figure 3 indicated that there i s l i t t l e or no i n h i b i t i o n of sucrose u t i l i z a t i o n by acetate i n c e l l s already induced f o r sucrose u t i l i z a t i o n . As seen i n Figure 3A, upon addition of acetate 14 and C-labelled sucrose to the culture, an immediate lag i n growth i s observed. In contrast to t h i s , only a s l i g h t i n t e r r u p t i o n i n growth i s 14 observed i n the culture receiving only C-labelled sucrose (Figure 3B). This observation indicates that a p h y s i o l o g i c a l shock r e s u l t s upon the addition of acetate to the culture. This p h y s i o l o g i c a l shock, however, is. only a transient e f f e c t as noted i n Figure 3A. Similar findings have been reported by Kurz e_t al. (41) . They observed a transient e f f e c t on i n vivo nitrogenase a c t i v i t y upon the addition of acetate to a nitrogen 39 Figure 3. E f f e c t of acetate on uptake and incorporation of [U-^C] sucrose i n cultures of Azotobacter v i n e l a n d i i induced for sucrose u t i l i z a t i o n . Duplicate cultures were grown i n 100 ml of Burk's medium containing 28 mM NH^+, 14 mM acetate and 7 mM sucrose. At the culture density indicated by the arrows 14 LU- CJsucrose (9 jiCl) and 29 pmoles of sodium acetate (Figure r 14 i 3A) or LU- CJsucrose (9 pCi) (Figure 3B) were added. Samples were c o l l e c t e d at times indicated and assayed for cpm remain-ing i n the medium (X X) and cpm incorporated into c e l l s ( A A ) a s described i n the Materials and Methods. B a c t e r i a l culture density also followed (• • ) . Hours 41 f i x i n g culture of A. v i n e l a n d i i . Acetate has been used to measure A pH, due to i t ' s a b i l i t y to permeate the membrane against a pH gradient (64). Thus, the observed p h y s i o l o g i c a l shock e f f e c t may r e s u l t from an in t e r r u p t i o n i n energy flow due to a depletion of &pH. As seen i n Figure 3, a lower i n i t i a l rate of incorporation of 14 C-labelled sucrose into c e l l s i s observed i n the culture which received 14 both acetate and C-labelled sucrose (Figure 3A). This i s followed by a rate approximately equal to that observed i n the culture which received 14 only C-labeled sucrose. The lower i n i t i a l rate of sucrose incorporation into c e l l s l a s t s f o r a time equivalent to the lag i n growth observed i n Figure 3A. Therefore, a p h y s i o l o g i c a l shock upon addition of acetate to a sucrose induced culture might also explain the lower i n i t i a l rate of 14 C-labelled sucrose incorporation observed. 14 Analysis of the disappearance of C-labelled sucrose from the medium revealed a s l i g h t l y slower rate i n the culture which received both sucrose and acetate. It i s known that both uptake and metabolism of acetate are c o n s t i t u t i v e i n A. v i n e l a n d i i (63). I f there i s no i n h i b i t i o n of sucrose u t i l i z i n g a c t i v i t y by acetate, then upon the addition of both sucrose and acetate to a culture induced f o r sucrose u t i l i z a t i o n , a lower rate of u t i l i z a t i o n of each of these carbon sources might be expected. This would be due to competition for the u t i l i z a t i o n of each of the carbon sources. This might explain the difference i n the 14 rates of disappearance of C-labelled sucrose observed i n Figure 3. These r e s u l t s suggest that the p r e f e r e n t i a l u t i l i z a t i o n o f acetate observed i n pre-lag c e l l s may be due to repression o f some protein^s) involved i n sucrose u t i l i z a t i o n . 42 II. Regulation of Con s t i t u t i v e Nitrogenase A. Nitrogenase A c t i v i t y A d e s c r i p t i o n of the s t r a i n s used and t h e i r phenotypes i s given i n Table 1. The wild type s t r a i n used i n these studies i s Azotobacter  v i n e l a n d i i OP (11) obtained from the American Type Culture C o l l e c t i o n and i s designated as A. v i n e l a n d i i s t r a i n JK. The Nif mutant s t r a i n UW2 described below, was derived from a d i f f e r e n t wild type s t r a i n designated as UW (25). It was noted that s t r a i n UW subsequently had undergone some modification r e s u l t i n g i n a decreased y i e l d of nitrogenase a c t i v i t y i n fermenter cultures (V.K. Shah, personal communication). Therefore, s t r a i n JK was used i n these studies as the wild type s t r a i n i n an attempt to more c l o s e l y mimic the o r i g i n a l w ild type s t r a i n UW. Wild type A. v i n e l a n d i i s t r a i n JK has an i n vivo nitrogenase a c t i v i t y of 0.35 nmoles G^H^ formed/min per ml per K l e t t unit when grown with N as nitrogen source. S t r a i n UW2 does not synthesize e i t h e r component of nitrogenase (25) and expresses no i n vivo nitrogenase a c t i v i t y when allowed to incubate i n N-free medium a f t e r transfer from N H ^ + containing medium. A revertant of s t r a i n UW2, UW59, synthesizes nitrogenase i n N-free medium but the a c t i v i t y i s only 50% of that found i n the wild type cultures. Consequently, growth with N as sole nitrogen source i s slower than that of the wild type (25). When grown i n medium containing 29 mM NH ~^*~ as ammonium acetate, UW59 has a maximum c o n s t i t u t i v e nitrogenase a c t i v i t y only 20% (0.07 nmoles ^^^2 f° r m ed/min per ml per Kl e t t unit) that of the f u l l y derepressed wild type l e v e l . A derivative of s t r a i n UW59, s t r a i n UW590, was selected f o r i t s a b i l i t y to grow more ra p i d l y using N 9 as nitrogen source (29). UW590 has approximately twice 43 the nitrogenase a c t i v i t y of UW59 when grown with e i t h e r N„ or NH.+ as ^ 4 nitrogen source. Thus, f u l l y derepressed nitrogenase a c t i v i t y i s present i n grown cultures. The maximum nitrogenase a c t i v i t y expressed when cultures of UW590 are grown i n medium containing 29 mM ammonium acetate i s 0.12 nmoles C^H^ formed/min per ml per K l e t t unit, approximately 34% of the f u l l y derepressed l e v e l . The a c t i v i t y expressed i s commensurate with the amount of nitrogenase protein synthesized (28). B. E f f e c t of pH on Derepressed Nitrogenase Synthesis When 14 mM ammonium s u l f a t e i s used as nitrogen source, the pH of the medium decreases dramatically as the density of the culture increases. As the pH decreases, the ^n vivo nitrogenase a c t i v i t y i n cultures of UW590 increases s i g n i f i c a n t l y as shown i n Figure 4, to a maximum a c t i v i t y of 0.20 nmoles formed/min per ml per K l e t t unit, representing a L7 fold greater a c t i v i t y than that observed when ammonium acetate i s used as N-source. Similar r e s u l t s are observed for s t r a i n UW59. There are several possible explanations f o r t h i s r e s u l t . One p o s s i b i l i t y i s that there i s stimula-t i o n of energy dependent nitrogenase a c t i v i t y because of an increased proton gradient across the membrane. •' A second p o s s i b i l i t y i s that nitrogenase synthesis i s stimulated by the decreasing pH. That there i s no stimulation of jLn vivo nitrogenase a c t i v i t y at low pH was shown by measuring the a c t i v i t y of the wild type s t r a i n JK and s t r a i n TJW590 at pH 5.9 and 7.3. The r e s u l t s are shown i n Table 7. When cultures of wild type and UW590 were grown with N^ as sole nitrogen source to a density of 75 K l e t t units, the pH of the culture medium was approximately 7.3. B a c t e r i a l c e l l s were harvested by f i l t r a t i o n , washed and resuspended to the same density i n fresh growth medium adjusted to appropriate pH values. The i n vivo nitrogenase a c t i v i t y was determined 44 Figure 4. The e f f e c t of culture pH on c o n s t i t u t i v e i n vivo nitrogenase a c t i v i t y . Azotobacter v i n e l a n d i i s t r a i n UW590 was grown i n Burk's sucrose medium containing ammonium s u l f a t e (14 mM) as nitrogen source. Both culture pH (0 0) and i n vivo nitrogenase a c t i v i t y were assayed at the times indicated. 2 4 6 Hours 46 Table 7. E f f e c t of pH on i n vivo nitrogenase a c t i v i t y . Nitrogenase A c t i v i t y Culture In vivo pH 5.9 pH 7.2 Stra i n N-Source pH Nitrogenase Sp. Act. t = 0 t = 10 t = 0 t = 10 JK N2 7.3 0.32 0 .00 0.02 0.17 0.25 UW590 N2 7.3 0.33 — 0.00 — 0.20 JK (NH 4)^0 4 5.3 0.00 0 .00 0.00 0.00 0.00 UW59 (NH 4)^0 4 5.7 0.10 0 .00 0.00 0.04 0.05 UW590 (NH 4)^0 4 5.6- 0.20 0 .11 0.14 0.19 0.20 UW590 CHoC00NH/ 3 4 7.2 0.10 0 .00 0.00 0.05 0.09 nmoles C„H formed/min-ml'Klett unit 47 immediately or a f t e r 10 min incubation at the pH indicated. Similar r e s u l t s were obtained when c e l l s were harvested and washed by centrifuga-tion. The nitrogenase a c t i v i t y was lowest i n medium of pH 5.9 and highest i n medium of pH 7.2. However, i t i s possible that the sudden decrease i n pH r e s u l t i n g when cultures at pH 7.3 are resuspended i n fresh growth medium at low pH may be i n h i b i t o r y . To ru l e out t h i s p o s s i b i l i t y , cultures were also grown i n medium containing ammonium su l f a t e as N-source so that the i n i t i a l culture pH would be low, then harvested and resuspended i n fresh medium of higher pH. The r e s u l t s were the same regardless of the proto-c o l used (Table 7): i n vivo nitrogenase a c t i v i t y of cultures resuspended i n medium of higher pH was greater than that of cultures resuspended i n medium of lower pH. Thus, the increased maximum nitrogenase a c t i v i t y observed i n cultures of UW590 (Figure 4) when grown i n medium containing 29 mM NH^+ as ammonium s u l f a t e i s not due to a stimulation of energy dependent nitrogenase a c t i v i t y by an increased proton gradient at low pH. The second p o s s i b i l i t y , that s y n t h e s i s of nitrogenase i s stimulated by the decreasing pH and i s thereby responsible for the increase i n observed a c t i v i t y , was examined by L a u r e l l I m m u n o e l e c t r o p h o r e s i s . The r e s u l t s , as seen i n Table 8, show that that amount of immunologically detectable p r o t e i n increases more t h a n 6-8-fold for TJW590 as the pH of the culture drops to pH 5.6 during growth on medium containing 29 mM NH^+ as ammonium su l f a t e . A corresponding increase i n in vivo nitrogenase a c t i v i t y was observed (Table 8), suggesting good agreement between i n vivo nitrogenase a c t i v i t y and the amount of protein present. Similar r e s u l t s were observed f o r s t r a i n UW59. 4 8 Table 8. Comparison of Nitrogenase a c t i v i t y and protein at d i f f e r e n t culture densities f o r c e l l s grown using ammonium su l f a t e as N-source. Str a i n Nitrogen Source Culture a Densxty Culture pH . % In vivo Nitrogenase A c t i v i t y % Nitrogenase Component % Nitrogenase Component I I d JK N 2 8 0 7 . 3 1 0 0 1 0 0 1 0 0 JK C H „ C O O ~ N H , + 3 4 8 0 7 . 2 0 . 0 0 JK ( N H 4 + ) 2 S 0 4 - 2 8 0 5 . 3 0 4 6 U W 5 9 0 ( N H 4 + ) 2 S O 4 - 2 2 5 6 . 2 4 6 7 U W 5 9 0 ( N H 4 + ) 2 S 0 4 _ 2 5 0 5 . 8 1 3 1 9 2 5 U W 5 9 0 ( N H 4 + ) 2 S 0 4 - 2 7 5 5 . 6 6 4 5 5 6 0 U W 5 9 ( N H 4 + ) 2 S 0 4 " 2 2 5 6 . 2 6 8 1 8 U W 5 9 ( N H 4 + ) 2 S 0 4 - 2 5 5 5 . 9 1 9 2 0 2 7 U W 5 9 ( N H 4 + ) 2 S 0 4 _ 2 7 5 5 . 4 3 5 3 6 4 8 Culture density i s reported i n K l e t t units 'l00% i n vivo nitrogenase a c t i v i t y i s 0.31 nmoles C 2H 4 formed/min per ml per K l e t t unit 100% l e v e l of component I i s 2 . 7 mm peak height/ug protein 'l00% l e v e l of component II i s 2.13 mm peak height/ug protein 49 Therefore, the increase i n maximum nitrogenase a c t i v i t y observed for s t r a i n UW590 when grown on medium containing 14 mM ammonium s u l f a t e as nitrogen source (Figure 4, Table 8) compared to that observed when 29 mM ammonium acetate was used as nitrogen source (Table 1) i s correlated with an increased synthesis of nitrogenase protein. An i n t e r e s t i n g observation i s that a small amount of both component I and component II i s detectable for the wild type s t r a i n JK when grown on medium containing 14 mM ammonium s u l f a t e as nitrogen source. However, no i n vivo nitrogenase a c t i v i t y i s detectable (Table 8). C. NH 4 + Uptake Synthesis of nitrogenase by the wild type A. v i n e l a n d i i i s known to be repressed i n the presence of NH^+ (77,82). The mutation(s) which r e s u l t s i n the p a r t i a l derepression of nitrogenase synthesis and a c t i v i t y i n the presence of NH^+ i n st r a i n s UW59 and UW590 has not as yet been characterized. I f the a b i l i t y of these s t r a i n s to take up NH.+ was 4 impaired at low pH they would accumulate less fixed nitrogen as the pH of the culture drops and should consequently synthesize more nitrogenase protein, as has been observed (Table 8). To investigate the p o s s i b i l i t y that these derepressed mutants might be impaired i n t h e i r a b i l i t y to take up NH^+ at low pH, the amount of NH^+ remaining i n the medium of cultures of the wild type s t r a i n JK and s t r a i n UW590 grown i n medium containing an i n i t i a l concentration of 100 jig N/ml as ammonium s u l f a t e were compared. Preliminary r e s u l t s com-p i l e d from two such experiments are shown i n Figure 5. The r e s u l t s i n d i -cate that as the pH of the culture decreases during growth, less NH^+ i s 50 t a k e n up by s t r a i n UW590 t h a n i s t a k e n up by t h e w i l d t y p e s t r a i n J K . I n a n o t h e r e x p e r i m e n t w h i c h does n o t depend on g r o w t h , t h e a b i l i t y o f s t r a i n UW590 t o t a k e up N H ^ + was a l s o compared t o t h a t o f s t r a i n J K by m e a s u r i n g t h e r a t e o f d i s a p p e a r a n c e o f NH^"*" f r o m t h e medium i n a 20 m i n a s s a y . The a s s a y was p e r f o r m e d a t pH 5.9 t o m a x i m i z e any d i f f e r e n c e s w h i c h m i g h t e x i s t be tween t h e s e s t r a i n s as i s s u g g e s t e d i n F i g u r e 5. P r e l i m i n a r y r e s u l t s c o m p i l e d f rom two s u c h a s s a y s a r e shown i n T a b l e 9. The d a t a i n d i c a t e t h e r a t e o f N H ^ + u p t a k e ( d i s a p p e a r a n c e ) f o r s t r a i n UW590 i s l o w e r t h a n t h a t o f s t r a i n J K , s u g g e s t i n g a d e f e c t i n t h e a b i l i t y o f s t r a i n UW590 t o a c c u m u l a t e N H ^ + a t l o w p H . From t h e s e p r e l i m i n a r y r e s u l t s i t a p p e a r s t h a t s t r a i n UW590 m i g h t be a l t e r e d i n i t s a b i l i t y t o t a k e up N H ^ + a t l o w pH and t h u s s y n t h e s i s o f n i t r o g e n a s e p r o t e i n i s f u r t h e r d e r e p r e s s e d w i t h d e c r e a s i n g c u l t u r e pH . However , t h e c o n t i n u e d N H ^ + u p t a k e r e q u i r e d t o q u a n t i t a t e t h e amount o f NH^"*" r e m a i n i n g i n t h e medium d u r i n g g r o w t h ( F i g u r e 5) and t h e r a t e o f + + NH^ d i s a p p e a r a n c e ( T a b l e 9) depend on a s s i m i l a t i o n o f NH^ . T h e r e f o r e , t h e p o s s i b i l i t y c a n n o t be r u l e d ou t t h a t t h e d e r e p r e s s e d s t r a i n UW590 may be a l t e r e d i n some a s p e c t o f ammonium a s s i m i l a t i o n . D. CH^NH + T r a n s p o r t The p o s s i b i l i t y t h a t t h e m u t a n t s UW59 and UW590 a r e a l t e r e d i n t h e i r a b i l i t y t o t r a n s p o r t N H ^ + a t l o w pH was e x a m i n e d by u s i n g t h e NH^"1" a n a l o g methylammonium (CH^NH^"1"). Methy lammonium and NH^"*" s h a r e a common b i n d i n g s i t e f o r t r a n s p o r t i n A. v i n e l a n d i i (2,27). T h i s s h o u l d make i t p o s s i b l e t o s t u d y t h e p r o p e r t i e s o f t h e NH^"*" t r a n s p o r t s y s t e m u s i n g methylammonium (CH^NH^"1") as s u b s t r a t e . The a d v a n t a g e o f u s i n g CH^NH + as s u b s t r a t e i s t h a t t h e i n i t i a l r a t e o f u p t a k e s h o u l d n o t depend on a s s i m i l a t i o n o f t h e Figure 5. Ammonium remaining i n the cu l ture medium of s t r a i n s JK and UW590 during growth. 0 0 UW590 • • JK 52 53 Table 9. Rate of NH uptake at pH 5 .9 . + S t r a i n NH 4 Uptake (nmoles/min per mg protein) JK . 66.3 UW590 31.4 54 s u b s t r a t e (2,27) w h e r e a s t h e c o n t i n u e d NH^T u p t a k e r e q u i r e d t o q u a n t i t a t e t h e r a t e o f NH^"*" d i s a p p e a r a n c e o r t h e amount o f NH^"*" r e m a i n i n g i n t h e medium d u r i n g g r o w t h does depend on a s s i m i l a t i o n . A c o m p a r i s o n o f C H ^ N H ^ u p t a k e r a t e s a t a s u b s t r a t e c o n c e n t r a t i o n o f 1 mM C H ^ N H ^ r e v e a l e d t h a t t h e d e r e p r e s s e d m u t a n t s UW59 and UW590 a r e d e f e c t i v e i n t h e i r a b i l i t y t o t a k e up CH^NH^ + a t b o t h pH 5 .9 and pH 7.2 ( T a b l e 10). C u l t u r e s were grown t o l o w pH i n medium c o n t a i n i n g 14 mM ammonium s u l f a t e as n i t r o g e n s o u r c e and a s s a y e d a t pH 5.9 t o m a x i m i z e any d i f f e r e n c e s p r e s e n t b e t w e e n s t r a i n s . S t r a i n s UW59 and UW590 were f o u n d t o t a k e up CH N H 3 + (1 mM) a t r a t e s 45% and 36% t h a t o b s e r v e d f o r t h e w i l d t y p e s t r a i n JK, r e s p e c t i v e l y . Methy lammonium u p t a k e r a t e s , a s s a y e d a t pH 7.3, were a l s o examined f o r c u l t u r e s grown i n medium c o n t a i n i n g 29 mM ammonium a c e t a t e as n i t r o g e n s o u r c e ( t h e f i n a l pH o f t h e c u l t u r e was 7.2). S t r a i n UW59 e x h i b i t e d a r a t e a p p r o x i m a t e l y 64% t h a t o b s e r v e d f o r t h e w i l d t y p e . P r e l i m i n a r y r e -s u l t s f r o m t h r e e a s s a y s o f s t r a i n TJW590 r e v e a l e d s i m i l a r f i n d i n g s . Methylammonium u p t a k e (1 mM CH^NH^"1") was a l s o a s s a y e d a t pH 5.9, a g a i n t o m a x i m i z e any d i f f e r e n c e s b e t w e e n s t r a i n s , f o r c u l t u r e s grown u s i n g 29 mM ammonium a c e t a t e as n i t r o g e n s o u r c e . No s i g n i f i c a n t d i f f e r e n c e s c o u l d b e d e t e c t e d b e t w e e n t h e m u t a n t s t r a i n s and t h e w i l d t y p e u n d e r t h e s e c o n d i t i o n s ( T a b l e 10). The r e a s o n f o r t h i s l a c k o f d i f f e r e n c e i n CH^NH^"1" u p t a k e i s as y e t unknown. No s i g n i f i c a n t d i f f e r e n c e was d e t e c t a b l e b e t w e e n s t r a i n UW2 and t h e 2 w i l d t y p e s t r a i n JK u n d e r any o f t h e c o n d i t i o n s a s s a y e d . T h u s , t h e n i f m u t a t i o n i s n o t r e s p o n s i b l e f o r t h e d e f e c t i v e CH^NH^"*" u p t a k e p h e n o t y p e o b -s e r v e d i n s t r a i n s UW59 and UW590. 55 Table 10. CH 3NH 3 + uptake rates. Nitrogen Source (i n growth Assay media) pH CH 3NH 3 + Uptake Rates (nmoles/min*mg protein) J K UW2 UW59 UW590 Large Colony Type £ Transformants' (NH 4) 2S0 4 NH,+CHoC00" 4 J NH,+CH C00~ 4 3 5.9 1.7±0.4 1.1+0.2 '/0..8±0.3 • 0.6+0.3 01.4+0.40 5.9 2.0+0.6 2.210.3 . 2..1+0.3 1.910.2 1.5±0.30 b 7.2 5.5±0.6 5.3±0.8 3.5±0.8 3.5±l.l b 4.9±0.34 b a R e f e r to Table 3. Cumulative data from three assays. 56 These observations ind i c a t e that the derepressed mutants UW59 and UW590 are defective i n t h e i r a b i l i t y to take up CH 3NH 3 +/NH 4 +. The data also suggests that t h i s defect i s further enhanced at low pH. This leads to the i n t e r e s t i n g p o s s i b i l i t y that the Nif phenotype might r e s u l t from an alte r e d a b i l i t y to take up NH +/CH NH + . Such a p o s s i b i l i t y would suggest a r o l e f or NH^/CH^NH^"1" uptake i n the regulation of nitrogenase. + , , + If NH / CH NH uptake i s involved i n the regulation of nitrogenase, then i t i s possible that a mutation i n t h i s uptake system might r e s u l t i n at l e a s t p a r t i a l derepression of nitrogenase synthesis i n the presence of + 2 NH^ . As previously shown, the n i f mutation alone ( s t r a i n UW2) does not confer a defective CH^ NH^ "*" uptake mechanism (Table 10) . However, i n 59 c mutants containing the n i f mutation (UW59 and UW590), both the Nif phenotype (Table 1) and a defective CH^ NH^ "1" uptake mechanism ex i s t 59 (Table 10). Therefore, i t appears that the n i f mutation i s responsible + + c for the defective NH^ /CH^NH^ uptake mechanism as we l l as the Nif pheno-type. 59 As shown i n Table 3, i t i s possible to separate the n i f mutation 590 from the n i f mutation of s t r a i n UW590 by transformation. Transformation 2 2 59 590 of s t r a i n UW2 ( n i f ) using UW590 (ni f , n i f , n i f ) DNA as donor r e s u l t s 2 i n two types of transformants: small colony type transformants ( n i f , 59 + c n i f ) which are s i m i l a r to UW59 phenotypically (Nif , Nif ) and :in 2 590 colony morphology; and large colony type transformants ( n i f , n i f ) which are s i m i l a r to UW590 phenotypically ( N i f + ) and i n colony morphology. Preliminary analysis of CH^ NH^ "1" uptake rates at a substrate concentration of 1 mM CH^ NH.^ " f o r the large colony type transformants i s presented i n Table 10. No s i g n i f i c a n t difference i n uptake rates between these large transformants and the wild type s t r a i n (JK) was detected. These r e s u l t s 57 59 also support the hypothesis that the n i f mutation i s responsible for c + + both the Nif phenotype and the defective NH /CH NH uptake system 4 3 3 observed i n s t r a i n s UW59 and UW590 at a substrate concentration of 1 mM CH^NH^. However, for a more complete analysis, CH^ NH^ "1" uptake i n the + c Nif Nif small colony type transformants needs to be assayed. 59 ' 2 The p o s s i b i l i t y that the n i f and the n i f mutations are both + c required for the defective CH^NH^ uptake and the Nif phenotype however can not be ruled out from t h i s analysis. E. CH^ NH^ "1" Uptake Under a Defined Atmosphere Another possible explanation for the r e s u l t s observed i n Table 10 i s that nitrogenase regulates NH^/CH^NH^"1" uptake a c t i v i t y . If t h i s p o s s i b i l i t y i s correct, as nitrogenase a c t i v i t y increases due to some stimulus, CH^NH^ uptake a c t i v i t y would be expected to decrease. To examine t h i s p o s s i b i l i t y , the e f f e c t of nitrogenase a c t i v i t y on CH^ NH^ "1" uptake a c t i v i t y was examined by comparing CH^ NH.^ " uptake rates under N^-fixing (N^/O^ atmosphere) and under non-N^-fixing (He/O^ atmos-phere) conditions as described i n the Materials and Methods. Preliminary r e s u l t s from a sing l e such experiment (Table 11) suggest that UW590 takes up". CH^ NH^ "1" (at pH 7.3) at the same rate regardless of the atmosphere. • Therefore, 1 CH^ NH^ "1" uptake i s probably no t r e g u l a t e d b y t h e a b i l i t y o f t h e c e l l t o f i x N 2 -I t i s i n t e r e s t i n g t o n o t e t h a t t h e r a t e o f u p t a k e i n c r e a s e d f o r b o t h t h e N ^ - f i x i n g and n o n - ^ - f i x i n g c u l t u r e s a f t e r e x c h a n g e o f t h e gas v o l u m e s . I t i s b e l i e v e d t h a t t h i s i n c r e a s e d r a t e o f CH^NH^"1" u p t a k e i s t h e r e s u l t o f a d d i t i o n a l oxygen a v a i l a b l e t o t h e c u l t u r e upon c h a n g i n g t h e gas vo lume f r o m p r e i n c u b a t i o n c o n d i t i o n s (He/0 2 a t m o s p h e r e ) t o N ^ - f i x i n g o r n o n - N ^ - f i x i n g c o n d i t i o n s . Table 11. Methylammonium uptake under defined atmospheres. 14 + moles CH^NH^ taken up Atmosphere per min per mg protein preincubation 3 0.046 He/0„ (80:20) b 0.121 '2 '2' J2 N o/0 o (80:20)° 0.110 Preincubat ion of both cultures was performed under a He/02 (^0*^0) atmosphere. Gas volumes were changed as indicated i n the Materials 14 and Methods at 10 min a f t e r addition of CH^NH^. Non-nitrogen f i x i n g conditions. Nitrogen f i x i n g conditions. 59 F. K i n e t i c s of CH 3NH 3 + Transport It appears that s t r a i n s UW59 and TJW590 have a phenotypic defect i n t h e i r a b i l i t y to take up CH^ NH^ "1" (1 mM) which i s enhanced at low pH. 59 It also appears that t h i s defect i s due to the n i f ' mutation which i s c also responsible f o r the N i f phenotype. A k i n e t i c analysis was therefore undertaken to further define t h i s defect. Rates were deter-mined f o r CH^ NH^ "*" uptake at concentrations ranging from 20 uM to 2 .5 mM CH 3NH 3 +. A plot of v e l o c i t y (V) versus substrate concentration ( S ) for s t r a i n TJW59 compared to that previously described f o r the wild type, s t r a i n JK, (27) can be seen i n Figure 6 (data f o r the wild type s t r a i n JK was provided by courtesy of Dr. J . Gordon). A comparison of the two curves suggests that s t r a i n UW59 i s al t e r e d i n i t s a b i l i t y to take up CH 3NH 3 +. A comparison of Lineweaver-Burk plots for s t r a i n JK and s t r a i n UW59 i s shown i n Figure 7. An approximate K value f o r low m CH 3NH 3 + concentrations (20 pK to 90 juM), as determined from an extrapola-t i o n of the Lineweaver-Burk plot of the observed data for TJW59 was found to be 89 uM. The r e c i p r o c a l of the y-intercept of t h i s portion of the curve describes a V of 3 .0 nmoles CH„NH +/min per mg protein. max 3 3 Determination of a K and V for high substrate concentrations (650 uM m max ' to 2 .5 mM) for s t r a i n UW59 i s d i f f i c u l t since the points do not f i t a st r a i g h t l i n e . However, extrapolation of a l i n e best f i t to the ob-served data r e s u l t s i n a K of 278 uM and a V of approximately 4 . 3 m max nmoles CH 3NH 3 +/min per mg protein. K and V values were also determined from the observed data m max using two single r e c i p r o c a l plots (Table 12) : a v/j^ s] versus v plot which weight points at high and low substrate concentrations more equally than 60 Figure 6. A p lo t of v e l o c i t y (v) versus substrate concentrat ion ( Data for s t r a i n JK compliments of Dr. J . Gordon. • • s t r a i n JK O O s t r a i n UW59 (U!9}cud 6uu-u|uu/sa|0uuu) A;JDO|9A 62 Figure 7. A Lineweaver-Burk pl o t f o r rates of CH^NH^ uptake. Data for s t r a i n JK compliments of Dr. J . K. Gordon. • • s t r a i n JK O O s t r a i n UW59 64 14 + Table 12. Ki n e t i c s of CH^NH^ uptake i n A. v i n e l a n d i i s t r a i n UW59 derived from four separate p l o t s . 20-80 jaM CH^NH^ K 3 V b nrzj— —max^  + 600 uM-2.5 mM CH^NH 1 j — j . b * + K V -max. Lineweaver-Burk plot (1/v vs 1/[S]) 86 3.0 278 1.3 Eadie-Scatcherd plot (v/,[S],vs v) 82 3.0 220 1.0 Hane's plot ( [S] /v vs [S.]) 77 2.8 210 1.3 Direct l i n e a r (v vs -[S.]) 70 2.6 260 1.7 *K units are IJM m by ,. units - are nmoles" CH NH +/min per mg protein max 3 3 *V ~~ maximum v e l o c i t y of "^C-la b e l l e d CH NH + uptake determined for max, - - - ' ', J 3 the concentration range 20 JJM to 80 JJM CH^NH^ 14 + *V = maximum v e l o c i t y f or C-labelled CH„NH. uptake determined f o r max. 3 4 1 + the concentration range 0.65 mM to 1.5 mM CH^NH^ . This value i s corrected for any contribution of V by subtracting the value deter-i n 3 . x mined for V from each of the v e l o c i t i e s observed at the higher con-maxi centrations assayed. 65 the double r e c i p r o c a l plot and a £s] /v versus [s] plot (Hanes plot) which i s thought to have an advantage over the double r e c i p r o c a l plot from a s t a t i s t i c a l point of view (35). A di r e c t l i n e a r plot (35), V versus -Qf| , was also used to determine K and V values from the observed data m max and i s also seen i n Table 12. A l l K and V values determined from m max these plots were found to agree very well with those determined from the double r e c i p r o c a l plot (Lineweaver-Burk p l o t ) . These r e s u l t s i n d i c a t e that there i s a s i g n i f i c a n t reduction i n the a b i l i t y of s t r a i n UW59 to take up CH^ NH^ "1" i n comparison to that of the wild type s t r a i n JK. Differences could be detected i n CH^NH^ up-take a b i l i t i e s at low (20 pM to 80 pM CH^NH^) and high (650 pM. to 2.5 mM CH^ NH^ "*") substrate concentrations. I l l . E f f e c t of Acetate on Constitutive Nitrogenase A c t i v i t y Understanding the mechanisms of regulation of the nitrogenase sys-tem i s greatly f a c i l i t a t e d by the chara c t e r i z a t i o n of d i f f e r e n t mutants i n the system. In the previous section i t was undertaken to characterize c — the Nif s t r a i n s TJW59 and TJW590 which were derived from the Nif s t r a i n c UW2. These Nif mutants were shown to be al t e r e d i n t h e i r a b i l i t y to + 59 take up CH^NH^ presumably due to the n i f mutation. c Another class of Nif mutants have been i s o l a t e d by s e l e c t i n g f o r methylalanine resistance (Mai ) from the wild type A. v i n e l a n d i i (79). Characterization of such mutants should also be b e n e f i c i a l i n further understanding the process by which nitrogenase i s regulated i n Azotobacter. A. I s o l a t i o n and Properties of Methylalanine Resistant Mutants The i n i t i a l report concerning a methylalanine r e s i s t a n t mutant of A. v i n e l a n d i i was published by Sorger (79). The mutant described was 66 i s o l a t e d a f t e r mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and p l a t i n g onto a nitrogen-free medium containing 0.2% methylalanine. Although i t was not reported how many colonies were tested, i t was reported that one i s o l a t e was p a r t i a l l y c o n s t i t u t i v e because extracts of mutant cultures grown with casamino acids exhibited higher nitrogenase a c t i v i t y than extracts of w i l d type cultures grown under the same conditions. St. John and B r i l l (72) showed that methylalanine i n h i b i t e d the growth of A. v i n e l a n d i i only when ei t h e r glucose or maltose was the sole carbon source. Therefore, i n t h i s lab, glucose was used as carbon source f o r the i s o l a t i o n of methylalanine r e s i s t a n t mutants. Aliquots of 0.1 ml volume from a ^ - f i x i n g culture (150 K l e t t units) were plated onto nitrogen-free medium containing 0.2% methylalanine. The volume plated contained about 4 x 10 ^  c e l l s . A f t e r incubation at 30°C, greater than 300 colonies were observed about a f a i n t lawn. Colonies were picked, restreaked several times f o r sing l e colony i s o l a t i o n , and then streaked i n patches onto medium containing ammonium. Individual i s o l a t e s were tested f o r c o n s t i t u t i v e nitrogenase synthesis i n an overnight acetylene reduction assay as described i n Materials and Methods. Wild type c u l -tures never produced ethylene i n t h i s assay. Isolates which did produce ethylene were retested with an assay which allowed quantitation of the nitrogenase s p e c i f i c a c t i v i t y . Of the colonies picked from methylalanine plates, less than 1% were c o n s t i t u t i v e i n the presence of excess ammonium. The r e s u l t s of the quantitative nitrogenase assays are shown i n Table 13. S t r a i n TJW590 i s also included i n Table 13 f o r comparison. The maximum c o n s t i t u t i v e nitrogenase a c t i v i t y observed i n the methylala-x nine r e s i s t a n t (Mai ) mutants ranged from 19 to 23% of the l e v e l observed i n N_-grown cultures of the wild type at a comparable density. 67 Table 13. Level of nitrogenase i n wild type and methylalanine r e s i s t a n t mutants. St r a i n Source of Nitrogen and Carbon i n vivo % Nitrogenase Component I i n v i t r o CRM : % % JK (wild type) JK N^ plus sucrose N^ plus sucrose and acetate 100 68 100 100 JK JK1 ammonium acetate and sucrose ammonium acetate and sucrose 0 20 23 22 JK2 JK3 JK4 UW590 ammonium acetate and sucrose ammonium acetate and sucrose ammonium acetate and sucrose ammonium acetate and sucrose 19 23 19 35 30 39 a -1 -1 -1 100% i s equal to 320 pmol ethylene produced min ml K l e t t unit b100% i s equal to 61 nmol ethylene produced min ^ mg protein CCRM i s cross reactive material measured by rocket Immunoelectrophoresis. 100% l e v e l of Component I i s 2.7 mm peak height per ug protein. '— means not determined. 68 B.. E f f e c t of Acetate on Constitutive Nitrogenase Ammonium acetate i s used ro u t i n e l y as a nitrogen source i n t h i s laboratory. An i n t e r e s t i n g observation was made when tes t i n g f or c o n s t i -r tutxve nitrogenase a c t i v i t y i n these Mai • mutants i n the presence of ammonium acetate. Variable r e s u l t s were observed which depended on the density of the culture. It was noted that cultures of the c o n s t i t u t i v e mutants or wi l d type exhibited diauxic growth when 29 mM ammonium acetate was used as nitrogen source i n a Burk's sucrose medium. This pattern of growth, r e s u l t s from sequential u t i l i z a t i o n of the two carbon substrates a v a i l a b l e , as previously described (Section I ) . Acetate u t i l i z a t i o n i n tire f i r s t growth phase (pre-lag) i s correlated with repression of sucrose transport and u t i l i z i n g enzymes. As seen i n Figure 8, the f i r s t growth x phase of the Mai mutant JK1 i s also characterized by the absence of co n s t i t u t i v e nitrogenase a c t i v i t y as measured i n vivo. Nitrogenase a c t i v i t y i s detectable only a f t e r the lag during the second phase of growth (Figure 8). The second growth phase has been shown to involve sucrose u t i l i z a t i o n (Section I ) . It was demonstrated i n Section I that reduction of the acetate concentration, without changing the ammonium concentration, resulted i n onset of the lag at a lower c e l l density (com-x pare Figures 1 and 2). Analysis of two Mai mutants, JK3 and JK4, revealed than an increase i n acetate concentration delayed the onset of detectable nitrogenase a c t i v i t y (Table 14). In a l l cases, ammonium was i n excess at the end of the experiment, i n d i c a t i n g that onset of nitrogenase was not correlated with depletion of ammonium from the medium. The behavior of another derepressed mutant was analyzed to deter-mine I f t h i s e f f e c t of acetate on c o n s t i t u t i v e nitrogenase a c t i v i t y i s a 69 Figure 8. Growth and c o n s t i t u t i v e nitrogenase a c t i v i t y i n the methylala-nine r e s i s t a n t s t r a i n JK1. Growth ( • • ) was followed together with rn vivo nitrogenase a c t i v i t y ( O O ). In v i t r o nitrogenase a c t i v i t i e s (histogram) were measured for cultures harvested at 35, 70 and 120 K l e t t u n i t s . Time zero on t h i s curve i s approximately 16 hours a f t e r i n o c u l a t i o n . 7 0 71 Table 14. Constitutive nitrogenase a c t i v i t y of methylalanine r e s i s t a n t mutants grown with various concentrations of acetate. S t r a i n JK4 JK3 Acetate Concentration 29 mM 14 mM 29 mM 14 mM 0 mM Culture Density 40~ K l e t t units 0 0 0 0 19 a 50 K l e t t units 0 0 0 0 65 60 K l e t t units 0 <1 0 1 110 70 K l e t t units 0 9 0 3 131 80 K l e t t units 0 25 0 - -90 K l e t t units - 47 - 12 -100 K l e t t units 5 - 1 - -110 K l e t t units 38 52 12 37 -140 K l e t t units 61 57 27 73 -In vivo nitrogenase a c t i v i t y i n pmol ethylene produced min -1 -,-1 ml K l e t t unit 1 . 72 property associated s o l e l y with the Mai mutation or i s a general charac-t e r i s t i c of c o n s t i t u t i v e mutants. S t r a i n UW590 was chosen because of i t s high nitrogenase a c t i v i t y and the l i k e l i h o o d that the mutations i n th i s s t r a i n are d i f f e r e n t from that i n s t r a i n JK1. As shown i n Figure 9, s t r a i n UW590 also exhibits the diauxic growth curve when grown on Burk's sucrose medium supplemented with 29 mM ammonium acetate as N-source. Nitrogenase a c t i v i t y , also shown i n Figure 9, i s detectable before the s h i f t from acetate to sucrose u t i l i z a t i o n , however, the a c t i v i t y increases dramatically a f t e r the s h i f t . Similar r e s u l t s are observed for s t r a i n TJW59. There are several possible explanations for t h i s observed pattern of nitrogenase a c t i v i t y during growth of UW590. One p o s s i b i l i t y i s that acetate has no e f f e c t on nitrogenase a c t i v i t y i n t h i s c o n s t i t u t i v e x mutant (UW590) and i s associated s o l e l y with the Mai mutants. Since one would expect the c o n s t i t u t i v e nitrogenase a c t i v i t y ( s p e c i f i c a c t i v i t y ) to remain constant during growth, some other factor(s) must then be a f f e c t i n g the nitrogenase during growth of the organism (such as oxygen concentra-t i o n , pH, e t c . ) . A second p o s s i b i l i t y i s that the i n i t i a l increase i n nitrogenase a c t i v i t y during pre-lag growth of s t r a i n UW590 r e s u l t s from decreasing concentrations of acetate during t h i s growth phase due to u t i l i z a t i o n of acetate as the preferred carbon source (Section I ) . C. E f f e c t of Acetate on Constitutive In V i t r o Nitrogenase A c t i v i t y To properly i n t e r p r e t the e f f e c t of acetate, i t was necessary to determine whether the e f f e c t was due to i n h i b i t i o n of nitrogenase a c t i v i t y or to repression of nitrogenase synthesis. It had been observed that 73 Figure 9. Growth and c o n s t i t u t i v e nitrogenase a c t i v i t y i n s t r a i n UW590. Growth ( • • ) was followed together with i n vivo nitrogenase a c t i v i t y ( O O ). In v i t r o nitrogenase a c t i v i t i e s (histograms) were measured for c u l -tures harvested at 35, 70 and 120 K l e t t u n i t s . Time zero on the curve i s approximately 16 hours a f t e r i n o c u l a t i o n . 74 75 addition of acetate to N^-fixing cultures of A. v i n e l a n d i i resulted i n transient i n h i b i t i o n of i n vivo nitrogenase a c t i v i t y (41). It i s l i k e l y that the a b i l i t y of acetate to permeate the membrane r e s u l t s i n a de-energized membrane and consequent i n t e r r u p t i o n of the flow of energy or reductant to nitrogenase. I f the absence of detectable nitrogenase a c t i v i t y i n a c e t a t e - u t i l i z i n g , pre-lag cultures were due only to interrup-t i o n of the flow of energy or reductant to nitrogenase, one would expect that an i n v i t r o assay of nitrogenase (performed with ATP and an a r t i f i c i a l e l e ctron donor) would y i e l d a c t i v i t y i n extracts of pre-lag cultures. To determine i f In. v i t r o nitrogenase a c t i v i t y was present or absent from pre-lag cultures, s t r a i n s JK1 and UW590 were grown with 29 mM ammonium acetate as nitrogen source and c e l l s were harvested when the culture reached a density of 35 K l e t t units (pre-lag) as well as 70 and 120 K l e t t units (post-lag). Anaerobic preparation of c e l l - f r e e extracts was followed by _in v i t r o assay of nitrogenase a c t i v i t y . The bars i n r Figure 8 depict the i n v i t r o a c t i v i t i e s of extracts of the Mai mutant JK1 prepared from the 35, 70, and 120 K l e t t unit cultures. The pattern of post-lag onset of nitrogenase a c t i v i t y i s apparent. Similar r e s u l t s were obtained for s t r a i n UW590 as seen i n Figure 9. However, i n t h i s c o n s t i t u t i v e mutant, a low l e v e l of both i n vivo and i n v i t r o a c t i v i t y i s observed i n pre-lag c e l l s which may be due to a lower s e n s i t i v i t y of nitrogenase synthesis to acetate i n t h i s s t r a i n as compared to s t r a i n JK1, as previously discussed. S t r a i n UW590 also exhibits a d r a s t i c reduction i n both i n vivo and i n v i t r o a c t i v i t y at high c e l l density (Figure 9). This may be due to a h y p e r s e n s i t i v i t y of t h i s mutant to the reduced oxygen concentration expected at high c e l l density i n batch cultures, 76 which would r e s u l t i n a decreased net amount of r e s p i r a t i o n occuring i n the culture. The data ind i c a t e that the presence of acetate or some aspect of acetate u t i l i z a t i o n may i n t e r f e r e with the synthesis of nitrogenase rather than the a c t i v i t y of the enzyme. D. E f f e c t of Acetate on Constitutive Synthesis of Nitrogenase Protein To d i r e c t l y examine whether acetate or some aspect of acetate u t i l i z a t i o n i n t e r f e r e s with the co n s t i t u t i v e synthesis of nitrogenase protein, the amount of nitrogenase component I protein was quantitated by L a u r e l l Immunoelectrophoresis i n extracts of s t r a i n s JK1 and UW590 (Table 15). Cultures were grown i n Burk's sucrose medium containing 29 mM ammonium acetate as nitrogen source. S t r a i n J K l was harvested at 35 and 120 K l e t t u n i t s . S t r a i n UW590 was harvested at 35 and 70 K l e t t u n i ts. Extracts were prepared anaerobically by osmotic shock or ae r o b i c a l l y with a french pressure c e l l . No immunologically detectable nitrogenase component I protein was observed i n extracts of s t r a i n J K l from the 35 K l e t t unit culture (pre-l a g ) . Cross reactive material was, however, present i n extracts prepared from the 120 K l e t t unit culture (Table 15). The c o r r e l a t i o n between nitrogenase a c t i v i t i e s observed and immunologically detectable protein measured i n extracts of s t r a i n J K l i s also presented i n Table 15. Nitrogenase a c t i v i t y and immunologically detectable nitrogenase component I were also found to correlate for s t r a i n UW590 (Table 15). Low nitrogenase a c t i v i t y (both ^n vivo and jLn v i t r o ) i n 35 K l e t t unit cultures corresponded with low nitrogenase cross reactive material. Therefore, the data indicates that acetate or some aspect of acetate u t i l i z a t i o n i n t e r f e r e s with c o n s t i t u t i v e synthesis or the s t a b i l i t y of nitrogenase protein. 77 Table 15. E f f e c t of acetate on c o n s t i t u t i v e synthesis of nitrogenase protein. S t r a i n Culture Density Nitrogenase . b i n vivo % • " b 'in v i t r o % Component I CRM % JK 100 100 100 100 J K l 35 0 0 0 J K l 120 20 23 22 UW590 35 3 6 12 UT590 70 34 30 30 Culture density expressed i n K l e t t units. 'Refer to Table 13. 78 DISCUSSION I. Regulation of Sucrose Transport and Metabolism by Acetate Diauxie growth has been previously observed with cultures of Escherichia c o l i provided with the sugars glucose and lactose as carbon sources (52). K l e b s i e l l a aerogenes, another enteric bacterium, has been found to display diauxie when grown on medium containing glucose and an organic acid (12,65). In both cases, glucose i s the preferred carbon source. In contrast, i n medium containing glucose and an organic acid, Pseudomonas aeruginosa exhibits "reverse" diauxie, using the organic acid as the preferred carbon source (33,88). This behavior has been found to be a r e s u l t of reduced glucose transport and metabolizing a c t i v i t i e s (36,51,53,55,88). One other bacterium which has been reported to use organic acids i n preference to a sugar i s Arthrobacter c r y s t a l l o p o i e t e s (40). These r e s u l t s show that Azotobacter v i n e l a n d i i also u t i l i z e s an organic a c i d , acetate, i n preference to sucrose when grown i n a medium containing both as p o t e n t i a l carbon sources. A t y p i c a l diauxie growth curve r e s u l t s , the pre-lag growth being supported by acetate, the post-lag growth by sucrose. P r e f e r e n t i a l u t i l i z a t i o n of acetate was f i r s t indicated by the observation that the onset of the lag i n the diauxie growth curve i s dependent on the concentration of acetate i n the medium (by comparison of Figures 1 and 2). Evidence that acetate i s the preferred carbon r 14 n source was obtained by following the disappearance of ; LU— -CJacetate and r 14 i LU- Cjsucrose from the medium. As shown i n Figure 2, acetate carbon disappears r a p i d l y from the medium of pre-lag cultures whereas sucrose carbon i s not u t i l i z e d u n t i l a f t e r the lag. The same r e s u l t s are seen 79 regardless of whether the i n i t i a l sucrose concentration i s '58 mM or 7 mM (Figures 2A and 2B). It was found that the medium from cultures provided with [U-^C] acetate s t i l l contained radioactive carbon a f t e r the lag when sucrose was present at a concentration of 7 mM. It i s not known i f the l a b e l l e d carbon i s i n the form of acetate or whether i t represents metabolic products excreted into the medium. Acetate u t i l i z a t i o n was found to be correlated with low sucrose transport and sucrose metabolic a c t i v i t i e s when f u l l (58 mM) sucrose was included i n the medium. The sucrose transport a c t i v i t y of pre-lag c e l l s was found to be only 5% that of post-lag c e l l s (Table 4). Because the presence of acetate i n the sucrose transport assay was not i n h i b i t o r y (Table 4), i t was concluded that the e f f e c t of acetate was due to repres-sion of some.protein (or proteins) involved i n sucrose transport. Stimula-t i o n of the sucrose uptake rate was noted when pre-lag c e l l s from cultures grown with f u l l sucrose (58 mM) were assayed i n the presence of acetate. This may be explained by an increase i n energy generation due to the presence of an exogenous u t i l i z a b l e energy source. On the basis of t h i s observation, i t i s expected that pre-lag c e l l s are d e f i c i e n t i n sucrose metabolism and are therefore somewhat energy starved i n the sucrose uptake assay. Acetate i s therefore l i k e l y to increase the rate of sucrose uptake i n the assay. The small stimulation of uptake rate r e s u l t i n g from the addition of acetate to post-lag c e l l s might also be due to increased energy generation. Thus, i n A. -vinelandii, regulation of sugar u t i l i z a t i o n i s brought about i n part by control of sugar uptake a c t i v i t y , as has been found f o r P_. aeruginosa (51, 53) and Arthrobacter c r y s t a l l o p o i e t e s (40). The Entner-Doudoroff pathway has been shown to be the major path-way f o r glucose/sucrose metabolism i n A. v i n e l a n d i i (81). The a c t i v i t y of 80 the metabolic enzymes of t h i s pathway, as well as that of pyruvate dehydrogenase, were found to be low i n extracts of acetate u t i l i z i n g pre-lag c e l l s which were grown i n medium containing f u l l (58 mM) sucrose (Table 5). Enzyme a c t i v i t i e s were corrected for any endogenous NADH oxidase a c t i v i t y present. The repression of sucrose transport and the lower l e v e l of a c t i v i t y of the metabolic enzymes i n acetate u t i l i z i n g pre-lag c e l l s would appear to be s u f f i c i e n t to explain the lack of sucrose u t i l i z a t i o n i n pre-lag c e l l s grown i n medium containing f u l l (58 mM) sucrose. It i s i n t e r e s t i n g that both sucrose transport and glucose-6-phosphate dehydro-genase a c t i v i t i e s are higher i n acetate u t i l i z i n g , pre-lag c e l l s grown i n medium containing acetate plus 7 mM sucrose as carbon sources than those l e v e l s observed i n sucrose u t i l i z i n g , post-lag c e l l s (compare Tables 4, 5 and 6). This observation suggests that regulation of sucrose metabolism by acetate or acetate metabolism occurs at a s i t e ( s ) other than these. A l i k e l y s i t e for regulation of sucrose metabolism by acetate would seem to be at 6-phosphogluconate dehydratase and/or 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase since only 17% of the post-lag sucrose u t i l i z i n g a c t i v i t y of the combined reaction i n v o l v i n g these enzymes i s observed i n pre-lag cultures grown i n medium containing e i t h e r 58 mM sucrose or 7 mM sucrose. I t i s possible that pyruvate dehydrogenase might also be involved i n the regulation of sucrose metabolism by acetate since t h i s enzyme was found not to derepress i n pre-lag cultures grown i n medium containing low sucrose concentrations (7 mM). However, only a low l e v e l of pyruvate dehydrogenase a c t i v i t y was detectable under sucrose u t i l i z i n g conditions which might indi c a t e a problem with the assay for th i s enzyme. To examine the p o s s i b i l i t y that acetate might be i n h i b i t i n g these enzymes and/or some other sucrose u t i l i z i n g a c t i v i t y i n vivo, acetate and 81 U- CJsucrose were added to sucrose u t i l i z i n g post-lag c e l l s . Disap-14 pearance of C-labelled carbon from the medium as well as incorporation into c e l l u l a r material was followed (Figure 3). Upon addition of acetate to the culture induced for sucrose u t i l i z a t i o n there occurred a lag i n growth and a s l i g h t i n h i b i t i o n of sucrose incorporation into c e l l material. As previously suggested, these may be due to a p h y s i o l o g i c a l shock r e s u l t i n g from the sudden addition of acetate (41). Upon completion of the lag, 14 incorporation of C-labelled sucrose was s i m i l a r to that seen i n the culture receiving no acetate. These preliminary r e s u l t s suggest that regulation of sucrose u t i l i z a t i o n occurs by repression and not i n h i b i t i o n of sucrose metabolic a c t i v i t i e s . Therefore, a preliminary model i s suggessted i n which repression of 6-phosphogluconate dehydrogenase and/or KDPG aldolase a c t i v i t y (and perhaps pyruvate dehydrogenase as well) occurs when a culture i s u t i l i z i n g acetate. This repression i s not r e l i e v e d under conditions where both sucrose transport and glucose-6-phosphate dehydrogenase are induced, and therefore r e s u l t s i n the observed diauxie growth at high (58 mM) and low (7mM) sucrose concentrations. This i s not unlike r e s u l t s reported for Pseudomonas aeruginosa. Growth of P_. aeruginosa i n c i t r a t e containing medium with a low l e v e l of glucose (10-15 mM) i s correlated with derepression of glucose transport and metaboliz-ing a c t i v i t i e s (13). However, i t was not reported whether glucose u t i l i z a t i o n i n P. aeruginosa accompanies the derepression of these a c t i v i t i e s . I t appears that the presence or u t i l i z a t i o n of acetate r e s u l t s i n repression of several c e l l u l a r a c t i v i t i e s involved i n sucrose u t i l i z a t i o n . It i s l i k e l y that other enzymatic a c t i v i t i e s associated with sugar catabolism are also repressed under these conditions, e s p e c i a l l y since derepression of sucrose transport and glucose-6-phosphate dehydrogenase i s not correlated with sucrose u t i l i z a t i o n when a low sucrose concentration (7 mM) i s used i n 82 the growth medium. The lag period observed i n the diauxie growth curve i s l i k e l y to be the time required f o r the synthesis of proteins required f o r sugar u t i l i z a t i o n . It should be noted that the lag period i n cultures grown with a low i n i t i a l concentration of sucrose (Figure 2B) i s not characterized by as severe a reduction i n growth rate as that seen i n cultures grown with high sucrose concentrations (Figure 2A). This would be expected i f several e s s e n t i a l a c t i v i t i e s such as sucrose transport and glucose-6-phosphate dehydrogenase were already derepressed. The molecular mechanism by which acetate or acetate u t i l i z a t i o n r e s u l t s i n regulation of sugar u t i l i z a t i o n i s not known. Addition of cyclic-AMP (1 mM or 5 mM), d i b u t y r y l cyclic-AMP (0.1 mM or 1.0 mM) or d i b u t y r y l cyclic-GMP (0.1 mM or 1.0 mM) to pre-lag c e l l s did not r e l i e v e diauxie (data not shown) suggesting that low c y c l i c nucleotide pools are probably not responsible f o r the lack of sucrose u t i l i z a t i o n . However, i t i s not known i f the addition of exogenous c y c l i c nucleotides to A. v i n e l a n d i i a l t e r s the i n t e r n a l pool s i z e . On the basis of these observations, i t i s possible to include A. v i n e l a n d i i with P. aeruginosa and Arthrobacter c r y s t a l l o p o i e t e s i n a group of organisms which e x h i b i t obligate aerobic metabolism and which use intermediates of the t r i c a r b o x y l i c a c i d cycle i n preference to sugars. In a l l three organisms, the sugar transport and metabolizing a c t i v i t i e s are repressed when an intermediate of the t r i c a r b o x y l i c a c i d cycle i s included i n the medium. This may suggest the evolution of s i m i l a r mechanisms for regulation of substrate u t i l i z a t i o n i n the obligate aerobic b a c t e r i a . The evolution of a s i m i l a r regulatory mechanism i n obligate aerobic bacterium as j u s t suggested, might be expected. It would seem l i k e l y that the t r i c a r b o x y l i c a c i d cycle i n obligate aerobes would be c o n s t i t u t i v e l y expressed. 83 It would also seem l i k e l y that t r i c a r b o x y l i c acid cycle intermediates would be able to repress u t i l i z a t i o n of carbon sources which require some i n i t i a l processing before being integrated i n t o the t r i c a r b o x y l i c a c i d cycle. One might speculate that the pool si z e of t r i c a r b o x y l i c acid cycle intermediates might play a regulatory role i n sugar u t i l i z a t i o n i n these organisms, but, there i s no evidence as yet to support t h i s hypothesis. I I . E f f e c t of NH^/CH^NH "*" Uptake on Constitutive Nitrogenase A c t i v i t y The genetic and biochemical properties of mutant s t r a i n s of A. vine-l a n d i i have been described. Although both s t r a i n s UW59 and UW590 are co n s t i t u t i v e f o r nitrogenase synthesis at pH 7.3, the a c t i v i t y increases s i g n i f i c a n t l y as the pH of the culture drops. This increased a c t i v i t y was found to be correlated with increased synthesis of nitrogenase protein (Table 8). Thus, high l e v e l s of c o n s t i t u t i v e nitrogenase a c t i v i t y i n these s t r a i n s i s pH con d i t i o n a l . There i s a report i n the l i t e r a t u r e of an NH^+ transport mutant of Hydrogenomonas eutropha i n which growth i s pH con d i t i o n a l (85). Wild type A. v i n e l a n d i i (JK), when grown with ammonium su l f a t e as nitrogen source, was found to have a low l e v e l of a n t i g e n i c a l l y detectable nitrogenase components I and II (Table 8). This observation was s u r p r i s i n g since no i n vivo nitrogenase a c t i v i t y was detectable. Also, no nitrogenase protein or a c t i v i t y i s detectable when ammonium acetate i s used as sole nitrogen source (Table 8). There are several possible explanations f o r t h i s r e s u l t . One p o s s i b i l i t y i s that the l e v e l of nitrogenase a c t i v i t y i n the wild type at low pH i s too low to be detected by the rn vivo assay. A second p o s s i b i l i t y i s that one (or both) of the nitrogenase components i s non-functional, yet immunologically 84 PLEASE NOTE: THERE IS NO PAGE 84 IN THIS THESIS. This page has been inserted to maintain the numbering sequence. 85 detectable. Another p o s s i b i l i t y i s that the low pH i s i n h i b i t o r y to nitroge-nase a c t i v i t y i n the wild type. This p o s s i b i l i t y i s also suggested from data seen i n Table 7, where less nitrogenase a c t i v i t y i s seen f o r s t r a i n UW590 at low pH than at high pH. Therefore, . increased synthesis of nitrogenase at low pH does not appear to be a function s o l e l y associated with the c o n s t i t u t i v e mutants UW59 and UW590. An as yet untested explanation i s that t h i s phenomenon observed at low pH re s u l t s from the p a r t i a l release of some regulatory function which i s normally Imposed on the organism when growing i n the presence of NH^+. Preliminary evidence from two d i f f e r e n t types of NH^+ disappearance assays suggested that there might be a defect i n the a b i l i t y of s t r a i n UW590 to take up NH^ "*" at low pH (Figure 5, Table 9). However, since both of these assays r e l y upon the a s s i m i l a t i o n of the NH^+ taken up i n order to detect any differences i n ammonium concentrations i n the medium, i t can not be conclusively determined from these analyses i f the defect i s i n NH^ "*" transport or a s s i m i l a t i o n . Investigation of the CH^ NH^ "1" uptake a c t i v i t i e s of the co n s t i t u t i v e mutants UW59. and UW590 was therefore undertaken since i n i t i a l rates of CH^NH + uptake are not believed to rely* on a s s i m i l a t i o n (2,27). The re s u l t s of such an analysis are shown i n Table 10. It i s apparent that both UW59 and UW590 are defective i n t h e i r a b i l i t i e s to take up CH 3NH 3 + (1 mM) r e l a t i v e to the wild type s t r a i n JK when grown to low pH on Burk's sucrose medium containing ammonium su l f a t e (14 mM) as N-source. S t r a i n UW59 i s also defective when ammonium acetate (29 mM) i s used as N-source and CH 3NH 3 + uptake i s assayed at pH 7.2. Preliminary r e s u l t s f o r s t r a i n UW590 were found to be s i m i l a r to those of s t r a i n UW59. It i s not known why 86 differences i n CH^ NH^ "*" uptake rates were not observed between the mutants UW59 and UW590 and the wild type s t r a i n JK when these s t r a i n s were grown on Burk's sucrose medium containing ammonium acetate as N-source and CH^ NH^ "*" uptake was assayed at pH 5.9. Due to the large standard deviation i n observed v e l o c i t i e s for s t r a i n UW2, i t i s d i f f i c u l t to determine if'UW2 i s s i g n i f i c a n t l y d i f f e r e n t from the w i l d type. The defective CH^ NH^ "1" uptake a b i l i t y of the st r a i n s UW59 and 59 UW590 was shown to be due to the n i f mutation by making use of the 59 590 a b i l i t y to separate the n i f and n i f mutations by transformation (Table 3). In agreement with t h i s i s the observation that the rates of CH 3NH 3 + uptake f o r TJW59 and TJW590 are s i m i l a r (Table 10) . 59 The n i f mutation appears to be responsible for both the c + + Nif phenotype and the defective NH4/CH3NH3 uptake mechanism. There are three possible explanations for how t h i s might occur. The f i r s t 59 p o s s i b i l i t y i s that n i f i s a mutation i n a regulatory s i t e involved + c i n repression of nitrogenase by NH^ r e s u l t i n g i n the Nif phenotype. This derepressed nitrogenase a c t i v i t y might i n turn regulate the amount of NH 4 +/CH 3NH 3 + taken up by regulation of the NH^ "*" transport system. However, analysis of CH 3NH 3 + uptake i n s t r a i n UW590 assayed under N^-fixing and n o n - ^ - f i x i n g conditions (Table 11) revealed that the amount of f ixation.occuring does not a f f e c t the a b i l i t y of the organism to take up CH 3NH 3 +. The second p o s s i b i l i t y i s that NH4+/CH3NH3+ uptake i s involved i n the regu-59 l a t i o n of nitrogenase and n i f i s a mutation which r e s u l t s i n a defective NH 4 +/CH 3NH 3 + uptake mechanism. I f NH^+ uptake i s involved i n regulation of nitrogenase i t would be expected that such a mutation might stimulate 59 the derepression of nitrogenase. A t h i r d p o s s i b i l i t y i s that n i f i s a 8 7 regulatory mutation which co-regulates NH^/CH^NH^"1" uptake and nitrogenase by a c t i v a t i n g nitrogenase synthesis and repressing NH^+ tranport. It i s not possible to d i s t i n g u i s h between the l a s t two p o s s i b i l i t i e s at t h i s time. In order to further examine the properties of the NH^ /CH^ NH^ "*" uptake system i n s t r a i n U W 5 9 , a k i n e t i c analysis was undertaken. It i s apparent from Figure 6 that s t r a i n U W 5 9 i s alte r e d i n i t s a b i l i t y to take up CH^ NH^ "1" r e l a t i v e to the wild type s t r a i n at a l l substrate concentrations assayed. A Lineweaver-Burk plot of these data (Figure 7 ) r e s u l t s i n a biphasic curve f o r s t r a i n U W 5 9 . This suggests two uptake mechanisms, one f o r lower substrate concentrations and the other for high substrate concentrations, as has been previously noted f o r the wild type s t r a i n JK ( 2 7 ) . K and V J m max values determined from t h i s Lineweaver-Burk pl o t and three other plots of the- observed v e l o c i t i e s for s t r a i n U W 5 9 are seen i n Table 1 2 . At low sub-s t r a t e concentrations ( 2 0 ^JM to 8 0 J J M CH^ NH^ "1") s t r a i n U W 5 9 was found to have an approximate K^ ranging from 7 0 juM to 8 6 juM and Vmayi ranging from 2 . 6 to 3 . 0 nmoles CH^ NH^ Vmin per mg protein, depending on the plot used (Table 1 2 ) . S t r a i n JK was previously found to have an approximate K^ of 6 1 uM and V of 3 . 7 nmoles CH„NH„ +/min per mg protein ( 2 7 , Dr. J . K. J max 3 3 Gordon, personal communication) at low substrate concentrations ( 5 }M to 8 0 JJM. CH^ NH^ "*"). Therefore, these data suggest that s t r a i n U W 5 9 has a reduced a f f i n i t y for CH_NH„ and a lower V for CH„NH„ uptake r e l a t i v e 3 3 max 3 3 to that of the wild type at low substrate concentrations. Examination of the i n i t i a l rates of CH^ NH^ "1" uptake at higher sub-s t r a t e concentrations ( 6 0 0 jM to 2 . 5 mM CH^ NH^ "1") revealed that the mutant, U W 5 9 , i s al t e r e d i n i t s a b i l i t y to take up CH NH + r e l a t i v e to s t r a i n JK at these substrate concentrations as well (Figure 6). The maximum v e l o c i t y observed f o r CH NH + uptake i s described by the r e c i p r o c a l of the y-intercej 3 3 + of a Lineweaver-Burk plot for concentrations greater than 600 jM CH 3NH 3 . 88 This maximum v e l o c i t y (calculated as 4.3 nmoles CH^NH^/min per mg protein), for s t r a i n UW59 i s composed of that contribution due to the V for low max CH^ NR.^ " concentrations (20 yM. to 80 jM) plus the contribution due to some other uptake mechanism with a lower a f f i n i t y for the substrate. Because the V calculated from observed v e l o c i t i e s of CH,,NH + uptake for low max 3 3 substrate concentrations, 20 11M to 80 joM CH^NH^ (3.0 nmoles CH^NH^/min per mg protein), makes up 70% of the maximum v e l o c i t y calculated from observed v e l o c i t i e s at high substrate concentrations, 600 jM to 2.5 mM CH^NH^ (4.3 nmoles CH^NH^ /min per mg protein), i t i s cl e a r that t h i s a d d i t i o n a l mechanism of CH^ NH^ "1" uptake plays a very small r o l e i n CH^NH^ uptake i n s t r a i n UW59 at high CH^ NH^ "1" concentrations. + In contrast, the V calculated for CH„NH„ uptake at low sub-max 3 3 stra t e concentrations for the wild type s t r a i n JK (27) makes up only 40% of the maximum v e l o c i t y calculated from the observed v e l o c i t i e s f or CH^ NH^ "1" concentrations ranging from 600 ^aM to 1.5 mM. Therefore, i n the wild type organism, the a d d i t i o n a l mechanism of CH^NH^ uptake observed at high substrate concentrations plays a very s i g n i f i c a n t r o l e i n CH^ NH^ "'" uptake, whereas, i n the mutant s t r a i n UW59, th i s a d d i t i o n a l mechanism appears to be defective. Because the CH^ NH^ "1" uptake mechanism described for low substrate concentrations i n s t r a i n JK (apparent observed K of 61 uM; V of 3.7 m ' max nmoles CH^NH^/min per mg protein) and the ad d i t i o n a l mechanism of CH^ NH^ "1" uptake both contribute l a r g e l y to uptake at high substrate concentrations, an i t e r a t i v e process for c a l c u l a t i n g the Michaelis-Menten constant was undertaken i n order to analyze each system independent of the other. A f t e r several i t e r a t i o n s , the K for that mechanism involved i n CH„NH„ + m 3 3 uptake at low substrate concentrations was found to remain constant at 55 JJM 89 CfLNHZ1". However, with each i t e r a t i o n the 1/V value for the a d d i t i o n a l 3 3 max mechanism of CH^NH^ uptake appears to approach zero value appears to approach i n f i n i t y ) . Therefore, the analysis of the data suggests that the v e l o c i t i e s observed at high substrate concentrations are composed of that contribution due to a high a f f i n i t y CH„NH_ + uptake system (V ) and ^ 3 3 J max a contribution from some other mechanism, the most probable being that of d i f f u s i o n (V ; . . _ ) . dxr r In agreement with t h i s analysis i s that observed i n Figure 10. At high substrate concentrations that contribution which the high a f f i n i t y system has on the observed v e l o c i t i e s should be constant and equal to the V of that system. I f d i f f u s i o n accounts for the difference between max the observed v e l o c i t i e s and the V of the high a f f i n i t y system, at max b high CH^ NH^ "1" concentrations, then one would expect to see a l i n e a r increase i n v e l o c i t y at high substrate concentrations. A l i n e a r regression analysis of the observed v e l o c i t i e s at high' CH NH^+ concentrations (200 yM. to 1.5 mM) determine these observed v e l o c i t i e s f i t very w e l l to a l i n e with slope 2.4 pmoles CH^NH^ per min per mg protein/uM CH^NH^4". When th i s proposed contribution due to d i f f u s i o n i s extrapolated from the o r i g i n and then subtracted from the observed v e l o c i t i e s , a new curve i s generated which i s independent of the proposed d i f f u s i o n e f f e c t s (Figure 10). A K of 55 L I M CH^ NH^ "1", derived from a Lineweaver-Burk plot of t h i s new curve (Figure 11) agrees with that of the i t e r a t i v e a n a l y sis. The V , deter-6 6 max mm ed from the r e c i p r o c a l of the y-intercept, i s approximated to be 3.4 nmoles CH^ NH^ Vmin per mg protein. Therefore, i t appears that the increased maximum v e l o c i t y for CH^ NH^ *" uptake, calculated from the observed v e l o c i t i e s at high substrate concentrations for wild type s t r a i n JK, i s due to a contribution of d i f f u s i o n of CH^ NH^ "*". This contribution of continued d i f f u s i o n to the 90 Figure 10. The k i n e t i c s of c a r r i e r transport of '"•^ CH^ NH T i n Azotobac-ter v i n e l a n d i i . V e l o c i t i e s for substrate concentrations ranging from 200 pM to 1.5 mM were analyzed by l i n e a r regression and found to f i t well to a l i n e with slope 2.4 pmoles CH 3NH 3 +-min - 1-mg - 1- pM - 1 CH 3NH 3 +. This c o n t r i -bution due to d i f f u s i o n can be extrapolated from the o r i g i n as the hatched str a i g h t l i n e and subtracted from the t o t a l rate of uptake ( 0 £ ) to y i e l d the component of c a r r i e r transport (X X ) • (u|a;oJd 6uuu!uu/s8|Ouuu) A^poiaA 92 Figure 1 1 . A Lineweaver-Burk pl o t f o r rates of "^CH^NH^' uptake i n Azotobacter v i n e l a n d i i s t r a i n JK ( • • ; data compliments of Dr. J. K. Gordon), and f o r rates cor-rected f o r a contribution due to d i f f u s i o n (A A ; r e f e r to Figure 1 0 ) . £6 94 rate of CH^ NH^ "1" uptake must be dependent on the metabolism of methyl-ammonium to some other metabolite, the organism thereby maintaining a con-centration gradient necessary f o r continued d i f f u s i o n . Work done by Barnes e_t al. ( 2 ) suggests that a sub s t a n t i a l portion of the CH^ NH^ "1" taken up by A. v i n e l a n d i i i s converted to less polar, u n i d e n t i f i e d metabolite. They suggest that transport i s rate l i m i t i n g i n wild type A. v i n e l a n d i i at the concentrations which they used i n t h e i r k i n e t i c analysis. However, these concentrations were not reported. It i s apparent that s t r a i n UW59 i s defective i n some aspect of i t s a b i l i t y to take up and/or metabolize CH^ NH^ "1". This defect r e s u l t s i n the observed v e l o c i t i e s f o r CH NH.j+ uptake being consistantly lower than those observed f o r the wild type s t r a i n JK (Figure 6). The high a f f i n i t y system for CH^ NH^ "*" uptake i n t h i s mutant appears to have a reduced a f f i n i t y for CH.NH„ + as we l l as a lower V f o r CH„NH„ + uptake. At high substrate con-3 3 max 3 3 centrations ( 6 5 0 LIM to 1.5 mM CH NH + ) where metabolism of CH^NH^ appears to be rate l i m i t i n g i n the wild type, the data suggest there i s some assimilatory or processing defect i n s t r a i n TJW59. A preliminary analysis of s t r a i n UW2 suggests that there are no differences i n the k i n e t i c s of CH^ NH^ "*" uptake between t h i s Nif mutant and the wild type s t r a i n JK (data not shown). Therefore, i t appears that the 59 + n i f mutation i s responsible for the observed defects i n CH^NH^ uptake and/or metabolism as well as the derepressed nitrogenase phenotype i n s t r a i n UW59. It i s possible that a defect i n an NH./CH„NH„ + assimilatory protein 4 3 3 might have an e f f e c t on the k i n e t i c s of an NH^/CH^NH^"1" uptake protein i f they are c l o s e l y associated i n the c e l l . The opposite case might also be hy-pothesized however, i t would be more d i f f i c u l t to explain the data for s t r a i n UW59 at high substrate concentrations (650 LIM to 1.5 mM CH^NH^) by t h i s hypothesis. I t i s also possible that a defective NH^ /CH^ NH^ "1" uptake/assimilatory system might have an e f f e c t on the regulation of N 2 f i x a t i o n > 95 Evidence suggesting that an ammonium assimilatory enzyme or pro-duct of NH^ "*" a s s i m i l a t i o n i s involved i n regulation of f i x a t i o n i n A. v i n e l a n d i i has been obtained using analogs of the amino acid glutamate (26). It was observed that i n h i b i t i o n of the c a t a l y t i c a c t i v i t y of gluta -mine synthetase by the addition of the glutamate analogs, methionine s u l -foximine (MSX) or methionine sulfone (MSF), resulted i n the a b i l i t y of A. v i n e l a n d i i to synthesize nitrogenase even i n the presence of excess NH^ "*". As previously stated, work done by Barnes e_t a l . (2) indicates that a s u b s t a n t i a l portion of the CH^ NH^ "1" taken up by A. v i n e l a n d i i i s converted to a less polar, u n i d e n t i f i e d metabolite. An analysis of CH^ NH^ "1" uptake i n Clostridium pasteurianum (39) suggests that CH^NH + taken up by t h i s bacterium i s also converted to a less polar compound, i n t h i s case i t i s thought to resemble methylglutamine. Research i s currently being done i n the lab of Dr. J . Gordon to determine whether methylamine i s indeed processed by wild type A. v i n e l a n d i i JK and the mutant s t r a i n UW59. The nature of the metabolite of such an as s i m i l a t i o n process i s also being investigated. Preliminary evidence suggests that CH^ NH^ "1" i s being assimilated to methylglutamine i n t h i s organism as we l l (Dr. J. K. Gordon and R. A. Moore, personal communication). I l l . E f f e c t of Acetate on Constitutive Nitrogenase A c t i v i t y It was found i n t h i s study that the technique of i s o l a t i n g methylalanine r e s i s t a n t (Mai ) mutants, o r i g i n a l l y described by Sorger (79), does i n fact y i e l d c o n s t i t u t i v e nitrogenase mutants, a l b e i t at low frequency. The Mai mutants described i n t h i s report are able to synthe-siz e nitrogenase i n the presence of excess ammonium at l e v e l s up to 23% of the f u l l nitrogenase l e v e l observed i n N^-grown cultures of wild type s t r a i n JK (Table 13). 96 It was found that screening of mutants f or a c o n s t i t u t i v e pheno-type i s complicated by the fa c t that factors such as the carbon source used and the density of the batch culture a f f e c t expression of nitrogenase c o n s t i t u t i v i t y . For example, acetate u t i l i z a t i o n by pre-lag cultures of the Mai mutants was found to correlate with the absence of c o n s t i t u t i v e nitrogenase synthesis. The r e s u l t s of t h i s study ind i c a t e that the presence or u t i l i z a t i o n of acetate i n t e r f e r e s , i n some way, with nitrogenase synthesis or s t a b i l i t y i n the Mai mutants. It was also found that acetate might have an e f f e c t on the c o n s t i t u t i v e mutant UW590, however, the r e s u l t s obtained f o r t h i s r mutant are not as cl e a r as those for the Mai mutants. The conclusion that acetate a f f e c t s nitrogenase synthesis or s t a b i l i t y i s based on the observation that derepressed mutant s t r a i n s of A. v i n e l a n d i i growing with ammonium acetate maintain low or n e g l i g i b l e l e v e l s of nitrogenase a c t i v i t y and p r o t e i n while u t i l i z i n g acetate as carbon source i n the presence of sucrose. At t h i s time i t i s not clear why nitrogenase synthesis i s affec t e d by acetate or acetate metabolism. One p o s s i b i l i t y i s that syn-thesis of nitrogenase might be regulated i n part by the amount of reductant a v a i l a b l e . The pool of reduced pyridine nucleotide might be expected to be lower when c e l l s are assimilating.acetate carbon than when u t i l i z i n g a sugar as carbon source and thus, nitrogenase synthesis might be somewhat repressed when acetate i s the carbon source. The density of the batch culture was also found to a f f e c t the r expression of nitrogenase c o n s t i t u t i v i t y . When cultures of Mai mutants were grown i n an acetate-free medium containing ammonium s u l f a t e , main-tained at pH 7.6, the c o n s t i t u t i v e nitrogenase a c t i v i t y did not remain 97 constant during growth as shown i n Table 14. Similar r e s u l t s were obtained for s t r a i n UW590 (data not shown). One possible explanation for t h i s behavior i s the accumulation during growth of some product which helps "induce" nitrogenase synthesis. Another possible explanation i s a change i n oxygen concentration which may play a r o l e i n regulation of c o n s t i t u -t i v e nitrogenase synthesis. It i s known that w i l d type ^ - f i x a t i o n i s s e n s i t i v e to oxygen, both i n vivo and i n v i t r o (9,17,82). Work done by Dr. J . Gordon suggests that i n acetate-free medium oxygen may a f f e c t nitrogenase at low culture densities (personal communication). Dr. Gordon has observed that the speed of r o t a t i o n of cultures during growth had an e f f e c t on the time of appearance of nitrogenase a c t i v i t y and the time at which maximum a c t i v i t y was observed. At any given culture density less, than 35 K l e t t u nits,cultures with the lowest aeration had the highest nitrogenase a c t i v i t y . At d e n s i t i e s greater than 35 K l e t t u nits, the 0^ concentration appears to become l i m i t i n g i n the most slowly rotated f l a s k and i n vivo a c t i v i t y i s lower than that i n f l a s k s r o t a t i n g at medium or higher rates. This oxygen e f f e c t may explain the observation that no detectable nitrogenase a c t i v i t y i s seen i n cultures at a density l e s s than 18 K l e t t units when grown on acetate-free medium. Another p o s s i b i l i t y i s that the e f f e c t of acetate i s to f a c i l i t a t e r uptake of ammonium. If the Mai mutants were defective i n ammonium uptake, a r o l e f o r acetate i n f a c i l i t a t i n g ammonium uptake might be put f o r t h as a possible explanation for the lack of c o n s t i t u t i v e nitrogenase synthesis i n the presence of acetate. Kleiner (38) reported that addi-t i o n of ammonium acetate to N ^ - f i x i n g chemostat cultures of A. v i n e l a n d i i , i n amounts causing 80-100% repression of nitrogenase, resulted i n immediate and complete uptake of the ammonium. However, when ammonium su l f a t e was added, immediate uptake to only 80% of the t o t a l added was observed. 98 A f t e r a period of time, the balance of the ammonium was taken up. His conclusion was that maximal uptake of ammonium su l f a t e required induction of a transport system not required f o r uptake of ammonium acetate. On the basis of Kleiner's observation, i t appears that acetate could f a c i l i -tate ammonium uptake by a mutant defective i n uptake of ammonium s u l f a t e . r However, no evidence for ammonium uptake defects was found i n the Mai mutant s t r a i n JK-105 (Dr. J . Gordon, personal communication). Therefore, one can not a t t r i b u t e the lack of c o n s t i t u t i v e nitrogenase synthesis i n pre-lag cultures to the Mai mutants to f a c i l i t a t i o n of ammonium uptake by acetate. Furthermore, acetate has been shown to a f f e c t nitrogenase a c t i v i t y i n ammonium-free cultures of wild type A. v i n e l a n d i i s t r a i n JK (Dr. J . Gordon, personal communication). The i n vivo nitrogenase a c t i v i t y was lower when wi l d type was grown with sucrose and acetate than when grown with sucrose alone. However, i t has not yet been determined i f the lower a c t i v i t y of the sucrose plus acetate culture i s a r e s u l t of i n h i b i t i o n of nitrogenase a c t i v i t y or repression of nitrogenase synthesis. The p o s s i b i l i t y that enzymes involved i n carbohydrate u t i l i z a t i o n might be required for nitrogenase synthesis could be put forward on the basis of the observation that the absence of nitrogenase synthesis by a c e t a t e - u t i l i z i n g pre-lag cultures of c o n s t i t u t i v e mutants i s correlated with the absence of a c t i v i t e s of c e r t a i n enzymes involved i n carbohydrate u t i l i z a t i o n . This p o s s i b i l i t y can be ruled out because acetate has been used as sole carbon source to support N 2 f i x a t i o n i n A. v i n e l a n d i i (20). In a recent f i n d i n g i n K l e b s i e l l a by Drs. Shah and B r i l l (personal communication), the n i f J product, involved i n electron t r a n s f e r to nitrogenase reductase, has been i d e n t i f i e d as an oxygen s e n s i t i v e pyruvate dehydrogenase. They speculate that because of i t s oxygen s e n s i t i v i t y t h i s 99 nif-coded pyruvate dehydrogenase functions s o l e l y i n combination with the n i f F product i n transfe r of electrons to nitrogenase reductase. If Azotobacter has a n i f gene s i m i l a r to n i f j i n K l e b s i e l l a , i t would be in t e r e s t i n g to speculate on the e f f e c t s which acetate might have on mutants defective i n t h i s gene. It might be expected that pyruvate dehydrogenase involved i n carbohydrate u t i l i z a t i o n could complement a defect i n a n i f - s p e c i f i c pyruvate dehydrogenase. As seen i n Table 5, when acetate i s used as the preferred carbon source during pre-lag growth, the l e v e l of pyruvate dehydrogenase i s reduced. This could r e s u l t i n an i n a b i l i t y i n a mutant lacking a n i f - s p e c i f i c pyruvate dehydrogenase to f i x nitrogen when growing on acetate as sole carbon source. 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