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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 o f B r i t i s h C o l u m b i a , 1967  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  In t h e Department o f of M icrob iology r  We a c c e p t t h i s t h e s i s as conforming  t o the  required standard  THE UNIVERSITY OF BRITISH COLUMBIA F e b r u a r y , 1971  In p r e s e n t i n g t h i s t h e s i s an  in p a r t i a l f u l f i l m e n t o f  requirements  advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree  the L i b r a r y  s h a l l make i t f r e e l y a v a i l a b l e f o r  I f u r t h e r agree t h a t p e r m i s s i o n f o r s c h o l a r l y purposes may by h i s r e p r e s e n t a t i v e s . of  the  f o r e x t e n s i v e copying o f  be granted by  the Head o f my  It i s understood that  t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not  written  permission.  Depa rtment The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  Date  reference and  Columbia  that  study.  this  thesis  Department  copying or  for  or  publication  be allowed without  my  i i  ABSTRACT  Pseudomonas a e r u g i n o s a was shown t o 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 dihydrolase  pathway t o o r n i t h i n e , which  was c o n v e r t e d both t o g l u t a m a t e and t o p u t r e s c i n e .  The c o n v e r s i o n  of o r n i t h i n e t o g l u t a m a t e appeared t o be t h e major r o u t e 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 was induced t o h i g h e r  activity  a f t e r growth o f t h e c e l l s w i t h a r g i n i n e as the s o l e source o f carbon and  nitrogen.  P_. a e r u g i n o s a d i d not f u r t h e r degrade  constitutively. in a p a r t i a l  However, growth o f t h e c e l l s  putrescine  in arginine  resulted  i n d u c t i o n o f 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 a n a b o l i c  ornithine  t r a n s c a r b a m y l a s e o f P_. aerug i nosa was r e p r e s s e d a f t e r growth o f the organism i n t h e presence o f a r g i n i n e . Pseudomonas p u t i d a and Pseudomonas f l u o r e s c e n s  a l s o possessed  the a b i l i t y t o c o n s t i t u t i v e l y c o n v e r t a r g i n i n e t o p u t r e s c i n e v i a the  i n t e r m e d i a t e s , c i t r u l l i n e and o r n i t h i n e .  However, these organ-  isms d i d not o x i d i z e a r g i n i n e t o the same e x t e n t as d i d P_. a e r u g i n o s a . P_. a e r u g i n o s a grew i n a m i x t u r e o f g l u c o s e and a r g i n i n e i n the presence o f ammonium ions w i t h o u t e x h i b i t i n g a d i a u x i e e f f e c t . G l u c o s e and a r g i n i n e were o x i d i z e d c o n c o m i t a n t l y when s u p p l i e d 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 s t u d i e s showed t h a t the two s u b s t r a t e s were  used t o s e r v e somewhat d i f f e r e n t b i o s y n t h e t i c Growth o f P_. aerug?nosa  needs.  i n a r g i n i n e caused an i n c r e a s e  i n the  r a t e s o f t r a n s p o r t o f 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 o f a r g i n i n e uptake demonstrated the presence o f two uptake systems w i t h 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 . studies  i n d i c a t e d t h a t a r g i n i n e was t r a n s p o r t e d  Inhibition  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 , a n d , w i t h a lower a f f i n i t y , for  o r n i t h i n e ; and a g e n e r a l permease, which t r a n s p o r t e d  b a s i c amino a c i d s .  Polyamines appeared t o be t r a n s p o r t e d  a l l the by an  uptake system which was induced t o h i g h e r l e v e l s a f t e r growth o f the c e l l s w i t h 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  as t h e s o l e s o u r c e  of carbon and n i t r o g e n . P_. a e r u g i n o s a was found t o m a i n t a i n a s t a b l e pool o f p u t r e s c i n e when s u p p l i e d w i t h exogenous  C-arginine  or  C - p u t r e s c i n e , even  when t h e organism had p r e v i o u s l y been induced t o degrade t h e s e substrates.  A physical f r a c t i o n a t i o n of the c e l l s  the major p o r t i o n o f t h i s pool was l o c a t e d  indicated  that  i n the s o l u b l e c y t o p l a s m .  IV  TABLE OF CONTENTS Page  INTRODUCTION  1  LITERATURE REVIEW  3  I. II. III.  Pathways of A r g i n i n e Degradation  C a t a b o l i t e R e p r e s s i o n of Amino A c i d Degradation The  Biological  I .  3  . . . .  12  in Microorganisms!!  Importance of Polyamines  in 16  Microorganisms IV.  T r a n s p o r t o f the B a s i c Amino A c i d s by M i c r o o r g a n i s m s .  MATERIALS AND I. I I. 111.  IV. V. VI. Vll.  VIII. IX.  METHODS  .  20 23  Organisms and Media  23  Growth of Cel Is  2k  P r e p a r a t i o n of C e l l Suspensions  2k  1.  Resting c e l l  2k  2.  C e l l suspensions f o r t r a n s p o r t s t u d i e s  suspensions f o r r e s p i r o m e t r y .„  25  P r e p a r a t i o n of C e l l - f r e e E x t r a c t s  25  Manometric Procedures  26  Uptake of L a b e l l e d Compounds  26  I n h i b i t i o n of T r a n s p o r t by Compounds S t r u c t u r a l l y Related to the S u b s t r a t e  28  Assay of S u c c i n i c Semialdehyde Dehydrogenase  28  Chemical F r a c t i o n a t i o n o f Whole C e l l s  29  V  T a b l e of Contents  (Continued)  Page  X. XI.  29  P h y s i c a l F r a c t i o n a t i o n of Whole C e l l s Chromatography of Supernatant F l u i d s from Warburg  Ik Cups C o n t a i n i n g XII. XIII. XIV.  Assay of O r n i t h i n e Transcarbamylase  32  A n a l y t i c a l Methods  33  Chemicals  33  RESULTS AND I.  3^  DISCUSSION  Pathways of A r g i n i n e Degradation 1.  2.  in P_. a e r u g i n o s a . . .  3h  ,  I n h i b i t o r y e f f e c t of T r i s b u f f e r 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  36  3.  O x i d a t i o n of i n t e r m e d i a t e s of a r g i n i n e d e g r a d a t i o n  39  *t.  C o n v e r s i o n of o r n i t h i n e to glutamate  kk  5.  S u c c i n i c semialdehyde dehydrogenase a c t i v i t y  R e p r e s s i o n of A r g i n i n e B i o s y n t h e s i s  . . .  50  in P_. a e r u g i n o s a  by Exogenous A r g i n i n e III.  3^  Accumulation of i n t e r m e d i a t e s of a r g i n i n e degradation  II.  30  C-labelled Substrates  53  The E f f e c t s of Glucose on the Degradation of A r g i n i n e by P_. a e r u g i n o s a .  Sh  1.  Sh  Growth in a m i x t u r e of g l u c o s e and a r g i n i n e . . . .  T a b l e of Contents  (Continued)  Page  2.  The  e f f e c t of g l u c o s e on  a r g i n i n e by r e s t i n g c e l l 3. IV.  the d e g r a d a t i o n  of  suspenstons-r  A s s i m i l a t i o n of a r g i n i n e and  59  glucose  Degradation of A r g i n i n e by P_. p u t i d a  65  and  P_. f 1 uorescens V.  68  Uptake of B a s i c Amino A c i d s and  Polyamines  by  P_. aerug ? nosa  73  1.  Induction of uptake .  73  2.  K i n e t i c s of a r g i n i n e uptake  76  3.  I n h i b i t i o n of t r a n s p o r t  79  a . B a s i c amino a c i d s  79  b. Polyamines  83  Pool formation  85  a. Arginine  85  k.  b. O r n i t h i n e and  5.  citrulline  . .  89  c. Putrescine  91  Location of  96  i n t r a c e l l u l a r p u t r e s c i n e pool . . . .  GENERAL DISCUSSION  100  LITERATURE CITED  105  V I I  LIST OF TABLES Page  Table  Table  Table  Table  Table  I.  Rates of oxygen uptake by P_. a e r u g i n o s a w i t h a r g i n i n e and suspected i n t e r m e d i a t e s as s u b s t r a t e s  I I . Degradation of o r n i t h i n e - ! C by a r g i n i n e and g l u c o s e grown c e l l s of P_. aerug i nosa . 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 o f induced and uninduced c e l l s of P_. a e r u g i n o s a IV.  V.  C o m p o s i t i o n of the s u p e r n a t a n t 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_. a e r u g i n o s a i n the presence and absence of g l u c o s e  h3  k6  52  6k  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 s u s p e n s i o n of IP. aerug i nosa  66 \k  Table  VI.  Table  VII.  Table  VIII.  Table  IX.  Table  A s s i m i l a t i o n of C - a r g i n i n e by a r e s t i n g c e l l s u s p e n s i o n o f P_. a e r u g i n o s a i n the presence and absence o f g l u c o s e 67 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 l u o r e s c e n s , and P_. p u t i d a . 71 I n d u c t i o n of t r a n s p o r t  7^  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  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  XI.  I n h i b i t i o n of p u t r e s c i n e t r a n s p o r t  84  Table  XII.  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 -arginine  98  viii  LIST OF  FIGURES  Page  Fig.  Fig.  Fig.  1.  2.  3-  Pathways of a r g i n i n e d i s s i m i l a t i o n organisms  in microk  Radioautogram of a t h i n - l a y e r chromatogram o f the supernatant f l u i d a f t e r the i n c u b a t i n g of g l u c o s e grown c e l l s w i t h -arginine for 30 minutes under c o n v e n t i o n a l Warburg c o n d i t i o n s Oxidation  of a r g i n i n e and  presence of T r i s and  putrescine  35  in the  phosphate b u f f e r s  37  Fig.  k.  Oxidation  of a r g i n i n e and  Fig.  5.  Oxidation  of p u t r e s c i n e  Fig.  6.  Oxidation  of c i t r u l l i n e and  Fig.  7.  Radioautogram of a t h i n - l a y e r chromatogram of the b a s i c f r a c t i o n of the supernatant f l u i d a f t e r i n c u b a1 tZ i o n of a r g i n i n e grown c e l l s w i t h o r n i t h i n e - 1 - * C f o r kO minutes  49  1 ij The u t i l i z a t i o n of C-glucose d u r i n g growth of P_. a e r u g i n o s a in a medium c o n t a i n i n g g l u c o s e , a r g i n i n e arid ammonium i o n s .  55  14 The u t i l i z a t i o n of C - a r g i n i n e d u r i n g the growth of P_. a e r u g i n o s a in a m i x t u r e of g l u c o s e and a r g i n i n e  56  The o x i d a t i o n of g l u c o s e , a r g i n i n e and mixture of g l u c o s e and a r g i n i n e  60  Fig.  Fig.  Fig.  Fig.  Fig.  8.  9.  10.  11.  12.  hO  ornithine  41 yaminobutyrate  kl  a  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 d u r i n g the i n c u b a t i o n of P_. a e r u g i n o s a with ^ C - a r g i n i n e under Warburg c o n d i t i o n s  62  O x i d a t i o n of a r g i n i n e by g l u c o s e grown c e l l s of P. a e r u g i n o s a , P. f l u o r e s c e n s and P. p u t i d a  70  IX  L i s t of F i g u r e s  (Continued)  Page  Fig.  13.  K i n e t i c s o f a r g f n i n e uptake o f - P > aerug N o s a  77  Fig.  Ik.  K i n e t i c s o f a r g i n i n e uptake by g l u c o s e cells. Lineweaver-Burk p l o t  78  Ftg.  Fig.  Fig.  Fig.  Fig.  Fig.  15-  16.  17-  18.  19.  20.  grown  Formation of an i n t r a c e l l u l a r pool u s i n g grown c e l l s s u p p l i e d w i t h ^ C - a r g i n i n e  glucose 86  Formation o f 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 w i t h 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  88  Formation of i n t r a c e l l u l a r pools o f o r n i t h i n e and c i t r u l l i n e by g l u c o s e grown c e l l s  90  Formation o f an i n t r a c e l l u l a r by g l u c o s e grown c e l l s  92  pool o f p u t r e s c i n e  Formation o f an 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 by p u t r e s c i n e grown c e l l s  93  Formation of an 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 by a r g i n i n e grown c e l l s  95  X  ACKNOWLEDGEMENTS  I would l i k e to e x p r e s s g r a t i t u d e t o my s u p e r v i s o r , D r . A.F. G r o n l u n d , f o r her encouragement, a d v i c e and c r i t i c i s m  throughout  the c o u r s e o f t h i s work. I am a l s o extremely g r a t e f u l t o D r . H.R. M a c M i l l a n and the H.R. M a c M i l l a n Family Fund f o r f i n a n c i a l a s s i s t a n c e d u r i n g the first  two y e a r s o f t h i s I would a l s o  study.  l i k e t o thank v e r y much Mrs.  I r i s Yu f o r her  help w i t h t r a n s p o r t e x p e r i m e n t s , and my c o l l e a g u e s and my f r i e n d s f o r t h e i r help throughout my graduate  s t u d i e s program.  1  INTRODUCTION  The enzymes o f a r g i n i n e d e g r a d a t i o n  have been shown t o be  i n d u c i b l e and t o be 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 i n s e v e r a l microorganisms (Ramos et_ aj_. 1967; L a i s h l e y and B e r n l o h r , 1968). Jacoby (1964) found t h a t the a b i l i t y o f Pseudomonas  fluorescens  to o x i d i z e eighteen  d i f f e r e n t amino a c i d s was r e p r e s s e d  by  glucose.  Kay (1968) o b t a i n e d  r e s u l t s which i n d i c a t e d t h a t 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 a e r u g i n o s a a t a r e l a t i v e l y rapid r a t e .  Kay (I969) found t h a t c e l l s o f P_. a e r u g i n o s a which were 14  supplied with  low e x t e r n a l c o n c e n t r a t i o n s  of  C-arginine  in glucose  minimal medium accumulated a l a r g e pool o f p u t r e s c i n e , which was e x t r e m e l y s t a b l e , being m a i n t a i n e d f o r p e r i o d s as long as twenty f o u r hours d u r i n g s t a r v a t i o n f o r an exogenous carbon s o u r c e . P u t r e s c i n e has been shown t o bind t o d e o 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 i n v i t r o and i s thought t o p l a y a r o l e i n 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  s t u d i e s have i n d i c a t e d t h a t p u t r e s c i n e may be r e q u i r e d f o r c e l l division  ( D a v i s , Lawless and P o r t , 1970; H i r s h f i e l d , et_ a l _ . 1970;  Inouye and P a r d e e , 1970) It was the o b j e c t o f t h i s  i n v e s t i g a t i o n t o determine the  pathway by which a r g i n i n e was degraded by P_. a e r u g i n o s a and the e f f e c t o f g l u c o s e on the c a t a b o l i s m o f a r g i n i n e by t h i s o r g a n i s m .  A further investigation of the basic amino acid uptake systems of P_. aerug i nosa , which were p a r t i a l l y characterized was also carried out.  by Kay (1968) ,  3  LtTERATURE REVIEW  t.  Pathways o f A r g i n i n e Degradation  The found  tn M i c r o o r g a n i s m s  pathways o f a r g i n i n e d i s s i m i l a t i o n  t o be v a r i e d and c o m p l e x  ( F i g . 1).  i n microorganisms tn N e u r o s p o r a  h a v e been  crassa  ( 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 , 1 9 6 5 ) , S a c c h a r o m y c e s cerevisiae  (Mtddelhoven,  1964), and t w o s p e c i e s o f B a c i 1 1 us (de  H a u w e r , L a v a l l e a n d Wiame, 1964; L a i s h l e y and B e r n l o h r , 1 9 6 8 ) , arginine  is first  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  d u c t i o n o f o r n i t h i n e and u r e a . ornithine-y-transaminase i n t e r m e d i a t e , which carboxylic acid.  Ornithine  i n the pro-  i s t h e n c o n v e r t e d by  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  spontaneously c y c l i z e s  to  -pyrroline-5  The l a t t e r compound may be c o n v e r t e d by a  d e h y d r o g e n a s e 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 . exception to the o r n i t h i n e transaminase  reaction  in 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 Y~semialdehyde dehydrogenase  ( C o s t i l o w and L a y c o c k ,  and  found  (NAD)-linked  1969).  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 in Bacillus  h a s been  An  i s converted t o glutamic-  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  Argtnase, ornithine  ornithine  -  i n d u c e d by a r g i n i n e o r by  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)  B a c i 1 l u s s u b t i l i s (de H a u w e r , L a v a l l e and Wiame, 1 9 6 4 ) .  Figure  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 microorganisms.  /"COOH  C0  2  + NH  proline  CHO \ CCH2)2  glutamate  CHNH 2 <r-  jJ^COOH  COOH glutamic-ysemialdehyde  A -pyrroline5-carboxylic actd NADP ^  NADPH  COOH :  (CHJ,  /  2  CHNH. COOH glutamate  N A D H  a - k e t o -5 glutarate  COOH  NADP  COOH  COOH  '(CHJ>  (  C H  2 2 }  CH2NH2 y-aminobutyrtc acid  > /  2  NADPH  2  CHO  succinic semialdehyde  2  / COOH  2  succ i nate  5  The  l a t t e r w o r k e r s f o u n d e v i d e n c e t h a t o r n i t h i n e was n o t t h e  actual first way,  i n d u c e r o f t h e s e e n z y m e s i n B_. s u b t ? l i s . converted  to arginine v i a the arginine  and a r g i n i n e  Ornithine  biosynthetic  then s e r v e d as t h e i n d u c e r .  They a l s o  was path-  isolated  a m u t a n t 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 arginase,  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  genes f o r t h e s e f u n c t i o n s carboxylate  may f o r m an o p e r o n .  dehydrogenase remained  transport,  the s t r u c t u r a l  A^-pyrroline~5  -  i n d u c i b l e , and t h u s m u s t be  under s e p a r a t e c o n t r o l . d e H a u w e r , L a v a l l e and Wiame  (1964) showed t h a t t h e  5-carboxylate dehydrogenase f u n c t i o n i n g  -pyrroline-  in arginine degradation i n  iL* s u b t ? 1 i s was a d i f f e r e n t enzyme f r o m t h e o n e f u n c t i o n i n g i n proline catabolism.  In t h i s o r g a n i s m , a r g i n i n 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 degrading enzymes.  induced o n l y  L a i s h l e y and B e r n l o h r  hand, found t h a t a r g i n i n e caused a p a r t i a l oxidase  the p r o l i n e  (1968), on t h e o t h e r induction of proline  i n B_. 1 i c h e n i f o r m i s , and p r o l i n e c a u s e d a p a r t i a l  of a r g i n a s e .  They t h e r e f o r e  hypothesized that  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 of  induced only t h e  induction  A^-pyrroline-5"  common t o t h e p a t h w a y s o f o x i d a t i o n  b o t h 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 t h e two  pathways. Organisms by  l a c k i n g an a r g i n a s e c a n c o n v e r t a r g i n i n e  the arginine dihydrolase  to c i t r u l l i n e  pathway.  by t h e enzyme a r g i n i n e  Arginine  isfirst  to ornithine converted  d e i m i n a s e , w h i c h h a s been  6  found  tn Pseudomonads, S t r e p t o c o c c t ,  1955).  T h e s e o r g a n i s m s a l s o c o n t a i n e d a n enzyme w h i c h , i n t h e  presence of substrate  amounts o f p h o s p h a t e , s p l i t  p r o d u c e o r n i t h i n e , NH^, and CO^. and  This  reaction  cttrulline to  required  e i t h e r a d e n o s i n e m o n o p h o s p h a t e (AMP) o r a d e n o s i n e  ( A D P ) , and r e s u l t e d (ATP).  i n the production of adenosine  O g i n s k y c a l l e d t h i s enzyme c i t r u l l i n e  Its a c t i v i t y , was  and C l o s t r t d i a ( O g t n s k y ,  in a cell-free  i n h i b i t e d by o r n i t h i n e .  triphosphate  aeruginosa,  Subsequent workers d i s c o v e r e d in this  that  reaction, and,  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  transcarbamylase  The c a r b a m y l p h o s p h a t e was d e g r a d e d by a  c a r b a m a t e k i n a s e , a n d t h i s was t h e ATP p r o d u c i n g Stalon  diphosphate  phosphorylase.  e x t r a c t o f Pseudomonas  c a r b a m y l p h o s p h a t e was an i n t e r m e d i a t e  ( M e i s t e r , 1965).  magnesium  et_ a_j_. (1967a)  P_. a e r u g i n o s a c o n t a i n e d  found  that  step.  Pseudomonas f l u o r e s c e n s  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  and  separable  by ammonium s u l p h a t e f r a c t i o n a t i o n .  One e n z y m e , a s s u m e d t o f u n c t i o n  in c a t a b o l i s m ,  i n the presence of a r g i n i n e ,  was i n d u c e d by g r o w t h  whereas t h e o t h e r ,  a s s u m e d t o h a v e an a n a b o l i c  repressed under these c o n d i t i o n s . was c o m p l e t e l y  for  but not i n v i v o  the a c t i v i t y  differed  Although the anabolic  enzyme  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  of c a t a l y s i n g both t h e s y n t h e s i s vitro  f u n c t i o n , was  and b r e a k d o w n o f c i t r u l l i n e i n  (Ramos e t _ a l .  1967).  o f t h e c a t a b o l i c enzyme  g r e a t l y , i t was h y p o t h e s i z e d  S i n c e t h e pH o p t i m a  i n t h e two d i r e c t i o n s  that  localization  i n an  7  acidic  c o m p a r t m e n t m i g h t p r e v e n t t h i s enzyme  in the a n a b o l i c offered of  d i r e c t i o n In v i v o .  an a l t e r n a t i v e e x p l a n a t i o n  the c a t a b o l i c o r n i t h i n e  the  c a t a b o l i c enzyme was  from  e t a l . (1967b)  However, S t a l o n f o r the i n vivo  transcarbamylase.  subject  functioning  irreversibility  They r e p o r t e d  to a l l o s t e r i c  inhibition  carbamyl phosphate at c o n c e n t r a t i o n s which saturated enzyme.  The c a t a b o l i c o r n i t h i n e  by ATP and was a c t i v a t e d  by  by  the  anabolic  t r a n s c a r b a m y l a s e was a l s o  that  Escherichia  col?  possessed  arginine  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 g r o w n a t l o w pH  Arginine  was d e c a r b o x y l a t e d  produced from o r n i t h i n e . was  5.0,  and P a r d e e  T h e pH o p t i m u m 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  but,  grown  k.O.  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 l o w pH a high  level of o r n i t h i n e  l a s e w i t h a pH o p t i m u m o f 5-3 was a l s o that  catabolic in excess.  present.  t h e c o n s t i t u t i v e enzyme  function, synthesizing putrescine,  decarboxylase  T h i s enzyme was p r e s e n t a t t h e same  under these c o n d i t i o n s ,  hypothesized  was  (1965) f o u n d t h a t when E_. c o l i was g r o w n i n  w i t h a pH o p t i m u m o f 7.5in cells  values.  decarboxylase  m i n i m a l 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  level  inducible  t o f o r m a g m a n t i n e , and p u t r e s c i n e  and t h a t o f a r g i n i n e  Morris  inhibited  ADP.  (19^0) d i s c o v e r e d  Gale  that  t h e normal c e l l u l a r  The  served a  values,  decarboxy-  authors  biosynthetic  concentration  of  w h e r e a s t h e i n d u c i b l e enzyme a p p e a r e d t o s e r v e a  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 Morris  and P a r d e e  i n the c e l l  (1966) f o u n d a s e c o n d p a t h w a y f o r t h e  8  synthesis of putrescine  i n E_. c o l i .  s t i t u t i v e arginine decarboxylase and  This c o n s i s t e d of a  converting a r g i n i n e to agmantine,  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 ,  t o p r o d u c e p u t r e s c i n e and  urea.  hydrolystng  flow through the  two  (1969) d i s c o v e r e d  Koffron  pathways v a r i e d c o n s i d e r a b l y  p r o d u c e d by  the d e c a r b o x y l a t i o n  the  is constitutive  t h a t the  relative  in v i v o .  c e l l s grown i n m i n i m a l medium, t h e m a j o r i t y of t h e was  agmantine  Although the s y n t h e s i s of  enzymes of both pathways of p u t r e s c i n e b i o s y n t h e s i s i n E_. c o l i , M o r r i s and  con-  With  putrescine  of o r n i t h i n e , whereas, in  p r e s e n c e of 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 a r g i n i n e was reported  the p r e f e r r e d r o u t e .  t h a t t h e o r n i t h i n e and  s t r a i n o f E_. c o l ? a u x o t r o p h i c and  feedback Gale  ability  by  Tabor  f o r o r n i t h i n e were both  p u t r e s c i n e and  of  (1969a)  a r g i n i n e decarboxylases  have:  of  a  repressed  spermidine.  (19^2) showed t h a t P_. a e r u g i n o s a  possessed the  constitutive  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 , a g m a n t i n e and  to completion and  inhibited  T a b o r and  tyramine.  oxidases  and  could  Zeller  be a d a p t e d t o p a r t i a l l y o x i d i z e  (1963) has  f r o m p l a n t , a n i m a l , and  reviewed the microbial  sources.  t h e o x i d a t i v e d e a m i n a t i o n o f a number o f  resulting  i n the formation  When p u t r e s c i n e was rapidly cyclized the c o n v e r s i o n  the corresponding  the s u b s t r a t e , the p r o d u c t , 1  to form A - p y r r o l i n e .  of p u t r e s c i n e  Evelyn  cadaverine histamine  p r o p e r t i e s of  catalysed  of  the  The  diamine  enzyme  diamines,  amine  aldehydes.  yaminobutyraldehyde, (1967a, b)  to A ^ - p y r r o l i n e , with  the  demonstrated,  concomitant  uptake of oxygen by c e l l - f r e e extracts of putrescine grown (1953) studied the oxidation of  Mycobacteria.  Satake and Fujita  putrescine and  histamine by a c e l l - f r e e extract of Achromobacter  and concluded that the a c t i v i t y 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  d i r e c t l y 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 Jakoby and Fredericks fluorescens  (1959) discovered  in the presence of putrescine  oxidase.  that growth of P_.  induced the  synthesis  of an enzyme, Y~aminobutyraIdehyde dehydrogenase, that oxidized A^-pyrroline to yaminobutyric acid, with the concomitant reduction of NAD. the conversion which was  Y" ' ' utyric-glutamate am  no  3  transaminase catalyzed  of yaminobutyric acid to succinic semialdehyde,  further oxidized to succinate by succinic semialdehyde  dehydrogenase.  Y~Aminobutyric-glutamlc transaminase was  by growth in either Y~aminobutyrate or putrescine. semialdehyde dehydrogenase was high l e v e l .  induced  Succinic  present c o n s t i t u t f v e l y at a f a i r l y  Growth in putrescine did not apprec?ably  increase  the a c t i v i t y of this enzyme; however, growth in y-aminobutyrate did increase i t 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 t h e E_. c o l i m u t a n t and M y c o b a c t e r i a ( E v e l y n , 1967) m e n t i o n e d grown  above.  Nakamura  (1960)  enzymes  dehydrogenases;  Padmanabhan and T c h e n i n a Pseudomonad.  dehydrogenase  by c h r o m a t o g r a p h y for succinic by g r o w t h  one l i n k e d  (1969)  have f u r t h e r s t u d i e d  The N A D P - l i n k e d  semialdehyde.  i n y-aminobutyrate  and y a m i n o b u t y r a l d e h y d e  yaminobutyrate.  succinic peaks  specific  or in putrescine.  levels The  a c t i n g o n 3~  in addition to succinic  and t h i s  t h e aminoenzyme  could  i n p u t r e s c i n e , but not i n  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  s t u d i e d by J a c o b y  and F r e d e r i c k s  activities  o f t h e NAD-1inked d e h y d r o g e n a s e s  all  conditions.  growth  into three  One p e a k was much more r e a c t i v e w i t h  i n d u c e d t o a h i g h l e v e l by g r o w t h  dehydrogenase  The NAD-1inked  dehydrogenases  these  constitutive,  T h i s enzyme was i n d u c e d t o h i g h  aldehydes than w i t h s u c c i n i c semialdehyde be  enzyme was  a c t i v i t y was r e s o l v e d  o t h e r two p e a k s w e r e a m i n o a l d e h y d e  semialdehyde.  t o NAD, t h e o t h e r  o n D E A E - S e p h a d e x , o n l y o n e o f w h i c h was  of the c e l l s  aminopropanal  compound  1960).  (Bachrach,  c o u l d n o t be i n d u c e d t o a h i g h e r l e v e l .  semialdehyde  this  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 semialdehyde t o NADP.  1962)  P_. a e r u g i r i b s a  i n the presence 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  to succinate v i a s u c c i n i c semialdehyde  and  ( K i m and T c h e n ,  (1959)•  Very low  were d e t e c t a b l e  under  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 h e  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  putrescine.  In summary, a r g i n i n e may i t 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 ,  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 o r by  a r g i n i n e d i h y d r o l a s e pathway. be c o n v e r t e d  B o t h a g m a n t i n e and  to p u t r e s c i n e , which  by some o r g a n i s m s . to  or  ornithine  the  can  c a n be f u r t h e r d e g r a d e d t o s u c c i n a t e  In a d d i t i o n , o r n i t h i n e c a n be d i r e c t l y  degraded  glutamate. An a d d i t i o n a l  scribed  p a t h w a y o f a r g i n i n e d e g r a d a t i o n h a s been  i n Streptomyces  griseus  ( v a n T h o a i , 1965).  In t h i s  a r g i n i n e undergoes d e c a r b o x y l a t i n g oxygenation r e s u l t i n g formation of y-guanidobutyramide, aminobutyrate v i a the  which  intermediate y-guanidobutyrate.  s y n t h e s i s , and arginine 1965).  catalysing  T h i s enzyme i s a l s o p r e s e n t C_. b o t u l inum ( M f t r u k a and  M o r e t h a n one  glycocyamine  W i l s o n and  to p u t r e s c i n e v i a agmantine  M i t r u k a and  griseus  Holden,  C o s t i l o w , 1967).  Holden  v i a o r n i t h i n e and  C o s t i l o w (l967) d e m o n s t r a t 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  be  operative  (1969a) h a v e r e p o r t e d  i n E_. c o l ? , k - 8% was  a r g i n i n e d i h y d r o l a s e pathway.  (Walker,  i n E_. c o l i ( W i l s o n and  t h a t , a l t h o u g h t h e m a j o r i t y o f e x o g e n o u s a r g i n i n e was  g l u t a m a t e , presumably  S.  in streptomycin bio-  p a t h w a y o f a r g i n i n e d e g r a d a t i o n may  in a s i n g l e organism.  t o y-  the t r a n s f e r of the g u a n i d i n e group of  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  1969a) and  organism,  in the  i s then converted  also contains a transamidinase functioning  des-  converted  converted  to  glutamic-y-semialdehyde. t h a t C_. b o t u l inum t o t h e enzymes o f  Moreover, t h i s organism  possessed  the  degraded  o r n i t h i n e by two w h i l e 75%  It.  was  p a t h w a y s , 20%  r  belng decarboxylated  degraded v i a 6 - a m i n o v a l e r i c  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  E p p s and  Gale  (19^2) were t h e f i r s t  acid.  Degradation  to study the r e p r e s s i o n  o f e n z y m e s c a t a b o l t z i n g a m t n o a c i d s , w h i c h was add t t ton o f g l u c o s e  t o the g r o w t h medium.  deaminases were s u b j e c t t o t h i s  a r g i n i n e , l y s i n e , and The  the  alantne, serine,  and  r e p r e s s i o n , whereas  htsttdtne decarboxylases  were u n a f f e c t e d .  i n d u c t i o n o f many e n z y m e s 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  of carbohydrates  and  T h i s phenomenon was 0 961)  c a u s e d by  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 glutamate  to putrescine,  who  o f amtno a c i d s t s r e p r e s s e d termed c a t a b o l t t e  considered  by  r e p r e s s i o n by  s e n s i t t v e e n z y m e s c o u l d be more r e a d i l y o b t a t n e d s i t u a t i o n where l i m i t a t t o n  accumulation  of catabolic  t h a t g l u c o s e and  from  glucose.  caused  an  (e.g., n i t r o g e n or  represston.  other  glucose-  of anabolism  tntermedtates  s t a r v a t i o n ) produced a s i m i l a r discovered  Magasantk  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 t h e c a t a b o l i t e s f o r m e d by t h e a c t i o n o f t h e  T h u s , any  glucose.  phosphate  R e c e n t l y , t t has  rapidly metaboltzable  carbon  s o u r c e s c a u s e 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 lowering  the  monophosphate  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 ( c y c l i c AMP).  The  adenosine  a d d i t i o n o f c y c l i c AMP  been  to  by  cultures of E. c o l i overcame the catabolite repression of tryptophanase, D-sertne deaminase, thymidine phosphorylase, and permeases and catafaoltc enzymes s p e c i f i c for several sugars,  (see review  by Pastan and Perlman, 1970). Jacoby (1964) found that glucose repressed the a b i l i t y of P_. fluoresceins to oxidize 18 different amino a c i d s .  He further  studied the oxidation of tyrosine and h i s t i d i n e and observed that glucose did not cause a decrease in the rate of uptake of these amino a c i d s .  However, the induction of enzymes s p e c i f i c for the  catabolism of these amino acids was repressed by glucose. Lessie and Neidhardt (1967) found that the hist idase a c t i v i t y 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 h i s t i d i n e was required as a source of carbon and energy and was somewhat reduced when h i s t i d i n e was required only as a nitrogen source.  It was severely repressed when a l l  of the products of h i s t i d i n e catabolism could be supplied by a more rapidly metabolizable energy source,  i t Is interesting to  note that the repression of a carbohydrate degrading enzyme, myoinositol dehydrogenase, was not affected by ammonium ions.  These  workers have proposed t h a t the compound t h a t  is physiologically  a c t i v e i n the c a t a b o l i t e r e p r e s s i o n of amino a c i 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.  C a s t a n a d a , M a r t u s c e l l i and Mora (1967) showed t h a t the presence of ammonium ions i n the growth medium of N_. c r a s s a decreased l e v e l t o which a r g i n a s e Wiame (1965) has  and  reported  the  o r n i t h i n e t r a n s a m i n a s e were i n d u c e d .  t h a t the presence of ammonium i o n s , but  not g l u t a m a t e , 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_. s u b t i 1 i s . Middelhoven (1970) has and  h y p o t h e s i z e d t h a t the l e v e l of  arginase  o r n i t h i n e t r a n s a m i n a s e i n 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 repressor.  The  i n d u c t i o n of t h e s e enzymes was  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 s u l p h a t e  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 . end-product r e p r e s s i o n due g l u t a m a t e i t s e l f was  not  t o the f o r m a t i o n  not a s t r o n g  T h i s was  inhibitor.  Moreover, s t a r v a t i o n ornithine  t r a n s a m i n a s e t o a l a v e l as high as t h a t induced by a r g i n i n e . resulted  i n 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 .  could  i n h i b i t e d by the a d d i t i o n of one  c o n t a i n i n g compounds.  I t was  This  Derepression  of a number of  nitrogen  s p e c i f i c f o r the a r g i n i n e d e g r a d i n g  enzymes; o t h e r enzymes i n v o l v e d t i o n were not  not  of g l u t a m a t e , s i n c e  of S^. c e r e v i s i a e f o r n i t r o g e n d e r e p r e s s e d a r g i n a s e and  be  and  i n p r o t e i n and  amino a c i d degrada-  affected.  L e s s i e and  N e i d h a r d t (1967) a l s o noted t h a t s u c c i n a t e , which  produced the most severe c a t a b o l i t e r e p r e s s i o n of h i s t i d a s e s y n t h e s i s  in P_. a e r u g i n o s a , 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 Hug, Roth and Hunter (1968) found t h a t s u c c i n a t e  in vivo.  competitively  i n h i b i t e d u r o c a n a s e , the second enzyme o f h i s t i d i n e d e g r a d a t i o n , in Pseudomonas p u t i d a . histidase.  Urocanate was a c o m p e t i t i v e  i n h i b i t o r of  T h u s , t h e presence of s u c c i n a t e caused an i n v i v o  r e p r e s s i o n o f h i s t i d a s e by s e q u e n t i a l  feedback.  However, Jensen and  N e i d h a r d t (1969) found t h a t , i n ' A . a e r o g e n e s , i n v i v o  inhibition  of h i s t i d a s e a c t i v i t y d i d not r e q u i r e the presence o f s u c c i n a t e , but c o u l d be caused by r e s t r i c t i n g growth i n h i s t i d i n e i n a chemos 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  t o be immediately r e l e a s e d 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 i n c r e a s e h i s t i d a s e l e v e l had i n c r e a s e d  i n growth r a t e b e f o r e t h e  significantly.  Thus, c a t a b o l i t e  i n h i b i t i o n appears t o a c t as a f i n e c o n t r o l mechanism s i m i l a r t o the feedback i n h i b i t i o n o f b i o s y n t h e t i c pathways.  Catabolite  i n h i b i t i o n of t h e u t i l i z a t i o n of a number o f sugars has been noted i n E_. c o l i (McGinnis and P a i g e n , 19^9) • As  f a r as the enzymes o f a r g i n i n e d e g r a d a t i o n a r e c o n c e r n e d ,  Ramos e t a 1. (1967) found t h a t a r g i n i n e d e i m i n a s e , carbamate k i n a s e , and t h e 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 were s u b j e c t to c a t a b o l i t e repression  i n P_. f 1 u o r e s c e n s .  These enzymes were  d e r e p r e s s e d when growth was l i m i t e d by the carbon s o u r c e , c i t r a t e . L a i s h l e y and B e r n l o h r (1968) showed t h a t t h e i n d u c t i o n o f a r g i 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 1  dehydrogenase  was r e p r e s s e d by g l u c o s e (1970) o b s e r v e d t h a t  i n JB. 1 t c h e n i f o r m i s ; h o w e v e r , M i d d e l h o v e n  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 w e r e h o t s u b j e c t t o c a t a b o l i t e Padmanabhan and T c h e n the  induction of succinic  butyrate.  dehydrogenase  semialdehyde dehydrogenase  by Y ~  inducer, Y~  am  a i T ,  n  i o-  aminoaldehyde  'nobutyraldehyde  the induction of succinic  b y p u t r e s c i n e , by l o w e r i n g am  and  These w o r k e r s t h e r e f o r e f e l t  the repression of Y~  inhibiting  hydrogenase  that  dehydrogenase,  semialdehyde de-  the concentration of the  n  i °butyrate.  The B i o l o g i c a l  Kay  semialdehyde dehydrogenase  were l o w e r e d .  g l u c o s e caused  lit.  (1969) f o u n d t h a t g l u c o s e d i d n o t r e p r e s s  H o w e v e r , when p u t r e s c i n e was t h e i n d u c e r , t h e l e v e l s  of both s u c c i n i c  thereby  repression.  (1969) f o u n d  Importance o f Polyamines  t h a t P_. a e r u g i n o s a , when s u p p l i e d  exogenous a r g i n i n e , formed  a high  intracellular  w h i c h was s t a b l e o v e r 2k h o u r s o f s t a r v a t i o n carbon s o u r c e .  Thus, p u t r e s c i n e appeared  product of a r g i n i n e degradation maintained wfthin  in Microorganisms  the c e l l  with  pool o f p u t r e s c i n e  f o r an exogenous  t o be an i m p o r t a n t  i n t h i s o r g a n i s m , and may be  i n a bound  state.  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 , which 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 present i n v a r y i n g concentratTons  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  to contain  k5% o f i t s p o l y a m i n e s a s  32% a s s p e r m i d i n e , and 23% a s s p e r m i n e  (Weaver and H e r b s t , 1 9 5 8 ) .  In E_. c o l ? , o n t h e o t h e r h a n d , p u t r e s c i n e total cell  polyamine, with  (Tabor and T a b o r , There portant  spermidine c o n s t i t u t i n g t h e remainder  evidence that  i n E_. c o l i .  i s t h e same i n c e l l s  p o l y a m i n e s p e r f o r m an im-  The i n t r a c e l l u l a r  T a b o r and T a b o r  continued to synthesize  the  the  i n growth  rate.  w i t h mutants  slightly  these c o n d i t i o n s ,  reduced.  o f E_. c o l i ( H i r s h f i e l d  et_ a l _ . 1 9 7 0 ) and  ( D a v i s , L a w l e s s and P o r t , 1 9 7 0 ) h a v e d e m o n s t r a t e d  g r o w t h 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 r e d u c e d when  synthesis  was r e p r e s s e d .  that c e l l  d i v i s i o n may h a v e been a f f e c t e d .  restored  putrescine  division  (1970)  putrescine  N o r m a l g r o w t h was  have p r e s e n t e d e v i d e n c e t h a t  t o s p e r m i d i n e may be a c r i t i c a l  i n E. c o l i .  that  Many s n a k e f o r m s w e r e o b s e r v e d , i n d i c a t i n g  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  Inouye and P a r d e e of  limitation  Although the  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 u n d e r  Studies  grown  Koffron,  p o l y a m i n e s , e v e n when o r n i t h i n e  s p e r m i d i n e l e v e l was o n l y  N_. c r a s s a  ( M o r r i s and  content  ( 1 9 6 9 a ) f o u n d t h a t an o r n i t h i n e a u x o t r o p h  caused a t h r e e - f o l d r e d u c t i o n putrescine  putrescine  g r o w n i n m i n i m a l medium a s i n c e l l s  in the presence o f a r g i n i n e or o r n i t h i n e 1969).  c o m p r i s e s 90% o f t h e  1964).  i s indirect  function  putrescine;  putrescine. the r a t i o  factor f o r cell  The b i o c h e m i c a l reviewed  (Stevens,  nucleic acid nucleotides  a c t i o n o f p o l y a m i n e s h a s been r e c e n t l y Polyamines bind  1970).  (DNA), r i b o n u c l e i c a c i d in vitro.  deoxyribo-  (RNA) , and s y n t h e t i c  They s t a b i l i z e  o f DNA, and a p p e a r t o c a u s e RNA  strongly to  the double h e l i c a l  polystructure  t o a s s u m e a more c o m p a c t s t r u c t u r e .  P o l y a m i n e s d o n o t a p p e a r 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 , b u t may  in the  intracellular resulted  i n an  concentration also.  concentration increased  RNA.  In E_. c o l ? , an  of spermidine,  r a t e o f RNA  but not p u t r e s c i n e ,  synthesis.  a p p e a r s t o be r e l a t e d t o RNA  increase  The  synthesis  spermidine i n animal  Moreover, the a d d i t i o n of polyamines to a c e l l - f r e e  c a u s e s an the  associate with  increase  i n RNA  polymerase a c t i v i t y , appearing  number o f a v a i l a b l e i n i t i a t i o n  tissues,  system  to  increase  sites.  Polyamines 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 subu n i t s and t h e b i n d i n g o f m e s s e n g e r RNA RNA  to ribosomes.  They can p a r t i a l l y  an  in vitro  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  s y n t h e s i s , H u r w i t z and R o s a n o p l a y an  important  t o many o f t h e s i t e s W e i s s and M o r r i s  role  transfer  r e p l a c e magnesium  p r o t e i n synthesizing system.  much l o w e r t h a n t h e c o n c e n t r a t i o n  may  and a m i n o a c y l  Since optimal  magnesium  required  ions i n growth  concentrations  for in vitro  (1967) h y p o t h e s i z e d  that  protein polyamines  in in vivo protein synthesis, binding  where magnesium  binds  i n the i n v i t r o  system.  (1970) f o u n d t h a t up t o 70% o f t h e m a g n e s i u m  bound t o r i b o s o m e s 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  putrescine  without a f f e c t i n g  their activity  However, a c r i t i c a l structural  l e v e l o f m a g n e s i u m was  and f u n c t i o n a l  be r e p l a c e d  in c e l l - f r e e  protein  required  in a l l the previously  i s spermine>spermidine>putrescine.  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 the  l a t t e r may  ing  t o n o t e t h a t , i n a Pseudomonad  the  binding  magnesium  of hydroxyputrescine, sites  concentration  I n v i t r o and  a c t as p o l y v a l e n t  reactions.  spermidine,  i n t h e same way a s d o e s s p e r m i d i n e t n 1969). t o and s t a b i 1 i z e DNA,  In v i v o .  cations  in v i t r o ,  RNA,  and  and  RNA  It i s p o s s i b l e that function  stabilizing  b i n d i n g , r e p l a c i n g many o f t h e m a g n e s i u m vitro  It i s interest-  w h i c h p o s s e s s e s t h e same number  a m i n e s do n o t h a v e a s i n g l e s p e c i f i c but may  spermidine,  unable t o synthesize  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 both  Thus,  as s p e r m i d i n e , t o ribosomes v a r i e s w i t h the  summary, p o l y a m i n e s b i n d  synthesis  than  be p h y s i o l o g i c a l l y m o r e i m p o r t a n t .  E_. c o l I CRosano and H u r w l t z , tn  not  by p o l y a m i n e s .  mentioned f u n c t i o n s  binding  f o r the  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  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  of  synthesis.  in cell  poly-  metabolism,  nucleotide-nucleotide  tons r e q u i r e d  f o r j_n_  20  IV.  T r a n s p o r t of the B a s i c Amino A c i d s by M i c r o o r g a n i s m s The t r a n s p o r t and a c c u m u l a t i o n o f amino a c i d s by m i c r o o r g a n i s m s  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  M c C l u r e , 1962; Kay, 1968; Kabak, 1970).  and  B a c t e r i a have been found  t o possess many t r a n s p o r t s y s t e m s , some of which a r e s p e c i f i c f o r s i n g l e amino a c i d s and o t h e r s o f which a r e 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 . such amino a c i d t r a n s p o r t systems  Kay  (1968) i d e n t i f i e d  11  i n P_. a e r u g i n o s a .  The t r a n s p o r t of the b a s i c amino a c i d s has been s t u d i e d i n several microorganisms.  S c h w a r t z , 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 i n the uptake of c a n a v a n i n e , 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 t h a t t h e s e  compounds must be t r a n s p o r t e d  by a s i n g l e permease.  Citrulline  uptake by a l s o a f f e c t e d somewhat by t h i s m u t a t i o n (Maas, 1965)• The s t u d i e s of W i l s o n and Holden  (1969a) i n d i c a t e d t h a t E_. c o l i  possessed a t l e a s t two systems f o r the uptake of b a s i c 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 w i t h a h i g h a f f i n i t y for  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 .  W i l s o n and  Holden  (1969b) i s o l a t e d f o u r p r o t e i n s , from the o s m o t i c shock f l u i d o f E_. c o l i , which bound a r g i n i n e , but showed no a f f i n i t y  for lysine.  Two o f t h e s e p r o t e i n s were s t u d i e d f u r t h e r , and were c a p a b l e 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 Kay  to transport  arginine.  (1968) o b t a i n e d e v i d e n c e f o r the e x i s t e n c e , i n P_. aerug 1 n o s a ,  of two systems f o r the t r a n s p o r t of b a s i c amino a c i d s : one  specific  for  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  for  lysine  and  histidine.  that  affinity  and l o w e r 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 , Grenson  (1966) and G r e n s o n et_ a j _ . (1966) d e m o n s t r a t e d  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 s y s t e m s f o r t h e u p t a k e o f  b a s i c a m i n o a c i d s : o n e was h i g h l y s p e c i f i c a very high a f f i n i t y  for lysine;  t h e o t h e r had  f o r a r g i n i n e , and a l o w e r a f f i n i t y  o r n i t h i n e and c a n a v a n i n e . sessed a b a s i c amino a c i d  Pall  for lysine,  (1970) showed t h a t N_. c_rassa_ p o s -  transport  system w i t h  a high 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 , a n d o r n i t h i n e , and a much l o w e r for  affinity  histidine. G r e n s o n , Hou a n d C r a b e e l  (1970) showed t h a t t h e o n l y  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  mechanism amino  a c i d p e r m e a s e , w h 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 a m i n o T h w a i t e s and P e n d y a l a transported  solely  acids.  (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  by t h e g e n e r a l a m i n o a c i d  transport  system o f  N_. c r a s s a . Ames  (1964) a n d Ames and R o t h  (1968) h a v e shown t h a t  typhimurium possessed a h i g h l y s p e c i f i c of h i s t i d i n e .  This  organism could  system f o r t h e t r a n s p o r t '  a l s o t a k e up h i s t i d i n e  general aromatic permease, which functioned tyrosine, phenylalanine, Kay with  (1968) f o u n d t h a t  and t r y p t o p h a n .  transported  concluded that  via a  i n the transport of  On t h e o t h e r  a l l b a s i c amino a c i d s  h i s t i d i n e , and t h e r e f o r e  Salmonella  competed  hand,  strongly  histidine  must be  m a i n l y by t h e b a s i c a m i n o a c i d u p t a k e s y s t e m s  in this  organism.  Pall  (1970)  h a s shown t h a t  histidine  v i a three  transport  system, the neutral  the  transport  g e n e r a l amino a c i d  systems:  N_. c r a s s a  transport  the b a s t e amino  amino a c i d  transport  can  system.  transport  acid  s y s t e m , and  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 s a l t s minimal medium and were checked p e r i o d i c a l l y for purity by streaking onto Difco plate count agar.  Cultures of P_. aeruginosa were also  checked for production of the species c h a r a c t e r i s t i c  pigment,  pyocyanine, by streaking onto Kings medium (King, Ward, and Raney, 1954). C e l l s were grown in a medium containing 0.3% NH^H^PO^, 0.2% K^PO^ and 0.5 ppm FeS0^.7H 2 0 at pH 7.2.  MgS0^.7H 0 and the 2  appropriate carbon source were s t e r i l i z e d separately and added a s e p t i c a l l y to give a f i n a l 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.  II.  Growth of C e l I s  F o r most e x p e r i m e n t s ; , E r l e n m e y e r w f t h 13 mm  test  medium w e r e  flasks  (250 m l )  t u b e 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  inoculated  to a final  concentration  cell  s u s p e n s i o n p r e v i o u s l y grown  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  Sctentific  C o . , New  r o t a t i n g a t 220  B r u n s w i c k , N.J.)  [equivalent  t o 0.7  Fullerton,  were grown  100 ml o f t h e a p p r o p r i a t e m e d i u m .  and t h e c e l l s  from  Preparation of Cel1  1.  (red f i l t e r ) 125  d e n s i t y u n i t s measured  (Beckman  Instruments  i n Roux f l a s k s  Resting c e l l  con-  A 1% i n n o c u l u m was  t h e e a r l y s t a t i o n a r y phase o f growth  h a r v e s t e d a f t e r 20 h o u r s a t 30  ill.  rev/mtn.  Calif.)].  F o r some e x p e r i m e n t s , c e l l s taining  Brunswick  colorimeter  ~ 0.8 o p t i c a l  660 nm w i t h a m o d e l B s p e c t r o p h o t o m e t e r  Inc.,  (New  f r o m t h e l o g a r i t h m i c p h a s e w e r e h a r v e s t e d a t 110 -  Klett units at  o f 2% w i t h a  t n t h e same medtum, and w e r e  G r o w t h was f o l l o w e d w t t h a K l e t t - S u m m e r s o n and c e l l s  equipped  were  C.  Suspensions  suspensions f o r respirometry  C e l l s w e r e 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.  T h e y w e r e washed  three times wtth cold  O.St  used,  NaCl  (pH 7.4) and resuspended  in cold 0.05 M Tris(hydroxymethyl)-  aminomethane-HCl (Tris) buffer (pH 7-4).  In some cases, c e l l s  were resuspended in 0.067 M potassium phosphate buffer (pH 7.4). The f i n a l c e l l concentration was approximately 5 mg of c e l l s (dry weight)/ml, unless otherwise s p e c i f i e d .  2.  Cell  suspensions f o r transport studies.  C e l l s in the logarithmic phase of growth  were harvested as  described above, but at room temperature, and washed twice with minimal s a l t s medium without carbon source.  They were resuspended  In glucose minimal medium to approximately 1.35 mg of c e l l s (dry weight)/ml and kept at room temperature until  required for  experimentation.  IV.  Preparation of C e l l - f r e e Extracts  C e l l s 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 c e l l p e l l e t s were stored at -70 C.  They were resuspended in cold  0.01 M potassium phosphate buffer (pH 7.4) containing 0.01% mercaptoethanol, to a f i n a l concentration of approximately 20 mg of c e l l s Xdry weight)/ml.  Deoxyribonuclease (Worthington Biochemical Corp.,  F r e e h o l d , N.J.) was a d d e d t o a f i n a l The  c e l l s w e r e b r o k e n by d r o p w t s e e x p u l s i o n f r o m a  French and  c o n c e n t r a t i o n o f 80 y g / m l .  pressure c e l l  u n d e r 15,000 p s i p r e s s u r e .  precooled  Unbroken  cells  l a r g e c e l l u l a r d e b r i s w e r e 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  The  Procedures  oxygen uptake o f c e l l s  respiring  endogenously or i nthe  p r e s e n c e o f e x o g e n o u s s u b s t r a t e s was f o l l o w e d manner w i t h t h e W a r b u r g r e s p i r o m e t e r . 1.0 ml c e l l  contained (dry  suspension  A typical  [approximately  w e i g h t ) / m l ] , 1.9 ml 0.05 M T r i s  reaction  b u f f e r (pH 7.4),  In t h e e n d o g e n o u s c o n t r o l , 0.1 ml d i s t i l l e d substrate.  F o r some e x p e r i m e n t s  M phosphate b u f f e r  mixture  5 mg o f c e l l s  i n 0.1 m l , and 0.15 ml 20% K0H i n t h e c e n t e r  substrate  VI.  i n the conventional  2.5 y m o l e s well.  water replaced the  t h e T r i s was r e p l a c e d by 0.067  (pH 7-4).  U p t a k e o f L a b e l l e d Compounds  14 The  incorporation of  C - l a b e l l e d compounds  protein,  and p o o l s was d e t e r m i n e d  procedure  o f B r i t t e n and M c C l u r e  i n t o whole  by t h e M i l l i p o r e (1962).  filtration  E x p e r i m e n t s were  o u t a t 30 C , i n 25 ml o r 50 ml E r l e n m e y e r f l a s k s  cells,  containing  carried glucose  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 , R o c h e s t e r , N.Y.) d r i v e n by a Lauda K2 c i r c u l a t i n g pump (Brinkman  I n s t r u m e n t s , Westbury, N.Y.).  Unless otherwise s p e c i f i e d ,  c e l l s were added 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 o f 0.135 mg o f c e l l s (dry  w e i g h t ) / m l , and s u b s t r a t e s were added t o g i v e an e x t e r n a l  -5 14 c o n c e n t r a t i o n o f 2.5 x 10 M, and 0.05 y c C/ml. Samples o f the c e l l s u s p e n s i o n were removed a t a p p r o p r i a t e time I n t e r v a l s and either filtered  immediately o r added t o an equal volume o f c o l d  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 o r t r i c h l o r o a c e t i c  i n s o l u b l e m a t e r i a l were f i l t e r e d on 0.45 y pore s i z e  acid-  filters  ( M i l l i p o r e C o r p . , B e d f o r d , Mass.) i n 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 w i t h 2 ml o f g l u c o s e minimal medium (whole c e l l s ) o r 2 ml o f d i s t i l l e d water (trichloroacetic acid-insoluble material).  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 p l a c e d i n v i a l s c o n t a i n i n g 5 ml o f scintillation fluid Mass.).  ( L i q u i f l u o r , New England N u c l e a r C o r p . , B o s t o n ,  The v i a l s were assayed f o r r a d i o a c t i v i t y  l i q u i d s c i n t i l l a t i o n spectrometer P l a i n e s , 111.).  i n a model 725  ( N u c l e a r Chicago C o r p . , Des  C o r r e c t i o n s were made f o r background.  In o r d e r  to reduce s t a t i s t i c a l d e v i a t i o n , a t l e a s t 1000 counts were r e corded.  The c o u n t i n g e f f i c i e n c y o f t h e i n s t r u m e n t was 80 per  c e n t under t h e c o n d i t i o n s employed.  28  VII.  I n h i b i t i o n of T r a n s p o r t to the  by Compounds S t r u c t u r a l l y  Related  Substrate  To determine  i n h i b i t i o n , the u n l a b e l led i n h i b i t o r was  added  -3 to a c o n c e n t r a t i o n of 2.5 x 10 of the  M immediately p r i o r to the a d d i t i o n  1k  C - l a b e l l e d s u b s t r a t e to a c o n c e n t r a t i o n o f 2.5 x 10  Uptake o f r a d i o a c t i v i t y  i n t o the whole c e l l s was  the degree o f i n h i b i t i o n was  -5  M.  f o l l o w e d , and  c a l c u l a t e d from the r e d u c t i o n  In  14 the r a t e of i n c o r p o r a t i o n of the to the a p p r o p r i a t e  VIII.  C-labelled substrate  control.  Assay of S u c c i n i c Semialdehyde  Dehydrogenase  S u c c i n i c semialdehyde dehydrogenase by Jakoby glutamic  relative  (1962) by c o u p l i n g transaminase.  was  measured as  the r e a c t i o n to Y ~  am  described  n  ' °butyrate-  The pH o f the r e a c t i o n m i x t u r e was  8.0  and 5 ymoles o f both y-amfnobutyrate and a - k e t o g l u t a r a t e were added.  The  r e d u c t i o n of 0.25 ymoles of NAD  or NADP was  at 340 nm a t 34 C in a model 2000 spectrophotometer Instrument L a b o r a t o r i e s  Incorp., O b e r l i n , Ohio).  measured  (Gilford  Appropriate  c o n t r o l s were c a r r i e d out to show the requirement of the r e a c t i o n f o r both y a m i n o b u t y r a t e and a - k e t o g l u t a r a t e .  Specific  are expressed as mymoles of s u b s t r a t e u t i l i z e d  per min per  (of p r o t e i n .  activities mg  IX.  Chemical Fractionation of Whole Cells  Cells were fractionated according to the procedure of Roberts et_al_. (1955) with the modification of C l i f t o n and Sobek (1961). The hot t r i c h l o r o a c e t i c f r a c t i o n was prepared by heating the sample at 90 C for 20 min rather than at 100 C for 30 min.  Samples  of the c e l l fractions were plated in duplicate onto stainless steel planchets, dried under an infra-red lamp, and counted at i n f i n i t e 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 s t a t i s t i c a l  d e v i a t i o n , at least 1000 counts were recorded when possible. The counting e f f i c i e n c y 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 resuspended in 0.05 M T r i s buffer (pH J.h)  containing 10 M MgCl 2  to approximately 20 mg of c e l l s (dry weight)/ml.  The suspension  was passed once through a French pressure c e l l and whole c e l l s and large debris were removed by centrifugation for 7 minutes at 5,000 x g_.  The resulting c e l l - f r e e extract was fractionated  a c c o r d i n g t o t h e procedure o f C a m p b e l l , Hogg and S t r a s d i n e ( 1 9 6 2 ) , o m i t t i n g t h e washing o f t h e i n i t i a l 25,000 x g_ pel l e t : . c  The  magnesium c o n c e n t r a t i o n o f the 25,000 x g_ s u p e r n a t a n t f l u i d was -3 a d j u s t e d t o 10  M before c e n t r i f u g a t i o n .  resuspended i n 0.05 M T r i s XI.  The p e l l e t s were  (pH 7 . 4 ) .  Chromatography o f Supernatant F l u i d s from Warburg Cups 14 Containing  C-labelled Substrates  P r o t e i n was p r e c i p i t a t e d from t h e s u p e r n a t a n t f l u i d s by t h e a d d i t i o n o f t r i c h l o r o a c e t i c a c i d t o a f i n a l c o n c e n t r a t i o n o f 5%-. A f t e r 20 min on i c e , t h e p r e c i p i t a t e d m a t e r i a l was removed by c e n t r i f u g a t i o n a t 10,000 x £ f o r 10 minutes a t 6 C.  The super-  natant f l u i d s were then e x t r a c t e d 6 times w i t h e t h y l e t h e r t o remove t r i c h l o r o a c e t i c a c i d , and evaporated  t o d r y n e s s u s i n g an  Evapomix instrument ( B u c h l e r I n s t r u m e n t s , F o r t L e e , N . J . ) . d r i e d samples were d i s s o l v e d  The  i n t h e d e s i r e d volume o f d i s t i l l e d  water. When phosphate was p r e s e n t i n t h e r e a c t i o n m i x t u r e , the sampl +  were a p p l i e d t o a column o f Dowex 50 H .  The column was washed  w i 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 t h e n e u t r a l and a c i d i c compounds, i n c l u d i n g p h o s p h a t e . The adsorbed  compounds were e l u t e d w i t h 4 M ammonium h y d r o x i d e ,  the e l u a t e was evaporated  t o d r y n e s s 4 times t o 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 c e l l u l o s e powder MN300 (Macherey and Nagel , Piiren, Germany) and the radioactive compounds were separated twodimensional ly by the method of Jones and Heathcote The chromatograms were then exposed X-ray f i l m  (1966)-  for one week to medical  (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 v i a l s which were subsequently f i l l e d with 10 ml of s c i n t i l l a t i o n f l u i d and assayed for r a d i o a c t i v i t y in a liquid s c i n t i l l a t i o n spectrometer.  The addition of from 5 to 60 mg of  c e l l u l o s e caused no increase in quenching under these conditions. In some instances, the presence of r a d i o a c t i v i t y  in aqueous  samples was assayed d i r e c t l y by l i q u i d s c i n t i l l a t i o n spectrometry. Up to 100 y l of the sample was placed in a v i a l containing 10 ml of a s c i n t i l l a t i o n f l u i d 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 comparable to those obtained with the previously mentioned lation f l u i d .  scintil-  32  XII.  Assay o f O r n i t h i n e  Transcarbamylase  The assay procedure o f S t a l o n e t ^ a j _ . (1967a) was f o l l o w e d . In o r d e r t o e s t a b l i s h a r e a c t i o n r a t e , t h e 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 o f c e l l - f r e e e x t r a c t , and 2.5 ml samples were removed a t v a r i o u s t i m e s , added t o an equal volume o f 1 N HC1 i n heavy-walled c e n t r i f u g e tubes s t a n d i n g i n i c e , and t r e a t e d as i n t h e procedure o f S t a l o n 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 o f g l u c o s e 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 o f c e l l s grown in the presence o f added a r g i n i n e were u s e d . 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 o f t h e method d e s c r i b e d by Oginsky  (1957) -  To t h e sample,contained  i n a 2.0 ml  volume, was added 1.0 ml o f a m i x t u r e o f 1 volume o f c o n c e n t r a t e d H^SO^ t o 3 volumes o f c o n c e n t r a t e d H^PO^, and 0.13 ml o f a 3% -  aqueous s o l u t i o n o f 2,3 butadionemonoxime. The c o n t e n t s o f t h e tubes were mixed v i g o r o u s l y and p l a c e d i n b o i l i n g water f o r 10 minutes  i n t h e d a r k , and c o o l e d i n i c e water i n t h e d a r k .  tube was removed immediately p r i o r t o being r e a d . was recorded a t 490 nm. citrul1ine.  Each  Optical density  T h i s assay measured 15 t o 50 yg o f  33  XIII.  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 _ . ( 1 9 5 1 ) . The presence o f ammonia i n Warburg s u p e r n a t a n t f l u i d s was determined by a m o d i f i c a t i o n o f t h e Conway (1950) m i c r o d i f f u s i o n t e c h n i q u e . V a l u e s between 0 and 20 yg o f ammonia c o u l d be measured under the c o n d i t i o n s used.  XIV.  Chemicals  A l l c h e m i c a l s used i n t h i s s t u d y were purchased from sources.  y-Aminobutyric acid-4-  commercial  C and u n i f o r m l y l a b e l l e d L-  C-  a r g i n i n e were o b t a i n e d from Schwartz B i o r e s e a r c h I n c . , Orangeburg, N.Y., and p u t r e s c i n e - ' ^ C  (tetramethylenediamine-1,4,-'\ dihydro14  c h l o r i d e ) and D L - o r n i t h i n e - 1 -  C from Amersham/Searle C o r p . , 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 for transport studies, DL-ornithine-5  -  C, were purchased from  New England N u c l e a r C o r p . , B o s t o n , Mass.  RESULTS AND  I.  DISCUSSION  Pathways of A r g i n i n e D e g r a d a t i o n  1.  Accumulation  i n P_. aerugtnosa  of i n t e r m e d i a t e s o f a r g i n i n e d e g r a d a t i o n  When g l u c o s e grown c e l l s of P_. a e r u g i n o s a were incubated w i t h 14 C - a r g i n i n e i n a c o n v e n t i o n a l Warburg a p p a r a t u s , subsequent t h i n l a y e r chromatography and  r a d l o a u t o g r a p h y o f the s u p e r n a t a n t  fluid  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 g l u t a m a t e  (Figure 2 ) .  Small amounts of a number  o f 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 a c i d s or i n t e r m e d i a t e s of the t r i c a r b o x y l i c a c i d present.  (TCA) c y c l e , were a l s o  Both c i t r u l l i n e and o r n i t h i n e were p r e s e n t a f t e r s h o r t  i n c u b a t i o n p e r i o d s (10 and 30 minutes) i n r e l a t i v e l y h i g h 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 a t l a t e r t i m e s .  These  r e s u l t s c o n f i r m e d the p r e s e n c e , i n t h i s s t r a i n of P_. a e r u g i n o s a , of the a r g i n i n e d i h y d r o l a s e pathway d e s c r i b e d by Oginsky  (1955).  Both p u t r e s c i n e and glutamate appeared t o accumulate i n the Warburg s u p e r n a t a n t f l u i d as p r o d u c t s o f a r g i n i n e d e g r a d a t i o n , i n d i c a t i n g t h a t the organism may  possess both an o r n i t h i n e d e c a r b o x y l a s e  the enzymes f o r the c o n v e r s i o n of o r n i t h i n e t o glutamate v i a g l u t a m i c -y-semiaIdehyde (Ramaley and B e r n l o h r , 1 9 6 6 ) .  and  35  F i g . 2.  Radioautogram o f a t h i n - l a y e r chromatogram of the s u p e r n a t a n t f l u i d a f t e r the i n c u b a t i o n of g l u c o s e grown c e l l s w i t h C - a r g i n i n e f o r 30 minutes under c o n v e n t i o n a l Warburg conditions.  1  ^glutamate citrulline z:  LU >  O +  arginine ornithine  LO  -+-» If)  origin 2nd S O L V E N T  putrescine  2.  I n h i b i t o r y e f f e c t o f T r i s b u f f e r on t h e o x i d a t i o n o f a r g i n i n e by whole  P r e l i m i n a r y manometric showed t h a t t h e i n i t i a l  cells  e x p e r i m e n t s , u s i n g a r g i n i n e as s u b s t r a t e ,  r a t e o f oxygen uptake o f a r g i n i n e grown  c e l l s suspended a t t h e usual c o n c e n t r a t i o n  [5 mg o f c e l l s ( d r y  w e i g h t ) / m l ] was so r a p i d t h a t d i f f u s i o n o f oxygen i n t o t h e r e a c t i o n m i x t u r e was p r o b a b l y 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 t h e r e a c t i o n m i x t u r e was 8.0. T h e r e f o r e , t h e c e l l was decreased by o n e - t h i r d , and t h e c o n c e n t r a t i o n increased  t o 0.067 M.  concentration  o f T r i s was  However, under t h e s e c o n d i t i o n s , oxygen  uptake was e x t r e m e l y s l o w , and stopped a t 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 o c c u r e d . T h u s , oxygen uptake i n t h e presence o f 0.067 M T r i s b u f f e r and  i n the presence o f 0.067 M phosphate b u f f e r  compared.  (pH 7.4)  (pH 7-4) were  With a r g i n i n e as s u b s t r a t e , both t h e r a t e o f oxygen  uptake and t h e t o t a l oxygen consumption were lower i n t h e presence of T r i s b u f f e r than i n t h e presence o f phosphate b u f f e r  (Figure 3A).  A l t h o u g h T r i s b u f f e r d i d not a f f e c t t h e t o t a l oxygen uptake when p u t r e s c i n e was t h e s u b s t r a t e , i t d i d cause a decreased r a t e o f oxygen consumption and a much longer p e r i o d o f a d a p t a t i o n r a p i d r a t e was reached ( F i g u r e 3B).  b e f o r e t h e most  R e p e t i t i o n o f t h i s experiment  14 using  C-arginine  as s u b s t r a t e showed t h a t , upon c o m p l e t i o n o f  oxygen u p t a k e , t h e s u p e r n a t a n t f l u i d from the r e a c t i o n m i x t u r e w i t h  Fig.  3.  Oxidation Arginine Each cup Note the  o f a r g i n i n e (A.) and p u t r e s c i n e (B) in the presence o f T r i s and phosphate b u f f e r s . grown c e l l s were used a t a c o n c e n t r a t i o n o f 1.6 mg o f c e l l s (dry w e i g h t ) / c u p . contained 2.5 ymoles o f s u b s t r a t e . Symbols: 0, T r i s b u f f e r ; phosphate b u f f e r . change in the o r d i n a t e in B.  M I N U T E S  T r i s b u f f e r c o n t a i n e d 3 times more r a d i o a c t i v i t y than t h a t from the r e a c t i o n w i t h phosphate b u f f e r . tography and  A l t h o u g h t h i n - l a y e r chroma-  r a d i o a u t o g r a p h y of the s u p e r n a t a n t f l u i d s showed t h a t  t h i s r a d i o a c t i v i t y was  not p r e s e n t as a s i n g l e  intermediate,  g l u t a m a t e accumulated t o much h i g h e r c o n c e n t r a t i o n s of T r i s b u f f e r than i n phosphate b u f f e r .  in the presence  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 r e s p o n s i b l e f o r d e g r a d i n g glutamate, causing  a d e c r e a s e i n 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  inhibitory  e f f e c t of T r i s on s e v e r a l 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 s u c c i n a t e by m i t o c h o n d r i a , has been d e s c r i b e d The may  decreased r a t e s of o x i d a t i o n  i n the presence of  have been caused by an a l t e r a t i o n  resulting  i n some l y s i s .  by Good e t a 1. ( 1 9 6 6 ) . Tris  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  Other workers have o b t a i n e d e v i d e n c e  that T r i s a f f e c t s c e l l permeability.  L e i v e and  Kollin  (1967)  found t h a t exposure of E_. c o l i to c o l d T r i s caused a d e c r e a s e i n the r a t e of RNA  s y n t h e s i s and  absorbing m a t e r i a l .  a l o s s of a c i d p r e c i p i t a b l e UV-  Neu, Ashman and  exposure of E_. c o l ? t o T r i s f o r one n u c l e o t i d e pool and and A s b e l l  P r i c e (1967) found t h a t hour caused a r e l e a s e of  d e g r a d a t i o n of RNA  and  nucleotides.  (1966) showed t h a t the a b i l i t y of o s m o t i c a l l y  P_. a e r u g i n o s a t o t r a n s p o r t and  oxidize c e r t a i n substrates  be r e s t o r e d  i n phosphate b u f f e r but not  •Ingram, and  Costerton  in T r i s b u f f e r .  the  Eagon fragile could Cheng,  (1970) found t h a t washing P_. a e r u g i n o s a  With T r i s b u f f e r caused p a r t i a l r e l e a s e 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 t h a t exogenous phosphate  may  s t i m u l a t e the r a t e of c o n v e r s i o n 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 . some r a d i o a c t i v e c i t r u l l i n e was  I t i s i n t e r e s t i n g t o note t h a t  p r e s e n t i n the s u p e r n a t a n t f l u i d  from the r e a c t i o n m i x t u r e c o n t a i n i n g T r i s b u f f e r , but not containing  3.  in that  phosphate b u f f e r .  Oxidation  of.intermediates  of a r g i n i n e d e g r a d a t i o n  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 ~ a m i n o b u t y r a t e were i n t e r m e d i a t e s  a r g i n i n e was  in the d e g r a d a t i o n of  o b t a i n e d by manometric s t u d i e 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 t h e s e s u b s t r a t e s was by growth of the c e l l s and  carbon ( F i g . k,  Glucose grown c e l l s  increased  i n a r g i n i n e as the s o l e source of  F i g . 6A,  Table I ) .  C i t r u l l i n e was  much more s l o w l y than the o t h e r s u b s t r a t e s , and c i t r u l l i n e as the s o l e carbon and Evidence w i l l be d e s c r i b e d may  cells  and  nitrogen  nitrogen  oxidized  growth w i t h  source was  a l s o poor.  l a t e r s u g g e s t i n g t h a t c i t r u l l i n e uptake  have been r a t e l i m i t i n g .  putrescine  5 to 8 f o l d  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  y - a m i n o b u t y r a t e more r a p i d l y than g l u c o s e grown  ( F i g . 5, F i g . 6 B , T a b l e I ) .  The  maximal r a t e of  putrescine  F i g . k.  O x i d a t i o n o f a r g i n i n e (A) and o r n i t h i n e ( B ) . Symbols: 0 , g l u c o s e grown c e l l s [ 3 . 8 mg of c e l l s grown c e l l s [ 1 . 5 mg o f c e l l s (dry w e i g h t ) / c u p ] .  (dry wei g h t ) / c u p ] ; • , a r g i n i n e Phosphate b u f f e r was u s e d .  2 0 0  3 UJ  OL  ZD  100  Z  LLI  o > X  o 30  60  9 0  M I N U T E S  F i g . 5.  O x i d a t i o n of p u t r e s c i n e . Symbols: 0, g l u c o s e grown c e l l s [3.8 mg o f c e l l s (dry w e i g h t ) / c u p ] ; • , a r g i n i n e grown c e l l s [1.5 mg o f c e l l s ( d r y w e i g h t ) / c u p ] ; 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 w e i g h t ) / c u p ] . Phosphate b u f f e r was u s e d .  120  Fig.  6.  O x i d a t i o n o f 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 ; • , a r g i n i n e grown c e l l s . C e l l c o n c e n t r a t i o n was 3.8 mg o f c e l l s (dry w e i g h t ) / c u p . Phosphate b u f f e r was used.  T a b l e I.  Rates of oxygen uptake by P_. a e r u g i n o s a w i t h a r g i n i n e and suspected i n t e r m e d i a t e s as s u b s t r a t e s .  Growth medium  Substrate  arginine-sa1ts  glucose-NH^ - s a l t s Q0 2  arginine  241  30  Ornithine  164  33  citrulline  19  3-7  putrescine  94  30  y-aminobutyrate  31  18  2.5  ymoles/Warburg v e s s e l .  S u b s t r a t e c o n c e n t r a t i o n was  oxidation  by g l u c o s e g r o w n c e l l s was r e a c h e d  w h e r e a s a r g i n i n e grown c e l l s cells This  a t t h e same c o n c e n t r a t i o n  (dry weight)/cup] demonstrated  [3.8 mg o f  a lagof only 5 minutes.  l a g was n o t d u e t o t h e i n d u c t i o n o f a n u p t a k e  p u t r e s c i n e , because subsequent was  o n l y a f t e r 35 m i n u t e s ,  experiments  system f o r  showed t h a t  transported c o n s t i t u t i v e l y at a high rate.  this  compound  Since a similar  lag period d i d not occur before t h e o x i d a t i o n o f Y~aminobutyrate, the  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  verting  putrescine  to Y~aminobutyrate.  o f t h e enzymes  The f a c t  con-  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 t h a n p u t r e s c i n e t h a t exogenous Y  _ a m  indicated  i n o b u t y r a t e was p o s s i b l y n o t a s a v a i l a b l e  f o r o x i d a t i o n a s 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 rate o f uptake o f t h i s  4.  substrate.  Conversion of o r n i t h i n e t o glutamate  14 Since  C-glutamate  centrations during  was r e c o v e r e d i n r e l a t i v e l y 14  the oxidation of  was made t o d e t e r m i n e  high con-  C - a r g i n i n e , an a t t e m p t  the presence, in a c e l l - f r e e  extract of  a r g i n i n e grown P_. a e r u g i n o s a , o f t h e enzymes c o n v e r t i n g to glutamate;  i e . ornithine  carboxylate dehydrogenase. reactions using  t r a n s a m i n a s e and  ornithine  -pyrroline-5~  An a t t e m p t was made t o c o u p l e t h e two  t h e c o n d i t i o n s o f R a m a l e y and B e r n l o h r ( 1 9 6 6 ) f o r  rfhe m e a s u r e m e n t o f o r n i t h i n e  t r a n s a m i n a s e , b u t by a d d i n g NADP o r  NAD  and measuring the r e d u c t i o n of the l a t t e r c o f a c t o r s a t 340  However, no a c t i v i t y was o b t a i n e d , a l t h o u g h the assay was  nm.  attempted  in both phosphate and T r i s b u f f e r s a t s e v e r a l pH v a l u e s . I t was t h e r e f o r e d e c i d e d t o d e t e r m i n e whether P_. aerug?nosa 14 degraded o r n i t h i n e - 1 -  14 C to  C-glutamate.  Resting c e l l suspensions  o f both a r g i n i n e grown and g l u c o s e grown c e l l s from the l o g a r i t h m i c phase of growth were i n c u b a t e d i n 50 ml Erlenmeyer f l a s k s  containing  6 ml volumes of a t y p i c a l Warburg r e a c t i o n m i x t u r e b u f f e r e d w i t h p h o s p h a t e , and s t i r r e d a t 30 C as d e s c r i b e d f o r uptake e x p e r i m e n t s . Two ymoles of u n l a b e l led g l u t a m a t e were added t o t r a p and the r e a c t i o n was  14  s t a r t e d by the a d d i t i o n o f 5 ymoles  C-glutamate, (15 y C i )  14 of o r n i t h i n e - 1 -  C.  Two ml samples were removed a t a p p r o p r i a t e  time i n t e r v a l s , added t o 2 ml of 10% t r i c h l o r o a c e t i c a c i d a t 0 C, and t r e a t e d as d e s c r i b e d f o r the t h i n - l a y e r chromatography Warburg s u p e r n a t a n t f l u i d s . c o u l d not become l a b e l l e d  of  I n t e r m e d i a t e s of p u t r e s c i n e d e g r a d a t i o n  i n t h i s e x p e r i m e n t , because the c o n v e r s i o n  14 of o r n i t h i n e - 1 -  C t o p u t r e s c i n e would r e s u l t  i n the l o s s of a l l the  14 l a b e l as  C02.  The r e s u l t s of t h i s experiment a r e summarized i n T a b l e I I . A p p r o x i m a t e l y the same amount o f r a d i o a c t i v i t y was  l o s t from the  s u p e r n a t a n t f l u i d a f t e r 45 minutes i n the presence of g l u c o s e grown c e l l s as was cells.  l o s t a f t e r 20 minutes i n the presence of a r g i n i n e grown  These r e s u l t s  i n d i c a t e d t h a t 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 a p p r o x i m a t e l y t w i c e 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 g l u c o s e grown c e l l s o f P. a e r u g i n o s a .  Growth  substrate  Arginine Rad i o a c t i v i t y  I n c u b a t i o n Time (min)  14 % of t o t a l C recovered in s u p e r n a t a n t f l u i d % l Zof supernatant t a C found a s :  Glucose  20  40  44%  17%  49.6% 20% 3-4% 7-4%  24.3% 35-5% 6.1% 10.3%  45  90  30%  fluid  ornithine a c i d i c and n e u t r a l glutamate proline  compounds  Supernatant f l u i d r a d i o a c t i v i t y unaccounted f o r i n t h i s was p r e s e n t as s e v e r a l ' u n i d e n t i f i a b l e compounds.  75% 10.8% 1.6% 0  table  74.4% 1.0% 4.6% 0  g l u c o s e grown c e l l s .  However, e x a m i n a t i o n o f t h e c o m p o s i t i o n o f t h e  s u p e r n a t a n t f l u i d s a t t h e s e times showed t h a t much more o f t h e o r i g i n a l o r n i t h i n e remained i n the presence o f t h e g l u c o s e grown cel1s. From t h e r e s u l t s o f t h e p r e v i o u s manometric e x p e r i m e n t s , i t was  expected t h a t a r g i n i n e grown c e l l s would have o x i d i z e d t h e  m a j o r i t y o f t h e o r n i t h i n e by hO minutes  ( F i g . 4 B ) , and o n l y h%  of t h e o r i g i n a l l a b e l was recovered as o r n i t h i n e a t t h i s  time.  G l u c o s e grown c e l l s should have o x i d i z e d t h e m a j o r i t y o f t h e o r n i t h i n e by 90 m i n u t e s ; however, 22% o f the o r i g i n a l l a b e l was recovered as o r n i t h i n e , a v a l u e c l o s e l y comparable t o t h e amount of l a b e l recovered a t 20 minutes  from  induced c e l l s .  Thus, i t  appeared t h a t the o x i d a t i o n o f o r n i t h i n e by g l u c o s e grown c e l l s was  s l o w e r under these c o n d i t i o n s than under c o n v e n t i o n a l Warburg  c o n d i t i o n s , perhaps due t o oxygen l i m i t a t i o n .  Moreover,  C-  o r n i t h i n e was s u p p l i e d as a m i x t u r e o f t h e D and L i s o m e r s , and it L  i s p o s s i b l e t h a t t h e D isomer was o x i d i z e d more s l o w l y t h a t t h e isomer. A r g i n i n e grown c e l l s accumulated  much more o f t h e added l a b e l  as g l u t a m a t e , p r o l i n e , and a c i d i c and n e u t r a l d e g r a d a t i o n p r o d u c t s than d i d g l u c o s e grown c e l l s .  These r e s u l t s i n d i c a t e d t h a t the  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 was r a t e l i m i t i n g  i n the  g l u c o s e grown c e l l s and t h a t 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 o t h e r hand, a r g i n i n e grown c e l l s appeared  t o c o n v e r t o r n i t h i n e to glutamate more r a p i d l y than t h e l a t t e r compound c o u l d be d i s s i m i l a t e d . T h i n - l a y e r chromatography and r a d i o a u t o g r a p h y o f t h e b a s i c f r a c t i o n s o f the s u p e r n a t a n t f l u i d s showed two u n i d e n t i f i e d s p o t s containing considerable r a d i o a c t i v i t y  (spots A and B, F i g . 7 ) . I t _  i s p o s s i b l e t h a t t h e s e 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 p r o d u c t s .  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 , f o r m i n g a t l e a s t two p r o d u c t s : one was formed both a t room  temperature  and a t -15 C, and t h e o t h e r , thought t o be a p o l y m e r i z a t i o n p r o d u c t , was formed o n l y a t -15 C. fluids  S i n c e treatment of the s u p e r n a t a n t  i n v o l v e d s t o r a g e a t -20 C, t r e a t m e n t s a t room  temperature,  and s e v e r a l e v a p o r a t i o n s a t 45 - 50 C, i t i s p o s s i b l e t h a t  break-  down p r o d u c t s o f t h i s compound were f o r m e d . The radioautograms  o f t h e t h i n - l a y e r chromatograms o f t h e b a s i c  f r a c t i o n s o f the s u p e r n a t a n t f l u i d s from g l u c o s e grown c e l l s showed low l e v e l s o f 8 t o 10 u n i d e n t i f i e d compounds.  also  These compounds  were a l s o p r e s e n t i n the 20 minute sample from a r g i n i n e grown c e l l s . These b a s i c compounds were not amino a c i d s and were presumed t o be b i o s y n t h e t i c p r o d u c t s or i n t e r m e d i a t e s . From t h e s e e x p e r i m e n t s , i t was concluded t h a t the c o n v e r s i o n of o r n i t h i n e t o glutamate was an important pathway o f a r g i n i n e d e g r a d a t i o n i n P_. a e r u g i n o s a .  The enzymes o f the pathway appeared  ,to be p r e s e n t i n g l u c o s e grown c e l l s , but were induced t o g r e a t e r a c t i v i t y a f t e r growth of the organism  in arginine.  Radioautogram o f a t h i n - l a y e r chromatogram o f the b a 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 w i t h o r n i t h i n e - 1 - ^ C f o r 40 m i n u t e s .  1  proline  glutamate  ornithine • origin  L 2nd SOLVENT  5.  S u c c i n i c semialdehyde dehydrogenase  activity  In an attempt t o a s s e s s the importance of p u t r e s c i n e as an i n t e r m e d i a t e of a r g i n i n e d e g r a d a t i o n i n P_. aerug i n o s a , 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 i n p u t r e s c i n e m e t a b o l i s m , was measured i n 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 g l u c o s e grown c e l l s ( u n i n d u c e d ) .  T h i s enzyme was assayed by l i n k i n g  i t to y-aminobutyrate t r a n s -  a m i n a s e , and thus enzyme a c t i v i t y was a c t u a l l y a measurement o f the am  r a t e o f c o n v e r s i o n of y ~ i n o b u t y r a t e t o s u c c i n a t e . It was p o s s i b l e , however, t h a t the i n d u c t i o n by a r g i n i n e of am  the enzymes d e g r a d i n g Y ~ i n o b u t y r a t e might o c c u r w i t h o u t the d e g r a d a t i o n of p u t r e s c i n e .  G a l e (1940) found t h a t growth of E_. c o l i  in the presence of g l u t a m a t e induced an enzyme which d e c a r b o x y l a t e d glutamate, forming y-aminobutyrate.  I f P_. a e r u g i n o s a possessed  the l a t t e r enzyme, y - a m i n o b u t y r a t e might be formed from g l u t a m a t e r a t h e r 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 o f the r a t e of 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 and the subsequent d e g r a d a t i o n of the l a t t e r compound, r a t h e r than of the o x i d a t i o n of p u t r e s c i n e . semialdehyde dehydrogenase  Therefore, succinic  a c t i v i t y was a l s o measured i n c e l l s  grown w i t h g l u t a m a t e as the s o l e s o u r c e of carbon and  nitrogen.  Nakamura (i960) found t h a t a s t r a i n o f P_. a e r u g i n o s a cont a i n e d two s u c c i n i c semialdehyde dehydrogenases: one l i n k e d t o NADP,  and t h e o t h e r t o NAD.  Unlike  t h e enzyme s t u d i e d  by Padmanabhan  and Tchen (1969) i n an u n i d e n t i f i e d Pseudomonad, t h e NADP l i n k e d s u c c i n i c semialdehyde dehydrogenase o f P_. a e r u g i n o s a appeared t o be i n d u c i b l e that synthesis stitutive  (Table I I I ) - Jakoby and F r e d e r i c k s  o f s u c c i n i c semialdehyde dehydrogenase was con-  i n P_. f 1 u o r e s c e n s , a l t h o u g h i t s a c t i v i t y was i n c r e a s e d  somewhat a f t e r growth  in y-aminobutyrate.  However, t h e s y n t h e s i s  of y - a m i n o b u t y r a t e t r a n s a m i n a s e was i n d u c i b l e  in this  and t h e s p e c i f i c a c t i v i t y o f t h i s enzyme was always of t h e dehydrogenase.  i s possible  organism,  lower than  that  S i n c e t h e assay used i n t h e p r e s e n t study  depended on y - a m i n o b u t y r a t e as a source o f s u c c i n i c it  (1959) found  semialdehyde,  t h a t t h e NADP l i n k e d s u c c i n i c semialdehyde  genase was c o n s t i t u t i v e , but t h a t  dehydro-  i t s a c t i v i t y was l i m i t e d by an  i n d u c i b l e y-aminobutyrate transaminase.  A comparison o f t h e  s p e c i f i c a c t i v i t i e s o f t h e NADP l i n k e d and NAD l i n k e d enzymes shows t h a t y - a m i n o b u t y r a t e t r a n s a m i n a s e was not r a t e l i m i t i n g  in the  assay o f t h e NAD l i n k e d s u c c i n i c semialdehyde dehydrogenase.  The  r a t i o s o f t h e s p e c i f i c a c t i v i t i e s o f t h e NADP l i n k e d t o t h e NAD l i n 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 , t h a t t h e two enzymes may have been c o o r d i n a t e l y  indicating  induced.  Both t h e NAD and t h e NADP l i n k e d enzymes were induced t o t h e h i g h e s t l e v e l s by growth  in putrescine.  P a r t i a l i n d u c t i o n o f both  enzymes was caused by growth w i t h e i t h e r a r g i n i n e or glutamate as  Table  III.  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 o f i n d u c e d and u n i n d u c e d c e l l s o f P. a e r u g i n o s a .  Specif ic a c t i v i t y ' Growth  putrescine  substrate  NADP  NAD  576  84  arginine  82.3  14.5  glutamate  58.5  7«3  glucose  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 a s mymoles o f s u b s t r a t e m i n ^ x mg ^ of p r o t e i n .  oxidized  x  the s o l e s o u r c e o f carbon and n i t r o g e n .  The f a c t t h a t growth  i n g l u t a m a t e induced o n l y 10% o f t h e a c t i v i t y in p u t r e s c i n e  indicated that decarboxylation  pathway o f g l u t a m a t e d e g r a d a t i o n c e l l s contained  significantly  induced by growth was not t h e major  i n P_. a e r u g i n o s a .  A r g i n i n e grown  h i g h e r l e v e l s o f both enzymes than  g l u t a m a t e grown c e l l s , and i t was c o n c l u d e d t h a t t h i s a c t i v i t y was due t o d e g r a d a t i o n catabolism.  increased  o f p u t r e s c i n e formed d u r i n g  G l u c o s e grown c e l l s c o n t a i n e d  arginine  a low c o n s t i t u t i v e  l e v e l o f t h e 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  II.  n e g l i g i b l e a c t i v i t y with  NAD.  Repress ion o f A r g i n ine B i o s y n t h e s i s i n P_. a e r u g i nosa by Exogenous A r g i n i n e  Radioautograms o f t h i n - l a y e r chromatograms of t h e s u p e r n a t a n t fluids  from t h e e x p e r i m e n t s examining t h e d e g r a d a t i o n  14  of o r n i t h i n e - 1 -  14 C showed t h a t g l u c o s e  grown c e l l s s y n t h e s i z e d  C-arginine  from  14 C - o r n i t h i n e , but t h a t a r g i n i n e grown c e l l s d i d n o t ; t h i s t h a t a r g i n i n e b i o s y n t h e s i s was r e p r e s s e d of a r g i n i n e .  indicated  d u r i n g growth i n t h e presence  Repression of the anabolic o r n i t h i n e transcarbamylase  of P_. a e r u g i n o s a and P. f l u o r e s c e n s by a r g i n i n e has been  reported  by S t a l o n et_ aj_. ( 1 9 6 7 a ) and Ramos et_ a l _ . ( 1 9 6 7 ) .  (1966)  found t h a t a r g i n i n e r e p r e s s e d d i f f e r e n t microorganisms.  Ukada  the s y n t h e s i s o f t h i s enzyme i n 1 9  In o r d e r t o c o n f i r m t h a t r e p r e s s i o n o f  t h i s enzyme o c c u r r e d i n 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 f o r 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 a c t i v i t y . in Roux f l a s k s  C e l l s were grown  i n g l u c o s e minimal medium i n the presence  and  absence of 0.05% a r g i n i n e , and were h a r v e s t e d from the l a t e i t h m i c phase of growth a t 14 h o u r s . 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  logar-  The s p e c i f i c a c t i v i t y of  three to four fold 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 w i t h o u t added a r g i n i n e than i n those grown i n the presence o f a r g i n i n e .  III.  The  E f f e c t s of G l u c o s e on the D e g r a d a t i o n of A r g i n i n e  by  P_. aerug ? nosa .  1.  Growth i n a m i x t u r e o f g l u c o s e and  arginine.  P_. a e r u g i n o s a grew i n a medium c o 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 w i t h o u t showing a d i a u x i e e f f e c t , d e s p i t e f a c t t h a t the inoculum had not been adapted  the  t o growth i n the presence  of a r g i n i n e  ( F i g . 8 and 9 ) . The d o u b l i n g time f o r growth i n t h i s  m i x t u r e was  the same as the. d o u b l i n g time i n g l u c o s e a l o n e ; i e . 1.25  hours. the  The growth r a t e was  not a f f e c t e d by p r i o r a d a p t a t i o n of  inoculum t o growth i n the presence of a r g i n i n e .  P_. aerug i nosa  grew somewhat more s l o w l y w i t h 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 d o u b l i n g time of a p p r o x i m a t e l y hours under these c o n d i t i o n s .  The  l a g p e r i o d was a l s o  1.6  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 d u r i n g growth of P_. a e r u g i n o s a in a medium c o 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 i o n s . Symbols: 0, t o t a l r a d i o a c t i v i t y i n the f l a s k ; O . r a d i o a c t i v i t y p r e s e n t i n 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 s u p e r n a t a n t 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 s u p e r n a t a n t fluid radioactivity.  56  H O U R S F t g . 9.  The u t i l i z a t i o n o f C - a r g i n i n e d u r i n g t h e growth o f P_. aerug i nosa i n a m i x t u r e of g l u c o s e and a r g i n i n e . Symbols: a r e the same as i n F i g . 8. Note t h a t 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 t h a t i n F i g . 8.  despite  p r i o r adaptation  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 e r f o r m e d t o d e t e r m i n e  whether glucose growth  of the c e l l s .  and a r g i n i n e were d e g r a d e d c o n c u r r e n t l y  i n the presence o f both s u b s t r a t e s .  o f ammonium s a l t s  C-arginine  Two s i d e - a r m  m i n i m a l medium, each c o n t a i n i n g  0.1 % a r g i n i n e , w e r e  inoculated with  was a d d e d t o o n e f l a s k  glucose  during flasks  0.1% g l u c o s e  and  grown P_. a e r u g i n o s a .  to give a final  concentration  14 o f 0.5 y C i p e r m l , a n d same c o n c e n t r a t i o n .  C-glucose t o the other f l a s k a t the  O p t i c a l d e n s i t y was f o l l o w e d  Summerson c o l o r i m e t e r e q u i p p e d w i t h were t a k e n a t one hour i n t e r v a l s  f l a s k , and were  a  Klett-  a r e d f i l t e r , a n d 1 ml s a m p l e s  and i m m e d i a t e l y f i l t e r e d  t r a n s p o r t s t u d i e s , and washed w i t h f l u i d s were c o l l e c t e d i n a t e s t  using  2 ml o f m e d i u m .  tube placed  as i n  The s u p e r n a t a n t  in the f i l t r a t i o n  l a t e r made up t o 5 ml v o l u m e s a n d a s s a y e d f o r  radioactivity. The  results  During growth  in either labelled  the whole cel1s The  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  increased  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  concomitantly  with  the optical  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  m i n e d by c a l c u l a t i n g density.  the r a t i o o f c e l l  During the f i r s t  three  radioactivity  h o u r s , s l i g h t l y more  t h a n a r g i n i n e was a s s i m i l a t e d p e r o p t i c a l d e n s i t y resulting  i n a c e l l u l a r glucose  8 and 9 .  to arginine  density. was  deter-  to optical glucose  unit of c e l l s ,  r a t i o o f 1.1.  As t h e  p t e r i o d o f t h e most r a p i d g r o w t h was r e a c h e d , t h e 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 g l u c o s e i n c r e a s e d  considerably.  During t h i s  period,  the r a t i o of g l u c o s 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 t o 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 was  1.9-  As the c e l l s e n t e r e d the s t a t i o n a r y phase of g r o w t h ,  they appeared t o 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 and  cells  a l m o s t h a l f of t h e i r a s s i m i l a t e d g l u c o s e ;  arginine,  t h u s , the  ratio  of g l u c o s 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 t o a r g i n i n e o p t i c a l d e n s i t y u n i t of c e l l s 1.2,  i n the e a r l y s t a t i o n a r y phase  s i m i l a r t o t h a t of c e l l s p r i o r t o the The  per was  i n i t i a t i o n of g r o w t h .  d e c r e a s e i n 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 s u b s t r a t e carbon l o s t as 14 d i o x i d e p r o d u c t i o n from g l u c o s e a t a time when an and  increase  c o n t i n u e d a t an  phase was  reached.  hours,  i n c r e a s i n g r a t e u n t i l one  hour b e f o r e s t a t i o n a r y  D e t e c t a b l e amounts of CC^ 14  d i d not appear to  h o u r s , a f t e r which time CC^  was  hour b e f o r e s t a t i o n a r y phase was  were degraded t o  apparent a f t e r two  Carbon  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 ,  l o s t from the medium c o n t a i n i n g  maximum i n c r e a s e  C was  CO^.  C-arginine  f o r the f i r s t  four  produced r a p i d l y u n t i l h a l f reached.  During the p e r i o d  i n o p t i c a l d e n s i t y , both a r g i n i n e and  be  an of  glucose  CO^.  14 than from C - g l u c o s e . However, the c e l l s d i d not grow t o as high A g r e a t e r p r o p o r t i o n of the l a b e l was l o s t from C-arginine 14  an o p t i c a l d e n s i t y i n the f l a s k c o n t a i n i n g l a b e l l e d 1  resulting  i n the a s s i m i l a t i o n of o n l y 21% of the 1  as compared t o 38% o f the  arginine,  k C-arginine  k C - g l u c o s e , a l t h o u g h the amount of  each s u b s t r a t 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 was  a p p r o x i m a t e l y the same.  a r g i n i n e was  cells  S i n c e a s m a l l e r p r o p o r t i o n of the  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^.  The e f f e c t of g l u c o s e on the d e g r a d a t i o n of a r g i n i n e  2.  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 g l u c o s e grown c e l l s from the s t a t i o n a r y phase of growth was and absence of g l u c o s e ( F i g . 10). c u r v e was  observed  harvested  compared i n the  presence  No break i n the oxygen uptake  when both s u b s t r a t e s were p r e s e n t , i n d i c a t i n g  t h a t g l u c o s e 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 p r e s e n t i n the s u p e r n a t a n t f l u i d measured a t v a r i o u s times d u r i n g oxygen u p t a k e . amount o f ammonia d e t e c t e d was  The maximum  i n the s u p e r n a t a n t f l u i d a t any  e q u i v a l e n t t o 2.k ymoles per ymole of a r g i n i n e  added.  was  time  originally  Because a r g i n i n e c o n t a i n s k yatoms of n i t r o g e n per y m o l e ,  the ammonia r e l e a s e d accounted  f o r o n l y 60% of the a r g i n i n e n i t r o g e n .  P r e s u m a b l y , the remaining k0% was  assimilated.  C e l l s which had been grown i n g l u c o s e minimal medium supplemented  60  30  6 0  9 0  M I N U T E S  Fig.  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 m i x t u r e of g l u c o s e and a r g i n i n e , A . Glucose grown c e l l s [ a p p r o x i m a t e l y 5 mg of c e l l s (dry w e i g h t ) / c u p ] were u s e d . Each cup c o n t a i n e d 2.5 ymoles of each s u b s t r a t e and T r i s buffer.  w i t h 0.1%  a r g i n i n e h a d r e l e a s e d t h i s maximum amount o f ammonia  a f t e r 30 m i n u t e s o f i n c u b a t i o n i i i a W a r b u r g c u p w i t h a r g i n i n e a s the o n l y s u b s t r a t e . time, i t i s l i k e l y  S i n c e o x y g e n u p t a k e was n o t c o m p l e t e by that, although  g r e a t e r than  this  50% o f t h e a r g i n i n e  ammonia h a d b e e n r e l e a s e d , ammonia p r o d u c t i o n was n o t 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 m a s k e d by a s s i m i l a t i o n .  Cells  O n l y 67%  w i t h o u t a d d e d a r g i n i n e r e l e a s e d ammonia more s l o w l y . o f t h e maximum v a l u e was r e a c h e d by 30 m i n u t e s , minutes. taining  The  glucose  induced  cells  induced  cells.  The by  of arginine  a t 30  o x i d a t i o n o f 2.5  The c h a n g e s  oxidized,  l o w e r c o n c e n t r a t i o n o f ammonia  i n the  minutes. ]imoles o f ^ C - a r g i n i n e  u n i n d u c e d c e l l s was e x a m i n e d  glucose.  therefore  i n the rate of degradation  i n c r e a s e d when g l u c o s e was b e i n g  in a slightly fluid  minutes.  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  supernatant  i n t h e Warburg c u p ,  d u r i n g a r g i n i n e o x i d a t i o n may  decrease  con-  rate of arginine degradation.  r e l e a s e d 83% o f t h e maximum amount by 30  presence o f glucose  resulting  a more r a p i d  g l u c o s e and a r g i n i n e w e r e p r e s e n t  have caused a s l i g h t by  and 92% by 60  T h u s , t h e a d d i t i o n o f a r g i n i n e t o a g r o w t h medium  When b o t h induced  grown  (1 u C i / i i m o l e )  i n t h e p r e s e n c e and a b s e n c e o f  i n the r a d i o a c t i v i t y  of the supernatant  f l u i d , w h o l e c e l l s , a n d C0^ i n t h e a b s e n c e o f a d d e d g l u c o s e a r e shown i n F i g u r e 11. were very s i m i l a r .  The v a l u e s o b t a i n e d when g l u c o s e was R a d i o a c t i v i t y was  lost  present  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 d u r i n g the i n c u b a t i o n o f P.\aeruginosa w?th C - a r g i n i n e under Warburg c o n d i t i o n s . Glucose grown c e l l s [ a p p r o x i m a t e l y 5 mg o f c e l l s (dry w e i g h t ) / c u p ] and T r i s b u f f e r were u s e d . Each cup c o n t a i n e d 2.5 ymoles o f C-arginine (specific a c t i v i t y = 1 yCi/ymole). Symbols: 0, C 0 ; • , s u p e r n a t a n t 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 g i v e n o f f as ^ C 0 was determined by measuring the r a d i o a c t i v i t y p r e s e n t i n the c e n t e r w e l l o f the Warburg c u p . 2  2  f l u i d throughout glucose.  the p e r i o d of o x i d a t i o n , d e s p i t e the presence  M o r e o v e r , 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  and the e v o l u t i o n of  CO^  a d d i t i o n of g l u c o s e . was  i n t o the c e l l s  were not a p p r e c i a b l y a l t e r e d by the  In both c a s e s , the r a t e of  slow f o r the f i r s t  of  14 CO^  evolution  20 m i n u t e s , a f t e r which time the amount of  14 CO2 r e l e a s e d i n c r e a s e d r a p i d l y . of  T h i r t y - f i v e to forty  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  first  percent  a s s i m i l a t e d w i t h i n the  10 m i n u t e s , and a s s i m i l a t i o n then proceeded a t a slower  r e a c h i n g the maximum v a l u e by 60 m i n u t e s . assimilated  Thus, a r g i n i n e  i n t o c e l 1 u l a r m a t e r i a l d u r i n g the i n i t i a l  o x i d a t i o n and was  s u b s e q u e n t l y degraded to CO^.  were s i m i l a r to those o b t a i n e d  i n the growth  T h i n - l a y e r chromatography and  rate,  was  s t a g e s of  These r e s u l t s  experiments.  r a d i o a u t o g r a p h y of the super-  n a t a n t f l u i d s from the 30 minute samples showed t h a t , a l t h o u g h the t o t a l r a d i o a c t i v i t y of the two samples was t h e v a r i o u s i n t e r m e d i a t e s were present IV).  e s s e n t i a l l y the same,  i n d i f f e r e n t amounts (Table  Much more c i t r u l l i n e and o r n i t h i n e were present  to which g l u c o s e had been added.  a c t i v i t y was present  cup  At a l l t i m e s , the p u t r e s c i n e  c o n t e n t o f the s u p e r n a t a n t f l u i d s was absence of g l u c o s e .  i n the  s l i g h t l y g r e a t e r i n the  By 90 m i n u t e s , the m a j o r i t y of the r a d i o -  p r e s e n t as glutamate and p u t r e s c i n e , w i t h some a l s o  i n unknown s p o t s 1 and 2.  Some o r n i t h i n e remained i n the  sample i n which g l u c o s e had been p r e s e n t .  Thus, i t i s p o s s i b l e  t h a t the a d d i t i o n of g l u c o s e d i d cause a s l i g h t d e c r e a s e r a t e of o r n i t h i n e d e g r a d a t i o n .  i n the  T a b l e IV.  C o m p o s i t i o n of the s u p e r n a t a n t f l u i d s a f t e r o x i d a t i o n of C - a r g i n i n e by P_. a e r u g i n o s a i n the presence and 3 absence o f g l u c o s e .  % of s u p e r n a t a n t  radioactivity'  3  Compound 14  arginine  . C-arginine  14  12 C-arginine +  39.2  34.0  citrul1ine  5-9  13.8  orn i th ine  5-3  15-2  p u t r e s c i ne  3.8  2.5  glutamate  6.7  7.1  1  1.0  1 .2  2  1.4  2.5  unknown  C-glucose  5 mg of c e l l s (dry weight) were incubated f o r 30 minutes under c o n v e n t i o n a l Warburg c o n d i t i o n s w i t h 0.05 M T r i s b u f f e r and 2.5 ymoles o f 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 y C i / y m o l e . the was  s u p e r n a t a n t r a d i o a c t i v i t y unaccounted f o r i n t h i s p r e s e n t i n a number of u n i d e n t i f i e d compounds.  table  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 c e l l s 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 a f f e c t the percentage d i s t r i b u t i o n of glucose between the various c e l l f r a c t i o n s . ethanol  More glucose was assimilated into the acid  soluble f r a c t i o n and less into the hot t r i c h l o r o a c e t i c acid  insoluble f r a c t i o n 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 e f f e c t 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 s o l e l y 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 s l i g h t 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 c e l l fractions was  ,  T a b l e V.  The e f f e c t o f a r g i n i n e and ammonium ions on the a s s i m i l a t i o 3n ^ C - g l u c o s e by a r e s t i n g c e l l s u s p e n s i o n o f P. a e r u g i n o s a .  % total Fraction  14 C-glucose  of  radioactivity  14 C-glucose + a r g i n i n e  14 + C-glucose + NH^  CO,  59.5%  51.9%  54.5%  supernatant f l u i d  15-7%  14.9%  13-9%  cel 1 s  27-7%  33.4%  30.8%  % cell  cold acid  radioactivity  trichloroacetic soluble  9.3%  9.4%  10.3%  a c i d ethanol s o l u b l e  26.3%  30.8%  28.1%  hot t r i c h l o r o a c e t i c acid soluble  13.6%  14.3%  12.4%  hot t r i c h l o r o a c e t i c acid precipitate  50.8%  45.5%  49.0%  a  5 mg o f c e l l s (dry w e i g h t ) were incubated f o r 90 minutes under c o n v e n t i o n a l Warburg c o n d i t i o n s w i t h 0.05 M T r i s b u f f e r . 2.5 ymoles o f each s u b s t r a t e were added. The s p e c i f i c a c t i v i t y o f the ^ C - g l u c o s e was 1 y C i / y m o l e .  Table V I .  A s s i m i l a t i o n of C - a r g i n i n e by a r e s t i n g c e l l s u s p e n s i o n3 of P_. aerug inosa i n the presence and absence of g l u c o s e .  % total  radioactivity  F r a c t ion 14  14 C-arginine  co  12 C-arginine +  55.1%  60.6%  supernatant f l u i d  18.8%  17.0%  cel 1 s  26.1%  24.2%  2  % cell  cold acid  radioactivity  trichloroacetic soluble  31.5%  35.4%  acid ethanol s o l u b l e  19.1%  13-3%  hot t r i c h l o r o a c e t i c acid soluble  12.5%  6.8%  hot t r i c h l o r o a c e t i c acid precipitate  36.9%  44.5%  E x p e r i m e n t a l c o n d i t i o n s were the a c t i v i t y of the 1 ^ C - a r g i n i n e was  C-glucose  same as g i v e n in T a b l e V. 1 uCi/ymole.  The  specific  n o t i c e a b l y a f f e c t e d by t h e presence o f g l u c o s e .  When g l u c o s e was  present, the proportion  into the cold  of arginine  incorporated  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 and  the proportion  incorporated  i n t o t h e a c i d e t h a n o l 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 d e c r e a s e d . 35% o f t h e r a d i o a c t i v i t y  increased,  G r e a t e r than  i n t h e a c i d e t h a n o l s o l u b l e f r a c t i o n was  lipid. T h u s , i n t h e presence o f both a r g i n i n e and g l u c o s e , P_. a e r u g i n o s a preferentially and  arginine  incorporated  g l u c o s e i n t o l i p i d and n u c l e i c a c i d s ,  i n t o p r o t e i n and t h e 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.  T h i s p a t t e r n o f u t i l i z a t i o n would prove economical f o r t h e c e l l , s i n c e g l u c o s e can be c o n v e r t e d 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 t o i n t e r m e d i a t e s  o f t h e TCA c y c l e .  On t h e o t h e r hand, many o f t h e amino a c i d s used i n p r o t e i n and  synthesis,  t h e amino a c i d s and o t h e r b a s i c substances p r e s e n t i n t h e c e l l  p o o l , may be o b t a i n e d more r e a d i l y from a r g i n i n e than from g l u c o s e .  IV.  D e g r a d a t i o n o f A r g i n i n e by P_. p u t i d a and P_. f l u o r e s c e n s  Kay  (1968) found t h a t £_. f l u o r e s c e n s  d i d not c a t a b o l i z e  a r g i n i n e a c t i v e l y , and t h a t t h e m a j o r i t y o f t h e r a d i o a c t i v i t y i n corporated into the c e l l s during incorporated  into protein.  arginine transport  s t u d i e s was  P_. put i d a , however, c a t a b o l i z e d 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 was  a s s i m i l a t i o n of  C-arginine  by these organisms  t h e r e f o r e s t u d i e d and compared t o t h a t by P_.  aeruginosa..  Manometric s t u d i e s showed t h a t 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 o n l y s l i g h t l y , t a k i n g up o n l y 1 ymole of oxygen in the presence of 2.5 ymoles of a r g i n i n e ( F i g . 1 2 ) .  P_. p u t i d a  demonstrated a g r e a t e r 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 r a t e and  f i n a l e x t e n t of o x i d a t i o n were much lower  than w i t h P_. a e r u g i n o s a ( F i g . 1 2 ) . The a s s i m i l a t i o n p a t t e r n s a f t e r i n c u b a t i o n of these t h r e e 1 ij organisms i n the presence of 2.5 ymoles of C - a r g i n i n e (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 - a r g i n i n e was  m e t a b o l i z e d to CO^  assimilated into c e l l u l a r material  and  by P_. aerug i nosa than by P_. put i d a , and  r a d i o a c t i v i t y remained i n the s u p e r n a t a n t f l u i d .  The  radioactivity  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 in t h e s e two o r g a n i s m s .  less  similar  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  i n the s u p e r n a t a n t f l u i d a f t e r i n c u b a t i o n of P_. f l u o r e s c e n s  with  14 C-arginine.  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 w e l l w i t h the low oxygen uptake  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  the c e l l s of t h i s organism was m a t e r i a l but remained i n the f  obtained  not  incorporated  intracellular pool.  are d i f f e r e n t from those o b t a i n e d  by Kay  into  into c e l l u l a r The  latter  results  (1968) ; however, r e s t i n g  . 12.  O x i d a t i o n of a r g i n i n e by g l u c o s e grown c e l l s of IP. a e r u g i n o s a , 0; P_. f 1 u o r e s c e n s , • ; and f_. p u t i d a , A . C e l l c o n c e n t r a t i o n s were 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 weight)/ml and T r i s b u f f e r was u s e d .  Table V l l .  Comparison of the a s s i m i l a t i o n of P. a e r u g i n o s a , P. f l u o r e s c e n s , and  % total  14  C - a r g i n i n e a by P. p u t i d a .  radioactivity  F r a c t ion P. aerug inosa  P. put ida  P.  fluorescens  46.7%  27.4%  8.8%  37.6%  61.8%  85.5%  cold t r i c h l o r o a c e t i c acid soluble  8.9%  8.4%  5.5%  cold t r i c h l o r o a c e t i c acid insoluble  10.3%  2.4%  0.2%  co  2  supernatant f l u i d  a  5 mg of c e l l s (dry w e i g h t ) were incubated f o r 100 minutes under c o n v e n t i o n a l Warburg c o n d i t i o n s w i t h 0.05M T r i s b u f f e r and 2.5 ymoles (2.5 y C i ) 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 h i g h s u b s t r a t e concentrations  were used  i n t h e s e e x p e r i m e n t s , 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 concentrations. f l u o r e s c e n s was confirming  The amount of r a d i o a c t i v i t y  low s u b s t r a t e  i n the pool of P_.  lower than t h a t i n P_. aerug i nosa and P_. put i d a ,  the r e s u l t s of Kay (1968)•  T h i n - l a y e r chromatography  and r a d i o a u t o g r a p h y of the s u p e r n a t a n t  f l u i d s showed t h a t a l l the r a d i o a c t i v i t y of the s u p e r n a t a n t f l u i d from P_. put ida was p r e s e n t as p u t r e s c i n e .  T h u s , 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 , s i n c e the  intra-  c e l l u l a r pool formed by t h e s e t h r e e organisms i n the presence o f a r g i n i n e has been shown to c o n s i s t of p u t r e s c i n e  (Kay, 1968).  In a d d i t i o n t o 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  citrulline  were found i n the s u p e r n a t a n t 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 t o c o n s t i t u t i v e l y c o n v e r t a r g i n i n e t o p u t r e s c i n e , presumably v i a c i t r u l l i n e ornithine. fluorescens.  IP. p u t i d a was more a c t i v e i n t h i s r e s p e c t  and  than P_.  S t a n i e r , P a l l e r o n i and Doudoroff (1966) d e s c r i b e d  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 t h a t P_. p u t i d a c o u l d f u r t h e r degrade a r g i n i n e , whereas P_. f l u o r e s c e n s almost i n a c t i v e i n t h i s r e s p e c t .  P_. a e r u g i n o s a was  was  the organism  *most a c t i v e i n the d e g r a d a t i o n of a r g i n i n e , and a l t h o u g h p u t r e s c i n e  was  t h e major r a d i o a c t i v e compound found i n t h e s u p e r n a t a n t f l u i d  t h i s o r g a n i s m , r a d i o a c t i v e g l u t a m a t e was a l s o p r e s e n t .  with  It i s  t h e r e f o r e p o s s i b l e t h a t t h e slow r a t e o f oxygen uptake by P_. p u t i d a was  due t o t h e f u r t h e r d e g r a d a t i o n o f p u t r e s c i n e , whereas t h e  f a s t e r 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 by P. a e r u g i n o s a was due t o the a d d i t i o n a l a b i l i t y o f t h i s organism t o degrade o r n i t h i n e v i a glutamate.  V.  Uptake o f B a s i c Amino A c i d s and Polyamines by P_. a e r u g i n o s a  1.  I n d u c t i o n o f uptake  de Hauwer, L a v a l l e , and Wiame (1964) found t h a t t h e r a t e o f t r a n s p o r t o f a r g i n i n e by B_. s u b t i 1 i s was g r e a t l y i n c r e a s e d growth i n t h e presence o f a r g i n i n e .  Growth o f P_. a e r u g i n o s a  a r g i n i n e as t h e s o l e s o u r c e o f carbon and n i t r o g e n five-fold  increase  after with  resulted in a  i n 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 , t h e r a t e s o f o r n i t h i n e and l y s i n e t r a n s p o r t were increased  seven-fold;  transport, three-fold. not o n l y  t h e r a t e s o f c i t r u l l i n e and p u t r e s c i n e T h u s , growth i n a r g i n i n e induced an  increase  i n t h e r a t e o f uptake o f a r g i n i n e , but a l s o i n t h a t o f  o t h e r b a s i c amino a c i d s and o f p u t r e s c i n e . P_. a e r u g i n o s a t r a n s p o r t e d the o t h e r b a s i c amino a c i d s .  c i t r u l l i n e a t a much lower r a t e than I t i s p o s s i b l e , however, t h a t t h e r a t e  of c i t r u l l i n e uptake as measured i n these e x p e r i m e n t s was lower than t h e a c t u a l  r a t e , because t h e l a b e l was i n t h e u r e i d o  carbon  Table V I I I .  Induction of transport  Rate o f t r a n s p o r t  Substrate  Growth medium  glucose  arginine  putrescine  10.0  52.1  4.9  orn i t h i ne  5-5  41.5  4.9  lysine  5.6  38.2  c i t r u 1 1 i ne  1.3  3.4  p u t r e s c i ne  11.0  31.5  a r g i n i ne  y-ami n o b u t y r a t e  0  0  b b 37.1 2.04  e x p r e s s e d as mym x min x mg o f c e l l s (dry w e i g h t ) . The r e a c t i o n m i x t u r e c o n t a i n e d 0.135 mg o f c e l l s ( d r y w e i g h t ) / m l and s u b s t r a t e s were added t o a f i n a l c o n c e n t r a t i o n o f 2.5 x 10~5M. A l l r a d i o a c t i v e s u b s t r a t e s had a s p e c i f i c a c t i v i t y of 2 y C i / y m o l e . not  tested  of t h e c i t r u l l i n e , and would be l o s t as of c i t r u l l i n e t o o r n i t h i n e .  CO^, upon the c o n v e r s i o n  S i n c e a r g i n i n e grown c e l l s  c i t r u l l i n e more r a p i d l y than g l u c o s e grown c e l l s  (Table  oxidized I),  i t is  l i k e l y t h a t t h e d i s c r e p a n c y between the a c t u a l r a t e o f c i t r u l l i n e t r a n s p o r t and t h e observed r a t e would be g r e a t e r  i n induced c e l l s .  The low r a t e s o f c i t r u l l i n e t r a n s p o r t may have been the  limiting  f a c t o r s r e s p o n s i b l e f o r the low r a t e s o f c i t r u l l i n e o x i d a t i o n , observed i n the r e s p i r o m e t r y  studies.  P_. aerug?nosa t r a n s p o r t e d  p u t r e s c i n e v e r y r a p i d l y , and the  r a t e o f t r a n s p o r t was induced t o a h i g h e r l e v e l a f t e r growth i n 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 r a t e o f t r a n s p o r t o f a l l  s u b s t r a t e s by p u t r e s c i n e grown c e l l s may be even more r a p i d than t h a t o b s e r v e d , because the c e l l s were c l u m p e d , a phenomenon t h a t was shown t o reduce d r a s t i c a l l y a r g i n i n e grown c e l l s . in a g r e a t e r  the r a t e o f a r g i n i n e t r a n s p o r t by  T h u s , growth i n p u t r e s c i n e p r o b a b l y r e s u l t s  i n d u c t i o n o f p u t r e s c i n e uptake than does growth i n  arginine. N e i t h e r g l u c o s e grown c e l l s nor a r g i n i n e grown c e l l s the c a p a c i t y t o t r a n s p o r t y - a m i n o b u t y r a t e . was t r a n s p o r t e d  s l o w l y by p u t r e s c i n e  However, t h i s  grown c e l l s .  possessed substrate  2.  K i n e t i c s of arginine  A study of was  c a r r i e d out  the by  uptake at v a r y i n g  kinetics  uptake  o f a r g i n i n e u p t a k e by  determining substrate  the  initial  rates of  concentrations  d i d not  r a t h e r , the  rate of a r g i n i n e uptake continued to  x 10  substrate  ^M  Glucose  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 ,  concentrations  p l o t demonstrated the 2.2  C-arginine  ( F i g . 13).  grown c e l l s  increasing  P_. a e r u g i n o s a  presence of  which functioned  at  ( F i g . 13A).  two  low  permeases:  increase A  but,  with  Michaelis-Menton one  with  a Km  arginine concentrations,  of  and  -6 one  with  a Km  centrations that  the  followed of  two  greater  low  x 10  M which functioned 5  t h a n 1.3  x 10~ M  similar kinetics.  kinetic  affinity  have been s t u d i e d  Mutants d e f e c t i v e  (Ames and  Roth, 1968).  ( 1 9 7 0 ) showed t h a t S t r e p t o c o c c u s components f o r the i s o l a t e d a mutant amino a c i d s  (Utech,  R e i d and  i n the  Holden, 1970).  i n S_.  transport  R e i d , U t e c h and  affinity  existence  for histidine,  faecal i s possessed  high  found  a e r u g i nosa  i n each o f the  t r a n s p o r t o f g l u t a m a t e and l a c k i n g the  P.  transport of h i s t i d i n e  permease s p e c i f i c  con-  (1968)  l e u c i n e by  permease w h i c h a l s o f u n c t i o n e d  the a r o m a t i c amino a c i d s .  Kay  (1964) d e m o n s t r a t e d the  components f o r the a high  affinity  Ames  at a r g i n i n e  ( F i g . 14).  u p t a k e o f g l u t a m a t e , p r o l i n e , and  typhimur?urn: a  o f 5.4  two  of  permeases  Holden kinetic  a s p a r t a t e , and  permease f o r  and  have  these  Fig.  13.  K i n e t i c s o f a r g i n i n e uptake by P. a e r u g i n o s a . A, g l u c o s e grown c e l l s ; Note t h a t the c o o r d i n a t e s have been changed i n B.  B, a r g i n i n e grown  cells.  77  g . 14.  K i n e t i c s o f a r g i n i n e uptake by g l u c o s e 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  a r g i n i n e grown c e l l s w e r e t o o r a p i d cel1 concentration  the  9 yg o f c e l l s  of arginine  t o be m e a s u r e d , and  by  therefore  decreased 15-fold t o approximately  was  (dry weight)/ml o f the f i n a l  The u p t a k e o f a r g i n i n e by t h e s e c e l l s  reaction  followed  mixture.  normal  saturation  kinetics  ( F i g . 1 3 B ) , and they d i d n o t a p p e a r t o p o s s e s s a low  affinity  permease a t h i g h s u b s t r a t e  is p o s s i b l e that  by  concentration  w h i c h was  not  used, or  that  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  induced c e l l s .  cells  The h i g h a f f i n i t y  had a Km o f a p p r o x i m a t e l y  to the value  obtained with  permease o f a r g i n i n e  1.7 x 10 ^M, w h i c h was  g l u c o s e grown c e l l s .  v e l o c i t y maximum (V - mymoles x mirt * x mg ' max u p t a k e by a r g i n i n e grown c e l l s o f g l u c o s e grown c e l l s a r g i n i n e caused but  However, i t  t h e a c t i v i t y o f t h e l a t t e r permease c o u l d  be d e t e c t e d a t t h e low c e l l this  concentrations.  was  increased  Inhibition of  a.  similar  However, the  ^) o f a r g i n i n e  was a p p r o x i m a t e l y 55, w h e r e a s  10.  Thus, growth o f the c e l l s  synthesis  d i d not induce the synthesis  3.  grown  of the high a f f i n i t y  o f a new  that  in permease,  permease.  transport  B a s i c amino  acids  The d i f f e r e n c e s  i n the degrees t o which the rates o f  transport o f the d i f f e r e n t substrates  were  induced  indicated  that  80  several  uptake systems were i n v o l v e d .  Kay  (1968) o b t a i n e d d a t a  which i n d i c a t e d t h a t P.. a e r u g i n o s a possessed two f o r the b a s i c amino a c i d s : a r g i n i n e and bas  had  h i s t i d i n e , i n o r d e r of  of t h e s e two  1, which was  a lower a f f i n i t y f o r o r n i t h i n e , and  2, which t r a n s p o r t e d  Inhibition  permease bas  transport  specific permease  lysine, arginine, ornithine, citrulline  the most e f f e c t i v e  presence  i n h i b i t o r of a r g i n i n e uptake in  uninduced c e l l s (Table I X ) .  Glucose grown c e l l s  appeared to t r a n s p o r t a r g i n i n e m a i n l y by the permease bas i n h i b i t i o n e x e r t e d by  c i t r u l l i n e and  l y s i n e was  h i s t i d i n e was  in a r g i n i n e resulted  low, and  negligible.  i n a r g i n i n e a l s o caused an  t r a n s p o r t of l y s i n e and  citrulline  1,  that exerted  since  by  However, growth of  i n a marked i n c r e a s e  i n h i b i t i o n e x e r t e d by these compounds. aerug inosa  and  affinity.  s t u d i e s r e p o r t e d here f u r t h e r c o n f i r m e d the  both induced and  cells  for  b a s i c amino a c i d uptake systems i n t h i s o r g a n i s m .  O r n i t h i n e was  the  systems  i n the degree of  Because growth of increase  the  P_.  in the r a t e s  (Table V I M ) ,  of  the r a t i o of  the a c t i v i t y of the g e n e r a l permease (bas 2) t o the s p e c i f i c permease (bas  1) must have i n c r e a s e d  O r n i t h i n e uptake was induced and inhibitorof  after  induction.  completely inhibited  uninduced c e l l s (Table X ) .  by a r g i n i n e  L y s i n e was  in both  a l s o a potent  o r n i t h i n e uptake w i t h both types of c e l l s , being  almost c o m p l e t e l y i n h i b i t o r y w i t h  induced c e l l s .  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  C i t r u l l i n e and  i n h i b i t o r s of o r n i t h i n e  Table IX.  Inhibition  of a r g i n i n e  transport.  %  3  inhibition  Growth s u b s t r a t e  Inhibitor  glucose  arginine  histidine  10  20  citrul1ine  11  25  lysine  20  52  orn i t h i n e  58  61  100  100  arginine  The r e a c t i o n m i x t u r e c o n t a i n e d 0.135 mg o f c e l l s ( d r y weight)/ml and I ^ C - a r g i n i n e 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 2.1 x 10~5M ( s p e c i f i c a c t i v i t y = 2.4 y C i / y m o l e ) . U n l a b e l l e d i n h i b i t o r s were added immediately p r i o r t o the a d d i t i o n o f the l a b e l l e d s u b s t r a t e t o a f i n a l c o n c e n t r a t i o n o f 2.5 x 10 M. -I>  T a b l e X.  Inhibition  of ornithine transport.  %  inhibition  Growth s u b s t r a t e  1nh? b i t o r  glucose  arginine  7  12  histidine  48  56  citrul1ine  69  72  lysine  84  97  arg i n i ne  100  99  orn i t h i n e  100  100  putrescine  C o n d i t i o n s were the same as f o r T a b l e IX w i t h the e x c e p t i o n t h a t ^ C - o5r n i t h i n e was added 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 o f 2.5 x 10~ M ( s p e c i f i c a c t i v i t y = 2 u C i / u m o l e ) .  u p t a k e , w i t h c i t r u l l i n e e x e r t i n g a somewhat s t r o n g e r than h i s t i d i n e .  These r e s u l t s  inhibition  i n d i c a t e d t h a t o r n i t h i n e was t r a n s -  p o r t e d by both permeases i n both types o f c e l l s ; c o n f i r m e d the i n c r e a s e i n t h e a c t i v i t y (bas 2) a f t e r growth o f the c e l l s  and they a l s o  o f t h e g e n e r a l permease  in arginine.  I t i s i m p o r t a n t t o note t h a t the c o n c e n t r a t i o n s o f 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 g r e a t e r than 2 x 10 ^M, and thus the low a f f i n i t y permease may have p l a y e d an important r o l e i n t r a n s p o r t .  b.  Polyamines  Tabor and Tabor (1966) found t h a t E. c o l i t r a n s p o r t e d p u t r e s c i n e very r a p i d l y , and spermine and s p e r m i d i n e more s l o w l y . They h y p o t h e s i z e d the e x i s t e n c e o f a t l e a s t two systems o f polyamine uptake  i n t h i s o r g a n i s m , one h a v i n g a h i g h a f f i n i t y f o r p u t r e s c i n e .  P u t r e s c i n e uptake  iti P_. a e r u g i n o s a was i n h i b i t e d o n l y 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 s p e r m i n e , and v e r y s t r o n g l y by s p e r m i d i n e  (Table X I ) .  a g e n e r a l polyamine  Thus, P_. a e r u g i n o s a p r o b a b l y  possesses  uptake system w i t h a h i g h a f f i n i t y f o r p u t r e s c i n e  and s p e r m i d i n e , and a lower a f f i n i t y f o r s p e r m i n e . that a p o r t i o n o f the i n h i b i t i o n  It is possible  e x e r t e d by spermine and s p e r m i d i n e  was due t o a d s o r p t i o n o f the l a t t e r compounds t o t h e c e l l s u r f a c e , a phenomenon t h a t was observed  t o o c c u r i n E_. c o l i by Tabor and  Table X I .  Inhibition of putrescine transport.  %  . Inhibitor  Inhibition  Growth s u b s t r a t e  Glucose  Putrescine  arginine  21%.  25%  orn i t h i ne  20%  21%  -  Y ami n o b u t y r a t e  _b  0  spermi ne  48%  ko%  spermidine  83%  60%  p u t r e s c i ne  100%  100%  a  T h e r e a c t i o n m i x t u r e c o n t a i n e d a p p r o x i m a t e l y 27 ug o f c e l l s (dry w e i g h t ) / m l and ^ C - p u t r e s c i n e 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 2.5 x 10"5M ( s p e c i f i c a c t i v i t y =? 2 y C i / u m o l e ) . Inhibitors were added immediately p r i o r t o the a d d i t i o n o f the l a b e l l e d s u b s t r a t e t o a f i n a l c o n c e n t r a t i o n o f 2.5 x 10~^M. Not t e s t e d .  Tabor ( 1 9 6 6 ) . may  The  i n h i b i t i o n e x e r t e d by a r g i n i n e and  have been n o n - c o m p e t e t i v e , because p u t r e s c i n e  uptake o n l y v e r y s l i g h t l y  (Table X ) .  ornithine  inhibited ornithine  Thus, i t i s u n l i k e l y t h a t  s i g n i f i c a n t p u t r e s c i n e uptake o c c u r r s v i a the b a s i c amino a c i d transport systems.  Growth of the c e l l s  in a s l i g h t d e c r e a s e in the degree of and  no s i g n i f i c a n t a l t e r a t i o n  in putrescine  i n h i b i t i o n e x e r t e d by  i n . t h e i n h i b i t i o n e x e r t e d by  of the o t h e r compounds, i n d i c a t i n g t h a t no new had  been i n d u c e d .  The  resulted  transport  spermidine any  systems  p o s s i b i l i t y t h a t growth i n spermine or  s p e r m i d i n e might induce t r a n s p o r t systems s p e c i f i c f o r these polyamines was  k.  Pool  a.  not  formation  Arginine  The was  investigated.  e f f e c t of  investigated using  C-arginine  p a t t e r n s of pool f o r m a t i o n In g l u c o s e grown c e l l s , the protein followed (Fig.15). the f i r s t  a  Thus, the  i n d u c t i o n on pool f o r m a t i o n as s u b s t r a t e .  The  d i f f e r e d i n induced and i n c o r p o r a t i o n of  and  stability  initial  uninduced  C-arginine  cells.  into  time c o u r s e s i m i l a r t o t h a t o f the t o t a l uptake i n t r a c e l l u l a r pool i n c r e a s e d  in size  during  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  t o c o n t i n u e d p r o t e i n s y n t h e s i s , and  then remained  T  50-  M I N U T E S  Fig.  15.  Formation of an i n t r a c e l l u l a r pool u s i n g g l u c o s e 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 o n c e n t r a t i o n was 0.135 mg of c e l l s (dry w e i g h t ) / m l , and the a r g i n i n e c o n c e n t r a t i o n was 2.1 x 10"5M ( s p e c i f i c a c t i v i t y = 2.4 y C i / y m o l e ) .  s t a b l e f o r t h e n e x t 40 m i n u t e s .  A r g i n i n e grown c e l l s , on  o t h e r h a n d , d e m o n s t r a t e d a l a g o f 90 s e c o n d s  the  b e f o r e t h e maximal  14 rate of (Fig.  i n c o r p o r a t i o n of  16).  This  C-arginine  lag presumably  i n t o p r o t e i n was  represented d i l u t i o n  p o o l o f u n l a b e l l e d a r g i n i n e , w h i c h had g r o w t h and  had  reached  through a  been e s t a b l i s h e d  n o t been removed by w a s h i n g .  during  T h u s , a f t e r 90  seconds  14 of  incubation  in  C - a r g i n i n e , induced c e l l s  extremely high p o o l , which decreased during  the f o l l o w i n g  incorporation total The  8 minutes of  measured a t c e r t a i n  r a d i o a c t i v i t y was  lost during Kay  for  decrease in the  the f i r s t  from a r g i n i n e  stable after  CO^,  during  original  the f i r s t  pool formed  A r g i n i n e grown c e l l s  m a i n t a i n a s t a b l e p o o l w h i c h was  in  uninduced c e l l s .  T h u s , under which were  15  a s i m i l a r amount  c o n s i s t e d o f p u t r e s c i n e , and was  to  was  incubation w i t h uninduced  the i n t r a c e l l u l a r  of a s i m i l a r  the  remaining in  of the whole c e l l s  i n d u c e d c e l l s , and  l o n g a s 24 h o u r s .  experiments, c e l l s  to oxidation.  A p p r o x i m a t e l y 45% o f t h e 14  30 m i n u t e s o f that  remained  radioactivity  l o s t , presumably as  incubation with  p e r i o d s as  The  after filtration  times.  (1969) f o u n d  aeruginosa  induced c e l l s  incubation.  supernatant f l u i d  minutes of  by a p p r o x i m a t e l y 50%  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 pool of  an  This decrease mainly represented  into protein, although a slight  intracellular  first the  cell  6 minutes.  in size  had a c c u m u l a t e d  size  the c o n d i t i o n s used  was cells.  by P_. stable  also  appeared  to that  formed  in these  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 o f a r g i n i n e by c e l l s grown w i t h a r g i n i n e as the s o l e source o f carbon and n i t r o g e n 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 o n c e n t r a t i o n s o f c e l l s and ^ C a r g i n i n e were the same as i n F i g . 15-  only  the same p r o p o r t i o n  tained  the  of a r g i n i n e as uninduced c e l l s , and  remainder in 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  Assuming t h a t p u t r e s c i n e was the p o o l , i t was arginine  l a b e l was  retained  in uninduced c e l l s .  due  to the  d i f f e r e n c e may  17A).  incorporated  significant  citrulline  an  initial  pattern  i n t o p r o t e i n as  i n t o p r o t e i n by a r g i n i n e grown c e l l s  s i m i l a r to that of  i t was  taken up  during  the f i r s t  These r e s u l t s were due  to the  s y n t h e s i s , and  Glucose grown c e l l s  are  A r g i n i n e grown c e l l s o f a r g i n i n e , and  in the  fundamental  for  two  induced f o r a r g i n i n e b i o -  incorporated  have been shown to be  repressed  17B).  5 minutes of  thus would r a p i d l y c o n v e r t o r n i t h i n e and  to a r g i n i n e , which would then be  was  incorporated  in the m e t a b o l i c f a t e of these compounds in the  types of c e l l s .  arginine  (Fig.  nor o r n i t h i n e were  these compounds were accumulated s o l e l y  intracellular pool.  of  incubation, c i t r u l l i n e  r a p i d l y as  the other hand, n e i t h e r c i t r u l l i n e  differences  be v e r y  involved.  A f t e r the f i r s t minute of  i n c u b a t i o n , and  not  and  uptake of o r n i t h i n e by g l u c o s e grown c e l l s  P_. a e r u g i n o s a f o l l o w e d  On  The  O r n i t h i n e and  The  r a d i o a c t i v e compound in  in t h i s pool in induced c e l l s ,  low c o n c e n t r a t i o n s  b.  pool.  c a l c u l a t e d t h a t a p p r o x i m a t e l y 23% of the o r i g i n a l  33%  (Fig.  the only  re-  citrulline  into p r o t e i n .  induced f o r the  its biosynthesis.  degradation  Thus, c i t r u l l i n e  F i g . 17.  Formation of i n t r a c e l l u l a r 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, t r i c h l o r o a c e t i c acid soluble pool. The c e l l concentration was 0.135 mg of c e l l s (dry weight)/ml, and the labelled substrates were 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).  and o r n i t h i n e would not be c o n v e r t e d t o p r o t e i n a r g i n i n e by t h e s e cel1s.  c.  Putrescine  The s t a b i l i t y o f p u t r e s c i n e p o o l s formed by induced and uninduced c e l l s o f P_. a e r u g i n o s a i n t h e presence o f e x t e r n a l C - p u t r e s c i n e was a l s o s t u d i e d .  The t o t a l uptake o f p u t r e s c i n e  i n t o g l u c o s e grown c e l l s reached a maximum a t 15 m i n u t e s , a f t e r which time 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 d i d not change ( F i g . 1 8 ) . However, a g r a d u a l i n c o r p o r a t i o n o f l a b e l i n t o  trichloroacetic a  a c i d i n s o l u b l e m a t e r i a l commenced a f t e r 20 m i n u t e s , r e s u l t i n g i n a concomitant d e c r e a s e i n t h e l e v e l o f t h e t r i c h l o r o a c e t i c  acid  soluble pool. 14 In t h e presence o f e x t e r n a l  C - p u t r e s c i n e , p u t r e s c i n e grown  c e l l s r a p i d l y accumulated a h i g h i n t r a c e l l u l a r p o o l , which  reached  a maximum a t 6 m i n u t e s , and dropped r a p i d l y f o r t h e next 4 m i n u t e s , due t o c o n t i n u e d r a p i d slight  incorporation into protein  ( F i g . 19).  lag i n incorporation of r a d i o a c t i v i t y into protein  The  presumably  r e p r e s e n t e d d i l u t i o n through a pool o f u n l a b e l l e d p u t r e s c i n e which had been e s t a b l i s h e d d u r i n g g r o w t h .  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 a p p r o x i m a t e l y 10 m i n u t e s , and then began t o d e c r e a s e s l o w l y , l o s i n g 64% o f i t s r a d i o a c t i v i t y d u r i n g t h e next 60 m i n u t e s . 'During t h i s time t h e r a d i o a c t i v i t y p r e s e n t i n t r i c h l o r o a c e t i c  acid  10  20  30  40  50  60  M I N U T E S  Fig.  18'.. Formation of an 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 by g l u c o s e 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 a c i d s o l u b l e p o o l ; A, p r o t e i n . The c e l l c o n c e n t r a t i o n 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 c o n c e n t r a t i o n was 2 . 5 x 10~5M ( s p e c i f i c a c t i v i t y = 2 y C i / y m o l e ) .  93  M I N U T E S  Fig.  1 9 . Formation of an 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 by p u t r e 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 a1 ic i d s o l u b l e p o o l . The c o n c e n t r a t i o n s o f c e l l s and o f * C - p u t r e s c i n e were the same as i n F i g . 1 8 .  i n s o l u b l e m a t e r i a l decreased by h3%, i n d i c a t i n g t h a t l y s i s had occurred.  However, the d e c r e a s e i n pool r a d i o a c t i v i t y was g r e a t e r  than t h a t due t o l y s i s , i n d i c a t i n g t h a t a p p r o x i m a t e l y 20% of the pool had been o x i d i z e d .  Measurements o f the r a d i o a c t i v i t y of the  s u p e r n a t a n t f l u i d s supported t h i s h y p o t h e s i s .  F o r t y - e i g h t per cent  of the added l a b e l had been l o s t from the r e a c t i o n m i x t u r e 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 m i n u t e s .  A s t a b l e pool  was m a i n t a i n e d from 80 t o 180 m i n u t e s . A r g i n i n e grown c e l l s a l s o accumulated a h i g h 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 a t 15 m i n u t e s , and then decreased d u r i n g  the next kS minutes  ( F i g . 20). The d e c r e a s e i n  pool r a d i o a c t i v i t y which o c c u r r e d d u r i n g  the f i r s t  t h i s p e r i o d was accounted f o r by an i n c r e a s e activity.  10 minutes o f  in protein radio-  However, the r a d i o a c t i v i t y o f the c e l l p r o t e i n  constant a f t e r the f i r s t  25 m i n u t e s , and thus the f u r t h e r d e c r e a s e  in pool r a d i o a c t i v i t y presumably intracellular putrescine.  remained  r e p r e s e n t e d the o x i d a t i o n o f  A s t a b l e pool was m a i n t a i n e d between  60 and 90 m i n u t e s , 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 m a t e r i a l decreased s l o w l y , i n d i c a t i n g t h a t 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  cell  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 o f the 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 showed t h a t 39% of the t o t a l l a b e l had been l o s t by 15 m i n u t e s , and 65% by 60 m i n u t e s .  After  3 0  20,  6 0 M I N U T E S  Formatton o f an I n t r a c e l l u l a r ,pdol d f p u 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 1s o l u b l e p o o l . The c o n c e n t r a t i o n s of c e l l s and o f ^ c - p u t r e s c i n e were the same as in F i g . 18.  3 h o u r s , 3% o f the o r i g i n a l l a b e l was in the s u p e r n a t a n t f l u i d , and  present  8.5%  i n the c e l l s ,  the remainder had  presumably been  14 l o s t as The  C02. s i z e o f 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 p o o l s  d i f f i c u l t t o d e t e r m i n e due  was  t o the e r r o r s a r i s i n g from the use  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 t h a t l y s i s had  of  occurred.  However, the c o n c e n t r a t i o n of p u t r e s c i n e i n the i n t r a c e l l u l a r water was  approximately 5.  2 t o 3 x 10  -2  M.  L o c a t i o n o f i n t r a c e l l u l a r p u t r e s c i n e pool  Kay aeruginosa  (1969) showed t h a t the s t a b l e pool formed i n P_.  from a r g i n i n e c o n s i s t e d of p u t r e s c i n e .  pool d i d not r e q u i r e energy f o r i t s m a i n t e n a n c e , he t h a t p u t r e s c i n e may (Kay, 1968).  this  hypothesized  be bound t o some component w i t h i n the  An attempt was  l o c a t i o n of t h i s  Since  cell  t h e r e f o r e made t o d e t e r m i n e the  i n t r a c e l l u l a r pool by p h y s i c a l  fractionation  procedures. C e l l s were h a r v e s t e d  from the s t a t i o n a r y phase of growth  in g l u c o s e minimal medium and 5 mg o f c e l l s were incubated 0.05 arm  (dry w e i g h t ) / m l .  resuspended t o a c o n c e n t r a t i o n Ten ml of the c e l l  under c o n v e n t i o n a l  of  suspension  Warburg c o n d i t i o n s w i t h 5 ml  of  M T r i s b u f f e r i n a l a r g e Warburg cup w i t h a s i n g l e s i d e 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 h a r v e s t e d 90 minutes a f t e r t h e a d d i t i o n o f t h e a r g i n i n e  and  were s u b j e c t e d  t o p h y s i c a l 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 t h e f r a c t i o n s was measured and compared w i t h t h a t of the c e l l - f r e e extract.  Samples o f each f r a c t i o n were e x t r a c t e d w i t h c o l d 10%  t r i c h l o r o a c e t i c a c i d , and t h e d i s t r i b u t i o n o f t h e r a d i o a c t i v i t y between t h e p r e c i p i t a t e and the s u p e r n a t a n t f l u i d was d e t e r m i n e d . The  r e s u l t s showed t h a t t h e m a j o r i t y o f t h e 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 i n t h e s o l u b l e c y t o p l a s m (Table X I I ) A l t h o u g h the membrane and ribosomal f r a c t i o n s a l s o c o n t a i n e d cons i d e r a b l e r a d i o a c t i v i t y , 65% t o 70% o f 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 i n c u b a t i o n o f the c e l l s w i t h  C-arginine.  Thus, 36% o f t h e t o t a l l a b e l was p r e s e n t i n the s o l u b l e c y t o p l a s m 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 o n l y 7-5% was p r e s e n t i n t h i s form i n t h e ribosomal f r a c t i o n , and a s i m i l a r amount i n the membrane f r a c t i o n .  Thin-layer  chromatography of t h e  t r i c h l o r o a c e t i c acid e x t r a c t of the s o l u b l e cytoplasm, followed by r a d i o a u t o g r a p h y , showed t h a t p u t r e s c i n e was the l a b e l l e d compound. The  s o l u b l e c y t o p l a s m has been found t o be the l o c a t i o n of  i n t r a c e l l u l a r putrescine 80%  in other organisms.  Kim (1966) found t h a t  t o 90% o f t h e i n t r a c e l l u l a r p u t r e s c i n e o f a Pseudomonad was  located  i n t h e s o l u b l e c y t o p l a s m , w i t h the remaining 10% t o 20%  located  i n the ribosomal f r a c t i o n .  s i m i l a r r e s u l t s w i t h E. c o l i ,  Tabor and K e l l o g g  (1967) o b t a i n e d  i n which 8% t o 11% o f t h e 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 physical f r a c t i o n a t i o n of c e l l s incubated i n t h e presence o f ^ C - a r g i n i n e .  % of t o t a l rad i o a c t i v i t y  F r a c t ion  % o f the l a b e l i n the f r a c t i o n which was e x t r a c t e d by c o l d t r i c h l o r o a c e t i c acid  membranes  23  35.4  r i bosomes  25  30.7  59  59  110,000 x  £  supernatant  fluid  polyamines were a s s o c i a t e d w i t h the r i b o s o m e s , and were l o c a t e d i n the s o l u b l e c y t o p l a s m .  the remainder  However, the l a t t e r workers  a l s o showed t h a t E_. c o l i ribosomes c o u l d take up or l o s e polyamines upon a l t e r a t i o n of the c o n c e n t r a t i o n of magnesium or amines i n the s u s p e n s i o n medium.  Thus, a l t h o u g h  a g r e a t e r p r o p o r t i o n of  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 u t r e s c i n e was i n P_. a e r u g i n o s a and  than was  a s s o c i a t e d w i t h ribosomes  observed i n a Pseudomonad by Kim  i n E_. c o l i by Tabor and  Kellogg  the c e l l , and  a  Cl966)  (1967), the l a t t e r workers used  a 10 f o l d h i g h e r magnesium c o n c e n t r a t i o n d u r i n g ribosome T h u s , polyamines a r e p r o b a b l y  the  isolation.  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  f r a c t i o n a t i o n r e s u l t s may  be  invalid.  GENERAL DISCUSSION  S t a n t e r , P a l l e r o n i , and  Doudoroff (1966) have shown t h a t  the  p o s s e s s i o n 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 examined in t h i s study were shown to e x c r e t e of t h i s pathway d u r i n g  the t h r e e  the  intermediates  the o x i d a t i o n of a r g i n i n e .  a l l t h r e e organisms s y n t h e s i z e d  In a d d i t i o n ,  p u t r e s c i n e 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 T h u s , i t i s l i k e l y t h a t the enzymes of the a r g i n i n e pathway may  species  system. dihydrolase  f u n c t i o n i n the b i o s y n t h e s i s of p u t r e s c i n e  i n these  organisms. There i s much i n d i r e c t e v i d e n c e i n the may  p l a y a r o l e i n t r a n s l a t i o n , and  l i t e r a t u r e t h a t polyamines  possibly also in t r a n s c r i p t i o n .  S e v e r a l workers have o b t a i n e d e v i d e n c e t h a t these compounds a r e n e c e s s a r y f o r the growth of E_. c o l i which s y n t h e s i z e s a h i g h pool of p u t r e s c i n e c o n s t i t u t i v e l y . synthesizes  It i s not known whether P_. a e r u g i n o s a  p u t r e s c i n e d u r i n g growth i n g l u c o s e minimal medium.  However, when s u p p l i e d w i t h exogenous a r g i n i n e , t h i s organism r e t a i n e d a l a r g e 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 d e g r a d a t i o n were f u l l y i s l i k e l y t h a t t h i s pool was has been found t o bind t o DNA, cond i t i o n s .  induced.  p r e s e n t i n a bound f o r m , s i n c e RNA,  and  It putrescine  ribosomes under i n v i t r o  Of the t h r e e Pseudomonads examined, o n l y P_. a e r u g i n o s a to have the c o n s t i t u t i v e a b i l i t y t o c o m p l e t e l y 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 and compound, presumably v i a c o n v e r s i o n  appeared  oxidize arginine,  f u r t h e r d e g r a d i n g the  to a - k e t o g l u t a r a t e .  latter  Cells  were unable t o 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 l a g b e f o r e s y n t h e s i s of p r o t e i n from p u t r e s c i n e commenced i n uptake e x p e r i m e n t s , and before  a long l a g  i n d u c t i o n o f the a b i l i t y t o o x i d i z e p u t r e s c i n e i n manometric  experiments. Growth of P_. a e r u g i n o s a carbon and  w i t h a r g i n i n e as the s o l e source of  n i t r o g e n r e s u l t e d i n the i n d u c t i o n of a g r e a t l y  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 r e p r e s e n t e d in the r a t e of d e g r a d a t i o n Presumably .higher  an  increased increase  of o r n i t h i n e v i a the g l u t a m a t e pathway.  l e v e l s of the enzymes of the a r g i n i n e d i h y d r o l a s e  system were a l s o i n d u c e d .  The  r e s u l t s of the s u c c i n i c semialdehyde  dehydrogenase a s s a y s showed t h a t growth i n a r g i n i n e r e s u l t e d i n a partial to  i n d u c t i o n of the enzymes o f y - a m i n o b u t y r a t e  degradation,  l e v e l s h i g h e r than those induced by growth in g l u t a m a t e .  results  i n d i c a t e d t h a t some p u t r e s c i n e was  growth i n a r g i n i n e .  These  being degraded d u r i n g  However a r g i n i n e grown c e l l s demonstrated a  s h o r t lag b e f o r e 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, was  i n uptake e x p e r i m e n t s , a s h o r t l a g b e f o r e p u t r e s c i n e carbon incorporated  i n t o . p r o t e i n . These lag p e r i o d s were much s h o r t e r  than those e x h i b i t e d by g l u c o s e grown c e l l s , and the r a t e o f p u t r e s 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 a r e s i m i l a r  t o those o b t a i n e d w i t h c e l l s which a r e induced f o r t h e 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 o f P_. a e r u g i n o s a t r a n s p o r t e d  putrescine very  rapidly.  T h u s , a r g i n i n e grown c e l l s had a g r e a t e r p o t e n t i a l a b i l i t y t o oxidize putrescine  than g l u c o s e grown c e l l s , but appeared t o be  b l o c k e d i n one s t e p .  I t i s p o s s i b l e t h a t a key enzyme o f p u t r e s c i n e  d e g r a d a t i o n was not induced d u r i n g growth on a r g i n i n e , o r t h a t 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 .  mechanism would a s s i s t putrescine The was  Such a c o n t r o l  i n t h e maintenance o f the l a r g e pool o f  found t o be formed from a r g i n i n e by P_. a e r u g i n o s a .  r a t e o f 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  r e l a t i v e l y high.  The d e g r a d a t i o n o f endogenously  synthesized  a r g i n i n e by t h e c o n s t i t u t i v e enzymes o f P_. a e r u g i n o s a would be a d i s a d v a n t a g e t o t h e c e l l , and i t i s t h e r e f o r e  l i k e l y t h a t the  a c t i v i t y o f t h e s e enzymes i s , i n some way, c o n t r o l l e d .  It i s  p o s s i b l e t h a t t h e a r g i n y l - t R N A s y n t h e t a s e has a much h i g h e r a f f i n i t y f o r a r g i n i n e than do the d e g r a d a t i v e enzymes, so t h a t , a t low endogenous c o n c e n t r a t i o n s , corporated into p r o t e i n .  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 On the o t h e r hand, t h e enzymes o f  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 incorporation  i n t o p r o t e i n may b e , i n some way, s e p a r a t e d from t h e enzymes responsible  f o r arginine degradation, preventing  endogenous a r g i n i n e  from being o x i d i z e d . for  S e r c a r z and G o r i n i (1964) o b t a i n e d e v i d e n c e  such a compartmenta 1 i z a t ion i n E_. c o l i , where endogenous l y  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  and exogenous a r g i n i n e f o r r e p r e s s o r  formation.  (1969b) showed t h a t E_. c o l ? s y n t h e s i z e d  synthesis,  Tabor and Tabor  putrescine  preferentially  from exogenous r a t h e r than endogenous a r g i n i n e , whereas p r o t e i n appeared t o be s y n t h e s i z e d  e q u a l l y w e l l from both s o u r c e s .  N_.  c r a s s a has been shown t o 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 m a i n l y 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 protein synthesis  ( C a s t a n e d a , M a r t u s c e l l i , and M o r a , 1967; D a v i s ,  1968). Ramos e t a 1.  (1967) and Jacoby (1964) found t h a t a r g i n i n e  d e g r a d a t i o n was s u b j e c t of P_. f l u o r e s c e n s .  to c a t a b o l i t e repression  in several s t r a i n s  However, t h e l a t t e r worker found t h a t t h e  o x i d a t i o n o f a r g i n i n e was r e p r e s s e d t o a l e s s e r e x t e n t t h a t o f o t h e r amino a c i d s . to catabol i t e repression  than was  A r g i n i n e d e g r a d a t i o n was not s u b j e c t  i n P_. a e r u g i n o s a ; both g l u c o s e 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 p r e s e n t as a m i x t u r e i n t h e presence of growing c e l l s or r e s t i n g c e l l s u s p e n s i o n s .  This  lack of repress!'  was p r o b a b l y due t o the high c o n s t i t u t i v e l e v e l o f t h e a r g i n i n e d e g r a d i n g enzymes.  Chemical f r a c t i o n a t i o n r e s u l t s d i d i n d i c a t e  t h a t , i n t h e presence o f a m i x t u r e o f a r g i n i n e and g l u c o s e , u t i l i z e d t h e two s u b s t r a t e s needs.  cells  t o 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  f_. aerug Inosa possessed a t l e a s t two systems f o r the t r a n s p o r t of a r g i n i n e .  K i n e t i c s t u d i e s demonstrated the presence of a low  a f f i n i t y system and a h i g h a f f i n i t y system i n g l u c o s e grown c e l l s . The  low a f f i n i t y  system c o u l d  not be measured i n a r g i n i n e grown  c e l l s ; however, t h e s e c e l l s possessed i n c r e a s e d a f f i n i t y system.  l e v e l s of t h e high  I n h i b i t i o n s t u d i e s a l s o demonstrated the presence  of two b a s i c amino a c i d t r a n s p o r t systems; one which was for  specific  a r g i n i n e a n d , w i t h 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  g e n e r a l system which t r a n s p o r t e d g e n e r a l uptake system was  a l l t h e b a s i c amino a c i d s .  induced t o a g r e a t e r e x t e n t  The  than was  the  s p e c i f i c system by growth of the organism i n 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 .  I t i s important t o note t h a t i n -  h i b i t i o n s t u d i e s were c a r r i e d out i n the c o n c e n t r a t i o n which the low a f f i n i t y cells.  permease was o p e r a t i v e  range i n  i n g l u c o s e grown  However, the degree t o which t h i s permease c o n t r i b u t e d  t o t o t a l uptake c o u l d  not be a s c e r t a i n e d , and t h e r e f o r e  d e f i n i t i v e c o r r e l a t i o n could  no  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 t u d i e s and those i d e n t i f i e d by  inhibition  studies. P u t r e s c i n e appeared t o be t r a n s p o r t e d permease i n P_. aerug i n o s a .  I t was  by a g e n e r a l polyamine  i n t e r e s t i n g t o note t h a t growth  of P_. aerug?nosa i n a r g i n i n e r e s u l t e d i n almost complete  induction  of p u t r e s c i n e  induction  transport.  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