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Ammonium and methylammonium uptake by the nitrogen-fixing bacterium Azotobacter vinelandii Moore, Richard Atwood 1983

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AMMONIUM AND METHYLAMMONIUM UPTAKE BY THE NITROGEN BACTERIUM AZOTOBACTER VINELANDII -FIXING by RICHARD ATWOOD MOORE B.SC, UNIVERSITY OF MASSACHUSETTS, 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA SEPTEMBER 1983 © RICHARD ATWOOD MOORE, 1983 In presenting t h i s thesis in p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t freely available for reference and study. I further agree that permission for extensive • copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his or her representatives.' It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of MICROBIOLOGY The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date: SEPTEMBER, 1983 Abstract Azotobacter v i n e l a n d i i , grown with ammonium as a nitrogen source, was shown to possess an active transport system which could concentrate ammonium 44 to 58 f o l d . Ammonium uptake was inhib i t e d by the glutamate analog methionine sulfone. The properties of the ammonium uptake system (transport and metabolism) were investigated using the ammonium analog methylammonium. The uptake of methylammonium was inhibited by arsenate indicating that phosphate bond energy was required. Methylammonium uptake was also inhibited by the electron transport i n h i b i t o r , cyanide, and the uncoupler, carbonyl cyanide- m-chlorophenyl hydrazone. However,it was shown that these agents served to deplete ATP pools in A_^  v i n e l a n d i i . Uptake of methylammonium was sensitive to a Tris-Mg +* shock treatment suggesting the possible involvement of a periplasmic binding protein, however, methylammonium-binding a c t i v i t y was not found in periplasmic extracts. A. v i n e l a n d i i was shown to exhibit a p o s i t i v e chemotactic response toward ammonium as well as acetate, glucose and sucrose. Comparison of outer membrane proteins from nitrogen-fixing c e l l s and ammonium-grown c e l l s revealed the production of a 44,000 dalton protein in membranes from nitrogen-fixing c e l l s . Inner membranes from nitrogen-fixing c e l l s contained a 41,000 dalton protein which was present in low amounts in the membranes of ammonium-grown c e l l s . It was shown that the outer membranes of ammonium-grown c e l l s contained a major protein which was "heat modifiable" in that i t s mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined by the temperature of s o l u b i l i z a t i o n prior to electrophoresis. Methylammonium was shown to be metabolized to N-methylglutamine. Strain JK301, an L-methionine-D,L-sulfoximine-resistant mutant of A. v i n e l a n d i i , was unable to catalyse N-methylglutamine synthesis in vivo or in c e l l - f r e e extracts and lacked detectable methylammonium uptake a c t i v i t y . Glutamine synthetase in c e l l - f r e e extracts of JK301 had a Km for glutamate approximately three-fold higher and a Vmax approximately four-f o l d lower than enzyme from the wild type s t r a i n . It was concluded that methylammonium uptake r e f l e c t s , in part, metabolism to N-methylglutamine by glutamine synthetase. Table of Contents Abstract i i L i s t of Tables v L i s t of Figures v i Acknowledgements v i i i I. INTRODUCTION 1 1. AMMONIUM TRANSPORT 2 2. REGULATION AND ENERGETICS OF AMMONIUM TRANSPORT ..4 3. AMMONIUM FROM NITROGEN FIXATION 5 4. AMMONIUM ASSIMILATION IN NITROGEN-FIXING BACTERIA 10 5. GLUTAMATE DEHYDROGENASE (GDH) 13 6. GLUTAMATE SYNTHASE (GOGAT) 14 7. GLUTAMINE SYNTHETASE (GS) 16 8. AIM OF THIS STUDY 19 II . MATERIALS AND METHODS 20 1. ORGANISM AND CULTURE CONDITIONS 20 2. MEASUREMENT OF AMMONIUM UPTAKE 21 3. DETERMINATION OF INTRACELLULAR AMMONIUM CONCENTRATION 22 4. METHYLAMMONIUM UPTAKE ASSAY 23 5. WHOLE-CELL PROTEIN DETERMINATION 24 6. ATP DETERMINATION 25 7. CHEMICAL SYNTHESIS OF N-METHYLGLUTAMINE 26 8. EXTRACTION AND PURIFICATION OF METABOLIZED METHYLAMMONIUM FROM WHOLE CELLS OF AZOTOBACTER VINELANDII 27 9. PAPER CHROMATOGRAPHY 28 10. CONJUGATION 29 11. BETA-LACTAMASE ASSAY 29 12. RELEASE OF PERIPLASMIC PROTEINS BY EDTA-LYSOZYME TREATMENT 30 13. TRIS-MG++ SHOCK TREATMENT OF WHOLE CELLS 31 14. AMMONIUM/METHYLAMMONIUM BINDING PROTEIN ISOLATION ATTEMPT 31 15. BINDING PROTEIN ASSAYS 33 16. CHEMOTAXIS STUDIES 35 17. PREPARATION OF INNER AND OUTER MEMBRANES OF AZOTOBACTER VINELANDII 35 18. SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) 37 19. TWO-DIMENSIONAL SDS-PAGE 37 20. TWO-DIMENSIONAL GEL ELECTROPHORESIS 38 21 . ENZYME ASSAYS 39 22. SNAKE VENOM PHOSPHODIESTERASE TREATMENT 43 I I I . AMMONIUM AND METHYLAMMONIUM TRANSPORT IN AZOTOBACTER VINELANDII , 44 1. AMMONIUM UPTAKE IN AZOTOBACTER VINELANDII 44 2. IS AMMONIUM UPTAKE AN ACTIVE TRANSPORT PROCESS? 47 3. INHIBITION OF AMMONIUM TRANSPORT BY METHIONINE SULFONE 49 i v 4. METHYLAMMONIUM UPTAKE IN AZOTOBACTER VINELANDII .49 5. EFFECT OF NITROGEN LIMITATION ON METHYLAMMONIUM UPTAKE 54 6. ENERGY REQUIREMENT FOR AMMONIUM AND METHYLAMMONIUM UPTAKE 56 7. SENSITIVITY OF METHYLAMMONIUM UPTAKE TO OSMOTIC SHOCK 66 IV. METHYLAMMONIUM METABOLISM IN AZOTOBACTER VINELANDII 72 1. DETECTION OF METHYLAMMONIUM METABOLITE 72 2. IDENTIFICATION OF THE PRODUCT OF METHYLAMMONIUM METABOLISM 75 3. INVOLVEMENT OF GLUTAMINE SYNTHETASE IN THE METABOLISM OF METHYLAMMONIUM 83 4. USE OF METHYLAMMONIUM UPTAKE ASSAYS TO DETECT GS DEFECTIVE MUTANTS 87 V. CHARACTERIZATION OF A GLUTAMINE SYNTHETASE DEFICIENT MUTANT 92 1. JK301, A GLUTAMINE SYNTHETASE DEFECTIVE MUTANT ..92 2. PROTEIN CONCENTRATION, 96 3. PH OPTIMUM .96 4. INCREASE IN GLUTAMINE SYNTHETHASE ACTIVITY IN RESPONSE TO NITROGEN LIMITATION 103 5. SNAKE VENOM PHOSPHODIESTERASE TREATMENT 104 6. KM DETERMINATION FOR GLUTAMATE 107 VI. CHEMOTAXIS TOWARDS AMMONIUM BY AZOTOBACTER VINELANDII 110 VII. MEMBRANE PROTEINS OF AZOTOBACTER VINELANDII 116 1. INNER AND OUTER MEMBRANES OF AZOTOBACTER VINELANDII 116 2. MEMBRANE AND PERIPLASMIC PROTEINS OF METHYLAMMONIUM RESISTANT MUTANTS OF AZOTOBACTER VINELANDII 119 3. HEAT MODIFIABLE OUTER MEMBRANE PROTEINS IN AZOTOBACTER VINELANDII 122 4. INFLUENCE OF CARBON SOURCE ON OUTER MEMBRANE PROFILE 127 VIII. DISCUSSION 131 IX. LITERATURE CITED 148 V L i s t of T a b l e s Table I A b i l i t y of A . v i n e l a n d i i to take up NH^ + against a c o n c e n t r a t i o n gradient I I I Page 48 I I E f f e c t + o f n i t r o g e n s t a r v a t i o n on r a t e of 55 CH3NH3 uptake E f f e c t of i n h i b i t o r s of energy generation on 57 uptake of CH3NH3 IV E f f e c t of arsenate and CCCP on i n t r a c e l l u l a r 63 ATP l e v e l s V Release of 8-lactamase from A. v i n e l a n d i i s t r a i n 68 JK (RP1) VI I d e n t i f i c a t i o n of in_ v i v o metabolite of C H 3 N H 3 + 80 VII Absence of i n h i b i t o r y molecules i n c e l l f r e e 93 e x t r a c t s of JK301 V I I I Y-glutamyl hydroxamate synt h e s i s a c t i v i t y i n c e l l 95 free e x t r a c t s of JK and JK301 IX E f f e c t of n i t r o g e n l i m i t a t i o n on glutamine synthetase 105 a c t i v i t y i n c e l l f r e e e x t r a c t s from JK and JK301 X E f f e c t of snake venom phosphodiesterase treatment 106 on glutamine synthetase a c t i v i t y i n c e l l f r e e e x t r a c t s from JK and JK301 v i L i s t o f F i g u r e s Figure 1. A model for regulation of nitrogen metabolism 2. Pathways of ammonia a s s i m i l a t i o n for production of glutamate and glutamine i n nitrogen f i x i n g b a c t e r i a 3. Disappearance of NH,+ i n cultures of A. v i n e l a n d i i JK (NH4 uptake) 4. I n h i b i t i o n of ammonium uptake by methionine sulfone 5. Uptake of 1^C-methylammonium by whole c e l l s of A. v i n e l a n d i i JK 6. I n h i b i t i o n of ammonium uptake by KCN and CCCP 7. A time course of arsenate i n h i b i t i o n of methylammonium uptake 8. A time course of the e f f e c t of arsenate on ATP pools in A. v i n e l a n d i i 9. . E f f e c t of Tris-Mg"*-1" shock treatment on 1 **C-methyl-ammonium and 1 ''C-fructose uptake i n 10. Retention of accumulated 1 "*C-methylammonium i n A. v i n e l a n d i i JK upon addition of excess unlabeled me thylammonium 11. Histogram representing paper chromatograph of c e l l free extracts containing metabolized "*C-methyl-• mmonium 12. The amino acids N-methylglutamine and N-methyl-glutamate 13. Paper chromatograph of acid hydrolysis products of chemically synthesized N-methylglutamine 14. N-methylglutamine synthesis i n c e l l free extracts of A. v i n e l a n d i i JK 15. N-methylglutamine synthesis and methylammonium uptake i n A. v i n e l a n d i i JK 16. Uptake of 1 "*C-methylammonium by whole c e l l s of A. v i n e l a n d i i JK and A. v i n e l a n d i i JK301 v i i L i s t of Figures(cont) Figure Page 17. Glutamine synthetase mediated glutamine formation 97 i n c e l l free extracts from JK and JK301 18. E f f e c t of c e l l free extract concentration on rate 99 of glutamine synthesis 19. pH p r o f i l e of glutamine synthetase a c t i v i t y i n 101 c e l l free extracts from JK and JK301 20. Lineweaver-Burk p l o t of glutamine synthetase 108 a c t i v i t y i n c e l l free extracts of JK and JK301 21. Chemotaxis of A. v i n e l a n d i i toward ammonium 112 22. Outer membrane proteins of A. v i n e l a n d i i 117 23. Inner membrane proteins from A. v i n e l a n d i i 119 24. Two-dimensional gel electrophoresis of periplasmic 122 proteins from A. v i n e l a n d i i JK and A. v i n e l a n d i i JK213 25. Two-dimensional SDS-polyacrylamide gel electrophoresis 125 of A. v i n e l a n d i i outer membrane proteins demonstrating the presence of a heat modifiable outer membrane protein 26. Outer membrane proteins from A. v i n e l a n d i i grown on 128 d i f f e r e n t carbon sources v i i i Acknowledgement I am sincerely grateful to my advisor , Dr. Joyce Gordon , for excellent instruction and thorough supervision. I would l i k e to give a special thanks to Lynn Moore. Her help was e s s e n t i a l . F i n a l l y , I would l i k e to acknowledge the following people for the i r assistance along the way : Marty Jacobson , Bob and L i z Hancock , Thalia Nicas, Kevin Strange , A l l i s o n Ottem and Hector Lizama. 1 I. INTRODUCTION Azotobacter vinelandi i i s a Gram negative, motile, heterotrophic bacterium belonging to the family Azotobacteraceae. This family belongs to the f r e e - l i v i n g nitrogen-fixing bacteria (those not intimately associated with a s p e c i f i c plant) and are found in a number of s o i l s worldwide. Members of Azotobacteraceae are most noted for their a b i l i t y to f i x nitrogen a e r o b i c a l l y . In addition, they are capable of pe r s i s t i n g in nutrient poor s o i l s by forming cysts which provide protection from desiccation and allow survival u n t i l nutrient conditions improve. Unlike Rhizobium species, most f r e e - l i v i n g nitrogen-fixing bacteria do not form symbiotic associations with plants. However, increasing attention i s being given to using free-l i v i n g nitrogen-fixing bacteria as a means of supplying plants with fixed nitrogen and as a method of generating ammonium on a commercial scale. To be useful as plant f e r t i l i z e r f r e e - l i v i n g nitrogen-fixers must be able to perform two essential tasks. F i r s t , they must excrete some of the fixed nitrogen in a form u t i l i z e d by the plant. Secondly, they must f i x nitrogen c o n s t i t u t i v e l y so that fluctuating nitrogen l e v e l s in the s o i l would not repress nitrogenase. Isolation of strains of A. vi n e l a n d i i possessing these features requires an understanding of ammonium transport and metabolism. The experiments described in t h i s thesis are part of an e f f o r t to obtain- that understanding. 2 1. AMMONIUM TRANSPORT An important component of the process of exogenous ammonium assimilation i s the transport of ammonium across the b a c t e r i a l membrane. Although ammonia (NH3) has been assumed to passively diffuse across b a c t e r i a l membranes, with a pKa of 9.25 greater than 99% would be present as impermeable ammonium ions (NH„*) under physiological growth conditions. Because ammonium i s often a l i m i t i n g nutrient in natural conditions, i t i s reasonable to expect that transport systems would evolve to sequester ammonium from the environment. Ammonium transport in nitrogen-fixing bacteria would possibly serve three roles. F i r s t , low concentrations of ammonium in the environment can be scavenged thereby preventing the energy-expensive use of nitrogenase to supply the c e l l with ammonia. Secondly, when the c e l l i s f i x i n g nitrogen, an e f f i c i e n t ammonium transport system might serve to recycle NH a + l o s t to the external environment as a res u l t of d i f f u s i o n . F i n a l l y , when encased in root nodules, bacteroids may transport ammonium outwards for use by the host plant. The study of ammonium transport in microorganisms has been greatly f a c i l i t a t e d by the discovery that methylammmonium (CH 3NH 3 +), in many cases, serves as a non-metabolizable ammonium analog. Methylammonium has been used to study ammonium transport in bacteria (Stevenson and S i l v e r , 1977; Kleiner and Fitzke, 1979; B e l l i o n et a l . , 1980; Barnes and Zimniak, 1981; Gordon and Moore, 1981; Kleiner, 1982; Hartmann and Kleiner, 1982; Gober and Kashket, 1983), yeast (Roon et a l ., 1975), 3 fungi (Hackette et a l ., 1970; Pateman et a l . , 1974; Cook and Anthony, 1978) and algae (Pelley and Bannister, 1979; Wheeler, 1978, 1980). It can be radioactively labeled thus allowing convenient measurement by l i q u i d s c i n t i l l a t i o n counting. A l t e r n a t i v e l y , 1 3 N H „ + or 1 SNH, + can be used. 1 3NH f t + ,however, requires a cyclotron f a c i l i t y for generating the isotope and, the very short half l i f e (10 min ) demands high l e v e l s of r a d i o a c t i v i t y and rapid analysis of a l l samples. 1 5NH f l + has not been popular for ammonium transport studies due to lack of s e n s i t i v i t y and a requirement for a mass spectrometer to analyze samples. Examination of NH„* transport using methylammonium i s complicated by the recent reports that methylammonium i s metabolized in several genera of bacteria (Kleiner, 1982; Kleiner and Fitzke, 1981; Yoch et a l . , 1983; Gober and Kashket, 1983; Moore and Gordon, submitted). When methylammonium i s metabolized, uptake becomes a two-part process consisting of transport across the b a c t e r i a l membrane and metabolism. It i s d i f f i c u l t under these conditions to determine meaningful transport rates because one does not know whether the transport event i s influenced by the rate of metabolism. Transport k i n e t i c s can s t i l l be defined, however, i f conditions are controlled so that transport i s the rate l i m i t i n g step of the transport-metabolism process. For example, Kleiner (1982) has provided evidence that transport becomes the rate l i m i t i n g step in uptake of methylammonium in K. pneumoniae when the assay temperature i s approximately 10°C. 4 With one exception, a l l of the nitrogen-fixing organisms studied to date have an ammonium transport system. Evidence for th i s includes energy dependent accumulation of methylammonium against a concentration gradient and competitive i n h i b i t i o n of accumulation of methylammonium by ammonium (demonstrating a common transport system for both compounds). Rhizobium melioti is the one N 2 - f i x i n g organism where an ammonium transport system has not been found. When grown aerobically, accumulation of methylammonium in t h i s organism i s not i n h i b i t e d by ammonium suggesting that methylammonium enters the c e l l by d i f f u s i o n or by an alternative transport system (Kleiner, 1982). This i s apparently not the case for a l l Rhizobium species as Gober and Kashket (1983) have demonstrated ammonium/methylammonium transport in Rhizobium sp. st r a i n 32H1. 2. REGULATION AND ENERGETICS OF AMMONIUM TRANSPORT Regulation of NH« + transport in N 2 - f i x i n g bacteria has not been examined in great d e t a i l . This i s probably due to the lack of suitable mutants and well defined genetic systems in the bacteria studied. Some information, however, i s available. The ammonium transport system in K. pneumoniae i s induced during nitrogen-limiting conditions and i s co-regulated with glutamine synthetase, nitrogenase and the enzymes for h i s t i d i n e u t i l i z a t i o n (Kleiner, 1982). This suggests that NH 4 + transport genes, l i k e nitrogenase genes, are under ntrA control. Inducible ammonium transport systems have also been found in Pseudomonas sp. s t r a i n MA (Be l l i o n and Wayland, 1981), Azospirilium brasilense (Hartman and Kleiner, 1982) and 5 Rhodospirilium rubrum (Alef and Kleiner, 1982) although i t i s i not known whether these transport systems are co-regulated with other nitrogen assimilatory enzymes. In contrast to these organisms, the ammonium uptake system in A. v i n e l a n d i i (Barnes and Zimniak,1981, Gordon and Moore, 1981) and E. c o l i (Stevenson and S i l v e r , 1977) is c o n s t i t u t i v e . Oxygen may also play a role in regulation of ammonium transport. This i s suggested by studies with Rhizobium sp. s t r a i n 32H1 (Gober and Kashket, 1983) which demonstrated that transport a c t i v i t y was present when c e l l s were grown under reduced oxygen tension but not when cultures were grown aerobical l y . Studies which have examined the energetics of methylammonium/ammonium transport suggest that the transmembrane e l e c t r i c a l potential (A\//) drives ammonium/methylammonium transport in bacteria. In Clostridium pasteurianum methylammonium transport can be driven by an a r t i f i c i a l l y induced membrane potential (Kleiner and Fitzke, 1982). Experiments with A. vinelandi i have produced similar results (Laane et a l . , 1980; Barnes and Zimniak, 1981). 3. AMMONIUM FROM NITROGEN FIXATION An a l t e r n a t i v e method of obtaining ammonium i s through nitrogen f i x a t i o n . The enzyme nitrogenase, by catalysing the reduction of atmospheric nitrogen to ammonia (Reaction 1), affords A. vinelandi i and other nitrogen-fixing bacteria independence from fixed nitrogen. 6 (1) N 2 + 12 ATP + 6e~ > 2 NH3 + 12 ADP + 12Pi Nitrogenase consists of two soluble proteins which are referred to as component I and component I I . Component I i s also known as the MoFe protein due to the presence of a molybdenum-iron cofactor (FeMoCo). It i s the actual s i t e of N 2 reduction and consists of two pairs of non-identical subunits. These subunits are the products of the nitrogen f i x a t i o n genes nifD and nifK. Component II i s also known as nitrogenase reductase because of the role i t has in supplying component I with electrons used for the reduction of N 2. It i s also c a l l e d the Fe-protein because i t contains a large amount of non-heme iron. Component II consists of two i d e n t i c a l subunits which are coded for by the nifH gene. In K l e b s i e l l a pneumoniae the genes coding for component I and component II comprise a single t r a n s c r i p t i o n a l unit: ni fHDK. Nitrogenase i s t i g h t l y regulated in response to fixed nitrogen. Figure 1 i l l u s t r a t e s the interactions of some of the gene products involved in the regulation of nitrogenase and other nitrogen assimilatory enzymes by fixed nitrogen. Studies with K. pneumoniae have shown that in the presence of excess NH4 + , nif encoded proteins and nitrogenase a c t i v i t y are not detectable (Roberts et a l . , 1978), although NH„ + i s not the actual effector of nitrogenase repression (Gordon and B r i l l , 1974). Conditions of nitrogen l i m i t a t i o n result in the synthesis of the ntrA (ntr = nitrogen regulation) gene product (also referred to as glnF). This protein i s believed to 7 Figure 1. A model for regulation of nitrogen metabolism (taken from Kustu et a l . , 1979 and Merrick, 1982). Under nitrogen l i m i t i n g conditions the ntrA gene product interacts with the ntrC, ntrB gene products to form an activator capable of stimulating expression of glnA (glutamine synthetase st r u c t u r a l genes) and the nifLA operon . The nifA gene product i s a pos i t i v e effector required for expression of a l l other n if operons except i t s own while the n i f L gene product mediates oxygen repression. 8 ntrA ntrCntrB Y repressor I qlnA activator nif L.nifA j activator of nif genes 9 interact with the ntrC gene product to form an activator of the nifLA operon. The nifA gene product serves as a posit i v e effector for the transcription of the st r u c t u r a l genes of nitrogenase ( nifHDK) and for a l l other n i f t r a n s c r i p t i o n a l units (MacNeil et a l . , 1979). In the presence of excess ammonium the ntrA product i s not synthesized (or i s modified) resulting in repression of the nifLA operon by ntrC and ntrB gene products. The positive and negative effector a c t i v i t i e s of ntrA, ntrB and ntrC gene products also regulate the trans c r i p t i o n of the structural genes for glutamine synthetase and four periplasmic amino acid binding proteins in S. typhimurium (Garcia et §_1. , 1977; Kustu et a l . , 1979; McFarland et a l . , 1981). 0 2 also plays an important role in the regulation of nitrogenase. Brief exposure to oxygen results in rapid and i r r e v e r s i b l e i n a c t i v a t i o n of component I and II (Bulen and LeComte, 1966). It i s not surprising then, that 0 2 has been found to repress expression of nitrogenase s t r u c t u r a l genes presumably by interacting with the n i f L gene product to form a trans c r i p t i o n repressor (Eady et a_l., 1978). The a b i l i t y of A. vine l a n d i i to f i x nitrogen aerobically i s made possible by protecting the oxygen l a b i l e enzyme nitrogenase from atmospheric 0 2. This i s accomplished in part, by the very high respiration a c t i v i t y of A. v i n e l a n d i i which serves to rapidly reduce 0 2 ( P h i l l i p s and Johnson, 1961). Under conditions where the rate of respiration i s slowed the enzyme i s protected by complexing with another protein (referred to as the Shethna protein or Fe-S 10 protein II) which functions to increase the s t a b i l i t y of nitrogenase in the presence of 0 2 (Haaker and Veeger, 1977; Veeger et a l . , 1978). Molybdenum, essential as a part of the iron-molybdenum cofactor of component I, shares a role in the regulation of nitrogenase. In A. v i n e l a n d i i , component I i s not synthesized in molybdenum deficient medium (Nagatani et a l . , 1974) while in K. pneumoniae, neither component I or component II i s synthesized in cultures depleted of molybdenum ( B r i l l et a l . , 1974). It has been suggested that molybdenum aff e c t s the synthesis of the nifHDK transcript and that f u l l expression of the operon requires both molybdenum and component I or some other "molybdo-protein" (Dixon et a l . , 1980). Recently, i t has been discovered that A. v i n e l a n d i i has an alternative N 2 - f i x a t i o n system (Bishop e_t a l . , 1980; Bishop et a l . , 1982). This alternative system, unlike conventional nitrogenase, i s expressed in the absence of molybdenum and may have evolved in response to molybdenum l i m i t i n g environments. To date, i t i s not known whether other nitrogen-fixers possess thi s a lternative system. 4. AMMONIUM ASSIMILATION IN NITROGEN-FIXING BACTERIA The major pathways of ammonium assimilation in nitrogen-f i x i n g bacteria are i l l u s t r a t e d in F i g . 2. Ammonium obtained by the nitrogen-fixing bacterium either by transport of the ammonium ion or by nitrogen f i x a t i o n is assimilated primarily into glutamine or glutamate. These amino acids then serve as nitrogen donors for the biosynthesis of amino acids, purines, 11 Figure 2 . Pathways of ammonia assimilation for production of glutamate and glutamine in nitrogen f i x i n g bacteria. 12 2-oxoglutarate glutamate dehydrogenase •> glutamate nitrogenase •* N H - , <- nitrate reductase glutamate glutamine synthetase } glutamine 2-oxoglutarate glutamate synthase glutamate 13 pyrimidines and other nitrogen containing compounds (Umbarger, 1978). The bulk of glutamate synthesis in most bacteria i s achieved in two ways. Under conditions of ammonium excess synthesis of glutamate i s catalysed primarily by the enzyme glutamate dehydrogenase (GDH). When c e l l s are grown in growth l i m i t i n g concentrations of ammonium or when c e l l s are f i x i n g nitrogen, glutamate i s formed via a coupled reaction involving glutamine synthetase and glutamate synthase (Meers et a l . , 1970). These enzymes are discussed in d e t a i l below (sections 5, 6 and 7). Glutamate can also be synthesized from transamination of 2-oxoglutarate or as a result of degradation of h i s t i d i n e , arginine, proline and other amino acids. Alanine dehydrogenase, which synthesizes alanine from pyruvate and NH„ + , serves as an alternative to the glutamate dehydrogenase pathway in Clostridium pasteurianum and in several species of Ba c i l l u s (Mortenson, 1978; Moat, 1979). 5. GLUTAMATE DEHYDROGENASE (GDH) Under conditions of ammonium excess, the glutamate dehydrogenase (GDH; L-glutamate: NADP oxidoreductase, EC 1.4.1.4) pathway (Reaction 2) is the primary ammonium assimilatory pathway in enteric bacteria (Tyler, 1978). (2) NH3 + 2-oxoglutarate + NADPH L-glutamate + NADP+ The involvement of GDH in ammonia assimilation under nitrogen l i m i t i n g conditions i s considered negligible due to the 14 r e l a t i v e l y high Km for 2-oxoglutarate and ammonia (Sakamoto et a l . , 1975; Coulton and Kapoor, 1973; Mantsala and Zalkin, 1976; M i l l e r and Stadtman, 1972). Regulation of GDH in response to nitrogen l i m i t a t i o n varies among b a c t e r i a l genera. In K. aerogenes, GDH expression i s repressed under nitrogen starvation conditions (Brenchly et a l . , 1973; Meers et a l . , 1970), whereas in E. c o l i (Mecke and Holzer, 1966) and S. typhimur ium (Brenchley et a_l., 1975) i t i s not. Mutants lacking GDH in K. aerogenes (Brenchley and Magasanik, 1974) have no d i s t i n c t phenotype presumably because the demand for glutamate i s f u l f i l l e d by the glutamine synthetase-glutamate synthase pathway. GDH in A. vi n e l a n d i i has not been well studied. Kleiner (1975) has reported only background levels of NADPH dependent GDH in N 2 _ f i x i n g and NH„ +-grown (non-N 2 _fixing) cultures of A. vinelandi i . Possibly, A. vinelandi i generates s u f f i c i e n t l e v e l s of glutamate v i a glutamate synthase to f u l f i l l protein synthesis requirements. This concept has precedence because B a c i l l u s megaterium (Elmerich and Aubert, 1971), Rhodopseudomonas capsulata (Johansson and Gest, 1976) and Caulobacter crescentus (Ely e_t §_1. , 1978) have a l l been reported to lack detectable GDH a c t i v i t y . 6. GLUTAMATE SYNTHASE (GOGAT) Glutamate synthase (L-glutamate: NADP oxidoreductase, EC 1.4.1.3; GOGAT - from the o r i g i n a l t r i v i a l name glutamine 2-oxoglutarate glutamate amino transferase) catalyzes the transfer of the amide group of glutamine to 2-oxoglutarate to form two 15 molecules of glutamate. This reaction i s i l l u s t r a t e d below (Reaction 3): (3) L-glutamine + 2-oxoglutarate + NADPH + H* > 2 glutamate + NADP* GOGAT, from either E. c o l i or K. aerogenes, i s a 53,000 dalton i r o n - s u l f i d e flavoprotein with two separate subunits. The response of GOGAT to nitrogen l i m i t a t i o n varies amongst bacteria. In K l e b s i e l l a pneumoniae, GOGAT i s found in equal quantities whether c e l l s are grown in minimal media containing excess or growth l i m i t i n g amounts of NH^ ,* (Nagatani et a l . , 1971). Studies with K. aerogenes, however, have resulted in c o n f l i c t i n g findings. In one s t r a i n , GOGAT a c t i v i t y was higher under ammonium l i m i t i n g conditions and lower with excess ammonia, while in another strain the reverse was found (Meers e_t a l . , 1 970; Brenchley et a l . , 1973). Tyler (1978) has suggested a model whereby a high internal concentration of glutamate or a low inte r n a l concentration of glutamine or NHft + result in repressed synthesis of the enzyme. Additional information i s needed, however, to test t h i s hypothesis. Mutants of K. aerogenes lacking glutamate synthase are unable to grow on glucose minimal medium containing low amounts of NH„ +. Presumably GDH cannot catalyse glutamate synthesis under these conditions because of i t s r e l a t i v e l y high Km for ammonia (Brenchley et a_l., 1973). 1 6 7. GLUTAMINE SYNTHETASE (GS) Under nitrogen l i m i t i n g conditions, ammonium i s primarily assimilated into glutamine by the enzyme glutamine synthetase (Glutamate: ammonia ligase, EC 6.3.1.2). Glutamine synthetase catalyzes glutamine biosynthesis as diagrammed in Reaction 4. (4) NH3 + L-glutamate + ATP > L-glutamine + ADP + Pi + H 20 Tronick et a l . (1973) have shown that antiserum against E. c o l i glutamine synthetase cross reacts with glutamine synthetase isolated from a number of Gram negative bacteria including A. v i n e l a n d i i . These authors suggest that glutamine synthetase proteins from a l l gram negative bacteria are an t i g e n i c a l l y related. The a c t i v i t y of glutamine synthetase i s regulated by covalent. modification, feedback i n h i b i t i o n and divalent cation concentration (Shapiro and Stadtman, 1970). Covalent modification involves the reversible adenylylation of ty r o s y l residues on each subunit of the enzyme. Because the enzyme has 12 subunits, the adenylylation state can range from completely adenylylated enzyme ( E 1 2 ) to a f u l l y deadenylylated enzyme ( E 0 ) . Highest enzyme a c t i v i t y i s obtained from the deadenylylated form and i s associated with N-limiting conditions. Adenylylation (attachment of AMP groups) and deadenylylation i s mediated by adenylyltransferase (ATase). This enzyme in turn, i s regulated by the PII protein. PII can exis t in two states, an unmodified form (PIIA) which stimulates adenylylation a c t i v i t y of the ATase 17 (resulting in lower glutamine synthetase a c t i v i t y ) or a modified form (PUD) which stimulates the deadenylylation a c t i v i t y of ATase. The modification of PII i s achieved by u r i d y l y l a t i o n of the protein by the enzyme uridy l y l t r a n s f e r a s e (UTase). Uridylyl-removing enzyme (UR) removes u r i d y l y l groups from the PII enzyme and may possibly be the same protein as UTase (Tyler, 1978). Growth in the presence of excess ammonium or high internal concentrations of glutamine result in increased adenylylation of glutamine synthetase as the result of stimulated UR a c t i v i t y and consequently increased ATase a c t i v i t y . When the enzyme is adenylylated i t i s more sensitive to feedback i n h i b i t i o n by ADP and several amino acids. In contrast, nitrogen l i m i t i n g conditions or high internal l e v e l s of 2-oxoglutarate result in greater deadenylylation of the enzyme as a result of stimulated UTase a c t i v i t y and increased ATase mediated deadenylylation a c t i v i t y . In E. c o l i (Tyler, 1978) an excess amount of a good nitrogen source represses synthesis of glutamine synthetase. In contrast, i t has been reported that synthesis of A. v i n e l a n d i i glutamine synthetase is not repressed under conditions of nitrogen excess (Lepo et a l .,1982). H i s t o r i c a l l y , glutamine synthetase was believed to be d i r e c t l y involved in the regulation of nitrogenase. This was suggested in part, by studies with glutamine synthetase mutants of K. pneumoniae. One class of these mutants, unable to synthesize glutamine synthetase, was also unable to synthesize nitrogenase. In addition, mutations which resulted in the 18 cons t i t u t i v e synthesis of glutamine-synthetase also resulted in con s t i t u t i v e synthesis of nitrogenase (Streicher et a l . , 1974; Tubb, 1974). An alternative interpretation of these results was made possible by the discovery of the ntrA, ntrB and ntrC l o c i in S. typhimur ium (Garcia et a_l. , 1977; Kustu et a l . , 1979; McFarland e_t a_l. , 1979). Genetic analysis has shown that genes coding for glutamine synthetase, nitrogenase and other nitrogen assimilatory enzymes are controlled by ntr gene products (deBruijn and Ausubel,1981; Espin et a l . , 1981) . These interactions are i l l u s t r a t e d in Fig. 2. The ntrA gene product is produced under conditions of nitrogen-limitation and interacts with the ntrC gene product to form a p o s i t i v e effector for expression of glutamine synthetase structural genes and for nifA (which i s required for the expression of a l l other n i f operons) and glnA, the s t r u c t u r a l gene for glutamine synthetase. The regulatory protein PII, in the de-uridylyated form, i s believed to act as a co-repressor preventing the ntrC gene product from acting as an activator (Foor et a l . , 1980; Bloom e_t a l . , 1978). Accordingly, mutants lacking PII have high levels of glutamine synthetase even when they are grown in the presence of excess ammonia (Foor e_t a_l., 1980). Glutamine synthetase from A. vinelandi i has been p u r i f i e d to homogeneity (Siedel and Shelton, 1979; Kleinschmidt and Kleiner, 1 978; Lepo et a_l. , 1979). As in E. c o l i , the enzyme consists of 12 subunits in two superimposed hexagonal ^r-ings and is regulated by adenylylation and deadenylylation (Siedel and Shelton, 1979; Kleinschmidt and Kleiner, 1978). The mutant in 19 thi s study, JK301, i s the only glutamine synthetase mutant of A. v i n e l a n d i i reported to date. Further study with t h i s mutant should help elucidate the genetics of glutamine synthetase in t h i s organism. 8. AIM OF THIS STUDY The aim of this study was to investigate ammonium uptake and assimilation in A. v i n e l a n d i i . The data reported here demonstrated that A. v i n e l a n d i i possesses an active transport system for ammonium. The ammonium uptake system (transport and metabolism) was characterized using the ammonium analog 1"C-methylammonium. It was shown that A. vinelandi i rapidly metabolizes methylammonium in a reaction mediated by the enzyme glutamine synthetase. In addition, a mutant resistant to the glutamate analog L-methionine-DL-sulfoximine was characterized. This mutant fixed N 2 c o n s t i t u t i v e l y in the presence of NH j, * and excreted NH a + into the growth medium. An examination of glutamine synthetase in c e l l - f r e e extracts of t h i s mutant revealed a Km for glutamate about 3-fold greater than that found in the the wild type s t r a i n and a Vmax about 4-fold lower than that found in the wild type s t r a i n , compared to enzyme from the wild type s t r a i n . 20 I I . MATERIALS AND METHODS 1. ORGANISM AND CULTURE CONDITIONS The wild type organism used was A. vinelandi i s t r a i n OP (Bush and Wilson, 1959) obtained from the American Type Culture Col l e c t i o n and is referred to as JK. Mutant strains UW1 (Fisher and B r i l l , 1969) and UW2 (Gordon and B r i l l , 1972) are derived from A. vi n e l a n d i i OP and are unable to f i x N 2. Cultures were grown in a modified Burk's medium (Strandberg and Wilson, 1968). Burk's medium was prepared by d i l u t i n g concentrated Burk's s a l t s solution d i r e c t l y into Burk's buffer. Burk's sal t s (10 f o l d concentrated stock solution) contain per l i t e r : MgS04-7H20, 2.0 g; CaCl 2-2H 20, 0.9 g; FeCl 3-6H 20, 48.8 mg; Na 2MoO«, 2.5 mg; and sucrose, 200 g, and was autoclaved separately. Burk's buffer contains, per l i t e r : KH2POa, 0.055 g and K2HPOa, 0.22 g. Ammonium grown cultures were provided with 400 Mg N/ml as ammonium acetate. The ammonium acetate (a 100-fold concentrated stock solution) was f i l t e r s t e r i l i z e d and added separately. A l l cultures were grown at 30°C in a New Brunswick environmental rotary shaker (250 rpm) in baffled flasks to increase aeration. C e l l density was followed with a Klett-Summerson colorimeter with a number 64 f i l t e r . With the instrument used, a density of 80 Klett units corresponds to a t o t a l protein content of 0.35 mg/ml. 21 2. MEASUREMENT OF AMMONIUM UPTAKE Ammonium uptake was measured by following the disappearance of ammonium from the medium. Cultures were grown to 80 Klett units, harvested by centrifugation (10,000 x g for 10 min) and washed twice with Burk's N-free medium. Alte r n a t i v e l y , c e l l s were co l l e c t e d on a 0.45 .unt-pore-size f i l t e r (Gelman, Inc., Ann Arbor, Mich.) and washed twice with Burk's N-free medium. The washed c e l l s were resuspended to the o r i g i n a l density in Burk's N-free medium and kept on ice u n t i l assayed. Immediately prior to assay of ammonium uptake, the c e l l suspension was incubated aerobically for 10 min to allow for depletion of remaining e x t r a c e l l u l a r ammonium. At the end of the incubation period, ammonium (as ammonium sulfate or ammonium acetate) was added to a f i n a l concentration of 0.8 mM. At periodic intervals, 1 ml samples were removed and f i l t e r e d through 0.45 -jxm-pore-size f i l t e r s . The f i l t r a t e was assayed in t r i p l i c a t e for ammonium content using a glutamate dehydrogenase assay (Strecker, 1955). The assay mixture consisted of 0.1 ml f i l t r a t e and 2.9 ml of potassium phosphate buffer, pH 7.8, containing 4.3 mM NADH, 22 mM ADP, 20 mM 2-oxoglutarate and approximately 100 Mg/ml of glutamate dehydrogenase (Sigma Chemical Co., St. Louis, Mo.). After 30 min incubation at 30°C the NADH concentration in each tube was determined by measuring absorbance at 340 nm. Control tubes received 0.1 ml of buffer in place of f i l t r a t e . The decrease in NADH concentration i s equivalent to the amount of ammonium present in the tube. Ammonium determinations for each time point were done in t r i p l i c a t e and the average values used 22 for determining rates- of ammonium disappearance. When ammonium assays were performed in the presence of carbonyl cyanide-m-chlorophenyl hydrazone (CCCP), arsenate, cyanide or L-methione sulfone (MSF), incubation for 5 min in the presence of the inhi b i t o r preceded the addition of ammonium. 3. DETERMINATION OF INTRACELLULAR AMMONIUM CONCENTRATION The i n t r a c e l l u l a r concentration of ammonium was determined using a modification of the procedure described by Kleiner (1975). Toluene (0.1%) was added to cultures (100-200 ml) to k i l l c e l l s thereby preventing . further metabolism of i n t r a c e l l u l a r ammonium. Control experiments showed that c e l l s treated with 0.1% toluene did not have lower ammonium pools than untreated c e l l s . C e l l s were c o l l e c t e d by centrifugation (12,000 x g for 5 min) and washed twice with Burk's N-free medium (containing 0.1% toluene). The c e l l p e l l e t from the f i n a l centrifugation was resuspended in 1 ml of Burk's phosphate buffer,transfered to a test tube , capped with a marble and placed in a b o i l i n g water bath for 5 min. The c e l l suspension was then subjected to ultrasonic disruption for 4 min with a Bronwill Biosonic ultrasonic o s c i l l a t o r at a setting of 55 to 70. C e l l debris was removed by centr-ifugation at 12,000 x g for 20 min and the ammonium concentration of the supernatant determined by a glutamate dehydrogenase assay. Concentration of i n t r a c e l l u l a r ammonium was determined assuming an i n t r a c e l l u l a r volume of 1.9 ul/ml of culture grown to 80 Klett units. This value was obtained by determining the volume of packed c e l l s from an 80 Klett unit culture and assuming that the i n t e r s t i t i a l 23 and c e l l bound water was approximately four times the dry weight of the packed c e l l s (Drozd et a l . , 1972). 4. METHYLAMMONIUM UPTAKE ASSAY Cultures were grown to a density of 80 Kl e t t units with ammonium acetate as an ammonium source. Because ammonium i s a potent i n h i b i t o r of methylammonium uptake, e x t r a c e l l u l a r ammonium was removed by washing c e l l s twice with Burk's N-free medium (washing was accomplished as described for ammonium uptake experiments). In some of the experiments designed to examine the effect of arsenate on methylammonium uptake, 5 mM glycylglycine buffer supplemented with Burk's s a l t s ( f i n a l pH 7.45) replaced Burk's N-free medium. Washed c e l l s , suspended in N-free medium to o r i g i n a l density, were stored on ice u n t i l assayed. C e l l s were preincubated aerobically for 10 min at 30°C in a gyrotary water bath before 1flC-methylammonium was added to i n i t i a t e the -reaction. The s p e c i f i c a c t i v i t y of the i aC-methylammonium was 1 to 5 MCi/ju,niol. Assays were performed in 20 ml glass s c i n t i l l a t i o n v i a l s in a t o t a l volume of 1.2 ml. When used, i n h i b i t o r s were added 5 min prior to the addition of 1 4C-methylammonium. At various times within a 5 minute time period, five 200 / i l samples 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 0.45 jum-pore-size f i l t e r s (25 mM diameter). The c e l l s on the f i l t e r were washed twice with approximately 2 ml of wash buffer. The wash buffer was maintained at the assay temperature of 30°C and consisted of Burk's N-free medium containing unlabeled methylammonium at a concentration equal to that in the assay. In some experiments an Amicon VFM f i l t e r 24 manifold was used to c o l l e c t c e l l s . When t h i s device was used, c e l l s c o l l e c t e d on the f i l t e r s were washed with approximately 5 ml of wash buffer. F i l t e r s containing b a c t e r i a l c e l l s were placed d i r e c t l y into p l a s t i c s c i n t i l l a t i o n v i a l s and dried at 55°C for 60 min. The f i l t e r s in the v i a l s were then covered with s c i n t i l l a t i o n f l u i d (10 ml, 4 g diphenyloxazole/1 toluene) and counted in 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 in a Beckman LS 7500 l i q u i d s c i n t i l l a t i o n counter. Uptake assays were performed in duplicate and rates of methylammonium uptake determined by linear regression analysis of data points. Data used for figures in this thesis represent the results of one of the duplicate assays performed for each experiment. 5. WHOLE-CELL PROTEIN DETERMINATION A portion of c e l l suspension used for uptake assays was centrifuged (10,000 x g for 10 min) and the p e l l e t washed once with Burk's buffer before being resuspended to the o r i g i n a l weight in d i s t i l l e d water. A 1 ml sample of the washed c e l l suspension was placed into a test tube containing 1 ml of 1N NaOH and the tube capped with a marble before being immersed in a b o i l i n g water bath for 5 min. The sample was cooled and a portion removed for determination of protein content using the modified Lowry procedure (Lowry et a l . , 1951) of Schacterle and Pollack (1973). 25 6. ATP DETERMINATION In t r a c e l l u l a r ATP levels were determined by the l u c i f e r i n -l u ciferase system of Kimmich et a l . (1975). Cultures were grown to 80 Klett units as described above and ATP extracted from c e l l s as described by Cole et al_ (1967). A 2 ml sample of c e l l s was rapidly transferred to a glass centrifuge tube containing 0.5 ml of ice cold 34% perchloric acid containing 67 mM EDTA, immediately mixed on a vortex mixer and placed on ice. The tubes were then centrifuged (20,000 x g for 15 min) at 4°C to remove c e l l debris. A 2 ml portion from each tube was adjusted to pH 7.0 with 50 mM Tris-H 2SO„ containing 1 M KOH and 83.3 mM K 2SO«. The tubes were then placed on ice for 1 hr to allow the potassium perchlorate (formed during neutralization) to p r e c i p i t a t e . A 2 ml portion was transferred to a glass centrifuge tube and centrifuged for 20 min at 20,000 x g at 4°C. A 1 ml portion of supernatant was then transferred to a new test tube and kept on ice u n t i l ATP determination was performed. Controls containing known amounts of ATP were subjected to the same extraction procedure used for c e l l samples. For ATP determination, 0.9 ml of a 200 mM glycylglycine buffer, pH 8.0, containing 5 mM sodium arsenate and 4 mM MgSO„ was dispensed into a 20 ml, glass s c i n t i l l a t i o n v i a l together with a 50 ul volume of sample. To start the reaction, 50 ul of f i r e f l y lantern extract (Sigma Chemical Co., St. Louis, Mo.) was added to the s c i n t i l l a t i o n v i a l . The v i a l was placed in a s c i n t i l l a t i o n counter (Nuclear Chicago Isocap 300-720 Series) operated at room temperature and lowered into the counting 26 chamber. Window settings were adjusted to maximum openings to detect a l l s c i n t i l l a t i o n s . Exactly 20 seconds after addition of the f i r e f l y lantern extract, s c i n t i l l a t i o n counting was i n i t i a t e d and recorded for 0.4 min. Counts per minute generated from the samples were compared to a standard curve (0-50 pmol/50 jul) for determination of ATP concentration. 7. CHEMICAL SYNTHESIS OF N-METHYLGLUTAMINE N-methylglutamine was synthesized according to the method of Lichtenstien (1942). The procedure for synthesis consisted of mixing 2.5 g of L-pyroglutamate (Sigma Chemicals, St. Louis, Missouri) with 30 ml of 17% aqueous methylammonium (Eastman Kodak) in an Erlenmeyer flask sealed with a rubber stopper, and incubating at 37°C for 10 days. The solution was then poured into a glass p e t r i dish and placed in a desiccator containing a beaker of concentrated H 2SO„ and a p e t r i dish of desiccant. The desiccator was evacuated and allowed to stand for 3 days at room temperature. A 20 ml volume of absolute ethanol was added to the reaction mixture res u l t i n g in the formation of a white p r e c i p i t a t e . The mixture containing the p r e c i p i t a t e was transferred to a glass bottle and placed at 4°C overnight. The p r e c i p i t a t e was then c o l l e c t e d on a piece of Whatman f i l t e r paper, washed three times with cold absolute ethanol and dried at room temperature. The melting point of the product agreed with that o r i g i n a l l y reported by Lichenstien for N-methylglutamine (192°C) and reacted strongly with ninhydrin. For confirmation that hydrolysis of the synthesized product would y i e l d methylammonium 27 and glutamate, a 5 mg/ml solution of product in 1M perchloric acid was heated at 90°C for 2 hours. The mixture was neutralized with an equal volume of 1M KHC03 and the precipitate removed by centrifugation. Approximately 5 ul of supernatant was spotted onto Whatman 3MM paper and chromatographed in a isopropanol: H 20: g l a c i a l acetic acid (70: 30: 1) solvent system together with standards (1 mg/ml solutions of glutamate, methylammonium-HCl, unhydrolysed product and N-methylglutamate). A sample of N-methylglutamate (5mg/ml, Sigma Chemical Co., St. Louis, Mo.) subjected to the same acid hydrolysis treatment as synthesized product was also run on the same chromatograph. Compounds were v i s u a l i z e d with ninhydrin. 8. EXTRACTION AND PURIFICATION OF METABOLIZED METHYLAMMONIUM  FROM WHOLE CELLS OF AZOTOBACTER VINELANDII Extraction of 1"C-methylammonium from whole c e l l s was accomplished as follows. C e l l s were prepared as described for 1"C-methylammonium uptake assays. 1"C-methylammonium was added to a f i n a l concentration of 20 pM ( s p e c i f i c a c t i v i t y = 10/iCi/Mniole) and the culture allowed to incubate an additional 3 min before 1 ml was transferred to a centrifuge tube containing 0.25 ml of an ice cold mixture of 41.25 g of bromododecane and 0.749 g of dodecane (Barnes and Zimniak, 1982). After centrifugation for 3 min in an Eppendorf 5412 centrifuge the supernatant and o i l were c a r e f u l l y removed and 50 ul of 1N perchloric acid was used to resuspend the p e l l e t . The mixture was neutralized by addition of 50 ul of 1N KHC03 and the p r e c i p i t a t e removed by centrifugation. A portion of the 2 8 supernatant was streaked along the bottom of a piece of Whatman 3MM chromatography paper and ascending chromatography performed in a phenol:H 20 solvent system (the lower phase of a phenol/H 20 mixture). The portion of the chromatograph corresponding to the metabolite of methylammonium was cut out and soaked in 10 ml of Burk's buffer to elute the metabolite. The eluate was ly o p h i l i z e d , resuspended in H 20 and chromatographed in several solvent systems to determine the ide n t i t y of the metabolite. 9. PAPER CHROMATOGRAPHY Paper chromatography was used to determine the product of methylammonium metabolism. A 5-10 M1 volume (5-10,000 cpms) of c e l l extract prepared from c e l l s incubated in the presence of 1"C-methylammonium was spotted onto Whatman 3MM chromatography paper. 1"C-methylammonium-HCl (Amersham Corporation, Oakville, Ontario) was used as a reference where required. Reference amino acids (5 mg/ml) were prepared in 1M perchloric acid, neutralized with an equal volume of 1M KHC03 and the supernatant, after centrifugation, used as standard. Approximately 5 M1 of standard was spotted onto the chromatography paper. In chromatography experiments designed to determine i f the product of methylammonium metabolism co-migrated with synthesized N-methylglutamine, the p u r i f i e d , radioactive metabolite was spotted d i r e c t l y on top of the N-methylglutamine standard. Chromatographs were developed u n t i l the solvent front had traveled 15-16 cm from the o r i g i n . Ninhydrin was used to v i s u a l i z e amino acid standards. Radioactive compounds were detected by cutting individual lanes 29 into 0.5 - 1.0 cm sections and counting in a l i q u i d s c i n t i l l a t i o n counter. I d e n t i f i c a t i o n of the compounds was achieved by comparing the position of radioactive peaks to the position of reference compounds in other lanes. 10. CONJUGATION The plasmid RP1 was introduced into A. v i n e l a n d i i s t r a i n JK by conjugation with Escherichia c o l i UB1936 (RP1) kindly provided by R. E. W. Hancock, University of B r i t i s h Columbia. The RP1 plasmid confers resistance to a m p i c i l l i n , c a r b e n i c i l l i n , neomycin, kanamycin and tetr a c y c l i n e (.Grinsted et a l . , 1972). Resistance to 0-lactam a n t i b i o t i c s i s mediated by a TEM-2 type /3-lactamase (Sykes and Mathew, 1976). A 0.5 ml volume of c e l l s , from a 80 Klett unit culture of A. vi n e l a n d i i was mixed with a 0.1 ml volume of an exponential culture of E. c o l i UB1636 (RP1) in a 250 ml flask containing Burk's medium (10 ml) supplemented with nutrient broth (8 g/1) and shaken gently overnight at 30°C. The following day the c e l l s were coll e c t e d by centrifugation, washed twice with Burk's N-free medium and plated onto Burk's N-free medium containing 1 mg/ml a m p i c i l l i n . Colonies a r i s i n g on these plates were p u r i f i e d by restreaking to the same medium a minimum of 2 times. 11. BETA-LACTAMASE ASSAY ^-lactamase was assayed using the chromogenic /3-lactam nitrocephin (O'Callaghan et a l . , 1972). A 1.5 mg portion of nitrocephin was dissolved in 100 M1 of dimethylsulfoxide and brought to 25 ml with sodium phosphate buffer, pH 7.0 ( f i n a l 3 0 phosphate concentration = 0.25 mg/ml). The nitrocephin solution was kept in a lightproof v i a l to prevent decomposition. A volume of 20 - 100 /ul of sample was added to 650 jul of the nitrocephin solution and the increase in absorbance at 540 nm monitored as nitrocephin was converted to n i t r o c e f o i c acid by /3-lactamase. Calculation of the nmol nitrocephin oxidized was based on a millimolar extinction c o e f f i c i e n t of 26.66 at 540 nm. 12. RELEASE OF PERIPLASMIC PROTEINS BY EDTA-LYSOZYME TREATMENT Periplasmic proteins examined by 2-dimensional gel electrophoresis were released by the EDTA-Lysozyme method of Cho et a l . , (1974), with s l i g h t modifications. Cultures (400 ml) were harvested at a density of 80 Klett units , washed twice with 10 mM Tris-HCl, pH 8.0, and the p e l l e t resuspended in (1 g wet weight/80 ml) 30 mM Tris-HCl, pH 8.0, containing 20% lactose and placed on ice. EDTA and lysozyme (Sigma Chemical Co., St. Louis, Mo.) were added to f i n a l concentrations of 10 mM and 0.5 mg/ml, respectively. Phenyl-methyl-sulfonyl fluoride (dissolved in isopropranol) was added to a f i n a l concentration of 1 mM (to i n h i b i t p r o t e o l y t i c enzymes) and the suspension gently s t i r r e d for 30 min. The c e l l suspension was centrifuged (10,000 x g for 1 hr) and the supernatant removed and f i l t e r e d through a Gelman 0.45 jum-pore-size f i l t e r . The f i l t e r e d supernatant was l y o p h i l i z e d to dryness and resuspended in 2 ml of d i s t i l l e d H 20. The sample was then placed in d i a l y s i s tubing and dialysed against several changes of Burk's buffer and stored at 0-4°C u n t i l used. 31 13. TRIS-MG++ SHOCK TREATMENT OF WHOLE CELLS Periplasmic proteins from A. v i n e l a n d i i were released by subjecting c e l l s to a Tris-Mg* + shock treatment as described by Hoshino (1979). C e l l s were harvested at a density of 80 Klett units and washed once with an equal volume of Tris-HCl, pH 7.4, containing 1 mM MgCl 2 and 1 mM KCl (TMK buffer). C e l l s were colle c t e d by centrifugation and resuspended in an equal volume of 10 mM Tris-HCl, pH 8.4, containing 0.2 M MgCl 2 (extraction buffer). After a 5 minute incubation at 30°C, the c e l l suspension was c h i l l e d in an ice bath to 4°C and incubated for 15 min. The hot-cold cycle was repeated and the c e l l s c o l l e c t e d by centrifugation (10,000 x g for 20 min). The supernatant, containing periplasmic proteins, was c a r e f u l l y removed and kept at 4°C u n t i l used. 14. AMMONIUM/METHYLAMMONIUM BINDING PROTEIN ISOLATION ATTEMPT An attempt was made to detect an ammonium/methylammonium-binding protein in periplasmic shock f l u i d from A. v i n e l a n d i i . C e l l s were grown in Burk's medium with ammonium acetate as a nitrogen source and 2% glucose as a carbon source . Glucose was used as a carbon source to determine i f a glucose binding protein could be induced in A. v i n e l a n d i i . However, glucose binding a c t i v i t y was not detected during the experiment. A t o t a l of 10 x 200 ml cultures were pooled and centrifuged (8,000 x g for 10 min). The p e l l e t was washed once in N-free medium'to-remove excess ammonium as th i s would presumably compete with 18C-methylammonium in subsequent binding assays. The culture 32 was then subjected to the Tris-Mg + + shock treatment described above. To i n h i b i t the action of proteolytic enzymes, phenylmethylsulfonyl fluoride (dissolved in isopropanol) was added to a concentration of 1 mM when c e l l s were resuspended in Tris-Mg + + buffer. To determine i f the shock procedure e f f e c t i v e l y reduced transport a c t i v i t y , c e l l s were assayed for 1"C-methylammonium uptake a c t i v i t y before and after shock treatment. After 2 cycles of warming to 30°C and cooling to 4°C the suspension was centrifuged (8,000 x g for 1 hr). The -supernatant (shock f l u i d ) was poured off and f i l t e r e d through a 0.45 ,um-pore-size membrane f i l t e r and concentrated to 30 ml in an Amicon u l t r a f i l t r a t i o n device equipped with a YMIO f i l t e r . After the concentrated shock f l u i d was removed, 10 ml of T r i s -HCl, pH 7.5, containing 300 mM NaCl was added to the u l t r a f i l t r a t i o n device chamber and s t i r r e d gently for 30 min to remove protein which may have been bound to the f i l t e r . The Tris-NaCl was removed and added to the 30 ml of concentrated shock f l u i d , placed in a d i a l y s i s bag and dialysed against 3 changes of reagent grade Tris-HCl, pH 7.5. D i a l y s i s tubing had previously been treated by b o i l i n g in d i s t i l l e d water containing 1% sodium carbonate and 1 mM EDTA for 15 min and washed extensively with d i s t i l l e d H 20. A volume of 35 ml of concentrated, dialysed shock f l u i d was loaded onto a 10 x 140 mm DEAE-Sephacel column equilibrated with 10 mM Tris-HCl, pH 7.5. Proteins were eluted off the column with a lin e a r gradient of NaCl (0 - 1 M) in 10 mM Tris-HCl, pH 7.5 and co l l e c t e d in 30 x 1.25 ml fractio n s . An elution p r o f i l e , obtained by pl o t t i n g the 33 absorbance at 280 nm of each fr a c t i o n against fracti o n number, revealed 3 major protein peaks. The fractions were placed in groups of 5 and a portion of each removed and pooled together in one test tube. The pooled column fractions were then tested for both 1 f tC-glucose and 1^methylammonium binding a c t i v i t y as described below. 15. BINDING PROTEIN ASSAYS (A) Membrane f i l t e r binding assay The membrane f i l t e r binding assay (Lever, 1972) was used in an attempt to detect 14C-methylammonium binding a c t i v i t y in pooled column fractions of periplasmic shock f l u i d . Because n i t r o c e l l u l o s e f i l t e r s bind protein n o n - s p e c i f i c a l l y , binding proteins bound to la b e l l e d substrate can be detected by f i l t e r i n g a mixture of binding protein and radioactive substrate through n i t r o c e l l u l o s e f i l t e r s and measuring r a d i o a c t i v i t y on the f i l t e r s (due to binding protein-substrate conjugates). The assay mix used in these experiments contained protein sample (1-200 jug protein/assay), 10 mM Tris-HCl, pH 7.5, and 1 M M 1 "C-methylammonium (56 /uCi/jumol). . For detection of glucose binding a c t i v i t y , 1"C-glucose was used at a concentration of 1 M M (210 MCi/Atmol). After incubation at room temperature for 5 min, volumes of 100 to 400 jul of assay mix were f i l t e r e d under vacuum through a 0.45 pun-pore-size n i t r o c e l l u l o s e f i l t e r (Gelman GN-6 "Metricel" membrane f i l t e r , Gelman Sciences, Inc., Ann Arbor, Michigan) and washed with 600 jil of 10 mM Tris-HCl, pH 7.5. To minimize rinsing off any protein:substrate complexes from the f i l t e r , a volume of assay mix was placed on the f i l t e r 34 with the vacuum off and the vacuum used only as long as required to p u l l the sample through the f i l t e r . The wash buffer was applied and f i l t e r e d in. the same manner. The f i l t e r s were removed, dried at 55°C for 1 hr and counted using a diphenyloxazole/toluene (4 g/1) s c i n t i l l a t i o n f l u i d . To estimate the amount of 1uC-methylammonium non-specifically bound to protein and to f i l t e r s , assay mix containing bovine gamma globulin instead of sample was f i l t e r e d through n i t r o c e l l u l o s e f i l t e r s and treated as described above. Background levels of ra d i o a c t i v i t y were also determined by adding 100 M M NH„C1 2 to the assay mix containing sample. This sample was then f i l t e r e d and treated as described above. Assuming that an ammonium binding protein would bind ammonium p r e f e r e n t i a l l y to 1"C-methylammonium the difference in 14C-methylammonium bound to f i l t e r s in the presence and absence of 100 M M NH„C1 should represent 1ftC-methylammonium bound to a s p e c i f i c ammonium binding s i t e . (B) Equilibrium d i a l y s i s binding assay Equilibrium d i a l y s i s was used as an alt e r n a t i v e method for detecting binding protein a c t i v i t y . Sample (0.3 ml containing 10-150 Mg protein) was dialysed overnight at 0-4°C against 10 mM Tris-HCl, pH 7.5, with 0.1, 1.0 or 10 nM 1"C-methylammonium or 10 nM 1"C-glucose. After d i a l y s i s , the r a d i o a c t i v i t y in a 50 M1 sample was counted and compared to the r a d i o a c t i v i t y in 50 M1 of external buffer. An accumulation of r a d i o a c t i v i t y inside the d i a l y s i s bag compared to the external buffer i s indicative of binding protein a c t i v i t y . 35 16. CHEMOTAXIS STUDIES Semi-solid agar plates (swarm plates) were used to detect chemotaxis towards ammonium and various carbon sources by A. vinelandi i . Ammonium swarm plates consisted of 0.3% agar plates containing Burk's medium supplemented with 0.25 mM (NHa)2SOj,. Sucrose, glucose or succinate were used as attractants in swarm plates at a concentration of 1 mM in place of 59 mM sucrose. Swarm plates were inoculated in the center with a stab using an ammonium-grown culture as a source of inoculum and incubated overnight at 30°C. A positive chemotactic response i s characterized by a c i r c u l a r spreading zone of growth with a d i s t i n c t edge. In experiments involving repeated transfer of c e l l s to new swarm plates, the purity and m o t i l i t y of the culture was monitored by examining c e l l s with a phase microscope. 17. PREPARATION OF INNER AND OUTER MEMBRANES OF AZOTOBACTER  VINELANDII Inner and outer membranes of A. vi n e l a n d i i were isolated as described by Hancock and Nikaido (1978). Cultures (5 x 200 ml), grown to 80 Klett units in Burk's medium were pooled, harvested by centrifugation (10,000 x g for 10 min) and the c e l l p e l l e t frozen at -20°C u n t i l used. Frozen c e l l p e l l e t s were thawed, resuspended in 5 ml of cold 20% (w/v) sucrose and DNase (50 ug/ml) in 10 mM Tris-HCl, pH 8.0 (Tri s buffer), and subjected to 2 treatments with a French pressure c e l l at 20,000 l b / i n 2 , 4°C. The disrupted c e l l s were centrifuged (1000 x g for 36 10 min, 4°C.) to remove whole c e l l s . The supernatant was layered onto a sucrose step gradient containing 1 ml of 70% (w/v) sucrose in T r i s buffer and 4 ml of 18 % (w/v) sucrose in Tr i s buffer and centrifuged for 1 hr at 183,000 x g at 4°C in a Beckman SW41 rotor. A Beckman L870 ultracentrifuge was used for a l l centrifugations. The bottom 2 ml was removed after centrifugation, mixed and layered onto another sucrose gradient containing 1 ml 70% (w/v) sucrose in T r i s buffer, and 3 ml each of 64% (w/v) sucrose, 58% (w/v) sucrose and 52% (w/v) sucrose in Tr i s buffer. After centrifugation (183,000 x g for 14 hr, 4°C) d i s t i n c t bands were v i s i b l e at each of the three interfaces of the sucrose gradient. The whitish bottom band represented the outer membrane fra c t i o n on the basis of high lipopolysaccharide content and low NADH oxidase a c t i v i t y (estimated by the method of Osborne et a_l. , 1972). The top red band represented the inner membrane fraction on the basis of low lipopolysaccharide content and high NADH oxidase a c t i v i t y . The middle band was primarily outer membrane with some inner membrane contamination. Each individual band was colle c t e d d i r e c t l y into Beckman 60Ti centrifuge tubes, which were subsequently f i l l e d with d i s t i l l e d H 20 to d i l u t e the sucrose and centrifuged at 177,000 x g for 1 hr at 4°C in a Beckman 60Ti rotor. After centrifugation, the supernatant was c a r e f u l l y removed and discarded and the p e l l e t resuspended in 0.3 ml of d i s t i l l e d H 20. Portions of the resuspended p e l l e t were then prepared for SDS-gel electrophoresis as described below. 37 18. SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS  (SDS-PAGE) SDS-PAGE was conducted according to Laemmli (1970). Samples of outer and inner membranes were s o l u b i l i z e d by heating at 88°C for 10 min in sample buffer consisting of 62.5 mM T r i s -HCl, pH 6.8 with 2% SDS, 10% glycerol and 5% 2-mercaptoethanol before application to the ge l . Slab gels were 0.75 mm thick and were run at 25 mA (constant current) using a PS 1200 Power Supply (Hoefer S c i e n t i f i c Instruments, San Francisco, U.S.A.). The protein bands were stained overnight in a solution containing 45% H20, 45% methanol, 10% g l a c i a l acetic acid and 0.1% (w/v) coomassie blue and destained by immersion in several changes of a solution containing 72.5% H20, 20% methanol, and 7.5% acetic acid. 19. TWO-DIMENSIONAL SDS-PAGE A heat modifiable outer membrane protein was i d e n t i f i e d by a s l i g h t l y modified two-dimensional SDS-PAGE procedure as described by Reithmeier and Bragg (1977). A sample of outer membranes, s o l u b i l i z e d at 37°C in sample buffer,was loaded onto a gel of 0.75 mm thickness and run as described above. The lane was cut out, placed into a screw-capped tube containing sample buffer, and heated at 100°C for 10 min. The lane was then placed on top of the stacking gel of a second gel, embedded with molten agarose (1% agarose (w/v) in sample buffer) .and run as described above. Because electrophoresis separates proteins on the basis of molecular weight, completion of electrophoresis in 38 the second dimension w i l l result in arrangement of the proteins on a diagonal l i n e . Proteins that are "heat modified" by the s o l u b i l i z a t i o n at 100°C run below or above the diagonal lin e depending on the nature of the modification. 20. TWO-DIMENSIONAL GEL ELECTROPHORESIS Two-dimensional gel elctrophoresis was performed as o r i g i n a l l y described by O'Farrell (1975) with modifications for use with membrane proteins as described by Ames and Nikaido (1976). Triton X-100 (2% f i n a l concentration) was used instead of NP-40 to s o l u b i l i z e membrane proteins. A f i n a l concentration of 2% ampholytes (LKB) was used to form a pH gradient ranging from approximately 4.0 to 7.0 in the i s o e l e c t r i c focusing gel (ampholytes , pH range 4-6, 6-8 and 3.5-10 were combined at a ra t i o of 2:2:1). I s o e l e c t r i c focusing gels (5 mm x 125 mm) were focused at 0.15 watt/tube for approximately 9 hr in a Bio-Rad Model 155 Gel electrophoresis c e l l . The SDS-PAGE used for the second dimension was performed as described above. The i s o e l e c t r i c focusing gel was immobilized on the top of the stacking gel with molten agarose (1% agarose w/v in sample buff e r ) . Proteins were located either by the s i l v e r stain method of Switzer et a l . (1979) with modifications described by Oakley et a_l. (1980) or with coomassie blue as described above. 39 21. ENZYME ASSAYS (A) Preparation of Cell-Free Extracts C e l l - f r e e extracts for in v i t r o enzyme assays described below were prepared as follows. Cultures (200 ml) grown to 80 Klett units were c h i l l e d rapidly to maintain GS adenylylation state by pouring into centrifuge bottles f i l l e d with crushed ic e . The c e l l s were washed twice with Burk's N-free medium and the p e l l e t resuspended in 2 ml of cold l y s i s buffer consisting of 100 mM imidazole buffer (pH 7.0 for glutamine biosynthetic assay, 7.8 for N-methylglutamine and 7-glutamylhydroxamate assays) containing 20 mM MgCl 2 and 2 mM d i t h i o t h r e i t o l . Extracts were prepared using a French pressure c e l l and unbroken c e l l s removed by centrifugation at 10,000 x g for 10 min. When used for the glutamine synthetase biosynthetic assay , extracts were routinely de-salted prior to use by gel f i l t r a t i o n with Bio-Gel P2 (Bio-Rad, Mississauga, Ontario). (B) Glutamine Synthetase Assays Three d i f f e r e n t assays were used to measure glutamine synthetase a c t i v i t y . The biosynthetic assay measured the rate of synthesis of glutamine from glutamate and ammonium. A second assay measured the rate of synthesis of 7-glutamylhydroxamate from glutamate and hydroxlamine, and the t h i r d assay measured the rate of synthesis of N-methylglutamine from glutamate and methylammonium. These assays are described below. Biosynthetic assay The assay to measure glutamine production was performed in 40 a volume of 0.3 ml at 37°C. The assay mixture contained 100 mM imidazole buffer, pH 7.0, 50 mM MgCl 2, 10 mM ATP, 50 mM sodium glutamate, 0.18 AiCi L-[U- 1 "C ]-glutamic acid (Amersham Corporation, Oakville, Ontario) and 15 ul of c e l l - f r e e extract containing approximately 150 nq protein. The reaction was i n i t i a t e d by addition of the c e l l - f r e e extract. Aliquots of 50 M1 were removed p e r i o d i c a l l y ( a t o t a l of 5/assay) to tubes containing 5 ul of 30% t r i c h o l o r a c e t i c acid and placed on i c e . The sample was neutralized by adding 0.5 ml of imidazole buffer (100 mM, pH 7.3). At the end of the assay, 0.5 ml of the neutralized sample was applied d i r e c t l y to a disposable anion exchange column (550 x 5 mm, Bio-Rad AG1X8, Cl'form) for separation of 1"C-glutamate and 1"C-glutamine as described by Prusiner and Milner (1970). The columns were washed with 1.0 ml of imidazole buffer (10 mM, pH 7.0) containing 30 mM glutamine and the eluate c o l l e c t e d d i r e c t l y into s c i n t i l l a t i o n v i a l s . Aquasol (10 ml, New England Nuclear, Lachine, Quebec) was added to each v i a l and the r a d i o a c t i v i t y determined by l i q u i d s c i n t i l l a t i o n counting in a Beckman Model LS7500 l i q u i d s c i n t i l l a t i o n counter. Background values from unbound 1"C-glutamate and radioactive impurities were obtained by running the assay as described above except that buffer replaced c e l l -free extract in the assay mix. Experiments aimed at determining the recovery of ^C-glutamine from the anion exchange columns demonstrated that 93 - 100% of 1"C-glutamine applied to the column was eluted after washing with imidazole buffer (10 mM, pH 7.0) containing 30 mM glutamine. In the assay described above 41 the anion exchange columns bound greater than 99.7% of the p u r i f i e d 1"C-glutamate applied to the column. 1"C-glutamate was p u r i f i e d p r i o r to use in the assay. A 200 M1 volume of 1 f tC-glutamate (approximately 30 nmol glutamate, 260 MCi/Aimol) was loaded onto a disposable anion exchange column (as described above) and the column rinsed with imidazole buffer (10 mM, pH 7.0) u n t i l the number of cpm in the eluate s t a b i l i z e d . The 1*C-glutamate was eluted with 0.01 M HC1 and neutralized with 1 M imidazole buffer, pH 7.0 (0.1 ml imidazole/0.9 ml eluate). Glutamyl Hydroxamate assay The 7-glutamyl hydroxymate assay (Lipman and Tuttle,l945) was performed in a f i n a l volume of 2.5 ml at 37°C. The assay mixture contained 50 mM Tris-maleate buffer, pH 8.0, 50 mM sodium glutamate, 10 mM ATP, 100 mM hydroxylamine and 40 mM MgCl 2. The reaction was i n i t i a t e d by adding 250 ul c e l l - f r e e extract. At periodic i n t e r v a l s , 0.5 ml aliquots were removed to centrifuge tubes containing 1.0 ml of "stop-solution" consisting of 55 g FeCl 3-6H 20, 20 g t r i c h l o r o a c e t i c acid, and 21 ml concentrated HC1 per l i t e r d i s t i l l e d H 20. The p r e c i p i t a t e was removed by centrifugation and the supernatant measured for absorbance at 540 nm. A volume of 0.5 ml of 2 mM 7-glutamyl hydroxamate (Sigma Chemical Co., St. Louis, Missouri) in assay mix together with 1 ml stop solution exhibits an absorbance at 540 nm of 0.391. N-methylglutamine Synthesis Assay A s l i g h t l y modified version of the N-methylglutamine assay described by Kung and Wagner (1966) was used to measure N-42 methylglutamine synthesis in c e l l - f r e e extracts. The assay was performed in a f i n a l volume of 1.2 ml at 37°C. The assay mixture contained 100 mM imidazole buffer, pH 7.8, 50 mM sodium glutamate, 50 mM methylammonium-HCl, 0.19 uCi 1*C-methylammonium, 50 mM MgCl 2 and 10 mM ATP. The reaction was i n i t i a t e d by adding 120 ul of extract. At periodic i n t e r v a l s , a 200 ul aliquot was removed to a centrifuge tube containing 20 ul of 30% t r i c h l o r o a c e t i c acid and placed on ice. The precipitate was removed by c e n t r i f ugation and 160 /ul of the supernatant loaded onto disposable cation exchange columns (20 x 5 mm, Dowex 50W, H* form). The columns were washed twice with 1 ml d i s t i l l e d H 20 to remove neutral and a c i d i c compounds. Amino acids were eluted off the column d i r e c t l y into s c i n t i l l a t i o n v i a l s by addition of 2 x 1 ml volumes of 2N NHj,OH. Aquasol (10 ml) was added to the v i a l s and the r a d i o a c t i v i t y determined by l i q u i d s c i n t i l l a t i o n counting. Background r a d i o a c t i v i t y (due to unbound 1"C-methylammonium and radioactive impurities) eluted from the columns was determined by running blank samples containing buffer instead of extract. The radioactive product eluted from the columns by NH„OH was i d e n t i f i e d as N-methylglutamine on the basis of co-migration with chemically synthesized N-methylglutamine in 3 solvent systems. The eluate from the NH„OH wash was l y o p h i l i z e d to dryness and resuspended in 200 ul of H 20. A 10 ul volume was spotted onto Whatman 3MM chromatography paper along with chemically synthesized N-methylglutamine as standard and chromatographed in 1) t-butyl alcohol - methylethyl ketone 43 formic acid - H 20 (40:30:15:15), 2) phenol - formic acid - H 20 (100:2.6:33.4) and 3) isopropanol - H 20 - g l a c i a l acetic acid (70:30:1). The radioactive metabolite and standards were located as described above (Section 8). 22. SNAKE VENOM PHOSPHODIESTERASE TREATMENT Snake venom phosphodiesterase (SVD, Sigma Chemical Co., St. Louis, Mo.) was used in an attempt to deadenylylate glutamine synthetase in crude c e l l - f r e e extracts. C e l l free extracts were prepared as described above for the biosynthetic assay except that 50 mM Tris-HCl, pH 8.0, replaced 100 mM imidazole buffer, pH 7.0 in the l y s i s buffer and extracts were not desalted u n t i l after SVD treatment. C e l l extracts (0.9 ml) received 0.1 ml of l y s i s buffer containing 3 mg/ml SVD. Control c e l l extracts received 0.1 ml of l y s i s buffer without SVD. Extracts were incubated for 3 hr at 37°C before desalting with a Bio-Rad P-2 column equilibrated with 50 mM Tris-HCl, pH 8.0. Desalted extracts were kept on ice u n t i l use. 44 II I . AMMONIUM AND METHYLAMMONIUM TRANSPORT IN AZOTOBACTER VINELANDII 1. AMMONIUM UPTAKE IN AZOTOBACTER VINELANDII Preliminary experiments were designed to develop an assay system which monitored the uptake of ammonium by A. v i n e l a n d i i . A method was adopted which measured the rate of ammonium disappearance from the external medium. This was accomplished as follows: c e l l s were washed and resuspended in ammonium-free medium and allowed to preincubate for 10 min. The assay was i n i t i a t e d by addition of ammonium sulf a t e to a f i n a l concentration of 800 or 400 uM ammonium. At periodic i n t e r v a l s , 1 ml aliquots were removed and f i l t e r e d through a microporous membrane f i l t e r . The concentration of ammonium in the f i l t r a t e was then determined by a glutamate dehydrogenase linked assay (Strecker, 1955). The results of a t y p i c a l uptake experiment are shown in F i g . 3. Similar rates of ammonium disappearance were obtained when either ammonium sulfate, ammonium acetate or ammonium chloride was used. The ammonium concentration of 800 M M was chosen because i t resulted in maximum velocity of the reaction and was high enough to allow detection of remaining e x t r a c e l l u l a r ammonium in samples taken within 10 min. The reaction rates obtained from these experiments (about 80 nmol of ammonium taken up/min/mg protein) represent a combination of transport across the b a c t e r i a l membrane and subsequent metabolism into amino acids. Under the conditions of thi s assay NH 4 + i s believed to be assimilated into glutamine via glutamine 45 Figure 3. Disappearance of NH,* in cultures of A. v i n e l a n d i i JK (NH„ + uptake). C e l l s were washed and resuspended in Burk's N-free medium as described in Materials and Methods. At 0 time NH„* (as ammonium sulfate) was added to a f i n a l concentration of approximately 800 jiM. Aliquots (1 ml) were removed at periodic i n t e r v a l s , f i l t e r e d through a 0.45 um pore size membrane f i l t e r and the f i l t r a t e analysed for NHS * content. 46 47 synthetase. 2. IS AMMONIUM UPTAKE AN ACTIVE TRANSPORT PROCESS? To es t a b l i s h whether NH„ + uptake in A. v i n e l a n d i i was an active transport process, experiments were designed to determine i f active transport c r i t e r i a were f u l f i l l e d . One such c r i t e r i o n i s that substrate i s taken up against a concentration gradient. To demonstrate th i s i t was necessary to show that ammonium uptake occurred when the external concentration of ammonium was less than the concentration of ammonium inside the c e l l . Uptake against a concentration gradient was demonstrated by the following experiment (Table I ) . C e l l s were washed and resuspended in medium containing 170 M M ammonium sulfate. At this point the internal ammonium pool was determined to be 2.9 mM. Disappearance of ammonium continued u n t i l a l l detectable ammonium (approximately 50 nmol/ml) was removed from the external medium. In these experiments the internal ammonium pool does not change s i g n i f i c a n t l y during the course of the experiment as determined by control experiments which measured ammonium pools during and after ammonium depletion from the medium. The results shown in Table I i l l u s t r a t e that ammonium is taken up against a gradient of at least 17 - 22 f o l d . It was estimated that the f i n a l concentration gradient achieved was at least 44 - 58 fo l d since linear ammonium uptake continued u n t i l the external ammonium concentration was at the lower l i m i t s of detection (about 50 nmol/ml). These results provide one l i n e of evidence for an active transport system for ammonium in A. vinelandi i . 48 TABLE I - A b i l i t y of A. v ine l and i i to take up NH4+ against a concentrat ion grad ient . I n i t i a l e x t r a c e l l u l a r N H 4 + I n t r a ce l l u l a r N H 4 + concentrat ion concentrat ion achieved Experiment [mMj [mM] 1 0 . 1 7 2 . 9 2 0 . 1 0 2 . 2 49 3. INHIBITION OF•AMMONIUM TRANSPORT BY METHIONINE SULFONE Because ammonium uptake i s a combination of transport and assimilation I attempted to block the assimilation component of this reaction to allow independent examination of the transport process. Assimilation was blocked by incubating c e l l s in the presence' of the glutamate analog methionine sulfone (MSF). MSF prevents ammonium assimilation by i n h i b i t i n g the a c t i v i t y of glutamine synthetase and glutamate synthase in A. vin e l a n d i i (Gordon and B r i l l , 1974). Treatment with MSF resulted in complete i n h i b i t i o n of ammonium uptake (Fig. 4) indicating that blocking assimilation does not result in continued accumulation of ammonium via the ammonium transport system. Therefore, examination of the transport component of ammonium uptake was not possible by thi s method. 4. METHYLAMMONIUM UPTAKE IN AZOTOBACTER VINELANDII Further examination of the ammonium uptake system was accomplished using the ammonium analog methylammonium. When ammonium-grown c e l l s were washed and placed into N-free medium as described in Materials and Methods, a linear rate of 1*C-methylammonium uptake was observed (Fig. 5). Methylammonium was determined to be a suitable analog for study of ammonium uptake on the basis of experiments which demonstrated ammonium was a competitive i n h i b i t o r of methylammonium uptake (Gordon and Moore, 1981). These studies also provided kinetic evidence for the existence of two uptake systems. One of these systems, designated as the low a f f i n i t y system, has an apparent Km for 50 Figure 4. Inhibition of ammonium uptake by methionine sulfone. C e l l s were prepared and assayed as described in Materials and Methods. Cel l s were incubated in the presence of 20 mg/ml methionine sulfone for 5 minutes before the assay was i n i t i a t e d by addition of NH„ + (as ammonium s u l f a t e ) . Symbols - MSF treated c e l l s (o), control c e l l s (no additions) (•). 51 52 Figure 5. Uptake of 1"C-methylammonium by whole c e l l s of A. vi n e l a n d i i JK. A, uptake of 1 mM 1"-methylammonium (5 AtCi/nmol). B, uptake of 20 M M ^-methylammonium (5 nCi/nmol). Uptake assays were performed as described in Materials and Methods. 53A A T 1 1 1 1 1 1 1 2 5 4 5 6 7 minutes 53B B 500-) T 1 1 1 1 1 1 2 5 4 5 6 m i n u t e s 54 methylammonium of 661 uM. The other "high a f f i n i t y " system has a Km of 61 M M . Because the high a f f i n i t y system would contribute most to the observed v e l o c i t y at low substrate concentrations i t i s possible to examine the high a f f i n i t y system independently by performing uptake assays at low substrate concentrations. Therefore, subsequent experiments examining methylammonium uptake were performed using 1"C-methylammonium at a concentration of 20 M M and 1 mM. 5. EFFECT OF NITROGEN LIMITATION ON METHYLAMMONIUM UPTAKE Experiments were performed to determine i f nitrogen l i m i t a t i o n caused increased rates of methylammonium uptake, thereby suggesting the presense of a repressable uptake system. Two mutant strains unable to f i x nitrogen, strain UW 1 (Fisher and B r i l l , 1969) and s t r a i n UW 2 (Gordon and B r i l l , 1972), were employed for these studies in order that nitrogen l i m i t a t i o n could e a s i l y be accomplished. When c e l l s grown in ammonium containing medium were transferred to N-free medium for a period of 3 hours the rate of uptake for 20 M M and 1 mM methylammonium increased approximately 2 f o l d (Table I I ) . In contrast, K. pneumoniae has been reported to increase methylammonium transport rates approximately nine f o l d in response to growth on a poor nitrogen source (Kleiner, 1982). The results reported here suggest that A. v i n e l a n d i i does not have an inducible uptake system for methylammonium. 55 TABLE II - E f fec t of nitrogen s ta rva t ion on rate of CH 3 NH 3 + uptake. Ratio of CH3NH3+ uptake a c t i v i t y (nmol/min/mg prote in ) of c e l l s starved for n i t rogen to c e l l s grown in NH 4 + -su f f i c i en t medium3 S t r a i n 5 Expt. No. Uptake of 1 mM CH 3 NH 3 + Uptake of 20 uM CH 3 NH 3 + UW2 1 2.12 2.63 2 1.76 2.36 3 2.84 1.38 UW1 1 1.73 1.39 a Cultures were grown to a density of 80 K le t t units in Burk's sucrose medium contain ing 29 mM ammonium acetate as an ammonium source. Nitrogen s tarvat ion was achieved by incubat ing washed c e l l s in N-free medium for 3 hours. Uptake assays were performed as described in Mater ia ls and Methods a f t e r resuspending c e l l s in N-free medium and again a f t e r 3 hrs . k UW2 and UW1 are mutant s t r a ins of A. v i ne l and i i unable to f i x n i t rogen . 56 6. ENERGY REQUIREMENT FOR AMMONIUM AND METHYLAMMONIUM UPTAKE The energy requirement for ammonium and methylammonium uptake was examined by use of various metabolic i n h i b i t o r s . Potassium cyanide (KCN), by virtue of i t s a b i l i t y to block respiration, w i l l result in the i n h i b i t i o n of ATP synthesis. Because respiration also functions to maintain a proton gradient, KCN treatment w i l l result in the dis s i p a t i o n of the proton gradient as well. The uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP), w i l l dissipate the proton gradient e x i s t i n g across the c e l l membrane. As a resu l t , ATP pools are depleted as the c e l l attempts to restore the proton gradient via the proton translocating Ca + +/Mg + + ATPase (Rosen and Kashket, 1978). Treatment of c e l l s with 0.2 mM cyanide resulted in 99% i n h i b i t i o n of 20 iM methylammonium uptake and 94% i n h i b i t i o n of uptake of 1 mM methylammonium (Table I I I ) . The same treatment resulted in strong i n h i b i t i o n of ammonium uptake as well (Fig. 6). Si m i l a r l y , 0.2 mM CCCP resulted in 99% inhibition' of methylammonium uptake (Table III) and complete i n h i b i t i o n of ammonium uptake. (Fig. 6). Because both KCN and CCCP w i l l result in the depletion of ATP and di s s i p a t i o n of a proton gradient, these results do not distinguish which i s required for ammonium/methylammonium uptake. However, by s e l e c t i v e l y i n h i b i t i n g ATP synthesis, one can determine whether a proton gradient alone i s s u f f i c i e n t to drive uptake. ATP synthesis can be s e l e c t i v e l y i n h i b i t e d without d i s s i p a t i n g the proton gradient (Klein and Boyer, 1972) by treatment with the phosphate analog, arsenate. Treatment 57 TABLE III - E f fec t of i n h i b i t o r s of energy generation on uptake of CH3NH 3 +. Addi t ion % Inh ib i t ion of CH 3 NH 3 + uptake rate Assay Buffer (mM) 20 uM C H 3 N H 3 + 1 mM CH 3 NH 3 + Burk's ( P 0 4 3 - ) None 3 0 0 KCN, 0.2 99 94 . KC1, 0.2 13 13 Na 2 As0 4 , 15 97 89 NaCl , 30 27 13 CCCP, 0.2 99 99 G lycy lg l yc ine None3 0 0 Na 2 As0 4 , 15 98 90 NaCl, 30 24 15 3 Control rates for cu l tures assayed in Burk's or g l y c y lg l y c i ne buf fer were 0.9 nmol/min per mg prote in (20 mM CH 3 NH 3 + ) and 5.3 nmol/min per mg prote in (1 mM C H 3 N H 3 + ) . 58 Figure 6. Inh i b i t i o n of ammonium uptake by KCN and CCCP. C e l l s were prepared and assayed as described in Materials and Methods. C e l l s were incubated for 5 minutes in the presence of 0.2 mM CCCP (O ) or 0.2 mM KCN (O) before the assay was i n i t i a t e d by addition of NH0* (as ammonium s u l f a t e ) . Control c e l l s (•) received no additions. 59 6 0 with 1 5 mM Na2AsOft resulted in strong i n h i b i t i o n of methylammonium uptake assayed at 2 0 M M and 1 mM substrate concentrations (Table I I I ) . The time required for arsenate i n h i b i t i o n was very rapid (Fig. 7 ) . Uptake rates of less than 1 1 % of control values were observed whether arsenate treatment and uptake assays were performed in glycylglycine or phosphate medium. Controls receiving equivalent amounts of phosphate ( 1 5 mM) or NaCl ( 3 0 mM) exhibited only s l i g h t i n h i b i t i o n (Table I I I ) . To confirm that arsenate was e f f e c t i v e l y reducing ATP pools, ATP was measured in whole c e l l s treated with arsenate using the f i r e f l y l u c i f e r a s e assay (Cole et a l . , 1 9 6 7 ) . The e f f e c t of CCCP on ATP pools was also determined (Table IV). The time required for reduction of ATP pools by arsenate i s very short as indicated in F i g . 8 . When arsenate was added to ammonium-grown c e l l s (washed and resuspended in Burk's N-free medium) a 5 1 % decrease of i n t r a c e l l u l a r ATP occured. within 1 0 seconds. Within 1 minute of treatment a 9 4 % decrease was observed. Arsenate also depleted ATP pools when treatment was performed in a non-phosphate containing medium (Burk's N-free medium, containing 6 mM glycylglycine instead of phosphate buffer) as i l l u s t r a t e d in Table IV. Barnes and Zimniak ( 1 9 8 1 ) have reported that treatment with 0 . 2 mM arsenate did not i n h i b i t methylammonium transport in A. vinelandi i . (Cultures were grown in N-free medium with succinate as a carbon source). To determine i f ATP was 1depleted under these conditions, ATP pools were measured in c e l l s grown 61 Figure 7. A time course of arsenate i n h i b i t i o n of methylammonium uptake. Measurement of 20 uH 1*C-methylammonium (5 MCi//umol), was performed as described in Materials and Methods. Arsenate was added to 15 mM at time indicated by arrow (A) or 5 minutes before sta r t of assay (o). Control cultures received no additions (•). 62 63 TABLE IV - E f fec t of arsenate and CCCP on i n t r a c e l l u l a r ATP l e v e l s . Carbon and Nitrogen Source fo r growth Buffer Addi t ion (mM) % Decrease in ATP Sucrose - NH 4 a Burk (P0 4 3~) None Na 2As04, 15 CCCP, 0.2 0 b 92 86 G lycy lg l yc ine None Na 2As04, 15 0 b 91 Sucrose - N 2 C Burk ( P 0 4 3 - ) None Na 2As04, 15 0 d 92 G lycy lg l yc ine None Na 2As04, 15 NaCl, 60 0 d 90 0 Succinate - N 2 e Glycy lg l yc ine None Na 2As04, ° « 2 Na 2As04, 15 0 f 0 12 Cultures were grown to a density of 80 K le t t uni ts in Burk's sucrose medium conta in ing 29 mM ammonium acetate as an ammonium source. Ce l l s were washed and resuspended in the appropriate buf fer supplemented with Burk's s a l t s . u ATP was approximately 17 nmol/mg p ro te in . c N2-grown cu l tures were grown to a densi ty of 120 Klet t uni ts in Burk's sucrose medium, washed and resuspended to 80 Klet t uni ts in appropriate buf fer supplemented with Burk's s a l t s . d ATP was approximately 15 nmol/mg p ro t e i n . e Cultures were grown to a density of approximately 35 K le t t un i ts in Burk's succinate medium, washed and resuspended to approximately 60 Klet t units in g l y cy lg l y c ine buf fer supplemented with Burk's s a l t s . f ATP was approximately 8 nmol/mg p ro te in . 64 Figure 8. A time course of the e f f e c t of arsenate on ATP pools in A. v i n e l a n d i i . NHi,+ grown c e l l s were washed twice, resuspended to o r i g i n a l volume in Burk's N-free medium and preincubated for 10 minutes at 30°C. At the times indicated, 2 ml aliquots were removed and the ATP content determined as described in Materials and Methods. At the time indicated by the arrow, Na2AsOi, was added to 15 mM. 65 66 and treated as described by these authors. As shown in Table IV, 0.2 mM and 15 mM arsenate did not s i g n i f i c a n t l y lower ATP pools in cultures grown in N-free medium with succinate as a carbon source. These results indicate that ammonium and methylammonium uptake are energy dependent processes requiring ATP or some other high energy compound. These experiments, however, do not rule out the involvement of an e l e c t r i c a l gradient (A\//) or a proton gradient (ApH) in methylammonium uptake. 7. SENSITIVITY OF METHYLAMMONIUM UPTAKE TO OSMOTIC SHOCK In many transport systems, proteins located in the periplasmic space are involved in the transport process (reviewed by Oxender and Quay, 1976). These systems are commonly referred to as shock sensitive transport systems because osmotic shock procedures, which function to s e l e c t i v e l y release periplasmic proteins, result in a sharp reduction of transport a c t i v i t y . In addition, these systems c h a r a c t e r i s t i c a l l y display an ATP requirement for transport of substrate. If the ATP dependence demonstrated by A. v i n e l a n d i i for ammonium and methylammonium uptake is indi c a t i v e of the energy requirement for transport across the inner membrane then transport of ammonium and methylammonium may f a l l into the class of transport systems which u t i l i z e periplasmic binding proteins. To determine i f periplasmic binding proteins were involved in methylammonium uptake, experiments were performed to esta b l i s h whether methylammonium uptake was shock se n s i t i v e . The T r i s -Mg + + shock procedure (Hoshino, 1979) was chosen to release 67 periplasmic binding proteins. Because a suitable periplasmic marker protein for determining release of periplasmic proteins has not been described in A. vinelandi, the plasmid RP1 encoding for the periplasmic TEM-2 /3-lactamase was transferred from E. c o l i to A. vi n e l a n d i i by conjugation. (3-lactamase a c t i v i t y was measured by following the hydrolysis of the chromogenic cephalosporin nitrocephin at 540 nm wavelength (O'Callaghan,1972). The a b i l i t y of the Tris-Mg + + procedure to release periplasmic /3-lactamase i s shown in Table V. The s e n s i t i v i t y of methylammonium uptake to shock treatment was determined by measuring uptake a c t i v i t y in c e l l s before and after treatment. When NH„ +-grown c e l l s were shocked as described in Materials and Methods, methylammonium uptake a c t i v i t y was completely abolished (Fig. 9). Under the same conditions fructose uptake was reduced only s l i g h t l y indicating that the shock treatment did not have an adverse effect on uptake a c t i v i t i e s per se. These results suggested that methylammonium uptake may involve a periplasmic binding protein. To examine this p o s s i b i l i t y further, an experiment was designed to test.the periplasmic shock f l u i d for methylammonium-binding a c t i v i t y . The experimental procedure i s outlined b r i e f l y below: 1. ) Tris-Mg + + shock of 2 1 of NH„ +- grown c e l l s 2. ) Concentration and d i a l y s i s of shock f l u i d * 3.) Fractionation of shock f l u i d via DEAE Sephacel column 4.) Assay column fractions for methylammonium binding 68 TABLE V - Release of B-lactamase from A. v i ne l and i i s t r a i n JK (RPI). % Total % Total % Total B-lactamase nuc le ic a c i d a NADH Oxidase Treatment a c t i v i t y re leased re leased a c t i v i t y French Pressure Ce l l 100% 100% 100% lOmM Tris-HCl-ImM MgCl2-lmM K C l b 9% 1.1% n d f lOmM T r i s - H C l c 62% 1.1% nd lOmM Tris-HCl-200mM M g C l 2 d 23% 1.0% nd lOmM Tris-HCl-200mM M g C l 2 e 102% 1.1% nd (with temperature sh i f t ) a Determined by OD 280/260. b A c e l l suspension (80 K l e t t uni ts ) was centr i fuged and resuspended in an equal volume of lOmM T r i s - H C l , pH 7.3, with ImM MgCl 2 and ImM KCl (TMK bu f f e r ) . The c e l l suspension was cent r i fuged (10,000 x g f o r 10 min) and the supernatant assayed fo r 3-lactamase a c t i v i t y . c A c e l l suspension was centr i fuged and resuspended in an equal volume of lOmM T r i s - H C l , pH 7.3. The c e l l suspension was cent r i fuged and the supernatant assayed fo r B-lactamase a c t i v i t y . d A c e l l suspension was cent r i fuged and washed once with an equal volume of TMK bu f fe r . The p e l l e t was resuspended in an equal volume of lOmM T r i s - H C l , pH 8 .5 , with 200 mM MgC l 2 . The c e l l suspension was cent r i fuged again and the supernatant assayed fo r B-lactamase a c t i v i t y . e As in d except that c e l l s resuspended in 10mM Tris-200mM MgCl 2 were heated to 30° f o r 5 minutes and cooled to 4 ° f o r 15 minutes. The temperature s h i f t was repeated once more, the c e l l suspension cent r i fuged and the supernatant assayed fo r B-lactamase a c t i v i t y . nd means not detec tab le . 69 Figure 9. Effect of Tris-Mg* + shock treatment on 1 4C-methylammonium and 1 4C-fructose uptake in A. vinelandi i . Cultures were grown in Burk's medium containing 2% fructose and 29 mM ammonium acetate to a density of 80 Klett units. Uptake assays were performed as described in Materials and Methods except that, after washing, c e l l s were resuspended to twice the o r i g i n a l density in Burk's buffer containing 29 mM sodium acetate as an energy source. After preincubation for 8 minutes at 30° C, c e l l s received either 1 4C-fructose ( f i n a l concentration 5 mM, s p e c i f i c a c t i v i t y = 5 MCi/Mmol) or 1 4C-methylammonium ( f i n a l concentration 1 mM, s p e c i f i c a c t i v i t y = 5 nCi/nmol). An equal volume of c e l l s from the same culture used for control assays was subjected to the Tris-Mg + + shock treatment as described in Materials and Methods. C e l l s were then prepared and assayed for uptake a c t i v i t y as described above. (A), 14C-methylammonium uptake in control and Tris-Mg + + shocked c e l l s , control (•), shocked c e l l s (o); (B), 1"C-fructose uptake in control and Tris-Mg* + shocked c e l l s , control (•), shocked c e l l s (o). 70A A 10000-i 8000 H 70B B -i , , , , 1 2 5 4 5 minutes 71 act i v i t y . Methylammonium binding protein a c t i v i t y was determined by a n i t r o c e l l u l o s e f i l t e r binding assay. This technique involves passing a mixture of presumptive binding protein and radioactive substrate through a n i t r o c e l l u l o s e membrane f i l t e r . The n i t r o c e l l u l o s e f i l t e r w i l l bind protein and thus any binding protein-substrate complexes can be detected by the presence of r a d i o a c t i v i t y on the f i l t e r in excess of that found on control f i l t e r s . Equilibrium d i a l y s i s was used as an alternative method to detect binding protein a c t i v i t y . This technique involves placing d i a l y s i s tubing containing presumptive binding protein into a buffered solution containing radioactive substrate. An accumulation of r a d i o a c t i v i t y inside the d i a l y s i s tubing r e l a t i v e to the buffered solution outside i s regarded as evidence for binding protein a c t i v i t y . Using both methods I was unable to demonstrate methylammonium-binding a c t i v i t y in periplasmic shock f l u i d from A. vinelandi i . It remains possible, however, that the a f f i n i t y of a binding protein for an analog of the natural substrate, in this case methylammonium, may be too low to be detected by the binding assays used in t h i s experiment. The use of 1 3NH f t + would be the best way to determine i f t h i s was the problem. 72 IV. METHYLAMMONIUM METABOLISM IN AZOTOBACTER VINELANDII 1. DETECTION OF METHYLAMMONIUM METABOLITE Two observations suggested that methylammonium was being metabolized in whole c e l l s of A. v i n e l a n d i i . F i r s t , attempts to chase methylammonium out of preloaded c e l l s by addition of excess methylammonium f a i l e d (Fig. 10), suggesting that accumulated methylammonium was being converted to a compound which i s unable to exit from the c e l l v ia the methylammonium transport system. Secondly, the glutamate analog L-methionine-DL-sulfoximine (MSX), which i n h i b i t s the c a t a l y t i c a c t i v i t y of glutamine synthetase and glutamate synthase, inhibited methylammonium uptake (J. Gordon, unpublished data). Although TCA pr e c i p i t a b l e material from c e l l s incubated in the presence of 1"C-methylammonium did not contain r a d i o a c t i v i t y (Gordon and Moore, 1981), indicating that methylammonium was not incorporated into c e l l u l a r components, i t was possible that methylammonium was metabolized into an amino acid not subsequently incorporated into protein. To examine t h i s p o s s i b i l i t y , extracts were prepared from c e l l s incubated in the presence of 14C-methylammonium and examined for a metabolite of methylammonium. Preparation of extracts was performed by centrifugation of c e l l s incubated in the presence of 1 f tC-methylammonium through an ice cold mixture of dodecane: bromododecane (Barnes and Zimniak, 1981) to insure maximum separation of c e l l s and medium. (The aqueous medium stays on top of the o i l after centrifugation while c e l l s form a p e l l e t in 73 Figure 10. Retention of accumulated 1aC-methylammonium in A. vin e l a n d i i JK upon addition of excess unlabeled methylammonium. Uptake of 20 M M 1*C-methylammonium (5 uCi/nmol) was measured as described in Materials and Methods except that a 2.4 ml volume of c e l l s was used. At the time indicated by the arrow, unlabeled CH 3NH 3 + was added to a f i n a l concentration of 44 mM. This represents an addition of greater than 2000 fo l d excess CH 3NH 3 +. 74 75 the bottom of the centrifuge tube). The p e l l e t was then extracted with perchloric acid, neutralized and the prec i p i t a t e removed by centrifugation. Paper chromatography of c e l l extracts in a solvent system consisting of isopropanol - H 20 -g l a c i a l acetic acid (70:30:1) revealed two radioactive compounds. One of the compounds co-chromatographed with non-radioactive methylammonium' while an unidentified, less polar compound formed a major spot (Fig. 11). This compound represents the only detectable metabolite of methylammonium in A vin e l a n d i i under these conditions. 2. IDENTIFICATION OF THE PRODUCT OF METHYLAMMONIUM METABOLISM It was assumed that methylammonium might be metabolized in a manner analogous to ammonium. If thi s were true, two l i k e l y products of methylammonium metabolism would be N-methylglutamate and N-methylglutamine (Fig. 12). N-methylglutamate was eliminated as a p o s s i b i l i t y because i t did not co-migrate with the metabolite in a solvent system consisting of t-butyl alcohol : methylethylketone: H 20: cone. NH„OH (Table VI). A similar experiment using N-methylglutamine was not possible because N-methylglutamine was not commercially available to run as a standard in chromatography experiments. Therefore, N-methylglutamine was chemically synthesized as described by Lichenstein (1942). Confirmation that the synthesized product was N-methylglutamine was achieved by i d e n t i f i c a t i o n of acid hydrolysis products as glutamate and methylammonium (Fig. 13). In addition, the melting point of the synthesized product (192°C) agreed with that o r i g i n a l l y reported by Lichtenstein. 76 Figure 11. Histogram representing paper chromatograph of c e l l free extracts containing metabolized 1"C-methylammonium. Whole c e l l s of A. v i n e l a n d i i JK were incubated in the presence of 1 *C-methylammonium (20 M M , 10 nCi/nmol) for approximately 3 minutes before extraction as described in Materials and Methods. Approximately 5 ul of extract was spotted to the bottom of Whatman 3MM chromatography paper and developed in an isopropanol: H 20: g l a c i a l acetic acid solvent system (70:30:1). The lane was cut into 1.0 cm sections and the r a d i o a c t i v i t y in each determined by l i q u i d s c i n t i l l a t i o n counting. 77 4000-1 metabolite 3000-1 CL 2 0 0 0 H o IOOO H methylammonium 2 ~T~ 4 T " 6 "T* 8 10 12 -r-14 16 DISTANCE FROM ORIGIN (cm) 78 Figure 12. The amino acids N-methylglutamine and N-methylglutamate. 79 CH 3 NH NH; C-CH-CH-CH-COO" II 2 2 0 N-methylglutamine CH 3 9" NH C-CHzCH-CH-COO' N-methylglutamate 80 TABLE VI - I d en t i f i c a t i on of in vivo metabol i te of CHoNH-,+ . Compound R f in solvent system 3 1 2 3 4 5 6 N-methylglutamine .43 .88 .65 .50 .80 .58 Me tabo l i t e 6 .43 .88 .65 .50 .80 .58 N-Methylglutamate .65 .23 Alanine .69 .39 .58 Glutamine .59 .30 .60 Glutamate .36 .14 .30 Methyl amine HC1 .60 .82 .65 .89 .79 3 Solvent 1, G l ac i a l ace t i c ac id-t-butyl a lcohol-H 2 0 (25:50:25) . Solvent 2, Phenol-formic acid-H 2 0 (500:13:167). Solvent 3, t-butyl alcohol-methyl ethyl ketone-formic acid-H 2 0 (40:30:15:15) . Solvent 4, t-butyl alcohol-methyl ethyl ketone-H 20-concentrated NH^OH (40:30:20:10) . Solvent 5, Phenol-H 20 (the lower phase from a 1:1 mixture of phenol and H 2 0. Solvent 6, Isopropanol-H 2 0-glacia l ace t i c ac id (70:30:1) . k C e l l f ree extracts conta in ing metabolized ^C^NHg" 1" were prepared as descr ibed in Mater ia ls and Methods. 81 Figure 13. Paper chromatograph of acid hydrolysis products of chemically synthesized N-methylglutamine. N-methylglutamine was synthesized and acid hydrolysed as described in Materials and Methods. Compounds were chromatographed in a solvent system containing isopropanol: H 20: g l a c i a l acetic acid (70:30:1) and vi s u a l i z e d by spraying with ninhydrin. The hatched c i r c l e in lane 2 indicates a faint ninhydrin reaction. Lane 1 chemically synthesized N-methylglutamine, Lane 2 - acid hydrolysis products of N-methylglutamine, Lane 3 methylammonium, Lane 4 - glutamate, Lane 5 - N-methylglutamate, Lane 6 - N-methylglutamate subjected to acid hydrolysis treatment. Abbreviations: mgln = N-methylglutamine; mglu = N-methylglutamate; ma = methylammonium; glu = glutamate. 82 SOLVENT FRONT 1 2 3 4 5 6 ma ma glu glu ORIGIN 83 Chromatography experiments showed that the metabolite of methylammonium co-migrated with chemically synthesized N-methylglutamine in 6 solvent systems (Table VI). On this basis the metabolite was i d e n t i f i e d as N-methylglutamine. 3. INVOLVEMENT OF GLUTAMINE SYNTHETASE IN THE METABOLISM OF METHYLAMMONIUM Demonstration that N-methylglutamine was the product of methylammonium metabolism strongly suggested that glutamine synthetase (GS) catalysed this reaction. Previous studies had shown that GS from sheep brain (Rowe et a l . , 1970) and pigeon l i v e r (Speck, 1949) i s capable of u t i l i z i n g methylammonium as substrate. Recently other workers have suggested that GS i s involved in the metabolism of methylammonium in various prokaryotic organisms (Kleiner and Fi t z e , 1981; Kleiner, 1982; Gober and Kashket, 1983; Yoch et a l . , 1983). If GS were responsible for in vivo N-methylglutamine synthesis in A. vine l a n d i i one would predict that c e l l - f r e e extracts could synthesize N-methylglutamine in a reaction mix containing glutamate, ATP, Mg + +, and methylammonium. As shown in Fig . 14 linear rates of N-methylglutamine synthesis were obtained in this reaction mix. Co-migration with chemically synthesized N-methylglutamine in 3 solvent systems (Solvent systems 2, 3 and 6, Table VI) i d e n t i f i e d the product of thi s reaction as N-methylglutamine. The Km for methylammonium in th i s reaction was determined to be between 60 and 120 mM using crude c e l l - f r e e extracts prepared from ammonium-grown WT c e l l s (data not shown). To examine further the involvement of GS in N-84 Figure 14. N-methylglutamine synthesis in c e l l free extracts of A. v i n e l a n d i i JK. The assay was performed as described in Materials and Methods. 85 86 C h r o m a t o g r a p h y e x p e r i m e n t s s h o w e d t h a t t h e m e t a b o l i t e o f m e t h y l a m m o n i u m c o - m i g r a t e d w i t h c h e m i c a l l y s y n t h e s i z e d N -m e t h y l g l u t a m i n e i n 6 s o l v e n t s y s t e m s ( T a b l e V I ) . On t h i s b a s i s t h e m e t a b o l i t e was i d e n t i f i e d a s N - m e t h y l g l u t a m i n e . 3 . INVOLVEMENT OF GLUTAMINE SYNTHETASE I N THE M E T A B O L I S M OF METHYLAMMONIUM D e m o n s t r a t i o n t h a t N - m e t h y l g l u t a m i n e w a s t h e p r o d u c t o f m e t h y l a m m o n i u m m e t a b o l i s m s t r o n g l y s u g g e s t e d t h a t g l u t a m i n e s y n t h e t a s e (GS) c a t a l y s e d t h i s r e a c t i o n . P r e v i o u s s t u d i e s h a d s h o w n t h a t GS f r o m s h e e p b r a i n (Rowe e t a l . , 1970 ) a n d p i g e o n l i v e r ( S p e c k , 1 9 4 9 ) i s c a p a b l e o f u t i l i z i n g m e t h y l a m m o n i u m a s s u b s t r a t e . R e c e n t l y o t h e r w o r k e r s h a v e s u g g e s t e d t h a t GS i s i n v o l v e d i n t h e m e t a b o l i s m o f m e t h y l a m m o n i u m i n v a r i o u s p r o k a r y o t i c o r g a n i s m s ( K l e i n e r a n d F i t z e , 1 9 8 1 ; K l e i n e r , 1 9 8 2 ; G o b e r a n d K a s h k e t , 1 9 8 3 ; Y o c h e t a l ' . , 1 9 8 3 ) . I f GS w e r e r e s p o n s i b l e f o r in_ v i v o N - m e t h y l g l u t a m i n e s y n t h e s i s i n A . v i n e l a n d i i o n e w o u l d p r e d i c t t h a t c e l l f r e e e x t r a c t s c o u l d s y n t h e s i z e N - m e t h y l g l u t a m i n e i n a r e a c t i o n m i x c o n t a i n i n g g l u t a m a t e , A T P , M g * * , a n d m e t h y l a m m o n i u m . A s shown i n F i g . 14 l i n e a r r a t e s o f N - m e t h y l g l u t a m i n e s y n t h e s i s w e r e o b t a i n e d i n t h i s r e a c t i o n m i x . C o - m i g r a t i o n w i t h c h e m i c a l l y s y n t h e s i z e d N -m e t h y l g l u t a m i n e i n 3 s o l v e n t s y s t e m s ( S o l v e n t s y s t e m s 2 , 3 a n d 6 , T a b l e V I ) i d e n t i f i e d t h e p r o d u c t o f t h i s r e a c t i o n a s N -m e t h y l g l u t a m i n e . T h e Km f o r m e t h y l a m m o n i u m i n t h i s r e a c t i o n was d e t e r m i n e d t o b e b e t w e e n 60 a n d 120 mM u s i n g c r u d e c e l l f r e e e x t r a c t s p r e p a r e d f r o m ammonium g r o w n WT c e l l s ( d a t a n o t s h o w n ) . To e x a m i n e f u r t h e r t h e i n v o l v e m e n t o f GS i n N -87 methylglutamine synthesis, a glutamine synthetase mutant ( A. vi n e l a n d i i s t r a i n JK301) was examined. C e l l free extracts of JK301 did not catalyse N-methylglutamine synthesis (see Table VII) strongly suggesting that GS i s the enzyme responsible for methylammonium metabolism in A. v i n e l a n d i i . 4. USE OF METHYLAMMONIUM UPTAKE ASSAYS TO DETECT GS DEFECTIVE  MUTANTS The results suggesting that intact c e l l s of A. v i n e l a n d i i metabolize methylammonium via glutamine synthetase to N-methylglutamine suggested the p o s s i b i l i t y of using methylammonium uptake assays as a simple screen for glutamine synthetase defective mutants. To determine i f the rate of methylammonium uptake i s representative of the rate of methylammonium metabolism, the ve l o c i t y for both processes was determined simultaneously (Fig. 15). These results show that the rate of methylammonium uptake i s equal to the rate of N-methylglutamine synthesis. JK301, a GS mutant which lacks N-methylglutamine synthesis a b i l i t y , has no detectable methylammonium uptake a c t i v i t y (Fig.16), thereby indicating that methylammonium uptake assays would reveal GS mutants similar to JK301. 8 8 Figure 15. N-methylglutamine synthesis and methylammonium uptake in A. vin e l a n d i i JK. Cultures were prepared for 1 f tC-methylammonium uptake assays as described in Materials and Methods except assays were performed using 125 ml Erlenmeyer flasks containing 12 ml of c e l l suspension. 1*C-methylammonium concentration was 20 /uM, (5 AiCi/nmol). At the times indicated 200 /ul of c e l l s were removed and f i l t e r e d for measurement of methylammonium uptake (•). For measurement of N-methylglutamine synthesis (o) 1 ml volumes were removed from the same flask and placed into centrifuge tubes containing 0.2 ml 30% TCA, the pre c i p i t a t e removed by centrifugation and the supernatants analysed for N-methylglutamine content using column chromotography as described in Materials and Methods. 89 mi mutts 90 Figure 16. Uptake of 1'C-methylammonium by whole c e l l s of A. vi n e l a n d i i JK (•) and A. vi n e l a n d i i JK301 (o). Uptake assays were performed as described in Materials and Methods except that 1*C-methylammonium concentration in the uptake assay was 200 MM (5 jzCi/Mmol). 91 92 V. CHARACTERIZATION OF A GLUTAMINE SYNTHETASE DEFICIENT MUTANT 1. JK301, A GLUTAMINE SYNTHETASE DEFECTIVE MUTANT Spontaneous mutants resistant to the glutamate analog L-methionine-DL-sulfoximine (MSX) were isolated in our lab as part of a fourth year student project (Gordon et a l . , submitted). It was f e l t that these mutants might display altered regulation of nitrogenase and therefore several were tested for const i t u t i v e nitrogenase production in the presence of ammonia. One i s o l a t e , JK301, displayed s l i g h t nitrogenase c o n s t i t u t i v i t y and was chosen for further study. When inoculated to a low c e l l density, t h i s mutant was unable to grow in N-free medium although i t could derepress nitrogenase and f i x nitrogen normally. When f i x i n g nitrogen, JK301 excretes ammonium into the growth medium (Gordon et a l . , submitted). The i n a b i l i t y to grow in N-free medium and excretion of ammonium suggested a defect in ammonium a s s i m i l i a t i o n . It was thought that JK301 might possess an altered glutamine synthetase since t h i s enzyme is sensitive to MSX (Gordon and B r i l l , 1974) and plays a major role in NHtt + assimilation under- N 2 - f i x i n g conditions. This idea is reinforced by the observation that JK301 was unable to catalyze N-methylglutamine synthesis (Table VII). The p o s s i b i l i t y that the absence of N-methylglutamine synthesis a c t i v i t y in JK301 extracts i s due to an in h i b i t o r not found in JK extracts was ruled out because experiments in which extracts from' JK and JK301 were mixed did not result in an i n h i b i t i o n of JK a c t i v i t y (Table VII). Because JK301 is not a glutamine 9 3 TABLE VII - Absence of i nh ib i t o r y molecules in c e l l f ree extracts of JK301. N-methylglutamine synthesis a c t i v i t y 3 Source of ext rac t untreated ext rac t desal ted extract 1 JK 5 .4 5.0 JK301 n d b nd 1:1 mixture of JK and JK301 2 .4 3.7 3 nmol N-methylglutamine produced/min/mg p ro t e i n . 6 ' n d ' means a c t i v i t y not detectab le . c Crude c e l l f ree ext rac t was passed through a Bio-Rad P2 column to remove small molecules as descr ibed in Mater ia ls and Methods. 94 auxotroph i t seemed possible that residual glutamine biosynthetic a c t i v i t y was present but not measurable when methylammonium was used as substrate. To test t h i s hypothesis, glutamine synthetase a c t i v i t y was compared in c e l l - f r e e extracts of JK and JK301 using a conventional assay procedure which measures the formation of 7-glutamylhydroxamate. This reaction proceeds as follows: Hydroxylamine + Glutamate * 7-Glutamylhydroxamate. Table VIII demonstrates that s i g n i f i c a n t differences in 7 -glutamylhydroxamate synthesis a c t i v i t y were not found between JK and JK301. These findings were in di r e c t contrast to experiments using methylammonium as substrate (Table VII) and therefore prompted examination of glutamine synthetase a c t i v i t y by a t h i r d method. This method measures 1"C-glutamine production and offers the best representation of GS a c t i v i t y because i t u t i l i z e s the natural substrates of the enzyme. The reaction assayed is the following: 1 f lC-glutamate + NH 4 + ) 1*C-glutamine The product of this reaction, 1*C- glutamine, i s separated from 1°C-glutamate by passing the reaction mixture through a Dowex 50(C1") anion exchange column at pH 7.0. This column binds glutamate and allows glutamine to pass through such that i t can be co l l e c t e d and counted by l i q u i d s c i n t i l l a t i o n . Preliminary 95 TABLE VIII - X-giiitamyl hydroxamate synthesis a c t i v i t y in c e l l f ree extracts of JK and JK301. B -glutamyl hydroxamate synthesis a c t i v i t y (nmol % -glutamyl hydroxamate synthesized/min/mg protein) experiment S t ra in 1 2 JK JK301 13.2 12.5 9.7 11.0 96 experiments using t h i s assay revealed that JK301 had about 30% the GS a c t i v i t y of JK (Fig. 17). Further experiments were performed with two main objectives. Because published procedures for using t h i s assay for A. v i n e l a n d i i GS do not exist the f i r s t objective was to optimize assay conditions. The second objective was to elucidate the nature of the defect in GS from JK301. 2. PROTEIN CONCENTRATION 1*C-glutamine synthesis increased at a linear rate in response to increased .protein concentration in both JK and JK301 (Fig. 18). When more than 200 Mg protein per assay was used the reaction v e l o c i t y did not increase in response to increased amounts of protein suggesting the presence of an i n h i b i t o r y compound or competing reaction. On the basis of these results a protein concentration of 150 Mg per assay was chosen for further experiments. 3. PH OPTIMUM Optimum pH for GS a c t i v i t y was examined in extracts of JK and JK301. Reaction rates measured over the range of pH 6.5 to 8.5 indicated a somewhat narrower pH optimum for JK enzyme as compared to JK301 enzyme (Fig. 19). On the basis of these re s u l t s , assays were performed at pH 7.0 to provide optimal a c t i v i t y . 97 Figure 17. Glutamine synthetase mediated glutamine formation in c e l l free extracts from JK and JK301. Symbols: JK •; JK301 o. Assays were performed as described in Materials and Methods.' 98 UO-i s e c o n d s 99 Figure 18. Effect of c e l l free extract concentration on rate of glutamine synthesis. Glutamine synthetase a c t i v i t y was determined as described in Materials and Methods except that amount of c e l l free extract per assay varied. Total assay volume was held constant. Symbols: JK •; JK301 o. 1 0 0 300 m i c r o g r a m s p r o t e i n i n a s s a y 101 Figure 19. pH p r o f i l e of glutamine synthetase a c t i v i t y in c e l l free extracts from JK and JK301. GS assays were performed as described in Materials and Methods except that Tris-maleate buffer OOOmM) was used in place of imidazole-HCl. Assay mix was adjusted to the appropriate pH with NaOH. Symbols: JK •; JK301 o. 102 J 1 1 1 r 1 1 6 6.5 7 7.5 8 8.5 9 PH 103 4. INCREASE IN GLUTAMINE SYNTHETHASE ACTIVITY IN RESPONSE TO  NITROGEN LIMITATION The e f f e c t of nitrogen l i m i t a t i o n on GS a c t i v i t y in JK and JK301 was examined. Glutamine synthetase, in a l l Gram negative bacteria tested to date, undergoes activation and deactivation by means of removal and covalent attachment of adenosine 5' monophosphate groups (Tronick et a l . , 1973). Poor or l i m i t i n g nitrogen sources lead to deadenylylation (activation) of the enzyme while s u f f i c i e n t amounts of good nitrogen sources result in an adenylylated, less active enzyme. One possible explanation for the low GS a c t i v i t y in JK301 i s that the enzyme is unable to undergo normal deadenylylation resulting in a highly adenylylated, less active form of the enzyme. To determine i f GS from JK301 was activated in response to nitrogen l i m i t a t i o n , as has been reported for wild type A. v i n e l a n d i i , (Tronick et a l . , 1973), the following experiment was performed. Both strains were shi f t e d from NH„* medium to N-free medium for a period of 3 hr. C e l l s were assayed for GS a c t i v i t y before and after the s h i f t to N-free medium. The r a t i o of GS a c t i v i t y in extracts prepared from the nitrogen l i m i t e d c e l l s to that in extracts from c e l l s grown in excess ammonium (before s h i f t ) should represent the a c t i v a t i o n of glutamine synthetase in response to nitrogen l i m i t a t i o n . If JK301 were unable to undergo normal response to nitrogen l i m i t a t i o n , the r a t i o should be lower than that observed for JK. It i s not l i k e l y that increased glutamine synthetase a c t i v i t y resulting from nitrogen l i m i t a t i o n would be due to increased synthesis of enzyme because 1 04 good nitrogen sources do not repress synthesis of glutamine synthetase in A. v i n e l a n d i i (Lepo et a_l., 1982). The results of these experiments, shown in Table IX, indicate that the rat i o s of nitrogen limited/nitrogen s u f f i c i e n t GS a c t i v i t y are the same for JK and JK301. It seems unlikely therefore, that JK301 i s defective in i t s a b i l i t y to deadenylylate GS in response to N-limitation. 5. SNAKE VENOM PHOSPHODIESTERASE TREATMENT Snake venom phosphodiesterase (SVD), by virt u e of i t s a b i l i t y to deadenylylate, has been reported to activate adenylylated GS in A. vinelandi i (Tronick et a_l. , 1973). To test the hypothesis that GS from JK301 was highly adenylylated and consequently less active than GS from JK, I attempted to determine whether deadenylylation of GS in JK301 by SVD treatment would restore the a c t i v i t y to the l e v e l observed in JK. Treatment of crude c e l l - f r e e extracts with SVD did not increase a c t i v i t y in either JK or JK301 (Table X). The GS a c t i v i t y measured in extracts of JK301 after SVD treatment was s i g n i f i c a n t l y lower than a c t i v i t y before treatment suggesting that the enzyme i s less stable to t h i s treatment. It was expected that treatment with SVD would increase GS a c t i v i t y in extracts from wild type. However, t h i s was not observed. Because neither JK nor JK301 showed an increase in GS a c t i v i t y i t i s impossible to draw conclusions from these experiments. 105 TABLE IX - E f f e c t of n i trogen l i m i t a t i o n on glutamine synthetase a c t i v i t y in c e l l f ree ext rac ts from JK and JK301. GS a c t i v i t y in c e l l f ree e x t r a c t s 3 Nitrogen l im i t ed C e l l s grown in NH4 Nitrogen l im i t ed a c t i v i t y / N H 4 + S t ra in s u f f i c i e n t medium c e l l s b s u f f i c i e n t a c t i v i t y JK .308 .612 1.99 JK301 .117 .205 1.75 a nmol ^C-glutamine produced/min/mg p ro t e i n . b 80 K l e t t , NH 4 + grown c e l l s were cent r i fuged and resuspended in N-free medium and allowed to incubate fo r 3 h rs . 106 TABLE X - E f f e c t of snake venom phosphodiesterase treatment on glutamine synthetase a c t i v i t y i n c e l l f ree ext rac ts from JK and JK301. GS a c t i v i t y (nmol glutamine produced/min/mg prote in) S t r a in c o n t r o l 3 SVD treated JK 463 374 JK301 148 68 a Ce l l f ree extracts used fo r cont ro ls were t reated i d e n t i c a l l y to SVD treated extracts except that buf fer replaced SVD s o l u t i o n . 107 6. KM DETERMINATION FOR GLUTAMATE Another possible explanation for the low a c t i v i t y observed in GS from JK301 is a structural a l t e r a t i o n which results in an enzyme with a lower a f f i n i t y for glutamate. Although i t i s possible that a str u c t u r a l defect might a l t e r the a f f i n i t y of the enzyme for any of i t s substrates (NH„ +, ATP, Mg+ + or glutamate) glutamate seemed a l i k e l y p o s s i b i l i t y because JK301 had been isolated by selection for resistance to the glutamate analog MSX. To test t h i s hypothesis, the Km for glutamate was determined in extracts from JK and JK301. Figure 20 i l l u s t r a t e s the marked difference in Lineweaver-Burk plots of data obtained from both s t r a i n s . The Km for glutamate derived from this plot is 8.0 mM for JK and 25.2 mM for JK301. The Vmax for GS from JK is 810 nmol/min/mg protein and from JK301, 200 nmol/min/mg protein. Using an Eadie-Hofstee plot the Km for glutamate was 7.7 mM for JK and 19.1 mM for JK301. The Vmax calculated from the plot are 790 nmol/min/mg protein for JK and 160 nmol/min/mg protein for JK301. These results indicate that the mutation in JK301 results in GS with an altered Km for glutamate and an altered Vmax. Although a mutation in a st r u c t u r a l gene might explain these results a regulatory mutation cannot be ruled out. 108 Figure 20. Lineweaver-Burk plot of glutamine synthetase a c t i v i t y in c e l l free extracts of JK and JK301. Glutamate concentrations ranged from 0.5 mM to 50 mM. The Vmax for the 2 curves are 814 nmol/min/mg protein (JK) and 200 nmoles/min/mg protein (JK301). The Km for glutamate was determined to be 8.0 mM (JK) and 25.2 mM (JK301). Glutamine synthetase assays were performed as described in Materials and Methods. Symbols: JK •; JK301 o. 109 500 Vfglutamate] mM 110 VI. CHEMOTAXIS TOWARDS AMMONIUM BY AZOTOBACTER VINELANDII It i s well established that periplasmic proteins serve a role in both chemotaxis and transport (reviewed by Koshland, 1979). Several reports have described the i s o l a t i o n of mutants with defective binding proteins which are unable to transport substrate and which are also defective in chemotaxis towards the substrate (Hazelbauer and Adler, 1971; Aksamat and Koshland, 1974; Stinson et a l . , 1977) It seemed possible, therefore, to obtain mutants with defective binding protein by selecting for str a i n s unable to respond chemotactically toward ammonium. Preliminary experiments were performed to determine i f A. vinelandi i exhibited a chemotactic response towards sucrose, glucose, succinate, acetate or ammonium. The assay system consisted of swarm plates (semi-solid agar plates with 0.3% agar, Adler, 1966) containing attractant at 1 mM (sucrose, glucose, succinate or acetate) or 0.5 mM (NH, +). Swarm plates were inoculated by stabbing the center of the plate with c e l l s from a l i q u i d culture and incubated overnight at 30°C. A pos i t i v e chemotactic response consisted of a d i s t i n c t , c i r c u l a r zone of spreading growth. This appearance i s a result of c e l l s depleting the immediate area of substrate and moving outwards toward areas of greater substrate concentration (Adler, 1966). A. v i n e l a n d i i demonstrated a chemotactic response to a l l of the compounds mentioned above. The a b i l i t y of A. vin e l a n d i i to respond chemotactically toward NH„* was demonstrated by inoculating the center of swarm plates containing minimal media supplemented with 0.5 mM 111 ammonium (as (NH f l) 2SO„). After overnight incubation these plates exhibited a c i r c u l a r zone of growth with a d i s t i n c t edge (Fig. 21). Plates containing high levels of ammonium (29 mM) did not exhibit a spreading zone of growth but rather a dense area of growth at the point of inoculation which, after prolonged incubation, spreads outward d i f f u s e l y . Swarm plates without NH„ + exhibited a d i f f u s e zone of growth lacking the d i s t i n c t edge found in plates containing 0.5 mM ammonium. Both JK and Nif" (unable to f i x nitrogen) strains displayed a similar chemotactic response in NH„ + swarm plates. A procedure described by Armstrong et al.,(l966) was used to attempt i s o l a t i o n of mutants unable to chemotactically respond to ammonium. C e l l s remaining in the middle of an NH,,* swarm plate (after overnight incubation) were transferred to a new swarm plate and allowed to incubate overnight. This pattern of transferring c e l l s from the center of the swarm plate to a new swarm plate was repeated 30 times. C e l l s in the middle of the l a s t swarm plate showed a reduced zone of spreading (Fig. 21) and were plated out for individual colonies, approximately 150 of which were tested for the a b i l i t y to respond chemotactically to ammonium in swarm plates. Presumptive chemotatic defective stains were isolated on the basis of s i g n i f i c a n t l y smaller zones of spreading in swarm plates when compared to the wild type s t r a i n . These stra i n s , however, exhibited a s i g n i f i c a n t l y smaller zone of spreading than the wild type s t r a i n in acetate or sucrose swarm plates indicating that these mutants possessed a generalized i n a b i l i t y to respond 112 Figure 21. Chemotaxis of A. v i n e l a n d i i toward ammonium. Ammonium swarm plates were prepared as described in Materials and Methods and contained 0.5 mM ammonium sulfate as an attractant. The center of each plate was inoculated with c e l l s (using a stab) and allowed to incubate overnight at 30° C. The center of the plate on the l e f t was inoculated with wild type c e l l s and i l l u s t r a t e s the d i s t i n c t zone of spreading in response to depletion of ammonium in the medium. The plate on the right is the f i n a l result of a selection for non-chemotactic mutants. 1 1 3 114 chemotactically rather than a s p e c i f i c i n a b i l i t y to respond toward ammonium. A l l of the strains tested had normal m o t i l i t y as determined by phase microscopy. Another attempt was made to is o l a t e mutants defective only in chemotaxis towards NHj, + . A Nif" s t r a i n , UW2 (Gordon and B r i l l , 1972), was used in case additional ammonium derived from nitrogen f i x a t i o n was suppressing the chemotactic response to ammonium in the swarm plate, thereby weakening the enrichment for chemotactic defective s t r a i n s . In addition, the procedure was modified such that enrichment for general non-chemotactic mutants could be avoided. The modified procedure consisted of transfe r r i n g c e l l s which had been passed through 5 NH,,* swarm plates to a swarm plate containing 1mM glucose as an attractant. C e l l s from the outer edge of the zone of growth were then transferred to the center of an NH„ + swarm plate. The entire process was repeated six times. Periodic use of c e l l s picked from the edge of a glucose swarm plate as inoculum for a series of NH„ + swarm plates should eliminate both non-motile and generalized non-chemotactant mutants. C e l l s from the center of the l a s t ammonium swarm plate showed a smaller zone of spreading compared to control swarm plates containing the parent s t r a i n . These c e l l s were plated out for individual colonies and tested for their a b i l i t y to respond chemotactically towards ammonium. As a means of quantitating the response toward ammonium in both putative mutants and the parent s t r a i n , the rate of zone growth was determined in ammonium swarm plates by measuring the diameter of the c i r c u l a r zone of growth over a period of 8-12 hr 1 15 approximately 12-14 hr after inoculation (Craven and Montie, 1981). Approximately 100 colonies were screened in this manner however none were detected which displayed a s i g n i f i c a n t reduction in the rate of swarming compared to wild type c e l l s . An average rate of zone growth in NH„ + swarm plates inoculated with parent str a i n c e l l s was calculated to be 2.5 mm/hr. The c a p i l l a r y tube method (Adler, 1973) was chosen as an alternative means of testing for a chemotaxis defective phenotype in mutants obtained from the i s o l a t i o n procedure described above. This method, which measures the number of bacteria which w i l l swim up a c a p i l l a r y tube in response to an attractant, should provide a more sensitive and quantitative measurement of chemotaxis than obtained by measuring zone growth in swarm plates. However, attempts to demonstrate chemotaxis towards NH„ + with th i s method f a i l e d , presumably because of the rapid depletion of oxygen within and surrounding the c a p i l l a r y tube due to the high respiratory a c t i v i t y of the bacteria. A. v i n e l a n d i i , being a s t r i c t aerobe, quickly becomes non-motile under these conditions. 1 16 VII. MEMBRANE PROTEINS OF AZOTOBACTER VINELANDII 1. INNER AND OUTER MEMBRANES OF AZOTOBACTER VINELANDII Another approach which can be used to id e n t i f y proteins involved in transport i s to compare membrane proteins of transport defective mutants and wild type. To i n i t i a t e t h i s approach, a preliminary examination of inner and outer membrane proteins of A. vin e l a n d i i was performed. As information on the is o l a t i o n of inner and outer membranes from A. vi n e l a n d i i was not available at the time t h i s work was i n i t i a t e d , procedures were adapted from those used for P. aeruginosa (Hancock and Nikaido, 1978). Comparision of outer membranes from nitrogen-fixing c e l l s and NH a + grown c e l l s revealed the increased production of a 44 kilodalton protein (Fig. 22) in membranes from nitrogen-fixing c e l l s . This protein was the only reproducible difference seen in major outer membrane proteins when c e l l s were grown under these two conditions. Page and von Tigerstrom (1982) have reported that a 44 kilodalton protein i s hyper-produced when c e l l s are starved for molybdenum and have suggested that t h i s outer membrane protein i s involved in molybdenum uptake. It seems l i k e l y that the increased production of thi s protein in nitrogen f i x i n g c e l l s , r e l a t i v e to NH4* grown c e l l s , i s due to an increased demand for molybdenum which i s required for the active center.of nitrogenase as part of an Fe-Mo cofactor. Inner membranes from nitrogen-fixing c e l l s and NHfl+ grown c e l l s were also compared. Examination using one dimensional 117 Figure 22. Outer membrane proteins of A. v i n e l a n d i i . Membranes were isolated as described in Materials and Methods. Proteins were s o l u b i l i z e d at 88° C and run on a 12% acrylamide g e l . Lane 1, outer membranes from c e l l s grown in Burk's nitrogen free medium ( N 2 - f i x i n g c e l l s ) . Lane 2, outer membranes from c e l l s grown in Burk's medium containing 29 mM ammonium acetate. The 44K marker indicates the position and approximate molecular weight of an outer membrane protein found in greater quantity in N 2 - f i x i n g c e l l s than in NH,+ grown c e l l s . 118 119 Figure 23. Inner membrane proteins from A. v i n e l a n d i i . Membranes were isolated as described in Materials and Methods. Proteins were s o l u b i l i z e d at 100 C and run on a 12% acrylamide gel. The 41K marker indicates the position and approximate molecular weight of a protein band which i s present in greater amounts in N 2 - f i x i n g c e l l s than in non-fixing c e l l s (NH„* grown). Lane 1, inner membrane proteins from A. v i n e l a n d i i JK grown in Burk's medium containing 29 mM ammonium acetate; Lane 2, inner membrane proteins from A. v i n e l a n d i i JK grown in Burk's nitrogen free medium (N 2 f i x i n g c e l l s ) . Lane 3, inner membrane proteins from the con s t i t u t i v e nitrogen f i x i n g mutant of A. vinelandi i s t r a i n UW 590 grown in Burk's medium containing 29 mM ammonium acetate 120 121 SDS-PAGE showed that nitrogen-fixing c e l l s contained a major protein (41 kilodaltons) which was only s l i g h t l y v i s i b l e in membranes from NH4 * grown c e l l s (Fig. 23). This protein was also present in inner membranes from s t r a i n UW 590, a mutant which c o n s t i t u t i v e l y produces nitrogenase in the presence of NH4 * (Fig. 23). Although one might speculate that t h i s protein has a role in nitrogen f i x a t i o n the true function of thi s protein i s not known. The 41 kilodalton protein represents the only reproducible difference in major proteins from inner membranes isolated from NH4* grown and nitrogen-fixing c e l l s . 2. MEMBRANE AND PERIPLASMIC PROTEINS OF METHYLAMMONIUM  RESISTANT MUTANTS OF AZOTOBACTER VINELANDII The inner and outer membranes from three methylammonium resistant mutants were compared to membranes from wild type c e l l s . In addition, the periplasmic proteins from one of the methylammonium resistant mutants and wild type were compared. These mutants had been isolated by selecting for c e l l s r esistant to 300 mM methylammonium (Gordon and Jacobson, 1983 ). Analysis of uptake data revealed that the mutants had a higher Km for methylammonium than the wild type s t r a i n One possible explanation for th i s phenotype was an a l t e r a t i o n in membrane proteins r e s u l t i n g in a decreased permeability of methylammonium or an a l t e r a t i o n in a periplasmic protein involved in methylammonium uptake. One dimensional SDS-PAGE revealed no major differences in proteins from inner and outer membranes from both mutant and 122 Figure 24. Two-dimensional gel electrophoresis of periplasmic proteins from A. v i n e l a n d i i JK and A. v i n e l a n d i i JK213 (a spontaneous methylammonium resistant derivative of JK obtained by selecting for growth on N-free plates containing 250 mM methylammonium (Gordon and Jacobson, 1983). Periplasmic proteins were released by producing spheroplasts as described in Materials and Methods. I s o e l e c t r i c focusing gels were loaded with 150 ug protein and focused for approximately 12 hours. The pH gradient of the i s o e l e c t r i c focusing gel was determined by running a blank gel along with gels containing sample. The blank gel was s l i c e d into 0.5 cm sections and each section placed into separate tubes containing 1 ml H 20. After standing at room temperature overnight, the pH of the water in each tube was determined. The SDS-PAGE (12% acrylamide) was performed at 55 v o l t s (constant voltage) u n t i l tracking dye had reached the running gel and then at 130 volts for approximately 4.5 hrs. Proteins were stained with coomassie blue. 1 - periplasmic proteins from A. v i n e l a n d i i JK. 2 - periplasmic proteins from A. vinelandi i JK213. 123 124 wild type strains (data not shown). In addition, two dimensional gel electrophoresis of periplasmic proteins isolated from one methylammonium resistant mutant (JK213) and the wild type s t r a i n revealed no s i g n i f i c a n t differences in major periplasmic proteins (Fig. 24). These results suggest that the al t e r a t i o n in these mutants r e s u l t i n g in a higher Km for methylammonium i s not due to a major a l t e r a t i o n in outer and inner membrane proteins or periplasmic proteins. 3. HEAT MODIFIABLE OUTER MEMBRANE PROTEINS IN AZOTOBACTER  VINELANDII The mobility in SDS-polyacrylamide gels of some outer membrane proteins i s influenced by temperature of membrane s o l u b i l i z a t i o n . For example, when outer membrane preparations of E. c o l i are heated at 37°C in a solution of SDS, urea and 2-mercaptoethanol, protein B migrates at a position corresponding to a MW of 28,500. When the same outer membrane preparation i s heated at 100°C, protein B migrates at a position corresponding to a MW of 33,400 (Bragg and Hou, 1972). Understandably, the phenomenon of heat m o d i f i a b i l i t y has caused considerable confusion in past outer membrane l i t e r a t u r e . Preliminary examination of outer membranes of A. vi n e l a n d i i revealed one heat modifiable protein. This protein had an apparent MW of 41 kilodaltons when membranes were s o l u b i l i z e d at 88°C in the presence of 2% SDS and 5% 2-mercaptoethanol. When s o l u b i l i z a t i o n was performed at 37°C the protein had an apparent molecular weight of 49 kilodaltons. The heat modification of t h i s protein i s best i l l u s t r a t e d in a 2-125 Figure 25. Two dimensional SDS-polyacrylamide gel electrophoresis of A. vinelandi i outer membrane proteins demonstrating the presence of a heat modifiable outer membrane protein. Outer membranes were is o l a t e d as described in Materials and Methods, s o l u b i l i z e d at 30°C and run on a 12% acrylamide gel (1st dimension). The appropriate lane was cut out, immersed in s o l u b i l i z a t i o n buffer and heated at 100°C for 10 minutes. The lane was then placed hori z o n t a l l y on top of the stacking gel of another 12% acrylamide gel, and run as described in Materials and Methods (2nd dimension). The markers indicate the position and approximate molecular weight of the heat modifiable protein when membranes are s o l u b i l i z e d at 30°C (41K) and at 100°C (49K). 126 4 9 K - • 41 K > # / 0 t • 127 dimensional SDS-polyacrylamide gel (Fig. 25). 4. INFLUENCE OF CARBON SOURCE ON OUTER MEMBRANE PROFILE A number of microorganisms synthesize new outer membrane proteins in response to growth on diff e r e n t carbon sources (Nakae, 1976; Hancock and Carey, 1980). The lam B protein of E. c o l i i s a well studied example. This outer membrane protein i s found in c e l l s grown with maltose as a carbon source and has been shown to f a c i l i t a t e the d i f f u s i o n of maltose and maltosedextrins across the outer membrane of E. c o l i (Nakae, 1976). The study of membrane proteins from A. vi n e l a n d i i was extended to include a preliminary examination for outer membrane proteins synthesized in response to growth on d i f f e r e n t carbon sources. Sucrose, glucose and mannitol (2% w/v) were chosen as carbon sources because they are most commonly used in A. vi n e l a n d i i growth medium. One dimensional SDS-PAGE did not reveal major protein differences in outer membranes from c e l l s grown on the three carbon sources (Fig. 26) suggesting that d i f f u s i o n of these carbon sources across the outer membrane of A vin e l a n d i i i s f a c i l i t a t e d by a c o n s t i t u t i v e l y synthesized outer membrane protein or that a l l three carbon sources induce synthesis of an outer membrane protein which i s able to mediate passage of these carbon sources across the outer membrane. An interesting, yet unexplained, observation made during these studies was that mannitol grown c e l l s were round (as determined by phase microscopy) as opposed to the very c h a r a c t e r i s t i c ovoid shape of A. v i n e l a n d i i grown in medium 128 Figure 26. Outer membrane proteins from A. vinelandi i grown on di f f e r e n t carbon sources. Outer membranes were isolated from c e l l s grown in Burk's medium with indicated carbon source (2%) and, unless noted otherwise, 29 mM ammonium acetate. Lane 2 sucrose (N-free). Lane 3 sucrose. Lane 4 glucose. Lane 5; mannitol. Lane 1 molecular weight standards (Bovine serum albumin, approximate molecular weight 66,000; egg albumin, approximate molecular weight 45,000; carbonic anhydrase, approximate molecular weight 29,000; Beta-lactoglobulin, approximate molecular weight 18,000; lysozyme, approximate molecular weight 14,000. 129 1 30 containing sucrose or glucose. 131 VIII. DISCUSSION This study provides evidence for the existence of an active transport system for ammonium in A. v i n e l a n d i i . Cultures of A vinelandi i , grown with ammonium as a nitrogen source, are able to take up ammonium against a concentration gradient of at least 58 f o l d . Kleiner (1975) has reported that N2-grown cultures of A. vin e l a n d i i are able to concentrate ammonium against a concentration gradient of approximately 100 f o l d . Ammonium uptake (transport and metabolism) in A. vinelandi i i s strongly inhibited by treatment with CCCP and KCN indicating a requirement for a proton gradient, ATP or both. It is l i k e l y that the ATP requirement i s , in part, for the synthesis of glutamine by the enzyme glutamine synthetase. Further examination of ammonium uptake was made using the ammonium analog methylammonium. Methylammonium has been shown to be a suitable ammonium analog by virtue of the fact that both compounds are transported by a common system in several eucaryotic and procaryotic microorganisms (Hackette et a l ., 1970; Roon et a_l., 1975; Breiman and Barash, 1980; Barnes and Zimniak, 1981; Gordon and Moore, 1981; Kleiner and Fitzke, 1981; Gober and Kashket, 1983). Ammonium i s a competitive i n h i b i t o r of methylammonium uptake in A. vi n e l a n d i i (Gordon and Moore, 1981) indicating that ammonium and methylammonium share at least one common s i t e in the uptake process. Because the uptake assay used in thi s study does not distinguish between transport across the c e l l membrane and metabolism by glutamine synthetase i t is not possible to determine whether the common s i t e i s a membrane 1 32 c a r r i e r , glutamine synthetase or both. Inhibitors which lower ATP pools result in i n h i b i t i o n of methylammonium uptake (Table I I I ) . A l i k e l y role for ATP in methylammonium uptake i s in the glutamine synthetase mediated synthesis of N-methylglutamine. Barnes and Zimniak (1981) have reported that methylammonium transport in A. v i n e l a n d i i does not require ATP on the basis that treatment with 0.2 mM arsenate does not i n h i b i t methylammonium accumulation. By measuring ATP pools however , i t was shown that 0.2 mM or 15 mM arsenate does not reduce ATP pools in c e l l s grown under the conditions used by these authors (succinate as carbon source, N 2 fixing) (Table IV). It i s not understood why succinate grown, N 2 f i x i n g c e l l s are resistant to arsenate treatment. Two observations suggest that c a t a l y t i c a l l y active glutamine synthetase is required for ammonium/methylammonium uptake. F i r s t , i n h i b i t i o n of glutamine synthetase by the glutamate analog MSF i n h i b i t s ammonium uptake (Fig. 4). Furthermore, when glutamine synthetase i s inhibited by MSF, methylammonium uptake a c t i v i t y i s not detectable (J. Gordon, unpublished data). these i n h i b i t o r s were chosen as a means of blocking the assimilation step of ammonium/methylammonium uptake in an e f f o r t to examine the transport step alone. A similar approach allows examination of amino acid transport by blocking assimilation with the protein synthesis i n h i b i t o r chloramphenicol (Ames and Lever, 1970). The second observation which indicates a requirement for glutamine synthetase in ammonium/methylammonium uptake i s that 1 33 JK301, a mutant with defective glutamine synthetase, does not have detectable methylammonium uptake a c t i v i t y (Fig. 16). It i s possible that methylammonium uptake i s not observed in these two situations because a small pool of methylammonium, lower than the detection l i m i t s of the uptake assay, rapidly forms in the c e l l and prevents further accumulation. A l t e r n a t i v e l y , when assimilation i s blocked (by MSF, MSX or a glutamine synthetase mutation), an ammonium pool generated as a result of amino acid degradation might block further methylammonium accumulation by competing for membrane c a r r i e r s i t e s . F i n a l l y , i t i s possible that MSF and MSX block ammonium/methylammonium uptake by i n h i b i t i n g the action of a membrane c a r r i e r . An alternative explanation for the i n h i b i t i o n of methylammonium and ammonium uptake by MSX and MSF i s that glutamine synthetase may be required for translocation of methylammonium and ammonium across the membrane in a manner analogous to the membrane proteins involved in potassium transport in E. c o l i (reviewed by Helmer et a l . , 1982). This idea i s supported by the observation that a s i g n i f i c a n t fraction of c e l l u l a r glutamine synthetase i s membrane associated. Kleinschmidt and Kleiner (1978) have reported that during i s o l a t i o n of glutamine synthetase from A. vinelandi i about 30% of the glutamine synthetase a c t i v i t y i s found associated with membranes when c e l l s were broken by a r e l a t i v e l y gentle osmotic shock procedure (Shah et a l . , 1972). The glutamine synthetase, from t h i s fraction was not s o l u b i l i z e d by treatment with n-134 butanol or with deoxyribonuclease. More recently, glutamine synthetase from the nitrogen-fixing phototroph Rhodospirillum  rubrum has been found to be d i s t r i b u t e d between soluble and membrane (chromatophore) fractions (Yoch et a l . , 1983). The membrane associated a c t i v i t y (approximately 60%) was not s o l u b i l i z e d by treatment with DNase, NaCl, Triton X-100 or deoxycholate. S o l u b i l i z a t i o n was accomplished by increasing ionic strength of the buffer or by adding 10 mM MgCl 2. Furthermore, the s o l u b i l i z e d a c t i v i t y could be reconstituted with membranes by dialysi n g the glutamine synthetase and the membranes together against low ionic strength buffer. These observations suggest that glutamine synthetase may have a membrane associated function. It i s interesting to consider the effect of Tris-Mg + +-shock treatment on methylammonium uptake in reference to membrane associated glutamine synthetase. Methylammonium uptake a c t i v i t y was undetectable in c e l l s subjected to Tris-Mg + + shock treatment while fructose uptake was reduced only s l i g h t l y (Fig. 9). One possible reason for the reduction in uptake a c t i v i t y is that the Tris-Mg + + treatment results in the dis s o c i a t i o n of glutamine synthetase from the inner membrane. Al t e r n a t i v e l y , the shock treatment may damage components of the electron transport chain or the membrane bound Ca + +/Mg + + ATPase, thereby reducing ATP pools required for methylammonium accumulation. Periplasmic shock f l u i d was examined for the presence of ammonium/methylammonium binding proteins because methylammonium uptake in A. v i n e l a n d i i was ATP dependent (Table IV) and shock 1 35 sensitive (Fig. 9). Both of these c h a r a c t e r i s t i c s have been shown to be associated with binding protein dependent transport systems (Koshland/ 1979). Although methylammonium-binding a c t i v i t y was not detected in the periplasmic shock f l u i d i t i s possible that an ammonium binding protein would not have detectable binding a c t i v i t y for methylammonium when measured by the techniques used in this study. The r e l a t i v e l y poor a b i l i t y of a binding protein to bind an analog of i t s natural substrate i s i l l u s t r a t e d in studies with glucose binding protein from Pseudomonas aeruginosa. . In the study reported the analogs 2-deoxyglucose and methyl-D-glucose inh i b i t e d binding of glucose by only 17% and 31% respectively when the analog concentration was 100 f o l d greater than glucose (Stinson et al.,1977). An additional problem may stem from the presence of contaminating ammonium resulting from the shock treatment. Ammonium may i n h i b i t methylammonium from binding to an ammonium binding protein because the protein would presumably have a much higher a f f i n i t y for ammonium than methylammonium. Binding protein for ammonium may not be present in A. vinelandi i . Some evidence e x i s t s which suggests that methylammonium transport in A. vin e l a n d i i i s mediated by a transmembrane potential (Ai£) (Laane et a l . , 1980, Barnes and Zimniak, 1981). However, because the method used to dissipate Ai// in these studies (addition of valinomycin and K +) has been reported to reduce ATP pools in E. c o l i (Plate, 1979), i t remains unclear whether A\//, ATP or both are required to drive methylammonium uptake. Transport systems energized solely by 136 th i s component of the proton motive force (A\//) have not generally been associated with the presence of binding proteins. Methylammonium uptake increased approximately 2 fo l d in response to nitrogen starvation (Table I I ) . The increase in uptake a c t i v i t y is probably a result of increased metabolism by glutamine synthetase as similar conditions of nitrogen l i m i t a t i o n result in an approximate 2 fold increase in glutamine synthetase a c t i v i t y (Table IX). Experiments in t h i s study demonstrate that methylammonium is rapidly metabolized to N-methylglutamine in whole c e l l s of A. v i n e l a n d i i . N-methylglutamine was i d e n t i f i e d as the product of methylammonium metabolism on the basis of co-migration with chemically synthesizied N-methylglutamine in 6 solvent systems. Methylglutamate was not detected as a product of methylammonium metabolism in A. vinelandi i suggesting that further metabolism of N-methylglutamine by glutamate synthase does not occur and that glutamate dehydrogenase i s not capable of using methylammonium as a substrate. It i s l i k e l y that N-methylglutamine is the only major product of methylammonium metabolism since paper chromotography reveals only one radioactive product. Two observations suggest that the conversion of methylammonium to N-methylglutamine in A. vinelandi i i s mediated by glutamine synthetase. F i r s t , a mutant which has defective glutamine synthetase i s —not able to catalyse N-methylglutamine synthesis in whole c e l l s (data not shown) or in c e l l - f r e e extracts (Table VII). Secondly, synthesis of N-1 37 methylglutamine can be accomplished in v i t r o in a reaction mix designed to measure glutamine synthetase a c t i v i t y (Fig. 14). These results are supported by studies which have reported glutamine synthetase from a number of sources i s able to accept methylammonium as substrate (Speck, 1949; Rowe e_t §_1. , 1 970). Rates of methylammonium uptake are equal to rates of methylammonium metabolism (Fig. 15). The mutant JK301, which is unable to synthesize N-methylglutamine in v i t r o (due to a defect in glutamine synthetase), also has no detectable methylammonium uptake a c t i v i t y . This finding suggests that a certain class of glutamine synthetase mutants in A. v i n e l a n d i i can be i d e n t i f i e d by examining rates of methylammonium uptake. This would have an important application as a screen for glutamine synthetase mutants in A. vinelandi i because conventional techniques aimed at i d e n t i f y i n g glutamine synthetase mutants involve selection for glutamine auxotrophs and to date amino acid auxotrophs have never been isolated in A. vinelandi i despite concerted e f f o r t s to achieve th i s goal (Sadoff et a_l, 1979). It must be noted, however, that screening for glutamine synthetase mutants by examining methylammonium uptake might id e n t i f y some false p o s i t i v e s . This i s i l l u s t r a t e d by the fact that A. v i n e l a n d i i UW590, which has a lower rate of methylammonium uptake than JK (M. R. Jacobson, MSc. Thesis, University of B r i t i s h Columbia, 1983), has about 150% of the in  v i t r o glutamine synthetase a c t i v i t y of JK (J. Gordon, unpublished data). A possible explanation for these results i s that UW590 i s p a r t i a l l y defective in methylammonium transport, 1 38 and therefore, methylammonium accumulation (and subsequent metabolism) i s lim i t e d by translocation of methylammonium across the membrane. A mutant such as th i s (transport defective, higher glutamine synthetase a c t i v i t y ) would not be detected by methylammonium uptake assays because higher glutamine synthetase a c t i v i t y would be masked due to lower levels of methylammonium inside the c e l l as a result of a slower rate of methylammonium transport across the c e l l membrane. Analysis of kinetic data from methylammonium uptake experiments predicts the existence of a membrane c a r r i e r involved in ammonium/methylammonium transport in A. v i n e l a n d i i . Jacobson (MSc. Thesis, University of B r i t i s h Columbia, 1983) has shown that an i t e r a t i v e analysis of methylammonium uptake data from A. v i n e l a n d i i reveals the presence of only one saturable uptake reaction with a Km of 61 MM. A Lineweaver-Burk plot of methylammonium uptake v e l o c i t i e s obtained over a substrate concentration of 10 MM to 1.5 mM reveals a biphasic curve (Gordon and Moore, 1 9 8 1 ) . This i s considered evidence for two transport systems. The Km for each system calculated from these curves was 61 *iM and 661 uM. It was concluded that the biphasic Lineweaver-Burk plot i s the result of active transport across the membrane and a second reaction composed of transport and d i f f u s i o n across the membrane. A possible interpretation of this finding i s that at low methylammonium concentrations, transport i s mediated primarily by a membrane c a r r i e r while at higher substrate concentrations, methylammonium enters the c e l l through the membrane c a r r i e r and via d i f f u s i o n through the 1 39 membrane. The Km that Jacobson calculated (61 MM) presumably i s the Km for membrane mediated transport, while the higher Km* value derived from the biphasic Lineweaver-Burk plot (660 MM) is possibly the Km for methylammonium in the glutamine synthetase mediated assimilation reaction. The fact that increasing methylammonium concentration in the uptake assay reveals another reaction (Km = 660 MM) suggests that at low substrate concentrations the rate l i m i t i n g reaction i s transport across the membrane. If t h i s interpretation proves correct then methylammonium i s a suitable probe for examining ammonium/methylammonium transport in A_^_ v i n e l a n d i i provided low concentrations of methylammonium are used. One problem with t h i s interpretation, however, i s that the Km of 660 juM i s far below the Km derived for methylglutamine synthesis in c e l l - f r e e extracts (60-120 mM, data not shown). It i s unlikely that lower substrate concentrations which might be found i_n vivo would result in.a lower Km value. It i s possible, however, that preparation of c e l l - f r e e extracts results in a form of glutamine synthetase with a much higher Km for methylammonium than found in glutamine synthetase i_n s i t u . JK301 represents the f i r s t glutamine synthetase mutant reported to date in A. v i n e l a n d i i . This mutant was isolated by selecting for resistance to the glutamate analog MSX and was found to f i x nitrogen c o n s t i t u t i v e l y in the presence of ammonium and excrete ammonium to high l e v e l s into the external medium (Gordon et a l ., submitted for publication). JK301 had approximately 15% of wild type glutamine synthetase a c t i v i t y 1 40 when determined by a biosynthetic assay which measures glutamine production from glutamate and ammonium. Glutamine synthetase in c e l l - f r e e extracts of JK301 had a Km for glutamate of approximately 18 mM vs. 6 mM for JK. Previous studies (Kleinschmidt and Kleiner, 1978; Lepo et a l . , 1982) using p u r i f i e d glutamine synthetase from A. v i n e l a n d i i , have reported a Km for glutamate of approximately 1.1 mM - 1.7 mM A yet unexplainable observation i s that JK301 and JK have nearly the same glutamine synthetase a c t i v i t y when measured via the formation of 7-glutamylhydroxamate from glutamate and hydroxylamine (Table VIII). Interestingly, a MSX resistant mutant of S. typhimurium (Miller and Brenchley, 1981), which has only 13% glutamine synthetase a c t i v i t y of the wild type st r a i n when measured by a biosynthetic glutamine synthetase assay also has 7-glutamylhydroxamate synthesis a c t i v i t y equal to that found in the wild type s t r a i n . Conceivably, the similar 7 -glutamylhydroxamate synthesis a c t i v i t i e s may be due to another enzyme a c t i v i t y . Ehrenfeld et a l . (1963) have reported an enzyme in an Azomonas sp. which catalyses 7-glutamylhydroxamate synthesis from glutamate and hydroxylamine in a reaction which does not require ATP or Mg + +. Al t e r n a t i v e l y , the defect in glutamine synthetase from JK301 may not affect the a b i l i t y of the enzyme to catalyse 7-glutamylhydroxamate synthesis. The 3 fol d difference in the apparent Km for glutamate i s not l i k e l y due to an a l t e r a t i o n in the adenylylation state of the enzyme because Kleinschmidt and Kleiner (1978) have shown that the Km for glutamate does not change as a result of a change in 141 adenylylation state. Furthermore, the lower glutamine synthetase a c t i v i t y in JK301 i s not l i k l y due to an i n a b i l i t y to deadenylylate the enzyme because the r a t i o of the glutamine synthetase a c t i v i t i e s in nitrogen-limited cultures and ammonium grown cultures i s not s i g n i f i c a n t l y d i f f e r e n t in JK and JK301 (Table X). The presence of i n h i b i t o r s is not l i k e l y to be responsible for the lower glutamine synthetase a c t i v i t y in JK301 because a c t i v i t y was not restored when small molecules were removed from the c e l l - f r e e extract (Table IX). In addition, mixing experiments demonstrated that c e l l - f r e e extracts from JK301 did not have an in h i b i t o r y e f f e c t on JK extracts (Table VII). It remains possible that the defect in glutamine synthetase from JK301 i s partly regulatory. This conclusion arises from the fact that the Vmax for glutamine biosynthesis i s about 3 fo l d lower in JK301 than in JK. This could be interpreted as meaning the l e v e l of enzyme in • JK301 is lower than in JK suggesting that glutamine synthetase i s not f u l l y expressed in th i s s t r a i n . However, because the Vmax determination was made using c e l l - f r e e extracts as opposed to p u r i f i e d enzyme, th i s p o s s i b i l i t y remains speculative. A l i k e l y explanation for the lower a c t i v i t y in JK301 arises from the observation that glutamine synthetase from t h i s s t r a i n has a Km for glutamate 3 f o l d greater than JK. This could be interpreted to mean that the enzyme has an altered active s i t e for glutamate which results in an enzyme with a lower a f f i n i t y for glutamate. This would provide an explanation why JK301 i s 142 resistant to the inhibitory e f f e c t of MSX; the same defect which results in a lower a f f i n i t y for the natural substrate, glutamate, may result in an active s i t e unable to bind (or bind well) an analog of the natural substrate (in t h i s case, MSX). It i s conceivable that the same defect results in an enzyme with a lower maximum velocity as well. The lack of detectable N-methylglutamine synthesis a c t i v i t y in JK301 (Table VII) may indicate a low a f f i n i t y for glutamate and for methylammonium. The combined effects of a low a f f i n i t y for both substrates may prevent any reaction from occurring at a l l . This would suggest that enzyme from JK301 might have an altered Km for ammonium as well as glutamate. Although the Km for ammonium was not determined i t i s worth noting that the MSX-resistant mutant described by M i l l e r and Brenchley (1981) had an altered Km for both ammonium and glutamate. The defect in glutamine synthetase from JK301 offers an explanation of other phenotypic c h a r a c t e r i s t i c s of t h i s mutant. These include i n a b i l i t y to grow in N- free medium, excretion of ammonium into the external medium, and low levels of con s t i t u t i v e nitrogenase synthesis (Gordon et a l . , submitted). The i n a b i l i t y to grow in N-free media i s possibly due to an i n a b i l i t y to assimilate the low lev e l s of ammonium produced from N 2 f i x a t i o n . . Presumably, because the ammonium i s not assimilated i t i s excreted. A l t e r n a t i v e l y , the mutant may not grow when f i x i n g nitrogen because of very low glutamine pools as a result of lower glutamine synthetase a c t i v i t y . This may result in reduced glutamate pools as well since glutamine serves 143 as substrate for glutamate synthase mediated glutamate biosynthesis. Low glutamate pools would further antagonize glutamine synthesis by glutamine synthetase because of the higher Km for glutamate in JK301 enzyme. Under these conditions supplementing c e l l s with glutamate or glutamine did not restore growth, however, i t i s not known whether JK301 can assimilate these amino acids from the external medium. A. vinelandi i exhibits a chemotactic response to low levels of ammonium in swarm plates (Fig. 21). Chemotaxis toward ammonium has been previously reported for Pseudomonas  aeruginosa (Moench and Konetzka, 1977) and Bdellovibrio  bacteriovorus (Straley et a_l.,l979). An attempt was made to isola t e ammonium transport mutants by selecting for individuals unable to respond chemotactically towards ammonium. This approach has been used successfully in the i s o l a t i o n of mutants of P^ aeruginosa defective in membrane transport of glucose (Stinson et al.,1977). In t h i s study, one class of mutants isolated was unable to respond chemotactically towards acetate and sucrose as well as ammonium. This suggests a general i n a b i l i t y to respond chemotactically towards a variety of substrates rather than a s p e c i f i c i n a b i l i t y to respond to ammonium. It i s possible that these mutants were defective in some component of the chemotaxis sensing system other than proteins which share a role in ammonium transport. Another attempt to is o l a t e mutants was designed to avoid i s o l a t i o n of generalized non-chemotactic mutants, however, th i s method did not prove successful. In E. c o l i there are at least nine genes 1 44 involved in chemotaxis (not including genes involved in f l a g e l l a or binding proteins) (Koshland, 1979). If A. vi n e l a n d i i has a similar number of genes involved in chemotaxis, i t i s not surprising that generalized chemotaxis defective mutants were the only c l a s s of mutants i s o l a t e d . A preliminary examination of outer and inner membrane proteins was performed as part of an attempt to ide n t i f y membrane proteins involved in transport of ammonium ahd methylammonium. Outer membranes isolated from nitrogen-fixing c e l l s contained a 44 kilodalton protein present in greater amounts than in ammonium-grown c e l l s (Fig. 22). Page (1982) has shown that this protein i s hyperproduced under conditions of molybdenum starvation and has suggested that the protein i s part of a siderchrome mediated uptake system for molybdenum. Using one dimensional SDS-PAGE, the 44K protein was the only difference in major outer membrane proteins observed between ammonium-grown and nitrogen-fixing c e l l s . Further examination of outer membrane proteins from A. vinel a n d i i revealed the presence of a heat modifiable outer membrane protein (Fig. 25). A number of heat modifiable proteins have been observed in E. c o l i and P. aeroginosa (Bragg and Hou, 1972; Dirienzo et a l . , 1978; Hancock and Carey, 1979). The single heat modifiable outer membrane protein in A. vi n e l a n d i i had an apparent molecular weight of approximately 41 kilodaltons when s o l u b i l i z e d at 37°C in a solution containing 2% SDS and 5% 2-mercaptoethanol. When s o l u b i l i z e d at 100°C, the protein ran at an apparent molecular weight of approximately 49 1 45 kilodaltons. The higher apparent molecular weight of the protein when s o l u b i l i z e d at 100°C probably represents an increased asymmetry in the shape of the protein which results in slower migration through the g e l . Growth of A. vine l a n d i i in glucose or mannitol did not appear to induce any outer membrane proteins not found in sucrose grown c e l l s (Fig. 26). Glucose has been shown to induce an outer membrane protein in P. aeruginosa (Hancock and Carey, 1980) which may be part of a high a f f i n i t y glucose transport system. In A. v i n e l a n d i i however, growth on 2% glucose (Fig. 26) and 10 mM glucose (data not shown) did not induce additional major outer membrane proteins not found in sucrose grown c e l l s . Membranes from strains of A. vinelandi i r esistant to high levels of methylammonium (Gordon and Jacobson, in press) were examined to determine i f resistance could be attributed to membrane protein a l t e r a t i o n s . Examination of outer and inner membranes from three resistant strains revealed no obvious differences as determined by one dimensional SDS-PAGE (data not shown). In addition, the periplasmic proteins from JK and one methylammonium resistant mutant were not s i g n i f i c a n t l y d i f f e r e n t (Fig. 24). These results suggest that resistance to high levels of methylammonium in t h i s s t r a i n i s not manifested by alterations in major membrane or periplasmic proteins. It i s possible that methylammonium resistant strains have an alt e r a t i o n other than a protein modification, which renders the outer membrane less permeable to methylammonium. Al t e r n a t i v e l y , 1 46 resistance to methylammonium might be explained- by a modification in the a b i l i t y of the c e l l to metabolize methylammonium or in the a b i l i t y to transport methylammonium across the inner membrane. In summary, t h i s study provides evidence for the existence of an active transport system for ammonium in A. vinelandi i . Ammonium and methylammonium uptake appear to be dependent on the presence of a functional glutamine synthetase, however, at this point i t i s not clear how the enzyme i s involved in the uptake process. Methylammonium i s rapidly metabolized by glutamine synthetase to N-methylglutamine in vivo and in c e l l - f r e e extracts. JK301, a MSX-resistant mutant of A. v i n e l a n d i i , was unable to catalyse N-methylglutamine synthesis in vivo or in c e l l - f r e e extracts and lacked detectable methylammonium uptake a c t i v i t y . Glutamine synthetase from JK301 was found to have a Km for glutamate approximately three fold higher than enzyme from JK and a Vmax approximately four fold lower than enzyme from JK as measured in c e l l - f r e e extracts. 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