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Amino acid transport and pool formation in Pseudomonas aeruginosa Kay, William Wayne 1968

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AMINO ACID TRANSPORT AND POOL FORMATION IN PSEUDOMONAS AERUGINOSA by WILLIAM WAYNE KAY B.S.A. (Microbiology), University of B r i t i s h Columbia, 1963 M.Sc. (Microbiology), University of B r i t i s h Columbia, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Microbiology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1968 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I ag ree tha t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and S tudy . I f u r t h e r agree tha t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y pu rposes may be g r a n t e d by the Head o f my Department or by hiis r e p r e s e n t a t i v e s . It i s u n d e r s t o o d tha t c o p y i n g or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f M j c r o b j o l o g y The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada Date May 3 , 1 9 6 8 ABSTRACT Pseudomonas aerugihosa has been shown to actively transport and accumulate twenty common amino acids by systems with enzymatic properties; that is the systems are energy dependent, temperature sensitive, are saturated at high amino acid concentrations and are lost by mutation. During growth on a synthetic, amino acid free medium this microorganism maintained a low, but s i g n i f i c a n t l y concentrated heterogeneous pool of amino acids for syntheses and this pool (native pool) was found to be in equilibrium with low levels of exogenous amino acids with at least one exception. Amino acid pools established from an exogenous source were found to behave d i f f e r e n t l y . Whereas some amino acids were unchanged during the passage through the i n t r a c e l l u l a r pool others underwent extensive degradation. Some amino acids or their degradation products were shown to be compartmentalized or made unavailable for metabolism. Proline did not form large pools under physiological conditions due to an imbalance between the rate of transport and the rate of protein synthesis. A m u l t i p l i c i t y of i n t r a c e l l u l a r proline pools was elucidated by inhibitors and studies at low temperatures. The amino acid transport systems operative at very low exogenous amino acid concentrations were shown to be strongly stereospecific. Several transport systems were elucidated by competitive i n h i b i t i o n studies and were found to recognize amino acids with si m i l a r chemical properties. Also very s p e c i f i c amino acid transport systems were demonstrated within the aromatic and basic amino acid families. The m u l t i p l i c i t y of amino acid c a r r i e r functions was confirmed by pool displacement studies and by the selection of appropriate transport negative (Tr ) mutants. Low a f f i n i t y amino acid permeases or carriers were shown to operate at high amino acid concentrations for most of the amino acids tested. Low and high a f f i n i t y permeases could be separately id e n t i f i e d by k i n e t i c studies. Amino acid transport was found to be induced to high levels by growth in the presence of the appropriate amino acid. Some evidence was presented to suggest that the control is coordinately linked to amino acid degradative enzymes. The constitutive levels of amino acid degradative enzymes were found to be lowered in the presence of glucose. With the exception of arginine, constitutive deaminases were inhibited by inorganic ammonia, whereas for the most part the constitutive transport functions were not changed. Induced transport levels were not markedly influenced by the presence of these nutrients. A novel mechanism for the transport and accumulation of amino acids was formulated. This mechanism provides for the accumulation of high and low i n t r a c e l l u l a r amino acid pools by an energy dependent mechanism. i v TABLE OF CONTENTS Page INTRODUCTION • . • . LITERATURE REVIEW . . . . • . . . . 2 I. Accumulation of Amino Acids . . . .• . .• . . . 2 II. The Transport of Amino Acids by Microorganisms. . . 6 I I I . Control of Amino Acid Transport. 7 IV. Amino Acid Transport in Microorganisms other than Bacteria 8 V. Location and Isolation of Transport Functions . . . 12 MATERIALS AND METHODS I. Organisms and Media 14 II . Preparation of Cell Suspensions. 15 I I I . Selection of Mutants . . 1 5 1. Mutagenesis 15 2. Amino acid auxotrophs . 16 3. Transport negative strains 16 IV. Uptake of Label led Amino Acids . . . . . . . . 17 V. Competitive Inhibition of Amino Acid Transport. . . 1 8 VI. Competitive Pool Displacement 19 VII. Chromatographic Techniques and Measurement of Amino Acids in the Pool and in the Culture Medium. . . . 19 VIII. Amino Acid Composition of C e l l u l a r Protein . . . . 21 IX. Chemical Fractionation of Whole C e l l s . . . . . . 22 X. Chemicals . 2 3 Table of Contents (Continued) Page RESULTS AND DISCUSSION 2k I. General Properties of Amino Acid Transport . .2k 1. Uptake of amino acids 2k 2. Precursor-pool relationships. . . . . . 2k 3. Comparative rates of transport 28 k. Distribution of radioactive amino acids in the eel 1 31 5. Energy requirement for amino acid incorporation 33 6. Effect of temperature on the rate of amino acid transport 36 7. Dependence of the rate of transport on substrate concentration 36 8. Concentration of amino acids in the i n t r a c e l l u l a r pool 38 9. Amino acid composition of the c e l l u l a r protein . kS I I . General Properties of Pool Formation and Ma i ntenance kb 1. Time course of pool formation . . . ke 2. The fate of intracel 1ular arginine. . . . 5 8 3. Pool formation and maintenance 72 a. Formation 72 b. Maintenance 7k vi Table of Contents (Continued) Page 4. Pool m u l t i p l i c i t y . 76 I I I . S p e c i f i c i t y of the AminoAcid Uptake Systems 83 1..Competition for amino acid uptake . . . . 8 3 • •2.. Kinetics,of competitive i n h i b i t i o n . . . . 88 3. Specific and general transport systems 89 a. Basic amino acids. . 93 b. A l i p h a t i c amino acids . . . . . . . 96 c. Aromatic amino acids . . . . . . . 99 4. The isol at ion and properti es of transportless mutants . 101 IV. Competition for the Amino Acid Pool. . . . . 108 V. Kinetics of Amino Acid Transport at High Substrate Concentrations. . . . . . . 1 1 4 VI. Control of Amino Acid Transport 116 VI I. Amino Acid Transport and Pool Formation in Starved Cell Suspensions . . . *. 120 1. Ami no aci d transport 120 2. Pool formation and maintenance during carbon or nitrogen starvation. .• . . . . 131 a. Time course of amino acid incorporation . . . 131 b. Fate of the amino acid pool 137 Table of Contents (Continued) Page VIM. The Mechanism of Amino Acid Transport and Accumulation 139 GENERAL DISCUSSION . 165 BIBLIOGRAPHY 170 vi i i LIST OF TABLES Page Table I. Rates of transport of amino acids into whole c e l l s of JP. aeruginosa; 32 Tab<le I I. Distribution of radioa c t i v i t y in the c e l l fractions of P. aeruginosa grown in the presence of ^C-amino acids 34 Table I I I . Composition of the amino acid pool and culture supernatant f l u i d of P_. aeruginosa during logarithmic growth 42 Table IV. Composition of the eel 1ular protein of P. aerug?nosa 47 14 Table V. Chromatographic analyses of C-amino acid pools 53 Table VI. L a b i l i t y of the putrescine pool 63 Table VII. Competitive i n h i b i t i o n of basic amino acid uptake 94 Table VIII. Competitive i n h i b i t i o n of a l i p h a t i c amino acid uptake 97 Table IX. Competitive i n h i b i t i o n of aromatic amino acid uptake 98 Table X. Competitive i n h i b i t i o n of neutral amino acid uptake 100 Table XI. Amino acid permeases of P.. aeruginosa 102 Table XI I. Transport negative (Tr ) mutants of P_. aerug ? nosa 106 Table XIII. Exchange of the a l i p h a t i c amino acid pools 110 Table XIV. Exchange of the preformed basic amino acid pools 111 Table XV. Exchange of the aromatic amino acids 113 Table XVI. Growth of P. aeruginosa on amino acids as carbon or nitrogen sources 121 Jx L i s t of Tables (Continued) Table XVII. Effect of carbon deprivation on amino acid transport 123 Table XVIII. Fate of ^C-amino acids incorporated into nitrogen starved c e l l s of P. aeruginosa 135 Table XIX. Fate of ^C-amino acids incorporated into nitrogen starved c e l l s 136 X LIST OF FIGURES Page Fig. 1. Increase in the rate.of proline uptake with increasing c e l l mass. 26 Fig. 2. Time course of tyrosine uptake and incorporation into protein by a growing suspension of IP. aeruginosa 27 14 Fig. 3. Incorporation of C-valine into the protein of P_. aeruginosa . 29 14 Fig. 4. Total uptake of C-glutamate and incorporation into protein of c e l l suspensions 30 Fig. 5. Effect of sodium azide and sodium azide plus^iodoacetamide on the active transport of C-proline 35 -6 14 Fig. 6. Rate of 1 x 10 M C-valine incorporation into whole c e l l s of P. aeruginosa as a function of temperature 37 Fig. 7. Saturation kinetics of phenylalanine incorporation by P_. aeruginosa 39 Fig. 8. Radioautograph of the nitrogen pool extracted from c e l l s growing logarithmically in the presence of U- C-glucose 41 -6 14 Fig. 9. Early time course of 1 x 10 M C-alanine uptake by a growing suspension of P_. aeruginosa 43 14 Fig. 10. Patterns of C-amino acid uptake into whole c e l l s 49 Fig. 11. Radioautograph of the amino acid pool established with C-isoleucine 50 Fig. 12. Radioautograph of the amino acid pool established with C-glutamate 51 Fig. 13. The i n h i b i t i o n of growth in minimal media as a function of the chloramphenicol (CM) concentration 52 xi L i s t of Figures (Continued) Page' Fig. 14. Effect of chloramphenicol on the rate of C-proline incorporation into whole c e l l s and eel 1 fractions of P. aeruginosa 56 Fig. 15. Effect of temperature on the rate of C-proline incorporation into whole c e l l s and c e l l fractions of f_. aeruginosa 57 Fig. 16. Time course of ^C-p r o l i n e uptake into whole c e l l s and eel 1 fractions of P_. aerug ? nosa 59 Fig. 17. The formation^and maintenance of the pool derived from C-arginine (2 x 10"!' M) by a growing culture of P. aeruginosa 60 Fig. 18. The displacement of the C-pool derived from 2 x 10~5 M ^C-arginine by 1 2C- arginine (2 x 10"'' M) or C-putrescine (2 x 10"3 M) added 6 min after the i n i t i a l 1^C-arginine 61 14 Fig. 19. The incorporation of C-arginine into c e l l fractions of P. aerug j nosa poi soned with 2 mg/ml chloramphenicol. 65 14 Fig. 20. P a r t i a l e f f l u x of the C-arginine C-putrescine pool in the presence of 30 mM NaN^ 66 Fig. 21. The exchange of the ^ C-arginine ^C- putrescine pool by 2 x 10~3 M ^ - a r g i n I n e 67 Fig. 22. Time course of ^C-arginine incorporation into c e l l fractions of P. aeruginosa and the disappearance of exogenous label 69 14 Fig. 23. The time course of C-arginine incorporation into whole c e l l s and c e l l fractions of P_. put i da and P_. f 1 uorescens 71 Fig. 24. Maximum pro!ine pool obtained at 10. C with 10~6 M ^C-pr o l i n e by f_. aeruginosa P22 growing in minimal medium 73 x i i L i s t of Figures (Continued) Page Fig. 25. S p e c i f i c i t y of.maintenance of the proline pool in P. aerugihosa P22. 75 Fig. 26. Exchange of a preloaded ^C-prol?ne pool of P. aeruginosa P22 with 10~ 4 M 1 2C- proline added at 60 min 77 Fig. 27. Efflux of a preformed leucine pool in the presence of 30 mM NaN, and 1 mM iodo acetamide 78 Fig. 28. P a r t i a l e f f l u x of the i n t r a c e l l u l a r proline pool of P. aerugihosa on the addition of 30 inM NaN^ . 79 Fig. 29. Efflux of the proline pool of P_. aeruginosa at 0 C 81 Fig. 30. Efflux of the proline pool of P_. aerugihosa at 5 C and i t s re- establishment at 10 C 82 Fig. 31. Competition for amino acid uptake in P_. aerug ? nosa 85 Fig. 32. Competition for a l i p h a t i c and aromatic amino acid uptake in P_. aeriigihosa 86 Fig. 33. Competitive i n h i b i t i o n of leucine uptake by C-valine 90 Fig. 34. Competitive i n h i b i t i o n of ^ C - l y s i n e uptake by C-arginine 91 Fig. 35. Competitive i n h i b i t i o n of ^^-phenyl alanine uptake by C-tyrosine 92 14 Fig. 36. .Inhibition of C-lysine uptake by C-arginine 95 14 + Fig. 37. Uptake of C-proline at 30 C by W and proline Tr" mutant strains of P_. aeriigi hosa 104 14 Fig. 38. The displacement of the C-valine pool by s t r u c t u r a l l y related amino acids 104 XI I I L i s t of Figures (Continued) Page Fig. 39. Kinetics of glutamate uptake a t ^ 0 C with varying concentrations of C- glutamate 115 Fig. 40. Time course of pool formation at 30 C with P. aeruginosa previously grown in minimal medium in the presence or absence of O'.U proline 118 Fig. 41. Kinetics of pro! ine uptake in P. aeruginosa grown in the presence of absence of 0.1% p ro1i ne 119 Fig. 42. The change irt ^ C-glutamate transport in P. aeruginosa during nutrient deprivation 124 14 Fig. 43. The change irt C-va1ine transport in P. aeruginosa during nutrient deprivation 125 Fig. 44. Rate of transport of ^ C - v a l i n e into whole c e l l s of P_. aeruginosa 126 Fig. 45. Proline transport in P_. aeruginosa P22 during carbon starvation 127 14 Fig. 46. Uptake of C-proline into whole c e l l s and protein of P_. aeruginosa during carbon starvation 129 14 Fig. 47. Uptake of C-proline into P_. aeruginosa c e l l s and c e l l fractions 130 14 Fig. 48. Time course of C-phenylalanine uptake into c e l l s and c e l l fractions during nutrient deprivation 132 14 Fig. 49. Time course of C-proline uptake in c e l l suspensions of P_. aeruginosa P22 during nutrient deprivation 133 Fig. 50. Model for an energy-coupled active transport system (designated as inside or outside the c e l l ) 141 Fig. 51. The c a r r i e r model for amino acid transport 143 xiv L i s t - o f Figures (Continued) Page Fig. 52. The uptake of 2 x 10 M C-proline at 30 C into NaN. treated and untreated c e l l s of wild-type and proline Tr" mutant (P5) f_. aeruginosa . 145 Fig. 53. Lineweaver-Burk plot of proline incorpo ration into c e l l s of P_. aeruginosa 147 14 Fig. 54. The incorporation of C-proline (10" M) into the pool of induced and non-induced c e l l s of P_. aeruginosa P22 at 10 C 148 • -5 ' 14 Fig. 55. Uptake of 2 x 10'.? M C-proline into NaN, treated and untreated c e l l s of previously grown P_. aeruginosa 150 Fig. 56. Lineweaver-Burk plot of proline uptake into induced and non-induced c e l l s of P_. aeruginosa 152 Fig. 57. The r a t i o of i n t r a c e l l u l a r to extra c e l l u l a r valine concentration as a function of the exogenous valine concentration 153 Fig. 58. The effect of the exogenous valine concentration on the valine pool size (double negative reciprocal log-log plot) 156 14 Fig. 59. Rate of e f f l u x of C-valine as a function of the i n t r a c e l l u l a r valine pool size 158 Fig. 60. Double c a r r i e r model for amino acid transport in P. aerugihosa 162 ACKNOWLEDGEMENTS I would f i r s t l i k e to express my sincere gratitude to Dr. A.F. Gronlund for her encouragement, d i r e c t i o n , and constructive c r i t i c i s m of both the research and writing of this thesis, and for her exhaustive editing of the manuscript. I would also l i k e to thank Dr. J.J.R. Campbell for his encouragement and interest and for editing the thesis. I would l i k e to thank Dr. R.A.J. Warren for his kind interest and his helpful suggestions concerning mutagenesis and selection of bacterial mutants and for other aspects of this work. Certainly not the least of my gratitude is extended to my wife Judith for her tolerance, forbearance and enthusiasm during the research and especially for her typing and exhaustive editing of this thesis. Lastly my thanks also go to my fellow-students, especially N. Medveczky, for aiding in my experiments, and to Mrs. Rita Rosbergen.for the typing of this f i n a l manuscript. INTRODUCTION The c e l l membrane has long been known to be se l e c t i v e l y permeable to a great variety of low molecular weight metabolites. The degree of permeability and s e l e c t i v i t y d i f f e r s markedly however, with different l i f e forms. Bacteria not only exhibit the properties of selective permeability but also are able to concentrate metabolites to a great extent from the external environment. In recent years investigators have t r i e d to elucidate the biochemical mechanisms involved in both the processes of s e l e c t i v i t y and accumulation common to most active transport systems. It was the object.of this investigation to study the transport and accumulation of amino acids in Pseudomonas aeruginosa, a microorganism which does not rely on exogenous amino acids for growth but is able to catabolize most ami no acids as a source of carbon or nitrogen. 2 LITERATURE REVIEW The permeability of c e l l s to low molecular weight metabolites such as amino acids has long been recognized as an important e e l l u l a r function. This preliminary step to metabolism could profoundly influence c e l l growth, the rate of substrate u t i l i z a t i o n , enzyme induction or repression and the feedback i n h i b i t i o n of biosynthetic enzymes. Consequently, the transport and accumulation of amino acids by microorganisms has received much attention in recent years and several excellent reviews have been written on this subject (Holden, 1962; Kepes and Cohen, 1362; Britten and McLure, 1962; Wilbrandt, 1963; Quastel, 1964). I. ' Accurhul at ion of Amino Acids The f i r s t observation that selective amino acid accumulating systems existed in microorganisms was made by E.F. Gale (1947). Gale showed that amino acids entered Staphylococcal c e l l s , not by a process of simple d i f f u s i o n , but rather by a uni-directional transfer mechanism which in the case of some ami no acids, requi red a metabolizable energy source. These amino acids were then concentrated into what Gale termed "amino acid pools". The properties of the systems which transport amino acids into microorganisms received l i t t l e attention un t i l the problem was reinvestigated by using labelled amino acids to follow the uptake 3 of these compounds into c e l l suspensions (Cohen and Rickenberg, 1956). The investigators at .the Pasteur Institute showed that Escherichia col? K12 accumulated amino acids by several d i s t i n c t stereospecific, energy-dependent systems, which could maintain i n t r a c e l l u l a r amino acid concentrations up to 500 times that of the external medium and which could also function in the absence of protein synthesis. As a result of their work a model - essent i a l l y the same as the model described for carbohydrate transport (Rickenberg, Cohen, Buttin, and Monod, 1957) - was postulated to describe the sequence of events mediating amino acid accumulation in E. c o l i . This model was subsequently modified by Kepes . (1'960), for carbohydrate transport, to include not only a stereospecific enzyme-like permease but also a less s p e c i f i c "transporter" for movement of metabolites across the c e l l membrane. At approximately this same time, investigators at the Carnegie Institute (Britten and McLure, 1962) developed a sensitive M i l l i p o r e f i l t r a t i o n technique to follow the rapid accumulation of amino acids by E. c o l i . These workers found several d i s t i n c t amino acid accumulating systems in E. c o l i and compiled a most comprehensive description of the properties of these amino acid transport systems. They found that many properties of amino acid pool behaviour were not commensurate with the permease model proposed by the workers at the Pasteur Institute. As a res u l t , an alternate model - the " c a r r i e r " model - was proposed to explain their observations of amino acid transport and pool maintenance. These workers also considered in some d e t a i l , the osmoregulation of the amino acid pools and found that maintenance and establishment of i n t r a c e l l u l a r amino acid pools were i n t r i n s i c a l l y affected by the osmolarity of the external medium. Some amino acid concentrating systems in E_. col i have been shown to be subject to loss by mutation. Schwartz, Mass and Simon (1959), isolated mutants of E. co1i W resistant to the arginine analogue, canavanine, or to the glycine analogue, D-serine. These mutants were subsequently found to be unable to concentrate the respective natural amino acids. Although the accumulation process for amino acids in microbial c e l l s has been shown to have r i g i d structural requirements, to obey saturation kinetics (Holden, 1962), and to be subject to loss by mutation, recent evidence has suggested that these observations may be secondary consequences of the a c t i v i t y of s p e c i f i c amino acid recognition sit e s or "permeases" residing at the c e l l membrane surface. By selecting E_. col i W auxotrophs which were unable to grow fn the presence of low concentrations of the required amino acids, Lubin, Budreau and Gross (1962), isolated several mutants defined as transport-negative (Tr ), which were linked to permease mutants of the B-galactoside system. These workers demonstrated that under conditions in which the primary means of entry of proline was by diffu s i o n (low temperature, high external concentration, or presence of 2 , 4-dinitrophenol), the rates of proline entry into both the wild type and mutant c e l l s were almost i d e n t i c a l . From comparative studies with the Tr mutant and the parent s t r a i n , these workers have argued against the "binding-site theory" of Britten and McLure (1962). Kessel and Lubin (1962), demonstrated that the process of exchange at 0 C was markedly reduced in the Tr s t r a i n , thereby firmly establishing a relationship between uptake and exchange processes. Kaback and Stadtman (I966), corroborated the observations of Kessel and Lubin 5 by carrying out experiments with isolated whole membrane preparations from both wild type (W6) and a proline transportless mutant (W157). Membranes from the wild type were able to incorporate and concentrate proline, whereas membranes from the mutant were unable to carry out these functions. In addition, membranes from the mutant were unable to exchange accumulated proline with exogenous proline at 0 C. Kessel and Lubin (1962), suggested that the mechanisms for proline uptake and exchange appeared to be closely related. Tristram and Neale (1968), selected a number of 3,4-dehydroproline and azetidine-2-carboxylic acid resistant strains of E; cOli K10, which had defective transport mechanisms. It was found that the rate of exchange at 0 C between labelled i n t r a c e l l u l a r and unlabel led e x t r a c e l l u l a r proline was independent of the i n i t i a l - 7 - i , external proline concentration in the range of 10 to 7.5 x 10 M. At 10 C the maximum i n t r a c e l l u l a r proline concentration was dependent upon the external proline concentration. This suggested that the pool size was a function of permease a c t i v i t y but that exchange at 0 C was not. However, a direct correlation was demonstrated between the abi1ity of proline analogues to i n h i b i t proline uptake and the a b i l i t y to affect change. Also, an azetidine-resistant s t r a i n , with a diminished proline permease, lacked the a b i l i t y to.exchange i n t r a c e l l u l a r and e x t r a c e l l u l a r proline, 3,4-dehydroproline or azetidine. This suggested that uptake and exchange are in fact related processes ih E. co1?. As the previous workers have shown, the systems of amino acid accumulation in micro organisms are exceedingly complex and recently no attempts have been made to further elucidate the proposed models or to formulate new 6 models. Currently, the attention of most investigators has been focused on a singular aspect of amino acid uptake systems, namely the s t e r e o s p e c i f i c i t y of the transport process. II . The Transport of Amino Acids by Microorganisms FerroLuzzi-Ames (1964), investigated the aromatic transport system of Salrhbhel la typhirhurium and from kin e t i c analyses of their system demonstrated the existence of both s p e c i f i c and non-specific permeases for these amino acids. Specific, h i g h - a f f i n i t y permeases, as well as a non-specific, low-affinity general permease, were found for phenylalanine, tyrosine, tryptophan and h i s t i d i n e . These results were confirmed by the i s o l a t i o n of Tr mutants of-S_. typhi murium deficient either in the general or in the h i s t i d i n e s p e c i f i c permease ( S h i f r i n , Ames, FerroLuzzi-Ames, 1966). Although most studies have indicated that bacterial amino acid-incorporating systems are s t r i c t l y stereospecific, recognizing only s t r u c t u r a l l y related amino acids, recent studies with Agrobacterium tiimefaciens have indicated that general non-specific permeases exist in this microorganism. Behki and Hochster (1966, 1967), compared both the valine and proline uptake systems in tumorogenic and non-tumorogenic strains of A. tumefaciens. The general properties of the accumulation process and the r i g i d structural requirements in the non-tumorogenic s t r a i n , with valine, were similar to those observed in Ei cb1i. The tumorogenic s t r a i n , however, displayed both an independence from an external energy requirement and a non-specific competition for transport by s t r u c t u r a l l y unrelated amino acids. Both 7 strains showed a marked lack of structural s p e c i f i c i t y for the proline incorporating system, as a number of unrelated amino acids inhibited the uptake of this amino acid. I I I . Control of Amino Acid Transport Halpern and Lupo (1965), investigated the glutamate transport of E. col?, strains H and K12, in a novel manner. These workers obtained mutants which, unlike the parent st r a i n s , could grow on glutamate as the sole carbon source. The mutation resulted in increased a b i l i t y of these c e l l s to transport glutamate and i t s analogues. Extensive ki n e t i c analyses of this phenomenon (Halpern and Even-Shoshan, 1967), led to the demonstration that the glutamate permease of E. c o l i was a l l o s t e r i c a l l y inhibited by glutamate. It was found that the s t r u c t u r a l l y similar compounds, a-ketoglutarate and aspartate, non-competitively inhibited transport a c t i v i t y . The authors suggested that these compounds also bind to the a l l o s t e r i c s i t e . Subsequently, Marcus and Hal pern (1967), mapped the gene determining glutamate transport (git C) in E. c o l i K12. The important observations that the transport gene was genetically similar to the glutamate decarboxylase gene (gad), and that the g i t C gene was probably a regulator gene for this uptake system, were made. Since both parent and mutant strains had sim i l a r a f f i n i t i e s for the substrate, the authors suggested that the glutamate permease was under genetic control, being partly repressed in the wild-type and derepressed in g i t C + mutants. There are only a few reports in the l i t e r a t u r e of inducible 8 transport systems. Bbezi and DeMoss (1961), demonstrated an inducible tryptophan transport system in E. cbl ? T3A, however, the induction inexplicably required the presence of casamino acids, indicating that induction by a singular inducer was not the mechanism. These workers demonstrated that amino acid transport systems, including the tryptophan transport system, were inhibited by pyruvate and stimulated by formate, thereby suggesting that the intermediary metabolism of the organism regulated the a c t i v i t y of the transport process. An inducible glutamate transport system in Mycobacterium tuberculosis and M. smegmatis has been demonstrated (Lyon, Rogers, Hall and Lichstein, 1967). It was found that glutamate-catabolizihg enzymes were constitutive in these organisms and that the oxidative lags exhibited by resting c e l l s were due to the induction of a s p e c i f i c glutamate permease. Inui and Akedo (1965), reported the f i r s t concrete evidence for the repressibi1ity of amino acid transport systems in E. cbl ? K10. These workers found that the uptake of cycloleucine and leucine was reduced when c e l l s had previously been grown in a medium containing cycloleucine, L-leucine or L-methionine, while other amino acid transport systems were unaffected. Kinetic studies of repressed c e l l s indicated that both the rate of influx and e f f l u x were altered. IV. Amino Acid Transport ?n Microorganisms Other Than Bacteria The mold Neurospora crassa and the yeast Saccharomyces cerevis iae show extensive s i m i l a r i t i e s to mammalian systems with respect to c e l l permeability. Many observations have suggested a lack of s p e c i f i c i t y 9 of amino acid transport and thus demonstrate the resemblance of the fungal to the mammalian systems. Recently, however, contrary evidence has been accumulating which indicates a m u l t i p l i c i t y of amino acid transporting systems both in Neurospora sp. and yeasts. Zalokar ( 1 9 6 1 ) , studied proline uptake ih N. crassa mycelial mats and demonstrated saturation kinetics for the uptake of this amino acid, indicating that a permease-1ike function existed. The kinetics and energy requirements for phenylalanine uptake into conidial suspensions were subsequently demonstrated by DeBusk and DeBusk ( 1 9 6 5 ) . However, competition studies between phenylalanine and twenty-three other amino acids showed that there existed a lack of s p e c i f i c i t y of the uptake process for phenylalanine. Lester ( 1 9 6 6 ) , found that mutants of N. crassa resistant to 4-methyl-tryptophan were defective not only in the transport of tryptophan and i t s methyl-analogues but also in the transport of a number of other unrelated amino acids. This implied a lack of s p e c i f i c i t y for the amino acid uptake systems. Stadtler ( 1 9 6 7 ) , q u a l i t a t i v e l y corroborated these results but also found that revertants of transport negative mutants resistant to 4-methyltryptophan consequently recovered the a b i l i t y to actively transport aromatic amino acids. These results were most simply explained by the occurrence of supressor mutations in another gene controlling a different transport system. It was suggested that the second mutation had modified a different uptake system, resulting in the expansion of i t s substrate range. Subsequently this second system was shown to govern the transport.of basic amino acids as had been demonstrated previously by Bauerle and Garner ( 1 9 6 4 ) . They found that N. crassa incorporated arginine, lysine, canavan-ine, 10 and c i t r u l l i n e with respectively decreasing a f f i n i t i e s for a single basic amino acid permease. Wiley and Matchett (1967), showed that N. crassa a c t i v e l y transported tryptophan by a stereospecific transport system which was s p e c i f i c for a family of neutral amino acids that were not necessarily s t r u c t u r a l l y related. These investigators found that an a-amino group next to a carboxyl group and an uncharged side chain were the minimal structural requirements for a c t i v i t y with the transport s i t e . It is important to r e a l i z e , as Kappy and Metzenberg (1967) have emphasized, that general transport defects can occur, not only through an a l t e r a t i o n of s p e c i f i c or general uptake systems, but also by membrane alterations affecting the a c t i v i t y of membrane-associated proteins such as permeases. These authors isolated a temperature conditional lethal mutant of N. crassa which was resistant to neutral amino acid analogues by virtue of a decreased a b i l i t y to transport these analogues and their natural cogenera across the c e l l membrane. They also showed, by several c r i t e r i a , that this mutant l i k e l y possessed a structural membrane defect which could account for the negative amino acid permeability. Hoi den (1965), showed that a defect in glutamate transport by a vitamin Bg requiring s t r a i n of Lactobacillus plantarum could be restored by the addition of acetate, ammonium ions, and vitamin Bg. It was suggested that the vitamin influences transport i n d i r e c t l y , either by modifying the t e r t i a r y structure of membrane components or by participating in their synthesis. Studies on the amino acid transport systems of the yeast Saccharorhyces cerevisiae by Surdin (1965), suggested that the substrate s p e c i f i c i t y of transport was considerably less than that observed with 11 bacterial systems. It was shown that although the accumulation "system could concentrate amino acids up to 1000 f o l d , a l l of the amino acids studied were concentrated by a single permease but at widely varying rates. Surdin also isolated a mutant with a ten fold lower level of permease and found that the s p e c i f i c i t y properties of this residual permease are essenti a l l y the same as those of the parent s t r a i n . Recently, conf1icting evidence has accumulated regarding the s p e c i f i c i t y of yeast amino acid permeation. Maw (1963), studied the 35 incorporation of S-labelled amino acids into brewers' yeast and found that the accumulation of the sulfur amino acids was inhibited by certain other amino acids having a close structural relationship to them. Grenson (1966), postulated that in S. cerevisiae both general and sp e c i f i c amino acid permeases were operative. Grenson found that only those am|no acids that were s t r u c t u r a l l y related to arginine would compete for that particular transport mechanism and the other naturally occurring amino acids had absolutely no effect on the incorporation of arginine. It was also shown by ki n e t i c analysis, that i s , the comparison of K and K. values, that the inhibitory amino acids were being m l ' 7 transported by the same mechanism as arginine. These results were confirmed by the selection of a transportless mutant common for the competitive amino acid i n h i b i t o r s . However, i t should be noted that the i r mutant recovered the a b i l i t y to transport arginine, without reversion, when ammonium ions were removed from the medium. It was suggested that this transport system was under metabolic control. Another mutant selected in this study was similar to the mutant isolated by Surdih et a l . (1965)> in that i t was affected at the level of general amino acid permeation. Grenson, however, objected to the conclusions 12 of Surdin e_t aj_., primarily because of the ten fold difference between the Km for transport of an amino acid and the apparent Michael is constant for the same amino acid when acting as an inhibitory agent (Kj) in the transport of another amino acid. Grenson (1966), has also demonstrated the existence of a s p e c i f i c lysine permease in yeast which doesnot recognize other basic amino acids. A s p e c i f i c lysine transportless mutant was isolated which did not affect the uptake of other basic amino acids. V. Locali zation and Isolation of Transport Functions Recently some progress has been made in the actual biochemical i d e n t i f i c a t i o n of the components of the active transport system. Kaback and Stadtman (1966), reported that carefully isolated membrane preparations from E. col 1 were able to acti v e l y incorporate and concentrate proline. In a comparative study with the wild-type (W6) and transportless strains (WI57) isolated by Lubin e_t aj_. (1962) , these workers found that the uptake process in the wild type c e l l membranes demonstrated saturation k i n e t i c s , whereas the amino acid passed through the mutant membranes only by simple d i f f u s i o n . Neu and Heppel (1965), and Nossal and Heppel (1966), showed that cold osmotic shock treatment of bacteria caused the loss of certain enzymes and proteins associated with the c e l l envelope. Piperno and Oxender (1966), used this procedure to isolate and purify a protein from El c o l i which would bind either leucine, isoleucine, or valine. The dissociation constants for the leucine and the isoleucine protein complexes were found to.be indistinguishable from their respective values for transport into whole c e l l s . These results suggest that the binding protein is a component of the transport system responsible for the concentrative uptake of leucine, isoleucine, and valine by E. c o l i KI2. A l l attempts to restore the transport process to shocked c e l l s by adding back the binding protein were unsuccessful. MATERIALS AND METHODS I. Organisms and Media Pseudomohas aeruginosa ATCC 9027 was used throughout most of this study. Also the following organisms were used: a h i s t i d i n e - requiring s t r a i n of P. aeruginosa WK4; P. aeruginosa P5 and P 6 , proline transport negative st r a i n s ; P. aeruginosa A5, IB9 and IBI0, TA3 and TA10'. which were arginine, isoleucine, and tyrosine transport negative s t r a i n s ; and P 2 2 , a str a i n unable to catabolize proline. Stock cultures were maintained at 6 C on ammonium salts minimal agar slants with glucose as the sole carbon source. Histidine (50 ug/ml) was added to.maintain WK4, the hi s t i d i n e auxotroph. Cells required for experimental procedures were grown from a 20 hr inoculum in Roux flasks at 30 C in a medium containing 0 .3% NH^PO^, 0.2% J^HPO^ and 0.5 ppm iron as FeS0^.7H20 at pH 7 . 4 , and supplemented with 50 yg/ml h i s t i d i n e for WK4. Glucose and MgS0i(.7H20 were added separately, after s t e r i l i z a t i o n , from 10% stock solutions to give f i n a l concentrations of 0.2% and 0.05% respectively (minimal medium). Cultures were routinely checked for purity and for production of the species char a c t e r i s t i c pigment pyocyanin by streaking c e l l s on King's medium (King, Ward and Raney, 1 9 5 4 ) . WK4 was routinely checked for reversion by streaking c e l l s on minimal medium. Transport negative strains were checked for reversion by streaking c e l l s on agar medium containing the appropriate amino acid as a sole carbon source. I I. Preparation of Cel1 Suspensions. Cells from late logarithmic phase were harvested by centrifugation at room temperature and were quickly resuspended to the desired concentration in minimal medium. The concentration of c e l l s was established by measuring the optical density at 650 my using a Model B Beckman spectrophotometer. The c e l l suspensions were s t i r r e d on a Troemner :submergeable magnetic s t i r r e r (Henry Troemner Inc., Philadelphia, Pa.) for 10 min prior to the addition of the radioactive amino acid. This procedure was found to give reproducible incorporation data for any one amino acid. I I I . Selection of Mutants I. Mutagenes i s Wild type logarithmic phase c e l l s were harvested from minimal medium by centrifugation at 13,000 x g for 10 min, resuspended in pH 5.5 c i t r a t e buffer to a density of approximately g 10 c e l l s per ml, and shaken at 30 C for 1 hr. N-methyl-N-nitro- N'-nitrosoguanidine was added to give a f i n a l concentration of 100 yg/ml and the c e l l s were shaken for kO min. The culture was quickly c h i l l e d to 0 C and centrifuged at 6 C. The c e l l s were washed twice with minimal medium, then resuspended to the o r i g i n a l volume in supplemented media to enrich for the desired mutant and then incubated at 37 C for 2-6 hr. 2. Amino acid auxotrophs The mutagenized culture was incubated for 6 hr in minimal medium supplemented with 50 yg/ml of the required amino acid. The c e l l s were diluted and plated d i r e c t l y on minimal medium containing 1 yg/ml of the desired amino acid. The plates were incubated for 48 hr and auxotrophs were detected as single minute colonies. These isolates were checked by patching on both minimal agar and minimal agar supplemented with 50 yg/ml h i s t i d i n e . 3. Transport negative strains Mutagenesis was carried out on c e l l s which had been f i r s t adapted to grow on the desired amino acid as the sole carbon source. After washing with medium the treated c e l l s were grown in minimal medium for 2 hr, then plated on s o l i d medium with 0.1% of the amino acid as the sole carbon source. After a 48 hr incubation, minute colonies were picked, washed in drops of saline, and patched onto both amino acid minimal and glucose minimal plates. After further incubation and daily scoring, those colonies which grew slowly on amino acid minimal plates and normally on glucose minimal plates were selected for further studies. These selected colonies were grown in minimal medium and amino acid uptake experiments were 14 carried out to determine the i n i t i a l (30 sec) rate of C-amino acid incorporation at a concentration of 10 ^ M. Growth curves on the mutants with defective transport systems were carried out to make sure no other genetic function had been altered. IV. Uptake.of Labelled Amino Acids 14 The incorporation of C-amino acids into c e l l s and c e l l u l a r constituents was studied by two different procedures, the choice of which depended upon the nature of the experiment. 14 In the f i r s t procedure, the incorporation of C-amino acids into whole eel Is, protein, and pools were determined by the M i l l i p o r e f i l t r a t i o n procedure of Britten and McLure (1962). Cells were f i l t e r e d on a Tracerlab E8B precipitation apparatus (Tracerlab, Waltham, Mass.) and immediately washed with 2 ml of minimal medium. This procedure did not remove pool amino acids. Dried f i l t e r s were placed in v i a l s containing 10 ml of s c i n t i 1 l a t i o n f l u i d ( L i q u i f l u o r , New England Nuclear Corporation) and the v i a l s were assayed for ra d i o a c t i v i t y in a Nuclear Chicago 1iquid s c i n t i 1 l a t i o n spectrometer model 725. In the second procedure, the rate of incorporation of amino acids into whole c e l l s was determined essen t i a l l y by the method of FerroLuzzi-Ames (1964), which was modified as follows: 1 ml of a vigorously s t i r r e d , heavy c e l l suspension was added to a test tube 14 containing 3 ml of minimal medium and the C-amino acid, at the desired s p e c i f i c a c t i v i t y . The reaction mixture was s t i r r e d on a Troemner magnetic s t i r r e r using miniature s t i r r i n g bars. Aliquots were removed for f i l t r a t i o n at 15 sec time intervals. F i l t e r s were clried and assayed for rad i o a c t i v i t y and rates of amino acid transport were calculated from the e a r l i e s t 1inear incorporation data. This method was used for the demonstration of saturation k i n e t i c s , and to determine the kinetics of competitive i n h i b i t i o n , since i t permitted a series of reactions to be completed within a short period of time and thereby eliminated variation in rates of transport due to eel 1 p r o l i f e r a t i o n . Experiments, designed to determine the rates of amino acid transport as a function of incubation temperature, were performed in water-jacketed reaction vessels maintained at the desired temperature with a Lauda K-2/R temperature-controlled c i r c u l a t i n g water pump (Brinkmann Instruments, Ine., Westbury, N.Y.). Cel1 suspensions were allowed to equi1ibrate at the desired temperature for 15 min prior to the i n i t i a t i o n of the transport experiment by the addition of the 14 C-amino acid. The reaction mixtures were continuously agitated with a magnetic s t i r r e r . Studies on the maintenance of amino acid pools at low temp eratures were carried out in the water-jacketed reaction vessels described for transport studies, which had been precooled. V. Corhpet ? tive Ihhib i t ion of Ami ho Aci d Transport The rate of uptake of 10 ^ to 10 ^ M 1^C-amino acids in the -4 -3 12 presence of 10 or 10 M C-amino acids was determined as follows: 3 ml of minimal medium, containing the amino acids to be tested for 14 competitive i n h i b i t i o n , and the test C-amino acid were added to an 18 mm test tube. These reaction tubes were equilibrated at 30 C and 1 ml of a dense cel1 suspension (O.k mg dry cells/ml) was added with continuous agitation to i n i t i a t e the reaction. Samples were collected with a 1 ml tuberculin syringe at 15 sec intervals and f i l t e r e d . The degree of competitive i n h i b i t i o n was calculated \k from the reduction in the rate of C-amino acid incorporation r e l a t i v e to the appropriate control. VI. Corhpeti ti v e Pool D i splacement Cell suspensions were preincubated with2 0 0 yg/ml chloram- Ak phenicol for 30 min at 30 C prior to the addition of C-amino -6 acid to a concentration of 10 M. Pool formation was followed and when maximal, the incubation temperature was reduced to 10. C. The point of maximal pool formation had been determined in preliminary experiments. Al1 preformed pools were found to be stable at this temperature and protein synthesis was negligible. To i n i t i a t e 12 the exchange reaction, a competitive C-amino acid was added to -k a f i n a l concentration of 10 M and the rate of exchange determined from the loss of pool label over a 15 min time in t e r v a l . VII. Chromatographic Techniques and Measurement of Amino Acids in the Cel1 Pool and in the Culture Mediurn A sensitive quantitative measurement of pool amino acids and amino acids excreted into the growth medium was devised as follows c e l l s were grown in 10 ml of minimal medium containing 111 ymoles of Ak uniformly labelled C-glucose with a s p e c i f i c a c t i v i t y of 2 mc/mmole 20 and logarithmic or stationary phase cultures were harvested by centrifugation at 10,000 x g_ at room temperature. The supernatant f l u i d was carefully decanted and the tube wiped free from adhering l i q u i d . The resulting packed c e l l s were extracted with 5 ml of 5% t r i c h l o r o a c e t i c acid (TCA) and the residual c e l l u l a r material was removed by centrifugation at 10,000 x £ for 10 min at 6 C. This procedure was repeated twice and the TCA extracts were combined. The growth medium was deproteiriized with cold 5% TCA in a similar manner. The TCA was removed from the fractions by extracting 5 times with cold ethyl ether. The resulting aqueous samples were evaporated to dryness in an a i r stream. Each dried sample was dissolved in d i s t i l l e d water and applied to a column (1.2 x 10 cm) of Dowex 50 (H + form). The column was washed w i t h : d i s t i l led water u n t i l no further r a d i o a c t i v i t y was eluted and the adsorbed compounds were then eluted with 100 ml of 4M ammonium hydroxide. The eluate was evaporated to dryness and the residue dissolved in 0.1 ml of d i s t i l l e d water. Samples were quantitatively applied to cellulose thin layer plates (eellulose powder MN 300) and the radioactive compounds were separated two-dimensionally by the method of Jones and Heathcote (1967)- The resulting chromatograms were exposed for 1 week to medical x-ray f i l m (Eastman Kodak Co., Rochester, N.Y.). The films were developed and the radioactive areas detected by this method were scraped loose from the plates and drawn, by vacuum, into s c i n t i 1 l a t i o n v i a l s which were subsequently f i l l e d with a toluene s c i n t i 1 l a t i o n f l u i d and assayed for r a d i o a c t i v i t y in a 1iquid s c i n t i 1 l a t i o n spectrometer. The addition of from 5 to 60 mg of eel 1ulose caused no increase in quenching under these conditions. It was found by this method that amino acids could 21 be detected in amounts as low as 1 x 10 ymoles. Confirmatory q u a l i t a t i v e amino acid analyses were carried out by two-dimensional chromatography on both thin layer and paper. Thin layer plates were coated with S i l i c a Gel G, spotted with the radioactive sample, and developed in a solvent system of chloroform- methanol-amnionia (2:2:1 v/v) and in the second dimension in phenol- water (75:25 v/v) (Randerath, 1963). Paper chromatograms, spotted with the radioactive samples, were developed in the f i r s t dimension in a solvent system of n-butanol-acetic acid-water (120:30:50 v/v) and in the second dimension in phenol-ammonia (200:1 v/v) (Smith, 1960). Chromatograms from both these methods were analyzed by radio- autography and by ninhydrin detection of the appropriate c a r r i e r 12. . . . " C-amino acids. Culture supernatant f l u i d s , which were collected by f i l t r a t i o n , were reacted with 10 M 2,4-dinitrophenylhydrazine in 2N HC1 for 1 hr at 38 C. The keto-acid hydrazohes were extracted into ethyl acetate and purified by extraction with 1 M tjnsj-(hydroxymethyl) ami no- methane buffer (Tris) pH 11.0. The acid hydrazohes were further extracted into e t h y l a c e t a t e , dried to a small volume, and the derivatives were separated by paper chromatography in a solvent system of n-butanol- ethanol-ammon ia (0.5 N) (70:10:20 v/v) as described by Smith (1960). VIM. AminO Acid Composition of Cel l u l a r Protein Cells from a culture in logarithmic phase were harvested by centrifugation and fractionated according to the procedure of Roberts e_t aj_. (1955). The protein residue remaining after extraction-with hot TCA was further extracted twice with ethyl ether to remove residual TCA and the samples were a i r - d r i e d . The protein was hydrolyzed with 6N HCI for 18 hr at 100 C in sealed glass v i a l s . After hydrolysis, the acid was removed by flash evaporation and the water-washed residue was dried and dissolved in 0.1 M citrate-buffer (pH 5.5). Amino acid analyses were carried out using a Beckman Model 120C amino acid analyzer. 14 Qualitative C-amino acid analyses of the protein of c e l l s which were grown in the presence of low concentrations of radioactive amino acids were carried out by paper chromatography as described above. IX. Chemical Fractionation of Whole Cel1s The chemical fractionation procedure used was es s e n t i a l l y that of Roberts e_ta_l_. (1955), with the modification by C l i f t o n and Sobek (1961). The hot TCA soluble fraction was prepared by heating the samples in5% TCA for 20 min at 90 C rather than at 100 C for 30 min. Aliquots of the c e l l fractions were plated in duplicate onto stainless steel planchets, dried under an infra-red lamp, and were counted at i n f i n i t e thinness with a thin end window Geiger tube attached to a Nuclear Chicago scaler model 181A equipped with an automatic gas- flow counter. Corrections were made for background. In order to reduce s t a t i s t i c a l deviation, at least 1000 counts were recorded when possible. X. Chemicals Amino acids used as carriers for C-amino acids were obtained from Nutritional Biochemicals Corp., Cleveland, Ohio. 14 L-tryptophan (methylene- C) was obtained from Nuclear-Chicago Corp., Des Plaines, 111. The other radioactive amino acids and C-glucose were purchased from Schwarz Bioresearch Inc., 14 Orangeburg, N.Y., and were obtained as the C uniformly labelled 3 3 product with the exception of L-hydroxyproline- H(hydroxyl- H). A l l radioactive amino acids were checked for purity by thin layer chromatography (Jones and Heathcote, 196?) and radioautography. N-methyl-N-nitro-N 1-nitrosoguanidine was obtained from Aldrich Chemical Co. Inc., Milwaukee, Wise. I. General Properties RESULTS AND DISCUSSION of AmihO Acid Transport 1. Uptake of amino acids The i n i t i a l rate of incorporation of amino acids into whole celIs during exponential growth was f i r s t order with respect to c e l l mass and was reproducible under the experimental conditions used (Fig. 1). The time course of amino acid uptake and d i s t r i b u t i o n within the c e l l s was studied. Figure 2 shows the results of an experiment measuring the uptake of C-tyrosine. Tyrosine was taken into the c e l l immediately and rapidly u n t i l the supply in the medium became exhausted. The entry of labelled tyrosine into TCA precipitable material was l i n e a r , and after two to three minutes continued at the expense of the amino acid pool. At three minutes, the tyrosine concentration in the pool was 13,350 times greater than the tyrosine concentration of the medium. This pattern of incorporation corresponds to the patterns of amino acid uptake into E. c o l i (Britten and McLure, 1962; Tristram and Neale, 1968). 2. Precursor-Pool relationships The incorporation of tyrosine (Fig. 2) was typical of 25 many of the amino acids studied. The negligible lag in the entry of amino acid into the protein fraction suggested either that passage of the amino acid through the pool was not obligatory for entry into protein, or possibly, that the amino acid concentration in the native pool of growing c e l l s was very low and thus had a negligible influence on the s p e c i f i c a c t i v i t y of the labelled amino acid which was transported into the c e l l s . Valine was selected as the amino acid to test these -6 p o s s i b i l i t i e s . When added to a concentration of 10 M, the amino acid was completely removed from the external medium by 90-120 sec, precisely the time at which the pool level was maximal (Fig. 3 , inset). In a similar experiment, a 90 sec preincubation 12 _£ with C-valine at 10 M was followed, at zero time, by a pulse \h of C-valine at the same concentration. The prolonged lag of Ah C-valine incorporation into TCA insoluble material (Fig. 3) was due therefore to the equi1ibration of the radioactive amino 12 acid with the C-valine pool. These results suggested that exogenously supplied amino acids entered the c e l l and accumulated in an i n t r a c e l l u l a r metabolic pool, and that entry into this pool was necessary for incorporation into protein. Similar results were obtained for the incorporation of 1h C-proline into E. to 1i (Britten and McLure, 1962). To account for 1h the negligible lag of C-amino acid entry into c e l l u l a r protein in the absence of a preloaded pool, concentrations of de novo synthesized amino acids in the native pool of growing c e l l s must, therefore, be r e l a t i v e l y low. 26 .120 0.25 O P T I C A L 0.50 0.75 1.0 DENSITY (650mu) Fig. 1. Increase in the. rate of proline uptake with increasing c e l l mass. External proline concentration was 10~° M; sp e c i f i c a c t i v i t y , 25 yc/ymole (O.D.,- of 0.50 equals 0.2 mg dry weight/ml). 27 4 0 r to 2 0 - 5 10 MINUTES Fig. 2. Time course of tyrosine uptake and incorporation into protein by a growing suspension of P_.' aeruginosa (0.2 mg dry weight/ml). External tyrosine concentration was 1 x 10 M; s p e c i f i c a c t i v i t y , 12.5 yc/ymole. 28 When the time course of C-amino acid uptake was determined for a l l the commonly occurring amino acids, i t was found that only the incorporation of glycine, serine, and glutamate exhibited a lag prior to entry into protein. Figure k i l l u s t r a t e s the incorporation of C-glutamate into whole c e l l s and into the TCA insoluble material of P. aeruginosa. With most \k amino acids tested, C entry into protein was ess e n t i a l l y linear from zero time. These results suggested that most amino acids were present in the pool of growing c e l l s at low leve l s , but that a few, such as glutamate, were present at r e l a t i v e l y high concentrations. Glutamate has also been reported to be present in r e l a t i v e l y high concentrations in the pool of _E. col i (Britten and McLure, 1962). 3. Comparative rates of transport From the results of rate studies of amino acid uptake, i t became obvious that a large variation in rates of transport for different Ak amino acids existed. Experiments to determine C-amino acid incorporation rates were performed for a l l the common amino acids by rapid sampling in order to obtain accurate, comparative values. From the results of Table I, i t can be seen that there is approximately a fo r t y - f o l d variation in the rates of transport; from a low of -k 1.92 x 10 umoles/min/mg dry weight of c e l l s for cysteine to -k ' ' 80 x 10.'. ymoles/min/mg dry weight for leucine. The basic and aromatic amino acids were incorporated much more rapidly than the a c i d i c or neutral amino acids. 8 - oo o 6 1o4 O A I" 2 Minutes 14, 4 6 MINUTES 8 1 0 Incorporation of C-valine Ingo-the protein .of P_. aeruginosa. Symbols: 0, 10 M C-valine; 4 , 10 M ^C-y a l i n e added 90 sec after preloading the r \ - D ii 1 2r_..~ l : i *.. : „-p eel 10 l i s with 10 u M 1^C-yaline. Insert: incorporation of ~° M C-valine into whole eel 1s, protein and pool 3 0 Total yProtein 2 3 MINUTES Fig. 4. Total uptakeof Oglutamate and incorporation into protein o f cel1.suspensions ( 6 . 1 mg dry weight.of cells/ml) External glutamate.concentration was 1 x 1 0 M; sp e c i f i c a c t i v i t y , 1 2 . 5 uc/umole. 31 The only amino acid tested to which these c e l l s were impermeable was hydroxy-L-proline. Experiments to detect uptake of this amino acid showed that no radioac t i v i t y was incorporated either into the amino acid pool or into the proteiniKaback and Stadtman (I965), reported that hydroxyproline could exchange 14 with C-proline concentrated in membrane preparations of E_. col ?. This suggested that the proline concentration mechanism also functioned with hydroxyproline. 4. Distribution of radioactive amino acids in the c e l l To establ ish whether or not the exogenously supplied 14 C-amino acids which were incorporated into the TCA insoluble material remained unchanged during passage through the i n t r a c e l l u l a r 14 pool, cultures were allowed to incorporate s p e c i f i c C-amino acids added at low concentrations and the d i s t r i b u t i o n of ra d i o a c t i v i t y within 14 the c e l l s was determined by chemical fractionation. For C-histidine, 14 14 14 C-isoleucine, C-phenylalanine, and C-proline, approximately 90 percent.of the rad i o a c t i v i t y was incorporated into the protein 14 14 14 fr a c t i o n , whereas with C-arginine, C-glycine and C-serine only 58.3 percent, 50.7 percent, and 56.7 percent respectively were found in the hot TCA insoluble fraction (Table I I ) . A s i g n i f i c a n t percentage of the label found in the cold TCA soluble fraction with the 14 14 C-arginine experiment accounted for this deviation. With C-glycine a large percentage of the added rad i o a c t i v i t y was found, understandably, in the nucleic acid frac t i o n . Also, the nucleic acid fraction Table I. Rates of transport of amino acids into whole c e l l of P. aeruginosa. AMINO ACID RATE OF TRANSPORT RELATIVE RATE" , umoles/min/mg dry weight 10" M x 10 _ Z f M hydroxyproli ne 0 0 cysteine 1.92 1.00 threonine 3.-5A 1.85 aspartate 4.11 2.14 seri ne 4.20 2.19 glutamate 4.67 2.43 p r o l i ne ••5.75 2.99 glyc i ne 5.90 3.08 tyrosine 14.40 7.50 methionine 19.90 9.90 valine 23.60 12.30 hi s t i d i n e 27.40 14.30 alanine 27.60 14.40 phenylalanine 32.30 16.80 lysine 33.60 17.50 isoleucine 39.20 20.42 tryptophan 52.00 27.10 arginine 80.00 41.60 leucine 80.00 41.60 33 accounted for a large percentage of the label when c e l l s were 14 grown in the presence of C-serine. This was attributed, presumably, to the presence of the transhydroxymethylase reaction. The r e l a t i v e l y low amounts of label found in the acid-ethanol ) soluble fractions were assumed to be present as alcohol soluble protein, however, the higher proportion of label in this fraction 14 from c e l l s labelled with C-serine was naturally attributed to the synthesis of l i p i d s or phospholipids. 5. Energy requirement for amino acid incorporation Most metabolite transport systems in microorganisms are sensitive to poisoning by metabolic inhibitors such as dinitrophenol, azide, or other uncouplers of energy metabolism. Cell suspensions of P. aeruginosa were tested for their a b i l i t y to transport amino acids when treated with such in h i b i t o r s . When c e l l s were preincubated for 30 min at 30 C in the presence of 30 mM sodium azide or with 30 mM sodium azide plus 1 mM iodoacetamide, a marked reduction in 14 amino acid transport a b i l i t y was observed as measured by C-proline incorporation into whole c e l l s (Fig. 5). Preincubation of the c e l l suspensions for 30 min with sodium azide inhibited the rate of incorporation of this amino acid by 97 percent and by 99 percent when both sodium azide and iodoacetamide were added to the preincubating mixture. This second, r e l a t i v e l y minor increase in in h i b i t i o n of proline uptake with iodoacetamide was presumably due to the termination of substrate level phosphorylation. Table II. Distribution of radioactivity in the cei l fractions of P_. aeruginosa grown in the presence of C-amino acids. FRACTION 14 C-AMINO ACID . T r n . cold TCA acid ethanol hot TCA residual soluble soluble soluble protein % of total radioactivity arginine •35.5 3.8 2.4 58.3 glycine 4.1 4.8 40.4 50.7 hist idine 2.5 •3.7 2.7 91.1 i soleuci ne ' 0.7 4.3 4.7 90 .3 phenylalanine 1.1 4.6 4.2 90.1 proline 2.0 6.5 •3.7 87.8 seri ne 6.3 11.9 25.1 56.7 Percent of total radioactivity incorporated into cel lu lar material. ( -35 MINUTES F ig . 5. Effect of sodium azlde and sodium azlde plus lodoacetamlde on the active transport of '^C-prolIne. Cells were preIncubated with the Inhibitors for 30 mtn at 30 C. The conclusion that the incorporation of amino acids was energy dependent was substantiated by the observation that 14 c e l l suspensions of P. aeruginosa did not incorporate C-proline at 0 C. Cell suspensions which were c h i l l e d to 0 C for 10 min 14 -6 prior to the addition of C-proline at a level of 10 M 14 showed no s i g n i f i c a n t amount of C-proline uptake over a 3 hr time in t e r v a l . 6. Effect of temperature on the rate of amino acid transport The uptake of amino acids into whole c e l l s of P. aerug?nosa 14 is temperature dependent. The rate of C-valine incorporation into eel 1 suspensions at various temperatures is shown in Figure 14 6. Below 10 C the rate of C-valine incorporation was negligible, but increased rapidly with increasing temperature to the maximum rate, exhibited at 45 C. At temperatures exceeding 45 C the rate 14 of C-valine uptake declined sharply, a chara c t e r i s t i c common to most enzyme systems. Incubation of whole c e l l s for 30 min at 55 C was found to inactivate permanently the valine transport system. The fact that transport of valine could be dissociated from growth was evident from the observation that the rate of valine transport was high at 45 C, whereas, growth of the organism does not occur at this temperature. The Q.^ for valine uptake was found to be approximately 2.75. 7. Dependence of the rate of transport on substrate concentration The rate of uptake, for most of the amino acids studied, 37 15- UJ 12 o _j 2 >* 2 V to ' o 6 2 0. « 3 \ 10 20 30 40 T E M P E R A T U R E 50 60 Fig. 6. Rate of 1 x 10** M C-val Ine Incorporation Into whole ce l ls of P_. aeruginosa as a function of temperature. Cells were equilibrated for 10 mtn at the appropriate temperature prior to the addition of label. Samples were removed at 30 sec Intervals to obtain Init ia l uptake ve loc i t ies . increased concomitantly with the exogenous amino acid concentration. The uptake system, however, tended to become saturated at high external amino acid concentrations. The 1V kinetics of incorporation of C-phenylalanine are shown in Figure 7. Michael i s 1 constants were of the order of 10 ^ M -6 to 10 M for most of the amino acids studied. The adherence of the uptake process to Michaelis-Menton k i n e t i c s , the dependence upon energy metabolism, temperature s e n s i t i v i t y , and the loss of this uptake process by mutation strongly suggest that the amino acid incorporation process is mediated by a protein, presumably a permease. 8. Concentration of amino acids in the i n t r a c e l l u l a r pool From the kinetics of amino acid incorporation into the protein fractions, i t was assumed that a wide variation in concentrations of the different amino acids existed within the native amino acid pool. Direct measurement of these amino acids was carried out to determine whether or not a correlation existed between native pool composition and the variation in rates of transport. The amino acid composition of the growth medium was also determined in order to establish the concentration gradients maintained between the i n t r a c e l l u l a r and e x t r a c e l l u l a r amino acids. The composition of the amino acid pool of c e l l s growing logarithmically was found to be extremely heterogeneous. Figure 8 represents the d i s t r i b u t i o n of 35 po s i t i v e l y charged compounds present in the pool of P. aeruginosa, only 11 of which were 39 Fig. 7. Saturation kinetics of phenylalanine incorporation by P. aeruginosa. C e l l s , in minimal'medium, were incubated for 30 sec at 30 C in the presence of increasing concentrations of C-phenylalanine. Inset: Lineweaver- Burk plot. i d e n t i f i e d as amino acids. The quantitative compositions of the pool, the culture medium, and the concentration ratios are l i s t e d in Table I I I . The linear amino acid incorporation kinetics into the protein fraction for most of the amino acids tested previously, suggested that the concentration of some amino acids in the native pool was low. Glycine, serine, alanine, and glutamate were present in concentrations approximately 7 to 30 fold higher than the other amino acids found in the native pool, and the incorporation data for glycine, serine, and glutamate (Fig. 4), demonstrate "precursor-pool" ki n e t i c s . However, as can be seen from Figure 9, alanine deviated from t h i s trend. This suggests that of the amino acids tested, alanine was the only one which did not f i r s t equi1ibrate with a l l of the native alanine amino acid pool prior to entry into protein. These results, therefore, suggested that i n t r a c e l l u l a r pools may be compartmentalized in different ways. From the amino acid concentration ratios (Table I I I ) , i t can be seen that concentration gradients of approximately 100 to 400 fold are maintained within the c e l l during logarithmic growth. An identical experiment, with c e l l s that had been in the stationary growth phase for 3 hr, demonstrated that a l l i n t r a c e l l u l and e x t r a c e l l u l a r amino acids had been u t i l i z e d with the exception of methionine, which was found in high concentration in the stationary phase culture medium. From the data of Tables I and I I I , i t can be seen that, 41 Fig. 8. Radioautograph of the nitrogen pool extracted from c e l l s groy^ng logarithmically in the presence of U- C-glucose 42 Table I I I . Composition of the amino acid pool and culture supernatant f l u i d of-P. aeruginosa during logarithmic growth. AMINO ACID POOL SUPERNATANT FLUID POOL SUPERNATANT FLUID ymoles/ml isoleucine 1.14 X 10-* 4.67 X 1 0 - 7 244 phenylalanine 1.25 x Id-* 4.53 X 10" 7 277 leucine 1.35 X IO"* 4.91 X i o " 7 232 v a l i ne 2.39 X •IO"* 1.41 X IO"* 1 69 tyrosine 2.55 X 10"* 0 threoni ne 3.72 X IO"* 2.00 X IO"* 186 glycine 7.78 X i o "* 5.60 X IO"* 139 serine 1.63 X IO" 3 3.80 X i o " 6 427 alanine 1.66 X 10- 3 6.99 X io": 6 237 glutamate 3.00 X 10- 3 7.73 X i o " 6 388 glutami ne 3.93 X 10- 3 0 g1ucosami ne 0 1.81 X 10"5 o MINUTE? Fig. 9. Early time course of 1 x 10~ M C-alanlne uptake by a growing suspension of P. aeruginosa (0.1 mg dry welght/ml). with the exception of alanine, amino acids present in the native i n t r a c e l l u l a r pool at high concentrations are transported at a r e l a t i v e l y low rate into P_. aeruginosa. In this organism the i n t r a c e l l u l a r concentrations of most amino acids in the native pool were found to be surprisingly low in relation to reported pool levels of other microorganisms (Holden, 1962), and some amino acids were not detectable by the sensitive method employed for their estimation. However, i t is important to emphasize that most investigators have used less direct methods to determine the levels of amino acids present in the pool and many analyses must be regarded with some caution since the pool may contain a great variety of nitrogenous compounds as has been demonstrated for the pool of P. aeruginosa (Fig. 8). The total amino acid pool levels for P. aeriiginosa ; when grown on minimal medium, are approximately one percent of those reported for E. cbl.i (Holden, 1962). Few reports are avai lable on the composition of the amino acid pool of Gram-negative bacteria and the data reported here represent the lowest native amino acid pool levels determined for any microorganism to date. Also, these data are the f i r s t reported for an obligate aerobe. When the individual components of the i n t r a c e l l u l a r pool were examined, s t r i k i n g differences in the amino acid concentrations were again observed. For instance, FerroLUzzi-Ames (1964), reported i n t r a c e l l u l a r concentrations of h i s t i d i n e of 0.06 umoles/ gm dry weight,of S. typhi mil r?urn but we have not been able to detect this amino acid in the native pool of P. aeruginosa. In fact, the only amino acid which approached this concentration was glutamate. Glutamate concentrations of 0.5 - 2 ymoles/gm dry weight of E. c b l i were reported by Britten and McLure (1962), and this is 10 fold greater than the glutamate pool level in P_, aeruginosa. Amino acids such as glutamate, alanine, glycine, and serine, which have multiple physiological roles, not only as direct precursors for protein, c e l l wall and c e l l membrane synthesis but also as precursors for other c e l l u l a r metabolites, would be expected to be present in the pool in higher concentrations than most other amino acids. One also would expect, on f i r s t analysis, that these amino acids would be transported more e f f i c i e n t l y than those for which the c e l l has a lower requirement. It is reasonable, however, that the low transport rates found for these amino acids are due to the fact that the c e l l does not rely on an exogenous supply of these amino acids, since they may be readily synthesized from the carbon skeletons available from the metabolism of glucose. 9. Amino acid composition of the e e l l u l a r protein In order to determine i f the composition of the c e l l u l a r protein reflected the efficacy of amino acid transport, protein was isolated from logarithmic phase c e l l s and submitted to amino acid analyses. The results (Table IV) were compared to the tabulated amino acid transport rates (Table I ) . Although the transport rates of some of the 18 amino acids 46 tested (proline, valine, leucine, and alanine) compared favourably with their r e l a t i v e abundance in the protein of P. aeruginosa, the majority of the amino acids showed no r i g i d correlation. However, when the data of Table III was compared with that of Table IV, i t was observed that those amino acids that are present in the native amino acid pool are essenti a l l y those present in highest concentrations in the protein. The amino acids present in the pool in r e l a t i v e l y low concentrations, that is leucine, isoleucine, valine, and phenylalanine were transported rapidly, whereas the amino acids present in r e l a t i v e l y high concentrations, were transported slowly. I I . General Properties of Pool Format ion and Ma i htenahce 1. Time course.of pool formation When experiments,designed to determine the rate of incorporation of amino acids, were performed for a l l of the amino acids commonly occurring in protein, s i g n i f i c a n t variations in these incorporation kinetics were found. Figure 10 represents the four patterns of incorporation found with these amino acids. Most of the a l i p h a t i c , aromatic, and basic amino acids demonstrated the pattern of uptake i l l u s t r a t e d by Figure 10A, and when pools were chromatographically analyzed the amino acid was found, for the most part, to maintain i t s chemical int e g r i t y . For example, chromatography and radioautography of the isoleucine pool is demonstrated in Figure 11. Most of these amino acids also 47 Table IV. Composition of the c e l l u l a r protein of P. aerug j hbsa. AMINO ACID * t L H I , v c AMINU A U U CONCENTRATION methionine 1.00 h i s t i d i n e 1.38 lys i ne 1.85 tyros ? ne 2.27 phenylalanine 3.09 isoleucine 3.76 p r o l i ne 3.95 seri ne 4.28 arginine 4.46 threonine 4.54 glutamic acid 5.48 valine 6.52 glycine 8.40 leucine 8.51 aspartic acid 8.94 alanine 11.95 demonstrated high transport rates (Table l ) , and the results agree with the kinetics of amino acid incorporation in E. c o l i (Britten and McLure, 1362). However, when incorporation studies were carried out under identical conditions, several amino acids were found to deviate from this general pattern. Figure 10B i l l u s t r a t e s the incorporation kinetics common to the amino acids glycine, serine, and glutamate. It can be seen that pool formation commences rapidly upon the addition of the labelled amino acid, but the pool is only p a r t i a l l y removed for protein synthesis; the remaining fraction being lost only very slowly over a long period of time - i f at a l l . To elucidate the nature of this phenomenon, pools were collected at time intervals indicated by arrows in Figure 10, and were analyzed by radioautography (Table V). Radioautography of compounds in the early glutamate pool is demonstrated in Figure 11. At 5 min, 66% of the rad i o a c t i v i t y in the pool was found to be glutamate and a "bound" or slowly metabolized compound was found to account for approximately 90% of the pool at a later time. This compound did not chromatograph discretely and resisted i d e n t i f i c a t i o n by several methods. Glycine and serine also formed heterogeneous pools and, in fact, these amino acids did not account for the majority of the r a d i o a c t i v i t y of the i n t r a c e l l u l a r pool. The major labelled component was found not to be an amino acid, purine, pyrimidine, nucleoside, or nucleotide, and did not form a hydrazone. This material proved to be heterogeneous and from i t s s o l u b i l i t y in chloroform and chloroform-NaC1 was assumed to be l i p i d and > Y-O < O < or C. Proline / T I M E >- > O < o < D. Arginine T I M E 14 Fig. 10. Patterns of C-amtno acid uptake Into whole ce l ls 0, protein 0, and the calculated pool A , of P. aeruginosa. 50 Fig. 11. Radioautograph of the amino acid pool with C-isoleucine established 51 Fig. 1 2 . Radioautograph of the amino acid pool established with 1^C-glutamate. 52 HQ (CM) ml MINUTES Fig. 13. The inhibition of growth In minimal media as a function of the chloramphenicol (CM) concentration. Table V. Chromatographic analyses of C-amino acid pools. ITf T4~ C-Amino Acid C Compounds Percent of Total Pool Added in the pool Radioactivity early pool late | isoleuci ne isoleucine 93.7 - p r o l i ne pr o l i ne 95.2 - glutamate 4.8 - glutamate glutamate 65.9 - compound X 13.4 89.5 alanine 0.7 -va1i ne 9.4 -isoleucine 0.4 -leucine 0.3 -p r o l i ne 0.2 - glycine compound Y 93.3 92.6 glycine 1.2 0.9 glutamate 1.2 1.6 threonine 0.9 1.2 seri ne 0.6 0.3 seri ne compound Z 86.8 89.2 glutamate 1.2 -aspartate 1.1 -threonine 1.0 0.6 seri ne 0.9 1.7 g l y c i ne 0.8 0.3 arginine glutamate 43.5 6.8 arginine 21.2 -putrescine 15.2 93.2 yami nobutyrate 2.6 -54 phospholipid. Since only a minor proportion of the added 14 C-amino acid was iden t i f i e d in the i n t r a c e l l u l a r pool, i t was concluded that under the conditions of the transport experiments, much of the amino acid was converted to l i p i d and phospholipid without f i r s t entering the free amino acid pool. It was f e l t that this conversion may have taken place at the c e l l membrane. 14 The third class of uptake kinetics was observed with C- proline and C-threonine (Fig. 10C). In this case, e s s e n t i a l l y no pool formation occurred during the course of the experiment; that i s , the amino acid entering the c e l l was immediately converted into TCA insoluble material. In order to investigate this deviation from normal pool behaviour, attempts were made to s e l e c t i v e l y i n h i b i t protein synthesis without a l t e r i n g the a b i l i t y of the c e l l s to transport proline. It was found (Fig. 13) that the degree of protein synthesis could be manipulated by the controlled addition of chloramphenicol (CM). At concentrations greater than 100 yg/ml, growth was completely inhibited. The 14 rates of C-proline incorporation into chloramphenicol treated c e l l suspensions as a function of the inhibitor concentration are shown in Figure 14. It can be seen that over the range of 14 chloramphenicol concentrations employed, the rate of C-proline incorporated into the c e l l was r e l a t i v e l y constant, whereas the rate of entry of the label into the protein fraction was inhibited in a manner proportional to the i n h i b i t o r concentration. As a result, accumulation of proline in the i n t r a c e l l u l a r pool increased s i g n i f i c a n t l y . 55 Preliminary experiments showed that the i n i t i a l rate of proline incorporation into whole c e l l s did not vary greatly as a function of temperature; however, the rate of growth diminished markedly at low temperatures. These observations were used to confirm the results obtained with chloramphenicol, since chloram phenicol has been shown to adversely affect pool formation in E. cb1i (Britten and McLure, 1962). When the incorporation of C-proline into c e l l suspensions was followed at different temperatures, results similar to those with chloramphenicol, were obtained (Fig. 15). At 10 C - 20 C, the rate of protein synthesis (as determined by incorporation of ^ C - p r o l i n e into TCA insoluble material) was s i g n i f i c a n t l y lowered. Low rates of protein synthesis were coincident with high proline pool levels. At higher temperatures, large pools were not formed due to the rapid incorporation of the label led ami no acid into protein. These results demonstrate that the rate of pool formation and the size of the i n t r a c e l l u l a r pool are functions of both the capacity of the c e l l to transport an amino acid and to u t i l i z e that amino acid for protein synthesis. Therefore, any demonstration of pool formation must be the result of a balance of the above variables, and not necessarily attributable to the pool forming 14 mechanism per se. Thus, i f the time course of C-proline uptake is followed at 20 C (Fig. 16A) or at 30 C when the rate of protein synthesis has been decreased due to starvation for a required amino acid, then pool formation (Fig. 16B) follows a course similar to that reported for E. c o l i (Britten and McLure, 1962). Chromatography of the labelled pool material, under these conditions, showed__that 56 -& : 1 a « • 25 5 0 75 100 125 150 C H L O R A M P H E N I C O L U G / i ^ L . Fig. 14. E f f e c t o f chloramphenicol on the rate.of C-proline incorporation into whole c e l l s and c e l l fractions of P. aeruginosa. Cells were pre incubated for 30 min at 30 C prior to the addition of C-proline and samples were removed at 30 sec time intervals to determine i n i t i a l incorporation v e l o c i t i e s . 57 TEMPERATURE C F i g . 15. Effect of temperature on the rate of C-proline incorporation into whole c e l l s and c e l l fractions of P. aeruginosa. Cells were equilibrated at the appropriate |emperatures for 10.min prior to the addition of C-proline and samples were removed at 30 sec intervals to determine i n i t i a l incorporation v e l o c i t i e s . the amino acid retained i t s chemical int e g r i t y . 58 2. The fate of i n t r a c e l l u l a r arginine The fourth class of uptake kinetics (Fig. 1OD), was observed with the amino acids arginine and cysteine. These amino acids formed a r e l a t i v e l y high pool level and this pool was maintained for extended periods of time. The arginine pool was studied further and Figure 17 demonstrates the kinetics and s t a b i l i t y of C-arginine pool formation over an extended period. Once formed, the i n t r a c e l l u l a r pool was stable and remained constant even during c e l l growth. During starvation for an exogenous carbon source, the pool was maintained for periods as long as 12 twenty-four hours. The pool was shown to exchange with C- arginine when the unlabelled amino acid was added after formation 14 and s t a b i l i z a t i o n of the C-pool. However, the pool exchanged at a greater rate when the polyamine, putrescine, was added to 12 the c e l l suspension (Fig. 18). No exchangebccurred with C- spermine. The results of these experiments suggested that the stable pool formed from arginine was not the or i g i n a l amino acid 14 but perhaps a degradation product. In addition, CO^ recoveries from these experiments indicated that the oxidation of arginine was constitutive in P. aeruginosa. It has been demonstrated that E_. c o l i cataboljzes both arginine and ornithine to putrescine (Morris and Pardee, 1966), and that P. fluorescens cataboljzes putrescine by way of Y"amin°butyrate and succinic semialdehyde to succinate (Jakoby and Fredericks, 1959). Consequently, the pools were extracted 10 20 30 MINUTES 10 20 30 MINUTES Fig. 16. Time course of l Z*C-prol ine uptake into whole c e l l s and c e l l fractions of P_. aeruginosa. A. At 20 C. B. At 30 C, by a his t i d i n e auxotroph (WK4) deprived of his t i d i n e for 30 min prior to the addition of 1 V-prol ine. U 3 60 16 00 -i 12 o ro 8 0 . 4 " 20 40 60 80 MINUTES 100 Fig. 17. The formation and maintenance of the pool derived from "/*C-arglnlne (2 x 10"5M) by a growing culture of P. aeruginosa. 61 C-Arginine {2 C~ Putr esciruT mtm I 10 2 0 30 40 MINUTES 50 14 Fig. 18. The displacement of the C-pool derived from 2 x 10"5M "'•C-arginine by , 2C-arglnlne (2 x 10"3M) or " 2C-putresclne (2 x 10"3M) added 6 min after the Init ia l '^C-arglnlne. 62 at the times indicated by the arrows in Figure 10D. Subsequent chromatography indicated that putrescine and -y-amino butyric acid were formed very quickly and that after the pool had s t a b i l i z e d , the majority of the radio a c t i v i t y was found to be present in putrescine. Putrescine was formed as a product of arginine catabolism and was not degraded further. Some investigators have implicated polyamines as structural components mediating the stabi1ization of the quaternary structure of the ribosomes (Silman, Artman and Engelberg, 1 9 6 5 ; Choi and Carr, 1967). This suggested that the i n t r a c e l l u l a r putrescine was not a "pool" of free putrescine, but rather putrescine bound t o . i n t r a c e l l u l a r structures, perhaps ribosomes or even nucleic acids, and that treatment with TCA may have neutralized the charge and permitted chemical extraction. However, when c e l l s containing 14 a C-putrescine pool were extracted with a variety of reagents (Table VI), i t appeared that the maintenance of the putrescine "pool" was also markedly dependent on c e l l u l a r i n t e g r i t y . The degree of extraction of the putrescine pool was commensurate with conditions which were known to cause eel 1 l y s i s ; that i s , low or high pH's or high alcohol concentrations. Since the putrescine pool was maintained during long periods of carbon starvation, i t was thought that energy may not have been required for i t s maintenance. To test this hypothesis, large 14 pools were allowed to form from C-arginine by f i r s t preincubating the c e l l s with high concentrations of chloramphenicol (Fig. 19). In the complete absence of protein synthesis (evidenced by the termination of the entry of ^*C-amino acid into protein) amino Table VI. L a b i l i t y of the putrescine pool. REAGENT PERCENTAGE OF THE POOL3 LOST 10 ml media 12.0 0.25% TCA 80.6 5.0 % TCA 100.0 10 % ETOH 30.3 20 % ETOH 60.2 40 % ETOH 98.0 pH 10.5 (NaOH) 122.0 pH 8.0 (NaOH) 100.0 pH 5.0 (HCI) 80.6 pH 3.0 (HCI) 85.0 pH 1.0 (HCI) 87.0 Samples of a c e l l suspension containing putrescine were added to reagents to give the conditions described in the f i r s t column. After 10 min at 0 C these suspensions were f i l t e r e d and the f i l t r a t e collected and assayed for ra d i o a c t i v i t y . The percentage of the pool lost was calculated from the increase ( i f any) in the radi o a c t i v i t y of the f i l t r a t e . acid pools are unstable and " e f f l u x " readily. However, the pool 14 formed froni C-arginine was stable under these conditions and esse n t i a l l y was unaffected by the addition of 30 mM NaN^ (Fig. 20). This treatment afforded only a minor loss of the pool which was attributed to unmetabolized arginine. This demonstrated that putrescine was not maintained in the i n t r a c e l l u l a r pool by an energy requiring mechanism, since energy deficient cel1s rapidly 12 lose their amino acid pools (Fig. 2 7 ) . Whert C-arginine, at high concentration, was added to the culture after this large pool had been formed the resulting exchange rate followed two isotherms (Fig. 21). The rapidly exchanging component was the minor one and was attributed to the presence of residual arginine in the pool, whereas the second more slowly exchanging component was the putrescine pool. Linear exchange rates previously i l l u s t r a t e d in Figure 18 occurred when the ^C-pool was homogeneous 14 as was found for pools formed from low levels of exogenous C- arginine. Unlike the normal amino acid pools ih P. aeruginosa, the putrescine pool was also shown to be stable at 0 C. From these kinds of data i t was concluded that i n t r a c e l l u l a r putrescine must be physically bound or oriented in an unusual manner to some i n t r a c e l l u l a r structural component and is not found free in the pool as in the case of other metabolic intermediates. 14 Exogenously supplied C-arginine has been shown to have a double metabol i c role;, the conversion to putrescine, and the incorporation into protein. When protein synthesis was inhibited with chloramphenicol, the majority of the incorporated arginine was converted to putrescine (Fig. 20). This demonstrated that the 65 F ig . 19. The Incorporation of C-arglnlne into ce l l fractions of P. aeruginosa poisoned with 2 mg/ml chloramphenicol. f 3 0 mM NaN- V ' Total o - ° - o — o o 120 180 i/IINUTES 210 Fig. 20. Partial eff lux of the C-arglnlne- *^C-putrescine pool In the presence of 30 mM NaN . 50 100 150 200 MINUTES Fig. 21. The exchange of the C-arglnlne- C-putrescIn pool by 2 x 10"3M * 2 C-arglnlne. fate of the arginine pool was a result of the competition between protein synthesis and catabolism. However, no putrescine was found in the pools extracted from c e l l s grown on high s p e c i f i c 1 Zf a c t i v i t y U- C-glucose. These results implied that arginine synthesized de novo was not as available for catabolism as for anabolism. To determine whether or not arginine which was synthesized de novo was unavailable for catabolism, an experiment was designed in which arginine synthesized de novo would be inhibited and at the same time an evaluation of the degree of exogenous arginine catabolism could be determined. At high exogenous arginine concentrations, arginine synthesized de novo would be terminated by feedback in h i b i t i o n of ornithine transcarbamylase. When a c e l l suspension was incubated with.0.4 mM Ah' \h C-arginine (Fig. 22), the accumulation of C-putrescine in the Ah pool was found to be low re l a t i v e to the incorporation of C- arginine into protein. This was expected due to the increased competition for pool arginine for the purpose of protein synthesis. However, during the time interval when the exogenous arginine was becoming exhausted (80-100 min), a greater proportion of the incorporated exogenous arginine was converted to putrescine. This coincides with the time at which arginine synthesized de novo would 12 again return to normal. Had the newly synthesized C-arginine \h equilibrated with C-arginine committed to catabolism, no "apparent" increase in the putrescine pool would have been observed since the s p e c i f i c a c t i v i t y of the putrescine synthesized from an Ah 12 equilibrium mixture of exogenous C-arginine and C-arginine 69 00 _I _J UJ o V X o /^Recovery Totol^J ' 18 >- or 15 o o r r 4 0 60 80 MINUTES 100 120 14 Fig. 22. Time course of C-arginine incorporation into c e l l fractions of P. aeruginosa and,the disappearance of exogenous label. C-arginine was present at 4 x 10"** M. synthesized de novo would have lessened. It was, therefore, concluded that arginine synthesized for incorporation into protein was unavailable for catabolism. Possibly this could be explained by a gross difference in a f f i n i t y . o f the arginine catabolizihg enzymes and the amino acid activating enzymes for the substrate arg i ni ne. To determine whether or not the formation,and presumably the binding, of i n t r a c e l l u l a r putrescine was peculiar to P. aeruginosa alone, C-arginine incorporation experiments were carried out with two different species of Pseudomonas;. P. piitida and P. f 1 uorescens. From the results of Figures 23A and 23B i t can be seen that this same phenomenon occurs in these species but to different degrees. From these incorporation experiments i t was evident that when the catabolism of arginine was most.active, evidenced by a loss.of label from the medium, more i n t r a c e l l u l a r putrescine was formed in P. putida; This stresses the significance of the observation made with P_. aeruginosa that the fate of i n t r a c e l l u l a r arginine was dependent on the competition between catabolism and anabolism. The formation of putrescine by the catabolism of arginine in Pseudomonads is undoubtedly ubiquitous; however, the results of this investigation suggest that putrescine has no essential physiological role, since i t was not formed in detectable amounts when c e l l s were grown on minimal medium. In the course of this investigation, several mutants were isolated, which were unable to catabolize arginine but these mutants grew normally on minimal medium. This observation emphasizes the non-essential nature of 6 5 4 ? 2 I 0 A. R putido. G / / / F ' V / o V b Total - o - o B. Protein -•A .o Pool 10 20 30 40 MINUTES R fluorescens Total O o o Protein © © — 40 60 MINUTES 14 Fig. 23. The time course of C-arginine incorporation into whole ce l ls and ce l l fractions of (A) P. put Ida and (B) P. fluorescens. 72 putrescine formation. It is possible, however, that putrescine biosynthesis may be important to structural integrity in the absence of magnesium. Cysteine was also found to accumulate i n t r a c e l l u l a r l y in P. aeruginosa; as much as 50% of the added rad i o a c t i v i t y was found in the TCA soluble fraction of the c e l l . However, unlike the pool 14 Ak formed from C-arginine, the pool formed from C-cysteine was found, by chromatography and radioautography, to retain i t s chemical identity. The nature of this i n t r a c e l l u l a r accumulation has not been investigated. 3. Pool formation and maintenance a. Formation Under optimum conditions for pool formation, that is when protein synthesis was p a r t i a l l y inhibited by low temperature without affecting the amino acid transport rate, amino acid pools were saturated at a part i c u l a r concentration determined by the level of the exogenous amino acid. Figure 2k demonstrated the maximum pool 1evel of proline obtained at an exogenous proline concentration -6 of 10 M with a proline oxidizeless mutant (P22) of P. aeruginosa. At.this concentration 40%.of the exogenous amino acid is incorporated into the c e l l u l a r pool. The concentration ratio established between the internal and external proline was found to be in the range of 1000 f o l d . At higher external proline concentrations the capacity of the pool became largely dependent on the external concentrations, 73 o 0 ' ' 1 * ' 20 4 0 6 0 8 0 MINUTES Fig. 2k. Max I m u r r r pro 1 i ne pool obtained at 10. C with 10 M C-proli ne by P.aeriig 1 hosa P22 grow?ng in minimal medium. Cells were preincubated at 30 C for 30 min with 200 yg/ml chloramphenicol and equ i l i b r a t e ^ for 10. min at 10 C prior to the addition of C-proline. and the resulting concentration ratios were found to decrease.., - 6 When 10. . M proline was allowed to form a maximum pool at 10 C (Fig. 25A), the addition of a l l other amino acids, each to a level of 10 M, resulted in no s i g n i f i c a n t change in the internal proline pool. Consequently, the formation of proline pools in this organism is s p e c i f i c . Also, a pre-established proline pool Ak did not greatly influence the incorporation of C-leucine at this temperature (Fig. 25B). Therefore, i t was concluded that the maintenance of large pools is s t r u c t u r a l l y • s p e c i f i c and that the maximum accumulation of one amino acid is en t i r e l y independent of the accumulation of another unrelated amino acid. Pool formation and maintenance were found to be closely related processes. At high external amino acid concentrations the pool capacity increased and an equilibrium was established between the in t r a  c e l l u l a r and ext r a c e l l u l a r • p r o l i n e . The pool did not increase without exchanging with preloaded i n t r a c e l l u l a r ' p r o l i n e . In the experiment represented by Figure 26, an i n t r a c e l l u l a r proline pool was established with 10 ^  M -^C-proline at 10 C, and then 1 2C-proline was added to a -k level of 10 M at the time indicated. Rapid exchange of the pre loaded pool occurred u n t i l the s p e c i f i c a c t i v i t y of external and internal proline were approximately the same. b. Maintenance The maintenance of high intracellular'amino'acid pools was found to be an energy dependent function. When leucine was allowed to accumulate at 15 C and NaN_ (30 mM) and iodoacetamide (1 mM) were B * 1 i — Q *— 1 i i i 0 4 0 6 0 8 0 100 4 8 12 16 MINUTES MINUTES Specif ic i ty of maintenance of the proline pool In P. aeruginosa P22. A. The time course of proline uptake and proline pool maintenance in the presence of a 10"*4N concentration of each of 18 amino acids added at 60 min. B. The time course of '^C-leucine uptake into ce l l s which had been preloaded with a maximum level of J 2 C-prol lne by previous incubation with lO'&M proline. Control has no preestab1Ished proline pool. added, an immediate eff1ux of the accumulated amino acid occurred (Fig. 27). 4. Pool m u l t i p l i c i ty Both:Britten and McLure (1962), and Kessel and Lubin (1962), postulated from exchange kinetics with "preloaded" E. c o l i pools, that a m u l t i p i i c i t y of pools existed in the organism. That i s , internal proline exchanged with external proline in a manner described by two c l a s s i c a l isotherms. P. aeruginosa pools are not stable to 0 C and such exchange reactions are impossible to perform with the same degree of precision. However, several other c r i t e r i a were established to demonstrate that the internal proline pool of P. aeruginosa was associated with the c e l l in different ways. The-.efflux .of " prol ine from the pool in the presence of 30 mM NaN^ and 1 mM iodoacetamide is shown in Figure 28. Unlike the leucine pool (Fig. 27), a s i g n i f i c a n t fraction of the proline pool was lost very slowly in the absence of energy production and this loss was due to the oxidation of the amino acid as evidenced by the loss of label from the reaction mixture. The NaN^ stable pool was 12 subsequently shown to be exchangeable with C-proline when added -k at 10 M. Control experiments demonstrated that the exchange rate was the same in the absence or presence of NaN^. Therefore, the NaN^ stable component of the pool was exchanged by a non-energy requiring mechanism. Perhaps this component of the proline pool is associated with some internal structure of the c e l l in such a way as not to be considered a "free" internal pool amino acid. 77 MINUTES Fig. 26. Exchange of a preloaded C-proline pool of P. aeruginosa P22 with 10-(,M 12c-p rolIne added at 60 minutes. Cells were preincubated with 200 ug/ml chloramphenicol prior to Init iat ion of the experiment and the Incornoratfon study was performed at in c. 78 10 3 hi O 8 r0 O Q. O A N a N \ 2 0 40 60 80 MINUTES F i g . 27. E f f l u x of a preformed leucine pool In the presence of 30 mH NaNj and 1 mM Iodoacetamide. C e l l s were prelncubated with lOO^/ml chloramphenicol f o r 30 min p r i o r to addition of 2 x 10'6M "C-leucine at 15 C 79 o 0 I L_ L I 1 20 40 60 80 M I N U T E S Fig. 28. Partial eff lux of the Intracellular proline pool of P. aeruginosa on the addition of 30 mM NaN^. Cells were preloaded by Incubating with 10"°M '^C-prollne after an Init ia l 30 min preincubation with 200 ug/ml of chloramphenicol. The experiment was conducted at 10 C. 80 However, even this "stable" proline pool was found to be effluxed at 0 C, although much more slowly than the majority of the pool. When the proline i n t r a c e l l u l a r pool was allowed to accumulate at 10. C and then the c e l l s quickly c h i l l e d to 0 C, rapid e f f l u x of most of the pool occurred within ten minutes of c h i l l i n g and this was followed by a slower e f f l u x of a second component of this pool (Fig. 29). Since one component of the proline pool was shown to be insensitive to NaN^ and since the proline pool was effluxed at two di s t i n c t rates at 0 C, i t was thought that perhaps a c r i t i c a l temperature may exist at which only one component of the pool would e f f l u x . When the proline pool was allowed to accumulate at 10 C and then the cel1 suspension quickly reduced to 5 C, only one major component of the proline pool was lost by efflu x (Fig. 30). The second pool remained stable for 90 min. However, when the temperature was brought back to 10 C the f i r s t pool quickly reformed. These data are interpreted as evidence for the existence .of .multiple proline pools ih P. aeruginosa. C h i l l i n g the c e l l suspensions to 0 C was not found to cause permanent damage to the pool forming mechanisms. When c e l l s were cooled to 0 C, held for 30 min, and then rewarmed to 30 C for 15 14 min, they were found to incorporate C-proline at the normal rate. 81 Fig. 29. Efflux of the proline pool of P. aeruginosa at 0 C. Chloramphenicol treated ce l ls were preloaded with I x 10" 6M '^C-proline at 10 C then placed In an Ice bath at 40 min. 82 10 2 0 3 0 4 0 5 0 M I N U T E S Fig. 30. Efflux of the proline pool of P_. aeruginosa at 5 C and Its re-establIshment at 10 C. Chloramphenicol treated ce l ls were preloaded with 1 x 10"6M l^C-prollne at 10 C. 83 I I I . Speci f i c i ty of the Amino Acid Uptake System 1. Competition for amino acid uptake Cohen and Rickenberg (1956) and Britten and McLure (1962), observed that amino acid pool formation was a competitive process for s t r u c t u r a l l y related amino acids. Both groups demonstrated that strong competitive interactions existed between the various a l i p h a t i c amino acids for the amino acid concentrating process. Britten and McLure (1962), have also shown that a s p e c i f i c concentrating mechanism is operative for the accumulation of proline in E. co1?. From the comparative rates of transport of amino acids at low substrate concentration (Table I ) , i t was observed that s t r u c t u r a l l y s i m i l a r amino acids, or those with similar properties, were transported at approximately the same rela t i v e rate. Thus the a l i p h a t i c , aromatic, and basic amino acids were transported more e f f i c i e n t l y than the a c i d i c , neutral, or sulfur containing amino acids. These results suggested that there may be "fami1ies" of transport systems operative in P_. aeruginosa at low external amino acid concentrations. In order to determine the s p e c i f i c i t y of the amino acid incorporating processes, competition experiments were carried out in a manner that would reveal reductions in the rate at which particular amino acids were transported into P. aeruginosa in the presence of high concentrations of other amino acids. When competitive experiments were performed i t became obvious that several interacting transport systems were present in the organism. A typical experiment is presented in Figure 31A, in which the -6 14 i n i t i a l rate of proline transport was rapid at 10 M C-proline. The addition of nineteen naturally occurring amino acids, including -k hydroxyproline but excluding proline, each present at 10 M caused no i n h i b i t i o n of uptake of the amino acid. These results demonstrated that proline uptake i h P. aerug1hosa ?s a s p e c i f i c process as has been shown previously in E. c o l i (Britten and McLure, 1962). Kaback and Stadtman (1965), found that hydroxy- proline was a competitive inhibitor of proline accumulation in E. cbl? membranes. This was not the case with P. aeruginosa whole -4 c e l l s since 10 M hydroxyproline exerted no competitive i n h i b i t i o n of proline transport. This result was expected as no uptake of hydroxyproline could be detected previously (Table l ) . Specific uptake mechanisms were also found for glutamic acid and aspartic acid, suggesting that these two processes were very s p e c i f i c indeed, in order to discriminate between such s t r u c t u r a l l y similar amino acids. Unlike the transport of proline, glutamic acid and aspartic acid, the transport of al1 other amino acids tested in this manner were competitively inhibited by s t r u c t u r a l l y related amino acids. Figure 31B demonstrates the i n h i b i t i o n of the incorporation of 14 -4 C-lysine by the basic amino acids. At concentrations of 10 M, only the basic amino acids inhibited lysine transport. S i m i l a r l y , amino acids classed as a l i p h a t i c (leucine, isoleucine, valine, and alanine) competitively inhibited the uptake of C-isoleucine (Fig. 32A), and amino acids c l a s s i f i e d here as aromatic (tryptophan, 15 30 45 60 15 30 45 60 SECONDS SECONDS 31. Competition for amino acid uptake In P. aeruginosa. The rate of Incorporation of 10 '^C-amino acid was followed In the presence or absence of lO ' ^ M '2c-amIno acids. x A. Competition for "^C-prolIne uptake. 12c-amIno aclds-lO '^M of a l l amino acids except proline. B . Competition for l^C-lyslne uptake. "2C-amlno acld-lO -^M of a l l .amino acids except Jpaslc amino acids. Control - 10"** M 1 2 C - l y s i n e . ^Control - 10"1* M " c - p r o l ine. 15 30 45 60 15 30 45 60 SECONDS SECONDS Fig. 32. Competition for a l i p h a t i c and aromatic amino acid uptake-iii P. aeruginosa. A. Isoleucine uptake. • - 1 , C-amino acids minus a l i p h a t i c amino acids. A - A , control contained 1^C-isoleucine plus 10 _ i t M 1 2C-isoleucine. B. Phenylalanine uptake, t - t , 1 2C-amino acids minus aromatic amino acids. A - A control contained C-phenylalanine plus 10" H M l z C - D h e h v l a l tyrosine, and phenylalanine), competitively inhibited the uptake of G-phenylalanine (Fig. 32B). In addition to these apparent fami1ies of transport systems, other interactions were found. A low molecular weight neutral amino acid transport system was also elucidated. This system was found to recognize alanine, glycine, serine, and threonine. Another more s p e c i f i c uptake system was found to recognize only cystine and cysteine, but not methionine. The incorporation of methionine appears to present a special case, as this amino acid was found to enter on the permease functioning for the a l i p h a t i c ami no acids, but very weakly, since i t caused only minor in h i b i t i o n of isoleucine uptake when present at 10 . M. However, methionine is incorporated r e l a t i v e l y e f f i c i e n t l y by P. aeruginosa (Table I ) , and i t was found that the a l i p h a t i c amino acids inhibited the uptake of this amino acid only to a moderate degree. It was concluded, therefore, that methionine is recognized by a s p e c i f i c methionine permease with high a f f i n i t y for this amino acid and also by the a l i p h a t i c permease which has a low a f f i n i t y for this amino acid. The reverse was also true, that i s , the a l i p h a t i c amino acids entered the c e l l c h i e f l y by a permease of high a f f i n i t y , however, these amino acids are recognized also by the methionine permease but with low a f f i n i t y . These competitions only become s i g n i f i c a n t at external amino acid -3 concentrations exceeding 10 M. Complicating the s p e c i f i c i t y of the permease systems even further, was the observation that the aromatic amino acids weakly -k inhibited isoleucine uptake at 10 M but at concentrations 88 -3 exceeding 10 M, the degree of competition was considerably more s i g n i f i c a n t . Again, the reverse.of this observation was shown to be true, but to a lesser degree. For example, the uptake of C-tyrosine was found to be very weakly inhibited by other amino acids, including the a l i p h a t i c amino acids. This i n h i b i t i o n was found to be additive and only s i g n i f i c a n t when -Li a l i p h a t i c amino acids were each present at 10 M. Thus, i t was demonstrated that amino acid s p e c i f i c and family s p e c i f i c permeases functioned at low amino acid concentrations but that the same degree.of s p e c i f i c i t y was not maintained at high substrate concentrations. Britten and McLure (1962), concluded from a study of the interactions of the a l i p h a t i c amino acid accumulating system of E. col? that there were s p e c i f i c mechanisms for the formation and maintenance of smal1 amino acid pools and less s p e c i f i c , or completely nonspecific, mechanisms for the formation of very large pools. However, i t should be noted that the "very large pools" referred to with E. co1i a re not found ih P. aerug ? nosa (Table I I I ) , and also that the competitions observed in this study are focused primarily on the transport systems elucidated by rapid sampling. In these experiments, the maximum pool capacity for any one amino acid tested was not reached. 2. Kinetics of competitive i n h i b i t i o n Since Boezl and DeMoss (1961), and Halpern and Lupo (1965), have shown that certain metabolites can non-competitively i n h i b i t the uptake of some amino acids into E. col1, i t was necessary t o d e m o n s t r a t e t h a t t he i n t e r a c t i o n s o b s e r v e d between groups oT s t r u c t u r a l l y s i m i l a r ami no a c i d s were c o m p e t i t i v e . When r a t e s o f amino a c i d i n c o r p o r a t i o n i n t o whole c e l l s were d e t e r m i n e d as a f u n c t i o n o f t h e e x t e r n a l amino a c i d c o n c e n t r a t i o n , s a t u r a t i o n k i n e t i c s were o b s e r v e d . For example, t h e k i n e t i c s o f p h e n y l a l a n i n e i n c o r p o r a t i o n i n t o g r o w i n g c e l l s o f IP. a e r u g i n o s a i n the p r e s e n c e o f s t r u c t u r a l l y s i m i l a r amino a c i d s has been d e m o n s t r a t e d ( F i g . 7 ) . 14 D o u b l e - r e c i p r o c a l p l o t s o f t h e C-amino a c i d i n c o r p o r a t i o n d a t a showed t h a t t h e o b s e r v e d i n h i b i t i o n s o f amino a c i d u ptake by s i m i l a r amino a c i d s , were c o m p e t i t i v e . The c o m p e t i t i v e i n h i b i t i o n o f l e u c i n e u ptake by v a l i n e a t two d i f f e r e n t v a l i n e c o n c e n t r a t i o n s i s shown i n F i g u r e 33. F i g u r e 34 d e m o n s t r a t e s the c o m p e t i t i v e 14 12 i n h i b i t i o n o f C - l y s i n e t r a n s p o r t by. C - a r g i n i n e and F i g u r e 14 35 d e m o n s t r a t e s t h e i n h i b i t i o n o f C - p h e n y l a l a n i n e u p t a k e by 12 C - t y r o s i n e . T h e r e f o r e , t h e o b s e r v e d i n t e r a c t i o n s between s t r u c t u r a l l y r e l a t e d amino a c i d s f o r t h e i r r e s p e c t i v e t r a n s p o r t systems was shown t o be c o m p e t i t i v e . 3. S p e c i f i c and g e n e r a l t r a n s p o r t systems F e r r o L u z z i - A m e s ( 1 9 6 4 ) , and Grenson (1966), d e m onstrated t h a t more than one permease may f u n c t i o n i n the t r a n s p o r t o f a p a r t i c u l a r amino a c i d i n m i c r o o r g a n i s m s . When the r e l a t i v e degrees o f c o m p e t i t i v e i n h i b i t i o n were examined i n l i g h t o f p r e d e t e r m i n e d t r a n s p o r t r a t e s , v a r i o u s i n c o n g r u i t i e s became a p p a r e n t . 90 Fig. 33- Competitive i n h i b i t i o n of leucine uptake by 1 2 C - v a l i n e . Rates of C-leucine incorporation were calculated from samples taken at 15 and 30 sec. 9 1 10 g 8 o \ 6 co UJ 4 _j o =* ^ 2 8 Lysine I MxlO" [ LY§] . .-6 12 16 14, Fig. 34. Competitiveinhibition of C-lysine uptake by C-arginine; •-• . ^ - l y s i n e uptake; 0-0, -^C-Jysine in the presence of 10"^ M C-arginine. 0 92 [PHE] 14 Fig. 35. Competitive^inhibition of (^phenylalanine uptake by C-^yrosine. - , C-pheny1alanine uptake. 0-0, C-phenylalanine uptake in the presence of 10"^ M 1 2C-tyrosine. 0-0, 1^C- phenylalanine uptake in the presence of 2 x 10-Z* M C-tyrosine. a) Basic amino acids Arginine transport was the most e f f i c i e n t system found with the basic amino acids (Table I ) , and arginine was incorporated nearly twice as fast as lysine. However, arginine inhibited lysine incorporation only s1ightly.(Fig. 31, Table VII). These results are construed as being evidence for the m u l t i p i i c i t y of high a f f i n i t y permeases operative with the basic amino acids. 12 Arginine does not compete as e f f e c t i v e l y as C-lysine for the lysine s p e c i f i c permease, but since arginine does show some competition for the permease, then arginine must also be recognized, with a lower a f f i n i t y , by the lysine transport system. The degree of competition of arginine for the lysine permease was, as expected, a 1inear function of the log of the arginine concentration (Fih. 36). By extrapolation, i t was concluded that k x 10. mumoles/ml of arginine would be necessary to competitively i n h i b i t lysine incorporation completely. These results confirm the existence of a separate basic amino acid permease which recognizes arginine with less a f f i n i t y than lysine. When the basic amino acids were used to compete against one another for their respective transport functions, various degrees of competition were observed. From Table VII i t can be seen that there exists two high a f f i n i t y permeases for the basic amino acids. It can also be seen from the 1ist of amino acids recognized by these permeases (Table XI) that for permease, bas 1, Table VII. Competitive i n h i b i t i o n of basic amino acid uptake. Percent decrease in the incorporation rate of ^C-Compet i tor 14 14 14 C-^Lysine C-Arginine C-Histidine 10" M 10" M Ornithine 33.4 31.6 83.2 C i t r u l l i n e 19.1 0 73.5 Arginine 66.7 99-5 87.7 Histidine 14.1 0 73.7 Lysine 99.7 0 64.9 95 Fig. 36. Inhibition of ^ C - l y s i n e uptake by C-arginine. C-lysine (10~ M) and ^ 2C-arginine at various concentrations were added simultaneously to a growing cel1 suspension. 96 which transports arginine, h i s t i d i n e , lysine, ornithine, and c i t r u l 1 i n e , the existence of a primary amine other than the a-amino group would seem to be the factor most important for recognition by this transport system. Permease, bas 2, appears to be s p e c i f i c for arginine and ornithine. However, the nature of the ornithine i n h i b i t i o n has not as yet been investigated and i t may, in fa c t , be non-competitive. It was concluded that h i s t i d i n e was transported p r i n c i p a l l y by both permeases since most of the 14 basic amino acids were more competitive for C-histidine transport 12 than was C-histidine i t s e l f . The transport of h i s t i d i n e i i i P. aeruginosa markedly d i f f e r s from that reported for S. typhirhurium (FerroLuzzi-Ames, 1964), where h i s t i d i n e transport was shown to be mediated by a s p e c i f i c high a f f i n i t y permease and a low a f f i n i t y , non-specific aromatic permease. b) A l i p h a t i c amino acids Again, incongruity was found between.the competition data for amino acids and the rate of transport (Table I) with the a l i p h a t i c amino acids. Of the a l i p h a t i c amino acids tested, leucine was transported at the fastest rate but i t was not as effective a competitive in h i b i t o r for isoleucine uptake as was valine or isoleucine. When the various a l i p h a t i c amino acids were tested against one another for re l a t i v e degrees of competition for the transport system, i t was found that the results.were not compatible with the existence of multiple permeases within this family of amino acids. The transport competition data• (Table'VIM), showed Table VIII. Competitive i n h i b i t i o n of a l i p h a t i c amino acid uptake. Percent decrease in the 1 ? incorporation rate of Competitor^ ] k ^ ^ C-Alanine C-1soleuci ne C-Valine C-Leuci ne - 4 . 10 M 1 0 " 6 M Alan i ne 99.1 59.1 62.1 . 4 7 . 6 Isoleucine 7 3 . 6 9 5 . 5 9 9 . 0 9 5 . 2 Valine 7 9 . 2 8 8 . 2 9 9 . 2 8 8 . 0 Leuci ne 41.6 5 6 . 9 6 0 . 2 2 3 . 8 Meth ioni ne _a) 19.4 - - Methionine 10-3 M - 40.0 Not tested. 98 Table IX. Competitive i n h i b i t i o n of aromatic amino acid uptake. 12 C-Competitor Percent decrease in the incorporation rate of 14 14 14 C-Phenylalanine C-Tyrosine C-Tryptophan -4 10 M 10" 6 M Phenylalanine Tyros ine Tryptophan 94.2 88.4 73.8 98.2 97.0 94.4 53.9 52.0 88.2 that a single a l i p h a t i c permease existed which strongly recognized isoleucine and valine and, to a lesser degree, alanine and leucine. The order of the a f f i n i t y of the a l i p h a t i c amino acids for the transport system is shown in Table IX. This interpretation, at f i r s t , would seem incongruous with -6 the rapid transport rate for leucine at 10 M (Table l ) ; however, these transport rates were determined at a single amino acid concentration and are of value as approximate comparisons between amino acids. As such, in no accurate way do these incorporation rates describe the rel a t i v e kinetic parameters for the uptake process. Competition data r e f l e c t r e l a t i v e orders of amino acid a f f i n i t y for the permease but do not accurately r e f l e c t r e l a t i v e transport rates since the turnover numbers for amino acids being transported into the c e l l by a common uptake system may d i f f e r by a large factor whereas the apparent a f f i n i t y constants may d i f f e r only • s l i g h t l y . Alanine was also shown to enter by a permease recognizing the neutral amino acids. From competition studies (Table X), i t was found that this transport mechanism recognized alanine most e f f i c i e n t l y and to a lesser degree glycine, serine, and threonine. c) Aromatic amino acids FerroLuzz?-Ames (1964), demonstrated that Salmohella  typhirhurium possessed s p e c i f i c high a f f i n i t y permeases for h i s t i d i n e , phenylalanine, tyrosine, and tryptophan as well as a general permease for aromatic amino acids. The results obtained Table X. Competitive i n h i b i t i o n of neutral amino acid uptake. Percent decrease in the incorporation rate of C-Compet i tor • 14 Ak Ak C-Alanine C-Glycine C-Serine -k -6 10 M 10 M Alanine 92.9 76.1 82.1 Glycine 73.9 77.6 75.1 Serine 50.2 17.7 74.2 Threonine 40.0 .10.2 53.4 101 with P_. aeruginosa d i f f e r largely with respect to the s p e c i f i c i t y of the permeases for aromatic amino acids. Of the aromatic amino acids, tryptophan exhibited the greatest transport rate in this organism (Table I ) ; however, this amino acid did not compete 14 14 for uptake of G-phenylalanine or C-tyrosine to the same 12 12 degree as C-phenylalanine or C-tyrosine. Again another permease, less s p e c i f i c for tryptophan, must be operative which mediates aromatic amino acid transport in P. aeruginosa. In accordance with the other permeases described so f a r , this permease w i l l be referred to as aro 2 (Table XI). Tryptophan does compete for the second aromatic permease, although not as e f f i c i e n t l y as tyrosine or phenylalanine. From the competition data of Table XI, i t can be seen that the simplest description of the a c t i v i t y of permease aro 2, is that i t recognizes phenylalanine and tyrosine with high a f f i n i t y and tryptophan with a lower a f f i n i t y . However, aro 1 was found to be more s p e c i f i c for tryptophan than for phenylalanine or tyrosine; although these l a t t e r two amino acids competitively inhibited i t s a c t i v i t y , they did not do so to the 12 same degree as C-tryptophan. 3. The iso l a t i o n and properties of transportless mutants The isolation of transport negative (Tr. ) mutants for amino acids was attempted by several methods. F i r s t , attempts to isolate a proline Tr mutant by the method of Lubin and Kessel (1962), proved f r u i t l e s s since P. aeruginosa was found to be strongly Table XI. Amino acid permeases of P. aerug?hosa. FAMILY PERMEASE AMINO ACIDS a) 102 Speci f i c Neutral Sulfur A l i p h a t i c Aromatic Bas ic sp 1 aspartate sp 2 glutamate sp 3 methionine sp h proline nt 1 alanine glycine serine threonine sul 1 cysteine cystine al 1 isoleucine valine alanine leucine a ro 1 phenylalanine tyros i ne tryptophan aro 2 tryptophan phenylalanine tyrosine bas 1 . arginine ornithine h i s t i d i n e bas 2 lysine ornithine arginine c i t r u l l i n e h i s t i d i n e Ami no acids are 1i sted in decreas i ng orders of a f f i n i t y for their respective permeases. 103 resistant to p e n i c i l l i n . Adaptation of the method by growing mutagenized cultures of a proline auxotroph of P. aeruginosa in low concentrations (1 yg/ml) of the required amino acid with subsequent enrichment by growth recycling in the presence of 400 yg/ml dihydrostreptomycin, only resulted in the isol a t i o n of strains resistant to the effects of the a n t i b i o t i c . Since some resistant strains of microorganisms have been shown to be transportless for the natural amino acid and the analogue, attempts were also made to isolate mutants resistant to the following amino acid analogues: thiazolidine-2-carboxylic acid (thioproline), 3,4-dehydroproline, canavanine, ethionine, thiosine, p-fluorophenylalanine, and 5-methyl tryptophan. However, growth experiments indicated that P. aeruginosa, unlike most microorganisms, was resistant to low concentrations of these analogues. Consequently, attempts were made to isolate strains resistant to very high concentrations of the analogues. Thirty resistant colonies isolated with each analogue were f i r s t tested for derepression of amino acid synthesis by cross-feeding experiments with the appropriate auxotroph. None of the mutants tested were derepressed for the synthesis of the amino acid and, therefore, were not excretor mutants. However, amino acid uptake experiments with these mutants did not reveal any s i g n i f i c a n t differences r e l a t i v e to the wild-type s t r a i n . No attempts were made to isolate strains resistant to 5-methyl-tryptophan because this organism was found to u t i l i z e the amino acid for growth even at very high concentrations. The resistance to most of these amino acid analogues was thought to be due to the presence of MINUTES ii> + Fig. 37. Uptake of _ C-prollne at 30 C by M and proline Tr mutant strains (P5, P6) of P. aeruginosa. constitutive oxidizing enzymes active on the analogues as well as the amino acids. Cell suspensions incubated with thioproline grew at a normal rate and catabolized this analogue. The t h i r d method tested was designed to take advantage of the c e l l s a b i l i t y to catabolize ami no acids. It was reasoned that c e l l s defective for the transport of a single amino acid would grow either slowly or not at a l l when plated on s o l i d media containing that amino acid as the sole carbon source. When colonies isolated in this manner were routinely screened for 14 the incorporation of C-amino acid added at low concentrations, i t was found that the mutants f e l l into two general classes. 14 -.6 Figure 37 represents the uptake of C-proline at 10 M by the wild type and two strains isolated by this method. One mutant, P5, in which the transport a b i l i t y for proline was altered approximately 10 f o l d , was isolated. Most mutants isolated by this method, however, exhibited transport rates approximately the same as mutant P6 (Fig. 37). Mutant P5 incorporated al1 14 other amino acids rapidly from a C-protein hydrolysate and only f a i l e d to incorporate proline at a s i g n i f i c a n t rate. Therefore, this mutation was considered s p e c i f i c for the proline transport system and, as such, agreed with amino acid competition data (Fig. 31B). Table XII l i s t s the mutants isolated by this method for the various groups of amino acids. Again, most transport defective mutants obtained in this manner were found to be only p a r t i a l l y defective when tested at low substrate concentration. Only one transport defective mutant (A5) was isolated for the basic amino 106 Table XII. Transport negative ( T r ) mutants of •£_. aerug i hosa; MUTANT DEFECTIVE PERMEASE P5 sp k (proline) P6 sp k P9 sp U P11 sp k AS bas 1 TA3 aro 2 TA10 aro 1 IB9 al 1 107 acids. When tested for the a b i l i t y to incorporate arginine, lysine, and h i s t i d i n e the mutant was found to be more defective for lysine transport than for arginine transport. Only pa r t i a l reduction of uptake with these amino acids was expected, however, due to the presence of multiple permeases. This mutant could, therefore, be c l a s s i f i e d as a lysine transportless mutant with a defect for the amino acid permease bas 1. Unexpectedly, A5 transported h i s t i d i n e more e f f e c t i v e l y than did the wild-type s t r a i n . The reason for this has not been determined, however, Grenson (1966), reported identical results with a canavanine insensitive, transport negative mutant of S. cerevisiae which was isolated by resistance to the arginine analogue canavanine. It was also shown by the competitive i n h i b i t i o n of arginine transport that, in this yeast, h i s t i d i n e was incorporated on the basic amino acid permease. Of the two aromatic transport mutants examined, TA3 behaved as a complete transport negative mutant analogous to P5, the proline Tr mutant. This mutant was most strongly defective for the tryptophan permease suggesting that the aromatic permease aro 2 was defective but that the test amino acid was slowly incorporated via permease aro 1. This interpretation was corroborated by the is o l a t i o n of Tr mutant TC10 which transported tryptophan at nearly the same rate as the wild type s t r a i n and was only moderately defective for phenylalanine or tyrosine transport. The transport defect could be most simply described as a mutation at the level of permease aro 1. Two mutants for the a l i p h a t i c uptake systems were also 108 isolated. The f i r s t , however, was found to be equally defective for a l l a l i p h a t i c amino acids as well as unrelated amino acids. This mutant, IBI6, may possess a general membrane defect affecting transport function (Stadtler, 1967), but i t was not analyzed in d e t a i l . The second mutant isolated, IB9, was found to be defective only for transport of the a l i p h a t i c amino acids. When multiple high a f f i n i t y permeases function to transport a particular amino acid, then i t is obvious that the transport rate observed with growing cultures would represent the cumulative rates of transport between at least two d i s t i n c t transport systems. IV. Competition for the Amino Acid Pool Britten and McLure (1962), and Rickenberg and Cohen (1956), showed that the st r u c t u r a l l y similar a l i p h a t i c amino acids could competitively displace one another from pre-established amino acid pools in E. co1i. This exchange process was carried out at 0 C where incorporation of the competitive amino acids was negligible and where preformed pools were stable. Without exception, the amino acid pools of P. aeruginosa rapidly effluxed at 0 C; however, the rate of transport of most amino acids with the exception df proline, was found to be markedly reduced at 10 C (Fig. 6), and exchange reactions were, therefore, performed at this temperature. Figure 38 demonstrates 14 12 12 the exchange of a preloaded C-valine .pool by C-valine, C-isoleucine, 12 12 12 -4 C-leucine, C-alanine, and C-methionine, each added at 10 M to the s t a b i l i z e d , preloaded culture. When rates of exchange were 4 6 8 1 0 M I N U T E S 14 F i g . 36. The displacement of the C-valIne pool by structural ly related amino aelds. «*C-amlno acids were added at 1Q~%1. The valine pools were pro-established tn the presence of chloramphenicol by extended incubations at 10 C, no Table XIII. Exchange of the a l i p h a t i c amino acid pools. % of Pool exchange/min with Pool amino acid Leucine Isoleucine Valine Alanine 10 M Leucine 0 10.7 4.3 0 Isoleucine 3.1 28.6 20.1 0 Valine 2.2 21.7 20.8 0 Table XIV. Exchange of the preformed basic amino acid pools. % of Pool exchanged/min with Pool amino acid Lysine Arginine Histidine 10 M Lysine 28.2 31.8 21.0 Arginine - - Histidine 14.3 23-3 17.2 Arginine pools were not formed due to the secondary effects of putrescine accumulation. calculated for pools preloaded and exchanged with the different a l i p h a t i c amino acids (Table XI l l ) , i t was obvious that a very close correlation existed between the amino acid uptake and competition data (Table VIII). Both valine and isoleucine exchanged readily with pools preloaded with these amino acids, and isoleucine exchanged with the C-leucine pool more readily than valine. Neither alanine, methionine, nor lysine exchanged with pre-established a l i p h a t i c amino acid pools. Clearly from these observations i t can be seen that the exchange process for the a l i p h a t i c amino acids was stereospecific for members of this family of amino acids, and followed the same competitive relation ships as the uptake process. Leucine was somewhat of an exception as, although i t was incorporated at a high rate in growing cultures (Table I) and was found to exchange isoleucine and valine pools rather poorly, i t would not exchange with i t s own preformed pool. Similar exchange data were obtained for the basic amino acids (Table XIV). The h i s t i d i n e pool was less e f f i c i e n t l y exchanged by hi s t i d i n e than by arginine. The lysine pool was most readily exchanged by arginine and lysine and to a lesser degree by h i s t i d i n e . The displacement of amino acid pools with the basic amino acids was shown to be family s p e c i f i c and closely correlated with the rel a t i v e a f f i n i t i e s for the uptake system. Aromatic amino acid pools were also displaced only by members of this family of amino acids. A l l three aromatic amino acids e f f e c t i v e l y displaced pre-established phenylalanine or tyrosine pools, but only tryptophan displaced i t s own pool (Table XV). It appeared, generally, that the displacement of an amino acid from .113 Table XV. Exchange of the aromatic amino acids. Pool amino acid % of Pool exchanged/10 min with Phenylalanine Tyrosine Tryptophan -4 10 M Phenylalanine 8 4 . 2 51.3 31.1 Tyrosine 78 . 3 7 7 . 9 2 6 . 7 Tryptophan 4 . 6 5.2 7 2 . 7 the pool of P. aeruginosa occurred by a process closely related to the uptake systems and seemed to be strongly influenced by both the structural relatedness of the exogenous amino acid, the pre-established ami no acid in the pool, and the a f f i n i t y constants of the transport system for the amino acids involved. Therefore, the displacement of amino acids from the pool is a function of the uptake process and also of a family s p e c i f i c e x i t process which may d i f f e r from the uptake process by i t s relative a f f i n i t i e s for the amino acids which constitute a family. Thus, whereas phenylalanine or tyrosine pools are displaced e f f e c t i v e l y by tryptophan, the tryptophan pool is displaced very slowly, i f at a l l , by these am?no acids. V. Kinetics of Amino Acid Transport at High Substrate Concentrations From a calculation of the maximum amount of amino acid capable of entering P. aeruginosa at concentrations saturating the high a f f i n i t y permeases ( i . e . at V ), i t was deduced that the amino ' max ' acid would not approximate the necessary carbon requirements for normal growth. The wild-type s t r a i n catabolized nearly a l l the commonly occurring amino acids (Table XVI), and i t was a natural assumption that to serve as growth substrates the amino acids would have to enter the c e l l perhaps by mechanisms other than those revealed at low amino acid concentrations. The kinetics of amino acid uptake determined at high substrate concentrations revealed the presence of a second permease with a greatly reduced a f f i n i t y for the amino acid. Figure 39 i l l u s t r a t e s the kinetics of both 115 ) 0 . 2 0 0. 15 0.10 0.05 / O i / - 5 1 2 E 6 o ^ 4 E ac E 10 20 30 40 50 mjjqnoles Glu/ml 0,05 .0.10 QI5 P E G 1 M X I 0 " 6 0.20 0,25 Fig. 39. Kinetics of glutamate uptake at 30 C with varying concentrations of C-glutamate. Lineweaver-Burk plot. Inset: saturation ki net ic s . 116 the high and low a f f i n i t y permeases for the transport of glutamic acid. At concentrations exceeding 10 ^ M, the second permeabi1ity mechanism becomes the more important means of amino acid entry into the c e l l . From the kinetics of amino acid uptake at high amino acid concentrations (exceeding 2 x 10 ^ M) , i t was found that low a f f i n i t y permeases or transport mechanisms were operative for proline, arginine, and leucine incorporation, but none was evident for phenylalanine incorporation. It was, then, reasonable to assume that these probably are the permeability mechanisms which supplied the c e l l with high amino acid concentrations for catabolism. FerroLuzzi-Ames (1964), also demonstrated a non-specific aromatic permease in S. typhirhurium which was operative at high amino acid concentrations. However, S. typhirhurium does not catabol ize the aromatic amino acids and the actual function of this permease remains a mystery. The nature of the ster e o s p e c i f i c i t y of the low a f f i n i t y permeases has not been investigated. It is postulated that permeability defects such as those i l l u s t r a t e d by.mutant P6, a proline Tr s t r a i n , are in fact mutations at the level of the low a f f i n i t y permease; however, at present no data are available to substantiate t h i s . VI. Control of Ami ho Acid Transport Very few clear demonstrations of the induction of permeability functions in bacteria have been reported. Lyon e t a j _ . (1967), found that glutamate transport was induced to high levels in 117 Mycobacteriurn smegma11s and M_. tuberculosis when grown In the presence of glutamic acid, but the nature and properties of the induced and uninduced glutamate transport systems were not thoroughly investigated. De Hauwer, Lavalle, and Wiame (1964), observed that the arginine incorporation process of Baci11 us  siibti 1 is was induced by growth en arginine and also that a mutant derepressed for the catabolism of arginine was coordinately derepressed for the arginine transport function. This suggested that the control of permeability was linked to catabolism, not anabolism. FerroLuzzi-Ames (1964), found that the h i s t i d i n e transport system was neither induced nor repressed by the presence of h i s t i d i n e , and concluded that the gene for the uptake system was not within the h i s t i d i n e operon. High rates of proline transport were found to be inducible in P. aeruginosa. Growth in minimal medium containing 0.1% proline increas'ed the rate of the transport of proline when tested at low substrate concentrations. The concomitant addition of high concentrations of chloramphenicol and proline to a logarithmic phase culture prevented the induction of proline transport above the constitutive l e v e l . Figure 40 demonstrates the incorporation 14 -6 rate of C-proline at 10 M into the pool of induced and noninduced aeruginosa. The control c e l l s were also preincubated with chloramphenicol prior to the i n i t i a t i o n of the transport experiment in order to normalize any secondary effects of the inh i b i t o r on the transport process. The kinetics of proline incorporation in induced and noninduced c e l l s are shown in Figure .41.. The a f f i n i t y constant for both induced and noninduced c e l l s was of the order-Proline o 1 2 3 4 5 M I N U T E S Fig. 40. Tim court* of pool tarnation at 30 C * ' t n f> ••roalnoia previously grown In minimal MCIIUM In the pretence or absence of 0.1) proline. 119 [PRO] ig. 41. Kinetics of proline uptake ih P_. aeruginosa. C - 8 , Cells were grown in minimal medium. 0-0, Cells were grown in minimal medium plus 0.1% prol ne. 120 of 2 x 10" 7 M. Although the control of the Incorporation mechanisms for other amino acids has not been investigated, some evidence gained from the study of transport mutants suggested that the control of amino acid transport i i i P. aeruginosa is quite general. Four mutants of P. aeriigihosa unab 1 e to catabolize arginine as a carbon source were able to transport arginine at increased rates re l a t i v e to the wi1d-type s t r a i n . Although the nature of these mutations has not been thoroughly investigated, i t is presumed that a lesion in the arginine degradative pathway resulted in the accumulation of an inducer for the transport system. S i m i l a r l y , two mutants (TC2 and TC12) were isolated which catabolized tyrosine poorly. These mutants also were able to transport tyrosine at a rate at least twice as fast as the wild-type s t r a i n . No investigation of the ste r e o s p e c i f i c i t y of these transport alterations has yet been attempted. VII. Amino Acid Transport and Pool Formation in Starved Cel1 Suspens ions 1. Amino acid transport When E. c b l i eel 1s were starved for glucose or maintained at 0 C, no si g n i f i c a n t pool losses were observed; in fac t , highly concentrated amino acid pools were found to be stable for many hours under these conditions (Britten and McLure, 1962). In direct contrast to these observations, the amino acid pools of P. aeruginosa were found to be rapidly depleted during carbon or nitrogen starvation. When c e l l s were prelabelled by growth on high s p e c i f i c 121 Table XVI. Growth of P. aeriigihbsa on amino acids as carbon or nitrogen sources. Amino Acid Nitrogen Carbon Source Source Alan i ne + + Arginine + + Asparag i ne + + Aspartate + + Cysteine + - Glutamate + + Glutamine + + Glycine + H i st id i ne + + 1soleuci ne + + Leuci ne + + Lysine + + Methionine - - Phenylalanine + + P r o l i ne + + Seri ne + - Threonine + - Tryptophan + + Tyros i ne + + Valine + + + no growth slow growth after 48 hr growth at 24 hr a c t i v i t y C-glucose, and then allowed to exhaust the exogenous glucose, the native amino acid pool was rapidly depleted. Radioautography of the extracted pool and culture supernatant f l u i d revealed that a l l amino acids, with the exception of methionine, had been removed from both the culture supernatant f l u i d and the i n t r a c e l l u l a r pool. Unlike most microorganisms, members of the genus Pseudomonas have very simple nu t r i t i o n a l requirements and catabolize a wide range of substrates (Stanier e_t aj_., 1966). P. aerugihosa can u t i l i z e any of the common amino acids as a sole nitrogen source and most of them as a sole carbon source (Table XVI). It was obvious then, that in the absence of glucose or other non-nitrogenous energy sources, P. aeruginosa undoubtedly catabolizes the residual amino acids present exogenously and in i t s pool. These preliminary results suggested that the catabolism and perhaps even the transport of amino acids might be influenced by the presence of an oxidizable energy source such as glucose. Transport rates for amino acids representative of various amino acid families were determined at frequent time intervals during periods of either carbon or nitrogen starvation. With most.of the amino acids tested, no drastic impairment of transport a b i l i t y was evident over the starvation period (Table XVI l ) . The rates of glycine, alanine, and glutamate transport increased to levels twice as high as those found with non-starved suspensions (Fig. Ml). The opposite effect was observed with members of the al i p h a t i c amino acids for the rate of transport of these amino Table XVII. Effect of carbon deprivation on amino acid transport. Rate of amino acid transport percent of control Amino acid Control Non-starved Starved 90 min Re-fed Alanine 100 172 92 Glutamate 100 222 110 Glycine 100 120 100 Leucine 100 42 101 Valine 100 64 104 Phenylalanine 100 106 100 Proline 100 95 102 Lysine 100 98 95 Glucose was added to the culture 60 sec prior to the determination of transport rate. a o \ z A oo 10 9 8 7 6 30 60 90 MINUTES 14, 120 60 90 MIN U T E S 120 -C- Fig. 42. The change in ' 'C-glutamate transport ih P_. aeruginosa during nutrient deprivation. Samples were removed at zero time and at various intervals during: A Carbon starvation; B Nitrogen starvation. 120 The change in were removed at 5 0 100 M I N U T E S 150 C-valine transport in P. aeruginosa during nutrient deprivation S a m n l c c zero time and at various exte various externals during: A carbon starvation;'B nit?ogIn starvation. 126 3 0 6 0 9 0 S E C O N D S Fig. M. Rate of transport of C-val In* Into who!* ea l U °' p« a*ruglnoaa. Symbols: t, nitrogen starved celTs; 8. nitrogen starved c e l l s with 7.5 K \0"h (NHjJjSCty added at zero tine. 127 100 a: 0, 8 0 6 0 or 4 0 ui < or 2 0 4 6 H O U R S S T A R V A T I O N 8 10 Fig. *5. Proline transport In P. aeruginosa P22 during carbon starvation. Rate* of '*C-prolIne transport wars dataralnad at Intervals during the starvation period and ara expressed as percentage of tha non- starved control. 128 acids decreased s i g n i f i c a n t l y during carbon or nitrogen starvation (Fig. 43). The amino acid transport rates demonstrated in Figure 42 and 43 were altered by a factor of approximately two. Extended periods of starvation caused no further change. From the results in Table XVII i t can be seen that short pre incubation with the deprived nutrient.effectively restored the transport rate to levels observed prior to starvation. The time 14 course for the restoration of C-valine transport by the addition of ammonium sulfate to c e l l s previously starved for nitrogen is shown in Figure 44. The nature of the maintenance of amino acid transport functions during extended periods of nutrient deprivation was investigated further. Cultures were deprived of an energy source and transport 14 a b i l i t y for C-proline was observed at time intervals up to ten hours. Only minor changes in the efficacy of proline transport were observed (Fig. 45), and this was presumably due to c e l l death. It was obvious from a consideration of protein turnover rates (Mandelstam, I960), that by the end of the starvation period i t was l i k e l y that over 50% of the c e l l u l a r protein had been subjected to turnover. Clearly then, the amino acid transport proteins in P. aeruginosa were exempt from turnover. It was reasonable to assume that the selective maintenance of these transport systems during extensive nutrient deprivation served some physiological function. As a resu l t , the influence of carbon or nitrogen starvation on pool behaviour was investigated. 129 Supernatant 20 30 TES Total Protein 40 50 F i g . 46. Uptake of C-prolln* Into whole c e l l s and protein of P. aerogloose during carbon s t a r v a t i o n . " C e l l suspensions were starved fo r carbon f o r 45 min p r i o r to the addition of 10*6M l«X-prol!ne. MINUTES Fig* *7« Uptake of C-prollna Into jP. aeruginosa calls and coll fractions. Calls ware starved for nitrogen for 45 « l n prior to the addition of l<r*M "C-protlne. 131 2. Pool formation and maintenance during carbon or nitrogen starvation a. Time course of amino acid incorporation When proline uptake was followed in cultures that were starved for carbon, the pools formed from the exogenous amino acid were found to be metabolically unstable (Fig. 46). The i n i t i a l incorporation of label was rapid and the resulting high pool level formed within 2.5 min was subsequently lost due to both catabolism and protein synthesis. The r e l a t i v e l y high loss of ra d i o a c t i v i t y was unusual and the results suggested that the catabolism of proline must be regulated in some manner by the presence of glucose. When cultures were starved for nitrogen and time course 14 experiments for C-proline incorporation carried out, quite different results were obtained (Fig. 47). Again the i n i t i a l uptake of proline was rapid, and a very large transient pool was formed at 2.5 min, due primarily to the reduction in the rate of protein synthesis. However, this large amino acid pool was not depleted by catabolism but a s i g n i f i c a n t fraction of the pool was excreted back into the medium. A l l the rad i o a c t i v i t y not incorporated into the c e l l could be accounted for in the culture supernatant f l u i d . It was obvious then that the fate.of the i n t r a c e l l u l a r amino acid pool differed depending on the nature of the starvation. These results 1 0 1 - -J - i U J o I/- Z 4 2 o M I N U T E S 10 15 M I N U T E S Fig. 48. Time course of 1*C-phenyla1anine uptake into c e l l s and c e l l fractions during nutrient deprivation. (A). During carbon starvation CB) Durinq nitroqen starvat ion. . MINUTES MINUTES Fig. 49. Tim course of C-proltne uptake In celt suspensions of £> aeruginosa F22 during nutrient deprivation. (A) During carbon starvation (B) During nitrogen starvation. 134 reinforced the observation that the catabolism of amino acids was controlled by the presence of glucose. Phenylalanine transport was also r e l a t i v e l y unaffected by carbon or nitrogen starvation; however, this amino acid is catabolized r e l a t i v e l y slowly by P. aeruginosa, and as a res u l t , phenylalanine was selected for pool behaviour studies during 14 nutrient deprivation. As was demonstrated for proline, the C- phenylalanine which entered the pool of carbon starved c e l l s was rapidly oxidized (Fig. 4 8 A ) , and only.31?'. of the added label was recovered at the end of the experiment. When a similar experiment was carried out under conditions of nitrogen starvation (Fig. 48B), 14 the results differed considerably from the experiment with C- proline. Quantitative recovery of ra d i o a c t i v i t y again indicated that the oxidation of phenylalanine was repressed by glucose or a product of glucose degradation. Approximately 10% of the added label was recovered in the c e l l s and the rest was found in the culture supernatant f l u i d . The pool was devoid of any ra d i o a c t i v i t y . Time course experiments with a mutant, P22, which was unable to catabolize proline as a carbon source, demonstrated that carbon or nitrogen deprivation caused no secondary alterations of the i n t r a c e l l u l a r pool (Fig. 49). With this organism, al1 the 14 incorporated r a d i o a c t i v i t y as C-proline was accounted for by the label found in the protein fraction at the end of the experiment either under conditions of carbon or nitrogen starvation. The pool was found to be stable under these starvation conditions. This mutant grew well on minimal medium, thereby indicating that the synthesis and catabolism of proline in P_. aerug?nosa are not Table XVIII. Fate of C-amino acids incorporated into nitrogen starved c e l l s of P. aeruginosa. Recovery 3' % C in Amino Acid of ^C Hydrazohes Hydrazones C-Glutamate 85.6 Pyruvate 81.4 a-ketoglutarate 18.6 4 C-Aspartate 92.5 Pyruvate 90.5 a-ketoglutarate 9-5 h C-Alanine 98.5 Pyruvate 89.3 a-ketoglutarate 10.7 C-Proline 90.5 Pyruvate 65.5 a-ketoglutarate 34.5 14 Percent recovery of C from supernatant f l u i d Table XIX. Fate of C-amino acids incorporated into nitrogen starved ce l l s . 14_ . . . . , % of Recovered 1*C % To t a l 3 ' C-Amino Acid n — Recovery Deaminated Non-deaminated Lysine 83.6 Histidine 31.1 Phenylalanine 88.0 Isoleucine 95.1 16.4 87.6 68.9 93.2 12.0 83.3 4.9 95.0 Percent recovery of total C from culture supernatant f l u id . 137 mediated by the same enzymes. b. Fate of the amino acid pool It was assumed that the amino acids added to nitrogen starved c e l l s were being deaminated. Two groups of amino acids were examined to determine the nature of the products excreted during nitrogen starvation. The f i r s t group of amino acids included proline, glutamate, aspartate, and alanine. These were selected as being amino acids which could be degraded easily due to their close relationship to main catabolic pathways in this microorganism. The second group of amino acids included lysine, h i s t i d i n e , phenyl alanine, and isoleucine, and these were considered to.be more remote from such pathways. Typical amino acid uptake experiments were carried out with each amino acid under conditions of nitrogen starvation. Culture supernatant f l u i d s were collected after centrifugation and were analyzed either by extraction of reacted 2,4-dinitrophenylhydrazohes, or by elution from a Dowex 50 (H + form) column. The results of these experiments are l i s t e d in Tables XVIII and XIX. A l l of the rad i o a c t i v i t y from the amino acids of the f i r s t group (Table XVIII) was extracted as the keto-acid hydrazones. It was, therefore, concluded that these amino acids were rapidly deaminated and the 14 12 resulting C-keto-acids were exchanged with exogenous C-keto- acids which had accumulated during glucose catabolism (McKelvie, 1965). 138 Culture supernatant f l u i d s from the second group of amino acids were passed through Dowex 50 (H + form) columns, and in each case, essent i a l l y al1 of the label passed through the column, indicating that the amino acids had undergone de amination (Table XIX). The only exception to this pattern was h i s t i d i n e , however, the ring nitrogen may not have been completely removed and, as a result, this amino acid would have adsorbed more strongly to the column. Thus, i t appeared that under conditions of carbon or nitrogen starvation, the controls which repressed the a c t i v i t y of enzymes concerned with the oxidation or deamination of incorporated amino acids were released. Control cultures which had been starved for nitrogen for f o r t y - f i v e minutes, were reincubated in the presence of 0.1% (NH^J^SO^ for the same time interval and then were allowed 14 to incorporate C-phenylalanine. It was found that the time course of uptake had been restored to the normal state. The f a i l u r e to excrete s i g n i f i c a n t quantities of label was taken as evidence that the presence of ammonium ions influences the a c t i v i t y of the enzymes which deaminate amino acids. Since amino acids synthesized from glucose accumulate in the native pool of P_. aeruginosa, a l b e i t at low levels, i t would be paradoxical physiologically to simultaneously catabolize these amino acids which are necessary for protein synthesis. Thus the organism undoubtedly evolved control mechanisms which regulate the constitutive levels of amino acid dissimilatory enzymes. However, these same controls were observed to be ineffectual with 139 respect to the high induced levels of enzymes involved in amino acid d i s s i m i l a t i o n , since these enzymes were induced even in the presence,of high glucose concentrations. This too would seem to be physiologically effective and as a result would allow the organism to degrade glucose for energy, pentoses, t r i o s e s , et cetera, and at the same time catabolize ami no acids to supply carbon skeletons for the synthesis of other essential metabolites with a related chemical structure. In conclusion, P. aeruginosa does not maintain the internal pool of amino acids during nutrient deprivation as has been shown for E. cb1i (Britten and McLure, 1962), but rather controls i t s levels of degradative enzymes in such a way as to allow maximum use of both i t s pool amino acids and also amino acids present in the environment in extremely low concentration. VIII. The Mechanism of Amino Acid Transport and Accumul at ion Unlike animal or plant c e l I s , bacteria are able to concentrate low molecular weight metabolites to a very great extent.over the external environment. For instance, Britten and McLure (1962), have reported amino acid concentration ratios greater than 28,000 for E. co1i. As a result of this unique a b i l i t y , concerted ef f o r t s have been directed at elucidating the mechanisms involved in this process. A l l the.avai1 able.evidence concerned with active transport of carbohydrates across biological membranes is consistent with the hypothesis that this process must consist of at least two 140 d i s t i n c t components. The basic components of this process have been outlined by Winkler and Wi1 son (1966), for the transport of g-galactosides into E. c b l i (Fig. 50). The system involves a substrate-specific membrane " c a r r i e r " which f a c i l i t a t e s movement of the substrate across the permeabi1ity b a r r i e r , and also, an unknown mechanism which couples metabolic energy to the c a r r i e r function which ultimately permits a net movement of substrate from the e x t r a c e l l u l a r environment into the c e l l against a concentration gradient. In the absence of energy, the c a r r i e r f a c i l i t a t e s the equilibration of internal and external substrate concentrations. The process is not regarded as simple di f f u s i o n since i t s t i l l demonstrates substrate s p e c i f i c i t y and saturation ki n e t i c s . The glucose transport systems of yeast (Burger, Hajmova, and K l e i n z e l l e r , 1959), and erythrocytes (Wil- brandt, 1963; Le Fevre, 1961), also have been demonstrated to operate in this manner and have been designated as carrier-mediated transport or f a c i l i t a t e d d i f f u s i o n . Various workers have shown, in both animal and bacterial eel 1s, that, when energy metabolism is terminated with s p e c i f i c inhibitors the membrane c a r r i e r remains intact, but the movement of substrates against a concentration gradient is prevented. Thus the active transport systems for carbohydrates were converted to f a c i l i t a t e d d i f f u s i o n . ( B i b l e r , Hawkins, and Crane, 1962; Koch, 1964; Winkler and Wilson, 1966). The transport and accumulation of amino acids in bacteria has received less attention than carbohydrate transport systems 141 OUTSiD! S oui MEMBRANE + C J Kt entry CS ~s INSIDE S in In the steady state.* ENTRY = EXIT Vmax S out Sout + Kt entry FOR FACILITATED DIFFUSION: = Vmax S in S in + Kt exit FOR ACTIVE TRANSPORT: Kt entry = Kt exit Kt.entry <^  Kt exit Fig. 50. Modal for an energy-uncoupled actEvo transport system. S, substrate (designated as Inside or outs Ido the c o l l ) ; C, roerabrane c a r r i e r ; CS, (seinbrene.carrler- .substrate complex through the membrane; Kt, equilibrium constant f o r th© reaction S • C—* CS. i t i s assuisad that (a) the chemical reactions at each Interface are much rcor© rapid than the d i f f u s i o n of c a r r i e r or substrate-carrier complex (b) a li n e a r concentration gradient of both C and CS exi s t s In the membrana (c) fciie d i f f u s i o n constants for C and CS are the 5eras. (Winkler and Wilson, 13&5)* l t : is obvious, however, that in this model Kt should be considered as the a f f i n i t y constant for amino acid-carrier formation, and not the equilibrium constant for this reaction. 142 as the processes have been found to be fraught with complexities. The amino acid accumulation a b i l i t y is less amenable to d e f i n i t i v e experimentation as more than one process is involved; that i s , the accumulation of amino acid pools ultimately is concerned with both the c e l l u l a r synthesis of amino acids and the incorporation of amino acids from the external environment. The active transport of amino acids across membranes has been shown to exhibit some properties common to carbohydrate transport, however, the process also was shown to possess many unique properties. The active transport system for amino acids as visualized for E. c b l i by Britten and McLure (1962), is shown in Figure 51. Similar to sugar transport, amino acids presumably are transported across the membrane by a stereospecific c a r r i e r to the i n t r a c e l l u l a r milieu, where they are then concentrated by association with s p e c i f i c s i t e s . The site-amino acid association was described as being the energy-dependent process in active transport. An even more complex system has been encountered with P_. aeruginosa. As described previously, the organism constantly maintains a low level of native pool amino acids for protein synthesis, at least during periods of nutrient abundance, but 1 i ke E. co1i, this organism can also concentrate amino acids to a very high level above the exogenous concentration. Unlike E . c b l i , however, these c e l l s c o n s t i t u t i v e l y catabolize the accumulated ami no acids especially duri ng carbon or n i trogen deprivation. 143 OUTSIDE A In the steady state- ENTRY(into pool) = EX IT(into protein) and AR+ C *-AC+ R reduces available C. FOR A C T I V E TRANSPORT: K , < < K 2 Flo. 51. The carrier model for amino acid transport. A, amino acid; C, membrane carrier; AC. mobile naembrane carrier- amino acid complex; R, non-mobile sites; Alt, amino ac Id- si t e complex (Britten and McLure, 1962). MEMBRANE +C I K 2 Ki j A C I NSIDE Energy coupled *H Z^ZZZZ AR + C K 4 . Preliminary studies on pool maintenance phenomena demonstrated that internal amino acid pools which resulted from the transport of amino acids added at low exogenous concentrations were effluxed in the absence of energy metabolism (Fig. 2 6 - 2 9 ) . This demonstrated that the a b i l i t y to maintain s i g n i f i c a n t concentration gradients had been interfered with. Such data do not provide insight into the nature of this e f f l u x phenomenon; however, rapid but limited i n i t i a l amino acid uptake was observed when c e l l s were poisoned with NaN^ (Fig. 5 ) . This suggested that the actual transport process may have been unaffected by the i n h i b i t o r and that the a b i l i t y to maintain amino acid pools had been lo s t . In order to pursue this observation further, comparisons of transport rates were made with c e l l s under normal conditions, c e l l s poisoned with NaN^ and iodoacetamide, and with c e l l s maintained at 0 C. Cell suspensions were exposed to a concentration of C-proline which saturated the high a f f i n i t y permease (2 x 10'. -^M) and then f i l t e r e d at f i v e second intervals during the incubation period. Transport against a concentration gradient was completely abolished in the c e l l s treated with NaN^ (Fig. 52). The amino acid, however, rapidly entered the c e l l u n t i l the i n t r a c e l l u l a r concentration was approximately the same as that in the incubation medium. Protein synthesis was completely inhibited by the preincubation with 30 mM NaN^ and 1 mM iodoacetamide. The proline Tr mutant, P5, also had a 1imited transport capacity at these proline concentrations. Preincubation with NaN did not S E C O N D S F i g . 52. Tha uptake of 2 x lo"5M 'Vprollne at 30 C Into HaN- treated and untreated c a l l s of %#l Id-type and proline Tr" mutant (P5) P. aeruginosa. . 146 s i g n i f i c a n t l y a l t e r the transport rate and these inhibited c e l l s only incorporated proline u n t i l the internal proline pool had equilibrated with the external medium. When kinetics of transport into poisoned wild-type c e l l s were determined (Fig. 5 3 ) , i t was observed from incorporation data obtained with fi v e and ten second sampling times, that the a f f i n i t y constant for uptake at low proline concentrations was s i g n i f i c a n t l y reduced, and also that V m a x was s i g n i f i c a n t l y lowered. This suggested that either the high a f f i n i t y transport function had been altered by treatment of the c e l l s with NaN^, or that i n i t i a l rates' were not being measured but rather, a composite of the entrance plus an additional ef f l u x component. The of amino acid entrance was reduced approximately 10 fold and the significance of this reduction in the for uptake w i l l be discussed later. These data are then commensurate with the hypothesis that the uptake or transport of proline occurs by an energy-independent mechanism, and that another energy-dependent function was involved, presumably for the accumulation of the amino acid. The incorporation of certain amino acids was previously shown to be mediated by both high and low a f f i n i t y permeases (Fig. 3 8 ) . Proline transport was not an exception, for this amino acid is incorporated into P. aeruginosa by permeases with widely varying values. It was also demonstrated previously that the a b i l i t y to transport proline was induced by growing c e l l s in the presence of the amino acid (Fig. 40). This presented a working model for studying the effect of increased transport levels on the a b i l i t y 147 o 2 CO p U J .o Km*2.2xlO"6 M 0.1 02 03 Mx 10 r 6 0.4 0.5 [ P R O ] Fig. 53. Lineweaver-Burk plot of proline incorporation into c e l l s of P. aeruginosa. Cell suspensions were preincubated with 30 mM NaN^ +1 mM iodoacetamide for 30 mi n at 30. C pr ior to the .add i t ion of ' C-proline. 148 Induced 20 INUTES 30 Fig. 54. 14 -6 The Incorporation of C-prollne (10 M) Into the pool of Induced and non-Induced ce l ls of P. aeruginosa P22 at 10 C. Induced ce l ls were grown In minimal medium with 0.1? proline. The ce l ls were pre-* Incubated at 30. C for 30 min with 200 ug/ml chloramphenicol prior to the addition of 1^C-prollne. 149 of the c e l l to concentrate amino acids. Mutant P22, a s t r a i n with a lesion in the catabolism of proline, was induced for this transport function, by growth of the organism in the presence of this amino acid. Subsequently, maximum -6 14 pool levels were established from 10 M C-proline in the absence of protein synthesis (Fig. 53). The induced celIs accumulated proline to levels twice those found with the uninduced c e l l s and the resulting concentration gradient increased approximately 10 f o l d . This indicated that the transport a b i l i t y and accumulation function were coordinately induced, or that the increased accumulation capacity was a consequence of increased amino acid transport a b i l i t y . According to the model visualized by Winkler and Wilson (1966) for g-galactoside transport (Fig. 49), an increased influx component would result in an increased concentration capacity of the c e l l , but in their investigation and in the studies of Koch (1964), the additional influx component would be ineffectual in establishing high concentration gradients when active transport was e f f e c t i v e l y converted to f a c i l i t a t e d d i f f u s i o n by poisoning the c e l l s with a metabolic in h i b i t o r such as NaN^. Figure 55 demonstrates that this was also found to be the case -5 14 when induced c e l l s were allowed to transport 2 x 10 M C-proline after preincubation with NaN^. The c e l l s did not accumulate in t r a  cel l u l a r prol ine against a concentration gradient and therefore, the increased transport capacity of P. aeruginosa did not permit 'I 150 S E C O N D S F i g . 55. Uptake of 2 K l<f 5K ! V - p r o I Ine Into t!eH3 treated and untreated c e l l s of previously grown P. aeruginosa. These cols were grown In* mint oat nedluw with the addition of 0.11 proline. i n t r a c e l l u l a r amino acid accumulation in inhibited c e l l s . Further investigation into the nature of the induced uptake system for proline was attempted. The kinetics of the low a f f i n i t y transport systems for both induced and uninduced c e l l s were determined and are demonstrated in Figure 56. The values for uptake were found to be identical with both kinds of c e l l s and V m a x was moderately increased with induced c e l l s , thereby indicating that the low a f f i n i t y permease was also inducible. However, i t was f e l t that the increase in V could have been max due to the influence of the greatly increased levels of the high a f f i ni ty p r o l i ne transport function. The Km values for amino acid incorporation determined with NaN^ poisoned c e l l s (Fig. 53), were found to be essenti a l l y the same as that of the low a f f i n i t y permease operative in non-poisoned c e l l s at high substrate concentrations. The effect of NaN^ on the a f f i n i t y constant for the high a f f i n i t y permease could not be determined since the equi1ibration of external and internal proline concentrations at low exogenous proline levels l i k e l y would have occurred at a rate impossible to measure by these methods. The increased concentrating a b i l i t y of c e l l s induced for amino acid transport can therefore be interpreted in li g h t of the model described in Figure 50. It is feasible that the rate of exit of an amino acid from the i n t r a c e l l u l a r pool is only moderately affected, i f at a l l , by induction, and that the rate of amino acid entrance into the c e l l at low exogenous amino acid concentrations was greatly increased due to the induced levels of the high a f f i n i t y permease (Fig. 54). Accordingly, this interpretation implicates 152 Fig. 56. Lineweaver-Burk plot of proline uptake into induced and non-induced c e l l s of 153 2 z O o r- p 300 < < CC oc H r- Z Z UJ UJ o u o § 2 0 0 1) 0 cr or < < _J _I LsJ rl 1 0 0 UJ O 0 < < OC rr \- r-z X UJ 0 Hh 1.43 1.67 2.00 2.50 330 5.00 » .(M/L) L06[VAL| Fie* 57. The ratio of Intracellular to extracellular vatIna concentration as a function of the exogenous valIne concentration. 154 the low a f f i n i t y permease as the entity mediating e f f l u x observed in Figures26-29, and i t operates in ef f l u x e s s e n t i a l l y only when high internal amino acid concentrations are present. To test the accuracy of this hypothesis, eff l u x and influx studies were carried out as functions of internal and external amino acid concentrations. If the i n t r a c e l l u l a r amino acid concentration is a function,of either permease, then differences in accumulating capacity of the microorganism would be revealed at external amino acid levels which would be conducive to the operation of a particular permease, since these permeases have greatly d i f f e r i n g entrance values. These studies were performed with the amino acid valine to obviate secondary effects of e f f l u x due to multiple pool components previously described for proline (Fig. 28-30). The experiment described in Figure 57 substantiates the v a l i d i t y of this hypothesis. Between 10 ^ and 10 ^ M external valine concentrations, the accumulating capacity as revealed by the internal to external proline concentration r a t i o s , increased -6 markedly. At valine concentrations exceeding 10 M the ratios decreased. The maximum concentration ratios observed were approx imately 300 f o l d , under the conditions of the experiment. Thus, at low external amino acid concentrations (where the majority of the, supplied ^C-amino acid enters via the high a f f i n i t y permease) the concentration ratio is high. At higher i n t r a c e l l u l a r amino acid concentrations (where effl u x via the low a f f i n i t y permeases becomes operative) the concentration ratio decreased concomitantly with increased exogenous ami no acid;presumably due to an increased component of ef f l u x . The precipitous decline in the internal to external amino acid r a t i o when the exogenous valine concentrations -5 ' exceeded 10 M, suggested that a large difference exists in the a b i l i t y to transport the amino acid out of the c e l l r e l a t i v e to transport into the c e l l . This difference in influx and ef f l u x capacity could feasibly be attributed to the a f f i n i t y for the transport protein i f the a f f i n i t y was d r a s t i c a l l y reduced on the inside of the membrane surface. Figure 58 shows the increase in pool size as a function of external amino acid concentrations. The pool increased with increasing exogenous amino acid concentration. Thus, at amino -6 acid concentrations exceeding 10 M, while the i n t r a c e l l u l a r to ext r a c e l l u l a r concentration: ;ratios decreased, the pool size increased. These data strongly suggested that at high in t r a  c e l l u l a r pool levels a difference in the a f f i n i t y of the amino acid for the transport function on the inner membrane surface would be overcome and would result in an increasing e f f l u x rate which ultimately would cause the observed decline in the concentration rat i o (Fig. 57). To determine accurately the contribution of each permease to the e f f l u x process, various pool levels were pre-established at different exogenous valine concentrations. The c e l l s were then immobilized on membrane f i l t e r s and continuously washed for pre determined time intervals. Rates of ef f l u x from high internal amino acid concentrations to low external concentrations were then calculated and plotted as a function of the pool size. From the results demonstrated in Figure 59, i t was observed that the rate of exit or ef f l u x of internal valine was a function 156 Fig. 58. Th© effect of the exogenous valine concentration on the valine pool stse (double negative reciprocal log-log plot). Cells wore preIncubated with 100 ug/ml chloramphenicol at 30 C for 30 rain then pulsed with the appropriate concentration of '*C-vallne at 15 C. Samples ware filtered at regular tine Intervals until the maximum pool level had been obtained In each case. of the pool valine at levels greater than 10 M; a concentration at which the low a f f i n i t y permease became operative. However, at lower pool concentrations, where the high a f f i n i t y permease was operative, no apparent changes in e f f l u x rates were observed. When a f f i n i t y constants for the exit process were calculated, very s t r i k i n g results.were found. Compared to the a f f i n i t y constants for valine uptake via the low a f f i n i t y permease the a f f i n i t y of the amino acid for the permease on the inside of the membrane was found to have been reduced by a factor of approximately 5000. Theoretically, this would permit concentration gradients of this magnitude to be formed. This has been found to be true for g-galactoside accumulation in E. c o l i (Winkler and Wilson, 1966). These investigators found that the maximum concentration ra t i o for 8-galactoside was 100 to 200 f o l d , and that q u a l i t a t i v e l y at least, the ratio of K of exit to K of entrance was substantially m m high. The magnitude of the difference in K of entrance and K m m of e x i t in this study is considered to be somewhat unique. The low a f f i n i t y permease has a of entrance approximately 10 fold less than the high a f f i n i t y permease, and as a result the.efflux rate would th e o r e t i c a l l y saturate at high internal valine concentrations. However, as already demonstrated (Fig. 52), there is in addition, an energy dependent function which is responsible for the accumulation of high levels of amino acid in the pool. This energy is visualized to act in a manner similar to the 3-galactoside accumulation system studied in E. c o l i ; that i s , that energy is 158 0.33 r - ^ 0.25 oc ° 0.20 CD 0J7 o I 0.12 o 0.11 o o 1.7 2.0 25 3.3 5.0 10.0 ! (M/L) LOG POOL VAL 14 Fig. 59. Rate of ef f l u x of C-valine as a function of the i n t r a c e l l u l a r valine pool size. Cells were pre incubated with 100ug/ml of chloramphenicol for 3Q.min at 30 C prior to the establishment of. C-valine pools of various sizes at 15 C. Efflux rates were determined by continuously washing c e l l s with amino acid free minimal medium for time intervals up to 3 min. 159 expended to prevent the amino acid on the inside.of the c e l l membrane from recombining with the transport " c a r r i e r " . As a resul t , the apparent of e f f l u x would be much greater than K for influx. m It is f e l t that in P. aeruginosa the combination of low a f f i n i t y o f the amino acid for the e f f l u x c a r r i e r and the energy function cause a concerted i n h i b i t i o n of e f f l u x . With the additional component of a high a f f i n i t y permease accelerating i n f l u x , the net result is a great i n t r a c e l l u l a r accumulation. Thus the transient high concentration gradients exhibited in transport experiments at low amino acid concentrations (Fig. 2) seemingly have a rational explanation. The data obtained so far explain the behaviour of r e l a t i v e l y large i n t r a c e l l u l a r amino acid pools. However, the maintenance of r e l a t i v e l y low i n t r a c e l l u l a r amino acid pools requires special consideration, since P. aeruginosa does not normally accumulate amino acids to very high levels when they are being synthes?zed de novo. From Figure 59, i t can be observed that at low pool levels valine is not eas i l y removed from the pool by washing and the rate of eff l u x does not greatly depend upon the i n t r a c e l l u l a r amino acid concentration. Furthermore, the amino acid present in the pool at low concentrations cannot be considered to be "bound" or compartmentalized (with the exception of proline), for i f they were immobilized in such a manner, then the amino acid theoretically would not be removed at a l l by excessive washing. A measurable rate of ef f l u x does occur at low amino acid concentrations (Fig. 59), thereby suggesting that perhaps the high a f f i n i t y 160 permease does contribute somewhat to the e f f l u x phenomenon. The mechanism for the maintenance of low amino acid pool levels at high concentration ratios is visualized as operating in a manner similar to the maintenance of high pool levels; that i s , that the a f f i n i t y of the amino acid for the high a f f i n i t y c a r r i e r must be d r a s t i c a l l y reduced on the inner surface of the c e l l membrane. The a b i l i t y of the c e l l to establish high concentration ratios at low exogenous amino acid levels (Fig. 57), further implies that the difference in the Km of influx r e l a t i v e to the for effl u x must be very large indeed to account for these data. This interpretation would seem to be borne out by the concentrations of amino acids in the native pool (Table I I I ) , and also by the apparent concentration ratios which are established between the native pool and the exogenous f l u i d . The average concentration for amino acids found in the native pool was 1.93 -6 x 10 M, and the average concentration ratio was found to be 255. These values are extremely close to the concentration data found experimentally for the amino acid valine (Fig. 57). It is possible that some degree of compartmentalization of i n t r a c e l l u l a r amino acids occurs at low concentrations and that such a process would prevent the recombination of the amino acid to the ca r r i e r which would inevitably result in a concentration phenomenon. However, i t is f e l t that this concept may unnecessarily complicate the actual pool forming mechanism in P_. aeruginosa. In E_. co 1 i , the pool maintenance a b i l i t y was attributed to a combination of 161 the amino acids with " s i t e s " which resulted in the compartment- a l i z a t i o n of the amino acid pool (Britten and McLure, 1962). This model was formulated largely to explain why pools in E. co1i are maintained under adverse conditions where they might be expected to leak out. P. aeruginosa pools are cer t a i n l y not maintained either at 0 C, in the absence of energy, during nutrient deprivation, or during excessive washing with amino acid free media. Tristram and Neale (1968) also have suggested that the i n t r a c e l l u l a r pool size in E. col i was a function of permease a c t i v i ty. Considerable variations between the a f f i n i t y constants for exit and entrance of amino acids in P. aeruginosa present an a t t r a c t i v e basis for a model for maintaining i n t r a c e l l u l a r pools. The i n t r a c e l l u l a r space of microorganisms consists partly of an accumulation of metabolic intermediates, any one of which might a l l o s t e r i c a l l y affect the function of a transport c a r r i e r in a way that would reduce the a f f i n i t y for the s p e c i f i c transport substrate. This hypothesis is not merely conjecture, since in E. c o l i , Halpern and Even-Shoshan (1967), demonstrated that the glutamate permease is non-competitively inhibited by aspartate and a-ketoglutarate. Also Boezi and DeMoss (1961), showed that pyruvate inhibited the transport of tryptophan in E_. co 1 i . These observations may explain some facets of the accumulation phenomenon in E_. co 1 i . The NaN^ s e n s i t i v i t y of pool maintenance in P. aeruginosa may indicate that a high energy intermediate is involved in the al t e r a t i o n of transport functions i n t r a c e l l u l a r l y . Fig. 60(A), Single c a r r i e r model f o r amino acid transport. A ^, amino acid in the external environment; out' C, membrane localized c a r r i e r protein; AC, amino acid-carrier complex; A. , amino acid in the internal amino acid pool; in' r » . K^ j - W rate constants for the formation and dissociation of the carrier-ami no acid complex. Fig. 60(B). Double c a r r i e r model for amino acid transport in P. aerug ? hosa. A l , amino acid present at low external concentrations (<10. M); Ah, amino acid present at high external concentrations (>10 M). Ah can react with both C^  and C^; C|, high a f f i n i t y permease or membrane c a r r i e r ; C^, low a f f i n i t y permease or membrane c a r r i e r ; AIC.J, amino acid-carrier complex formed at low amino acid concentrations; AhC^, high a f f i n i t y carrier-amino acid complex formed at high amino acid concentrations; A h^, low a f f i n i t y carrier-amino acid complex formed at high ami no acid concentrations; K^entry and K'^entry, equilibrium constants for the reaction A+C AC at the outer-membrane surface; K^exit and K ^ e x i t , equilibrium constants for the reaction A+C AC at the inner-membrane surface; Diffusion constants for occupied and unoccupied carriers are presumed to be equal. During metabolic energy production, K texit and K ^ e x i t are e s s e n t i a l l y negligible possibly through a l l o s t e r i c deactivations of C, and C. ca r r i e r functions. OUTSIDE I MEMBRANE INSIDE (A) A0 Uf}C K. K2 ±AC K a. *C + A t n In the steady state: Entry = Exit For facilitated diffusion: K| = K 4 assuming K2= K3or K2cannot^ K 3 For active transport: A C ^ - A + C K 4 K ^ K. due to inactivation of SC. 4 ^ 1 4 by a product of energy metabolism. Similarly for a double carrier system: (B) Al in energy function in Figure 60 i l l u s t r a t e s a hypothetical model devised to explain the amino acid transport and accumulation phenomenon in P. aeruginosa. The amino acid is visualized to c o l l i d e with stereospecific mobile c a r r i e r s , to be transported into the c e l l , and released at the inner membrane surface. The c a r r i e r is prevented from recombining with the i n t r a c e l l u l a r amino acid by an energy dependent process and returns to the outer membrane surface unoccupied. This model provides for essenti a l l y a unidirectional transport process at low amino acid concentrations with an increasing component of ef f l u x at high amino acid concentrations. 165 GENERAL DISCUSSION The enzymatic nature of metabolite incorporation into c e l l s , f i r s t demonstrated with carbohydrates by Rickenberg et a l . ( 1 9 5 6 ) , has been extended in recent years to encompass the amino acids as well as other compounds. The amino acid transport systems of P. aeruginosa are no exception. These systems have a l l the properties ascribed to active transport systems; that i s , transport is seemingly energy dependent, temperature sensitive, saturated at high substrate concentrations, is lost by mutation, and results in the accumulation of the incorporated metabolite at concentrations far exceeding those of the external environment. The amino acid transport system, as well as accumulation and catabolism, are under genetic control and these functions may possibly constitute an operon. The property of substrate s t e r e o s p e c i f i c i t y is also demonstrated by bacterial transport systems. However, this property varies greatly with the nature of the microorganism. Bacteria have now been shown to possess a high degree of substrate s e l e c t i v i t y , or s t e r e o s p e c i f i c i t y , with regards to both sugars and amino acids (Kepes and Cohen, 1 9 6 5 ; Britten and McLure, 1 9 6 2 ; FerroLuzzi-Ames, 1 9 6 4 ; Behkr and Hochster, 1 9 6 7 ) . It would seem that this special ization diminished with the evolution of the procaryotic to the eucaryotic c e l l s . Whereas the bacteria maintain highly specialized 166 permeabi1ity functions, Neurospora and Saccharomyces species tend to lose this property of s p e c i f i c i t y (Maw, 1963; Grenson, 1966; DeBusk and DeBusk, 1965)- This change has been observed even within one species of bacter?um, Agrobacterum turhefaciens (Behki and Hochster, 1967) - The p a r a s i t i c s t r a i n has seemingly lost the s t e r e o s p e c i f i c i t y of amino acid transport demonstrated by the non-parasitic s t r a i n . Apparently animal c e l l s do not possess the high a f f i n i t y permeases demonstrated for microorganisms. Although several d i s t i n c t , but strongly interacting, amino acid transport systems have recently been demonstrated with animal c e l l s (Begin and Scholefield, 1965; Eavenson and Christensen, 1967; Christensen, Liang, and Archer, 1967), the a f f i n i t y constants measured are high (10 to 10 M) re l a t i v e to bacterial a f f i n i t y constants (10 to 10"7 M). These animal permeation systems would seem to correspond more to the low a f f i n i t y bacterial permeases, which function at high amino acid concentrations (FerroLuzzi-Ames, 1964), or to bacterial sugar transport systems (Winkler and Wilson, 1966). The i n t r a c e l l u l a r metabolic pools of microorganisms are extremely complex. These pools contain a l l the soluble intermediates of anabolism and catabolism and i t is not unreasonable that this multitude of compounds is oriented in different ways within the c e l l depending on their metabolic fate. Thus, P. aeruginbsa has been shown to "compartmentalize" putrescine, proline, and probably alanine intracel 1ularly. It has also been suggested that proline is compart mentalized in E. co1i (Britten, 1965). Secarz and Gorini (1964), postulated that endogenous and exogenous arginine contributed d i f f e r e n t l y toward repressor formation ih E. co 1i. Two d i s t i n c t metabolically active pools of tryptophan were demonstrated in N. crassa (Matchett and DeMoss, 1 9 6 4 ) . Biosynthetically generated tryptophan was used pr e f e r e n t i a l l y for protein synthesis and exogenous tryptophan was pr e f e r e n t i a l l y oxidized via the tryptophan cycle. The maintenance of high i n t r a c e l l u l a r concentrations of amino acids would seem to be a function of the degradative capacity of the particular microorganism. Whereas P. aerug?hbsa was shown to maintain low internal amino acid concentrations during growth, E. cdT i and some fungi maintain high pool levels. However, iE. c o l i does not deplete i t s i n t r a c e l l u l a r amino acid pool under conditions of nutrient deprivation. Hoch and DeMoss ( 1 9 6 6 ) , showed that a constitut ive tryptophanase in Bac?11 us alve? kept the i n t r a c e l l u l a r pool level of this amino acid to low levels. P. aerugihbsa can concentrate amino acids many thousand times that of the external environment demonstrating that this organism can express a high amino acid accumulation capacity tra n s i e n t l y , but i t normally does not do so. The observation that nutrient deprivation (carbon or nitrogen) does not s i g n i f i c a n t l y a l t e r the amino acid transport capacity of P. aerug1hbsa may underline the true significance of the existence of high a f f i n i t y amino acid, or other metabolite, permeases. McGrew and Malette ( 1 9 6 2 ) , and Marr, Wilson, and Clark ( 1 9 6 3 ), demonstrated that E_. col i u t i l i z e d small amounts of glucose for energy in 168 maintaining v i a b i l i t y without concomitant growth. Since then P. aeruginosa catabolizes most amino acids, i t would seem logical that under carbon or nitrogen starvation conditions the high a f f i n i t y permeases function to maintain v i a b i 1 i t y , but not to support growth. In this role, these permeases should indeed be considered b i o l o g i c a l l y advantageous to the survival of the species. The formation and maintenance of i n t r a c e l l u l a r pools of metabolites is a ubiquitous phenomenon amongst microorganisms. Mechanisms for concentrating metabolites must have been an early evolutionary cha r a c t e r i s t i c among l i f e forms, since the ready a v a i l a b i l i t y of precursors for growth would certainly be advantageous for rapid m u l t i p l i c a t i o n . It is feasible that the evolution of the accumulation process paralleled the decline in available nutrients which is presumed to have existed in the primordial soup. However, in the experimental elucidation of transport mechanisms for amino acids studied in P. aeruginosa, a conscious attempt was made to formulate a mechanism which was not merely commensurate with recent models devised for carbohydrate transport, but which also embodied the facets of s i m p l i c i t y and physiological f e a s i b i l i t y . Thus the unknown "energy function" operating on the inner membrane surface was postulated to be a high energy intermediate such as ATP perhaps acting as an a l l o s t e r i c i n h i b i t o r of the amino acid complexing a c t i v i t y of the c a r r i e r . Koch (1964), calculated that one molecule of ATP was required to permit the active transport of one molecule of 3-galactoside in E_. cbl 1. 169 This may be true, but such a mechanism would certainly seem to waste energy and would l i k e l y be lost in l i e u of a less wasteful mechanism during the course of evolution. Perhaps one of the most puzzling and as yet unsolved properties of almost a l l models of carrier-mediated transport is the property of c a r r i e r mobility. The distance to be transversed across the membrane exceeds 70 A and i t is d i f f i c u l t to v i s u a l i z e how the carrier-amino acid complex moves across the structural barrier of the bacterial membrane. The multifunctional nature of the bacterial c e l l membrane underlines not only i t s importance to the integrity of the c e l l but also emphasizes i t s staggering complexity. A large number of s p e c i f i c transport systems for metabolites have now been demonstrated in microorganisms. In addition to the rather large number.of permeases described here for amino acids, several have been demonstrated, and in some cases isolated, for carbohydrates (Ganesan and Rotman, 1965), nucleic acids (Peterson and Koch, 1966), polyamines (Tabor and Tabor, 1966), and ions (Pardee, I966; Peters and Warren, 1968). As more low molecular weight metabolites are tested, increasing numbers of transport functions w i l l undoubted ly be found. Since more than one function has been implicated in the transport of some metabolites, for example the sugars (Winkler and Wilson, 1966), the complexity of the bacterial membrane appears overwhelming. 170 LITERATURE CITED Bauerle, R.H., and H.R. Garner. 1 9 6 4 . The assimilation of arginine and lysine in canavanine resistant and sensitive strains of Neurospora crassa; Biochim. Biophys. Acta 9_3_: 316-322 . Behki, R.M. 1968. Metabolism of amino acids in Agrobacteriurn  tumefaciens. I l l Uptake of L-proline. Can. J. Biochem. 4 5 : I8I9-I83O. 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Elsevier Publishing Co., Amsterdam. 26. Holden, J.T. .'1965. Restoration of normal glutamic acid transport in vitamin B^-deficient Lactobaci1lus pi antarum by acetate, ammonium and vitamin Bg. Biochim. Biophys. Acta J 0 4 : 121-138. 27. Holden, J.T., and J. Holman. 1959. Accumulation of freely extractable glutamic acid by l a c t i c acid bacteria. J. B i o l . Chem; 234: 865-871. 28. Inui, Y., and 29. 30. 31. 32. col i 1 n 1 9 6 5 . Amino acid uptake by Escherichia the presence of amino acids. Evidence for the repressibi 1ity of amino acid uptake. Biochim. Biophys.Acta 9 4 : 1 4 3 - 1 5 2 . H. Akedo grown Jakoby, W.B., and J. Fredericks. 1959. Pyrrolidine and putrescine metabolism:' Y" a minobutyraldehyde dehydrogenase. J. B i o l . Chem. 234: 2145-215O. Jones, K., and J.G. Heathcote. 1966. The rapid resolution of natura1ly occurring amino acids by thin-layer chromatography. J. Chromatog. 2 4 : 106-111. Kaback, H.R., and E.R. Stadtman. 1966. 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Genetic control of amino acid permeability i h Neurospora crassa. J. Bacter i o l . 9J_: 677-684. 39. Lubin, M., D.H. Kessel, A. Budreau, and J.D. Gross. I960. The isolation of bacterial mutants defective in amino acid transport. Biochim. Biophys. Acta 42: 535-538. 40. Lyon, R.H., P. Rogers, W.H. H a l l , and H.C. Lichstein. 1967. Inducible glutamate:transport in Mycobacteria and it s relation to glutamate oxidation. J. Bacteriol. 94:92-100. 41. McKelvie, R.M. 1965. The endogenous respiration of Pseudbrhbhas aeruginosa during periods of prolonged starvation. Ph.D. Thesis, University of B r i t i s h Columb i a . 42. Mandelstam, J.L. 1960. The i n t r a c e l l u l a r turnover of proteins and nucleic acids and i t s role in biochemical d i f f e r e n t i a t i o n . Bacteriol. Rev. 24: 289-308. 43- Marr, A.G., E.H. Nilson, and D.J. Clark. 1963- The maintenance requi rement of Escherichia c b l i . Ann. N.Y. Acad. S c i . 102: 536-548. 44. Matchett, W.H., and J.A. DeMoss. 1964. 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