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Catabolism and transport of arginine by Pseudomonas aeruginosa Cook, Kathleen Anne 1971

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THE CATABOLISM AND TRANSPORT OF ARGININE BY PSEUDOMONAS AERUGINOSA by KATHLEEN ANNE COOK B.Sc. , U n i v e r s i t y of B r i t i s h Columbia, 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In the Department of of M icrob iology r We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February, 1971 In presenting t h i s thesis in p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Depa rtment The University of B r i t i s h Columbia Vancouver 8, Canada Date i i ABSTRACT Pseudomonas aeruginosa was shown to c o n s t i t u t i v e l y degrade a r g i n i n e v i a the a r g i n i n e d i h y d r o l a s e pathway to o r n i t h i n e , which was converted both to glutamate and to p u t r e s c i n e . The conversion of o r n i t h i n e to glutamate appeared to be the major route of a r g i n i n e degradation in t h i s organism, and was induced to higher a c t i v i t y a f t e r growth of the c e l l s w ith a r g i n i n e as the sol e source of carbon and n i t r o g e n . P_. aeruginosa did not f u r t h e r degrade putrescine c o n s t i t u t i v e l y . However, growth of the c e l l s in a r g i n i n e r e s u l t e d in a p a r t i a l induction of s u c c i n i c semialdehyde dehydrogenase, an enzyme f u n c t i o n i n g in putrescine degradation. The anabo l i c o r n i t h i n e transcarbamylase of P_. aerug i nosa was repressed a f t e r growth of the organism in the presence of a r g i n i n e . Pseudomonas putida and Pseudomonas flu o r e s c e n s a l s o possessed the a b i l i t y to c o n s t i t u t i v e l y convert a r g i n i n e to putrescine v i a the intermediates, c i t r u l l i n e and o r n i t h i n e . However, these organ-isms did not o x i d i z e a r g i n i n e to the same extent as did P_. aerugi nosa. P_. aeruginosa grew in a mixture of glucose and a r g i n i n e in the presence of ammonium ions without e x h i b i t i n g a d i a u x i e e f f e c t . Glucose and a r g i n i n e were o x i d i z e d concomitantly when supplied as a mixed s u b s t r a t e , by both growing c e l l s and r e s t i n g c e l l suspensions. However, a s s i m i l a t i o n studies showed that the two substrates were used to serve somewhat d i f f e r e n t b i o s y n t h e t i c needs. Growth of P_. aerug?nosa in a r g i n i n e caused an increase in the rates of transpo r t of a r g i n i n e , l y s i n e , o r n i t h i n e and c i t r u l l i n e . K i n e t i c s t u d i e s of a r g i n i n e uptake demonstrated the presence of two uptake systems with d i f f e r e n t a f f i n i t i e s f o r a r g i n i n e . I n h i b i t i o n s t udies indicated that a r g i n i n e was transported by two uptake systems: a permease s p e c i f i c f o r a r g i n i n e , and, with a lower a f f i n i t y , f o r o r n i t h i n e ; and a general permease, which transported a l l the basic amino a c i d s . Polyamines appeared to be transported by an uptake system which was induced to higher l e v e l s a f t e r growth of the c e l l s with e i t h e r a r g i n i n e or putrescine as the sol e source of carbon and n i t r o g e n . P_. aeruginosa was found to maintain a s t a b l e pool of putrescine when supplied with exogenous C-arginine or C-putrescine, even when the organism had p r e v i o u s l y been induced to degrade these s u b s t r a t e s . A physical f r a c t i o n a t i o n of the c e l l s i n d i c a t e d that the major po r t i o n of t h i s pool was located in the s o l u b l e cytoplasm. I V TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW 3 I. Pathways of Arginine Degradation in Microorganisms!! I . 3 I I . Catabolite Repression of Amino Acid Degradation . . . . 12 I I I . The B i o l o g i c a l Importance of Polyamines in Microorganisms 16 IV. Transport of the Basic Amino Acids by Microorganisms. . 20 MATERIALS AND METHODS 23 I. Organisms and Media 23 I I. Growth of Cel Is 2k 111. Preparation of Cel l Suspensions 2k 1. Resting c e l l suspensions for respirometry .„ 2k 2. Cell suspensions for transport studies 25 IV. Preparation of C e l l - f r e e Extracts 25 V. Manometric Procedures 26 VI. Uptake of Labelled Compounds 26 V l l . I nhibition of Transport by Compounds S t r u c t u r a l l y Related to the Substrate 28 V I I I . Assay of Succinic Semialdehyde Dehydrogenase 28 IX. Chemical Fractionation of Whole C e l l s 29 V Table of Contents (Continued) Page X. Physical Fractionation of Whole C e l l s 29 XI. Chromatography of Supernatant F l u i d s from Warburg Ik Cups Containing C-labelled Substrates 30 X I I . Assay of Ornithine Transcarbamylase 32 X I I I . A n a l y t i c a l Methods 33 XIV. Chemicals 33 RESULTS AND DISCUSSION 3^ I. Pathways of Arginine Degradation in P_. aeruginosa. . . 3^ 1. Accumulation of intermediates of arginine degradation , 3h 2 . Inhibitory e f f e c t of T r i s buffer on the oxidation of arginine by whole c e l l s 36 3 . Oxidation of intermediates of arginine degradation 39 *t. Conversion of ornithine to glutamate kk 5. Succinic semialdehyde dehydrogenase a c t i v i t y . . . 50 I I . Repression of Arginine Biosynthesis in P_. aeruginosa by Exogenous Arginine 53 I I I . The Effects of Glucose on the Degradation of Arginine by P_. aeruginosa. Sh 1. Growth in a mixture of glucose and a r g i n i n e . . . . Sh Table of Contents (Continued) Page 2. The e f f e c t of glucose on the degradation of arginine by resting c e l l suspenstons-r 59 3. A s s i m i l a t i o n of arginine and glucose 65 IV. Degradation of Arginine by P_. putida and P_. f 1 uorescens 68 V. Uptake of Basic Amino Acids and Polyamines by P_. aerug ? nosa 73 1. Induction of uptake . 73 2. Kinetics of arginine uptake 76 3. Inhibition of transport 79 a. Basic amino acids 79 b. Polyamines 83 k. Pool formation 85 a. Arginine 85 b. Ornithine and c i t r u l l i n e . . 89 c. Putrescine 91 5. Location of i n t r a c e l l u l a r putrescine pool . . . . 96 GENERAL DISCUSSION 100 LITERATURE CITED 105 V I I LIST OF TABLES Page Table I. Rates of oxygen uptake by P_. aeruginosa with a r g i n i n e and suspected intermediates as substrates h3 Table I I . Degradation of o r n i t h i n e - ! - C by a r g i n i n e and glucose grown c e l l s of P_. aerug i nosa . k6 Table I I I . S u c c i n i c semialdehyde dehydrogenase a c t i v i t i e s of c e l l - f r e e e x t r a c t s of induced and uninduced c e l l s of P_. aeruginosa 52 Table IV. Composition of the supernatant f l u i d s a f t e r o x i d a t i o n of - a r g i n i n e by P_. aeruginosa in the presence and absence of glucose 6k Table V. The e f f e c t of a r g i n i n e and ammonium ions on the a s s i m i l a t i o n of '^C-glucose by a r e s t i n g c e l l suspension of IP. aerug i nosa 66 \k Table V I . A s s i m i l a t i o n of C-arginine by a r e s t i n g c e l l suspension of P_. aeruginosa in the presence and absence of glucose 67 Table V I I . Comparison of the a s s i m i l a t i o n of - a r g i n i n e by P_. aerug inosa , P_. f luorescens , and P_. p u t i d a . 71 Table V I I I . Induction of t r a n s p o r t 7^ Table IX. I n h i b i t i o n of a r g i n i n e t r a n s p o r t 81 Table X. I n h i b i t i o n of o r n i t h i n e t r a n s p o r t 82 Table X I . I n h i b i t i o n of p u t r e s c i n e tra n s p o r t 84 Table X I I . D i s t r i b u t i o n of r a d i o a c t i v i t y a f t e r p h y s i c a l f r a c t i o n a t i o n of c e l l s incubated fn the presence of - a r g i n i n e 98 viii LIST OF FIGURES Page F i g . 1. Pathways of arginine d i s s i m i l a t i o n in micro-organisms k F i g . 2. Radioautogram of a thin-layer chromatogram of the supernatant f l u i d a f t e r the incubating of glucose grown c e l l s with -arginine for 30 minutes under conventional Warburg conditions 35 F i g . 3- Oxidation of arginine and putrescine in the presence of T r i s and phosphate buffers 37 F i g . k. Oxidation of arginine and ornithine hO F i g . 5. Oxidation of putrescine 41 F i g . 6. Oxidation of c i t r u l l i n e and yaminobutyrate kl F i g . 7. Radioautogram of a thin-layer chromatogram of the basic f r a c t i o n of the supernatant f l u i d a f t e r incubation of arginine grown c e l l s with ornithine-1-1 Z*C for kO minutes 49 1 ij F i g . 8. The u t i l i z a t i o n of C-glucose during growth of P_. aeruginosa in a medium containing glucose, arginine arid ammonium ions. 55 14 F i g . 9. The u t i l i z a t i o n of C-arginine during the growth of P_. aeruginosa in a mixture of glucose and arginine 56 F i g . 10. The oxidation of glucose, arginine and a mixture of glucose and arginine 60 F i g . 11. D i s t r i b u t i o n of r a d i o a c t i v i t y during the incubation of P_. aeruginosa with ^ C - a r g i n i n e under Warburg conditions 62 F i g . 12. Oxidation of arginine by glucose grown c e l l s of P. aeruginosa, P. fluorescens and P. putida 70 I X L i s t of Figures (Continued) Page F i g . 13 . K i n e t i c s of argfnine uptake of-P> aerug Nosa 77 F i g . Ik. Kinetics of arginine uptake by glucose grown c e l l s . Lineweaver-Burk plot 78 Ftg. 15- Formation of an i n t r a c e l l u l a r pool using glucose grown c e l l s supplied with ^ C - a r g i n i n e 86 F i g . 16. Formation of an i n t r a c e l l u l a r pool of arginine by c e l l s grown with arginine as the sole source of carbon and nitrogen 88 F i g . 17- Formation of i n t r a c e l l u l a r pools of ornit h i n e and c i t r u l l i n e by glucose grown c e l l s 90 F i g . 18. Formation of an i n t r a c e l l u l a r pool of putrescine by glucose grown c e l l s 92 F i g . 19. Formation of an i n t r a c e l l u l a r pool of putrescine by putrescine grown c e l l s 93 F i g . 20 . Formation of an i n t r a c e l l u l a r pool of putrescine by arginine grown c e l l s 95 X ACKNOWLEDGEMENTS I would l i k e to express gratitude to my supervisor, Dr. A.F. Gronlund, for her encouragement, advice and c r i t i c i s m throughout the course of t h i s work. I am also extremely grateful to Dr. H.R. MacMillan and the H.R. MacMillan Family Fund for f i n a n c i a l assistance during the f i r s t two years of t h i s study. I would a l s o l i k e to thank very much Mrs. I r i s Yu for her help with transport experiments, and my colleagues and my friends for t h e i r help throughout my graduate studies program. 1 INTRODUCTION The enzymes of a r g i n i n e degradation have been shown to be i n d u c i b l e and to be subject to c a t a b o l i t e repression in several microorganisms (Ramos et_ aj_. 1967; L a i s h l e y and Bernlohr, 1968). Jacoby (1964) found that the a b i l i t y of Pseudomonas fluorescens to o x i d i z e eighteen d i f f e r e n t amino aci d s was repressed by glucose. Kay (1968) obtained r e s u l t s which i n d i c a t e d that a r g i n i n e was degraded c o n s t i t u t i v e l y by Pseudomonas aeruginosa at a r e l a t i v e l y rapid r a t e . Kay (I969) found that c e l l s of P_. aeruginosa which were 14 supplied with low external concentrations of C-arginine in glucose minimal medium accumulated a large pool of p u t r e s c i n e , which was extremely s t a b l e , being maintained f o r periods as long as twenty four hours during s t a r v a t i o n f o r an exogenous carbon source. Putrescine has been shown to bind to deo x y r i b o n u c l e i c a c i d and r i b o n u c l e i c a c i d in v i t r o and i s thought to play a r o l e in t r a n s -l a t i o n and, p o s s i b l y , in t r a n s c r i p t i o n (Stevens, 1970). Several studies have in d i c a t e d that p u t r e s c i n e may be required f o r c e l l d i v i s i o n (Davis, Lawless and P o r t , 1970; H i r s h f i e l d , et_ al_. 1970; Inouye and Pardee, 1970) It was the object of t h i s i n v e s t i g a t i o n to determine the pathway by which a r g i n i n e was degraded by P_. aeruginosa and the e f f e c t of glucose on the catabolism of a r g i n i n e by t h i s organism. A further investigation of the basic amino acid uptake systems of P_. aerug i nosa , which were partially characterized by Kay (1968) , was also carried out. 3 LtTERATURE REVIEW t . Pathways o f A r g i n i n e D e g r a d a t i o n tn M i c r o o r g a n i s m s The pathways o f a r g i n i n e d i s s i m i l a t i o n i n m i c r o o r g a n i s m s have been found t o be v a r i e d and complex ( F i g . 1 ) . tn Neurospora c r a s s a ( C a s t a n a d a , M a r t u s c e l l i , and M o r a , 1967; M e f s t e r , 1965), Saccharomyces  c e r e v i s i a e ( M t d d e l h o v e n , 1964), and two s p e c i e s o f Baci11 us (de Hauwer, L a v a l l e and Wiame, 1964; L a i s h l e y and B e r n l o h r , 1968), a r g i n i n e i s f i r s t h y d r o l y s e d by a r g i n a s e , r e s u l t i n g i n t h e p r o -d u c t i o n o f o r n i t h i n e and u r e a . O r n i t h i n e i s then c o n v e r t e d by o r n i t h i n e - y - t r a n s a m i n a s e t o g l u t a m i c - y - s e m i a l d e h y d e , an u n s t a b l e i n t e r m e d i a t e , w h i c h s p o n t a n e o u s l y c y c l i z e s t o - p y r r o l i n e - 5 -c a r b o x y l i c a c i d . The l a t t e r compound may be c o n v e r t e d by a dehydrogenase t o g l u t a m a t e , o r by a r e d u c t a s e t o p r o l i n e . An e x c e p t i o n t o t h e o r n i t h i n e t r a n s a m i n a s e r e a c t i o n has been found i n C l o s t r i d i u m b o t u l i n u m , where o r n i t h i n e i s c o n v e r t e d t o g l u t a m i c -Y ~ s e m i a l d e h y d e by a n i c o t i n e a d e n i n e d i n u c l e o t i d e ( N A D ) - l i n k e d dehydrogenase ( C o s t i l o w and L a y c o c k , 1969). A r g t n a s e , o r n i t h i n e t r a n s a m i n a s e , and A ' - p y r r o l i n e - 5 - c a r b o x y -l a t e dehydrogenase a r e c o i n c i d e n t l y induced by a r g i n i n e or by o r n i t h i n e i n B a c i l l u s 1 i c h e n i f o r m i s ( L a i s h l e y and B e r n l o h r , 1968) and B a c i 1 l u s s u b t i l i s (de Hauwer, L a v a l l e and Wiame, 1964). F i g u r e 1. Pathways o f a r g i n i n e d i s s i m i l a t i o n i n m i c r o o r g a n i s m s . C0 2 + NH /"COOH g l u t a m a t e CHO \ C C H2)2 CHNH2 <r-COOH g l u t a m i c - y -s e m i a l d e h y d e p r o l i n e jJ^COOH A - p y r r o l i n e -5 - c a r b o x y l i c a c t d NADP ^ NADPH COOH : ( C H J , / 2 CHNH. COOH g l u t a m a t e N A D H COOH > ( C H 2 } 2 CH2NH2 a - k e t o -5 g l u t a r a t e COOH ' ( C H J -> / 2 2 CHO NADP NADPH y-amino-b u t y r t c a c i d s u c c i n i c s e m i a l d e h y d e COOH / 2 2 COOH su c c i n a t e 5 The l a t t e r w o r k e r s found e v i d e n c e t h a t o r n i t h i n e was not t h e a c t u a l i n d u c e r o f t h e s e enzymes i n B_. sub t ? l i s . O r n i t h i n e was f i r s t c o n v e r t e d t o a r g i n i n e v i a t he a r g i n i n e b i o s y n t h e t i c p a t h -way, and a r g i n i n e then s e r v e d as the i n d u c e r . They a l s o i s o l a t e d a mutant o f B_. s u b t i 1 i s w h i c h was c o n s t i t u t i v e f o r a r g i n i n e t r a n s p o r t , a r g i n a s e , and o r n i t h i n e t r a n s a m i n a s e , i n d i c a t i n g t h a t t he s t r u c t u r a l genes f o r t h e s e f u n c t i o n s may form an o p e r o n . A ^ - p y r r o l i n e ~ 5 -c a r b o x y l a t e dehydrogenase remained i n d u c i b l e , and thus must be under s e p a r a t e c o n t r o l . deHauwer, L a v a l l e and Wiame (1964) showed t h a t t h e - p y r r o l i n e -5 - c a r b o x y l a t e dehydrogenase f u n c t i o n i n g i n a r g i n i n e d e g r a d a t i o n i n iL* s u b t ?1 i s was a d i f f e r e n t enzyme from t h e one f u n c t i o n i n g i n p r o l i n e c a t a b o l i s m . In t h i s o r g a n i s m , a r g i n i n e induced o n l y t h e a r g i n i n e d e g r a d i n g enzymes, and p r o l i n e induced o n l y the p r o l i n e d e g r a d i n g enzymes. L a i s h l e y and B e r n l o h r (1968), on the o t h e r hand, found t h a t a r g i n i n e caused a p a r t i a l i n d u c t i o n o f p r o l i n e o x i d a s e i n B_. 1 i c h e n i f o r m i s , and p r o l i n e caused a p a r t i a l i n d u c t i o n o f a r g i n a s e . They t h e r e f o r e h y p o t h e s i z e d t h a t A ^ - p y r r o l i n e - 5 " c a r b o x y l i c a c i d , an i n t e r m e d i a t e common t o the pathways o f o x i d a t i o n o f both p r o l i n e and a r g i n i n e , was t h e a c t u a l i n d u c e r o f the two pathways. Organisms l a c k i n g an a r g i n a s e can c o n v e r t a r g i n i n e t o o r n i t h i n e by t h e a r g i n i n e d i h y d r o l a s e pathway. A r g i n i n e i s f i r s t c o n v e r t e d t o c i t r u l l i n e by t h e enzyme a r g i n i n e d e i m i n a s e , w h i c h has been 6 found tn Pseudomonads, S t r e p t o c o c c t , and C l o s t r t d i a ( O g t n s k y , 1955). These o r g a n i s m s a l s o c o n t a i n e d an enzyme w h i c h , i n t h e p r e s e n c e o f s u b s t r a t e amounts o f p h o s p h a t e , s p l i t c t t r u l l i n e t o produce o r n i t h i n e , NH^, and CO^. T h i s r e a c t i o n r e q u i r e d magnesium and e i t h e r a d e n o s i n e monophosphate (AMP) o r a d e n o s i n e d i p h o s p h a t e (ADP), and r e s u l t e d i n t h e p r o d u c t i o n o f a d e n o s i n e t r i p h o s p h a t e ( A T P ) . Oginsky c a l l e d t h i s enzyme c i t r u l l i n e p h o s p h o r y l a s e . I t s a c t i v i t y , i n a c e l l - f r e e e x t r a c t o f Pseudomonas a e r u g i n o s a , was i n h i b i t e d by o r n i t h i n e . Subsequent w o r k e r s d i s c o v e r e d t h a t carbamyl phosphate was an i n t e r m e d i a t e i n t h i s r e a c t i o n , a n d , t h e r e f o r e , t h e enzyme was a c a t a b o l i c o r n i t h i n e t r a n s c a r b a m y l a s e ( M e i s t e r , 1965). The carbamyl phosphate was degraded by a carbamate k i n a s e , and t h i s was t h e ATP p r o d u c i n g s t e p . S t a l o n et_ a_j_. (1967a) found t h a t Pseudomonas f l u o r e s c e n s and P_. a e r u g i n o s a c o n t a i n e d two o r n i t h i n e t r a n s c a r b a m y l a s e s s e p a r a b l e by ammonium s u l p h a t e f r a c t i o n a t i o n . One enzyme, assumed t o f u n c t i o n i n c a t a b o l i s m , was induced by growth i n t h e p r e s e n c e o f a r g i n i n e , whereas t h e o t h e r , assumed t o have an a n a b o l i c f u n c t i o n , was r e p r e s s e d under t h e s e c o n d i t i o n s . A l t h o u g h the a n a b o l i c enzyme was c o m p l e t e l y i r r e v e r s i b l e , t h e c a t a b o l i c enzyme was c a p a b l e o f c a t a l y s i n g both t h e s y n t h e s i s and breakdown o f c i t r u l l i n e i n  v i t r o but not i n v i v o (Ramos e t _ a l . 1967). S i n c e t h e pH opti m a f o r t he a c t i v i t y o f t h e c a t a b o l i c enzyme i n t h e two d i r e c t i o n s d i f f e r e d g r e a t l y , i t was h y p o t h e s i z e d t h a t l o c a l i z a t i o n i n an 7 a c i d i c compartment might p r e v e n t t h i s enzyme from f u n c t i o n i n g i n t h e a n a b o l i c d i r e c t i o n In v i v o . However, S t a l o n e t a l . (1967b) o f f e r e d an a l t e r n a t i v e e x p l a n a t i o n f o r t h e i n v i v o i r r e v e r s i b i l i t y o f t he c a t a b o l i c o r n i t h i n e t r a n s c a r b a m y l a s e . They r e p o r t e d t h a t t h e c a t a b o l i c enzyme was s u b j e c t t o a l l o s t e r i c i n h i b i t i o n by carbamyl phosphate a t c o n c e n t r a t i o n s which s a t u r a t e d t h e a n a b o l i c enzyme. The c a t a b o l i c o r n i t h i n e t r a n s c a r b a m y l a s e was a l s o i n h i b i t e d by ATP and was a c t i v a t e d by ADP. G a l e (19^0) d i s c o v e r e d t h a t E s c h e r i c h i a c o l ? p o s s e s s e d i n d u c i b l e a r g i n i n e and o r n i t h i n e d e c a r b o x y l a s e s when grown a t low pH v a l u e s . A r g i n i n e was d e c a r b o x y l a t e d t o form a g m a n t i n e , and p u t r e s c i n e was produced from o r n i t h i n e . The pH optimum o f o r n i t h i n e d e c a r b o x y l a s e was 5.0, and t h a t o f a r g i n i n e d e c a r b o x y l a s e was k.O. M o r r i s and Pardee (1965) found t h a t when E_. c o l i was grown i n minimal medium, i t c o n t a i n e d a c o n s t i t u t i v e o r n i t h i n e d e c a r b o x y l a s e w i t h a pH optimum o f 7.5- T h i s enzyme was p r e s e n t a t the same l e v e l i n c e l l s grown i n t h e p r e s e n c e o f o r n i t h i n e a t low pH v a l u e s , b u t , under t h e s e c o n d i t i o n s , a h i g h l e v e l o f o r n i t h i n e d e c a r b o x y -l a s e w i t h a pH optimum o f 5-3 was a l s o p r e s e n t . The a u t h o r s h y p o t h e s i z e d t h a t the c o n s t i t u t i v e enzyme s e r v e d a b i o s y n t h e t i c f u n c t i o n , s y n t h e s i z i n g t he normal c e l l u l a r c o n c e n t r a t i o n o f p u t r e s c i n e , whereas the i n d u c i b l e enzyme appeared t o s e r v e a c a t a b o l i c f u n c t i o n , d e g r a d i n g o r n i t h i n e when p r e s e n t i n t h e c e l l i n e x c e s s . M o r r i s and Pardee (1966) found a second pathway f o r the 8 s y n t h e s i s o f p u t r e s c i n e i n E_. c o l i . T h i s c o n s i s t e d o f a con-s t i t u t i v e a r g i n i n e d e c a r b o x y l a s e c o n v e r t i n g a r g i n i n e t o a g m a n t i n e , and a c o n s t i t u t i v e agmantine u r e o h y d r o l a s e , h y d r o l y s t n g agmantine t o produce p u t r e s c i n e and u r e a . A l t h o u g h t h e s y n t h e s i s o f t h e enzymes of both pathways o f p u t r e s c i n e b i o s y n t h e s i s i s c o n s t i t u t i v e i n E_. c o l i , M o r r i s and K o f f r o n (1969) d i s c o v e r e d t h a t the r e l a t i v e f l o w t h r o u g h t h e two pathways v a r i e d c o n s i d e r a b l y i n v i v o . W i t h c e l l s grown i n minimal medium, t h e m a j o r i t y of t h e p u t r e s c i n e was produced by t h e d e c a r b o x y l a t i o n o f o r n i t h i n e , w h e r e a s , i n t h e p r e s e n c e o f exogenous a r g i n i n e , the d i r e c t d e c a r b o x y l a t i o n o f a r g i n i n e was the p r e f e r r e d r o u t e . Tabor and Tabor (1969a) have: r e p o r t e d t h a t t h e o r n i t h i n e and a r g i n i n e d e c a r b o x y l a s e s o f a s t r a i n o f E_. c o l ? a u x o t r o p h i c f o r o r n i t h i n e were both r e p r e s s e d and f e e d b a c k i n h i b i t e d by p u t r e s c i n e and s p e r m i d i n e . G a l e (19^2) showed t h a t P_. a e r u g i n o s a p o s s e s s e d t h e c o n s t i t u t i v e a b i l i t y t o o x i d i z e t h e d i a m i n e s p u t r e s c i n e , agmantine and c a d a v e r i n e t o c o m p l e t i o n and c o u l d be adapted t o p a r t i a l l y o x i d i z e h i s t a m i n e and t y r a m i n e . Z e l l e r (1963) has reviewed t h e p r o p e r t i e s o f d i a m i n e o x i d a s e s from p l a n t , a n i m a l , and m i c r o b i a l s o u r c e s . The enzyme c a t a l y s e d t h e o x i d a t i v e d e a m i n a t i o n of a number o f d i a m i n e s , r e s u l t i n g i n t h e f o r m a t i o n o f the c o r r e s p o n d i n g amine a l d e h y d e s . When p u t r e s c i n e was the s u b s t r a t e , t h e p r o d u c t , y a m i n o b u t y r a l d e h y d e , r a p i d l y c y c l i z e d t o form A1- p y r r o l i n e . E v e l y n (1967a, b) d e m o n s t r a t e d , the c o n v e r s i o n o f p u t r e s c i n e t o A ^ - p y r r o l i n e , w i t h the c o n c o m i t a n t uptake of oxygen by cell-free extracts of putrescine grown Mycobacteria. Satake and Fujita (1953) studied the oxidation of putrescine and histamine by a cell-free extract of Achromobacter and concluded that the activity was the result of the action of two substrate-specific enzymes rather than a single enzyme reacting with both diamines. Moreover, the enzymes were dehydro-genases linked to the electron transport system, rather than oxidases.reacting directly with molecular oxygen. Kim and Tchen (1962) found that a mutant of E_. col? capable of degrading putrescine catalysed the conversion of putrescine to yamino-butyraIdehyde by a transaminase rather than by an oxidase. Jakoby and Fredericks (1959) discovered that growth of P_. fluorescens in the presence of putrescine induced the synthesis of an enzyme, Y~aminobutyraIdehyde dehydrogenase, that oxidized A^-pyrroline to yaminobutyric acid, with the concomitant reduction of NAD. Y" a m' n o' 3utyric-glutamate transaminase catalyzed the conversion of yaminobutyric acid to succinic semialdehyde, which was further oxidized to succinate by succinic semialdehyde dehydrogenase. Y~Aminobutyric-glutamlc transaminase was induced by growth in either Y~aminobutyrate or putrescine. Succinic semialdehyde dehydrogenase was present constitutfvely at a f a i r l y high level. Growth in putrescine did not apprec?ably increase the activity of this enzyme; however, growth in y-aminobutyrate did increase it somewhat. These three enzymes also function in p u t r e s c i n e d e g r a d a t i o n i n the E_. c o l i mutant (Kim and T c h e n , 1962) and M y c o b a c t e r i a ( E v e l y n , 1967) mentioned above. P_. a e r u g i r i b s a grown i n the p r e s e n c e o f y a m i n o b u t y r a t e a l s o o x i d i z e d t h i s compound t o s u c c i n a t e v i a s u c c i n i c s e m i a l d e h y d e ( B a c h r a c h , 1960). Nakamura (1960) d i s c o v e r e d t h a t P. a e r u g i n o s a p o s s e s s e d two s u c c i n i c s e m i a l d e h y d e d e h y d r o g e n a s e s ; one l i n k e d t o NAD, t h e o t h e r t o NADP. Padmanabhan and Tchen (1969) have f u r t h e r s t u d i e d t h e s e enzymes i n a Pseudomonad. The NADP-linked enzyme was c o n s t i t u t i v e , and c o u l d not be induced t o a h i g h e r l e v e l . The NAD-1inked s u c c i n i c s e m i a l d e h y d e dehydrogenase a c t i v i t y was r e s o l v e d i n t o t h r e e peaks by chromatography on DEAE-Sephadex, o n l y one o f w h i c h was s p e c i f i c f o r s u c c i n i c s e m i a l d e h y d e . T h i s enzyme was induced t o h i g h l e v e l s by growth o f the c e l l s i n y-aminobutyrate o r i n p u t r e s c i n e . The o t h e r two peaks were aminoaldehyde dehydrogenases a c t i n g on 3~ aminopropanal and y a m i n o b u t y r a l d e h y d e i n a d d i t i o n t o s u c c i n i c s e m i a l d e h y d e . One peak was much more r e a c t i v e w i t h t h e amino-a l d e h y d e s than w i t h s u c c i n i c s e m i a l d e h y d e and t h i s enzyme c o u l d be i nduced t o a h i g h l e v e l by growth i n p u t r e s c i n e , but not i n y a m i n o b u t y r a t e . T h u s , i t may c o r r e s p o n d t o t h e y a m i n o b u t y r a l d e h y d e dehydrogenase s t u d i e d by Jacoby and F r e d e r i c k s (1959)• Very low a c t i v i t i e s o f the NAD-1inked dehydrogenases were d e t e c t a b l e under a l l growth c o n d i t i o n s . I t was t h e r e f o r e h y p o t h e s i z e d t h a t t he o r g a n i s m was c o n t i n u o u s l y s y n t h e s i z i n g and d e g r a d i n g p u t r e s c i n e . In summary, a r g i n i n e may be d e c a r b o x y l a t e d t o a g m a n t i n e , o r i t may be c o n v e r t e d t o o r n i t h i n e , e i t h e r by an a r g i n a s e or by t h e a r g i n i n e d i h y d r o l a s e pathway. Both agmantine and o r n i t h i n e can be c o n v e r t e d t o p u t r e s c i n e , w h i c h can be f u r t h e r degraded t o s u c c i n a t e by some o r g a n i s m s . In a d d i t i o n , o r n i t h i n e can be d i r e c t l y degraded t o g l u t a m a t e . An a d d i t i o n a l pathway o f a r g i n i n e d e g r a d a t i o n has been de s -s c r i b e d i n S t r e p t o m y c e s g r i s e u s (van T h o a i , 1965). In t h i s o r g a n i s m , a r g i n i n e undergoes d e c a r b o x y l a t i n g o x y g e n a t i o n r e s u l t i n g i n t h e f o r m a t i o n o f y - g u a n i d o b u t y r a m i d e , w h i c h i s then c o n v e r t e d t o y-a m i n o b u t y r a t e v i a t h e i n t e r m e d i a t e y - g u a n i d o b u t y r a t e . S. g r i s e u s a l s o c o n t a i n s a t r a n s a m i d i n a s e f u n c t i o n i n g i n s t r e p t o m y c i n b i o -s y n t h e s i s , and c a t a l y s i n g t h e t r a n s f e r o f t h e g u a n i d i n e group o f a r g i n i n e t o g l y c i n e , f o r m i n g o r n i t h i n e and g l y c o c y a m i n e ( W a l k e r , 1965). T h i s enzyme i s a l s o p r e s e n t i n E_. c o l i ( W i l s o n and H o l d e n , 1969a) and C_. b o t u l inum ( M f t r u k a and C o s t i l o w , 1967). More than one pathway o f a r g i n i n e d e g r a d a t i o n may be o p e r a t i v e i n a s i n g l e o r g a n i s m . W i l s o n and Holden (1969a) have r e p o r t e d t h a t , a l t h o u g h the m a j o r i t y o f exogenous a r g i n i n e was c o n v e r t e d t o p u t r e s c i n e v i a agmantine i n E_. c o l ?, k - 8% was c o n v e r t e d t o g l u t a m a t e , presumably v i a o r n i t h i n e and g l u t a m i c - y - s e m i a l d e h y d e . M i t r u k a and C o s t i l o w (l967) d e m o n s t r a t e d t h a t C_. b o t u l inum p o s s e s s e d an a r g i n i n e t r a n s a m i d i n a s e i n a d d i t i o n t o t h e enzymes o f t h e a r g i n i n e d i h y d r o l a s e pathway. M o r e o v e r , t h i s o r g a n i s m degraded o r n i t h i n e by two pathways, 20% b e l n grd e c a r b o x y l a t e d t o p u t r e s c i n e , w h i l e 75% was degraded v i a 6 - a m i n o v a l e r i c a c i d . I t . C a t a b o l t t e R e p r e s s i o n o f Amtno A c i d D e g r a d a t i o n Epps and G a l e (19^2) were t h e f i r s t t o s t u d y t h e r e p r e s s i o n o f enzymes c a t a b o l t z i n g amtno a c i d s , w h i c h was caused by the add t t ton o f g l u c o s e t o the g r o w t h medium. In E_. c o l t , o r n t t h t n e d e c a r b o x y l a s e , t r y p t o p h a n a s e , a s p a r t a s e and a l a n t n e , s e r i n e , and g l u t a m a t e deaminases were s u b j e c t t o t h i s r e p r e s s i o n , whereas a r g i n i n e , l y s i n e , and h t s t t d t n e d e c a r b o x y l a s e s were u n a f f e c t e d . The i n d u c t i o n o f many enzymes r e s p o n s i b l e f o r t h e c a t a b o l ism o f c a r b o h y d r a t e s and o f amtno a c i d s t s r e p r e s s e d by g l u c o s e . T h i s phenomenon was termed c a t a b o l t t e r e p r e s s i o n by Magasantk 0 961) who c o n s i d e r e d i t t o be a t y p e o f e n d - p r o d u c t r e p r e s s t o n , s i n c e the c a t a b o l i t e s formed by t h e a c t i o n of t h e g l u c o s e -s e n s i t t v e enzymes c o u l d be more r e a d i l y o b t a t n e d from g l u c o s e . T h u s , any s i t u a t i o n where l i m i t a t t o n o f a n a b o l i s m caused an a c c u m u l a t i o n o f c a t a b o l i c t n t e r m e d t a t e s ( e . g . , n i t r o g e n o r phosphate s t a r v a t i o n ) produced a s i m i l a r r e p r e s s t o n . R e c e n t l y , t t has been d i s c o v e r e d t h a t g l u c o s e and o t h e r r a p i d l y m e t a b o l t z a b l e c a r b o n s o u r c e s cause t h e r e p r e s s t o n o f enzyme s y n t h e s i s tn E_. c o l t by l o w e r i n g t h e i n t r a c e l l u l a r c o n c e n t r a t i o n of c y c l i c a d e n o s i n e monophosphate ( c y c l i c AMP). The a d d i t i o n of c y c l i c AMP t o cultures of E. coli overcame the catabolite repression of trypto-phanase, D-sertne deaminase, thymidine phosphorylase, and permeases and catafaoltc enzymes specific for several sugars, (see review by Pastan and Perlman, 1970). Jacoby (1964) found that glucose repressed the ab i l i t y of P_. fluoresceins to oxidize 18 different amino acids. He further studied the oxidation of tyrosine and histidine and observed that glucose did not cause a decrease in the rate of uptake of these amino acids. However, the induction of enzymes specific for the catabolism of these amino acids was repressed by glucose. Lessie and Neidhardt (1967) found that the hist idase activity of P_. aeruginosa was, subject to repression by a number of carbon sources, but that partial derepression occurred when ammonium ions were omitted from the medium. A similar derepression has been demonstrated in Aerobacter aerogenes (Neidhardt and Magasanik, 1957); however ammonium ions did not repress the synthesis of histidase in the absence of glucose. Thus, histidase production was maximal when histidine was required as a source of carbon and energy and was somewhat reduced when histidine was required only as a nitrogen source. It was severely repressed when all of the products of histidine catabolism could be supplied by a more rapidly metabolizable energy source, it Is interesting to note that the repression of a carbohydrate degrading enzyme, myo-inositol dehydrogenase, was not affected by ammonium ions. These workers have proposed that the compound that i s p h y s i o l o g i c a l l y a c t i v e in the c a t a b o l i t e repression of amino aci d s i s a nitrogenous compound which i s r e a d i l y formed from the c a t a b o l i t e s of glucose. Castanada, M a r t u s c e l l i and Mora (1967) showed that the presence of ammonium ions in the growth medium of N_. crassa decreased the l e v e l to which arginase and o r n i t h i n e transaminase were induced. Wiame (1965) has reported that the presence of ammonium i o n s , but not glutamate, a f f e c t e d the u t i l i z a t i o n of a r g i n i n e by B_. subti 1 i s . Middelhoven (1970) has hypothesized that the l e v e l of arginase and o r n i t h i n e transaminase in S_. c e r e v i s i a e i s c o n t r o l l e d by a nitrogenous r e p r e s s o r . The induction of these enzymes was not a f f e c t e d by g l u c o s e , but was i n h i b i t e d by ammonium sulphate and a few of the more r e a d i l y a s s i m i l a b l e amino a c i d s . This was not end-product repression due to the formation of glutamate, since glutamate i t s e l f was not a strong i n h i b i t o r . Moreover, s t a r v a t i o n of S^ . c e r e v i s i a e f o r nitrogen derepressed arginase and o r n i t h i n e transaminase to a l a v e l as high as that induced by a r g i n i n e . This r e s u l t e d in a d e p l e t i o n of the c e l l u l a r a g i n i n e p o o l . Derepression could be i n h i b i t e d by the a d d i t i o n of one of a number of nitrogen c o n t a i n i n g compounds. It was s p e c i f i c f o r the a r g i n i n e degrading enzymes; other enzymes involved in p r o t e i n and amino a c i d degrada-t i o n were not a f f e c t e d . L e s s i e and Neidhardt (1967) a l s o noted that s u c c i n a t e , which produced the most severe c a t a b o l i t e repression of h i s t i d a s e synthesis in P_. aeruginosa, a l s o i n h i b i t e d the h i s t i d a s e a c t i v i t y in v i v o . Hug, Roth and Hunter (1968) found that succinate c o m p e t i t i v e l y i n h i b i t e d urocanase, the second enzyme of h i s t i d i n e d e g radation, in Pseudomonas p u t i d a . Urocanate was a competitive i n h i b i t o r of h i s t i d a s e . Thus, the presence of succinate caused an in v i v o r e p r e s s i o n of h i s t i d a s e by sequential feedback. However, Jensen and Neidhardt (1969) found t h a t , in'A. aerogenes, in v i v o i n h i b i t i o n of h i s t i d a s e a c t i v i t y d i d not requ i r e the presence of s u c c i n a t e , but could be caused by r e s t r i c t i n g growth in h i s t i d i n e in a chemo-s t a t by l i m i t i n g an e s s e n t i a l n u t r i e n t . This i n h i b i t i o n appeared to be immediately released when the b i o s y n t h e t i c r e s t r i c t i o n was r e l e a s e d , a l l o w i n g an immediate increase in growth rate before the h i s t i d a s e l e v e l had increased s i g n i f i c a n t l y . Thus, c a t a b o l i t e i n h i b i t i o n appears to act as a f i n e c o n t r o l mechanism s i m i l a r to the feedback i n h i b i t i o n of b i o s y n t h e t i c pathways. C a t a b o l i t e i n h i b i t i o n of the u t i l i z a t i o n of a number of sugars has been noted in E_. col i (McGinnis and Paigen, 19^9) • As f a r as the enzymes of a r g i n i n e degradation are concerned, Ramos et a 1. (1967) found that a r g i n i n e deiminase, carbamate k i n a s e , and the c a t a b o l i c o r n i t h i n e transcarbamylase were subject to c a t a b o l i t e repression in P_. f 1 uorescens. These enzymes were derepressed when growth was l i m i t e d by the carbon source, c i t r a t e . L a i s h l e y and Bernlohr (1968) showed that the induction of a r g i n a s e , o r n i t h i n e transaminase, and A 1 - p y r r o l i n e - 5 ~ c a r b o x y l a t e dehydrogenase was r e p r e s s e d by g l u c o s e i n JB. 1 t c h e n i f o r m i s ; however, M i d d e l h o v e n (1970) o b s e r v e d t h a t t h e a r g i n a s e and o r n i t h i n e t r a n s a m i n a s e o f S.- c e r e v t s i a e were h o t s u b j e c t t o c a t a b o l i t e r e p r e s s i o n . Padmanabhan and Tchen (1969) found t h a t g l u c o s e d i d not r e p r e s s the i n d u c t i o n o f s u c c i n i c s e m i a l d e h y d e dehydrogenase by Y ~a i T ,ino -b u t y r a t e . However, when p u t r e s c i n e was the i n d u c e r , t h e l e v e l s o f b o t h s u c c i n i c s e m i a l d e h y d e dehydrogenase and aminoaldehyde dehydrogenase were l o w e r e d . These w o r k e r s t h e r e f o r e f e l t t h a t g l u c o s e caused t h e r e p r e s s i o n o f Y ~a m' n o b u t y r a l d e h y d e d e h y d r o g e n a s e , t h e r e b y i n h i b i t i n g t h e i n d u c t i o n o f s u c c i n i c s e m i a l d e h y d e de-hydrogenase by p u t r e s c i n e , by l o w e r i n g t h e c o n c e n t r a t i o n o f the i n d u c e r , Y ~a min°b u t y r a t e . l i t . The B i o l o g i c a l Importance of P o l y a m i n e s i n M i c r o o r g a n i s m s Kay (1969) found t h a t P_. a e r u g i n o s a , when s u p p l i e d w i t h exogenous a r g i n i n e , formed a h i g h i n t r a c e l l u l a r pool o f p u t r e s c i n e w h i c h was s t a b l e o v e r 2k hours o f s t a r v a t i o n f o r an exogenous c a r b o n s o u r c e . T h u s , p u t r e s c i n e appeared t o be an i m p o r t a n t p r o d u c t o f a r g i n i n e d e g r a d a t i o n i n t h i s o r g a n i s m , and may be m a i n t a i n e d w f t h i n t h e c e l l i n a bound s t a t e . P u t r e s c i n e and t h e h i g h e r p o l y a m i n e s , s p e r m i d i n e and s p e r m i n e , w h i c h a r e s y n t h e s i z e d from p u t r e s c i n e , a r e p r e s e n t i n v a r y i n g c o n c e n t r a t T o n s i n m i c r o o r g a n i s m s , p l a n t s , and a n i m a l s . P_. a e r u g i n o s a 17 has been r e p o r t e d t o c o n t a i n k5% o f i t s p o l y a m i n e s as p u t r e s c i n e ; 32% as s p e r m i d i n e , and 23% as spermine (Weaver and H e r b s t , 1 9 5 8 ) . In E_. c o l ?, on the o t h e r hand, p u t r e s c i n e c o m p r i s e s 90% o f the t o t a l c e l l p o l y a m i n e , w i t h s p e r m i d i n e c o n s t i t u t i n g t h e remainder (Tabor and T a b o r , 1 9 6 4 ) . There i s i n d i r e c t e v i d e n c e t h a t p o l y a m i n e s p e r f o r m an im-p o r t a n t f u n c t i o n i n E_. c o l i . The i n t r a c e l l u l a r p u t r e s c i n e c o n t e n t i s t h e same i n c e l l s grown i n minimal medium as i n c e l l s grown i n t he p r e s e n c e o f a r g i n i n e o r o r n i t h i n e ( M o r r i s and K o f f r o n , 1 9 6 9 ) . Tabor and Tabor (1969a) found t h a t an o r n i t h i n e a u x o t r o p h c o n t i n u e d t o s y n t h e s i z e p o l y a m i n e s , even when o r n i t h i n e l i m i t a t i o n caused a t h r e e - f o l d r e d u c t i o n i n growth r a t e . A l t h o u g h t h e p u t r e s c i n e c o n t e n t was m a r k e d l y d e c r e a s e d under t h e s e c o n d i t i o n s , t h e s p e r m i d i n e l e v e l was o n l y s l i g h t l y r e d u c e d . S t u d i e s w i t h mutants o f E_. c o l i ( H i r s h f i e l d et_ a l _ . 1970) and N_. c r a s s a ( D a v i s , L a w l e s s and P o r t , 1970) have d e m o n s t r a t e d t h a t t h e g rowth r a t e o f t h e s e o r g a n i s m s was g r e a t l y reduced when p u t r e s c i n e s y n t h e s i s was r e p r e s s e d . Many snake forms were o b s e r v e d , i n d i c a t i n g t h a t c e l l d i v i s i o n may have been a f f e c t e d . Normal growth was r e s t o r e d by t h e a d d i t i o n o f s p e r m i n e , s p e r m i d i n e , o r p u t r e s c i n e . Inouye and Pardee (1970) have p r e s e n t e d e v i d e n c e t h a t t he r a t i o o f p u t r e s c i n e t o s p e r m i d i n e may be a c r i t i c a l f a c t o r f o r c e l l d i v i s i o n i n E. c o l i . The b i o c h e m i c a l a c t i o n o f p o l y a m i n e s has been r e c e n t l y r e v i e w e d ( S t e v e n s , 1970). P o l y a m i n e s b i n d s t r o n g l y t o d e o x y r i b o -n u c l e i c a c i d (DNA), r i b o n u c l e i c a c i d (RNA) , and s y n t h e t i c p o l y -n u c l e o t i d e s i n v i t r o . They s t a b i l i z e t h e d o u b l e h e l i c a l s t r u c t u r e o f DNA, and appear t o cause RNA t o assume a more compact s t r u c t u r e . P o l y a m i n e s do not appear t o be p r e f e r e n t i a l l y a s s o c i a t e d w i t h DNA i n v i v o , but may a s s o c i a t e w i t h RNA. In E_. c o l ?, an i n c r e a s e i n t h e i n t r a c e l l u l a r c o n c e n t r a t i o n o f s p e r m i d i n e , but not p u t r e s c i n e , r e s u l t e d i n an i n c r e a s e d r a t e o f RNA s y n t h e s i s . The s p e r m i d i n e c o n c e n t r a t i o n a p p e a r s t o be r e l a t e d t o RNA s y n t h e s i s i n animal t i s s u e s , a l s o . M o r e o v e r , t h e a d d i t i o n o f p o l y a m i n e s t o a c e l l - f r e e system c a u s e s an i n c r e a s e i n RNA polymerase a c t i v i t y , a p p e a r i n g t o i n c r e a s e t h e number o f a v a i l a b l e i n i t i a t i o n s i t e s . P o l y a m i n e s a l s o promote t h e a s s o c i a t i o n o f r i b o s o m a l sub-u n i t s and t h e b i n d i n g o f messenger RNA and amino a c y l t r a n s f e r RNA t o r i b o s o m e s . They can p a r t i a l l y r e p l a c e magnesium i o n s i n an i n v i t r o p r o t e i n s y n t h e s i z i n g s y s t e m . S i n c e o p t i m a l growth of E_. c o l i o c c u r e d a t i n t r a c e l l u l a r magnesium c o n c e n t r a t i o n s much lower than t h e c o n c e n t r a t i o n r e q u i r e d f o r i n v i t r o p r o t e i n s y n t h e s i s , H u r w i t z and Rosano (1967) h y p o t h e s i z e d t h a t p o l y a m i n e s may p l a y an i m p o r t a n t r o l e i n i n v i v o p r o t e i n s y n t h e s i s , b i n d i n g t o many o f t h e s i t e s where magnesium b i n d s i n the i n v i t r o s y s t e m . Weiss and M o r r i s (1970) found t h a t up t o 70% of t h e magnesium bound t o ribosomes c o u l d be r e p l a c e d by s p e r m i d i n e o r p u t r e s c i n e w i t h o u t a f f e c t i n g t h e i r a c t i v i t y i n c e l l - f r e e p r o t e i n s y n t h e s i s . However, a c r i t i c a l l e v e l o f magnesium was r e q u i r e d f o r t h e s t r u c t u r a l and f u n c t i o n a l i n t e g r i t y o f r i b o s o m e s , and c o u l d not be r e p l a c e d by p o l y a m i n e s . The o r d e r o f a c t i v i t y o f p o l y a m i n e s i n a l l t h e p r e v i o u s l y mentioned f u n c t i o n s i s s p e r m i n e > s p e r m i d i n e > p u t r e s c i n e . T h u s , a l t h o u g h E_. co_lj_ c o n t a i n s much more p u t r e s c i n e t h a n s p e r m i d i n e , t h e l a t t e r may be p h y s i o l o g i c a l l y more i m p o r t a n t . I t i s i n t e r e s t -i n g t o n o t e t h a t , i n a Pseudomonad u n a b l e t o s y n t h e s i z e s p e r m i d i n e , t h e b i n d i n g o f h y d r o x y p u t r e s c i n e , w h i c h p o s s e s s e s t h e same number o f b i n d i n g s i t e s as s p e r m i d i n e , t o ribosomes v a r i e s w i t h t h e magnesium c o n c e n t r a t i o n i n the same way a s does s p e r m i d i n e tn E_. c o l I CRosano and H u r w l t z , 1969). tn summary, p o l y a m i n e s b i n d t o and s t a b i 1 i z e DNA, RNA, and r i b o s o m e s . . They s t i m u l a t e p r o t e i n s y n t h e s i s i n v i t r o , and RNA s y n t h e s i s both In v i t r o and In v i v o . I t i s p o s s i b l e t h a t p o l y -amines do not have a s i n g l e s p e c i f i c f u n c t i o n i n c e l l m e t a b o l i s m , but may a c t as p o l y v a l e n t c a t i o n s s t a b i l i z i n g n u c l e o t i d e - n u c l e o t i d e b i n d i n g , r e p l a c i n g many of t h e magnesium tons r e q u i r e d f o r j_n_ v i t r o r e a c t i o n s . 20 IV. Transport of the Basic Amino Acids by Microorganisms The t r a n s p o r t and accumulation of amino aci d s by microorganisms has been reviewed e x t e n s i v e l y (Kepes and Cohen, 1962; B r i t t e n and McClure, 1962; Kay, 1968; Kabak, 1970). B a c t e r i a have been found to possess many transport systems, some of which are s p e c i f i c f o r s i n g l e amino aci d s and others of which are s p e c i f i c f o r " f a m i l i e s " of 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 . Kay (1968) i d e n t i f i e d 11 such amino a c i d tr a n s p o r t systems in P_. aerugi nosa. The transport of the basic amino aci d s has been studied in several microorganisms. Schwartz, Maas, and Simon (1959) i s o l a t e d a mutant of E_. col_i_ which was d e f e c t i v e in the uptake of canavanine, a r g i n i n e , o r n i t h i n e , and l y s i n e , and t h e r e f o r e concluded that these compounds must be transported by a s i n g l e permease. C i t r u l l i n e uptake by a l s o a f f e c t e d somewhat by t h i s mutation (Maas, 1965)• The studies of Wilson and Holden (1969a) in d i c a t e d that E_. col i possessed at l e a s t two systems f o r the uptake of basic amino a c i d s : one h i g h l y s p e c i f i c f o r a r g i n i n e , and one with a high a f f i n i t y f o r l y s i n e and a lower a f f i n i t y f o r a r g i n i n e . Wilson and Holden (1969b) i s o l a t e d four p r o t e i n s , from the osmotic shock f l u i d of E_. col i , which bound a r g i n i n e , but showed no a f f i n i t y f o r l y s i n e . Two of these p r o t e i n s were studied f u r t h e r , and were capable of r e s t o r i n g the a b i l i t y of shocked c e l l s to transport a r g i n i n e . Kay (1968) obtained evidence f o r the e x i s t e n c e , in P_. aerug 1 nosa, of two systems for the transp o r t of b a s i c amino a c i d s : one s p e c i f i c f o r a r g i n i n e and o r n i t h i n e , and t h e o t h e r h a v i n g a h i g h a f f i n i t y f o r l y s i n e and lower a f f i n i t i e s f o r a r g i n i n e , o r n i t h i n e , c i t r u l l i n e , and h i s t i d i n e . Grenson (1966) and Grenson et_ a j _ . (1966) dem o n s t r a t e d t h a t S_. c e r e v i s i a e a l s o p o s s e s s e d two systems f o r t h e uptake o f b a s i c amino a c i d s : one was h i g h l y s p e c i f i c f o r l y s i n e ; t h e o t h e r had a v e r y h i g h a f f i n i t y f o r a r g i n i n e , and a lower a f f i n i t y f o r l y s i n e , o r n i t h i n e and c a n a v a n i n e . P a l l (1970) showed t h a t N_. c_rassa_ pos-s e s s e d a b a s i c amino a c i d t r a n s p o r t system w i t h a h i g h a f f i n i t y f o r a r g i n i n e , l y s i n e , c a n a v a n i n e , and o r n i t h i n e , and a much lower a f f i n i t y f o r h i s t i d i n e . G r e n s o n , Hou and C r a b e e l (1970) showed t h a t t h e o n l y mechanism o f c i t r u l l i n e u p t a k e by S_. c e r e v i s i a e was v i a t h e g e n e r a l amino a c i d permease, wh i c h was c a p a b l e o f t r a n s p o r t i n g most amino a c i d s . T h w a i t e s and P e n d y a l a (1969) d e m o n s t r a t e d t h a t c i t r u l l i n e was a l s o t r a n s p o r t e d s o l e l y by t h e g e n e r a l amino a c i d t r a n s p o r t system o f N_. c r a s s a . Ames (1964) and Ames and Roth (1968) have shown t h a t S a l m o n e l l a  t y p h i m u r i u m p o s s e s s e d a h i g h l y s p e c i f i c system f o r the t r a n s p o r t ' o f h i s t i d i n e . T h i s o r g a n i s m c o u l d a l s o t a k e up h i s t i d i n e v i a a g e n e r a l a r o m a t i c permease, wh i c h f u n c t i o n e d i n t h e t r a n s p o r t o f t y r o s i n e , p h e n y l a l a n i n e , and t r y p t o p h a n . On the o t h e r hand, Kay (1968) found t h a t a l l b a s i c amino a c i d s competed s t r o n g l y w i t h h i s t i d i n e , and t h e r e f o r e c o n c l u d e d t h a t h i s t i d i n e must be t r a n s p o r t e d m a i n l y by the b a s i c amino a c i d u p t a k e systems i n t h i s o r g a n i s m . P a l l (1970) has shown t h a t N_. c r a s s a can t r a n s p o r t h i s t i d i n e v i a t h r e e t r a n s p o r t s y s t e m s : the b a s t e amino a c i d t r a n s p o r t s y s t e m , t h e n e u t r a l amino a c i d t r a n s p o r t s y s t e m , and th e g e n e r a l amino a c i d t r a n s p o r t s y s t e m . MATERIALS AND METHODS I. Organisms and Media Pseudomonas aeruginosa (ATCC 9027) was used throughout this study. Pseudomonas fluorescens A.3.12 (W.A. Wood) and Pseudomonas  put Ida (ATCC 4359) were also used for one experiment. Stock cultures were maintained at 6 C in glucose ammonium salts minimal medium and were checked periodically for purity by streaking onto Difco plate count agar. Cultures of P_. aeruginosa were also checked for production of the species characteristic pigment, pyocyanine, by streaking onto Kings medium (King, Ward, and Raney, 1954). Cells were grown in a medium containing 0.3% NH^ H^ PO^ , 0.2% K^PO^ and 0.5 ppm FeS0^.7H20 at pH 7.2. MgS0^.7H20 and the appropriate carbon source were sterilized separately and added aseptically to give a final concentration of 0.05% and 0.2% respectively. When putrescine or arginine were used as sources of both nitrogen and carbon, NH^PO^ was replaced by 0.35% KH^O^ When arginine was used as the sole carbon source, 1 ml of 1M potassium phosphate buffer (pH 7.0) was added to 20 ml of the minimal medium described above, to prevent an increase in pH during growth. I I . Growth o f C e l I s For most experiments;, Erlenmeyer f l a s k s (250 ml) equipp e d w f t h 13 mm t e s t tube s t d e a r m s and c o n t a f n f n g 30 ml q u a n t i t i e s o f medium were i n o c u l a t e d t o a f i n a l c o n c e n t r a t i o n o f 2% w i t h a c e l l s u s p e n s i o n p r e v i o u s l y grown tn t h e same medtum, and were then t n c u b a t e d a t 30 C i n a model G77 w a t e r b a t h (New B r u n s w i c k S c t e n t i f i c Co., New B r u n s w i c k , N.J.) r o t a t i n g a t 220 r e v / m t n . Growth was f o l l o w e d w t t h a Klett-Summerson c o l o r i m e t e r ( r e d f i l t e r ) and c e l l s f r o m t h e l o g a r i t h m i c phase were h a r v e s t e d a t 110 - 125 K l e t t u n i t s [ e q u i v a l e n t t o 0.7 ~ 0.8 o p t i c a l d e n s i t y u n i t s measured a t 660 nm w i t h a model B s p e c t r o p h o t o m e t e r (Beckman I n s t r u m e n t s I n c . , F u l l e r t o n , C a l i f . ) ] . For some e x p e r i m e n t s , c e l l s were grown i n Roux f l a s k s con-t a i n i n g 100 ml o f t h e a p p r o p r i a t e medium. A 1% innoculum was u s e d , and t h e c e l l s from the e a r l y s t a t i o n a r y phase o f growth were h a r v e s t e d a f t e r 20 h o u r s a t 30 C. ill. P r e p a r a t i o n o f C e l 1 S u s p e n s i o n s 1. R e s t i n g c e l l s u s p e n s i o n s f o r r e s p i r o m e t r y C e l l s were h a r v e s t e d by c e n t r i f u g a t t o n a t 10,000 x g_ f o r »7 m i n u t e s a t 6 C. They were washed t h r e e t i m e s w t t h c o l d O.St NaCl (pH 7.4) and resuspended in cold 0.05 M Tris(hydroxymethyl)-aminomethane-HCl (Tris) buffer (pH 7-4). In some cases, cells were resuspended in 0.067 M potassium phosphate buffer (pH 7.4). The final c e l l concentration was approximately 5 mg of cells (dry weight)/ml, unless otherwise specified. 2. Cell suspensions for transport studies. Cells in the logarithmic phase of growth were harvested as described above, but at room temperature, and washed twice with minimal salts medium without carbon source. They were resuspended In glucose minimal medium to approximately 1.35 mg of cells (dry weight)/ml and kept at room temperature until required for experimentation. IV. Preparation of Cell-free Extracts Cells were harvested from the logarithmic phase of growth by centrifugation at 10,000:.x g_ for 7 minutes at room temperature, and washed twice with 0.9% NaCl (pH 7-4). When necessary, dry cell pellets were stored at -70 C. They were resuspended in cold 0.01 M potassium phosphate buffer (pH 7.4) containing 0.01% mercapto-ethanol, to a final concentration of approximately 20 mg of cells Xdry weight)/ml. Deoxyribonuclease (Worthington Biochemical Corp., F r e e h o l d , N.J.) was added t o a f i n a l c o n c e n t r a t i o n o f 80 y g / m l . The c e l l s were broken by dropwtse e x p u l s i o n from a p r e c o o l e d F r e n c h p r e s s u r e c e l l under 15,000 p s i p r e s s u r e . Unbroken c e l l s and l a r g e c e l l u l a r d e b r i s were removed by c e n t r t f u g a t t o n a t 10,000 x £ f o r 7 m i n u t e s a t 6 C. V. Manometric P r o c e d u r e s The oxygen u p t a k e o f c e l l s r e s p i r i n g e n d ogenously o r i n the p r e s e n c e o f exogenous s u b s t r a t e s was f o l l o w e d i n t h e c o n v e n t i o n a l manner w i t h t h e Warburg r e s p i r o m e t e r . A t y p i c a l r e a c t i o n m i x t u r e c o n t a i n e d 1.0 ml c e l l s u s p e n s i o n [ a p p r o x i m a t e l y 5 mg o f c e l l s ( d r y w e i g h t ) / m l ] , 1.9 ml 0.05 M T r i s b u f f e r (pH 7.4), 2.5 ymoles s u b s t r a t e i n 0.1 m l , and 0.15 ml 20% K0H i n t h e c e n t e r w e l l . In t h e endogenous c o n t r o l , 0.1 ml d i s t i l l e d w a t er r e p l a c e d t h e s u b s t r a t e . For some e x p e r i m e n t s t he T r i s was r e p l a c e d by 0.067 M phosphate b u f f e r (pH 7-4). V I . Uptake o f L a b e l l e d Compounds 14 The i n c o r p o r a t i o n o f C - l a b e l l e d compounds i n t o whole c e l l s , p r o t e i n , and p o o l s was d e t e r m i n e d by the M i l l i p o r e f i l t r a t i o n p r o c e d u r e o f B r i t t e n and M c C l u r e (1962). E x p e r i m e n t s were c a r r i e d out a t 30 C, i n 25 ml o r 50 ml Erlenmeyer f l a s k s c o n t a i n i n g g l u c o s e minimal medium, s t i r r e d w i t h a Mag-Jet underwater s t i r r e r (Bronwel1 S c i e n t i f i c , Rochester, N.Y.) dr i v e n by a Lauda K2 c i r c u l a t i n g pump (Brinkman Instruments, Westbury, N.Y.). Unless otherwise s p e c i f i e d , c e l l s were added to give a f i n a l c oncentration of 0.135 mg of c e l l s (dry weight)/ml, and substrates were added to give an external -5 14 concen t r a t i o n of 2.5 x 10 M, and 0.05 yc C/ml. Samples of the c e l l suspension were removed at appropriate time I n t e r v a l s and e i t h e r f i l t e r e d immediately or added to an equal volume of cold 10% t r i c h l o r o a c e t i c a c i d . Whole c e l l s or t r i c h l o r o a c e t i c a c i d -i n s o l u b l e m a t e r ial were f i l t e r e d on 0.45 y pore s i z e f i l t e r s ( M i l l i p o r e Corp., Bedford, Mass.) in an E8B p r e c i p i t a t i o n apparatus ( T r a c e r l a b , Waltham, Mass.) and immediately washed with 2 ml of glucose minimal medium (whole c e l l s ) or 2 ml of d i s t i l l e d water ( t r i c h l o r o a c e t i c a c i d - i n s o l u b l e m a t e r i a l ) . F i l t e r s were d r i e d under an i n f r a - r e d lamp and placed in v i a l s c o n t a i n i n g 5 ml of s c i n t i l l a t i o n f l u i d ( L i q u i f l u o r , New England Nuclear Corp., Boston, Mass.). The v i a l s were assayed f o r r a d i o a c t i v i t y in a model 725 l i q u i d s c i n t i l l a t i o n spectrometer (Nuclear Chicago Corp., Des P l a i n e s , 111.). C o r r e c t i o n s were made f o r background. In order to reduce s t a t i s t i c a l d e v i a t i o n , at l e a s t 1000 counts were re-corded. The counting e f f i c i e n c y of the instrument was 80 per cent under the c o n d i t i o n s employed. 28 VII. Inhibition of Transport by Compounds S t r u c t u r a l l y Related  to the Substrate To determine i n h i b i t i o n , the unlabel led i n h i b i t o r was added -3 to a concentration of 2.5 x 10 M immediately p r i o r to the addition 1 k -5 of the C-labelled substrate to a concentration of 2.5 x 10 M. Uptake of r a d i o a c t i v i t y into the whole c e l l s was followed, and the degree of i n h i b i t i o n was calculated from the reduction In 14 the rate of incorporation of the C-labelled substrate r e l a t i v e to the appropriate c o n t r o l . V I I I . Assay of Succinic Semialdehyde Dehydrogenase Succinic semialdehyde dehydrogenase was measured as described by Jakoby (1962) by coupling the reaction to Y ~a m'n°b u t y r a t e -glutamic transaminase. The pH of the reaction mixture was 8.0 and 5 ymoles of both y-amfnobutyrate and a-ketoglutarate were added. The reduction of 0.25 ymoles of NAD or NADP was measured at 340 nm at 34 C in a model 2000 spectrophotometer ( G i l f o r d Instrument Laboratories Incorp., O b e r l i n , Ohio). Appropriate controls were ca r r i e d out to show the requirement of the reaction for both yaminobutyrate and a-ketoglutarate. S p e c i f i c a c t i v i t i e s are expressed as mymoles of substrate u t i l i z e d per min per mg (of p r o t e i n . IX. Chemical Fractionation of Whole Cells Cells were fractionated according to the procedure of Roberts et_al_. (1955) with the modification of Clifton and Sobek (1961). The hot trichloroacetic fraction was prepared by heating the sample at 90 C for 20 min rather than at 100 C for 30 min. Samples of the cell fractions were plated in duplicate onto stainless steel planchets, dried under an infra-red lamp, and counted at infinite thinness with an automatic low background planchet counting system (Model 43^2, Nuclear Chicago Corp., Des Plaines, 111.). Corrections were made for background. In order to reduce statistical deviation, at least 1000 counts were recorded when possible. The counting efficiency of the instrument was 28%. X. Physical Fractionation of Whole Cells Cells were harvested by centrifuging the contents of the Warburg cups at 10,000 x g_ for 7 minutes at 6 C, and were re-suspended in 0.05 M Tris buffer (pH J.h) containing 10 M MgCl2 to approximately 20 mg of cells (dry weight)/ml. The suspension was passed once through a French pressure cell and whole cells and large debris were removed by centrifugation for 7 minutes at 5,000 x g_. The resulting cell-free extract was fractionated according to the procedure of Campbell, Hogg and Strasdine (1962), o m i t t i n g the washing of the i n i t i a l 25,000 x g_ pel l e t : .c The magnesium con c e n t r a t i o n of the 25,000 x g_ supernatant f l u i d was -3 adjusted to 10 M before c e n t r i f u g a t i o n . The p e l l e t s were resuspended in 0.05 M T r i s (pH 7.4). X I . Chromatography of Supernatant F l u i d s from Warburg Cups 14 Containing C - l a b e l l e d Substrates P r o t e i n was p r e c i p i t a t e d from the supernatant f l u i d s by the a d d i t i o n of t r i c h l o r o a c e t i c a c i d to a f i n a l c o n c e n t r a t i o n of 5%-. A f t e r 20 min on i c e , the p r e c i p i t a t e d m a t e r ial was removed by c e n t r i f u g a t i o n at 10,000 x £ f o r 10 minutes at 6 C. The super-natant f l u i d s were then extracted 6 times w i t h e t h y l ether to remove t r i c h l o r o a c e t i c a c i d , and evaporated to dryness using an Evapomix instrument (Buchler Instruments, Fort Lee, N.J.). The d r i e d samples were d i s s o l v e d in the desired volume of d i s t i l l e d water. When phosphate was present in the r e a c t i o n m i x t u r e , the sampl were app l i e d to a column of Dowex 50 H+. The column was washed wi t h d i s t i l l e d water u n t i l no f u r t h e r r a d i o a c t i v i t y was e l u t e d , removing the neutral and a c i d i c compounds, i n c l u d i n g phosphate. The adsorbed compounds were eluted with 4 M ammonium hydroxide, the e l u a t e was evaporated to dryness 4 times to remove ammonia, and the residue was dissolved in the desired amount of d i s t i l l e d water. Samples were quantitatively applied to thin layer plates spread with cellulose powder MN300 (Macherey and Nagel , Piiren, Germany) and the radioactive compounds were separated two-dimensional ly by the method of Jones and Heathcote (1966) -The chromatograms were then exposed for one week to medical X-ray film (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 l -lation vials which were subsequently f i l l e d with 10 ml of sc i n t i l l a t i o n fluid and assayed for radioactivity in a liquid s c i n t i l l a t i o n spectrometer. The addition of from 5 to 60 mg of cellulose caused no increase in quenching under these conditions. In some instances, the presence of radioactivity in aqueous samples was assayed directly by liquid s c i n t i l l a t i o n spectrometry. Up to 100 yl of the sample was placed in a vial containing 10 ml of a s c i n t i l l a t i o n fluid consisting of kO volumes of methanol and 60 volumes of toluene plus luquifluor. Values obtained by this method were multiplied by 1.3 to convert them into values com-parable to those obtained with the previously mentioned s c i n t i l -lation f l u i d . 32 X I I . Assay of O r n i t h i n e Transcarbamylase The assay procedure of Stalon et^aj_. (1967a) was f o l l o w e d . In order to e s t a b l i s h a r e a c t i o n r a t e , the r e a c t i o n was c a r r i e d out in a t o t a l volume of 25 ml c o n t a i n i n g 1 ml of c e l l - f r e e e x t r a c t , and 2.5 ml samples were removed at various times, added to an equal volume of 1 N HC1 i n heavy-walled c e n t r i f u g e tubes standing in i c e , and treated as in the procedure of Stalon et_ a_l_. (1967a). Sampling times were as f o l l o w s : 2, k, 6, 8, 10, 15, 20 and 30 minutes when c e l l - f r e e e x t r a c t s of glucose grown c e l l s were used; and 5, 10, 15, 20, 25 and 30 minutes when c e l l - f r e e e x t r a c t s of c e l l s grown in the presence of added a r g i n i n e were used. C i t r u l l i n e was assayed by a m o d i f i c a t i o n of the method described by Oginsky (1957) - To the sample,contained in a 2.0 ml volume, was added 1.0 ml of a mixture of 1 volume of concentrated H^SO^ to 3 volumes of concentrated H^PO^, and 0.13 ml of a 3% aqueous s o l u t i o n of 2,3-butadionemonoxime. The contents of the tubes were mixed v i g o r o u s l y and placed in b o i l i n g water f o r 10 minutes in the dark, and cooled in ice water in the dark. Each tube was removed immediately p r i o r to being read. O p t i c a l d e n s i t y was recorded at 490 nm. This assay measured 15 to 50 yg of c i t r u l 1 i n e . 33 X I I I . A n a l y t i c a l Methods P r o t e i n was determined by the method of Lowry et_ a l _ . (1951). The presence of ammonia in Warburg supernatant f l u i d s was determined by a m o d i f i c a t i o n of the Conway (1950) m i c r o d i f f u s i o n technique. Values between 0 and 20 yg of ammonia could be measured under the c o n d i t i o n s used. XIV. Chemicals A l l chemicals used in t h i s study were purchased from commercial sources. y-Aminobutyric acid-4- C and uniformly l a b e l l e d L- C-a r g i n i n e were obtained from Schwartz Bioresearch Inc., Orangeburg, N.Y., and putrescine-'^C (tetramethylenediamine-1,4,-'\ dihydro-14 c h l o r i d e ) and DL-ornithine-1- C from Amersham/Searle Corp., 14 Des P l a i n e s , 111. L - c ? t r u l 1 i n e - u r e i d o - C and the o r n i t h i n e used 14 f o r transport s t u d i e s , D L - o r n i t h i n e - 5- C, were purchased from New England Nuclear Corp., Boston, Mass. RESULTS AND DISCUSSION I. Pathways of A r g i n i n e Degradation in P_. aerugtnosa 1. Accumulation of intermediates of a r g i n i n e degradation When glucose grown c e l l s of P_. aeruginosa were incubated with 14 C-arginine in a conventional Warburg apparatus, subsequent t h i n -layer chromatography and radloautography of the supernatant f l u i d demonstrated the presence of r a d i o a c t i v e c i t r u l l i n e , o r n i t h i n e , p u t r e s c i n e and glutamate (Figure 2 ) . Small amounts of a number of u n i d e n t i f i e d r a d i o a c t i v e compounds, which were not amino acids or intermediates of the t r i c a r b o x y l i c a c i d (TCA) c y c l e , were a l s o present. Both c i t r u l l i n e and o r n i t h i n e were present a f t e r short incubation periods (10 and 30 minutes) in r e l a t i v e l y high c o n c e n t r a t i o n s , but were v i r t u a l l y absent at l a t e r times. These r e s u l t s confirmed the presence, in t h i s s t r a i n of P_. aeruginosa, of the a r g i n i n e d i h y d r o l a s e pathway described by Oginsky (1955). Both p u t r e s c i n e and glutamate appeared to accumulate in the Warburg supernatant f l u i d as products of a r g i n i n e degradation, i n d i c a t i n g that the organism may possess both an o r n i t h i n e decarboxylase and the enzymes f o r the conversion of o r n i t h i n e to glutamate v i a glutamic -y-semiaIdehyde (Ramaley and B e r n l o h r , 1966). 35 F i g . 2. Radioautogram of a t h i n - l a y e r chromatogram of the supernatant f l u i d a f t e r the incubation of glucose grown c e l l s w i th C-arginine f o r 30 minutes under conventional Warburg c o n d i t i o n s . 1 ^ g l u t a m a t e z : LU > O + LO -+-» If) citrulline arginine ornithine putrescine origin 2nd SOLVENT 2. I n h i b i t o r y e f f e c t of T r i s buffer on the o x i d a t i o n of a r g i n i n e by whole c e l l s P r e l i m i n a r y manometric experiments, using a r g i n i n e as s u b s t r a t e , showed that the i n i t i a l rate of oxygen uptake of a r g i n i n e grown c e l l s suspended at the usual concentration [5 mg of c e l l s (dry weight)/ml] was so rapid that d i f f u s i o n of oxygen i n t o the r e a c t i o n mixture was probably r a t e - l i m i t i n g . In a d d i t i o n , the f i n a l pH of the r e a c t i o n mixture was 8.0. The r e f o r e , the c e l l concentration was decreased by o n e - t h i r d , and the concentration of T r i s was increased to 0.067 M. However, under these c o n d i t i o n s , oxygen uptake was extremely slow, and stopped at a lower l e v e l than would be expected i f normal o x i d a t i o n and a s s i m i l a t i o n had occured. Thus, oxygen uptake in the presence of 0.067 M T r i s buffer (pH 7.4) and i n the presence of 0.067 M phosphate b u f f e r (pH 7-4) were compared. With a r g i n i n e as s u b s t r a t e , both the rate of oxygen uptake and the t o t a l oxygen consumption were lower in the presence of T r i s buffer than in the presence of phosphate buffer (Figure 3A). Although T r i s b u f f e r did not a f f e c t the t o t a l oxygen uptake when putres c i n e was the s u b s t r a t e , i t did cause a decreased rate of oxygen consumption and a much longer period of adaptation before the most rapid rate was reached (Figure 3B). R e p e t i t i o n of t h i s experiment 14 using C-arginine as substrate showed t h a t , upon completion of oxygen uptake, the supernatant f l u i d from the r e a c t i o n mixture with F i g . 3. Oxidation of arginine (A.) and putrescine (B) in the presence of T r i s and phosphate b u f f e r s . Arginine grown c e l l s were used at a concentration of 1.6 mg of c e l l s (dry weight)/cup. Each cup contained 2.5 ymoles of substrate. Symbols: 0, T r i s buffer; phosphate b u f f e r . Note the change in the ordinate in B. M I N U T E S T r i s b u f f e r contained 3 times more r a d i o a c t i v i t y than that from the r e a c t i o n with phosphate b u f f e r . Although t h i n - l a y e r chroma-tography and radioautography of the supernatant f l u i d s showed that t h i s r a d i o a c t i v i t y was not present as a s i n g l e i n t e r m e d i a t e , glutamate accumulated to much higher concentrations in the presence of T r i s buffer than in phosphate b u f f e r . Thus T r i s may have had a d i r e c t i n h i b i t o r y e f f e c t on the enzymes responsible f o r degrading glutamate, causing a decrease in the t o t a l oxygen consumption when a r g i n i n e , but not p u t r e s c i n e , was the s u b s t r a t e . The i n h i b i t o r y e f f e c t of T r i s on several enzyme r e a c t i o n s , i n c l u d i n g the o x i d a t i o n of succinate by mitochondria, has been described by Good et a 1. ( 1 9 6 6 ) . The decreased rates of o x i d a t i o n in the presence of T r i s may have been caused by an a l t e r a t i o n in c e l l p e r m e a b i l i t y , p o s s i b l y r e s u l t i n g in some l y s i s . Other workers have obtained evidence that T r i s a f f e c t s c e l l p e r m e a b i l i t y . Leive and K o l l i n (1967) found that exposure of E_. col i to c o l d T r i s caused a decrease in the rate of RNA synthesis and a loss of a c i d p r e c i p i t a b l e UV-absorbing m a t e r i a l . Neu, Ashman and P r i c e (1967) found that exposure of E_. col ? to T r i s f o r one hour caused a release of the n u c l e o t i d e pool and degradation of RNA and n u c l e o t i d e s . Eagon and A s b e l l (1966) showed that the a b i l i t y of o s m o t i c a l l y f r a g i l e P_. aeruginosa to tr a n s p o r t and o x i d i z e c e r t a i n substrates could be restored in phosphate buffer but not in T r i s b u f f e r . Cheng, •Ingram, and Costerton (1970) found that washing P_. aeruginosa With T r i s buffer caused p a r t i a l release of the p e r i p l a s m i c enzyme, a l k a l i n e phosphatase. The p o s s i b i l i t y a l s o e x i s t s that exogenous phosphate may s t i m u l a t e the r a t e of conversion of c i t r u l l i n e to o r n i t h i n e , which i s a phosphate r e q u i r i n g r e a c t i o n . It i s i n t e r e s t i n g to note that some r a d i o a c t i v e c i t r u l l i n e was present in the supernatant f l u i d from the r e a c t i o n mixture c o n t a i n i n g T r i s b u f f e r , but not in that c o n t a i n i n g phosphate b u f f e r . 3. Oxidation o f . i n t e r m e d i a t e s of a r g i n i n e degradation Further evidence that o r n i t h i n e , c i t r u l l i n e , p u t r e s c i n e , and Y~aminobutyrate were intermediates in the degradation of a r g i n i n e was obtained by manometric s t u d i e s . Glucose grown c e l l s o x i d i z e d a r g i n i n e , o r n i t h i n e , and c i t r u l l i n e c o n s t i t u t i v e l y ; however, the r a t e of o x i d a t i o n of these substrates was increased 5 to 8 f o l d by growth of the c e l l s in a r g i n i n e as the s o l e source of nitrogen and carbon ( F i g . k, F i g . 6A, Table I ) . C i t r u l l i n e was o x i d i z e d much more slowly than the other s u b s t r a t e s , and growth with c i t r u l l i n e as the s o l e carbon and nitrogen source was a l s o poor. Evidence w i l l be described l a t e r suggesting that c i t r u l l i n e uptake may have been ra t e l i m i t i n g . A r g i n i n e grown c e l l s a l s o o x i d i z e d putrescine and y-aminobutyrate more r a p i d l y than glucose grown c e l l s ( F i g . 5, F i g . 6B , Table I ) . The maximal rat e of p u t r e s c i n e F i g . k. Oxidation of arginine (A) and ornithine (B). Symbols: 0 , glucose grown c e l l s [ 3 . 8 mg of c e l l s (dry wei ght)/cup]; • , arginine grown c e l l s [ 1 . 5 mg of c e l l s (dry weight)/cup]. Phosphate buffer was used. 2 0 0 3 UJ OL ZD Z LLI o > X o 1 0 0 3 0 6 0 9 0 M I N U T E S 1 2 0 F i g . 5. Oxidation of p u t r e s c i n e . Symbols: 0, glucose grown c e l l s [3.8 mg of c e l l s (dry weight)/cup]; • ,argi n i n e grown c e l l s [1.5 mg of c e l l s (dry weight)/cup ] ; A, a r g i n i n e grown c e l l s [3.8 mg of c e l l s (dry weight)/cup] . Phosphate buffer was used. F i g . 6. Oxidation of c i t r u l l i n e (A) and y-am\nobutyrate (B). Symbols: 0, glucose grown c e l l s ; • , arginine grown c e l l s . Cell concentration was 3.8 mg of c e l l s (dry weight)/cup. Phosphate buffer was used. Table I. Rates of oxygen uptake by P_. aeruginosa with a r g i n i n e and suspected intermediates as s u b s t r a t e s . Growth medium Substrate a r g i n i n e - s a 1 t s glucose-NH^ - s a l t s Q02 a r g i n i n e 241 30 O r n i t h i n e 164 33 c i t r u l l i n e 19 3-7 p u t r e s c i n e 94 30 y-aminobutyrate 31 18 Substrate concentration was 2.5 ymoles/Warburg v e s s e l . o x i d a t i o n by g l u c o s e grown c e l l s was reached o n l y a f t e r 35 m i n u t e s , whereas a r g i n i n e grown c e l l s a t t h e same c o n c e n t r a t i o n [3.8 mg o f c e l l s ( d r y w e i g h t ) / c u p ] d e m o n s t r a t e d a l a g o f o n l y 5 m i n u t e s . T h i s l a g was not due t o the i n d u c t i o n o f an up t a k e system f o r p u t r e s c i n e , because subsequent e x p e r i m e n t s showed t h a t t h i s compound was t r a n s p o r t e d c o n s t i t u t i v e l y a t a h i g h r a t e . S i n c e a s i m i l a r l a g p e r i o d d i d not o c c u r b e f o r e t h e o x i d a t i o n o f Y ~ a m i n o b u t y r a t e , t h e l a g may r e p r e s e n t a p e r i o d o f i n d u c t i o n o f t h e enzymes con-v e r t i n g p u t r e s c i n e t o Y ~ a m i n o b u t y r a t e . The f a c t t h a t Y~amino-b u t y r a t e was o x i d i z e d a t o a much s l o w e r r a t e than p u t r e s c i n e i n d i c a t e d t h a t exogenous Y_ a mi n o b u t y r a t e was p o s s i b l y not as a v a i l a b l e f o r o x i d a t i o n as endogenous Y ~ a m i n o b u t y r a t e , p o s s i b l y due t o a s l o w r a t e o f up t a k e o f t h i s s u b s t r a t e . 4 . C o n v e r s i o n o f o r n i t h i n e t o g l u t a m a t e 14 S i n c e C - g l u t a m a t e was r e c o v e r e d i n r e l a t i v e l y h i g h con-14 c e n t r a t i o n s d u r i n g t he o x i d a t i o n o f C - a r g i n i n e , an a t t e m p t was made t o d e t e r m i n e t he p r e s e n c e , i n a c e l l - f r e e e x t r a c t o f a r g i n i n e grown P_. aerug i n o s a , o f t h e enzymes c o n v e r t i n g o r n i t h i n e t o g l u t a m a t e ; i e . o r n i t h i n e t r a n s a m i n a s e and - p y r r o l i n e - 5 ~ c a r b o x y l a t e d e h y d r o g e n a s e . An a t t e m p t was made t o c o u p l e t h e two r e a c t i o n s u s i n g t h e c o n d i t i o n s o f Ramaley and B e r n l o h r (1966) f o r rfhe measurement o f o r n i t h i n e t r a n s a m i n a s e , but by a d d i n g NADP o r NAD and measuring the reduction of the l a t t e r c o f a c t o r s at 340 nm. However, no a c t i v i t y was obtained, although the assay was attempted in both phosphate and T r i s b u f f e r s at several pH v a l u e s . It was t h e r e f o r e decided to determine whether P_. aerug?nosa 14 14 degraded o r n i t h i n e - 1 - C to C-glutamate. Resting c e l l suspensions of both a r g i n i n e grown and glucose grown c e l l s from the l o g a r i t h m i c phase of growth were incubated in 50 ml Erlenmeyer f l a s k s c o n t a i n i n g 6 ml volumes of a t y p i c a l Warburg r e a c t i o n mixture buffered with phosphate, and s t i r r e d at 30 C as described f o r uptake experiments. 14 Two ymoles of unlabel led glutamate were added to trap C-glutamate, and the r e a c t i o n was s t a r t e d by the a d d i t i o n of 5 ymoles (15 yCi) 14 of o r n i t h i n e - 1 - C. Two ml samples were removed at appropriate time i n t e r v a l s , added to 2 ml of 10% t r i c h l o r o a c e t i c a c i d at 0 C, and treated as described f o r the t h i n - l a y e r chromatography of Warburg supernatant f l u i d s . Intermediates of putrescine degradation could not become l a b e l l e d in t h i s experiment, because the conversion 14 of o r n i t h i n e - 1 - C to putrescine would r e s u l t in the loss of a l l the 14 label as C02. The r e s u l t s of t h i s experiment are summarized in Table I I . Approximately the same amount of r a d i o a c t i v i t y was l o s t from the supernatant f l u i d a f t e r 45 minutes in the presence of glucose grown c e l l s as was l o s t a f t e r 20 minutes in the presence of a r g i n i n e grown c e l l s . These r e s u l t s i n d icated that under these c o n d i t i o n s , a r g i n i n e ^grown c e l l s o x i d i z e d o r n i t h i n e approximately twice as r a p i d l y as 14 Table I I . Degradation of o r n i t h i n e - 1 - C by a r g i n i n e grown and glucose grown c e l l s of P. aeruginosa. Growth substrate Rad ioact i v i ty Ar g i n i n e Glucose Incubation Time (min) 20 40 45 90 14 % of t o t a l C recovered in supernatant f l u i d % of supernatant f l u i d lZtCa found as: o r n i t h i n e a c i d i c and glutamate p r o l i n e neutral compounds 44% 49.6% 20% 3-4% 7-4% 17% 24.3% 35-5% 6.1% 10.3% 75% 10.8% 1.6% 0 30% 74.4% 1.0% 4.6% 0 Supernatant f l u i d r a d i o a c t i v i t y unaccounted f o r in t h i s t a b l e was present as several' u n i d e n t i f i a b l e compounds. glucose grown c e l l s . However, examination of the composition of the supernatant f l u i d s at these times showed that much more of the o r i g i n a l o r n i t h i n e remained in the presence of the glucose grown c e l 1 s . From the r e s u l t s of the previous manometric experiments, i t was expected that a r g i n i n e grown c e l l s would have o x i d i z e d the ma j o r i t y of the o r n i t h i n e by hO minutes ( F i g . 4B), and only h% of the o r i g i n a l label was recovered as o r n i t h i n e at t h i s time. Glucose grown c e l l s should have o x i d i z e d the m a j o r i t y of the o r n i t h i n e by 90 minutes; however, 22% of the o r i g i n a l label was recovered as o r n i t h i n e , a value c l o s e l y comparable to the amount of label recovered at 20 minutes from induced c e l l s . Thus, i t appeared that the o x i d a t i o n of o r n i t h i n e by glucose grown c e l l s was slower under these c o n d i t i o n s than under conventional Warburg c o n d i t i o n s , perhaps due to oxygen l i m i t a t i o n . Moreover, C-o r n i t h i n e was supplied as a mixture of the D and L isomers, and i t i s p o s s i b l e that the D isomer was o x i d i z e d more slowly that the L isomer. A r g i n i n e grown c e l l s accumulated much more of the added label as glutamate, p r o l i n e , and a c i d i c and neutral degradation products than did glucose grown c e l l s . These r e s u l t s indicated that the conversion of o r n i t h i n e to glutamate was rate l i m i t i n g in the glucose grown c e l l s and that glutamate was d i s s i m i l a t e d as r a p i d l y as i t was formed. On the other hand, a r g i n i n e grown c e l l s appeared to convert o r n i t h i n e to glutamate more r a p i d l y than the l a t t e r compound could be d i s s i m i l a t e d . T h i n - l a y e r chromatography and radioautography of the ba s i c f r a c t i o n s of the supernatant f l u i d s showed two u n i d e n t i f i e d spots c o n t a i n i n g c o n s i d e r a b l e r a d i o a c t i v i t y (spots A and B, F i g . 7). It is p o s s i b l e that these compounds were A ^ - p y r r o l i n e - 5 _ c a r b o x y l i c a c i d and/or i t s breakdown products. Strecker (1960) found that s o l u t i o n s of A ^ - p y r r o l i n e-5 - c a r b o x y l i c a c i d were u n s t a b l e , forming at l e a s t two products: one was formed both at room temperature and at -15 C, and the o t h e r , thought to be a p o l y m e r i z a t i o n product, was formed only at -15 C. Since treatment of the supernatant f l u i d s involved storage at -20 C, treatments at room temperature, and several evaporations at 45 - 50 C, i t i s p o s s i b l e that break-down products of t h i s compound were formed. The radioautograms of the t h i n - l a y e r chromatograms of the basic f r a c t i o n s of the supernatant f l u i d s from glucose grown c e l l s a l s o showed low l e v e l s of 8 to 10 u n i d e n t i f i e d compounds. These compounds were a l s o present in the 20 minute sample from a r g i n i n e grown c e l l s . These basic compounds were not amino acids and were presumed to be b i o s y n t h e t i c products or intermediates. From these experiments, i t was concluded that the conversion of o r n i t h i n e to glutamate was an important pathway of a r g i n i n e degradation in P_. aeruginosa. The enzymes of the pathway appeared ,to be present in glucose grown c e l l s , but were induced to greater a c t i v i t y a f t e r growth of the organism in a r g i n i n e . Radioautogram of a t h i n - l a y e r chromatogram of the ba s i c f r a c t i o n of the supernatant f l u i d a f t e r incubation of a r g i n i n e grown c e l l s with o r n i t h i n e - 1 - ^ C f o r 40 minutes. 1 proline glutamate ornithine • origin 2nd SOLVENT L 5. S u c c i n i c semialdehyde dehydrogenase a c t i v i t y In an attempt to assess the importance of putrescine as an intermediate of a r g i n i n e degradation in P_. aerug i nosa, the a c t i v i t y of s u c c i n i c semialdehyde dehydrogenase, an enzyme p a r t i c i p a t i n g in put r e s c i n e metabolism, was measured in a r g i n i n e grown c e l l s , p u t r e s c i n e grown c e l l s (induced) and glucose grown c e l l s (uninduced). This enzyme was assayed by l i n k i n g i t to y-aminobutyrate t r a n s -aminase, and thus enzyme a c t i v i t y was a c t u a l l y a measurement of the rat e of conversion of y ~a mi n o b u t y r a t e to s u c c i n a t e . It was p o s s i b l e , however, that the induction by a r g i n i n e of the enzymes degrading Y ~a mi n o b u t y r a t e might occur without the degradation of p u t r e s c i n e . Gale (1940) found that growth of E_. col i in the presence of glutamate induced an enzyme which decarboxylated glutamate, forming y-aminobutyrate. If P_. aeruginosa possessed the l a t t e r enzyme, y-aminobutyrate might be formed from glutamate rather than from p u t r e s c i n e , and s u c c i n i c semialdehyde dehydrogenase a c t i v i t y would be i n d i c a t i v e of the rate of conversion of o r n i t h i n e to glutamate and the subsequent degradation of the l a t t e r compound, rather than of the o x i d a t i o n of p u t r e s c i n e . T herefore, s u c c i n i c semialdehyde dehydrogenase a c t i v i t y was a l s o measured in c e l l s grown with glutamate as the s o l e source of carbon and n i t r o g e n . Nakamura (i960) found that a s t r a i n of P_. aeruginosa con-tai n e d two s u c c i n i c semialdehyde dehydrogenases: one l i n k e d to NADP, and the other to NAD. Un l i k e the enzyme studied by Padmanabhan and Tchen (1969) in an u n i d e n t i f i e d Pseudomonad, the NADP li n k e d s u c c i n i c semialdehyde dehydrogenase of P_. aeruginosa appeared to be i n d u c i b l e (Table I I I ) - Jakoby and Fr e d e r i c k s (1959) found that synthesis of s u c c i n i c semialdehyde dehydrogenase was con-s t i t u t i v e in P_. f 1 uorescens, although i t s a c t i v i t y was increased somewhat a f t e r growth in y-aminobutyrate. However, the syn t h e s i s of y-aminobutyrate transaminase was i n d u c i b l e in t h i s organism, and the s p e c i f i c a c t i v i t y of t h i s enzyme was always lower than that of the dehydrogenase. Since the assay used in the present study depended on y-aminobutyrate as a source of s u c c i n i c semialdehyde, i t i s p o s s i b l e that the NADP link e d s u c c i n i c semialdehyde dehydro-genase was c o n s t i t u t i v e , but that i t s a c t i v i t y was l i m i t e d by an in d u c i b l e y-aminobutyrate transaminase. A comparison of the s p e c i f i c a c t i v i t i e s of the NADP link e d and NAD linked enzymes shows that y-aminobutyrate transaminase was not rate l i m i t i n g in the assay of the NAD li n k e d s u c c i n i c semialdehyde dehydrogenase. The r a t i o s of the s p e c i f i c a c t i v i t i e s of the NADP li n k e d to the NAD lin k e d enzymes were s i m i l a r under a l l growth c o n d i t i o n s , i n d i c a t i n g that the two enzymes may have been c o o r d i n a t e l y induced. Both the NAD and the NADP link e d enzymes were induced to the highest l e v e l s by growth in p u t r e s c i n e . P a r t i a l induction of both enzymes was caused by growth with e i t h e r a r g i n i n e or glutamate as T a b l e I I I . S u c c i n i c s e m i a l d e h y d e dehydrogenase a c t i v i t i e s o f c e l l - f r e e e x t r a c t s o f induced and uninduced c e l l s o f P. aerug i n o s a . S p e c i f i c a c t i v i t y ' Growth s u b s t r a t e NADP NAD p u t r e s c i n e 576 84 a r g i n i n e 82.3 14.5 g l u t a m a t e 58.5 7«3 g l u c o s e 16.1 <2.5 S p e c i f i c a c t i v i t y e x p r e s s e d as mymoles o f s u b s t r a t e o x i d i z e d x m i n-^ x mg-^ o f p r o t e i n . the s o l e source of carbon and n i t r o g e n . The f a c t that growth in glutamate induced only 10% of the a c t i v i t y induced by growth in putrescine i n d i c a t e d that decarboxylation was not the major pathway of glutamate degradation in P_. aeruginosa. A r g i n i n e grown c e l l s contained s i g n i f i c a n t l y higher l e v e l s of both enzymes than glutamate grown c e l l s , and i t was concluded that t h i s increased a c t i v i t y was due to degradation of putrescine formed during a r g i n i n e c a t a b o l i s m . Glucose grown c e l l s contained a low c o n s t i t u t i v e l e v e l of the NADP l i n k e d enzyme, and e x t r a c t s from these c e l l s had n e g l i g i b l e a c t i v i t y with NAD. I I . Repress ion of Argi n ine Biosynthes i s in P_. aerugi nosa by Exogenous A r g i n i n e Radioautograms of t h i n - l a y e r chromatograms of the supernatant f l u i d s from the experiments examining the degradation of o r n i t h i n e - 1 -14 14 C showed that glucose grown c e l l s synthesized C-arginine from 14 C - o r n i t h i n e , but that a r g i n i n e grown c e l l s did not; t h i s i n d i c ated that a r g i n i n e b i o s y n t h e s i s was repressed during growth in the presence of a r g i n i n e . Repression of the anabolic o r n i t h i n e transcarbamylase of P_. aeruginosa and P. fluorescens by a r g i n i n e has been reported by Stalon et_ aj_. ( 1 9 6 7 a ) and Ramos et_ al_. ( 1 9 6 7 ) . Ukada ( 1 9 6 6 ) found that a r g i n i n e repressed the synthesis of t h i s enzyme in 19 d i f f e r e n t microorganisms. In order to confirm that repression of t h i s enzyme occurred in P_. aerug i n o s a , c e l l - f r e e e x t r a c t s were assayed for o r n i t h i n e transcarbamylase a c t i v i t y . C e l l s were grown in Roux f l a s k s in glucose minimal medium in the presence and absence of 0.05% a r g i n i n e , and were harvested from the l a t e logar-ithmic phase of growth at 14 hours. The s p e c i f i c a c t i v i t y of o r n i t h i n e transcarbamylase was three to four f o l d greater in c e l l -f r e e e x t r a c t s of c e l l s grown without added a r g i n i n e than in those grown in the presence of a r g i n i n e . I I I . The E f f e c t s of Glucose on the Degradation of A r g i n i n e by P_. aerug ? nosa . 1. Growth in a mixture of glucose and a r g i n i n e . P_. aeruginosa grew in a medium co n t a i n i n g g l u c o s e , ammonium i o n s , and a r g i n i n e without showing a d i a u x i e e f f e c t , d e s p i t e the f a c t that the inoculum had not been adapted to growth in the presence of a r g i n i n e ( F i g . 8 and 9). The doubling time f o r growth in t h i s mixture was the same as the. doubling time in glucose alone; i e . 1.25 hours. The growth rate was not a f f e c t e d by p r i o r adaptation of the inoculum to growth in the presence of a r g i n i n e . P_. aerug i nosa grew somewhat more slowly with a r g i n i n e as the s o l e source of carbon and n i t r o g e n , having a doubling time of approximately 1.6 hours under these c o n d i t i o n s . The lag period was a l s o longer, 55 H O U R S F i g . 8. The u t i l i z a t i o n of C-glucose during growth of P_. aeruginosa in a medium co n t a i n i n g g l u c o s e , a r g i n i n e and ammonium io n s . Symbols: 0, t o t a l r a d i o a c t i v i t y in the f l a s k ; O . r a d i o a c t i v i t y present in whole c e l l s ; A, o p t i c a l d e n s i t y ; #, r a d i o a c t i v i t y of the supernatant f l u i d . C e l l r a d i o a c t i v i t y has been p l o t t e d on a d i f f e r e n t s c a l e from t o t a l and supernatant f l u i d r a d i o a c t i v i t y . 56 H O U R S F t g . 9. The u t i l i z a t i o n of C-arginine during the growth of P_. aerug i nosa in a mixture of glucose and a r g i n i n e . Symbols: are the same as in F i g . 8. Note that c e l l r a d i o a c t i v i t y has been p l o t t e d on a d i f f e r e n t s c a l e than that i n F i g . 8. d e s p i t e p r i o r a d a p t a t i o n o f t h e c e l l s . The f o l l o w i n g e x p e r i m e n t s were t h e r e f o r e p erformed t o d e t e r m i n e whether g l u c o s e and a r g i n i n e were degraded c o n c u r r e n t l y d u r i n g growth i n the p r e s e n c e o f both s u b s t r a t e s . Two s i d e - a r m f l a s k s o f ammonium s a l t s m i nimal medium, each c o n t a i n i n g 0.1% g l u c o s e and 0.1 % a r g i n i n e , were i n o c u l a t e d w i t h g l u c o s e grown P_. a e r u g i n o s a . C - a r g i n i n e was added t o one f l a s k t o g i v e a f i n a l c o n c e n t r a t i o n 14 o f 0.5 y C i per m l , and C - g l u c o s e t o t h e o t h e r f l a s k a t the same c o n c e n t r a t i o n . O p t i c a l d e n s i t y was f o l l o w e d u s i n g a K l e t t -Summerson c o l o r i m e t e r e q u i p p e d w i t h a red f i l t e r , and 1 ml samples were t a k e n a t one hour i n t e r v a l s and i m m e d i a t e l y f i l t e r e d as i n t r a n s p o r t s t u d i e s , and washed w i t h 2 ml o f medium. The s u p e r n a t a n t f l u i d s were c o l l e c t e d i n a t e s t tube p l a c e d i n t h e f i l t r a t i o n f l a s k , and were l a t e r made up t o 5 ml volumes and as s a y e d f o r r a d i o a c t i v i t y . The r e s u l t s o f t h e s e e x p e r i m e n t s a r e shown i n F i g u r e s 8 and 9 . D u r i n g growth i n e i t h e r l a b e l l e d s u b s t r a t e , the r a d i o a c t i v i t y i n t h e whole c e l 1 s i n c r e a s e d c o n c o m i t a n t l y w i t h t h e o p t i c a l d e n s i t y . The degree o f a s s i m i l a t i o n o f the r a d i o a c t i v e s u b s t r a t e s was d e t e r -mined by c a l c u l a t i n g t he r a t i o o f c e l l r a d i o a c t i v i t y t o o p t i c a l d e n s i t y . D u r i n g t he f i r s t t h r e e h o u r s , s l i g h t l y more g l u c o s e t h a n a r g i n i n e was a s s i m i l a t e d per o p t i c a l d e n s i t y u n i t o f c e l l s , r e s u l t i n g i n a c e l l u l a r g l u c o s e t o a r g i n i n e r a t i o o f 1.1. As the pteriod o f the most r a p i d growth was r e a c h e d , the amount o f a r g i n i n e per o p t i c a l d e n s i t y u n i t of c e l l s decreased s l i g h t l y , whereas the amount of glucose increased c o n s i d e r a b l y . During t h i s p e r i o d , the r a t i o of glucose a s s i m i l a t e d per o p t i c a l d e n s i t y u n i t of c e l l s to a r g i n i n e a s s i m i l a t e d per o p t i c a l d e n s i t y u n i t of c e l l s was 1.9- As the c e l l s entered the s t a t i o n a r y phase of growth, they appeared to u t i l i z e a p o r t i o n of t h e i r a s s i m i l a t e d a r g i n i n e , and almost h a l f of t h e i r a s s i m i l a t e d glucose; t h u s , the r a t i o of glucose per o p t i c a l d e n s i t y u n i t of c e l l s to a r g i n i n e per o p t i c a l d e n s i t y u n i t of c e l l s in the e a r l y s t a t i o n a r y phase was 1.2, s i m i l a r to that of c e l l s p r i o r to the i n i t i a t i o n of growth. The decrease in t o t a l r a d i o a c t i v i t y was used as a measurement 14 of the amount of l a b e l l e d substrate carbon l o s t as CO^. Carbon 14 d i o x i d e production from glucose- C was apparent a f t e r two hours, at a time when an increase in o p t i c a l d e n s i t y became n o t i c e a b l e , and continued at an i n c r e a s i n g rate u n t i l one hour before s t a t i o n a r y phase was reached. Detectable amounts of CC^ did not appear to be 14 l o s t from the medium co n t a i n i n g C-arginine f o r the f i r s t four hours, a f t e r which time CC^ was produced r a p i d l y u n t i l h a l f an hour before s t a t i o n a r y phase was reached. During the period of maximum increase in o p t i c a l d e n s i t y , both a r g i n i n e and glucose were degraded to CO^. 14 A greater proportion of the label was l o s t from C-arginine 14 than from C-glucose. However, the c e l l s did not grow to as high an o p t i c a l d e n s i t y in the f l a s k c o n t a i n i n g l a b e l l e d a r g i n i n e , 1 k r e s u l t i n g in the a s s i m i l a t i o n of only 21% of the C-arginine 1 k as compared to 38% of the C-glucose, although the amount of each substrate a s s i m i l a t e d per o p t i c a l d e n s i t y u n i t of c e l l s was approximately the same. Since a smaller proportion of the a r g i n i n e was a s s i m i l a t e d , more was a v a i l a b l e f o r complete degradation to CO^. 2. The e f f e c t of glucose on the degradation of a r g i n i n e by r e s t i n g c e l l suspensions. The o x i d a t i o n of a r g i n i n e by glucose grown c e l l s harvested from the s t a t i o n a r y phase of growth was compared in the presence and absence of glucose ( F i g . 10). No break in the oxygen uptake curve was observed when both substrates were present, i n d i c a t i n g that glucose and a r g i n i n e were o x i d i z e d c o n c u r r e n t l y . The amount of ammonia present in the supernatant f l u i d was measured at various times during oxygen uptake. The maximum amount of ammonia detected in the supernatant f l u i d at any time was equivalent to 2.k ymoles per ymole of a r g i n i n e o r i g i n a l l y added. Because a r g i n i n e contains k yatoms of nitrogen per ymole, the ammonia released accounted f o r only 60% of the a r g i n i n e n i t r o g e n . Presumably, the remaining k0% was a s s i m i l a t e d . C e l l s which had been grown in glucose minimal medium supplemented 60 3 0 6 0 9 0 M I N U T E S F i g . 10. The o x i d a t i o n of g l u c o s e , 0; a r g i n i n e , • ; and a mixture of glucose and a r g i n i n e , A . Glucose grown c e l l s [approximately 5 mg of c e l l s (dry weight)/cup] were used. Each cup contained 2.5 ymoles of each substrate and T r i s b u f f e r . w i t h 0.1% a r g i n i n e had r e l e a s e d t h i s maximum amount o f ammonia a f t e r 30 minutes o f i n c u b a t i o n i i i a Warburg cup w i t h a r g i n i n e as the o n l y s u b s t r a t e . S i n c e oxygen uptake was not c o m p l e t e by t h i s t i m e , i t i s l i k e l y t h a t , a l t h o u g h g r e a t e r than 50% o f the a r g i n i n e ammonia had been r e l e a s e d , ammonia p r o d u c t i o n was not c o m p l e t e , and t h a t f u r t h e r r e l e a s e was masked by a s s i m i l a t i o n . C e l l s grown w i t h o u t added a r g i n i n e r e l e a s e d ammonia more s l o w l y . Only 67% o f the maximum v a l u e was reached by 30 m i n u t e s , and 92% by 60 m i n u t e s . T h u s , the a d d i t i o n o f a r g i n i n e t o a growth medium con-t a i n i n g g l u c o s e induced a more r a p i d r a t e o f a r g i n i n e d e g r a d a t i o n . When bot h g l u c o s e and a r g i n i n e were p r e s e n t i n t h e Warburg c u p , induced c e l l s r e l e a s e d 83% o f the maximum amount by 30 m i n u t e s . The p r e s e n c e o f g l u c o s e d u r i n g a r g i n i n e o x i d a t i o n may t h e r e f o r e have caused a s l i g h t d e c r e a s e i n the r a t e o f d e g r a d a t i o n o f a r g i n i n e by i n d u c e d c e l l s . However, i t i s a l s o p o s s i b l e t h a t t h e a s s i m i l a -t i o n o f ammonia was i n c r e a s e d when g l u c o s e was b e i n g o x i d i z e d , r e s u l t i n g i n a s l i g h t l y lower c o n c e n t r a t i o n o f ammonia i n the s u p e r n a t a n t f l u i d a t 30 m i n u t e s . The o x i d a t i o n o f 2.5 ]imoles o f ^ C - a r g i n i n e (1 u C i / i i m o l e ) by uninduced c e l l s was examined i n t h e p r e s e n c e and absence o f g l u c o s e . The changes i n t h e r a d i o a c t i v i t y o f the s u p e r n a t a n t f l u i d , whole c e l l s , and C0^ i n the absence o f added g l u c o s e a r e shown i n F i g u r e 11. The v a l u e s o b t a i n e d when g l u c o s e was p r e s e n t were v e r y s i m i l a r . R a d i o a c t i v i t y was l o s t from t h e s u p e r n a t a n t 62 M I N U T E S F i g . 11. D i s t r i b u t i o n of r a d i o a c t i v i t y during the incubation of P.\aeruginosa w?th C-arginine under Warburg c o n d i t i o n s . Glucose grown c e l l s [approximately 5 mg of c e l l s (dry weight)/cup] and T r i s b u f f e r were used. Each cup contained 2.5 ymoles of C-arginine ( s p e c i f i c a c t i v i t y = 1 yCi/ymole). Symbols: 0, C0 2 ; • , supernatant f l u i d ; A, whole c e l l s . The r a d i o a c t i v i t y given o f f as ^C0 2 was determined by measuring the r a d i o a c t i v i t y present in the center well of the Warburg cup. f l u i d throughout the period of o x i d a t i o n , d e s p i t e the presence of glucose. Moreover, the a s s i m i l a t i o n of r a d i o a c t i v i t y i n t o the c e l l s and the e v o l u t i o n of CO^ were not a p p r e c i a b l y a l t e r e d by the 1 4 a d d i t i o n of g l u c o s e . In both cases, the rate of CO^ e v o l u t i o n was slow f o r the f i r s t 20 minutes, a f t e r which time the amount of 14 CO2 released increased r a p i d l y . T h i r t y - f i v e to f o r t y percent of the f i n a l c e l l u l a r r a d i o a c t i v i t y was a s s i m i l a t e d w i t h i n the f i r s t 10 minutes, and a s s i m i l a t i o n then proceeded at a slower r a t e , reaching the maximum value by 60 minutes. Thus, a r g i n i n e was a s s i m i l a t e d i n t o c e l1 u l a r material during the i n i t i a l stages of o x i d a t i o n and was subsequently degraded to CO^. These r e s u l t s were s i m i l a r to those obtained in the growth experiments. T h i n - l a y e r chromatography and radioautography of the super-natant f l u i d s from the 30 minute samples showed t h a t , although the t o t a l r a d i o a c t i v i t y of the two samples was e s s e n t i a l l y the same, the various intermediates were present in d i f f e r e n t amounts (Table IV). Much more c i t r u l l i n e and o r n i t h i n e were present in the cup to which glucose had been added. At a l l times, the p u t r e s c i n e content of the supernatant f l u i d s was s l i g h t l y greater in the absence of g l u c o s e . By 90 minutes, the m a j o r i t y of the r a d i o -a c t i v i t y was present as glutamate and p u t r e s c i n e , with some a l s o present in unknown spots 1 and 2. Some o r n i t h i n e remained in the sample in which glucose had been present. Thus, i t i s p o s s i b l e that the a d d i t i o n of glucose did cause a s l i g h t decrease in the ra t e of o r n i t h i n e degradation. Table IV. Composition of the supernatant f l u i d s a f t e r o x i d a t i o n of C-arginine by P_. aeruginosa in the presence and absence of g l u c o s e .3 % of supernatant r a d i o a c t i v i t y '3 Compound 14 . 14 12 C-arginine C-arginine + C-glucose a r g i n i n e 39.2 c i t r u l 1 i n e 5-9 orn i th ine 5-3 p u t r e s c i ne 3.8 glutamate 6.7 unknown 1 1.0 2 1.4 34.0 13.8 15-2 2.5 7.1 1 .2 2.5 5 mg of c e l l s (dry weight) were incubated f o r 30 minutes under conventional Warburg c o n d i t i o n s with 0.05 M T r i s buffer and 2.5 ymoles of s u b s t r a t e . The s p e c i f i c a c t i v i t y of the ^ C - a r g i n i n e was 1 yCi/ymole. the supernatant r a d i o a c t i v i t y unaccounted f o r in t h i s t a b l e was present in a number of u n i d e n t i f i e d compounds. 3. Assimilation of arginine and glucose The effects of arginine on the assimilation of C-glucose, 14 and of glucose on the assimilation of C-arginine, were examined. The conditions were the same as in the previously described experiments, and the cells were fractionated at 90 minutes, after the rate of oxygen uptake in the presence of arginine had decreased. The data presented in Table V showed that the addition of arginine caused an increase in the amount of glucose assimilated, and a decrease in the amount released as CO^. The addition of arginine did not greatly affect the percentage distribution of glucose between the , various cell fractions. More glucose was assimilated into the acid ethanol soluble fraction and less into the hot trichloroacetic acid insoluble fraction in the presence of arginine. The addition of 2.5 umoles of NH^Cl affected the assimilation of glucose iii a similar way; however, the effect exerted by arginine was stronger. This was to be expected, because arginine contains 4 yatoms of nitrogen per ymole and could, therefore, provide four times as much nitrogen. However, arginine was not acting solely as a nitrogen source in the presence of glucose, because arginine carbon was assimilated under these conditions (Table VI). The addition of glucose caused a slight decrease in the amount of arginine assimilated, with a concomitant increase in the amount released as CO^i However, the pattern of arginine incorporation into the cell fractions was Table V. The e f f e c t of a r g i n i n e and ammonium ions on the a s s i m i l a t i o n of ^ C - g l u c o s e by a r e s t i n g c e l l suspension of P. aeruginosa.3 F r a c t i o n % t o t a l r a d i o a c t i v i t y 14 14 14 + C-glucose C-glucose + a r g i n i n e C-glucose + NH^ CO, supernatant f l u i d cel 1 s 59.5% 15-7% 27-7% 51.9% 14.9% 33.4% 54.5% 13-9% 30.8% % c e l l r a d i o a c t i v i t y cold t r i c h l o r o a c e t i c a c i d s o l u b l e a c i d ethanol s o l u b l e hot t r i c h l o r o a c e t i c a c i d s o l u b l e hot t r i c h l o r o a c e t i c a c i d p r e c i p i t a t e 9.3% 26.3% 13.6% 50.8% 9.4% 30.8% 14.3% 45.5% 10.3% 28.1% 12.4% 49.0% a 5 mg of c e l l s (dry weight) were incubated f o r 90 minutes under conventional Warburg c o n d i t i o n s with 0.05 M T r i s b u f f e r . 2.5 ymoles of each substrate were added. The s p e c i f i c a c t i v i t y of the ^ C - g l u c o s e was 1 yCi/ymole. Table V I . A s s i m i l a t i o n of C-arginine by a r e s t i n g c e l l suspension of P_. aerug inosa in the presence and absence of g l u c o s e .3 % t o t a l r a d i o a c t i v i t y Fract ion 14 14 12 C-arginine C-arginine + C-glucose co2 supernatant f l u i d cel 1 s 55.1% 18.8% 26.1% 60.6% 17.0% 24.2% cold t r i c h l o r o a c e t i c a c i d s o l u b l e a c i d ethanol s o l u b l e hot t r i c h l o r o a c e t i c a c i d s o l u b l e hot t r i c h l o r o a c e t i c a c i d p r e c i p i t a t e % c e l l r a d i o a c t i v i t y 31.5% 19.1% 12.5% 36.9% 35.4% 13-3% 6.8% 44.5% Experimental c o n d i t i o n s were the same as given in Table V. The s p e c i f i c a c t i v i t y of the 1^C-arginine was 1 uCi/ymole. n o t i c e a b l y a f f e c t e d by the presence of glucose. When glucose was present, the proportion of a r g i n i n e incorporated i n t o the c o l d t r i c h l o r o a c e t i c a c i d s o l u b l e pool and i n t o p r o t e i n was incr e a s e d , and the proportion incorporated i n t o the a c i d ethanol and hot t r i c h l o r o a c e t i c a c i d s o l u b l e f r a c t i o n s was decreased. Greater than 35% of the r a d i o a c t i v i t y in the a c i d ethanol s o l u b l e f r a c t i o n was l i p i d . Thus, in the presence of both a r g i n i n e and gl u c o s e , P_. aeruginosa p r e f e r e n t i a l l y incorporated glucose into l i p i d and n u c l e i c a c i d s , and a r g i n i n e i n t o p r o t e i n and the t r i c h l o r o a c e t i c a c i d s o l u b l e p o o l . This pattern of u t i l i z a t i o n would prove economical f o r the c e l l , s i nce glucose can be converted i n t o l i p i d and pentoses more r e a d i l y than a r g i n i n e , which i s degraded to intermediates of the TCA c y c l e . On the other hand, many of the amino aci d s used in p r o t e i n s y n t h e s i s , and the amino acids and other basic substances present in the c e l l p o o l , may be obtained more r e a d i l y from a r g i n i n e than from glucose. IV. Degradation of A r g i n i n e by P_. putida and P_. fluorescens Kay (1968) found that £_. fluorescens did not catabol i z e a r g i n i n e a c t i v e l y , and that the ma j o r i t y of the r a d i o a c t i v i t y i n -corporated i n t o the c e l l s during a r g i n i n e t r a n s p o r t studies was incorporated into p r o t e i n . P_. put i d a , however, catabol ized more of the a r g i n i n e and formed a l a r g e r i n t r a c e l l u l a r p o o l . The 14 o x i d a t i o n and a s s i m i l a t i o n of C-arginine by these organisms was therefore studied and compared to that by P_. aeruginosa.. Manometric s t u d i e s showed that uninduced c e l l s of P_. f l u o r e s c e n s o x i d i z e d a r g i n i n e only s l i g h t l y , taking up only 1 ymole of oxygen in the presence of 2.5 ymoles of a r g i n i n e ( F i g . 12). P_. putida demonstrated a greater c o n s t i t u t i v e c a p a c i t y to o x i d i z e a r g i n i n e , but the i n i t i a l rate and f i n a l extent of o x i d a t i o n were much lower than with P_. aeruginosa ( F i g . 12). The a s s i m i l a t i o n patterns a f t e r incubation of these three 1 ij organisms in the presence of 2.5 ymoles of C-arginine (1 yCi/ymole) f o r 100 minutes under Warburg c o n d i t i o n s were compared (Table V I I ) . 14 More C-arginine was a s s i m i l a t e d into c e l l u l a r material and metabolized to CO^ by P_. aerug i nosa than by P_. put i d a , and less r a d i o a c t i v i t y remained in the supernatant f l u i d . The r a d i o a c t i v i t y of the t r i c h l o r o a c e t i c a c i d s o l u b l e e x t r a c t a b l e pool was s i m i l a r in these two organisms. The m a j o r i t y of the r a d i o a c t i v i t y remained in the supernatant f l u i d a f t e r incubation of P_. fluorescens with 14 C - a r g i n i n e . Only 8.8% of the r a d i o a c t i v i t y was recovered as 14 CO2, and t h i s c o r r e l a t e d well with the low oxygen uptake obtained with t h i s organism. The m a j o r i t y of the r a d i o a c t i v i t y taken into the c e l l s of t h i s organism was not incorporated into c e l l u l a r m a t e r i al but remained in the i n t r a c e l l u l a r p o o l . The l a t t e r r e s u l t s fare d i f f e r e n t from those obtained by Kay (1968) ; however, r e s t i n g . 12. Oxidation of a r g i n i n e by glucose grown c e l l s of IP. aeruginosa, 0; P_. f 1 uorescens, • ; and f_. p u t i d a , A . C e l l concentrations were approximately 5 mg of c e l l s (dry weight)/ml and T r i s b u f f e r was used. 14 Table V l l . Comparison of the a s s i m i l a t i o n of C-arginine by P. aeruginosa, P. f l u o r e s c e n s , and P. p u t i d a .a Fract ion % t o t a l r a d i o a c t i v i t y P. aerug inosa P. put ida P. fluorescens co2 supernatant f l u i d cold t r i c h l o r o a c e t i c a c i d s o l u b l e c o l d t r i c h l o r o a c e t i c a c i d i n s o l u b l e 46.7% 37.6% 8.9% 10.3% 27.4% 61.8% 8.4% 2.4% 8.8% 85.5% 5.5% 0.2% a 5 mg of c e l l s (dry weight) were incubated f o r 100 minutes under conventional Warburg c o n d i t i o n s with 0.05M T r i s buffer and 2.5 ymoles (2.5 yCi) of a r g i n i n e . c e l l s from the s t a t i o n a r y phase of growth and high substrate con-c e n t r a t i o n s were used in these experiments, whereas Kay (19^8) used c e l l s from the l o g a r i t h m i c phase of growth and low substrate c o n c e n t r a t i o n s . The amount of r a d i o a c t i v i t y in the pool of P_. fluorescens was lower than that in P_. aerug i nosa and P_. put i d a , confirming the r e s u l t s of Kay (1968)• Thin-layer chromatography and radioautography of the supernatant f l u i d s showed that a l l the r a d i o a c t i v i t y of the supernatant f l u i d from P_. put ida was present as p u t r e s c i n e . Thus, 70% of the t o t a l r a d i o a c t i v i t y was accounted f o r as p u t r e s c i n e , since the i n t r a -c e l l u l a r pool formed by these three organisms in the presence of a r g i n i n e has been shown to c o n s i s t of putrescine (Kay, 1968). In a d d i t i o n to p u t r e s c i n e , r a d i o a c t i v e a r g i n i n e and c i t r u l l i n e were found in the supernatant f l u i d from P_. f l u o r e s c e n s . Thus, both P_. put ida and P_. f 1 uorescens had the c a p a c i t y to c o n s t i t u t i v e l y convert a r g i n i n e to p u t r e s c i n e , presumably v i a c i t r u l l i n e and o r n i t h i n e . IP. putida was more a c t i v e in t h i s respect than P_. f l u o r e s c e n s . S t a n i e r , P a l l e r o n i and Doudoroff (1966) described the presence of a c o n s t i t u t i v e a r g i n i n e d i h y d r o l a s e pathway as c h a r a c t e r i s t i c of f l u o r e s c e n t Pseudomonads. Both the oxygen uptake and a s s i m i l a t i o n data showed that P_. putida could f u r t h e r degrade a r g i n i n e , whereas P_. fluorescens was almost i n a c t i v e in t h i s r e s p e c t . P_. aeruginosa was the organism *most a c t i v e in the degradation of a r g i n i n e , and although putrescine was the major r a d i o a c t i v e compound found in the supernatant f l u i d with t h i s organism, r a d i o a c t i v e glutamate was a l s o present. It i s there f o r e p o s s i b l e that the slow r a t e of oxygen uptake by P_. putida was due to the f u r t h e r degradation of p u t r e s c i n e , whereas the f a s t e r rate of a r g i n i n e degradation by P. aeruginosa was due to the a d d i t i o n a l a b i l i t y of t h i s organism to degrade o r n i t h i n e v i a glutamate. V. Uptake of Basic Amino Acids and Polyamines by P_. aeruginosa 1. Induction of uptake de Hauwer, L a v a l l e , and Wiame (1964) found that the rat e of transport of a r g i n i n e by B_. subti 1 i s was g r e a t l y increased a f t e r growth in the presence of a r g i n i n e . Growth of P_. aeruginosa with a r g i n i n e as the s o l e source of carbon and nitrogen r e s u l t e d in a f i v e - f o l d increase in the rate of a r g i n i n e transport (Table V I I I ) . I n , a d d i t i o n , the rates of o r n i t h i n e and l y s i n e transport were increased seven-fold; the rates of c i t r u l l i n e and putre s c i n e t r a n s p o r t , t h r e e - f o l d . Thus, growth in a r g i n i n e induced an increase not only in the rate of uptake of a r g i n i n e , but a l s o in that of other basic amino aci d s and of p u t r e s c i n e . P_. aeruginosa transported c i t r u l l i n e at a much lower rate than the other basic amino a c i d s . It i s p o s s i b l e , however, that the rate of c i t r u l l i n e uptake as measured in these experiments was lower than the actual r a t e , because the label was in the ureido carbon Table V I I I . Induction of transport Rate of transpo r t Substrate Growth medium glucose a r g i n i n e putrescine arg i n i ne 10.0 52.1 4.9 orn i th i ne 5-5 41.5 4.9 l y s i n e 5.6 38.2 b c i tru11i ne 1.3 3.4 b put r e s c i ne 11.0 31.5 37.1 y-ami nobutyrate 0 0 2.04 expressed as mym x min x mg of c e l l s (dry w e i g h t ) . The r e a c t i o n mixture contained 0.135 mg of c e l l s (dry weight)/ml and substrates were added to a f i n a l concentration of 2.5 x 10~5M. A l l r a d i o a c t i v e substrates had a s p e c i f i c a c t i v i t y of 2 yCi/ymole. not tested of the c i t r u l l i n e , and would be l o s t as CO^, upon the conversion of c i t r u l l i n e to o r n i t h i n e . Since a r g i n i n e grown c e l l s o x i d i z e d c i t r u l l i n e more r a p i d l y than glucose grown c e l l s (Table I), i t i s l i k e l y that the discrepancy between the actual r a t e of c i t r u l l i n e t r a n s p o r t and the observed ra t e would be greater in induced c e l l s . The low rates of c i t r u l l i n e t r a n s p o r t may have been the l i m i t i n g f a c t o r s r e s p o n s i b l e f o r the low rates of c i t r u l l i n e o x i d a t i o n , observed in the respirometry s t u d i e s . P_. aerug?nosa transported putrescine very r a p i d l y , and the rate of t r a n s p o r t was induced to a higher l e v e l a f t e r growth in e i t h e r a r g i n i n e or p u t r e s c i n e . The rate of transpo r t of a l l substrates by putr e s c i n e grown c e l l s may be even more rapid than that observed, because the c e l l s were clumped, a phenomenon that was shown to reduce d r a s t i c a l l y the ra t e of a r g i n i n e t r a n s p o r t by a r g i n i n e grown c e l l s . Thus, growth in pu t r e s c i n e probably r e s u l t s in a greater induction of putr e s c i n e uptake than does growth in a r g i n i n e . Neither glucose grown c e l l s nor a r g i n i n e grown c e l l s possessed the c a p a c i t y to transpo r t y-aminobutyrate. However, t h i s s u bstrate was transported s l o w l y by putrescine grown c e l l s . 2. K i n e t i c s o f a r g i n i n e uptake A s t u d y o f t h e k i n e t i c s o f a r g i n i n e u p t a k e by P_. a e r u g i n o s a was c a r r i e d o u t by d e t e r m i n i n g the i n i t i a l r a t e s o f C - a r g i n i n e u p take a t v a r y i n g s u b s t r a t e c o n c e n t r a t i o n s ( F i g . 1 3 ) . G l u c o s e grown c e l l s d i d not d e m o n s t r a t e normal s a t u r a t i o n k i n e t i c s , b u t , r a t h e r , the r a t e o f a r g i n i n e u p t a k e c o n t i n u e d t o i n c r e a s e w i t h i n c r e a s i n g s u b s t r a t e c o n c e n t r a t i o n s ( F i g . 1 3 A ) . A M i c h a e l i s - M e n t o n p l o t d e m o n s t r a t e d the p r e s e n c e o f two permeases: one w i t h a Km o f 2.2 x 10 M^ w h i c h f u n c t i o n e d a t low a r g i n i n e c o n c e n t r a t i o n s , and -6 one w i t h a Km o f 5.4 x 10 M w h i c h f u n c t i o n e d a t a r g i n i n e con-c e n t r a t i o n s g r e a t e r than 1 .3 x 10~5M ( F i g . 1 4 ) . Kay (1968) found t h a t the u ptake o f g l u t a m a t e , p r o l i n e , and l e u c i n e by P. a e r u g i nosa f o l l o w e d s i m i l a r k i n e t i c s . Ames (1964) de m o n s t r a t e d the e x i s t e n c e o f two k i n e t i c components f o r the t r a n s p o r t o f h i s t i d i n e i n S_. typhimur?urn: a h i g h a f f i n i t y permease s p e c i f i c f o r h i s t i d i n e , and a low a f f i n i t y permease w h i c h a l s o f u n c t i o n e d i n t h e t r a n s p o r t o f the a r o m a t i c amino a c i d s . Mutants d e f e c t i v e i n each o f t h e permeases have been s t u d i e d (Ames and R o t h , 1 9 6 8 ) . R e i d , Utech and Holden (1970) showed t h a t S t r e p t o c o c c u s f a e c a l i s p o s s e s s e d two k i n e t i c components f o r the t r a n s p o r t o f g l u t a m a t e and a s p a r t a t e , and have i s o l a t e d a mutant l a c k i n g t h e h i g h a f f i n i t y permease f o r t h e s e amino a c i d s ( U t e c h , R e i d and H o l d e n , 1 9 7 0 ) . F i g . 13. K i n e t i c s of a r g i n i n e uptake by P. aeruginosa. A, glucose grown c e l l s ; B, a r g i n i n e grown c e l l s . Note that the coordinates have been changed in B. 77 g. 14. K i n e t i c s of a r g i n i n e uptake by glucose grown c e l l s . Lineweaver-Burk p l o t . The r a t e s o f u p t a k e o f low c o n c e n t r a t i o n s o f a r g i n i n e by a r g i n i n e grown c e l l s were t o o r a p i d t o be measured, and t h e r e f o r e the c e l1 c o n c e n t r a t i o n was d e c r e a s e d 15-fold t o a p p r o x i m a t e l y 9 yg o f c e l l s (dry w e i g h t ) / m l o f the f i n a l r e a c t i o n m i x t u r e . The uptake o f a r g i n i n e by t h e s e c e l l s f o l l o w e d normal s a t u r a t i o n k i n e t i c s ( F i g . 1 3 B ) , and they d i d not appear t o p o s s e s s a low a f f i n i t y permease a t h i g h s u b s t r a t e c o n c e n t r a t i o n s . However, i t i s p o s s i b l e t h a t the a c t i v i t y o f the l a t t e r permease c o u l d not be d e t e c t e d a t the low c e l l c o n c e n t r a t i o n w h i c h was u s e d , o r t h a t t h i s a c t i v i t y was o b s c u r e d due t o t h e r a p i d d e g r a d a t i o n o f a r g i n i n e by induced c e l l s . The h i g h a f f i n i t y permease o f a r g i n i n e grown c e l l s had a Km o f a p p r o x i m a t e l y 1.7 x 10 ^M, wh i c h was s i m i l a r t o the v a l u e o b t a i n e d w i t h g l u c o s e grown c e l l s . However, the v e l o c i t y maximum (V - mymoles x mirt * x mg ^) o f a r g i n i n e ' max uptake by a r g i n i n e grown c e l l s was a p p r o x i m a t e l y 55, whereas t h a t o f g l u c o s e grown c e l l s was 10. T h u s , growth o f the c e l l s i n a r g i n i n e caused i n c r e a s e d s y n t h e s i s o f the h i g h a f f i n i t y permease, but d i d n o t i n d u c e the s y n t h e s i s o f a new permease. 3. I n h i b i t i o n o f t r a n s p o r t a . B a s i c amino a c i d s The d i f f e r e n c e s i n t h e degrees t o w h i c h t h e r a t e s o f t r a n s p o r t o f the d i f f e r e n t s u b s t r a t e s were induced i n d i c a t e d t h a t 80 several uptake systems were i n v o l v e d . Kay (1968) obtained data which indicated that P.. aeruginosa possessed two transport systems f o r the basic amino a c i d s : permease bas 1, which was s p e c i f i c f o r a r g i n i n e and had a lower a f f i n i t y f o r o r n i t h i n e , and permease bas 2, which transported l y s i n e , a r g i n i n e , o r n i t h i n e , c i t r u l l i n e and h i s t i d i n e , in order of a f f i n i t y . I n h i b i t i o n s tudies reported here f u r t h e r confirmed the presence of these two basic amino a c i d uptake systems in t h i s organism. O r n i t h i n e was the most e f f e c t i v e i n h i b i t o r of a r g i n i n e uptake in both induced and uninduced c e l l s (Table IX). Glucose grown c e l l s appeared to transport a r g i n i n e mainly by the permease bas 1, since the i n h i b i t i o n exerted by l y s i n e was low, and that exerted by c i t r u l l i n e and h i s t i d i n e was n e g l i g i b l e . However, growth of the c e l l s in a r g i n i n e r e s u l t e d in a marked increase in the degree of i n h i b i t i o n exerted by these compounds. Because growth of P_. aerug inosa in a r g i n i n e a l s o caused an increase in the rates of transport of l y s i n e and c i t r u l l i n e (Table V I M ) , the r a t i o of the a c t i v i t y of the general permease (bas 2) to the s p e c i f i c per-mease (bas 1) must have increased a f t e r i n d u c t i o n . O r n i t h i n e uptake was completely i n h i b i t e d by a r g i n i n e in both induced and uninduced c e l l s (Table X ) . Lysine was a l s o a potent i n h i b i t o r o f o r n i t h i n e uptake with both types of c e l l s , being almost completely i n h i b i t o r y with induced c e l l s . C i t r u l l i n e and h i s t i d i n e were a l s o r e l a t i v e l y e f f e c t i v e i n h i b i t o r s of o r n i t h i n e Table IX. I n h i b i t i o n of a r g i n i n e t r a n s p o r t .3 % i n h i b i t i o n I n h i b i t o r Growth substrate glucose a r g i n i n e h i s t i d i n e 10 20 c i t r u l 1 i n e 11 25 l y s i n e 20 52 orn i t h i n e 58 61 a r g i n i n e 100 100 The r e a c t i o n mixture contained 0.135 mg of c e l l s (dry weight)/ml and I^C-arginine was added to a f i n a l c oncentration of 2.1 x 10~5M ( s p e c i f i c a c t i v i t y = 2.4 yCi/ymole). Unlabelled i n h i b i t o r s were added immediately p r i o r to the a d d i t i o n of the l a b e l l e d s u b s t r a t e to a f i n a l c o n c e n t r a t i o n of 2.5 x 10-I>M. Table X. I n h i b i t i o n of o r n i t h i n e t r a n s p o r t . % i n h i b i t i o n 1nh? b i tor Growth substrate glucose a r g i n i n e p u t r e s c i n e 7 12 h i s t i d i n e 48 56 c i t r u l 1 i n e 69 72 l y s i n e 84 97 arg i n i ne 100 99 orn i t h i n e 100 100 Conditions were the same as f o r Table IX with the exception that ^ C - o r n i t h i n e was added to give a f i n a l c oncentration of 2.5 x 10~5M ( s p e c i f i c a c t i v i t y = 2 uCi/umole). uptake, with c i t r u l l i n e e x e r t i n g a somewhat stronger i n h i b i t i o n than h i s t i d i n e . These r e s u l t s i n d i c a t e d that o r n i t h i n e was trans-ported by both permeases i n both types of c e l l s ; and they a l s o confirmed the increase in the a c t i v i t y of the general permease (bas 2) a f t e r growth of the c e l l s i n a r g i n i n e . It i s important to note that the concentrations of l a b e l l e d a r g i n i n e and o r n i t h i n e used i n these s t u d i e s were greater than 2 x 10 ^M, and thus the low a f f i n i t y permease may have played an important r o l e in t r a n s p o r t . b. Polyamines Tabor and Tabor (1966) found that E. c o l i transported put r e s c i n e very r a p i d l y , and spermine and spermidine more s l o w l y . They hypothesized the existence of at l e a s t two systems of polyamine uptake i n t h i s organism, one having a high a f f i n i t y f o r p u t r e s c i n e . Pu t r e s c i n e uptake iti P_. aeruginosa was i n h i b i t e d only s l i g h t l y by a r g i n i n e and o r n i t h i n e , more s t r o n g l y by spermine, and very s t r o n g l y by spermidine (Table X I ) . Thus, P_. aeruginosa probably possesses a general polyamine uptake system with a high a f f i n i t y f o r put r e s c i n e and spermidine, and a lower a f f i n i t y f o r spermine. I t i s p o s s i b l e that a p o r t i o n of the i n h i b i t i o n exerted by spermine and spermidine was due to adsorption of the l a t t e r compounds to the c e l l s u r f a c e , a phenomenon that was observed to occur in E_. col i by Tabor and Table X I . I n h i b i t i o n of putrescine t r a n s p o r t . % I n h i b i t i o n . Growth su b s t r a t e I n h i b i t o r Glucose Putrescine a r g i n i n e 21%. 25% orn i t h i ne 20% 21% Y-ami nobutyrate _b 0 spermi ne 48% ko% spermidine 83% 60% p u t r e s c i ne 100% 100% aThe r e a c t i o n mixture contained approximately 27 ug of c e l l s (dry weight)/ml and ^ C - p u t r e s c i n e was added to a f i n a l concentration of 2.5 x 10"5M ( s p e c i f i c a c t i v i t y =? 2 yCi/umole). I n h i b i t o r s were added immediately p r i o r to the a d d i t i o n of the label led sub s t r a t e to a f i n a l concentration of 2.5 x 10~^M. Not t e s t e d . Tabor (1966). The i n h i b i t i o n exerted by a r g i n i n e and o r n i t h i n e may have been non-competetive, because putrescine i n h i b i t e d o r n i t h i n e uptake only very s l i g h t l y (Table X ) . Thus, i t i s u n l i k e l y that s i g n i f i c a n t p u t r e s c i n e uptake occurrs v i a the basic amino a c i d t r a n s p o r t systems. Growth of the c e l l s in putrescine r e s u l t e d in a s l i g h t decrease in the degree of i n h i b i t i o n exerted by spermidine and no s i g n i f i c a n t a l t e r a t i o n in.the i n h i b i t i o n exerted by any of the other compounds, i n d i c a t i n g that no new t r a n s p o r t systems had been induced. The p o s s i b i l i t y that growth in spermine or spermidine might induce transp o r t systems s p e c i f i c f o r these polyamines was not i n v e s t i g a t e d . k. Pool formation a. A r g i n i n e The e f f e c t of induction on pool formation and s t a b i l i t y was i n v e s t i g a t e d using C-arginine as s u b s t r a t e . The i n i t i a l patterns of pool formation d i f f e r e d in induced and uninduced c e l l s . In glucose grown c e l l s , the i n c o r p o r a t i o n of C-arginine i n t o p r o t e i n followed a time course s i m i l a r to that of the t o t a l uptake ( F i g . 1 5 ) . Thus, the i n t r a c e l l u l a r pool increased in s i z e during the f i r s t 10 minutes of i n c u b a t i o n , a f t e r which time i t decreased . s l i g h t l y due to continued p r o t e i n s y n t h e s i s , and then remained T 5 0 -M I N U T E S F i g . 15. Formation of an i n t r a c e l l u l a r pool using glucose grown c e l l s supplied with ^ C - a r g i n i n e . Symbols: 0, whole c e l l s ; • , p r o t e i n ; A, t r i c h l o r o a c e t i c a c i d s o l u b l e p o o l . The c e l l c oncentration was 0.135 mg of c e l l s (dry weight)/ml, and the a r g i n i n e concentration was 2.1 x 10"5M ( s p e c i f i c a c t i v i t y = 2.4 yCi/ymole). s t a b l e f o r t h e next 40 m i n u t e s . A r g i n i n e grown c e l l s , on t h e o t h e r hand, d e m o n s t r a t e d a l a g o f 90 seconds b e f o r e t h e maximal 14 r a t e o f i n c o r p o r a t i o n of C - a r g i n i n e i n t o p r o t e i n was reached ( F i g . 1 6 ) . T h i s l a g presumably r e p r e s e n t e d d i l u t i o n t h r o u g h a pool of u n l a b e l l e d a r g i n i n e , w h i c h had been e s t a b l i s h e d d u r i n g growth and had not been removed by w a s h i n g . Thus, a f t e r 90 seconds 14 of i n c u b a t i o n i n C - a r g i n i n e , induced c e l l s had a c cumulated an e x t r e m e l y h i g h p o o l , w h i c h d e c r e a s e d i n s i z e by a p p r o x i m a t e l y 50% d u r i n g the f o l l o w i n g 6 m i n u t e s . T h i s d e c r e a s e m a i n l y r e p r e s e n t e d i n c o r p o r a t i o n i n t o p r o t e i n , a l t h o u g h a s l i g h t d e c r e a s e i n t h e t o t a l c e l l r a d i o a c t i v i t y a l s o o c c u r r e d , p r o b a b l y due t o o x i d a t i o n . The i n t r a c e l l u l a r pool o f induced c e l l s remained s t a b l e a f t e r t h e f i r s t 8 m i n u t e s of i n c u b a t i o n . The r a d i o a c t i v i t y r e m a i n i n g i n the s u p e r n a t a n t f l u i d a f t e r f i l t r a t i o n o f the whole c e l l s was measured a t c e r t a i n t i m e s . A p p r o x i m a t e l y 45% o f the o r i g i n a l 14 r a d i o a c t i v i t y was l o s t , presumably as CO^, d u r i n g the f i r s t 15 m i n u t e s o f i n c u b a t i o n w i t h induced c e l l s , and a s i m i l a r amount was l o s t d u r i n g the f i r s t 30 m i nutes o f i n c u b a t i o n w i t h uninduced c e l l s . Kay (1969) found t h a t t h e i n t r a c e l l u l a r pool formed by P_. a e r u g i n o s a from a r g i n i n e c o n s i s t e d o f p u t r e s c i n e , and was s t a b l e f o r p e r i o d s as lon g as 24 h o u r s . A r g i n i n e grown c e l l s a l s o appeared t o m a i n t a i n a s t a b l e pool which was o f a s i m i l a r s i z e t o t h a t formed i n u ninduced c e l l s . T h u s , under the c o n d i t i o n s used i n t h e s e e x p e r i m e n t s , c e l l s which were induced f o r a r g i n i n e d e g r a d a t i o n degraded 16. Formation of an i n t r a c e l l u l a r pool of a r g i n i n e by c e l l s grown with a r g i n i n e as the s o l e source of carbon and nitrogen Symbols: 0, whole c e l l s ; • , p r o t e i n ; A , t r i c h l o r o a c e t i c a c i d s o l u b l e p o o l . The concentrations of c e l l s and ^C-a r g i n i n e were the same as in F i g . 15-only the same proportion of arginine as uninduced c e l l s , and re-tained the remainder in the t r i c h l o r o a c e t i c acid soluble pool. Assuming that putrescine was the only radioactive compound in the pool, i t was calculated that approximately 23% of the o r i g i n a l arginine label was retained in t h i s pool in induced c e l l s , and 33% in uninduced c e l l s . The d i f f e r e n c e may not be very s i g n i f i c a n t due to the low concentrations involved. b. Ornithine and c i t r u l l i n e The uptake of ornithine by glucose grown c e l l s of P_. aeruginosa followed an i n i t i a l pattern s i m i l a r to that of arginine ( F i g . 1 7 A ) . After the f i r s t minute of incubation, c i t r u l l i n e was incorporated into protein as rapidly as i t was taken up ( F i g . 17B). On the other hand, neither c i t r u l l i n e nor ornithine were incorporated into protein by arginine grown c e l l s during the f i r s t 5 minutes of incubation, and these compounds were accumulated s o l e l y in the i n t r a c e l l u l a r pool. These results were due to the fundamental differences in the metabolic fate of these compounds in the two types of c e l l s . Glucose grown c e l l s are induced for arginine bio-synthesis, and thus would rapidly convert ornithine and c i t r u l l i n e to a r g i n i n e , which would then be incorporated into p r o t e i n . Arginine grown c e l l s have been shown to be induced for the degradation of a r g i n i n e , and repressed for i t s biosynthesis. Thus, c i t r u l l i n e Fig. 17. Formation of intracellular pools of ornithine (A) and c i t r u l l i n e (B) by glucose grown c e l l s . Symbols: 0, whole c e l l s ; • , protein; A, trichloroacetic acid soluble pool. The cell concentration was 0.135 mg of cells (dry weight)/ml, and the labelled substrates were added to a final concentration of 2.5 x 10"5M (specific activity = 2 yCi/umole). and o r n i t h i n e would not be converted to p r o t e i n a r g i n i n e by these c e l 1 s . c. Putrescine The s t a b i l i t y of putrescine pools formed by induced and uninduced c e l l s of P_. aeruginosa in the presence of external C-putrescine was a l s o s t u d i e d . The t o t a l uptake of putr e s c i n e i n t o glucose grown c e l l s reached a maximum at 15 minutes, a f t e r which time the t o t a l c e l l r a d i o a c t i v i t y did not change ( F i g . 18). However, a gradual i n c o r p o r a t i o n of label i n t o t r i c h l o r o a c e t i c a a c i d i n s o l u b l e m a t e rial commenced a f t e r 20 minutes, r e s u l t i n g in a concomitant decrease in the lev e l of the t r i c h l o r o a c e t i c a c i d s o l u b l e p o o l . 14 In the presence of external C-putrescine, putrescine grown c e l l s r a p i d l y accumulated a high i n t r a c e l l u l a r p o o l , which reached a maximum at 6 minutes, and dropped r a p i d l y f o r the next 4 minutes, due to continued rapid i n c o r p o r a t i o n i n t o p r o t e i n ( F i g . 19). The s l i g h t lag in in c o r p o r a t i o n of r a d i o a c t i v i t y i n t o p r o t e i n presumably represented d i l u t i o n through a pool of unlabel led pu t r e s c i n e which had been e s t a b l i s h e d during growth. The i n t r a c e l l u l a r pool remained s t a b l e f o r approximately 10 minutes, and then began to decrease s l o w l y , l o s i n g 64% of i t s r a d i o a c t i v i t y during the next 60 minutes. 'During t h i s time the r a d i o a c t i v i t y present in t r i c h l o r o a c e t i c a c i d 10 20 30 40 50 60 M I N U T E S F i g . 18'.. Formation of an i n t r a c e l l u l a r pool of putrescine by glucose g rown c e l l s . Symbols: 0 , whole c e l l s ; CI , t r i c h l o r o a c e t i c acid soluble pool; A, p r o t e i n . The c e l l concentration was 27 yg of c e l l s (dry weight)/ml , and the ^ C - p u t r e s c i n e concentration was 2.5 x 10~5M ( s p e c i f i c a c t i v i t y = 2 yCi/ymole). 93 M I N U T E S F i g . 19 . Formation of an i n t r a c e l l u l a r pool of putrescine by putre s c i n e grown c e l l s . Symbols: 0 , whole c e l l s ; • , p r o t e i n ; A, t r i c h l o r o a c e t i c a c i d s o l u b l e p o o l . The concentrations of c e l l s and of 1 i*C-putrescine were the same as in F i g . 18 . i n s o l u b l e material decreased by h3%, i n d i c a t i n g that l y s i s had occurred. However, the decrease in pool r a d i o a c t i v i t y was greater than that due to l y s i s , i n d i c a t i n g that approximately 20% of the pool had been o x i d i z e d . Measurements of the r a d i o a c t i v i t y of the supernatant f l u i d s supported t h i s hypothesis. F o r t y - e i g h t per cent of the added label had been l o s t from the r e a c t i o n mixture a f t e r 10 minutes of i n c u b a t i o n , and 76% a f t e r 60 minutes. A s t a b l e pool was maintained from 80 to 180 minutes. A r g i n i n e grown c e l l s a l s o accumulated a high i n t r a c e l l u l a r pool of p u t r e s c i n e , which reached a maximal l e v e l at 15 minutes, and then decreased during the next kS minutes ( F i g . 20). The decrease in pool r a d i o a c t i v i t y which occurred during the f i r s t 10 minutes of t h i s period was accounted f o r by an increase in p r o t e i n r a d i o -a c t i v i t y . However, the r a d i o a c t i v i t y of the c e l l p r o t e i n remained constant a f t e r the f i r s t 25 minutes, and thus the f u r t h e r decrease in pool r a d i o a c t i v i t y presumably represented the o x i d a t i o n of i n t r a c e l l u l a r p u t r e s c i n e . A s t a b l e pool was maintained between 60 and 90 minutes, a f t e r which the r a d i o a c t i v i t y of the t r i c h -l o r o a c e t i c a c i d i n s o l u b l e material decreased s l o w l y , i n d i c a t i n g that l y s i s was o c c u r r i n g . During t h i s p e r i o d , the t o t a l c e l l r a d i o a c t i v i t y decreased r a p i d l y , and no i n t r a c e l l u l a r pool remained a f t e r 3 hours of i n c u b a t i o n . Measurements of the r a d i o -a c t i v i t y of the supernatant f l u i d showed that 39% of the t o t a l label had been l o s t by 15 minutes, and 65% by 60 minutes. A f t e r 3 0 6 0 M I N U T E S 20, Formatton of an I n t r a c e l l u l a r ,pdol df pu t r e s c i n e by a r g i n i n e grown c e l l s . Symbols: 0, whole c e l l s ; • , p r o t e i n ; A, t r i c h -l o r o a c e t i c a c i d s o l u b l e p o o l . The concentrations of c e l l s and of 1^c-putrescine were the same as in F i g . 18. 3 hours, 3% of the o r i g i n a l label was present in the c e l l s , 8.5% in the supernatant f l u i d , and the remainder had presumably been 14 l o s t as C02. The s i z e of the s t a b l e i n t r a c e l l u l a r p u t r e s c i n e pools was d i f f i c u l t to determine due to the e r r o r s a r i s i n g from the use of low c e l l c o n c e n t r a t i o n s , and the f a c t that l y s i s had o c c u r r e d . However, the concentration of p u t r e s c i n e in the i n t r a c e l l u l a r water -2 was approximately 2 to 3 x 10 M. 5. Location of i n t r a c e l l u l a r putrescine pool Kay (1969) showed that the s t a b l e pool formed in P_. aeruginosa from a r g i n i n e consist e d of p u t r e s c i n e . Since t h i s pool did not r e q u i r e energy f o r i t s maintenance, he hypothesized that p u t r e s c i n e may be bound to some component w i t h i n the c e l l (Kay, 1968). An attempt was t h e r e f o r e made to determine the l o c a t i o n of t h i s i n t r a c e l l u l a r pool by physical f r a c t i o n a t i o n procedures. C e l l s were harvested from the s t a t i o n a r y phase of growth in glucose minimal medium and resuspended to a concentration of 5 mg of c e l l s (dry weight)/ml. Ten ml of the c e l l suspension were incubated under conventional Warburg c o n d i t i o n s with 5 ml of 0.05 M T r i s buffer in a large Warburg cup with a s i n g l e s i d e -arm to which was added 15 ym a r g i n i n e ( s p e c i f i c a c t i v i t y 0.67 yCi/ymo The c e l l s were harvested 90 minutes a f t e r the a d d i t i o n of the a r g i n i n e and were subjected to physical f r a c t i o n a t i o n . The r a d i o a c t i v i t y of the f r a c t i o n s was measured and compared with that of the c e l l - f r e e e x t r a c t . Samples of each f r a c t i o n were extracted with cold 10% t r i c h l o r o a c e t i c a c i d , and the d i s t r i b u t i o n of the r a d i o a c t i v i t y between the p r e c i p i t a t e and the supernatant f l u i d was determined. The r e s u l t s showed that the m a j o r i t y of the t r i c h l o r o a c e t i c a c i d e x t r a c t a b l e pool was present in the s o l u b l e cytoplasm (Table XII) Although the membrane and ribosomal f r a c t i o n s a l s o contained con-s i d e r a b l e r a d i o a c t i v i t y , 65% to 70% of t h i s was t r i c h l o r o a c e t i c a c i d p r e c i p i t a b l e m a t e r i a l , presumably p r o t e i n , which would have been l a b e l l e d during the incubation of the c e l l s with C - a r g i n i n e . Thus, 36% of the t o t a l label was present in the s o l u b l e cytoplasm as t r i c h l o r o a c e t i c a c i d e x t r a c t a b l e m a t e r i a l , whereas only 7-5% was present in t h i s form in the ribosomal f r a c t i o n , and a s i m i l a r amount in the membrane f r a c t i o n . Thin-layer chromatography of the t r i c h l o r o a c e t i c a c i d e x t r a c t of the s o l u b l e cytoplasm, followed by radioautography, showed that putrescine was the l a b e l l e d compound. The s o l u b l e cytoplasm has been found to be the l o c a t i o n of i n t r a c e l l u l a r p utrescine in other organisms. Kim (1966) found that 80% to 90% of the i n t r a c e l l u l a r p utrescine of a Pseudomonad was located in the s o l u b l e cytoplasm, with the remaining 10% to 20% located in the ribosomal f r a c t i o n . Tabor and Kellogg (1967) obtained s i m i l a r r e s u l t s with E. c o l i , in which 8% to 11% of the i n t r a c e l l u l a r Table X I I . D i s t r i b u t i o n of r a d i o a c t i v i t y a f t e r p h y s i c a l f r a c t i o n a t i o n of c e l l s incubated in the presence of ^ C - a r g i n i n e . Fract ion % of t o t a l rad i o a c t i v i ty % of the label in the f r a c t i o n which was extracted by cold t r i c h l o r o a c e t i c acid membranes r i bosomes 110,000 x £ supernatant f l u i d 23 25 59 35.4 30.7 59 polyamines were associated with the ribosomes, and the remainder were located in the s o l u b l e cytoplasm. However, the l a t t e r workers a l s o showed that E_. col i ribosomes could take up or lose polyamines upon a l t e r a t i o n of the concentration of magnesium or amines in the suspension medium. Thus, although a greater proportion of the t r i c h l o r o a c e t i c a c i d s o l u b l e putrescine was associated with ribosomes in P_. aeruginosa than was observed in a Pseudomonad by Kim Cl966) and in E_. col i by Tabor and Kellogg (1967), the l a t t e r workers used a 10 f o l d higher magnesium concentration during ribosome i s o l a t i o n . Thus, polyamines are probably r e d i s t r i b u t e d a f t e r d i s r u p t i o n of the c e l l , and f r a c t i o n a t i o n r e s u l t s may be i n v a l i d . a GENERAL DISCUSSION S t a n t e r , P a l l e r o n i , and Doudoroff (1966) have shown that the possession of a c o n s t i t u t i v e a r g i n i n e dihydrola'se pathway i s a c h a r a c t e r i s t i c of f l u o r e s c e n t Pseudomonads, and the three species examined in t h i s study were shown to excrete the intermediates of t h i s pathway during the o x i d a t i o n of a r g i n i n e . In a d d i t i o n , a l l three organisms synthesized putrescine from o r n i t h i n e , the end-product of the a c t i o n of the a r g i n i n e d i h y d r o l a s e system. Thus, i t i s l i k e l y that the enzymes of the a r g i n i n e d i h y d r o l a s e pathway may f u n c t i o n in the b i o s y n t h e s i s of putrescine in these organisms. There i s much i n d i r e c t evidence in the l i t e r a t u r e that polyamines may play a r o l e in t r a n s l a t i o n , and p o s s i b l y a l s o in t r a n s c r i p t i o n . Several workers have obtained evidence that these compounds are necessary f o r the growth of E_. c o l i which synthesizes a high pool of putrescine c o n s t i t u t i v e l y . It i s not known whether P_. aeruginosa synthesizes putrescine during growth in glucose minimal medium. However, when supplied with exogenous a r g i n i n e , t h i s organism retained a large p o r t i o n as a s t a b l e pool of p u t r e s c i n e , even when the enzymes of a r g i n i n e degradation were f u l l y induced. It i s l i k e l y that t h i s pool was present in a bound form, s i n c e putrescine has been found to bind to DNA, RNA, and ribosomes under in v i t r o cond i t i o n s . Of the three Pseudomonads examined, only P_. aeruginosa appeared to have the c o n s t i t u t i v e a b i l i t y to completely o x i d i z e a r g i n i n e , converting o r n i t h i n e to glutamate and f u r t h e r degrading the l a t t e r compound, presumably v i a conversion to a - k e t o g l u t a r a t e . C e l l s were unable to c o n s t i t u t i v e l y degrade the p u t r e s c i n e formed from a r g i n i n e , as they e x h i b i t e d a long lag before synthesis of p r o t e i n from putrescine commenced in uptake experiments, and a long lag before induction of the a b i l i t y to o x i d i z e putrescine in manometric experiments. Growth of P_. aeruginosa with a r g i n i n e as the s o l e source of carbon and nitrogen r e s u l t e d in the induction of a g r e a t l y increased r a t e of a r g i n i n e o x i d a t i o n , which p r i m a r i l y represented an increase in the rate of degradation of o r n i t h i n e v i a the glutamate pathway. Presumably .higher l e v e l s of the enzymes of the a r g i n i n e dihydrolase system were a l s o induced. The r e s u l t s of the s u c c i n i c semialdehyde dehydrogenase assays showed that growth in a r g i n i n e r e s u l t e d in a p a r t i a l induction of the enzymes of y-aminobutyrate degradation, to l e v e l s higher than those induced by growth in glutamate. These r e s u l t s i n d i cated that some putrescine was being degraded during growth in a r g i n i n e . However a r g i n i n e grown c e l l s demonstrated a short lag before o x i d i z i n g p u t r e s c i n e under Warburg c o n d i t i o n s , and, in uptake experiments, a short lag before putrescine carbon was incorporated i n t o . p r o t e i n . These lag periods were much shorter than those e x h i b i t e d by glucose grown c e l l s , and the rat e of putres c i n e o x i d a t i o n was much h i g h e r . These r e s u l t s are s i m i l a r to those obtained with c e l l s which are induced f o r the o x i d a t i o n of a compound but not f o r i t s uptake; however, a r g i n i n e grown c e l l s of P_. aeruginosa transported putrescine very r a p i d l y . Thus, a r g i n i n e grown c e l l s had a greater p o t e n t i a l a b i l i t y to o x i d i z e p u t r e s c i n e than glucose grown c e l l s , but appeared to be blocked in one s t e p . It i s p o s s i b l e that a key enzyme of putrescine degradation was not induced during growth on a r g i n i n e , or that i t was synthesized but i t s a c t i v i t y was i n h i b i t e d . Such a c o n t r o l mechanism would a s s i s t in the maintenance of the large pool of put r e s c i n e found to be formed from a r g i n i n e by P_. aeruginosa. The r a t e of c o n s t i t u t i v e a r g i n i n e o x i d a t i o n by P_. aerug inosa was r e l a t i v e l y h i g h . The degradation of endogenously synthesized a r g i n i n e by the c o n s t i t u t i v e enzymes of P_. aeruginosa would be a disadvantage to the c e l l , and i t i s ther e f o r e l i k e l y that the a c t i v i t y of these enzymes i s , in some way, c o n t r o l l e d . It i s p o s s i b l e that the arginyl-tRNA synthetase has a much higher a f f i n i t y f o r a r g i n i n e than do the degradative enzymes, so t h a t , at low endogenous c o n c e n t r a t i o n s , a r g i n i n e would be p r e f e r e n t i a l l y i n -corporated i n t o p r o t e i n . On the other hand, the enzymes of a r g i n i n e b i o s y n t h e s i s and those r e s p o n s i b l e f o r i t s i n c o r p o r a t i o n i n t o p r o t e i n may be, in some way, separated from the enzymes resp o n s i b l e f o r a r g i n i n e degradation, preventing endogenous a r g i n i n e from being o x i d i z e d . Sercarz and G o r i n i (1964) obtained evidence fo r such a compartmenta 1 i z a t ion in E_. c o l i , where endogenous ly synthesized a r g i n i n e was used p r e f e r e n t i a l l y f o r p r o t e i n s y n t h e s i s , and exogenous a r g i n i n e f o r repressor f o r m a t i o n . Tabor and Tabor (1969b) showed that E_. col? synthesized putrescine p r e f e r e n t i a l l y from exogenous rather than endogenous a r g i n i n e , whereas p r o t e i n appeared to be synthesized e q u a l l y well from both sources. N_. crassa has been shown to u t i l i z e exogenous a r g i n i n e and o r n i t h i n e mainly f o r c a t a b o l i s m , and endogenous a r g i n i n e and o r n i t h i n e f o r p r o t e i n synthesis (Castaneda, M a r t u s c e l l i , and Mora, 1967; D a v i s , 1968). Ramos et a 1. (1967) and Jacoby (1964) found that a r g i n i n e degradation was subject to c a t a b o l i t e repression in several s t r a i n s of P_. f l u o r e s c e n s . However, the l a t t e r worker found that the o x i d a t i o n of a r g i n i n e was repressed to a l e s s e r extent than was that of other amino a c i d s . A r g i n i n e degradation was not subject to catabol i t e repression in P_. aeruginosa; both glucose and a r g i n i n e were o x i d i z e d c o n c u r r e n t l y when present as a mixture in the presence of growing c e l l s or r e s t i n g c e l l suspensions. This lack of repress!' was probably due to the high c o n s t i t u t i v e level of the a r g i n i n e degrading enzymes. Chemical f r a c t i o n a t i o n r e s u l t s d id i n d i c a t e t h a t , in the presence of a mixture of a r g i n i n e and gl u c o s e , c e l l s u t i l i z e d the two substrates to serve s l i g h t l y d i f f e r e n t b i o s y n t h e t i c needs. f_. aerug Inosa possessed at l e a s t two systems f o r the transport of a r g i n i n e . K i n e t i c studies demonstrated the presence of a low a f f i n i t y system and a high a f f i n i t y system in glucose grown c e l l s . The low a f f i n i t y system could not be measured in a r g i n i n e grown c e l l s ; however, these c e l l s possessed increased l e v e l s of the high a f f i n i t y system. I n h i b i t i o n s tudies a l s o demonstrated the presence of two b a s i c amino a c i d tr a n s p o r t systems; one which was s p e c i f i c f o r a r g i n i n e and, with a lower a f f i n i t y , f o r o r n i t h i n e , and a second general system which transported a l l the basic amino a c i d s . The general uptake system was induced to a greater extent than was the s p e c i f i c system by growth of the organism in a r g i n i n e as the s o l e source of carbon and n i t r o g e n . It i s important to note that i n -h i b i t i o n s tudies were c a r r i e d out in the concentration range in which the low a f f i n i t y permease was o p e r a t i v e in glucose grown c e l l s . However, the degree to which t h i s permease con t r i b u t e d to t o t a l uptake could not be a s c e r t a i n e d , and t h e r e f o r e no d e f i n i t i v e c o r r e l a t i o n could be made between the two permeases i d e n t i f i e d by k i n e t i c s tudies and those i d e n t i f i e d by i n h i b i t i o n s t u d i e s . Putrescine appeared to be transported by a general polyamine permease in P_. aerug i nosa. It was i n t e r e s t i n g to note that growth of P_. aerug?nosa in a r g i n i n e r e s u l t e d in almost complete induction of putrescine t r a n s p o r t . This may have been a r e s u l t of induction ©f the polyamine transp o r t system by the large i n t r a c e l l u l a r pool of putrescine which was formed during growth in a r g i n i n e . 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