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The biosynthesis of N-putrescinylthymine in bacteriophage φW-14 infected Pseudomonas acidovorans Karrer, Earl 1973

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THE BIOSYNTHESIS OF N-PUTRESCINYLTHYMINE IN BACTERIOPHAGE 0W-14  INFECTED Pseudomonas  acidovorans  by  EARL KARRER  B.Sc.  (Honours, M i c r o b i o l o g y )  U n i v e r s i t y o f B r i t i s h Columbia, 1971  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  IN THE DEPARTMENT OF MICROBIOLOGY  WE ACCEPT THIS THESIS AS CONFORMING TO THE REQUIRED STANDARD  THE UNIVERSITY OF BRITISH COLUMBIA JULY, 1973  In p r e s e n t i n g an the  advanced degree at Library  I further for  this thesis  shall  the  of  this thesis  written  University  of B r i t i s h  representatives.  be  g r a n t e d by  for f i n a n c i a l gain  permission.  Department  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  the  I t i s understood  Columbia  shall  the  requirements  Columbia,  for reference  permission for extensive  s c h o l a r l y p u r p o s e s may his  f u l f i l m e n t of  make i t f r e e l y a v a i l a b l e  agree that  by  in partial  that  not  and  copying of Head o f my  be  I agree  that  study.  this  thesis  Department  copying or  for  or  publication  allowed without  my  !  •i  ABSTRACT  The b i o s y n t h e s i s of N - p u t r e s c i n y l t h y m i n e d i n e which o c c u r s  (NpT), a m o d i f i e d  i n the DNA o f phage 0W-14, was s t u d i e d .  P r i o r to t h i s  study, the metabolism o f a r g i n i n e and polyamines i n the host Pseudomonas  a c i d o v o r a n s 29,  organism,  was i n v e s t i g a t e d .  P_. a c i d o v o r a n s t r a n s p o r t e d Neither  pyrimi-  o r n i t h i n e and a r g i n i n e but n o t p u t r e s c i n e .  amino a c i d was used as a s o l e source o f n i t r o g e n .  Thus a r g i n i n e  cannot be c a t a b o l i z e d to p u t r e s c i n e and o r n i t h i n e i s not c a t a b o l i z e d t o Y-aminobutyrate.  O r n i t h i n e was s y n t h e s i z e d  from glutamate but the  o p e r a t i o n o f t h i s pathway was not i n h i b i t e d by h i g h c o n c e n t r a t i o n s ginine.  Since o r n i t h i n e was d e c a r b o x y l a t e d  route f o r the s y n t h e s i s  of ar-  to putrescine, this single  o f polyamines i s unusual i n t h a t o r n i t h i n e b i o -  synthesis i s c o n s t i t u t i v e . P_. a c i d o v o r a n s and 0W-14  c o n t a i n an unusual complement o f polyamines:  s p e r m i d i n e , p u t r e s c i n e and 2 - h y d r o x y p u t r e s c i n e . concentrations crease  Their respective b a c t e r i a l  (mM) were: 3-5, 50 and 45, phage i n f e c t i o n l e d to an i n -  i n the p r o p o r t i o n of p u t r e s c i n e .  14  C-Ornithine  was used to l a b e l NpT i n phage DNA.  14  C-5-0rnithine  14 e x c l u s i v e l y l a b e l l e d NpT, whereas i n this capacity. tuent  ineffective  Hence, the c a r b o x y l group of o r n i t h i n e i s not a c o n s t i -  o f the NpT m o l e c u l e . L a b e l l i n g of NpT w i t h  (THF)  C - l - o r n i t h i n e was t o t a l l y  14  C-3-serine  involvement i n i t s b i o s y n t h e s i s .  demonstrated a t e t r a h y d r o f o l a t e  3  The use o f  H-2,3-serine showed  t h a t N^^methylene THF was the carbon donor f o r the p y r i m i d i n e p r e c u r s o r o f  ii  NpT.  I t i s proposed t h a t NpT i s s y n t h e s i z e d i n the f o l l o w i n g  way:  5-hydroxymethyl d e o x y u r i d i n e monophosphate i s formed by the i n t e r a c t i o n of N^^methylene THF and d e o x y u r i d i n e monophosphate; p u t r e s c i n e then condenses w i t h the hydroxymethyl group of the n u c l e o t i d e to y i e l d thymine.  N-putrescinyl-  iii  TABLE OF CONTENTS  Page INTRODUCTION I.  II.  1 A r g i n i n e metabolism  1  A.  Arginine biosynthesis....  1  B.  Arginine catabolism  4  C.  C o n t r o l of a r g i n i n e b i o s y n t h e s i s . . . .  Polyamine metabolism  II. III. IV. V. VI.'  14  D i s t r i b u t i o n o f polyamines  14  B.  Synthesis  16  C.  C o n t r o l of polyamine s y n t h e s i s  17  D.  D e r i v a t i v e s of polyamines  19  E.  Catabolism  F.  S i g n i f i c a n c e of polyamines  24  1.  polyamines and b a c t e r i o p h a g e . . . . . . . . . . .  24  2.  polyamines and b a c t e r i a . . . .  24  o f polyamines  of polyamines  Polyamine: DNA i n t e r a c t i o n s  M o d i f i e d bases  MATERIALS AND METHODS I.  . .•  A.  G. III.  11  20  . ...  26 27 30  Organisms  30  Media  30  Growth of b a c t e r i a . . . .  30  Phage t i t r a t i o n  31  P r e p a r a t i o n of phage s t o c k s  31  P u r i f i c a t i o n of phage  31  iv Table of Contents (cont'd) VII. VIII. IX. X.  XI. XII.  XIII. XIV. XV. XVI. XVII.  XVIII. XIX.  Page  Osmotic s e n s i t i v i t y of 0W-14  31  U t i l i z a t i o n of compounds as nitrogen source  32  Uptake of radioactive compounds  32  I s o l a t i o n of polyamines  . 33  A.  From b a c t e r i a .  33  B.  From phage  34  Preparation of dansylated polyamines  34  Chromatography  34  A.  Thin layer (TLC)  34  1.  polyamines  34  2.  dansylated polyamines  35  3.  DNA  35  hydrolysates  Detection of polyamines  36  Quantitation of polyamines  36  I s o l a t i o n of 2-hydroxyputrescine  36  NMR  37  of 2-hydroxyputrescine  I s o l a t i o n of DNA  37  A.  From b a c t e r i a  37  B.  From phage  38  Hydrolysis of DNA  38  Measurement of r a d i o a c t i v i t y  38  A.  Chromatographed polyamines or DNA  hydrolysates 38  B.  TCA soluble materials  38  C.  TCA precipitable materials  38  V,  Table of Contents (cont'd) Page  D. XX. XXI.  Counting r a d i o a c t i v i t y  38  Chemicals  39  Radiochemicals  39  RESULTS AND DISCUSSION I. II. III. IV. V. VI. VII. VIII.  U t i l i z a t i o n of compounds as sole source of nitrogen...  40  Transport of radioactive substrates  40  Polyamines of P. acidovorans  40  L a b e l l i n g of polyamines....  49  Quantitation of polyamines  53  Polyamines of 0W-14  54  Osmotic s e n s i t i v i t y of 0W-14  55  The biosynthesis of NpT  55  SUMMARY  61  LITERATURE CITED  63  vi  LIST OF FIGURES Figure  1.  Page  Pathway A, A r g i n i n e b i o s y n t h e s i s i n h i g h e r organisms  1.  2  Pathways B and C, A r g i n i n e b i o s y n t h e s i s i n bacteria  2.  3  A r g i n i n e c a t a b o l i s m i n h i g h e r organisms and B a c i l l u s sp  5  3.  The a r g i n i n e d i h y d r o l a s e pathway  7  4.  A r g i n i n e c a t a b o l i s m i n S_. g r i s e u s and P_. p u t i d a  8  5.  A r g i n i n e c a t a b o l i s m i n E_. c o l i  10  6.  Spermidine b i o s y n t h e s i s i n E_. c o l i  18  7A&,  Polyamine c a t a b o l i s m :  putrescine  21  7B5.  Polyamine c a t a b o l i s m :  spermidine  23  8.  The t r a n s p o r t o f "^C-U-arginine  9.  The t r a n s p o r t o f " ^ C - 5 - o r n i t h i n e by P_. a c i d o v o r a n s . . ..  10.  The t r a n s p o r t of  14  by P_. a c i d o v o r a n s  C-3,4-putrescine  by P_. a c i d o v o r a n s .  C-3,4-putrescine  by P_.  42 43 44  14 11.  The t r a n s p o r t of  i n f e c t e d w i t h 0W-14 12.  100 MHz NMR  45  spectrum o f a D 0  2-hydroxyputrescine  acidovorans  2  s o l u t i o n of  dihydrochloride  48  vii  LIST OF TABLES Table  I. II.  Page  Common polyamines U t i l i z a t i o n o f compounds  15 as s o l e source o f  nitrogen III. IV.  41  T h i n l a y e r chromatography o f polyamines  47  L a b e l l i n g o f polyamines  50 14  V.  D i s t r i b u t i o n o f l a b e l (CPM) i n  C-glutamate  l a b e l l e d polyamine e x t r a c t s VI. VII. VIII.  Osmotic s e n s i t i v i t y o f phage 0W-14 14 L a b e l l i n g o f 0W-14 DNA w i t h C-ornithine L a b e l l i n g of 0W-14 DNA w i t h r a d i o a c t i v e s e r i n e . .  52 56 57 59  viii  ACKNOWLEDGEMENTS  To Dr. R.A.J. Warren, f o r h i s t h o u g h t f u l comments, g u i d a n c e , encouragement and w i t d u r i n g t h i s gratitude.  s t u d y , I express my s i n c e r e  Tony's t r u t h f u l approach to s c i e n c e i s admirable and w i l l  remain w i t h me, always.  Dr. R.A. K e l l n , an e t e r n a l source o f s t i m u -  l a t i n g d i s c u s s i o n , good humour and exuberance,  I owe thanks, as w e l l .  I am i n d e b t e d t o , Dr. R.J. Bose f o r h i s o p e r a t i o n of the NMR  spectro-  meter, a d v i c e and eagerness t o p a r t i c i p a t e i n the p r o j e c t and a l s o t o Dr. J.B. Farmer, f o r the use o f h i s F l u o r o m e t e r . Mrs. P a t Waldron, f o r your s k i l l f u l i n d e c i p h e r i n g my penhand.  Thank you,  also,  t y p i n g o f t h i s t h e s i s and p a t i e n c e  1  INTRODUCTION  I.  ARGININE METABOLISM A.  Arginine Biosynthesis  C e l l u l a r routes l e a d i n g to a r g i n i n e are i l l u s t r a t e d Although  i n F i g u r e 1.  some f e a t u r e s o f a r g i n i n e b i o s y n t h e s i s a r e common t o a l l c e l l s ,  t h e r e i s c o n s i d e r a b l e v a r i a t i o n i n the o v e r a l l pathway.  In higher o r -  ganisms, p r o l i n e i s t h e i n i t i a l p r e c u r s o r f o r a r g i n i n e anabolism Handler  (White,  and Smith, 1968); and o r n i t h i n e i s formed v i a A p y r r o l i n e - 5 J  c a r b o x y l i c a c i d and g l u t a m i c acid-y-semialdehyde. o r n i t h i n e i n b a c t e r i a d i f f e r s , however. Bonner, 1956), P r o t e u s m i r a b i l i s  The f o r m a t i o n of  Escherichia c o l i  (Vogel and  (Prozesky, 1967), S e r r a t i a marcescens,  o t h e r e n t e r i c s (Udaka, 1966) and B a c i l l u s s u b t i l i s  (Vogel and V o g e l ,  1963)  c o n v e r t g l u t a m i c a c i d t o N - a c e t y l o r n i t h i n e , which i s then h y d r o l y z e d to ornithine  ( F i g . 1, pathway B ) .  fluorescens  Micrococcus  g l u t a m i c u s , Pseudomonas  (Udaka, 1966), Pseudomonas a e r u g i n o s a  1972) and Pseudomonas p u t i d a (Chou and Gunsalus,  (Isaac and Holloway, 1971) a l s o form N - a c e t y l o r n i -  t h i n e from glutamate but convert i t t o o r n i t h i n e by t r a n s a c e t y l a t i o n of the a c e t y l group to g l u t a m i c a c i d  (Udaka, 1966), ( F i g . 1, pathway C ) .  Acety-  l a t i o n i n these two pathways, presumably, p r e v e n t s an i n t r a m o l e c u l a r cyclization reaction.  Glutamic  acid-y-semialdehyde  c y c l i z e s r e a d i l y to  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 which i n h i b i t s the subsequent 1  transamination  step. The  c o n v e r s i o n o f o r n i t h i n e to a r g i n i n e i n b a c t e r i a mimics the u r e a  c y c l e o f h i g h e r organisms (White, Handler  and Smith, 1968).  In the  2  CITRULLINE  ASPARTATE  ARGININE-*  —  ARGINOSUCCINATE  / , ' FUMARATE  Number  p  1 2 3 4 5 6  p r o l i n e oxidase ornithine-6-transaminase o r n i t h i n e transcarbamylase arginosuccinate synthetase arginosuccinase carbamyl phosphate s y n t h e t a s e  FIGURE 1, PATHWAY A.  Enzyme  Arginine biosynthesis i n higher  organisms  3  N-ACETYLGLUTAMIC:  N-ACET YL-iC-GLUtAM YL  2f-SEMI ALDEHYDE  PHOSPHORIC AG ID  ATP  N-ACETYLORNITHINE  N-ACETYLGLUTAMATE11  GLUTAMIC ACID  —ORN THINE  6,7,8  pathway B: pathway C:  ARGININE Numb e r 1 2 3 4 5  6,7,8  Enzyme N-acetylglutamate synthetase N-acetyl-y-glutamokinase N - a c e t y l g l u t a m i c - Y - s e m i a l d e h y d e dehydrogenase acetylornithine-6-transaminase acetylornithinase as i n h i g h e r organisms acetylglutamate-acetylornithine transacetylase  FIGURE 1, PATHWAYS B & C.  Arginine biosynthesis i n bacteria  4  presence o f o r n i t h i n e transcarbamylase  (OTCase), o r n i t h i n e and carbamyl  phosphate r e a c t t o produce c i t r u l l i n e .  Carbamyl phosphate, common t o  b o t h a r g i n i n e and p y r i m i d i n e s y n t h e s e s ,  i s d e r i v e d from CC^ and NH^ i n  a r e a c t i o n c a t a l y s e d by carbamyl phosphate s y n t h e t a s e . accomplished 1965)  by two isozymes i n Saccharomyces  and Neurospora c r a s s a (Bernhardt  This reaction,  cerevisiae  ( L a c r o u t e et a l . ,  and D a v i s , 1972) and by a s i n g l e  enzyme i n E. c o l i ( P i e r a r d and Wiame, 1964), i s s u b j e c t to end product inhibition  by a r g i n i n e and u r i d i n e t r i p h o s p h a t e (UTP) (Thuriaux e t a l . ,  1972).  B.  Arginine  Catabolism  The u r e a c y c l e i n h i g h e r organisms i s d r i v e n by the enzyme a r g i n a s e , which h y d r o l y z e s a r g i n i n e t o u r e a and o r n i t h i n e ( F i g . 2 ) . B_. s u b t i l i s c o n t a i n s a r g i n a s e and a l s o an o r n i t h i n e - 6 - t r a n s a m i n a s e which o r n i t h i n e to g l u t a m i c acid-y-semialdehyde;  converts  f o l l o w i n g spontaneous  cycliza-  t i o n t o 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 , e i t h e r p r o l i n e i s formed by A 1  1  p y r r o l i n e - 5 - c a r b o x y l i c a c i d r e d u c t a s e , o r glutamate i s formed by a dehydrogenase c a t a l y z e d o x i d a t i o n . 1964)  I n B. s u b t i l i s  and B a c i l l u s l i c h e n i f o r m i s  ornithine-S-transaminase induced  (de Hauwer, L a v a l l e and Wiame,  ( L a i s h l e y and B e r n l o h r , 1968), a r g 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 i c dehydrogenase a r e 1  s i m u l t a n e o u s l y by a r g i n i n e .  The f i r s t  two enzymes appear t o be  g e n e t i c a l l y l i n k e d w i t h an a r g i n i n e permease i n B_. s u b t i l i s was i s o l a t e d i n which a l l t h r e e a c t i v i t i e s a r e c o n s t i t u t i v e . genase remains i n d u c i b l e and i s , a p p a r e n t l y , u n l i n k e d and Wiame, 1964).  s i n c e a mutant The dehydro-  (de Hauwer, L a v a l l e  5  G L U T A IVIIC ACID  ARGININE  UREA  G L U T A M I C ACIDORNITHINE  V-SEMIALDEHYDE  A 1 PYRROLINE-5-  PROLINE-  C A R B O X Y L I C ACID  Number 1 2 3  4  FIGURE 2.  Enzyme argxnase ornithine-6-transaminase , Apyrroline-5-carboxylic acid g l u t a m i c acid-y-semialdehyde  reductase dehydrogenase  A r g i n i n e c a t a b o l i s m i n h i g h e r organisms and B a c i l l u s s p .  6  An a l t e r n a t i v e route arginase  i s the a r g i n i n e d i h y d r o l a s e  C l o s t r i d i a sp. (Mitruka (Oginsky and G e h r i g ,  i n organisms l a c k i n g  pathway ( F i g . 3) which o c c u r s i n  and C o s t i l o w ,  1967), S t r e p t o c o c c u s  1952), Pseudomonas  Halobacterium s a l i n a r i u m first  f o r arginine catabolism  faecalis  sp. ( S t a l o n et a l . , 1967), and  (Dundas and H a l v o r s o n , 1966).  deiminates a r g i n i n e to c i t r u l l i n e .  Arginine  deiminase  OTCase, i n a p h o s p h o r l y t t i c  r e a c t i o n , generates carbamyl phosphate and o r n i t h i n e from c i t r u l l i n e and i n o r g a n i c phosphate.  In a d d i t i o n to an a n a b o l i c  OTCase, a s p e c i f i c  b o l i c enzyme o c c u r s i n P_. a e r u g i n o s a and P_. f l u o r e s c e n s 1967).  cata-  (Stalon et a l . ,  In c o n t r a s t , o n l y one, amphibolic OTCase i s found i n H.  salinarium  (Dundas, 197,2). The two enzymes, h a v i n g pH optima of 7.1-7.4 and 8.5, are e a s i l y separable  by ammonium s u l f a t e f r a c t i o n a t i o n .  l i n e a c t i v i t y i s repressed  I n P_. a e r u g i n o s a , the a l k a -  by growth i n the presence o f a r g i n i n e , a s i t u a -  t i o n which augments the n e u t r a l a c t i v i t y . a l k a l i n e a c t i v i t y represents  I t was thus reasoned t h a t the  the b i o s y n t h e t i c enzyme w h i l e the c a t a b o l i c  OTCase has the n e u t r a l pH optimum and i s c o n t r o l l e d c o o r d i n a t e l y w i t h a r g i n i n e deiminase and carbamate k i n a s e  (Ramos e t a l . , 1967).  The t h i r d enzyme o f the a r g i n i n e d i h y d r o l a s e nase, s p l i t s via  substrate  pathway, carbamate k i - .•  carbamyl phosphate i n t o NH^ and C O 2 , w i t h one mole o f ATP formed l e v e l phosphorylation  per mole of c i t r u l l i n e  hydrolyzed.  T h i s r e a c t i o n enables Pseudomonads, n o r m a l l y immotile when grown c a l l y i n minimal medium, to r e g a i n m o t i l i t y i f a l s o s u p p l i e d w i t h  anaerobiarginine  ( S t a n i e r , P a l l e r o n i and D o u d o r o f f , 1966). The pathway of a r g i n i n e d e g r a d a t i o n i n Streptomyces grisfeus i s shown in  F i g . 4, pathway A.  Arginine  is first  decarboxylated  to  Y~guanidino-  7  INORGANIC  Number  PHOSPHATE  Enzyme  1 2 3  a r g i n i n e deiminase o r n i t h i n e transcarbamylase carbamate k i n a s e  FIGURE 3.  The a r g i n i n e d i h y d r o l a s e pathway  8  ARGININE  1  ot-KETOARGININE  ^-GUANIDINOBUTYRIC  ACID  UREA-  y - A M I N O B U T Y R I C ACID  Enzyme a r g i n i n e decarboxyoxidase a r g i n i n e deaminase guanidinobutyramidase a-ketoarginine decarboxylase y-guanidinoamidino h y d r o l a s e  A r g i n i n e c a t a b o l i s m i n _S. g r i s e u s and P_. p u t i d a .  9  butyramide  ( T h o a i , Thome-Beau and Pho,  of O2 per mole of CO^ needs FAD  1962), the r e a c t i o n uses one mole  l i b e r a t e d and the enzyme, a r g i n i n e decarboxyoxidase,  as a p r o s t h e t i c group ( T h o a i , Thome-Beau and O.lomucki,  1966).  The  enzymes o f t h i s pathway are c o o r d i n a t e l y induced by a r g i n i n e , but to d i f f e r e n t l e v e l s , s u g g e s t i n g t h a t the genes are not l i n k e d Beau and Pho,  ( T h o a i , Thome-  1962) .  Growth o f P_. p u t i d a 7^ on a r g i n i n e r e s u l t s i n the i n d u c t i o n o f a s i m i l a r d e g r a d a t i v e sequence ( F i g . 4, pathway B ) , ( M i l l e r and 1971).  I n t h i s microbe, however, the l o s s o f NH^  Rodwell,  precedes d e c a r b o x y l a t i o n .  Hence, the f i r s t p r o d u c t , a - k e t o a r g i n i n e , i s subsequently d e c a r b o x y l a t e d to y - g u a n i d i n o b u t y r a t e (yGBA).  a-Ketoarginine decarboxylase  (Rodwell  2+ Gaby, 1973) tors.  r e q u i r e s thiamine pyrophosphate  Optima f o r t h i s enzyme are at pH 8.5  and e i t h e r Mg and 70°C.  one mole of yGBA and one mole o f an " a c y l o i n - l i k e " adduct  2+ o r Mn  Two  and  as c o f a c CO2,  moles o f are  produced  f o r e v e r y t h r e e moles of a - k e t o a r g i n i n e consumed (Rodwell and Gaby, 1973). yGBA accumulates  i n c u l t u r e s grown on a r g i n i n e a t pH  o f the t h i r d enzyme, y-guanidinoamidino  7.0.  Synthesis  h y d r o l a s e , i s induced by growth  on a r g i n i n e or yGBA (Chou and Rodwell, 1972).  The p u r i f i e d enzyme  (mole-  2+ c u l a r weight: 178,000 to 190,000) r e q u i r e s Mn 10 and 50°C.  shows optima  In view of the d e c r e a s e i n a c t i v i t y a t pH 7.0  m u l a t i o n o f yGBA, Chou and Rodwell amidino h y d r o l a s e a c t i v i t y may The p a t i e n c e of Gale  (1972) suggested  that  at pH  and the accu-  y-guanidino-  be r a t e l i m i t i n g i n t h i s pathway.  (1940) r e v e a l e d t h a t i n E_. c o l i  pH supplemented w i t h c a s e i n d i g e s t , a r g i n i n e was The  and  c u l t u r e s of  low  d e c a r b o x y l a t e d ( F i g . 5 ).  enzyme, termed the i n d u c i b l e a r g i n i n e d e c a r b o x y l a s e , has s i n c e been  10  A R G NINE  CO.  -PUTRESCINE  AGMATINE  UREA  Number  Enzyme  1 2  arginine decarboxylase agmatine u r e o h y d r o l a s e  FIGURE 5.  A r g i n i n e c a t a b o l i s m i n E. c o l i  11  p u r i f i e d and  s t u d i e d i n t e n s i v e l y by  pentamer o f m o l e c u l a r  The  In c u l t u r e s of n e u t r a l pH,  Morris  cannot be d e t e c t e d ,  and Pardee (1965, 1966)  optimum pH  is  5.2.  a c o n d i t i o n i n which the i n d u c i b l e E.coli.„continues to d e c a r b o x y l a t e solved  T h i s enzyme, a t e t r a m e r , has  dearginine.  t h i s dilemma by d e m o n s t r a t i n g  presence of a second a r g i n i n e d e c a r b o x y l a s e , enzyme,,  It is a  weight 820,000, c o n t a i n i n g ten moles of p y r i d o x a l  phosphate p e r mole o f enzyme--  carboxylase  Boeker:, et al.=, (1971).  the  the s o - c a l l e d b i o s y n t h e t i c  an a b s o l u t e  requirement f o r p y r i d o x a l  2+ phosphate and Mg  .  I t s pH  optimum i s 8.4  l a c k i n g t h i s enzyme have been i s o l a t e d L e i f e r and P o i n d e x t e r , the g e n e t i c l o c i parated C.  1970).  (Wu  and M o r r i s , 1973).  ( M o r r i s and J o r s t a d , 1970  Mutants and Maas,  P r e l i m i n a r y mapping s t u d i e s i n d i c a t e d t h a t  f o r the i n d u c i b l e and b i o s y n t h e t i c enzymes are w i d e l y  (Maas, L e i f e r and P o i n d e x t e r , C o n t r o l of A r g i n i n e  se-  1970).  Biosynthesis  P h e n o t y p i c a l l y , a r g i n i n e b i o s y n t h e s i s i n E_. c o l i K comprises e i g h t steps for  ( F i g . 1).  G e n o t y p i c a l l y , however, n i n e genes (arg A to arg I) code  these f u n c t i o n s .  Arg  I and .arg F determine the s y n t h e s i s of two  zymes of OTCase i n E_. c o l i K12 E l s e v i e r s , Cunin and Salmonella 1971).  ( G l a n s d o r f f , Sand and V e r h o e f , 1967  G l a n s d o r f f , 1972).  and E_. c o l i B  Only f o u r o f the n i n e genes are c l u s t e r e d arg H  c o n t r o l l e d coordinately with  ( G l a n s d o r f f , 1965). arg C,B,H.  and  Only a s i n g l e OTCase i s found i n  typhimurium (Syvanen and Roth, 1972)  arg E, arg C, a r g B and  iso-  (Jacoby,  the sequence  being:  However, arg E i s not  Nonsense mutations i n a r g C or  arg B are p o l a r f o r arg H but nonsense mutations i n arg E are n o n - p o l a r for  the remaining genes (Jacoby, 1972).  and  Glansdorff  (1972) i n d e p e n d e n t l y  Jacoby  (1972) and  Elseviers,  proposed, t h e r e f o r e , t h a t arg ECBH  Cunin  12  represents gently  two  operons, arg E and  arg CBH,  which are t r a n s c r i b e d d i v e r -  from an i n t e r n a l operator-promoter complex.  the u n l i n k e d opposite  gene arg I , are u n u s u a l i n t h a t they are o r i e n t e d i n  d i r e c t i o n to the other genes o f the a r g i n i n e r e g u l o n ,  t e r clockwise  (Jacoby, 1971,  W there  i s a r e g u l a t o r y p r o t e i n (Jacoby and and V o g e l , 1970). i t s own  cess a r g i n i n e has  In arg R  +  with the  i . e . coun-  1972).  In E_. c o l i s t r a i n s B,C,K, and  represses  Arg E, t o g e t h e r  i s a gene, arg R, whose product  G o r i n i , 1969,  Udaka, 1970  s t r a i n s of s t r a i n s K,W,  biosynthesis  ( G o r i n i , 1962).  o r C,  and  Hirvonen  excess  arginine  In s t r a i n B, however,  a s l i g h t i n d u c t i v e e f f e c t ( G o r i n i , 1962)!  catabolite repression i s also operative  ex-  A form o f  i n t h i s s t r a i n s i n c e g l u c o s e grown  c e l l s c o n t a i n much lower enzyme l e v e l s than g l y c e r o l grown c e l l s ( G o r i n i , and  Gundersen, 1961). R e p r e s s i o n i n s t r a i n K r e q u i r e s both the arg R p r o t e i n and  arginine  s i n c e arg R mutants c o n t a i n d e r e p r e s s e d enzyme l e v e l s even i n the presence of a r g i n i n e  ( G o r i n i , 1962).  K transductant  Jacoby and  G o r i n i (1969) found t h a t a  r e c e i v i n g the a r g R l o c u s from s t a i n B has  t i o n i n c l u d i n g low  l e v e l i n d u c t i o n by  B-type  strain  regula-  a r g i n i n e , whereas a s t r a i n B r e -  c i p i e n t of the arg R l o c u s from s t a i n K becomes a r g i n i n e r e p r e s s i b l e . Karlstrom  and  G o r i n i (1969) noted t h a t s t r a i n B i s r e p r e s s i b l e by  when grown at temperatures above 39°C. (1969) r e p o r t e d  c o n t r o l of a r g i n i n e biosynthesis  similar.  Gorini  t h a t s i n g l e amino a c i d changes i n the arg R p r o t e i n o f  s t r a i n B produces K-type r e p r e s s i b i l i t y . the  Subsequently, Jacoby and  arginine  In s t r a i n K,  a r g i n i n e and  I t was  i n the two  proposed, t h e r e f o r e , strains i s basically  the R p r o t e i n i n t e r a c t to  repress  that  13  enzyme f o r m a t i o n . and  I n s t r a i n B, the R p r o t e i n , alone, mediates  repression  i n t e r a c t i o n o f the p r o t e i n w i t h a r g i n i n e o r an a r g i n i n e d e r i v a t i v e  decreases t h i s a b i l i t y .  Presumably, p o i n t mutations i n the R p r o t e i n  o f s t r a i n B a l t e r s the i n t e r a c t i o n w i t h a r g i n i n e so that i t o n l y b i n d s t o the o p e r a t o r s  i n the presence o f the amino a c i d (Jacoby and G o r i n i , 1969).  Recently, arg R p r o t e i n . should  also  repressor  Urm e t al.(1973) have r e p o r t e d T h i s development should  facilitate  an i n v i t r o assay f o r  the  now enable i t s p u r i f i c a t i o n and  s t u d i e s o f the r e l a t i v e b i n d i n g  o f a r g i n i n e to the  from s t r a i n s B and K.  Regulation  of arginine biosynthesis  i n E_. c o l i i n v o l v e s both  t i o n of enzyme a c t i v i t y and r e p r e s s i o n of enzyme s y n t h e s i s .  inhibi-  Udaka (1966)  suggested t h a t i n a l l organisms d e r i v i n g o r n i t h i n e by t r a n s a c e t y l a t i o n , the second enzyme, N - a c e t y l - y - g l u t a m o k i n a s e i s i n h i b i t e d by a r g i n i n e , whereas i n a l l organisms forming o r n i t h i n e by d i r e c t h y d r o l y s i s o f N - a c e t y l o r n i t h i n e , i t i s the f i r s t  enzyme o f the pathway, N - a c e t y l g l u t a m a t e s y n t h e t a s e  which i s i n h i b i t e d by a r g i n i n e . The  r e g u l a t i o n o f enzyme s y n t h e s i s  as w e l l as t r a n s c r i p t i o n a l c o n t r o l s .  i n E_. c o l i i n v o l v e s t r a n s l a t i o n a l  The a d d i t i o n o f r i f a m p i c i n  by excess a r g i n i n e t o p r e v i o u s l y d e r e p r e s s e d , . c u l t u r e s r o f i n a decrease i n the r a t e o f s y n t h e s i s sis  (Vogel  e t a l . , 1971).  r i f a m p i c i n alone,  implying  followed  E._,c6li W r e s u l t s  o f the enzymes o f a r g i n i n e  biosynthe-  T h i s d e c r e a s e i s n o t observed i n the presence o f that i n the presence o f the arg R p r o t e i n and  a r g i n i n e , the t r a n s l a t i o n o f mRNA from the a r g i n i n e genes i s  retarded.  M c C e l l a n and V o g e l (1972), V o g e l , K n i g h t and V o g e l (1972) have shown a l s o t h a t mRNA from the arg ECBH c l u s t e r , decays a t a f a s t e r r a t e d u r i n g sion.  Furthermore, s t r e p t o m y c i n  o r t e t r a c y c l i n e reduces the  repres-  differential  14  rate of N-acetylornithine-6-transaminase  s y n t h e s i s o n l y under c o n d i t i o n s  of d e r e p r e s s i o n , l e a d i n g V o g e l e t a l . , (1971) to reason  t h a t some f a c e t of  the t r a n s l a t i o n a l machinery must be  altered during repression.  Maas (1971) and  (1969) showed t h a t a r g i n y l tRNA i s  probably  L e i s i n g e r and  not m o d i f i e d  from c e l l s h a r v e s t e d  Vogel  Celis  and  d u r i n g r e p r e s s i o n s i n c e p u r i f i c a t i o n of tRNA s p e c i e s i n the derepressed  or r e p r e s s e d  s t a t e s produced  identi-  c a l column chromatographic e l u t i o n p r o f i l e s . T r a n s c r i p t i o n a l c o n t r o l over the enzymes o f a r g i n i n e b i o s y n t h e s i s has  r e c e n t l y been shown by h y b r i d i z a t i o n experiments.  ECBH mRNA t r a n s c r i b e d d u r i n g d e r e p r e s s i o n sion  (Kryzek  and  Rogers, 1972,  The  amount of arg  exceeds t h a t made d u r i n g  Rogers et a l . , 1971).  repres-  Transcriptional  c o n t r o l i n genes o t h e r than the arg ECBH c l u s t e r has not been observed because of t e c h n i c a l d i f f i c u l t i e s .  II.  POLYAMINE METABOLISM A.  D i s t r i b u t i o n of Polyamines  Polyamines are low m o l e c u l a r the s y s t e m a t i c 1972).  and  weight o r g a n i c bases.  Table  I  common names of s e v e r a l polyamines (Tabor and  provides Tabor,  S y s t e m a t i c Name  Common Name  1.3- diaminopropane  -  1.4- diaminobutane  putrescine  1.5- diaminopentane  cadaverine  N-(3-aminopropyl)-1,4-diaminobutane  spermidine  N ,N ''-Bis (3-aminopropyl) -1,4-diaminobutane  TABLE I .  Common polyamines  spermine  16  Polyamines a r e u b i q u i t o u s l y d i s t r i b u t e d i n n a t u r e .  The h i g h e s t  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 s r e p o r t e d so f a r a r e i n b a c t e r i a and f u n g i . For example, i n E_. c o l i , s p e r m i d i n e and p u t r e s c i n e are p r e s e n t  a t con-  c e n t r a t i o n s o f 1-2 mM and 10-20'r..mM r e s p e c t i v e l y (Cohen, 1971). while  Spermine,  common i h h i g h e r organisms, so f a r has been found i n two b a c t e r i a :  B a c i l l u s stearothermophillus (Weaver and H e r b s t ,  1958).  (Stevens  and M o r r i s o n ,  1968) and P_. a e r u g i n o s a  Once thought t o occur i n o n l y a few gram p o s i -  t i v e b a c t e r i a , such as B_. s u b t i l i s and Staphylococcus  aureus  Weaver and K e i s t e r , 1958)-,more s e n s i t i v e d e t e c t i o n techniques  (Herbst, should now  enable t h e d i s c o v e r y of polyamines i n o t h e r gram p o s i t i v e s p e c i e s . v e r y h i g h l e v e l s o f spermidine a r e p r e s e n t lag  i n L a c t o b a c i l l u s casei during  phase, c e l l u l a r growth d i l u t e s t h i s p o o l to a v e r y low l e v e l  and M i c h a e l s o n ,  B.  Though  (Elliott  1969).  Synthesis  of Polyamines  E_. c o l i can s y n t h e s i z e p u t r e s c i n e by e i t h e r of two r o u t e s . mentioned e a r l i e r  ( F i g . 5 ) , i n v o l v e s the d e c a r b o x y l a t i o n  d i r e c t l y , p u t r e s c i n e can a r i s e from the d e c a r b o x y l a t i o n (1940) d e s c r i b e d an o r n i t h i n e d e c a r b o x y l a s e  The f i r s t ,  of a r g i n i n e .  More  of o r n i t h i n e .  Gale  which appeared i n o r n i t h i n e  supplemented E_. c o l i c u l t u r e s under c o n d i t i o n s of low pH and poor a e r a t i o n . Termed the i n d u c i b l e o r n i t h i n e d e c a r b o x y l a s e ,  t h i s p r o t e i n i s heat  and has a pH optimum o f 5.3 (Tabor and Tabor, 1972).  labile  C e l l s of E_. c o l i  grown i n unsupplemented minimal media a t n e u t r a l pH a l s o c o n t a i n a b i o s y n t h e t i c o r n i t h i n e decarboxylase  (Morris et al.1970).  T h i s enzyme has a  pH optimum of 8.4; i t i s i n h i b i t e d by p u t r e s c i n e and spermidine  (Morris  17  et  a l , . ,1970), but i t s a c t i v i t y i s enhanced by guanosine o r deoxy guano s i n e  triphosphate  ( H o l t t a , Janne and P i s p a , 1972).  Both o r n i t h i n e  l a s e s have an o b l i g a t e requirement f o r p y r i d o x a l phosphate. mutants  l a c k i n g e i t h e r a c t i v i t y have not been  decarboxyTo d a t e ,  isolated.  The b i o s y n t h e s i s o f s p e r m i d i n e i s i l l u s t r a t e d i n P i g . 6. methionine (SAM)  S-Adeno:syl-  d e c a r b o x y l a s e i s an octamer o f 113,000 m o l e c u l a r weight  (Wickner, Tabor and Tabor, 1970); i t c o n t a i n s p y r u v a t e not p y r i d o x a l 2+ phosphate, as a p r o s t h e t i c group, r e q u i r e s Mg SAM  , i s specific for L-(-)-  and i s n o t s t i m u l a t e d by p u t r e s c i n e ( Z a p p i a e t a l . ,  1969).  Propylamine t r a n s f e r a s e has been p u r i f i e d from E. c o l i W (Bowman, Tabor and Tabor, 1973).  The enzyme i s a dimer; i t has no c o f a c t o r  ment and i t s pH optimum is;iO.3 w i t h p u t r e s c i n e as s u b s t r a t e .  require-  Spermidine  and c a d a v e r i n e are a l s o s u i t a b l e s u b s t r a t e s f o r the enzyme i n vitro-. V Dion ;  and Cohen (1972) d e t e c t e d c a d a v e r i n e and a s p e r m i d i n e analogue, N-C3-ami.nopropyl)-l,5-diaminopentane, i n a polyamine d e p l e t e d mutant o f E_. c o l i grown i n the presence of l y s i n e . s u b s t r a t e f o r the enzyme i n v i v o . d e c a r b o x y l a s e have not been C.  Cadaverine, then, i s also a s u i t a b l e Mutants l a c k i n g  t h i s a c t i v i t y or  SAM  isolated.  C o n t r o l o f Polyamine  Tabor and Tabor  K12  Synthesis  (1969) r e p o r t e d t h a t , d e s p i t e s e v e r e a r g i n i n e r e -  s t r i c t i o n , up t o 18% o f the a v a i l a b l e a r g i n i n e i s used f o r polyamine s y n t h e s i s i n an E_. c o l i a r g  strain.  Such data argue t h a t polyamines, a l -  though t h e i r f u n c t i o n i s u n c e r t a i n , are important i n c e l l u l a r  metabolism.  The e v o l u t i o n o f two r o u t e s f o r t h e i r s y n t h e s i s s t r e n g t h e n s t h i s premise. A c l a s s i c a l means o f r e g u l a t i o n , however, awaits d i s c o v e r y .  18  METHIONINE  S-ADENOSYLMETHIONINE + ATP  2  SPERMIDINE  DECARBOXYLATED S - A D E N O S Y L METHIONINE  METHYLTHIOADENOSINE  PUTRESCINE  Enzyme  Number  S-adenosylmethionine s y n t h e t a s e S-adenosylmethionine d e c a r b o x y l a s e propylamine t r a n s f e r a s e  1 2 3  FIGURE 6.  Spermidine b i o s y n t h e s i s i n E_. c o l i .  19  When growing i n minimal media, E_. c o l i from o r n i t h i n e ( M o r r i s and K o f f r o n , 1968).  d e r i v e s 95% o f i t s polyamines T h i s route i s e n e r g e t i c a l l y  more p r a c t i c a l , s i n c e ATP i s expended i n the arginine  (Fig. 1).  anabolism o f o r n i t h i n e t o  Only i f a r g i n i n e i s i n excess a r e polyamines synthe-  s i z e d from a r g i n i n e ( M o r r i s and K o f f r o n , 1968).  T h i s suggests t h a t  poly-  amine s y n t h e s i s i s r e g u l a t e d , Tabor and Tabor (1969) demonstrated t h a t exogenous r a t h e r than endogenous a r g i n i n e was s e l e c t i v e l y c a t a b o l i z e d t o polyamines i n E_. c o l i .  The genes spe A ( b i o s y n t h e t i c a r g i n i n e decarboxy-  l a s e ) , spe B (agmatine u r e o h y d r o l a s e ) and met K (SAM s y n t h e t a s e ) a r e c l u s t e r e d i n E_. c o l i and perhaps form an operon (Maas, 1972). for  A r g P,  coding  an a r g i n i n e permease, l i e s w i t h i n one minute o f t h i s c l u s t e r on t h e  E_. c o l i map  ( T a y l o r , 1970).  the c h a n n e l i n g  Maas (1972) h a s , t h e r e f o r e , s p e c u l a t e d  that  o f exogenous a r g i n i n e i n t o polyamines may be due t o a c o -  o r d i n a t e c o n t r o l o f these genes. In t h e i r study, the Tabors (1969) a l s o i n f e r r e d t h a t a homeostatic b a l a n c e e x i s t s between p u t r e s c i n e and spermidine. t i o n o f an a r g i n i n e auxotroph, the p u t r e s c i n e s i g n i f i c a n t l y while  D.  limita-  c o n c e n t r a t i o n was reduced  the spermidine l e v e l remained normal.  D e r i v a t i v e s o f Polyamines  First  r e p o r t e d by Dubin and R o s e n t h a l (1960), N - a c e t y l a t e d  were thought to be normal c o n s t i t u e n t s of E_. c o l i . s i s , however, r e v e a l e d handling  During a r g i n i n e  A more c a u t i o u s  analy-  t h a t these d e r i v a t i v e s a r e a c t u a l l y a r t i f a c t s o f  p r o c e d u r e s , and 98% o f t h e polyamines a r e not n o r m a l l y  (Tabor, 1968).  polyamines  T h i s phenomenon occurs  i n cells either chilled  acetylated during  20  h a r v e s t i n g o r grown i n the presence o f excess p u t r e s c i n e , spermidine and/or spermine  (Tabor and Hobbs, 1970).  A Bseudomonad i s o l a t e d by Kim  (1966), l a c k s s p e r m i d i n e but c o n t a i n s  p u t r e s c i n e and 2 - h y d r o x y p u t r e s c i n e ( T o b a r i and Tchen, 1971).  The amount 2+  of  t h i s d e r i v a t i v e bound to ribosomes v a r i e s i n v e r s e l y w i t h the Mg  c o n c e n t r a t i o n , as does the spermidine bound to E_. c o l i , and H u r w i t z , 1969). t h r e e hydrogen spermidine. S^. aureus  Hence, i t was  ribosomes  (Rosano  proposed t h a t 2 - h y d r o x y p u t r e s c i n e , w i t h  bonding s i t e s , c o u l d assume the r o l e s n o r m a l l y p l a y e d by  2-Hydroxyputrescine has now  been found i n B a c i l l u s megatherium,  (Tehen, p e r s o n a l communication) and P_. a c i d o v o r a n s ( K a r r e r , Bose  and Warren, 1973).  S t u d i e s on the h y d r o x y l a t i o n r e a c t i o n have not been  reported. At  the end o f l o g a r i t h m i c growth, E_. c o l i n o r m a l l y c o n v e r t s a l l of  i t s spermidine and 50% of i t s g l u t a t h i o n e to g l u t a t h i o n y l s p e r m i d i n e and Tabor, 1972).  The molecule has the f o l l o w i n g sequence:  cysteinylglycylspermidine.  Y gl -  u  When s t a t i o n a r y phase c e l l s are d i l u t e d  f r e s h media (pH 7.0), t h e r e i s a r a p i d l o s s o f t h i s d e r i v a t i v e w i t h an i n c r e a s e i n f r e e s p e r m i d i n e .  Since l i t t l e  (Tabor t  a  m  yl  -  into  coincident  i s known o f the f u n c -  t i o n s of spermidine o r g l u t a t h i o n e i n v i v o , i t i s not s u r p r i s i n g t h a t the adduct i s even more e n i g m a t i c . E.  Catabolism of  P u t r e s c i n e may  Polyamines  be o x i d a t i v e l y deaminated  to y-aminobutyraldehyde  p u t r e s c i n e o x i d a s e i n Mycoplasma ( E v e l y n , 1967) (Fig.  7A, pathway A ) .  and M i c r o c o c c i  (De Sa,  by 1972).  In M i c r o c o c c u s rubens, p u t r e s c i n e o x i d a s e i s a  f l a v o p r o t e i n c o n t a i n i n g FAD.  The enzyme i s unusual among f l a v o p r o t e i n  dases i n t h a t i t c o n t a i n s o n l y one mole o f FAD  oxi-  per mole o f enzyme (De Sa,  21  PUTRESCINE ©C-KETOGLUTARATE  °2 \ /  1  GLUTAMATE 2T-AMINOBUTYR A L D E H Y D E  PYRROLINE  +NAD ^-AMINOBU  YRIC  ACID <*KG  4  •GLU SUCCINIC S MIALDEHYDE  pathway A: pathway B:  +NADP  SUCCINIC ACID  Enzyme  Number  putrescine  1 2 3 4 5  oxidase  p u t r e s c i n e - a - k e t o g l u t a r a t e transaminase Y-aminobutyraldehyde dehydrogenase Y-aminobutyrate-a-ketoglutarate transaminase s u c c i n i c semialdehyde dehydrogenase  FIGURE 7A:  Polyamine c a t a b o l i s m :  putrescine  22  1972).  I n c e r t a i n pseudomonads and E_. c o l i ,  c a t a b o l i z e d non-o'xidatively t o Y (Fig.  7A, pathway B ) .  tive reactions, Y  - a m  - a m  i °b n  u t  y  r a  however, p u t r e s c i n e i s  ldehyde  (Bachrach, 1970)  The product of both t h e o x i d a t i v e and non-oxida-  i ° b u t y r a l d e h y d e , i s u n s t a b l e and c y c l i z e s  spontan-  n  eously to A p y r r o l i n e . 1  Y-Aminobutyraldehyde  dehydrogenase  i s induced i n P_. f l u o r e s c e n s ,  by growth on p u t r e s c i n e (Jakoby and F r e d e r i c k s , 1959).  Kim (1963),  iso-  l a t e d a mutant of E_. c o l i B which u t i l i z e s p u t r e s c i n e as the s o l e source of  carbon and n i t r o g e n .  I n t h i s mutant, p u t r e s c i n e - a - k e t o g l u t a r a t e  transaminase i s c o n s t i t u t i v e w h i l e the enzymes c o n v e r t i n g dehyde t o s u c c i n i c a c i d a r e i n d u c i b l e .  _ a m  i °butyraln  These enzymes o c c u r a l s o i n  P_. a e r u g i n o s a (Nakamura, 1960) and E_. c o l i K12 In  Y  (Dover and H a l p e r n , 1972).  the l a t t e r organism, the enzymes a r e e i t h e r r e p r e s s e d o r induced c o -  o r d i n a t e l y under a v a r i e t y o f growth c o n d i t i o n s , i m p l y i n g a common c o n t r o l (Dover and H a l p e r n , 1972). When N e i s s e r i a per f l a v a , S. marsescens  and M.' rub ens a r e grown i n the  prescence o f polyamines, spermidine i s o x i d i z e d (Fig. et  7 B, r e a c t i o n A ) .  is  The S e r r a t i a enzyme has been p u r i f i e d by Campello  al-.,(1965) and r e q u i r e s FAD as c o f a c t o r .  a l s o o x i d i z e ^spermidine.  Pseudomonads and Mycoplasma  I n doing s o , p u t r e s c i n e and n o t 1,3-diaminopropane  t h e u l t i m a t e diamine formed  these organisms  (Tabor and Tabor, 1972)  ( F i g . 7B, r e a c t i o n B ) .  The i n a b i l i t y o f  to o x i d i z e the p r i m a r y amino groups o f s p e r m i d i n e  undoubted-  l y r e f l e c t s the b a c t e r i a l t o x i c i t y of t h e mono and d i a l d e h y d e p r o d u c t s (Cohen,  1971).  23  SPERMIDINE NH2(CH2)3NH(CH2)4NH2  ^-AMINOBUTYR A L D E H Y D E  6 -AMINOPROPI O N A L D E H Y D E  r e a c t i o n A: r e a c t i o n B:  FIGURE 7B:  Polyamine c a t a b o l i s m :  spermidine  24 F.  S i g n i f i c a n c e o f Polyamines 1.  Polyamines  In 1957, Hershey positive, identified  are  noted t h a t b a c t e r i o p h a g e T2 c o n t a i n s two n i n h y d r i n  non amino a c i d  compounds.  (1958)  f o r 40-50% of the i d e n t i f i a b l e c a t i o n s w i t h i n the phage Ames and Dubin  (1960) s t a t e d t h a t almost a l l o f the polyamines  d i s p l a c e d from a permeable 0  result  Ames, Dubin and R o s e n t h a l  these substances as p u t r e s c i n e and s p e r m i d i n e and showed t h a t  they account particles.  and b a c t e r i o p h a g e  i m p l i e d t h a t polyamines  mutant of T4 by washing.  a r e unnecessary  Although  this  f o r i n f e c t i v i t y , phage  r e p r o d u c t i o n i s s t r o n g l y i n f l u e n c e d by these bases  (Cohen,  1971).  As i s w e l l known, T4 r l l mutants a r e unable to m u l t i p l y i n E_. c o l i K12( A) (Benzer, 1957) u n l e s s spermidine i s p r e s e n t e x t e r n a l l y Ames and Ames, 1965).  (Ferroluzzi-  S i n c e i n f e c t i o n l e a d s to a leakage of p u t r e s c i n e ,  the a d d i t i o n o f s p e r m i d i n e , presumably,  r e s t o r e s the i n t r a c e l l u l a r p o l y -  amine b a l a n c e . Ordinarily,  when T4DD mutants i n f e c t E_. c o l i B, the onset o f phage  DNA s y n t h e s i s i s d e l a y e d  (Dion and Cohen, 1971).  I n f e c t i o n i n the p r e -  sence o f s p e r m i d i n e but n o t i n the presence o f p u t r e s c i n e , s h o r t e n s t h i s d e l a y c o n s i d e r a b l y (Dion and Cohen, 1971). d e p l e t e d i n polyamine  content r e s u l t s  (Dion and Cohen, 1972).  T4 i n f e c t i o n o f E_. c o l i mutants  i n v e r y r e t a r d e d phage development  The a d d i t i o n o f p u t r e s c i n e , c a d a v e r i n e o r s p e r -  midine j u s t p r i o r t o i n f e c t i o n , however, promotes s u c c e s s f u l phage r e p r o duction  (Dion and Cohen, 1972).  i n f e c t i o n o f E_. c o l i 2.  i s t o be normal, polyamines  Polyamines  H e r b s t and S n e l l  From t h e s e d a t a , i t i s c l e a r  t h a t i f T4  are v i t a l .  and b a c t e r i a (1948) demonstrated  t h a t Haemophillus  parainfluenziae  25  i s unable to grow u n l e s s s u p p l i e d w i t h polyamines. c o n d i t i o n a l polyamine their  The i s o l a t i o n of  a u x o t r o p h i c s t r a i n s o f E_. c o l i has been a c h i e v e d ;  growth under r e s t r i c t i v e c o n d i t i o n s i s v e r y slow  J o r s t a d , 1970  and Maas, L e i f e r  the c e l l s form snakes connection  :  and P o i n d e x t e r , 1970).  (Maas, L e i f e r  •:• between polyamines  Inouye and Pardee  ( M o r r i s and In one  and P o i n d e x t e r , 1970).  and c e l l d i v i s i o n was  strain,  A stronger  established  by  (1970) : growth of an a r g i n i n e auxotroph o f E_. c o l i  b l o c k e d b e f o r e o r n i t h i n e i s synchronous n i n e to an a r g i n i n e - s t a r v e d c u l t u r e .  f o l l o w i n g the a d d i t i o n of a r g i -  In c o n t r a s t , another a r g  mutant,  b l o c k e d a f t e r o r n i t h i n e d i v i d e s a s y n c h r o n o u s l y when a r g i n i n e i s added t o a starved culture.  I t was  suggested that an adequate  a prerequisite for c e l l division t a i n e d by heat shock  i n E_. c o l i .  (Smith and Pardee, 1970).  polyamine  Synchrony  c r e a t e synchrony.  I t was  may  distort  1972).  ob-  (1970)  and p o s t u l a t e d  l a t e r reported that  an enzyme concerned w i t h methionine b i o s y n t h e s i s i n E_. c o l i (Lomnitzer and Ron,  a l s o be  Smith and Pardee  noted the e x i s t e n c e of a heat l a b i l e p r o t e i n i n E_. c o l i t h a t i t s s e n s i t i v i t y might  may  pool i s  i s heat  I t i s p o s s i b l e , t h e r e f o r e , t h a t heat  labile  shock  the polyamine p o o l (see F i g . 6 ) .  In b a c t e r i a , s p e r m i d i n e i s p h y s i o l o g i c a l l y more a c t i v e than p u t r e scine.  D u r i n g polyamine  d e p l e t i o n i n E. c o l i , a h o m e o s t a t i c b a l a n c e  m a i n t a i n s a spermidine p o o l a t the expense o f p u t r e s c i n e (Tabor and 1969).  In Myxococcus' • xanthus, m i c r o c y s t f o r m a t i o n i s b l o c k e d by  dine but enhanced by p u t r e s c i n e ( W i t k i n and Rosenburg, 1970). sis  of s p e r m i d i n e and RNA  methionine  t o E. c o l i  appear  to be l i n k e d i n E_. c o l i .  s t i m u l a t e s b o t h RNA  The  Tabor,  spermisynthe-  The a d d i t i o n of  and spermidine syntheses  (Raina  26  Jansen and for  Cohen, 1967).  a r g i n i n e , RNA  When a r e l a x e d s t r a i n of E_. c o l i TAU  synthesis continues  and  i s starved  the spermidine l e v e l but not  p u t r e s c i n e l e v e l r i s e s d u r i n g t h i s time (Cohen e t a l . , 1967). however, does not  Spermidine,  accumulate when the organism i s s t a r v e d f o r u r a c i l .  S u r p r i s i n g l y , i n a s t r i n g e n t s t r a i n o f E_. c o l i TAU,  the a d d i t i o n of exo-  genous spermidine d u r i n g a r g i n i n e s t a r v a t i o n e f f e c t s an i n c r e a s e i n synthesis—  as i f the s t r a i n were r e l a x e d  Moreover, spermidine,  of the phosphogluconate pathway and i n E_. c o l i  (Lanning  and  RNA  (Raina, Jansen and Cohen, 1967).  at p h y s i o l o g i c a l concentrations, stimulates  6-phosphate dehydrogenase (Sanwal, 1970)  way  the  glucose-  which c a t a l y z e s the f i r s t  step  ribose i s derived mainly v i a t h i s  Cohen, 1954).  midine or the s p e r m i d i n e r p u t r e s c i n e  I t would seem, then,  r a t i o i s important  i n RNA  that  pathsper-  synthesis  and  b a c t e r i a l metabolism g e n e r a l l y .  G.  Polyamine: DNA  Interactions  The  d i s c o v e r y of. polyamines i n T-even b a c t e r i a l v i r u s e s i n d i c a t e d  t h a t polyamines c o u l d b i n d to DNA  i n vivo  (Hershey, 1957).  t r a t i o n s observed, polyamines c o u l d n e u t r a l i ze one T4 DNA  (Ames, Dubin and R o s e n t h a l ,  1958).  The  At the concen-  t h i r d to one  nature  h a l f of  of t h i s b i n d i n g i s  i o n i c and polyamines can be d i s p l a c e d i n s o l u t i o n s o f h i g h osmotic Mahler and Mehrotra (1963) showed t h a t polyamines i n c r e a s e the Tm and  t h a t of those  t e s t e d , cadaverine  p u t r e s c i n e b e i n g almost as e f f e c t i v e .  strength. of  DNA  produces the l a r g e s t increment, w i t h The  X-ray c r y s t a l l o g r a p h i c s t u d i e s  of L i q u o r i et a l . , (1967) l e d them to propose t h a t s p e r m i d i n e and  spermine form i o n i c b r i d g e s  a c r o s s the narrow groove of  h e l i x i v A Mandel (1962) Vtheori'zed^tfr  the  the  DNA spermine  27  r e f l e c t s the DNA base c o m p o s i t i o n , A-T r i c h DNA m o l e c u l e s were thought., to p r e f e r e n t i a l l y b i n d spermine more s t r o n g l y than A-T poor DNA et a l . ,  1967).  I n d i s p u t i n g t h i s p r o p o s a l , Hirschman  et a l . ,  (Liquori  (1967),  found t h a t the n e t charge o f spermine v a r i e d w i t h temperature.  When t h e  DNA:spermine i n t e r a c t i o n was measured by e q u i l i b r i u m d i a l y s i s , no c o r r e l a t i o n between spermine and DNA base c o m p o s i t i o n was observed et a l . ,  1967).  The d e m o n s t r a t i o n o f p o l y a m i n e : n u c l e i c a c i d  i n v i v o has y e t t o be made.  The i s o l a t i o n o f polyamines  t o g e t h e r , t h e r e f o r e , c o u l d merely be an a r t i f a c t  III.  (Hirschman  interactions  and n u c l e i c  acids  of extraction.  MODIFIED BASES Though common i n tRNA, m o d i f i e d bases o f DNA, i n comparison,  usual..5-Hydroxymethylcytosine  (HMC) which o c c u r s i n t h e DNA o f T-even  phages as a g l u c o s y l a t e d d e r i v a t i v e , was f i r s t and Cohen, 1952).  a r e un-  HMC t o t a l l y r e p l a c e s  d i s c o v e r e d i n 1952  (Wyatt  Cytosine i n T-even phage DNA.  T h i s i s brought about by two enzymes: c y t o s i n e deaminase, which  deaminates  d e o x y c y t i d i n e monophosphate (dCMP) to d e o x y u r i d i n e monophosphate (dUMP) and c y t o s i n e hydroxymethylase  (Cohen, 1968).  p o o l and i s p o l y m e r i z e d as such.  HMC, then, e x i s t s i n t h e  A further modification,  o c c u r s o n l y a f t e r t h e T-even DNA i s s y n t h e s i z e d (Cohen,  glucosylation,  1968).  C e r t a i n B_.. s u b t i l i s phages, such as 0e, SPOr-1 and SP8, c o n t a i n 5hydroxymethyluracil  (HMU) i n p l a c e o f thymine  (Roscoe and Tucker, 1966).  The b i o s y n t h e s i s o f HMU i n phage i n f e c t e d c e l l s a l s o o c c u r s at the nucleotide level.  The i n c o r p o r a t i o n o f thymine  i n t o phage DNA i s p r e v e n t e d  by a thymidine t r i p h o s p h a t e n u c l e o t i d o h y d r o l a s e which h y d r o l y z e s deoxythymidine t r i p h o s p h a t e (dTTP) t o dteoxythymidine monophosphate (dTMP) (Roscoe, 1969).  I n i t s absence, thymine  i s i n c o r p o r a t e d i n t o 0e DNA w i t h o u t a  28  loss of i r i f e c t i v i t y  (Marcus and Newlon, 1971).  PBS-1, another B_. s u b t i l i s phage, c o n t a i n s u r a c i l i n s t e a d of thymine (Tomita  and Takahashi,  1969).  A n o v e l enzyme, d e o x y c y t i d i n e t r i p h o s p h a t e  deaminase, which deaminates dCTP to dUTP, i s r e s p o n s i b l e f o r the i n the p o o l (Tomita and Takahashi, s i n e appears i n PBS-1  DNA  1969).  latter  The q u e s t i o n of j u s t how  cyto-  has not been answered but r e g u l a t i o n of dCTP  deaminase i s i n d i c a t e d . The B y s u b t i l i s phage, SP-15, has r e c e n t l y been shown to c o n t a i n d i — hydroxypentyluracil  (DHPU.) (Marmur e t a l . , 1972  and Brandon e t a l . , 1972).  The base o n l y p a r t i a l l y r e p l a c e s thymine and e x i s t s i n the DNA  i n a glu-  c o s y l a t e d form. Since t h e i r b i o s y n t h e s e s  can be complex (Cohen, 1968) , these m o d i f i e d  bases presumably e v o l v e d f o r a reason or reasons. m o d i f i c a t i o n i s p r o b a b l y the p r o t e c t i o n of DNA vivo.  In the T-even phages, HMC  molecule  ing  are unable  h o s t s supports  major f u n c t i o n of  from n u c l e a s e d i g e s t i o n i n  serves as a d i s t i n c t i v e s i t e on the  at which g l u c o s y l a t i o n can o c c u r .  g l u c o s y l a t e d DNA  One  That phages c o n t a i n i n g non-  to m u l t i p l y i n r e s t r i c t i v e or n u c l e a s e c o n t a i n -  this idea.  O f t e n , work l e a d i n g to the i d e n t i f i c a t i o n of m o d i f i e d bases was t i a t e d by a common c u r i o s i t y . not correspond of  ylthymine  0W-14 (NpT)  phage,  ini-  That i s , the Tin and the buoyant d e n s i t y d i d  to the same p e r c e n t  the P_. a c i d o v o r a n s  vation.  DNA  GC  value.  In c h a r a c t e r i z i n g the  0W-14, K r o p i n s k i (1970; made a s i m i l a r  c o n t a i n s , i n a d d i t i o n to the r e g u l a r f o u r bases,  DNA.  obser-  N-putrescin^  ( K r o p i n s k i , Bose and Warren, 1973), which r e p l a c e s  h a l f o f the thymine r e s i d u e s i n the phage  DNA  almost  29  An obvious q u e s t i o n which a r o s e was the b i o s y n t h e t i c o r i g i n o f NpT i n phage i n f e c t e d c e l l s .  I t was the g o a l o f t h i s t h e s i s , then,  b l i s h the o r i g i n o f the p u t r e s c i n e moiety of the base.  to e s t a -  An e s s e n t i a l p r e -  l i m i n a r y to t h i s work was the e l u c i d a t i o n o f a r g i n i n e and polyamine metab o l i s m i n the u n i n f e c t e d  host.  30  MATERIALS AND METHODS  .1.  ORGANISMS Pseudomonas a c i d o v o r a n s 29, o b t a i n e d from R.Y. S t a n i e r , was used  e x c l u s i v e l y throughout p l a t e s supplemented e v e r y two months.  t h i s study.  I t was m a i n t a i n e d on minimal  w i t h disodium s u c c i n a t e . B a c t e r i o p h a g e 0W-14  agar  A f r e s h p l a t e was prepared  stocks^; were s t o r e d i n complex  media a t 4°C over c h l o r o f o r m .  I I . MEDIA M a n n i t o l L u r i a b r o t h (MLB) c o n t a i n e d ( g / 1 ) : t r y p t o n e , 10.0; y e a s t e x t r a c t , 5.0; N a C l , 2.5; and d - m a n n i t o l , 1.0. (DMLB) was MLB d i l u t e d t e n f o l d .  D i l u t e mannitol L u r i a broth  007 minimal medium  modified to contain (g/1): ( N H ^ S O ^  , 2.0; N a H P 0 , 6.0; K H P 0 , 3.0; 2  4  M g C l , 0.4; F e C l - 6 H 0 , 0.0008; and C a C l ; 0.017. 2  3  2  ( C l a r k , 1968) was  2  2  Disodium s u c c i n a t e  (4 g/1) was added s e p a r a t e l y as a s t e r i l e 20% s o l u t i o n . Bacto-Agar  4  F o r s o l i d media,  ( D i f c o ) was added t o the a p p r o p r i a t e medium 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 1.5%.  III.  GROWTH OF BACTERIA All  c u l t u r e s were grown a t 30°C.  Routinely, l i q u i d  a g i t a t e d on a M e t a b o l y t e G-77 shaker water b a t h Co., New Brunswick,  c u l t u r e s were  (New Brunswick  Scientific  N.J.) s e t a t 250 r e v o l u t i o n s per minute (RPM) .  1 c u l t u r e s were grown i n a fermenter  Ten  (Fermentation D e s i g n , A l l e n t o w n , P a . ) ,  a d j u s t e d t o 250 RPM and a flow r a t e of 5000 cc o f a i r p e r minute.  Cell  31  d e n s i t y was  determined w i t h a Klett-Summerson c o l o r i m e t e r  t u r i n g Co.,  New  York, N.Y.)  equipped w i t h a number 54  were c o n v e r t e d to o p t i c a l d e n s i t y  (OD)  u n i t s by  ( K l e t t Manufac-  filter.Klett  reference  units  to a s t a n d a r d  curve.  IV.  PHAGE TITRATION The  agar l a y e r technique of Adams (1959) was  forming u n i t s  (PFU)/ml of l y s a t e .  l a y e r s c o n t a i n e d 0.75  V.  PREPARATION OF  and  1.5%  U s i n g MLB  used to assay plaque  p l a t e s , the  top and  bottom  agar, r e s p e c t i v e l y .  PHAGE STOCKS  Q Cultures  were grown to 2.5-5.0 x 10  c e l l s / m l and  s u f f i c i e n t phage  added t o g i v e a m u l t i p l i c i t y o f i n f e c t i o n (m.o.i.) of 0.1. c o n t i n u e d u n t i l l y s i s was  complete".  Incubation  T i t e r s o f 10^PFU/ml were  was  generally  obtained. VI.  PURIFICATION OF PHAGE D e o x y r i b o n u c l e a s e (DNase) 1 was  added to l y s a t e s at 1 ug/ml and  c u b a t i o n c o n t i n u e d f o r 60 min.  B a c t e r i a l d e b r i s was  tion  The  (10,000 x g f o r 10 m i n ) .  x g f o r 2 hrs  and  were performed at  VII.  the p e l l e t resuspended i n DMLB.  0.1  ml  s u s p e n s i o n was  transferred  d i l u t e d one  centrifugations  made i n 5 M NaCI.  t o 0.2  ml  5 M NaCI.  thousand f o l d i n water.  p l a t i n g , f u r t h e r d i l u t i o n s were a l s o made i n water. of d i l u t i o n s was  All  at 14,000  0W-14  sample of phage i n DMLB was  A f t e r 16 h r s , the  centrifuged  centifuga-  4°C.  OSMOTIC SENSITIVITY OF An  supernatant was  removed by  in-  As  For  a control, a series  PFU's were determined as i n s e c t i o n  IV.  32  VIII.  UTILIZATION OF COMPOUNDS AS NITROGEN SOURCE M i n i m a l agar p l a t e s were used except t h a t MgSO^ and  (Oxoid) r e p l a c e d MgC^ omitted.  and Bacto-Agar, r e s p e c t i v e l y ,  B a c t e r i a were washed w i t h  0.05  Ionagar No.  (NH^^SO^  M KH^PO^ b u f f e r (pH  2  was  7.0).,prior  g to  plating.  P l a t i n g was  flamed g l a s s r o d .  performed by s p r e a d i n g  Approximately 1.0  p l a c e d i n the c e n t e r of each p l a t e . ble IX.  i f growth was  mg  2.0  x 10  c e l l s with  of the t e s t compound was  A compound was  a  then  c o n s i d e r e d non  utiliza-  not observed w i t h i n t h r e e days.  UPTAKE'OF RADIOACTIVE COMPOUNDS g C u l t u r e s were grown to a d e n s i t y of 2.5  f i e d medium.  x 10  c e l l s / m l i n (.007  Then the a p p r o p r i a t e compound ( n o n - r a d i o a c t i v e ) was  modi-  added to  g i v e a c o n c e n t r a t i o n of 20 ug/ml. I n c u b a t i o n was c o n t i n u e d and when the g c e l l d e n s i t y was 5.0 x 10 c e l l s / m l , the r a d i o a c t i v e compound was added to g i v e 0.25  uc/ml ( i . e . 0.25  For the t r a n s p o r t o f c i e n t phage was  uc/20ug o f compound). 14  C-putrescine  by phage i n f e c t e d c e l l s , s u f f i 8 added to a c u l t u r e at 5 x 10 c e l l s / m l to g i v e a m.o.i. of 14  10.  A f t e r a l l o w i n g 1.0  min  for adsorption,  C-putrescine  was  added  as  d e s c r i b e d above. Ten  seconds a f t e r adding the r a d i o a c t i v e t e s t s u b s t r a t e , a 1.0  sample was 60 min One  removed; f u r t h e r samples were taken every 5 min  for uninfected cultures.  h a l f ml  of each sample was  F i l t e r Corp., B e d f o r d , then washed w i t h 2.0 to  0.5  the TCA  ml  thereafter for  I n f e c t e d c u l t u r e s were sampled f o r 80  passed through a 0.45  urn f i l t e r  The  remaining  0.5  ml was  t r i c h l o r o a c e t i c a c i d (TCA); a f t e r 20 min.  p r e c i p i t a b l e m a t e r i a l was  c o l l e c t e d on a 0.45  um  min.  (Millipore  Mass.)pre-^wetted w i t h minimal medium; the f i l t e r  ml o f minimal medium.  i c e c o l d 10%  ml  filter  was  added  on i c e , previously  33  moistened  w i t h 5% TCA;  which was  a l s o passed through the  X.  the tube was  r i n s e d w i t h 2.0 ml o f c o l d 5%  TCA  filter.  ISOLATION OF POLYAMINES A.  From b a c t e r i a  The method of R a i n a polyamines.  (1963.) was  adopted  C e l l s were c e n t r i f u g e d and washed i n a h a l f volume of 0.05  KR^PO^ (pH 7.0)  buffer.  D u r i n g these p r o c e d u r e s , c h i l l i n g was  The washed p e l l e t s were resuspended min.  f o r the r o u t i n e e x t r a c t i o n of  i n c o l d 5% TCA  F o l l o w i n g c e n t r i f u g a t i o n at 4°C, the TCA  t h r e e times w i t h an equal volume of e t h e r . n a t a n t was added 2.5  a d j u s t e d t o 12-13  w i t h 5 N NaOH.  volume o f Ji^-butanol.  4  The b u t a n o l e x t r a c t i o n was  supernatant was e x t r a c t e d  (62.5 g:9.0  g) and an equal  shaken at room temperature  min. , a f t e r which the b u t a n o l phase was 6N HC1.  on i c e f o r 20  To 10 ml of supernatant were  2  The mixture was  avoided.  The pH o f the e x t r a c t e d super-  g o f a Na^O^: N a P 0 » 1 2 H 0 s a l t mix 3  and l e f t  removed and a c i d i f i e d  r e p e a t e d t h r e e times.  f o r 30  to pH 2 w i t h  The b u t a n o l  e x t r a c t s were pooled and evaporated to dryness u s i n g a r o t a r y f l a s h ator.  The r e s i d u e was  M  evapor-  washed i n d i s t i l l e d water and f i n a l l y d i s s o l v e d i n  a s m a l l volume of water.  The polyamine  s o l u t i o n s o b t a i n e d were s t o r e d a t  o.:?cv.. The  e x t r a c t i o n method o f Dion and Cohen (1972) was  used  for quantita-  9 tive studies.  A t o t a l o f 10  c e l l s was  c o l l e c t e d on 0.45  urn f i l t e r s  8 (5 x 10  c e l l s / f i l t e r ) pre-wetted w i t h 007 medium.  w i t h 2.0 ml 007 medium then t r a n s f e r r e d t o 1.0 perchloric acid u s i n g 0.45  um  (PCA).  f i l t r a t e s were s t o r e d at  0°C.  diameter  f i l t e r s were washed  ml o f i c e c o l d 0.2  A f t e r 20 min on i c e , the PCA  f i l t e r s o f 13 mm  The  N  s o l u t i o n s were f i l t e r e d  ( M i l l i p o r e F i l t e r Corp.).  PCA  34  B.  From Phage  3.0 x l O ^ p u r i f i e d phage p a r t i c l e s were washed which c o n t a i n e d  (g/1):Na HP0 •12 H 0, 2  4  K S 0 , 5.0; MgS0 -7H G, 0.50; 2  4  4  p e l l e t was  2  2  7.4; VNaH^PO^, 1.5; NaCI,  CaCl '2H 0, 2  (3x) w i t h T2 b u f f e r  2  4.0;  0.0194 and g e l a t i n ; 0.01.  e x t r a c t e d w i t h 0.5 ml o f i c e c o l d 0.2 N PCA.  PCA  The  filtrates  c o n t a i n i n g polyamines were o b t a i n e d i n a manner i d e n t i c a l to that described for bacteria.  XI.  PREPARATION OF DANSYLATED POLYAMINES To 0.2 ml PCA f i l t r a t e were added 50 mg N a C 0 2  chloride  (2 mg/ml i n a c e t o n e ) .  dued l i g h t w i t h 0.5 ml benzene.  (100 mg/ml) was  The s o l u t i o n was  added and i n c u b a t i o n  then e x t r a c t e d 3x i n sub-  The p o o l e d e x t r a c t s were evaporated w i t h  a g e n t l e stream of a i r ; the aqueous r e s i d u e was i n vacuo over NaOH.  and 1.0 ml o f d a n s y l  A f t e r 16 h r s i n the dark at room tempera-  t u r e , 0.1 ml of a p r o l i n e s o l u t i o n c o n t i n u e d f o r another 30 min.  3  The r e s i d u e was  taken to complete dryness  d i s s o l v e d i n 1.0 ml benzene.  f e r e n c e s , 0.2 N PCA s o l u t i o n s of p u t r e s c i n e  As r e -  (0.00056 M) and spermidine  (0.00034 M) were t r e a t e d i n an i d e n t i c a l manner.  XII.  CHROMATOGRAPHY A.  T h i n L a y e r (TLC) 1.  Polyamines  S o l v e n t s f o r development were: A (Hammond and H e r b s t , 1968), d i e t h y l e n e g l y c o l m o n o e t h y l e t h e r / p r o p i o n i c acid/H 0.(70/15/15, V/V/V) s a t u r a t e d w i t h 2  NaCI; B (Holder and Bremer, 1966), i s o p r o p a n o l / c o n c e n t r a t e d HC1/H 0 (80/30/ 2  20, V/V/V), C ( T o b a r i and Tchen, 1971), m e t h a n o l / c o n c e n t r a t e d NH^OH (7/3, V/V)  and D ( S t a h l , 1969), a c e t o n e / d i e t h y l a m i n e / H 0 (30/6/15, V/V/V). 2  35  C e l l u l o s e sheets  (Eastman Chromogram Sheets, 6064, w i t h o u t f l u o r e s c e n t  i n d i c a t o r , Eastman Organic Chemicals, R o c h e s t e r , N.Y.) s o l v e n t s A, B and D.  were used  with  S i l i c a g e l sheets (Eastman Chromogram Sheets,  6061,  f l u o r e s c e n t i n d i c a t o r ) were used w i t h s o l v e n t C_.  without  2.  Dansylated  Polyamines  S o l v e n t E_ (Dion and Cohen, 1972), e t h y l a c e t a t e / c y c l o h e x a n e (1/2, and  s i l i c a g e l G sheets  ( g l a s s backed, 1011,  ware) were used t o e f f e c t  A n a l t e c h I n c . , Newark, D e l e -  s e p a r a t i o n of d a n s y l a t e d polyamine e x t r a c t s and  d a n s y l a t e d polyamine standards.Chromatography was 3. DNA sheets  DNA  V/V)  performed  i n the  dark.  Hydrolysates  h y d r o l y s a t e s and  r e f e r e n c e standards, were a p p l i e d t o  (Eastman Chromogram Sheets,  6064, without  cellulose  fluorescent indicator).  S o l v e n t s f o r development were: F_ (Bendich, 1957), i s o p r o p a n o l / c o n c e n t r a t e d HC1/H 0 (65/17/18, V/V/V/); G ( K r o p i n s k i e t a l . , 1973), 2  t r a t e d NH 0H/H 0 (70/10/20, V/V/V) and H 4  2  methyl e t h y l k e t o n e / c o n c e n t r a t e d Chromato Vue  ( C l i n e et a l . , 1959), t - b u t a n o l /  HC1/H 0 (40/30/10/20, V/V/V/V).  ( U l t r a - V i o l e t Products  I n c . , San G a b r i e l , C a l i f . ) was  used  to  chromatograms.  Column chromatography  Polyamines were s e p a r a t e d on Dowex 50W-X2 (100-200 mesh) L a b o r a t o r i e s , Richmond, C a l i f . ) . d i e n t of HC1 Tabor,  A  2  d e t e c t u l t r a - v i o l e t a b s o r b i n g areas on developed  B.  isopropanol/concen-  1958).  t i o n s were  Polyamines were e l u t e d w i t h a l i n e a r g r a -  (0-2.5 N.HC1; t o t a l volume 600 ml) A flow r a t e o f 1.0  collected.  (Bio-Rad  ml/min was  (Tabor, R o s e n t h a l  maintained  and  3.0  ml  and frac-  36  XIII.  DETECTION OF POLYAMINES Free polyamines were d e t e c t e d on developed  chromatograms by s p r a y i n g  w i t h n i n h y d r i n as follows.:" the f o l l o w i n g s o l u t i o n s were p r e p a r e d : (a) 50 ml of 0.25% n i n h y d r i n i n a b s o l u t e e t h a n o l , 10 ml o f g l a c i a l a c e t i c and  acid  2.0 ml o f 2,4 , 6 - t r i m e t h y l p y r i d i n e , (b) 1% C u ( N 0 ) • 3 H 0 i n a b s o l u t e 3  ethanol.  F r e s h l y prepared  p e r i o d a t e spray  detect 2-hydroxyputrescine.  a t 100°C f o r 5 min.  (Lemieux and Bauer, 1954) was used t o  The chromatagram was sprayed w i t h a mixture o f  4 p a r t s aqueous sodium metaperiodate 2  2  J u s t p r i o r t o u s e , 50 p a r t s (a) were mixed w i t h 3 p a r t s ( b ) .  A f t e r s p r a y i n g , chromatograms were heated  Na C0.j.  2  and 1 p a r t 1% KMnO^ d i s s o l v e d i n 2%  A f t e r the p e r i o d a t e p o s i t i v e s p o t s developed,  the chromatograms were  washed w i t h water.  XIV.  QUANTITATION OF POLYAMINES Developed chromatograms o f d a n s y l a t e d e x t r a c t s and standards were  scanned u s i n g a Turner Model .11-1 F l u b r o m e t e r -equipp.ed-with'-a TLC-scanner and  a r e c o r d e r (G.K. Turner A s s o c i a t e s , Palo A l t o , C a l i f . ) .  Dansylated  polyamines p r e s e n t i n e x t r a c t s were q u a n t i t a t e d by r e f e r e n c e to a standard curve prepared  XV.  from scans of the r e f e r e n c e , d a n s y l a t e d  polyamines.  ISOLATION OF 2-HYDROXYPUTRESCINE The polyamines were e x t r a c t e d from the c e l l p e l l e t o b t a i n e d from a  10 1 c u l t u r e , h a r v e s t e d at a d e n s i t y o f 10 gation.  c e l l s / m l , by S h a r p i e s  centrifu-  The polyamines were s e p a r a t e d by chromatography on a column (2 cm  x 12 cm) o f Dowex (see M a t e r i a l s Methods X I I B ) .  Amine p o s i t i v e  fractions  were d e t e c t e d by s p o t t i n g a l i q u o t s on Whatman no. 1 f i l t e r paper and  37  spraying with ninhydrin. to determine  f r a c t i o n s c o n t a i n i n g pure 2 - h y d r o x y p u t r e s c i n e .  hydroxyputrescine t o a second  XVI.  T h i n l a y e r chromatography ( s o l v e n t C) was used  f r a c t i o n s were p o o l e d , reduced  Impure 2-  i n volume and a p p l i e d  column (0.9 cm x 13.5 cm) and the p u r i f i c a t i o n  repeated.  NMR OF 2-HYDROXYPUTRESCINE An NMR spectrum  was o b t a i n e d o f a D^O s o l u t i o n o f  u s i n g an HA-100 instrument  2-hydroxyputrescine  ( V a r i a n A s s o c i a t e s , P a l o Alto., C a l i f . ) .  Chemi-  c a l s h i f t s were r e c o r d e d r e l a t i v e t o an e x t e r n a l t e t r a m e t h y l s i l a n e (TMS) standard.  XVII.  ISOLATION OF DNA A.  From b a c t e r i a  DNA was i s o l a t e d by the sodium d o d e c y l s u l f a t e of K o z i n s k i and L i n (1965). 0.05 M K H P 0 2  4  B a c t e r i a were c e n t r i f u g e d , washed once w i t h  b u f f e r (pH 7.0) and resuspended  0.15 M NaCl/0.15 M e t h y l e n e d i a m i n e (TNE b u f f e r ) .  (SDS) - phenol method  tetraacetic acid  L y s i s was e f f e c t e d by adding  then h e a t i n g at 45°C f o r 15 min.  i n 2.0 ml 0.01 M TRIS-HC1/ (EDTA) b u f f e r , pH 7.6  SDS t o a c o n c e n t r a t i o n o f 1%  The volume was i n c r e a s e d t o 5.0 ml by  the a d d i t i o n o f 0.15 M NaCl/0.015 M t r i s o d i u m c i t r a t e , pH 7.0 ( s t a n d a r d s a l i n e c i t r a t e , SSC). DNA was e x t r a c t e d by adding an e q u a l volume o f w a t e r - s a t u r a t e d phenol t o the s u s p e n s i o n .  The emulsion was separated by  c e n t r i f u g a t i o n at 4°C, t h e aqueous l a y e r removed, and r e - e x t r a c t e d w i t h water-saturated phenol.  R e s i d u a l phenol was removed from the aqueous l a y e r  by c a r e f u l e x t r a c t i o n w i t h e t h e r  ( 3 x ) . I f t h e DNA was r a d i o a c t i v e , i t was  d i a l y s e d a g a i n s t 2,2:1 volumes 0.1 M SSC f o r 24 h r s . chilled  The DNA s o l u t i o n was  and 2 volumes o f i c e c o l d 95% e t h a n o l were added.  Nucleic acid  38  p r e c i p i t a t e s were c o l l e c t e d on a g l a s s  r o d by s t i r r i n g .  washed i n acetone, d r i e d and t r a n s f e r r e d B.  P r e c i p i t a t e s were  to g l a s s ampoules.  From phage  Phage were p u r i f i e d a c c o r d i n g to s e c t i o n V I . a c t i v e , they were washed t h r e e times w i t h DMLB.  I f t h e phage were  radio-  The phage p e l l e t was r e -  suspended i n 2.0 ml TNE b u f f e r p r i o r to the a d d i t i o n o f 3.0 mis o f SSC. Phage l y s i s and DNA e x t r a c t i o n were a c h i e v e d s i m u l t a n e o u s l y by adding an equal volume o f w a t e r - s a t u r a t e d p h e n o l . described  XVIII.  The DNA was p u r i f i e d f u r t h e r as  f o r b a c t e r i a l DNA.  HYDROLYSIS OF DNA  A f t e r 0.2 ml o f 6N HC1 was added to the g l a s s DNA, the ampoule was s e a l e d at 100°C f o r 90 min. i n vacuo over NaOH.  XIX.  ampoule c o n t a i n i n g t h e  under reduced p r e s s u r e and t h e DNA h y d r o l y z e d  The s o l u t i o n was c o o l e d The r e s i d u e  was d i s s o l v e d  and evaporated to dryness i n 0.1 N HC1.  MEASUREMENT OF RADIOACTIVITY A.  Chromatographed polyamines o r DNA h y d r o l y s a t e s  Chromatograms c o n t a i n i n g  r a d i o a c t i v e m a t e r i a l were s l i c e d i n t o  sec-  t i o n s and each s e c t i o n then t r a n s f e r r e d t o an i n d i v i d u a l s c i n t i l l a t i o n B.  TCA s o l u b l e  Aliquots  materials  of a c i d s o l u b l e m a t e r i a l were s p o t t e d  (Reeve. A n g e l , C l i f t o n , N.J.) and d r i e d t h o r o u g h l y . placedci i n s c i n t i l l a t i o n C.  TCA p r e c i p i t a b l e  um) and d r i e d t h o r o u g h l y . vials.  on g l a s s f i b e r f i l t e r s The f i l t e r s were then  vials. materials  Acid p r e c i p i t a b l e materials  tion  vial.  were c o l l e c t e d on M i l l i p o r e f i l t e r s (0.45  The f i l t e r s were then t r a n s f e r r e d  to s c i n t i l l a -  39  D.  Counting r a d i o a c t i v i t y  A f t e r t r a n s f e r of the samples tillation fluid  to s c i n t i l l a t i o n v i a l s ,  ( L i q u i f l u o r , New England N u c l e a r , Boston, Mass.) was added  and the r a d i o a c t i v i t y measured w i t h an Isocap/300  liquid  spectrometer  111.).  XX.  5.0 ml o f s c i n -  (Nuclear Chicago Corp., Des P l a i n e s ,  scintillation  CHEMICALS P u t r e s c i n e d i h y d r o c h l o r i d e , spermidine t r i h y d r o c h l o r i d e ,  c y t o s i n e , thymine Calif.).  and adenine were o b t a i n e d from Calbiochem  guanine  (San Diego,  D a n s y l c h l o r i d e was purchased from N u t r i t i o n a l B i o c h e m i c a l Co.  (Cleveland, Ohio).  Worthington B i o c h e m i c a l Corp.  ( F r e e h o l d , N.J.) s u p p l i e d  DNase 1.  XXI.  RADIOCHEMfCALS " 3 All  r a d i o c h e m i c a l s , w i t h the e x c e p t i o n o f  Mann, Orangeburg,  H-2,3-serine  (Schwarz  N.Y.) were purchased from New England N u c l e a r .  40  RESULTS AND I.  DISCUSSION  UTILIZATION OF COMPOUNDS AS Table  I I shows the u t i l i z a t i o n of v a r i o u s compounds as the  source o f n i t r o g e n . gested  SOLE SOURCE OF NITROGEN sole  P_. a c i d o v o r a n s d i d not u t i l i z e a r g i n i n e , which sug-  t h a t the organism c o u l d n e i t h e r deaminate a r g i n i n e to  nor d e i m i n a t e i t to c i t r u l l i n e .  a-ketoarginine  P_. a c i d o v o r a n s s t r a i n s , however, were known  to l a c k a d i h y d r o l a s e pathway ( S t a n i e r , P a l l e r o n i and D o u d o r o f f , 1966). S i n c e u r e a and Y  - a m  inobutyrate  (yABA) were e f f e c t i v e l y u t i l i z e d ,  c o u l d not have been c a t a b o l i z e d t o these compounds, e i t h e r .  arginine  T h i s means  t h a t , i n c o n t r a s t to E_. c o l i and B a c i l l i , P_. a c i d o v o r a n s cannot d e r i v e p u t r e s c i n e or o r n i t h i n e from a r g i n i n e . t h a t the organism was  II.  however, assumed  permeable to a r g i n i n e .  TRANSPORT OF RADIOACTIVE SUBSTRATES The  Since  d e m o n s t r a t i o n of an a r g i n i n e t r a n s p o r t  s a t u r a t i o n occurred  facilitated.  a v e r y l a r g e Km  III.  the p r o c e s s  s i n c e s a t u r a t i o n was or o r n i t h i n e was  the o t h e r hand, was  host  at 25 min,  system i s shown i n F i g . appeared to be  F i g . 9 demonstrates t h a t o r n i t h i n e was  P_. a c i d o v o r a n s ;  on  This reasoning,  ( F i g . 10 and  carrier  a l s o taken up  by  not observed, e i t h e r the c a r r i e r  taken up by p a s s i v e d i f f u s i o n .  t r a n s p o r t e d n e i t h e r by  8.  had  Putrescine,  the u n i n f e c t e d nor i n f e c t e d  11).  POLYAMINES OF P. ACIDOVORANS As a p r e l u d e  composition  to d i r e c t l y s t u d y i n g  of the host was  determined.  the o r i g i n of NpT, Three TLC  the polyamine  systems were used to  show the presence of t h r e e polyamines: s p e r m i d i n e , p u t r e s c i n e and  a third  41  Compound  (NH ) S0 4  2  Used as N i t r o g e n Source  +  4  proline  +  glutamic acid  +  arginine ornithine citrulline putrescine spermidine spermine  +  urea Y-aminobutyric a c i d  (YABA)  +  TABLE I I . U t i l i z a t i o n of compounds as s o l e source o f n i t r o g e n . P_. a c i d o v o r a n s was p l a t e d on n i t r o g e n - f r e e minimal agar p l a t e s . Approximately 1 mg of the t e s t s u b s t r a t e was p l a c e d i n the c e n t e r of each p l a t e . Compounds were c o n s i d e r e d n e g a t i v e i f growth was not observed a f t e r t h r e e days.  42  10  20  30  40  SO  MINUTES  FIGURE 8. The t r a n s p o r t o f C - U - a r g i n i n e by P_. a c i d o v o r a n s . . . ; > • ' I n c o r p o r a t i o n i n t o whole c e l l s , o ; i n c o r p o r a t i o n i n t o TCA p r e c i p i t a b l e m a t e r i a l , a . See M a t e r i a l s and Methods ( S e c t i o n I X ) , f o r p r o t o c o l . Since c u l t u r e s were a c t i v e l y growing, the CPM/ml were c a l c u l a t e d by d i v i d i n g the a c t u a l CPM/ml v a l u e s by a f a c t o r p r o p o r t i o n a l to the growth r a t e . ;  60  0  10  20  30  40  50  60  MINUTES  14 FIGURE 9.  ^  The t r a n s p o r t o f C - 5 - o r n i t h i n e by P. a c i d o v o r a n s . , I n c o r p o r a t i o n i n t o whole c e l l s , e ; i n c o r p o r a t i o n i n t o TCA p r e c i p i t a b l e m a t e r i a l , • . See M a t e r i a l s and Methods ( S e c t i o n IX) f o r p r o t o c o l . Since c u l t u r e s were a c t i v e l y growing, the CPM/ml were c a l c u l a t e d by d i v i d i n g the a c t u a l CPM/ml v a l u e s by a f a c t o r p r o p o r t i o n a l t o the growth r a t e . 1  0  10  20  30  40  50  60  MINUTES  FIGURE 10.  14 i The t r a n s p o r t o f C - 3 , 4 - p u t r e s c i n e by P. a c i d o v o r a n s . I n c o r p o r a t i o n i n t o whole c e l l s , • ; i n c o r p o r a t i o n i n t o TCA p r e c i p i t a b l e m a t e r i a l , a . See M a t e r i a l s and Methods ( S e c t i o n IX) f o r p r o t o c o l . Since c u l t u r e s were a c t i v e l y growing, the CPM/ml were c a l c u l a t e d by d i v i d i n g the a c t u a l CPM/ml v a l u e s by a f a c t o r p r o p o r t i o n a l t o t h e growth r a t e .  MINUTES  FIGURE 11.  The t r a n s p o r t o f C - 3 , 4 - p i i t r e s c i n e by P_. a c i d o v o r a n s i n f e c t e d w i t h 0W-14. I n c o r p o r a t i o n i n t o whole c e l l s , •©., i n c o r p o r a t i o n i n t o TCA p r e c i p i t a b l e * m a t e r i a l . , See M a t e r i a l s and Methods ( S e c t i o n IX) f o r p r o t o c o l .  46  compound which, because  o f i t s Rf v a l u e s (Table I I I ) c o u l d have been  e i t h e r 1,3-diaminopropane or 2 - h y d r o x y p u t r e s c i n e .  I t was  d e c i d e d to  a s c e r t a i n c o n c l u s i v e l y the i d e n t i t y of t h i s unknown s u b s t a n c e . Firstly,  the unknown polyamine  r e a c t e d w i t h n i n h y d r i n to g i v e a  p u r p l e c o l o r ; 1,3-diaminopropane, i n comparison,  produced  a red c o l o r .  As expected o f an amine w i t h a v i c i n a l h y d r o x y l group, i t a l s o r e a c t e d w i t h periodate.1,3-Diaminopropane The  t h i r d polyamine The  was  o x i d i z e d o n l y s l i g h t l y by p e r i o d a t e .  could be l a b e l l e d by  14  C - 6 r n i t h i n e (Table I V ) .  o n l y r a d i o a c t i v e p r o d u c t t h a t c o u l d a r i s e from the o x i d a t i o n o f  14 C-omithine  l a b e l l e d spermidine would be Y  -am  i obutyraldehy.de'. n  Fig.  7B, r e a c t i o n A ) ; 1,3-diaminopropane would be n o n - r a d i o a c t i v e v i a such a cleavage.  R i g o r o u s p r o o f o f the compound's i d e n t i t y was  spectroscopy.  Approximately  compound were p u r i f i e d  22 mgs  from a t e n 1 b a t c h c u l t u r e .  (4H, -CH N-) and 4.37  2-hydroxyputrescine  The NMR  S i g n a l s a t : 2.25  spectrum  ( T o b a r i and Tchen,  of  ^H.-CE^) ,  (IH, -CH0-) confirmed t h a t the compound  2  NMR  o f the d i h y d r o c h l o r i d e s a l t of the  t h i s m a t e r i a l i s p r e s e n t e d i n F i g . 12. 3.49  o b t a i n e d by  was  1971).  The o c c u r r e n c e o f 2 - h y d r o x y p u t r e s c i n e , p u t r e s c i n e and spermidine t o g e t h e r , i s to my knowledge, the f i r s t  r e p o r t e d case o f such a  combination.  Kim's (1966) Pseudomonad c o n t a i n e d 2 - h y d r o x y p u t r e s c i n e and p u t r e s c i n e but lacked spermidine. d i n e may  Rosano and Hurwitz  not occur i n c e l l s  t h a t these polyamines t i o n , S e c t i o n IID) .  may  (19'69), i n t h e o r i z i n g t h a t  spermi-  c o n t a i n i n g 2 - h y d r o x y p u t r e s c i n e , suggested  also  have s i m i l a r m e t a b o l i c f u n c t i o n s (see I n t r o d u c -  The o c c u r r e n c e of both compounds i n P_. a c i d o v o r a n s  suggests t h a t they may  play d i s t i n c t  roles.  47  Compound  Rf Values A  B  spermidine  0.24  0.25  0.10  putrescine  0.40  0.38  0.22  P_. a c i d o v o r a n s extract  TABLE I I I .  0.20, 0.32 0.39  0.29,0.39  0.11, 0.24 0.38  T h i n l a y e r chromatography of polyamines. See M a t e r i a l s and Methods ( S e c t i o n XIIA1) f o r a d e s c r i p t i o n of s o l v e n t s A, B and C, and the c o n d i t i o n s employed.  48  49  IV.  LABELLING OF POLYAMINES In an attempt t o c r e a t e an i n t e r n a l p o o l of r a d i o a c t i v e p u t r e s c i n e ,  v a r i o u s compounds were t e s t e d as p o t e n t i a l polyamine p r e c u r s o r s  (Table  14 IV).  The i n a b i l i t y o f  C-putrescine  the concept t h a t P_. acidovorans s m a l l amount o f r a d i o a c t i v i t y s c i n e a r e a was p r o b a b l y which was p r e s e n t  The  arginine.  c o u l d not t r a n s p o r t t h i s diamine.  (156 CPM) t h a t was c o n f i n e d t o t h e p u t r e -  due t o t h e presence o f c o n t a m i n a t i n g  C-arginine i n l a b e l l i n g  the n o t i o n t h a t P_. acidovorans  input  label  the polyamines a l s o  could n o t o b t a i n u r e a  Presuming t h a t a r g i n i n e i s degraded a t a l l ,  p o s s i b l e : agmatine o r y - g u a n i d i n o b u t y r a m i d e . of these  The  i n t h e polyamine e x t r a c t . 14  i n e f f i c a c y of  strengthened  t o l a b e l the polyamines r e i t e r a t e d  two compounds d i f f e r s ,  from  two c a t a b o l i t e s a r e  E n z y m a t i c a l l y , the f o r m a t i o n  so t h a t enzyme assays c o u l d determine i f  a r g i n i n e can be degraded and i f s o , to which c a t a b o l i t e . 14 rCnQrnithine  e f f e c t i v e l y l a b e l l e d t h e polyamines.  t h e r e f o r e , must possess an a m i t h i n e d e c a r b o x y l a s e . not u t i l i z e d  as a s o l e source  of nitrogen  S i n c e o r n i t h i n e was  (Table I I ) , p u t r e s c i n e was  a p p a r e n t l y not c a t a b o l i z e d t o yABA ( F i g . 7A).  S t a n i e r , P a l l e r o n i and  Doudoroff (1966) have r e p o r t e d t h a t , of o r n i t h i n e and l a t t e r i s s u i t a b l e as a s o l e source i n o t h e r P_. acidovorans  The organism,  o f carbon.  yABA, o n l y the  Hence, i n s t r a i n 29, as  s t r a i n s , o r n i t h i n e i s not c o n v e r t e d  t o yABA.  14 C-Glutamic a c i d a l s o l a b e l l e d t h e polyamines s i g n i f i c a n t l y IV) i n d i c a t i n g t h a t a glutamate to o r n i t h i n e pathway i s o p e r a t i v e .  (Table In  14 using  C-glutamate, a l a r g e p r o p o r t i o n o f r a d i o a c t i v i t y was d e t e c t e d i n  the 2-hydroxyputrescine have s i m i l a r Rf v a l u e s  area.  S i n c e a r g i n i n e and  i n s o l v e n t C, the  2-hydroxyputrescine  presence o f r a d i o a c t i v e a r g i n i n e  Precursor  14 14 14 14 14 14  Supplement  ,Phage Infection  L a b e l (CPM) i n Polyamines spermidine putrescine  2-hydroxyputrescine  C-3,4-putrescine  nil  32  156  79  C-U-arginine  nil  20  31  50  C-5-ornithine  nil  67  631  641  C-5-ornithine  nil  45  1216  441  C-3,4-glutamate  nil  83  244  627  64  228  637  C-3,4-glutamate  arginine  +  (1 mg/ml)  TABLE., I V . ^ L a b e l l i n g o f polyamines. L a b e l (0.25 uc/20 ug precursor and 20 ug p r e c u r s o r / m l ) was added to 25 mi c i i l L u r e s ; c u l t u r e s were harvested a t a d e n s i t y g f 10 c e l l s / m l . Where a p p r o p r i a t e , phage was added a t a m.o.i. of 10 t o c u l t u r e s a t a d e s i t y o f 5 x 10 c e l l s / m l ; the i n f e c t e d c u l t u r e s were h a r v e s t e d 20 min a f t e r i n f e c t i o n . Polyamines were e x t r a c t e d by t h e Raina method. A l i q u o t s o f polyamine e x t r a c t s were a p p l i e d to c e l l u l o s e sheets and chromatography performed i n s o l v e n t C. Areas c o n t a i n i n g polyamines were d e t e c t e d by n i n h y d r i n , e x c i s e d and counted i n d i v i d u a l l y .  o  51  was  suspected.  The  polyamine e x t r a c t s were, t h e r e f o r e , chromatographed  i n s o l v e n t D, which e f f e c t i v e l y separated 2 - h y d r o x y p u t r e s c i n e and  spermidine.  a r g i n i n e from p u t r e s c i n e ,  In t h i s system, the a r g i n i n e  region  14 was  e s s e n t i a l l y non-radioactive,  thus a l a r g e p e r c e n t a g e of the  glutamate l a b e l l e d polyamines was  t r u e l y due  C-  to 2 - h y d r o x y p u t r e s c i n e  (Table  V). Whether or not P_. a c i d o v o r a n s forms o r n i t h i n e v i a t r a n s a c e t y l a t i o n or not  i s unknown.  o n l y one  I t would seem, however, t h a t the organism possesses 14  pathway f o r polyamine s y n t h e s i s .  Since  the polyamines even i n the presence of a r g i n i n e g i n i n e d i d not was  repress o r n i t h i n e biosynthesis  C-glutamate l a b e l l e d (1 mg/ml), exogenous a r -  (Table I V ) .  Such a r e s u l t  somewhat unexpected, because i t means t h a t the r e g u l a t i o n of a r g i n i n e  synthesis i s unusual. On On  the one  hand, a r g i n i n e b i o s y n t h e s i s c o u l d be  the o t h e r hand, i f o r n i t h i n e i s s y n t h e s i z e d  c y c l e i n the  be  the second enzyme  Arginine,  i n d u c t i v e e f f e c t on the enzymes concerned  T h i s i s mere s p e c u l a t i o n f o r i t i s not  an arg R l o c u s e x i s t s i n P_. a c i d o v o r a n s .  Also  even known  feasible is a situation  i n which the enzymes of o r n i t h i n e b i o s y n t h e s i s are u n e f f e c t e d by but  the o r n i t h i n e to a r g i n i n e steps are i n h i b i t e d by  assays c o u l d e a s i l y prove or d i s p r o v e Since  there i s apparently  to  A l t e r n a t i v e l y , r e g u l a t i o n of  s i m i l a r to t h a t seen i n E_. c o l i B.  t h e r e f o r e , would have a s l i g h t with i t s biosynthesis.  and not  pathway would thus c o n t i n u e  presence of t h i s amino a c i d .  a r g i n i n e b i o s y n t h e s i s may  if  The  unregulated.  v i a the t r a n s a c e t y l a t i o n  r o u t e , then perhaps, through m u t a t i o n , the f i r s t ( F i g . 1) i s i n h i b i t e d by a r g i n i n e .  totally  this  o n l y one  arginine.  arginine Enzyme  point.  route  f o r polyamine b i o s y n t h e s i s  52  Supplement  Precursor  D i s t r i b u t i o n of Label arginine  14 14  C-3,4-glutamate C-3,4-glutamate  TABLE V.  nil arginine  (1 mg/ml)  (CPM)  polyamines  32  955  22  923  14 D i s t r i b u t i o n of l a b e l (CPM) i n C-glutamate l a b e l l e d polyamine e x t r a c t s . A l i q u o t s o f polyamine e x t r a c t s were a p p l i e d to c e l l u l o s e s h e e t s and chromatography performed i n s o l v e n t D. N i n h y d r i n p o s i t i v e areas were e x c i s e d and counted i n d i v i d u a l l y .  53  i n t h i s bacterium,  the c o n s t i t u t i v i t y of a r g i n i n e s y n t h e s i s may  be  r a t i o n a l i z e d as f o l l o w s : polyamines appear to be e s s e n t i a l f o r normal c e l l u l a r metabolism and  an i n h i b i t i o n of a r g i n i n e and  polyamine b i o s y n -  theses would be d e t r i m e n t a l to metabolism.  V.  QUANTITATION OF POLYAMINES The  v e r y s e n s i t i v e d a n s y l technique  (Dion and  to q u a n t i t a t e the polyamines o f P_. a c i d o v o r a n s . concentrations 50 and  45,  of s p e r m i d i n e ,  p u t r e s c i n e and  Cohen, 1972)  The  was  used  internal millimolar  2-hydroxyputrescine  were  r e s p e c t i v e l y , assuming t h a t the volume o f P_. a c i d o v o r a n s  that o f E_. c o l i and  the i n t r a c e l l u l a r water content  3-5,  equalled  of P_. acidovorans  was  70%. The  a b s o l u t e polyamine c o n c e n t r a t i o n s  spermidine,  p u t r e s c i n e and  corresponded to a r e l a t i v e  2-hydroxyputrescine  r a t i o o f 1: 10:9;  which  approximated the r a t i o of 1: 9.4:9.6, o b t a i n e d by l a b e l l i n g i n f e c t e d c e l l s 14 with curve  C-ornithine  (Table I V ) .  f o r the d e t e r m i n a t i o n  s y n t h e t i c standard) seemed Kelln  Hence, the use o f a p u t r e s c i n e  of 2-hydroxyputrescine  to a l a c k of a  reasonable.  (1973) a s c e r t a i n e d t h a t phage DNA  post i n f e c t i o n .  (due  s y n t h e s i s begins  to the s t a r t o f phage DNA  synthesis  (Table I V ) .  of p u t r e s c i n e suggested t h a t phage DNA  c u r s o r of NpT  at 20 14  To examine t h e e f f e c t ( s ) o f phage i n f e c t i o n on  t h i n e l a b e l l e d polyamine p o o l s , i n f e c t e d c e l l s were h a r v e s t e d  f o r t h i s diamine.  standard  The  just  DNA.  C-orniprior  increased proportion  s y n t h e s i s imposed a g r e a t e r demand  In other words, i t i m p l i e d t h a t p u t r e s c i n e was  i n phage  min  a pre-  54  Phage i n f e c t i o n c o u l d d i s t o r t the polyamine p o o l i n any one s e v e r a l ways.  The  of  phage genome might code f o r an o r n i t h i n e d e c a r b o x y l a s e  so t h a t , s i m p l y , a gene dose e f f e c t would be o p e r a t i v e .  Phage i n f e c t i o n  might l e a d to the f o r m a t i o n of a p o s i t i v e e f f e c t o r f o r the h o s t ' s thine decarboxylase.  Conversely,  a n e g a t i v e e f f e c t o r of t h i s enzyme  might be n e u t r a l i z e d as a r e s u l t of phage i n f e c t i o n . are extreme by v i s c o u s ; an i n d i c a t i o n t h a t h o s t DNA ing i n f e c t i o n .  orni-  L y s a t e s of  0W-14  i s not degraded  The phage, t h e r e f o r e , c o u l d s y n t h e s i z e ai; r j - l i k e  dur-  factor  which s p e c i f i c a l l y enhanced the t r a n s c r i p t i o n o f the h o s t ' s gene f o r o r n i t h i n e decarboxylase.  One  o r more of these means, then, c o u l d  f o r the e l e v a t e d p o o l of p u t r e s c i n e observed phage  VI.  account  p r i o r to the s y n t h e s i s of  DNA.  POLYAMINES OF The  0W-14  d a n s y l method (Dion and Cohen, 1972)  was  a l s o used to  the polyamines p r e s e n t i n p u r i f i e d phage p a r t i c l e s . of s p e r m i d i n e , p u t r e s c i n e and tography  (solvent E).  2-hydroxyputrescine  The  dansyl d e r i v a t i v e s  were r e s o l v e d by chroma-  Though not q u a n t i t a t e d , the phage polyamine e x t r a c t  c o n t a i n e d a q u a l i t a t i v e l y l a r g e r p r o p o r t i o n of spermidine i n the u n i n f e c t e d or i n f e c t e d h o s t . c e n t r a t i o n i s much lower  than  observed  Since the b a c t e r i a l spermidine  con-  than the c o n c e n t r a t i o n s of p u t r e s c i n e o r 2-hydroxy-  p u t r e s c i n e , t h i s suggested the g r e a t e s t a f f i n i t y  analyze  t h a t , o f the t h r e e polyamines,  f o r 0W-14  DNA.  The presence  the t r i a m i n e had  of polyamines,  even  a f t e r thorough ." washing of the phage p a r t i c l e s , i m p l i e d t h a t the phage was  osmotically sensitive.  55  VII.  OSMOTIC SENSITIVITY OF The  effect  i n T a b l e VI.  of an osmotic shock on the v i a b i l i t y o f 0W-14  The  i s shown  s i g n i f i c a n t drop i n t i t e r as a r e s u l t of osmotic shock  i n d i c a t e d t h a t 0W-14 occurrence  0W-14  contained  a semi-permeable membrane so t h a t  of f r e e polyamines i n the p a r t i c l e s i s f e a s i b l e .  the  I f the phage  were not exposed to 5M NaCI f o r 16 hrs p r i o r to the shock, a much lower drop i n t i t e r was  VIII.  THE Of  observed  BIOSYNTHESIS OF  (C. Spencer, p e r s o n a l  communication).  NpT  the p o s s i b l e polyamine p r e c u r s o r s  (Table I V ) , o r n i t h i n e  deemed the most a p p r o p r i a t e f o r l a b e l l i n g NpT.  was  C e l l s were i n f e c t e d i n  14 the presence of  DNA  purified.  C-5-ornithine,  the progeny phage r e c o v e r e d 14  R a d i o a c t i v i t y from  C - 5 - o r n i t h i n e was  (Table V I I ) , so t h a t the p u t r e s c i n y l moiety o f NpT  could,  Since P_. a c i d o v o r a n s  to p u t r e s c i n e , t h i s problem was  resolved.  permeable to p u t r e s c i n e seemed h o p e l e s s .  Any  The  was  impermeable  s e l e c t i o n of a mutant  s e l e c t i o n procedure would of n i t r o g e n .  As d i s c u s s e d e a r l i e r , t h i s i s not p o s s i b l e i n P_. a c i d o v o r a n s ,  thus  c o n s i d e r e d h i g h l y improbable.  of phage i n f e c t e d , t o l u e n i z e d c e l l s may  NpT  theoretically,  r e q u i r e the organism to u t i l i z e p u t r e s c i n e as a s o l e source  s e l e c t i o n of a m u l t i p l e mutant was  their  detected only i n  come from o r n i t h i n e or p u t r e s c i n e . not  and  provide  the The  use  an answer, however.  To r e i n f o r c e the concept t h a t p u t r e s c i n e , r a t h e r than o r n i t h i n e , p r e 14 cursed NpT,  C - l - o r n i t h i n e was  utilized.  The  i n t e r n a l p o o l of  should be n o n - r a d i o a c t i v e , i n t h i s i n s t a n c e , and A n a l y s i s o f the phage DNA VII).  putrescine  s i m i l a r l y , so should  r e v e a l e d a l l bases to be n o n - r a d i o a c t i v e  T h i s suggested t h a t the c a r b o x y l group of o r n i t h i n e d i d not  NpT.  (Table  Situation  Titer  + NaCI ( c o n t r o l ) - NaCI  (PFU/ml x 10  14.5 1.0  TABLE V I .  Osmotic s e n s i t i v i t y o f phage 0W-14. Phage were p l a c e d i n 5M NaCI f o r 16 h r s d i l u t e d i n water and p l a t e d . A c o n t r o l , i n which d i l u t i o n s were performed i n 5M NaCI, was a l s o done.  )  L a b e l l e d Compound Added  Label  (CPM) i n the N u c l e i c A c i d Bases of Progeny Phage  A  C  G  T  NpT  C-5-ornithine  24  20  42  31  1680  C-l-ornithine  38  66  30  49  43  TABLE V I I .  L a b e l l i n g o f 0W-14 DNA w i t h C-ornithine. ^ C - o r n i t h i n e was added to 50 ml c u l t u r e s a t a d e n s i t y o f 2.5 x 10^ c e l l s / m l such t h a t t h e r e were 0.25 uc/20 ug o r n i t h i n e and 20 ug o r n i t h i n e / m l . C u l t u r e s were i n fected at a d e n s i t y o f 1 0 c e l l s / m l with a m.o.i 10. Progeny phage were r e c o v e r e d 6 h r s post i n f e c t i o n . Phage DNA was p u r i f i e d and h y d r o l y z e d . A l i q u o t s o f DNA h y d r o l y z a t e s were a p p l i e d to c e l l u l o s e sheets and chromatography performed i n s o l vent F. Areas c o n t a i n i n g bases were d e t e c t e d by u l t r a - v i o l e t l i g h t , e x c i s e d and counted i n d i v i d u a l l y . 9  58  c o n t r i b u t e to the s t r u c t u r e o f NpT but d i d not e l i m i n a t e the p o s s i b i l i t y t h a t o r n i t h i n e was  a p r e c u r s o r to NpT.  The c a r b o x y l group c o u l d  function  to i n c r e a s e the r e a c t i v i t y o f the a d j a c e n t secondary amine, thereby f a cilitating Kelln  the attachment o f t h a t amino group to the p y r i m i d i n e p r e c u r s o r . (1973) showed the p y r i m i d i n e h a l f o f the n o v e l base to be  d e r i v e d from d e o x y u r i d i n e ; n e i t h e r thymine nor thymidine were i n v o l v e d i n this was  capacity.  The consequences  of t h i s r e s u l t were t w o - f o l d : 0W-14  DNA  s y n t h e s i z e d from f i v e bases and the methylene group, which l i n k s the  p u t r e s c i n e moiety to the r i n g o f NpT, of carbon atoms.  A tetrahydrofolate  must a r i s e from a m e t a b o l i c donor (THF) involvement was  immediately  suspected. S e r i n e t r a n s h y d r o x y m e t h y l a s e t r a n s f e r s the hydroxymethyl group o f s e r i n e t o THF,  forming N m e t h y l e n e THF. 10  Kelln  (1973) c o n f i r m e d the THF 14  involvement by s u c c e s s f u l l y l a b e l l i n g NpT w i t h Moreover, t h e NpT/T l a b e l l i n g r a t i o u s u a l l y observed f o r phage DNA. related—but  how,  exactly?  Was  donor the same i n both cases?  (0.88) was  C - 3 - s e r i n e (Table V I I I ) . the same as the NpT/T r a t i o  The o r i g i n of dTMP and the NpT  seemed  the o x i d a t i o n s t a t e o f the THF carbon 3 The use of  H-2,3-serine c l a r i f i e d  the s i -  t u a t i o n somewhat. In t h i s c a s e , N^^formyl THF,  f o r example, would c o n t a i n one  less  t r i t i u m atom than N"^methylene THF.  T h e r e f o r e , i f the former compound  was  then the NpT/T l a b e l l i n g  used i n the b i o s y n t h e s i s o f NpT,  be about 0.45, would be about  ratio  would  i f N^^methylene THF were used f o r b o t h compounds, the r a t i o 0.90.  L a b e l l e d Compound Added  Label  (CPM) i n the N u c l e i c A c i d Bases of Progeny Phage  A  14  C-3-serine  *  3 H-2,3-serine  C  G  T  NpT  Ratio  NpT/T  3624  20  10493  2133  1796  0.88  1095  16  800  639  579  0.90  Experiment performed by R.A. K e l l n ( K e l l n , 1973).  TABLE V I I I .  L a b e l l i n g o f 0W-14 DNA w i t h r a d i o a c t i v e serine. H-2,3-Serine was added t o 20 ml c u l t u r e s supplemented w i t h 20 ug methionine/ml at a d e n s i t y o f 5 x 10^ c e l l s / m l such t h a t there were 2.5 uc/20 ug s e r i n e and 20 ug serine/ml. A t a d e n s i t y o f 7.5 x 10^ c e l l s / m l , the c u l t u r e s were i n f e c t e d w i t h a. m.o.i of 10. Progeny phage were r e c o v e r e d 6 h r s post infection. Phage DNA was p u r i f i e d and hydrol y z e d . A l i q u o t s o f DNA h y d r o l y z a t e s were a p p l i e d t o c e l l u l o s e s h e e t s and chromatography performed i n two dimensions (one: s o l v e n t H, two: s o l v e n t G). Areas c o n t a i n i n g bases were d e t e c t e d by u l t r a - v i o l e t l i g h t , e x c i s e d and counted i n d i v i d u a l l y 1 3  60  A n a l y s e s o f h y d r o l y s a t e s o f 0W-14 DNA l a b e l l e d w i t h y i e l d e d an NpT/T r a t i o o f 0.90 (Table V I I I ) .  Therefore,  H-2,3-serine N methylene 10  THF i s an i n t e r m e d i a t e i n the b i o s y n t h e s i s of the n o v e l base.  The a c c u r -  acy o f t h i s i n t e r p r e t a t i o n i s supported by t h e d i s t r i b u t i o n o f r a d i o a c t i v i t y i n adenine and guanine (Table V I I I ) .  These v a l u e s a r e p r e c i s e l y  3 those expected i n l a b e l l i n g  the phage DNA w i t h  H-2,3-serine.  To s p e c u l a t e , t h e f o l l o w i n g sequence can be c o n s t r u c t e d : THF i n t e r a c t s w i t h dUMP t o y i e l d  N^^methylene  5-HMdUMP which condenses w i t h p u t r e s c i n e  to produce NpT. In t h e b i o s y n t h e s i s of HMC i n T4 i n f e c t e d E_. c o l i , is  the donor f o r t h e hydroxymethyl group  (Cohen, 1968).  N m e t h y l e n e THF 10  Thiamine b i o s y n -  t h e s i s i n microbes i n v o l v e s t h e r e a c t i o n of an amine w i t h a hydroxymethyl group t o y i e l d a C-N bond  (White, Handler and Smith, 1968).  These  reac-  t i o n s , then, p a r a l l e l those proposed f o r t h e b i o s y n t h e s i s o f NpT, and l e n d f e a s i b i l i t y t o the proposed sequence.  61  SUMMARY  The  metabolism of a r g i n i n e and  polyamines was  i n v e s t i g a t e d as a  p r e l i m i n a r y to examining the b i o s y n t h e s i s of NpT.  F_. a c i d o v o r a n s ,  though  permeable to a r g i n i n e , i s l i m i t e d i n i t s a b i l i t y to c a t a b o l i z e t h i s amino a c i d . nitrogen.  That i s , a r g i n i n e was O r n i t h i n e was  u n s u i t a b l e as a s o l e source of  converted  to p u t r e s c i n e but not  glutamate to o r n i t h i n e pathway was  demonstrated but  to yABAr  A  i t s operation  was  unusual because a r g i n i n e , the u l t i m a t e end p r o d u c t , d i d not b l o c k thine biosynthesis.  T h i s seems reasonable,  appears to have o n l y one  orni-  however, s i n c e the organism  route f o r the s y n t h e s i s o f polyamines ( i . e .  a r g i n i n e , as i n E_. c o l i , cannot be c a t a b o l i z e d t o p u t r e s c i n e ) . An unusual combination of polyamines occurs 0W-14: s p e r m i d i n e , p u t r e s c i n e and b a c t e r i a l concentrations was  (mM)  i n P_. a c i d o v o r a n s  2-hydroxyputrescine.  were: 3-5,  i d e n t i f i e d c o n c l u s i v e l y by NMR  50 and  45.  spectroscopy.  expanded a f t e r phage i n f e c t i o n , perhaps e n s u r i n g the s y n t h e s i s of  2-Hydroxyputrescine  an adequate supply  pool  for  NpT.  i n g the o r i g i n of the n o v e l base. i s impermeable to p u t r e s c i n e ,  and  respective,  Only the p u t r e s c i n e  An i n t e r n a l p o o l of r a d i o a c t i v e p u t r e s c i n e was  polyamines.  The  and  14  Unfortunately,  n e c e s s a r y f o r study-  s i n c e P_. a c i d o v o r a n s  to be used to l a b e l the 14 When c e l l s were i n f e c t e d i n the presence o f C-5-ornithine  the progeny phage DNA  C - o r n i t h i n e had  analyzed,  NpT  was  the o n l y r a d i o a c t i v e base.  14 In a p a r a l l e l experiment, phage DNA,  C - l - o r n i t h i n e was  so t h a t the c a r b o x y l  ineffective in labelling  group of t h i s amino a c i d was  not  the  directly  62  i n v o l v e d i n the s y n t h e s i s The THF  o f NpT.  s u s p i c i o n t h a t the b i o s y n t h e s i s o f the n o v e l base i n v o l v e d a  carbon donor was confirmed by l a b e l l i n g the NpT i n phage DNA w i t h  "^C-3-serine.  The u s e of ^H-2,3-serine showed N"*"^methylene THF t o be a  l i k e l y intermediate.  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