UBC Theses and Dissertations

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UBC Theses and Dissertations

Some aspects of phenolic acid and iron metabolism in selected bacterial strains Walsh, Barry L. 1969

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SOME A S P E C T S IRON  METABOLISM  OF  PHENOLIC  IN S E L E C T E D  ACID  AND  BACTERIAL  STRAINS  by  BARRY L.  B.Sc.  (Agr.)  McGill  A THESIS  SUBMITTED  THE  in Microbiology 1967  University,  IN P A R T I A L  REQUIREMENT  FOR  M A S T E R OF  in  WALSH  FULFILMENT  THE  DEGREE  OF  OF  SCIENCE  the Department  of  Microbiology  We  accept  this  thesis  required  THE  U N I V E R S I T Y OF  as conforming  to the  standard  BRITISH  September,  1969  COLUMBIA  In p r e s e n t i n g  this thesis  in p a r t i a l  f u l f i l m e n t o f the  requirements for  an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e I f u r t h e r agree tha  permission  f o r s c h o l a r l y p u r p o s e s may by h i s r e p r e s e n t a t i v e s .  for extensive  Department  of  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, C a n a d a  Columbia  thesis  Department  It i s understood that copying or  permission.  study.  copying of this  be g r a n t e d by t h e H e a d o f my  o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not written  and  that  or  publication  be a l l o w e d w i t h o u t  my  ii  ABSTRACT  B a c i l l u s s u b t i l i s and  Micrococcus lysodeikticus  when s u b j e c t e d t o c o n d i t i o n s were washed. c h r o m e and  phenolic  acids.  d i a m i n e t e t r a a c e t i c a c i d and g r o w t h o f JB;  subt i 1 i s but  The  stimulated  chelating  stimulated  high levels of a carbon source, with glucose serving  only  of those tested.  benzoylglycine  acid early  in the  l a t e l o g and  under iron d e f i c i e n t c o n d i t i o n s ! produced phenolic  acids  relatively as t h e  most  produced  _  phenolic  peptide  B_. s u b t i 1 i s WB7*t6 p r o d u c e d  2 , 3 d i h y d r o x y b e n z o i c a c i d w h e r e a s s t r a i n B1A71  unidentified  effect  conditions.  a c i d p r o d u c t i o n by B_. s u b t j l i s r e q u ? r e d  effective substrate  ferri-  l y s o d e i k t i c u s . The  a n t i b i o t i c a l b o m y c i n d e p e n d e d on g r o w t h  inocula  agents ethylene-  n i t r i l o t r i a c e t i c acid  n o t o f -M..  by  o f t h e s e c o m p o u n d s as a n t a g o n i s t s t o t h e a c t i o n o f t h e  Phenolic  poorly  of l i m i t i n g iron n u t r i t i o n a f t e r  G r o w t h o f b o t h o r g a n i s m s was  various  grew  i n l o g p h a s e and  an  2,3~dihydroxy-  early stationary  phases of  growth,  M u t a n t s t r a i n s o f B_. s u b t ? 1 ?s  in the absence or p r e s e n c e of i r o n in  the  g r o w t h medium. DHB  synthesizing  e n z y m e s w e r e r e p r e s s e d by g r o w t h o f B^. s u b t ? 1 ?s  in the p r e s e n c e of i r o n , f e r r i c h r o m e or the a r o m a t i c amino Active  d i h y d r o x y b e n z o i c a c i d s y n t h e t a s e was  compounds.  The  DHB  not a f f e c t e d  s y n t h e t a s e s y s t e m f r o m B_. s u b t i 1 i s was  p u r i f i e d by D E A E - c e l 1 u l o s e c o l u m n c h r o m a t o g r a p h y and  acids.  by  these partially  sucrose  gradient  c e n t r i f u g a t i o n ; the r e s u l t suggested  the e x i s t e n c e of a  multienzyme  complex. I r o n u p t a k e by B_. s u b t i 1 i s and be e n e r g y - d e p e n d e n t of adequate appeared  iron.  t y p h i m u r i um was  a n d r e p r e s s i b l e by g r o w t h i n t h e The  shown t o  presence  i r o n uptake c a p a c i t y of M . l y s o d e i kt i cus  t o be i n d u c i b l e , w i t h a c h e l a t i n g a g e n t o r an F e : c h e l a t e  c o m p l e x s e r v i n g as t h e i n d u c e r . a c i d appeared organisms,  F e r r i c h r o m e and  dihydroxybenzoic  t o s e r v e as i r o n t r a n s p o r t f a c t o r s f o r a l l t h r e e  whereas e t h y l e n e d i a m i n e t e t r a a c e t i c ac|d i n h i b i t e d  iron  uptake. 3 ~ F 1 u o r o b e n z o i c a c i d , a d i h y d r o x y b e n z o i c a c i d a n a l o g , had e f f e c t on g r o w t h , b y , and  but d i d reduce both p h e n o l i c a c i d  i r o n u p t a k e c a p a c i t y o f , B a c i 1 1 us  subtilis.  production  little  T A B L E OF CONTENTS Page INTRODUCTION  1  MATERIALS AND METHODS . . . .  5  I. B a c t e r i a l  5  Strains  1. W i l d t y p e B_. s u b t i 1 i s s t r a i n s  5  2. M u t a n t B_. s u b t i 1 i s s t r a i n s  5  a) S p o n t a n e o u s m u t a n t s  5  b) N N G - i n d u c e d  6  mutants  3. W i l d t y p e M_. l y s o d e i k t i c u s  6  k.  6  W i l d t y p e ' S_. t y p h i m u r i u m  5. M u t a n t S_. t y p h i m u r i u m s t r a i n s II. Media and C u l t u r e C o n d i t i o n s  . . . . . . . .  6 8  1 . General  8  2. EL s u b t i 1 i s  8  3. M^. l y s o d e i k t i c u s  9  k.  S_. t y p h i m u r i u m  .  10  III. Determination  10  1. G r o w t h  10  2. P h e n o l i c a c i d s  10  a) DHB(G)  ^10  b) O t h e r p h e n o l i c s  10  c) Chromatography  11  i) Paper chromatography  11  V  Table o f Contents  (Continued) Page  i i ) Thin layer chromatography . 3. C o p r o p o r p h y r i n k.  .  .  .  .  .  III  .11 12 \k  Protein  IV. A n t a g o n i s m S t u d i e s  .  . \k  1 . L i qu i d med i um \k  2. S o l i d medi um V. P r e p a r a t i o n s o f C e l l s a n d C e l l - f r e e E x t r a c t s .  .  .  .  VI . E n z y m e ' A s s a y . S y n t h e s i s o f DHB  .15 16  V I I . Enzyme P u r i f i c a t i o n . P a r t i a l P u r i f i c a t i o n o f DHB  16  1. D E A E - C e l l u l o s e 2. S u c r o s e VIII.  column chromatography  .  .  .  .  .  .17 18  gradient centrifugation  Iron T r a n s p o r t ' S t u d i e s  18  1 . I ron uptake  18  2. I r o n e f f l u x  19  IX. C h e m i c a l s  20  RESULTS I. G r o w t h F a c t o r s , A l b o m y c i n Introduction 1. M. l y s o d e i k t i c u s  22 and A l b o m y c i n - A n t a g o n i s m .  .  .22 22 .22  vi  Table of Contents  (Continued) Page  a) Growth  23  b) G r o w t h f a c t o r s  23  c) Growth i n h i b i t i o n and a l b o m y c i n a n t a g o n i s m  .  .  .  25  2. B_. s u b t i 1 i s  25  a) G r o w t h  25  b) G r o w t h i n h i b i t i o n a n d a l b o m y c i n a n t a g o n i s m  . . .  30  Discussion of Results  30  I I . Some G e n e r a l C h a r a c t e r i s t i c s o f G r o w t h , P h e n o l i c A c i d and C o p r o p o r p h y r i n P r o d u c t i o n i n B_. s u b t i l i s  35  Introduction  35  1.  Wi Id t y p e s t r a i n s  36  a) E f f e c t o f c a r b o n s o u r c e  36  b) C h a n g e s i n pH d u r i n g g r o w t h  38  c) Time o f o n s e t o f p h e n o l i c a c i d p r o d u c t i o n  .  .  .  38  2. M u t a n t S t r a i n s  h0  Discussion of Results  ^2  I I I . E f f e c t o f A n a l o g s a n d C h e l a t i n g A g e n t s on P h e n o l i c A c i d P r o d u c t i o n by B_. s u b t i 1 i s Introduction .  .  k(> ^6  vi i  Table o f Contents  (Continued) Page  1.  E f f e c t o f v a r i o u s a g e n t s on p h e n o l i c a c i d production  2.  . . . . 47  by g r o w i n g c e l l s  a) A n a l o g s o f DHB  47  b) C h e l a t i n g a g e n t s  47  In v j t r o A s s a y s  47  a) P r e l i m i n a r y e x p e r i m e n t s  47  b) Enzyme r e p r e s s i o n  52  c ) Enzyme i n h i b i t i o n  52  d) Enzyme r e a c t i v a t i o n  52 55  Discussion of Results  58  IV. P a r t i a l P u r i f i c a t i o n o f DHB S y n t h e t a s e  58  Introduction 1.  DEAE- C e l l u l o s e c o l u m n c h r o m a t o g r a p h y  . . . . .  2. S u c r o s e g r a d i e n t c e n t r i f u g a t i o n  59 .59 61  Discussion of Results V. I r o n T r a n s p o r t  61  Introduction  61  1.  B. s u b t i l i s s t r a i n B1471  63  a) P r e l i m i n a r y e x p e r i m e n t s  63  b) E x p e r i m e n t s  with i r o n d e f i c i e n t c u l t u r e s  . . .  64  i) Iron uptake as a f u n c t i o n o f p h y s i o l o g i c a l age  64  vi i i  T a b l e o f C o n t e n t s (Cont i nued) Page  6k  ii) Effect of metabolic inhibitors  68  i i i ) Effect of glucose iv)  Iron u p t a k e as a f u n c t i o n  o f membrane. 68  integrity  68  v) S p e c i f i c i t y o f i r o n u p t a k e b) E x p e r i m e n t s w i t h i r o n s u f f i c i e n t c u l t u r e s  71  . . .  71  i ) E f f e c t on u p t a k e c a p a c i t y c) E x p e r i m e n t s w i t h c o n t r o l l e d  levels of  i r o n . Growth i n c h e m i c a l l y e x t r a c t e d  . medium  .  .  75  i) E f f e c t o f p h e n o l i c a c i d p r o d u c t i o n on. 75  iron uptake capacity d)  I r o n u p t a k e as a f u n c t i o n  of chelating  i) E f f e c t o f d e s f e r r i f e r r i c h r o m e , and  .  75  DHBG 75  EDTA  ii) Effect o f varying  concentrations of 78  DHBG 2.  agents.  B. s u b t i 1 i s m u t a n t s t r a i n s  78 mutant  78  b) S t r e p t o m y c i n r e s i s t a n t m u t a n t s  78  c) A l b o m y c i n r e s i s t a n t m u t a n t  78  a) S h i k i m i c a c i d  requiring  ix  Table of Contents  (Continued) Page  d) C o b a l t r e s i s t a n t m u t a n t  83  e) Hemin r e s i s t a n t m u t a n t  83  f) Iron r e s i s t a n t mutants  83  g) Hemin and  84  Discussion 3.  M_.  Iron r e s i s t a n t mutant  of Results  84 87  lysodeiktlcus  a) P r e l i m i n a r y  87  experiments  b) E f f e c t o f g r o w t h w i t h o r w i t h o u t i r o n  .... 87  on u p t a k e c a p a c i t y c) E f f e c t o f g r o w t h w i t h c h e l a t i n g  agents  .... 88  on u p t a k e c a p a c i t y . d) E f f e c t o f c h e l a t i n g  a g e n t s on  iron 88  u p t a k e \n_ v i t r o e) E f f e c t o f a l b o m y c i n on of Results  95  S_. t y p h i m u r i u r n s t r a i n LT-2  97  a)  97  Discussion 4.  95  iron uptake  I ron u p t a k e i) Growth under i r o n - d e f i c i e n t  conditions  a) E f f e c t o f m e t a b o l i c i n h i b i t o r s on  .  .  .  .  . 97  i ron u p t a k e b) E f f e c t o f  iron-chelating  c) S p e c i f i c i t y o f t h e  97  system  agents.  ...  99 99  X  Table of Contents  (Continued) Page  i i ) Growth under i r o n s u f f i c i e n t c o n d i t i o n s . . E f f e c t on u p t a k e c a p a c i t y .  5.  101  b) I r o n e f f l u x  101  S^. t y p h i m u r ? u m m u t a n t s t r a i n s  101  a) P r e l i m i n a r y e x p e r i m e n t s  101  b) C o m p a r i s o n w i t h w i l d t y p e s t r a i n  104  c) E f f e c t o f c h e l a t i n g a g e n t s o n u p t a k e  104  Discussion of Results  104  VI. E f f e c t o f 3 f 1 u o r o b e r i z o i c A c i d on G e n e r a l -  P r o p e r t i e s o f EL s u b t i 1 i s BT471 Introduction 1. G r o w t h .  109 .109  .  109  2.  Phenolic acid production  109  3.  Iron uptake  109  Discussion of Results.  112  GENERAL D I S C U S S I O N  114  L I S T OF REFERENCES  120  XI  L I S T O F FIGURES Page Figure Figure Figure  1.  E f f e c t o f w a s h i n g i n o c u l u m o f M_. lysbdeikticus  2k  2.  E f f e c t o f v a r i o u s compounds o n g r o w t h o f M_. l y s o d e i k t i c u s  2k  3.  E f f e c t o f v a r i o u s compounds o n g r o w t h o f B_. s u b t ? 1 i s  Figure  Figure  k. . V a r i a t i o n w i t h t i m e o f g r o w t h , p h e n o l i c a c i d p r o d u c t i o n a n d pH o f t h e c u l t u r e medium o f B. s u b t i 1 i s 5. 6.  f r o m B. s u b t i 1 i s  Figure  7.  Figure  m u t a n t s t r a i n s o f B. s u b t i 1 i s WB746 8. E n z y m a t i c c o n v e r s i o n o f c h o r i s m i c a c i d to 2,3-dihydroxybenzoic a c i d by c e l l - f r e e e x t r a c t s o f B_. s u b t ? l i s W B 7 4 6 9.  F i g u r e 10.  k3  Growth and p h e n o l i c a c i d p r o d u c t i o n by  C o n v e r s i o n o f c h o r i s m i c a c i d t o 2,3d i h y d r o x y b e n z o i c a c i d as a f u n c t i o n o f protein concentration U l t r a v i o l e t spectra o f substrate, product and i n t e r m e d i a t e s i n t h e e n z y m a t i c c o n v e r s i o n of chorismic a c i d t o 2,3-dihydroxybenzoic a c i d by c e l l - f r e e e x t r a c t s o f B_. s u b t i 1 i s WB746  F i g u r e 11.  kl  Growth and p h e n o l i c a c i d p r o d u c t i o n b y m u t a n t s t r a i n s o f B. s u b t i 1 i s BU71  Figure  39  Thin layer chromatography o f e x t r a c t s o f culture supernatants  Figure  31  D E A E - c e l 1 u l o s e c h r o m a t o g r a p h y o f DHBs y n t h e t a s e a c t i v i t y i n an e x t r a c t o f B. s u b t i l i s W B 7 4 6  k3  kS 50  51  60  xi i  List of Figures  (Continued) Page  F i g u r e 12.  F i g u r e 13.  F i g u r e 14. F i g u r e 15. F i g u r e 16. F i g u r e 17. F i g u r e 18.  F i g u r e 19. F i g u r e 20.  F i g u r e 21. F i g u r e 22. F i g u r e 23. F i g u r e 24.  S u c r o s e - g r a d i e n t c e n t r i f u g a t i o n o f DHBsynthetase a c t i v i t y in a crude extract of i - s u b t i l i s WB746  62  I r o n u p t a k e by B. s u b t i l l s B1 47 1 as a f u n c t i o n o f p h y s i o l o g i c a l age and p h e n o l i c acid production  66  E f f e c t o f m e t a b o l i c i n h i b i t o r s and s u l f h y d r y l r e a g e n t s on i r o n u p t a k e by B_. s u b t i 1 i s B1471  67  E f f e c t o f g l u c o s e on i r o n u p t a k e c a p a c i t y o f B. s u b t i l i s B1471  69  E f f e c t o f l i p o l y t i c compounds on u p t a k e by 13. s u b t i 1 i s B1471  70  iron  S p e c i f i c i t y o f the iron uptake system o f B_. s u b t i l i s B1471  72  Repression of the iron uptake capacity o f B. s u b t i 1 i s B1471 by g r o w t h w i t h a d d e d i ron  73  E f f e c t o f growth w i t h added u p t a k e by B_. s u b t i 1 i s B1471  74  i r o n on  iron  I r o n u p t a k e by B_. s u b t i 1 i s B1471 grown with different levels of iron in extracted medium  76  E f f e c t o f v a r i o u s i r o n - b i n d i n g a g e n t s on i r o n u p t a k e by B. s u b t i 1 i s B1471  77  E f f e c t o f i t o i c a c i d on i r o n u p t a k e by B. s u b t i 1 i s B1471  79  E f f e c t of various iron-binding agents on i r o n u p t a k e by EL s u b t i 1 ? s l471y  80  I r o n u p t a k e by two S t r e p t o m y c i n - r e s i s t a n t m u t a n t s o f B. s u b t i l i s B1471  81  XI I I  L i s t o f F? g i i r e s ( C o n t i n u e d ) Page Figure  25.  E f f e c t o f g r o w t h w i t h v a r i o u s c o m p o u n d s on i r o n u p t a k e c a p a c i t y o f H. 1 y s b d e i k t ? c u s  89  Figure  26.  I r o n u p t a k e by M. l y s o d e i k t i c u s medium w i t h o u t c i t r a t e  90  Figure  27.  Effect of chelating b y M_. l y s o d e i k t i c u s  Figure  28.  E f f e c t o f h y d r o x a m a t e on i r o n u p t a k e b y M_. l y s b d e i k t i c u s  92  Figure  29.  E f f e c t o f p h e n o l i c compounds on u p t a k e b y M_. l y s o d e i k t i c u s  93  Figure  30.  Effect of varying  grown i n  a g e n t s on i r o n  uptake  iron  concentrations of  a c i d o n i r o n u p t a k e b y M.  91  itoic  Sk  lysbdeikticus  Figure  31.  E f f e c t o f a l b o m y c i n on i r o n u p t a k e by M_. l y s o d e i k t i c u s  96  Figure  32.  E f f e c t o f m e t a b o l i c i n h i b i t o r s on u p t a k e by S_. t y p h i m u r i u m LT-2  98  Figure  33-  E f f e c t o f c h e l a t i n g a g e n t s on i r o n b y S_. t y p h iriiuri urn LT-2  Figure  34.  Repression o f iron uptake capacity o f S. t y p h i m u r i u m LT-2 b y g r o w t h w i t h a d d e d i r o n , and t h e e f f e c t o f i t o i c a c i d on iron uptake  102 105  iron uptake  Figure  35.  Iron uptake c a p a c i t y and mutant s t r a i n s  Figure  36.  E f f e c t o f c h e l a t i n g a g e n t s on i r o n by S_. t y p h i m u r i u m m u t a n t f e r - 1  uptake  Figure  37-  E f f e c t o f c h e l a t i n g a g e n t s on i r o n by S_. t y p h i m u r i u m m u t a n t f e r D19  uptake  o f S. t y p h i m u r i u m  LT-2  100  10.6. 107  xi v  List of Figures  (Continued) Page  Fi gure  38.  E f f e c t o f 3 ~ f l u o r o b e n z o i c a c i d on g r o w t h B_. s u b t i l i s B1471  Figure  39-  E f f e c t o f 3 " f l u o r o b e n z o i c a c i d on p h e n o l i c a c i d p r o d u c t i o n by B. s u b t i 1 i s B1471  111  Figure  40.  E f f e c t o f 3 " f 1 u o r o b e n z o i c a c i d on i r o n u p t a k e c a p a c i t y o f EL s u b t i 1 i s B1471  113  Fi gure  41.  Diagrammatic i l l u s t r a t i o n s o f and f e r r i c h r o m e  116  Figure  42.  D i a g r a m m a t i c i l l u s t r a t i o n s o f Fe(DHBG) and Fe(EDTA) complexes  117  Figure  43.  Intermediates in the b i o s y n t h e s i s o f 2,3"dihydroxybenzoic acid  119  albomycin  of  110  XV  L I S T OF T A B L E S Page Table  I.  Table  II.  Table  III.  Description of bacterial strains  7  Rf v a l u e s and c h a r a c t e r i s t i c r e a c t i o n s o f some p h e n o l i c a c i d s i n c h r o m a t o g r a p h y  13  E f f e c t o f i r o n - b i n d i n g compounds on growth o f M_. l y s b d e i k t i c u s  26  Table  IV.  E f f e c t o f v a r i o u s compounds as a l b o m y c i n a n t a g o n i s t s w i t h M_. l y s o d e i k t i c u s  27  Table  V.  E f f e c t o f v a r i o u s compounds as a l b o m y c i n a n t a g o n i s t s w i t h M. l y s b d e i k t i c u s u s i n g the paper s t r i p method  28 31  Table  VI.  E f f e c t o f v a r i o u s compounds as a n t a g o n i s t s w i t h B_. s u b t i 1 i s  albomycin  Table  VII.  E f f e c t o f v a r i o u s compounds as a n t a g o n i s t s w i t h B_. s u b t i 1 i s  albomycin  Table VIII.  32  E f f e c t o f v a r i o u s c a r b o n s o u r c e s on g r o w t h , p h e n o l i c a c i d and c o p r o p o r p h y r i n p r o d u c t i o n by IB. s u b t i 1 i s  37  Table  IX.  E f f e c t o f DHB a n a l o g s o n p h e n o l i c a c i d p r o d u c t i o n by B. s u b t i 1 i s  48  Table  X.  E f f e c t o f c h e l a t i n g a g e n t s on p h e n o l i c a c i d p r o d u c t i o n b y B^. s u b t i 1 i s  48  R e p r e s s i o n o f DHB s y n t h e t a s e o f B_. s u b t ? 1 i s by v a r i o u s c o m p o u n d s  53  I n h i b i t i o n o f DHB s y n t h e t a s e a c t i v i t y B_. s u b t i 1 ?s by v a r i o u s c o m p o u n d s  54  Table Table  XI. XII.  Table XIII.  from  Attempted r e s t o r a t i o n o f DHB-synthetase a c t i v i t y t o e x t r a c t s o f B_. s u b t i 1 i s g r o w n in the presence o f iron  56  xv i  List of Tables  (Continued) Page  Table  XIV.  Table  XV.  Table  XVI.  E f f e c t o f w a s h i n g c e l l s on i r o n c a p a c i t y o f B. s u b t i 1 i s B1471  uptake  65  U p t a k e o f r a d i o a c t i v e i r o n by m u t a n t s t r a i n s o f B. s u b t i 1 i s B1471  82  E f f e c t o f v a r i o u s compounds on e f f l u x f r o m S. t y p h i m u r i u m  103  xv i i  GLOSSARY OF A B B R E V I A T I O N S  Coproporphyrin  III  Cop I I I  2,3-dihydroxybenzoic acid 2,3-dihydroxybenzoylglycine  DHB (itoic acid)  DHBG  2,3-dihydroxybenzoylserine  DHBS  2-N,6-N-Di-(2,3-dihydroxybenzoyl)-L-lysine  DHBL  ethylenediaminetetraacetic acid  EDTA  n i t r i l o t r i a c e t i c acid  NTA  4,5-di h y d r o x y - m - b e n z e h e d i s u l f o n i c a c i d  T i ron  Albomycin  Alb  Ferrichrome  Fc  if  INTRODUCTION  When grown u n d e r c o n d i t i o n s o f i r o n d e f i c i e n c y a n d aeration, Bacillus subtilis alters  i t s metabolism  adequate  i n s u c h a way (45).  t h a t p h e n o l i c a c i d s and p o r p h y r i n compounds a r e e x c r e t e d  T h e f o r m e r h a v e b e e n i d e n t i f i e d a s DHB, DHBG o r b o t h o f t h e s e compounds (DHB(G)), consideration. (DHBS) (1),  depending  upon t h e s t r a i n o f EL s u b t i 1 i s u n d e r  Under s i m i l a r c o n d i t i o n s , E s c h e r i c h i a c o l i  Aerobacter aerogenes  bacter v i n e l a n d i i produces  DHB (49,  produces  b o t h DHB a n d  has been i d e n t i f i e d a s c o p r o p o r p h y r i n  DHBL.  III  72)  (12).  produces  and A z o t o The p o r p h y r i n  (33).  In B_. s u b t i l i s , a n i n v e r s e r e l a t i o n s h i p e x i s t s b e t w e e n t h e l e v e l o f i r o n a d d e d t o t h e c u l t u r e medium a n d t h e l e v e l o f DHB(G) by g r o w i n g  cells  (45).  In a d d i t i o n , i r o n d e f i c i e n c y p r e v e n t s  f u n c t i o n i n g o f t h e heme p a t h w a y , r e s u l t i n g t h e g r o w t h medium o f c o p r o p o r p h y r i n o g e n t o C o p I I I (33).  normal  in the accumulation in  III which  Compounds o t h e r t h a n i r o n  produced  is rapidly oxidized  (or those c o n t a i n i n g  i r o n , s u c h a s f e r r i c h r o m e ) a f f e c t DHB(G) a n d c o p r o p o r p h y r i n p r o d u c t i o n . Of t h e s e , t h e m o s t p o t e n t  inhibitors of phenolic acid  are meta-substituted d e r i v a t i v e s of benzoic acid which Cop  i n h i b i t normal  production  (44).  f u n c t i o n i n g o f t h e TCA c y c l e cause  Compounds decreased  I I I p r o d u c t i o n when c u l t u r e s a r e g r o w n u n d e r c o n d i t i o n s o f i r o n  deficiency  (44).  2  The m e t a b o l i c  a l t e r a t i o n ( s ) in the aromatic  pathway caused  i r o n d e f i c i e n c y w h i c h r e s u l t i n DHB(G) p r o d u c t i o n characterized.  74)  have not been  fully (72)  W o r k i n g w i t h A. a e r o g e n e s , Y o u n g and c o - w o r k e r s  h a v e shown t h a t DHB (73,  by  is synthesized  by way  o f a t l e a s t two  intermediates  f r o m c h o r i s m i c a c i d , t h e common b r a n c h p o i n t i n t h e  s y n t h e t i c pathway of aromatic  compounds.  B r o t , G o o d w i n and  bio(1)  Fales  h a v e shown t h a t c e l l - f r e e e x t r a c t s made f r o m E_. c o l ? g r o w n u n d e r i r o n d e f i c i e n t conditions are capable (44)  presented  of converting  preliminary evidence  DHB(G) by e x t r a c t s o f EL s u b t i l i s . the  in vi t r o formation  The e v i d e n c e duction strong  (2,  45)  DHB  t o DHBS.  f o r the in v i t ro f o r m a t i o n  c a r r i e d out  in the present  that iron appears to control phenolic acid and  the hypothesis  m i g h t s e r v e as i r o n - s e q u e s t e r i n g  t h e r o l e p l a y e d by  u p t a k e , w i t h Q_. s u b t i 1 i s.  them (39),  investigated in t h i s organism.  the t r a n s f e r o f m e t a b o l i c  membranes (65,  66)  ,  i t was  agents study  chelating  lysodeikticus,  In a d d i t i o n , s i n c e  i s t h o u g h t t o a c t by b l o c k i n g  i n s e r t i o n of i r o n i n t o the p r o t o p o r p h y r i n f e r i n g with  are  iron-binding agents in iron  c o m p o u n d s s e r v e as " g r o w t h f a c t o r s " f o r M i c r o c o c c u s  the p e p t i d e a n t i b i o t i c albomycin  study.  led to a  Furthermore, since various  r i n g (75)  precursors  the  o r by i n t e r -  through  of  pro-  that phenolic a c i d s , which  f o r the b e n e f i t of the organisms producing o f i r o n u p t a k e , and  of  A f u r t h e r more i n t e n s i v e s t u d y  o f DHB(G) was  iron-binding agents,  i r o n u p t a k e was  Peters  bacterial  of i n t e r e s t to i n v e s t i g a t e the r o l e of  3  v a r i o u s i r o n - s e q u e s t e r i n g agents as p o s s i b l e a n t a g o n i s t s t o a l b o mycin, and  as agents involved  i n i r o n m e t a b o l i s m o f b o t h B_. s u b t i 1 ?s  a n d M_. l y s o d e i k t i c u s .  As t h e w o r k o n i r o n u p t a k e w i t h t h e t w o o r g a n i s m s  mentioned  a b o v e was n e a r i n g c o m p l e t i o n , t h e e x i s t e n c e o f s t r a i n s o f S a l m o n e l l a typhimurium requiring t o my a t t e n t i o n  i r o n a n d / o r DHB a s g r o w t h f a c t o r s was b r o u g h t  ( D r . B. Ames, p e r s o n a l c o m m u n i c a t i o n ) .  sequence o f t h i s ,  As a con-  iron uptake s t u d i e s were performed w i t h  typhimurium and a r e i n c l u d e d i n t h i s  thesis.  T h e l i t e r a t u r e c o n t a i n s f e w r e p o r t s o n c a t i o n u p t a k e by microorganisms and  until very recently the majority o f those publish-  ed c o n c e r n e d t h e u p t a k e o f d i v a l e n t c a t i o n s b y y e a s t c e l l s . E v i d e n c e p r e s e n t e d b y Conway a n d c o - w o r k e r s R o t h s t e i n group and!Ni  + +  (21,  52),  (9,  10,  11)  a n d by t h e  indicated that Mg , C o , Z n , + +  + +  c r o s s t h e c e l l membrane v i a a c t i v e t r a n s p o r t  + +  Mn  + +  systems.  T h e o b s e r v a t i o n t h a t c a t i o n a c c u m u l a t i o n i s b a l a n c e d by a n e q u i v a lent secretion of H (77),  +  i o n s i n y e a s t (9),  a n d i n E^. c o l i (54)  in Streptococcus faecal is  l e d t o the hypothesis that ion accumulation  i s p r i m a r i l y a p r o c e s s o f i o n e x c h a n g e (15).  Whether t h i s exchange  is s i m p l y p a s s i v e , f a c i l i t a t e d o r energy dependent depends  largely  on t h e c r i t e r . i a u s e d t o d i s t i n g u i s h b e t w e e n t h e s e p o s s i b i l i t i e s a n d o n t h e whim o f t h e i n v e s t i g a t o r .  There i s evidence  t h a t b a c t e r i a l t r a n s p o r t s y s t e m s , i n common  w i t h o t h e r e n z y m e s y s t e m s , a r e s u b j e c t t o r e g u l a t i o n (25, 56). The  uptake o f M n  + +  and M g  + +  was i n h i b i t e d b y o t h e r c a t i o n s i n Gram-  p o s i t i v e but not i n Gram-negative species  (Webb, 69, 70).  Tungstate  i n h i b i t e d m o l y b d a t e u p t a k e i n A. v i n e l a n d 1 i ' b u t d i d n o t i n h i b i t i t s intracellular function; F e  + +  also inhibited Mo  + +  u p t a k e (3, 30, 31).  T h r e e v e r y r e c e n t papers i n d i c a t e t h a t E. c o l i p o s s e s s e s a r a t e , s p e c i f i c a c t i v e t r a n s p o r t mechanisms f o r M n Mg  + +  (37, 57).  In a d d i t i o n , e v i d e n c e  m e c h a n i s m i n E_. c o l ? i n w h i c h C r published  (67, 68).  + + +  + +  (58) a n d f o r  f o r an a c t i v e i r o n uptake ions i n t e r f e r e has j u s t been  Preliminary evidence  suggested  the existence  o f a n a c t i v e u p t a k e m e c h a n i s m f o r i r o n i n B_. s u b t i 1 i s (46). work has been e x t e n d e d i n t h e p r e s e n t  study.  This  In a d d i t i o n , i r o n  uptake has been examined i n o t h e r b a c t e r i a l s p e c i e s t o g a i n an u n d e r s t a n d i n g  sep-  o f iron uptake processes  i n an a t t e m p t  in bacteria.  MATERIALS AND METHODS  B a c t e r i a 1 S t r a i ns  1.  W i l d T y p e S t r a i n s o f J3. s u b t i 1 i s S t r a i n B1471 was o b t a i n e d  of C a l i f o r n i a a t Berkeley. M a r b u r g s t r a i n ) was o b t a i n e d of Washington, 2.  from Dr. J.B. Neilands,  University  S t r a i n WB746 ( a d e r i v a t i v e o f t h e f r o m D r . E.W. N e s t e r ,  University  Seattle.  M u t a n t S t r a i n s o f B_. s u b t i 1 i s Mutant s t r a i n s were s e l e c t e d as spontaneous d e r i v a t i v e s o r  as N - m e t h y 1 - N - n i t r o - N - n i t r o s o g u a n i d i n e ( N N G ) - i n d u c e d (a)  Spontaneous Mutants Strains  of F e  + + +  containing final  r e s i s t a n t t o l o w (2 mM) o r h i g h  (100 mM)  , C o , hemin, albomycin o r streptomycin  by s e r i a l  and  derivatives,  + +  t r a n s f e r o f t h e wild type s t r a i n increasing concentrations  were  to t h e desired  compound.  o f t h e compounds.  The plates  and t e s t e d f o r r e s i s t a n c e  Resistant  c o l o n i e s were  on m i n i m a l m e d i u m a n d s i n g l e c o l o n i e s stra ins.  obtained  i n m i n i m a l medium  c u l t u r e s were d i l u t e d and spread on minimal a g a r  s i n g l e c o l o n i e s were picked  levels  picked  streaked  as r e s i s t a n t  6  (b)  NNG-Induced Mutants The  obtain  m e t h o d o f L o r e i i c e a n d N e s t e r (35) was f o l l o w e d  NNG-induced mutants auxotrophic  acids or their biosynthetic All  f o r a r o m a t i c amino  precursors.  B_. s u b t ? 1 i s s t r a i n s w e r e s t o r e d a t room t e m p e r a t u r e  on TAM s l a n t s a f t e r o v e r n i g h t 3.  incubation  Salton,  f r o m D r . M.R.J.  New Y o r k U n i v e r s i t y , New Y o r k , a n d u s e d i n a l l e x p e r i -  ments i n v o l v i n g m i c r o c o c c i . s l a n t s and t r a n s f e r r e d  I t was s t o r e d a t k C on T S Y - a g a r  monthly.  W i l d T y p e S t r a i n o f S_. t y p h i m u r i um S_. t y p h i m u r i u m s t r a i n LT-2 was o b t a i n e d  University o f C a l i f o r n i a at Berkeley. TSY-agar s l a n t s and t r a n s f e r r e d 5.  a t 37 C.  W i l d T y p e S t r a i n o f M, l y s o d e i k t i c u s ML l y s o d e i k t i c u s s t r a i n ML-1 was o b t a i n e d  h.  to  f r o m D r . B.N. Ames,  I t was s t o r e d a t k C on  monthly.  M u t a n t S t r a i n s o f S_. t y p h i m u r i u m M u t a n t s t r a i n s o f S_. t y p h i m u r i u m r e q u i r i n g Fe o r DHB f o r g r o w t h  were a l s o obtained The T a b l e 1.  properties  f r o m D r . Ames. o f a l l b a c t e r i a l s t r a i n s used a r e o u t l i n e d i n  7  Table  1.  Parent  Description of Bacterial Strains  Organism  j3. s u b t ? 1 i s B1471  Mutant Stra i n  Characteristics  I471y  requires shikimic a c i d f o r growth  H1  r e s i s t a n t t o 1 mM hemin  Fe J  r e s i s t a n t t o 100 FeCl  R  Source  present research  mM  3  —iFe RH R  r e s i s t a n t t o 1 mM  U  h e m i n a n d 100 FeCl  mM  3  123 Co  R  r e s i s t a n t t o 100 Albomyci n  mM  r e s i s t a n t t o 100 CoCl  mM  2  A B_. s u b t i 1 i s  Fe  WB746  LT-2  R  9  r e s i s t a n t t o 50 ug/ml Streptomyci n r e s i s t a n t t o 100 FeCl  mM  3  H typhimurium  & A  5  R  r e s i s t a n t t o 1 mM hemi n  fer-1  r e q u i r e s Fe f o r growth  fer-3  r e q u i r e s Fe f o r g r o w t h  f e r D19  r e q u i r e s Fe o r f o r growth  DHB  f e r D9  r e q u i r e s Fe o r f o r growth  DHB  D r . B.N.  Ames  M e d i a and C u l t u r e C o n d i t i o n s  1.  General B a c t e r i a w e r e g r o w n r o u t i n e l y a t 37 C i n E r l e n m e y e r  f i t t e d with sidearms. r e c e i v e d a 0.5% hour  M e d i u m ( 1 0 % by v o l u m e o f f l a s k c a p a c i t y )  i n o c u l u m f r o m a 10 - 14 h o u r c u l t u r e (24 - 36  i n t h e c a s e o f Mu  1ysode?kticus) growing  in Trypticase-Soy-  Y e a s t E x t r a c t (TSY) b r o t h , u n l e s s s t a t e d o t h e r w i s e . s h a k e n a t 250 rpm Brunswick w a r e was  flasks  F l a s k s were  i n a m o d e l G77 M e t a b o l y t e W a t e r B a t h  S c i e n t i f i c C o . , New  Brunswick,  New  Jersey).  (New A l l glass  autoclaved twice in g l a s s - d i s t i l l e d water p r i o r to  use to e l i m i n a t e r e s i d u a l g l a s s - d i s t i l l e d water.  iron.  A l l m e d i a w e r e made up w i t h  Sugar s o l u t i o n s were s t e r i l i z e d  a r a t e l y a s c o n c e n t r a t e d s o l u t i o n s (20 - 50%)  sep-  in g l a s s - d i s t i l l e d  water.  T h e f o l l o w i n g c o m p o u n d s w e r e s t e r i l i z e d a s s o l u t i o n s by  passage  t h r o u g h 0.45  U Mi 1 l i p o r e f i I t e r s  f o r d , M a s s . ) : a l b o m y c i n , c a t e c h o l , DHB,  ( M i l l i p o r e Corp.,  Bed-  DHBG, f e r r i c h r o m e , h e m i n  anthranilic acid, 3~hydroxyanthrani1ic acid,  3 hydroxybenzoic -  acid, shikimic acid, salicylamide, N-hydroxymethylsalicylamide. 2.  B a c ? 1 1 us  subti1is  The media used f o r growth  and m a i n t e n a n c e  o f B_. s u b t ? 1 i s  w e r e t h o s e d e s c r i b e d by P e t e r s and W a r r e n ( 4 5 ) , e x c e p t t h a t y e a s t e x t r a c t was Minimal  n o t i n c l u d e d r o u t i n e l y i n t h e b a s a l medium.  m e d i u m c o n t a i n e d , i n g / l i t e r : KH^PO^, 3.0;  I^SO^,  1.0;  9  NH^  a c e t a t e , 3-0;  c i t r i c a c i d , 1.0;  MgS0^.,7H 0, 0.08;  spora t r a c e element s o l u t i o n of Vogel T h e medium was  adjusted  A c c u m e t M o d e l 220  t o pH 7-4  (63),  0.01%  Neuro-  by v o l u m e .  w i t h 5N NaOH, u s i n g a F i s h e r  pH m e t e r ( F i s h e r S c i e n t i f i c Co.)  by a u t o c l a v i n g a t 121 C f o r 15 m i n u t e s .  and  sterilized  S t e r i l e glucose  added s e p a r a t e l y to the d e s i r e d c o n c e n t r a t i o n  was  (routinely \ % ) .  M i n i m a l a g a r p l a t e s f o r m u t a n t s e l e c t i o n and f o r a n t a g o n i s m s t u d i e s (see below) were prepared  according  T h e medium c o n t a i n e d ,  ( N H ^ S O ^ , 2.0;  K H P 0 , 6.0; 2  s o d i u m c i t r a t e . 2 H 0 , 1.0;  /(  2  g l u c o s e , 5.0. ution.  in g / l i t e r ;  The  g l u c o s e was  toSpizizen  (62).  I^HPO^,  M g S O ^ H ^ , 0.2;  14.0; and  a d d e d s e p a r a t e l y as a s t e r i l e  F u r t h e r a d d i t i o n s w e r e made i m m e d i a t e l y  p r i o r to  sol-  the  pouring of plates. Chemically Vogel 3.  (63)  e x t r a c t e d medium was  using  Micrococcus  prepared  by t h e m e t h o d o f  8-hydroxyquinoline. lysodeikticus  T h e medium u s e d f o r t h e g r o w t h o f M_. l y s o d e i k t i c u s was of Salton 0.1;  (53)  N H ^ C l , 1.0;  and c o n t a i n e d ,  i n g / l i t e r : K H ^ O ^ , 6.8;  s o d i u m g l u t a m a t e , 10.0;  M n C l . 4 H 0 , 0 . 0 0 2 ; and B i o t i n , 0 . 0 0 0 5 . 2  t o pH 7-5 prepare  2  p r i o r to autoclaving.  NH^  acetate,  T h e medium was  A g a r (1.5%) was  minimal agar p l a t e s where i n d i c a t e d .  that  MgSO^H^, 10,0; adjusted  added to  4.  Salmonella  typhimurium  S_. t y p h i m u r i u m Bonner (64) w i t h o u t  was g r o w n i n t h e b a s a l m e d i u m o f V o g e l a n d added t r a c e elements.  T h e medium c o n t a i n e d ,  i n g / l i t e r : ^ H P O ^ , 1 0 . 0 ; c i t r i c a c i d , 2 . 0 ; M g S O ^ h ^ O , 0.2; Na ( N H j ^ P O ^ . k U ^ O , 3-5. Sterile  T h e medium was pH 7-0 b e f o r e a u t o c l a v i n g .  g l u c o s e was a d d e d s e p a r a t e l y t o a f i n a l c o n c e n t r a t i o n o f  0. 5..  Determinations  1.  Growth G r o w t h was f o l l o w e d t u r b i d i m e t r i c a l l y u s i n g a K l e t t - S u m m e r s o n  c o l o r i m e t e r with a green converted  ( 5 4 0 nm) f i l t e r .  K l e t t r e a d i n g s were  t o v i a b l e c e l l s p e r ml b y u s e o f p r e p a r e d  standard  g  curves. (cfu)  A v i a b l e c e l l d e n s i t y o f 10  colony forming  p e r ml g a v e a K l e t t r e a d i n g o f 40 u n i t s f o r J3_. s u b t i l i s ,  36 u n i t s f o r H_. l y s o d e i k t i c u s a n d 32 u n i t s f o r S_. 2.  units  typhimurium.  Phenolic Acids (a)  DHB(G) a s s a y was p e r f o r m e d  a c c o r d i n g t o Peters and  Warren ( 4 5 ) . (b)  When l o o k i n g f o r a d d i t i o n a l p h e n o l i c a c i d s  produced  i n c u l t u r e s u p e r n a t a n t s , 5 0 0 ml v o l u m e s o f c u l t u r e s g r o w n in 2 - l i t e r Erlenmeyer  f l a s k s without  f i t t e d sidearms  were  c e n t r i f u g e d f o r 24 m i n u t e s a t 18,000 x £ u s i n g t h e A - 2 8  r o t o r o f a n IEC m o d e l E>60 c e n t r i f u g e ( I n t e r n a t i o n a l E q u i p ment C o . , Needham H t s . , M a s s . ) .  The supernatants were a d -  j u s t e d t o pH 2 w i t h 6N HC1 a n d c l a r i f i e d b y f u r t h e r c e n t r i fugation.  T h e c l e a r l i q u i d was t h e n e v a p o r a t e d u n d e r  i n a 500 ml c a p a c i t y C a n L a b r o t a r y f l a s h e v a p o r a t o r  vacuum (Chem-  Q u i p C o . , B e r k e l e y , C a l i f o r n i a ) t o a b o u t 50 m l , a n d e x t r a c t e d with 3 volumes o f e t h y l a c e t a t e . was  The e t h y l acetate f r a c t i o n  c o n c e n t r a t e d o n t h e e v a p o r a t o r t o about 3 ml. The aqueous  f r a c t i o n was f u r t h e r e x t r a c t e d w i t h 2 v o l u m e s o f e t h e r , a n d the e t h e r e x t r a c t c o n c e n t r a t e d by e v a p o r a t i o n t o about 3 ml. The e t h y l a c e t a t e and e t h e r e x t r a c t s were examined by paper and t h i n l a y e r c h r o m a t o g r a p h y  and were a l s o scanned f o r  absorption of ultraviolet  l i g h t u s i n g a U n i c a m SP800 r e c o r d i n g  spectrophotometer  Instruments  (Unicam  L t d . , Cambridge,  England) . (c)  Chromatography (i)  Paper  Chromatography  The f o l l o w i n g s o l v e n t systems were 1. 2.  n-butanol:acetic acid:water  used:  (k:1:5)(v/v/v).  (27).  t-butanol:methylethylketone:water:diethylamine  ( I 0 : 1 0 : 5 : l ) ( v / v / v / v ) . (27). ( i i ) Thin Layer Chromatography P l a t e s (20 cm x 20 cm) w e r e p r e p a r e d w i t h gel G ( E . Merck and Co.).  Silica  The f o l l o w i n g s o l v e n t systems were u s e d : 3.  benzene:methanol:acetic  4.  same as #2 a b o v e .  5.  benzene:acetic water.  (7).  (27).  a c i d (20:5)(v/v)  saturated  with  (12).  ( i i i ) Detection The  a c i d (10:2 : 1 ) ( v / v / v ) .  Systems  f o l l o w i n g d e t e c t i o n systems were used f o r both  p a p e r and  t h i n layer chromatography:  1.  Evans reagent,  used to d e t e c t o - d i h y d r i c phenols  2.  S u l f a n i l i c a c i d , t o d e t e c t p h e n o l i c compounds  3>  Potassium  (60).  f e r r i c y a n i d e , t o d e t e c t i r o n b i n d i n g com-  pounds ( 8 ) . 4.  S i l v e r n i t r a t e , t o d e t e c t mono- and phenols  5.  F e r r i c c h l o r i d e in water:acetone ( 4 : 1 ) ( v / v ) , to  Chromatb-vve cabinet  R.p v a l u e s and  are given  Coproporphyrin Cop  I I I was  ultraviolet  r e a c t i o n s w i t h some o f t h e  i n T a b l e 2 f o r DHB,  run r o u t i n e l y as  (48).  ( U l t r a - v i o l e t Products,  San G a b r i e l , C a l . ) w h i c h a b s o r b The  polyhydric-  (60).  d e t e c t i r o n b i n d i n g compounds 6.  (13).  Inc., light.  reagents  DHBG and some common  analogs  standards.  II I  determined  by t h e m e t h o d o f L e s s i e S  Sistrom  u s i n g t h e e x t i n c t i o n c o e f f i c i e n t g i v e n by R i m i n g t o n  (50).  T a b l e 2.  R^. v a l u e s a n d c h a r a c t e r i s t i c r e a c t i o n s o f some common p h e n o l i c a c i d s i n paper and t h i n - l a y e r  ^ Compound  chromatography.  Reaction with: Evans Reagent FeCl  Paper*  f TLC**  2 , 3 d i h y d r o x y b e h z o y 1 g 1 y c i ne  0.42  0 .31  pink  +  2,3-dihydroxybenzoic  acid  0.64  0 .48  pink  +  2,4-dihydroxybenzoic  acid  0 .49  p i nk  +  2,5 dihydroxybenzoic  acid  0 .44  p i nk  +  2,6-dihydroxybehzoic  acid  0 .18  pink  +  m-hydroxybenzoate  0 .57  y e 11ow  p-hydroxybenzoate  0 .63  y e 11ow  o-hyd roxypheny1 a c e t a t e  0 .55  y e 11ow  -  o-hydroxyh i ppurate  0 .51  p i nk  +  catechol  0 .60  pink  +  0 .75  green  +  0 .75  y e 11ow  -  -  _  salicylic  acid  anthrani1ic  acid  *  **  Solvent system:  t-butanol:methylethy1 ketone:water: d i e t h y l a m i n e ( 1 0 : 1 0 : 5 : 1 ) ( v / v / v / v ) (27)  Solvent system: benzene:methanol:acetic ( v / v / v ) (7)  acid  (10:2:1)  14  0. 48 u m o l e s o f Cop 4.  Protein  p e r ml = 0 . 1  O.D.  ( E j JJ  M  (400 nm)  =  0.1)  Determination  P r o t e i n was  assayed  and S e l l s (26). standard.  III  a c c o r d i n g t o the method o f G r o v e s ,  C r y s t a l l i n e beef serum albumin  was  Davis  used as a  The m e t h o d g a v e r e s u l t s c o m p a r a b l e t o t h o s e o f L o w r y ,  R o s b r o u g h , F a r r and R a n d a l l o r g a n i s m s u n d e r s t u d y and was  (36), using standard more e a s i l y a n d  BSA  and  the  r a p i d l y run  under  routine conditions.  IV.  Antagonism  Studies  Compounds w h i c h o v e r c o m e t h e i n h i b i t o r y a c t i o n o f t h e b i o t i c albomycin albomycin 1.  are said to antagonize  anti-  the drug, or to serve  as  antagonists. L i q u i d Medium C u l t u r e s o f B_. s u b t i 1 ?s o r M_. l y s o d e i k t i c u s w e r e grown i n  l i q u i d m i n i m a l m e d i a as d e s c r i b e d p r e v i o u s l y .  At the time  of  i n o c u l a t i o n , a d d i t i o n s o f t h e c o m p o u n d s t o be t e s t e d w e r e made to the f l a s k s at the d e s i r e d c o n c e n t r a t i o n s . as d e s c r i b e d 2.  G r o w t h was  followed  above.  S o l i d Med i urn A n t a g o n i s m on s o l i d medium was  determined  e s s e n t i a l l y accord-  i n g t o t h e m e t h o d o f Z a h n e r , B a c h m a n n , H u t t e r and Nu'esch ( 7 6 ) . C u l t u r e s o f B. s u b t ? 1 i s o r M.  l y s o d e ? k t i c u s growing in minimal  medium were s p r e a d o v e r m i n i m a l a g a r p l a t e s .  Strips of sterile  b i b u l o u s p a p e r (60 mm x 6 mm), i m p r e g n a t e d w i t h s o l u t i o n s o f t h e compounds t o be t e s t e d , were o v e r l a i d on t h e a g a r s u r f a c e i n T h e p l a t e s w e r e t h e n i n c u b a t e d a t 37 C a n d  the form o f a c r o s s . examined  at intervals.  Zones o f growth and i n h i b i t i o n were  recorded.  V.  P r e p a r a t i o n s o f C e l l s a n d C e l l - F r e e E x t r a c t s f o r Enzyme A s s a y s  A f t e r 12 - 14 h o u r s g r o w t h a t 37 C, 200 ml o r 500 ml v o l u m e s o f B_. s u b t ? 1 i s c u l t u r e s w e r e h a r v e s t e d b y c e n t r i f u g a t i o n a t 8000 x £ f o r 20 m i n u t e s a t k C i n a S o r v a l l S S 4 c e n t r i f u g e ( I v a n S o r v a l l  Inc.,  Norwalk, C o n n e c t i c u t ) . T h e c e l l s were washed two o r t h r e e t i m e s i n m i n i m a l medium w i t h o u t * c a r b o n - s o u r c e a n d r e s u s p e n d e d a t a c o n c e n t r a t i o n o f 1 g w e t w e i g h t o f c e l l s p e r 2 ml o f 0.1 M T r i s / H C l b u f f e r , pH 8.1. L y s o z y m e (5 x 10  (100 u g / m l ) , DNase (10 u g / m l ) a n d M g C l  2  M) w e r e a d d e d a n d t h e m i x t u r e i n c u b a t e d f o r 30 m i n u t e s  a t 37 C o r f o r k5 m i n u t e s a t 30 C. T h e r e s u l t i n g e x t r a c t was c e n t r i f u g e d a t 20,000 x £ f o r 30 m i n u t e s a t k C i n a S o r v a l l S S I c e n t r i fuge. assays.  T h e s u p e r n a t a n t f l u i d was u s e d d i r e c t l y f o r r o u t i n e e n z y m e  VI.  Enzyme A s s a y .  Synthes is of  The f o r m a t i o n o f  DHB  DHB.  f r o m c h o r i s m i c a c i d was m e a s u r e d by (72),  m e t h o d o f Y o u n g , Cox a n d G i b s o n (a) 0.1  ml o f IN HC1  (b) T h e  r e a c t i o n p r o d u c t was  was  with the f o l l o w i n g m o d i f i c a t i o n s  used t o t e r m i n a t e the enzyme r e a c t i o n .  ( c ) T h e e t h e r e a l l a y e r was  e x t r a c t e d i n t o 2.0  ml o f e t h e r .  s e p a r a t e d f r o m t h e a q u e o u s l a y e r by  c e n t r i f u g a t i o n a t low s p e e d GLC-1  the  (200  x g_) f o r 3 m i n u t e s  in a  c e n t r i f u g e ( G e n e r a l L a b o r a t o r i e s C e n t r i f u g e , Ivan  Sorvall  Inc., Norwalk,  f r o m 300  nm t o 350  nm  photometer.  C a r e was  the o r i g i n a l  volume.  Conn.),  then scanned  immediately  i n a U n i c a m SP800 r e c o r d i n g s p e c t r o taken to a d j u s t the e t h e r l a y e r to  (d) A r e a c t i o n b l a n k was  run f o r each  i d e n t i f i c a t i o n purposes,  in a d d i t i o n to the u l t r a v i o l e t scan, a  p o r t i o n o f t h e e x t r a c t was a u t h e n t i c DHB  individual assay.  chromatographed  (see Chromatography,  For  and c o m p a r e d w i t h  above).  DHB  production  was  q u a n t i t a t e d r o u t i n e l y from a standard curve prepared u s i n g commercial  DHB.  U n d e r t h e s e c o n d i t i o n s , 0.1  r e p r e s e n t s 0.075 y m o l e s  V I I . Enzyme P u r i f i c a t i o n .  o f DHB  p e r ml  (E^  5  O.D. yH  (318  ( 3 l 8 nm)  P a r t i a l P u r i f i c a t i o n o f DHB  nm) =  0.1).  Synthetase  A c t i v e c e l l - f r e e e x t r a c t s o f B. s u b t ? 1 i s WB746 w e r e p r e p a r e d  a s d e s c r i b e d a b o v e u s i n g 0.05 1 mM  M T r i s / H C l b u f f e r , pH 8.1,  e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (EDTA) and 0.1  ethanol.  mM  containing mercapto-  T h e s e w e r e i n c l u d e d t o h e l p s t a b i l i z e t h e enzyme o v e r  p e r i o d o f s e v e r a l days d u r i n g column chromatography. e x t r a c t was  treated i n i t i a l l y  The  a  crude  i n one o f t h r e e w a y s : e i t h e r (a)  alter-  n a t e f r e e z i n g ( i n d r y - i c e / e t h a n o l ) and t h a w i n g (10 m i n u t e s a t 30  C)  f o r two c y c l e s , t h e n c e n t r i f u g a t i o n a t 2 0 , 0 0 0 x g_ f o r 5 m i n u t e s t o remove d e n a t u r e d  p r o t e i n o r (b) a d j u s t m e n t  t o pH 4.9  i n an  u s i n g 1.0 M a c e t i c a c i d , f o l l o w e d by c e n t r i f u g a t i o n as above; o r (c) f r a c t i o n a t i o n w i t h s o l i d  ice-bath  described  ( N H ^ ^ S O ^ a t 4 C, w i t h  the  f r a c t i o n p r e c i p i t a t i n g b e t w e e n 30 and 6 0 % s a t u r a t i o n b e i n g d i s s o l v e d i n 0.05  M T r i s / H C l b u f f e r c o n t a i n i n g EDTA and m e r c a p t o e t h a n o l  described.  In e a c h c a s e , t h e e n z y m e p r e p a r a t i o n was  passage through  then d e s a l t e d  a 1.0 x 10 cm c o l u m n o f S e p h a d e x G-10.  ing s o l u t i o n s were c o n c e n t r a t e d  by d i a l y s i s a g a i n s t  6 0 0 0 , t h e n p u r i f i e d f u r t h e r by D E A E - c e l l u l o s e  as by  The r e s u l t -  Polyethyleheglycol  chromatography or  sucrose gradient c e n t r i f u g a t i o n .  1.  DEAE-Cellulose  Chromatography  A l l o p e r a t i o n s were performed processed Peterson  and c o l u m n s (2 x 20 cm) and S o b e r ( 4 7 ) .  o r 45 mg o f p r o t e i n , was was  a t 4 C.  DEAE-cel1ulose  were packed a c c o r d i n g  A maximum o f 2.5  ml o f c r u d e  was to  extract,  a p p l i e d t o t h e c o l u m n and t h e p r o t e i n  e l u t e d w i t h a l i n e a r g r a d i e n t o f NaCl  (0 - 1.0 M)  i n 0.05  M  T r i s / H C l b u f f e r , pH 8.2.  F r a c t i o n s c o n t a i n i n g 45 o r 99  w e r e c o l l e c t e d i n 16 x 150 mm collector  (LKB P r o d u k t e r  o f 15 ml p e r h o u r was  AB,  t e s t tubes  u s i n g an LKB f r a c t i o n  Stockholm, Sweden).  maintained.  The  A flow  f r a c t i o n s were  f o r e n z y m e a c t i v i t y w i t h i n 12 h o u r s o f t h e b e g i n n i n g run.  T h e e f f l u e n t was  a U n i c a m SP800 r e c o r d i n g 2.  Sucrose  f o r a b s o r b a n c e a t 280  monitored  drops  rate  assayed of each nm  using  spectrophotometer.  Grad i e n t C e n t r i f u g a t i o n  Exponential  s u c r o s e g r a d i e n t s f r o m 5% t o 25%  were  prepared  i n 15 ml p o l y a l l o m e r c e n t r i f u g e t u b e s a c c o r d i n g t o G2 D a t a #42  o f t h e I n t e r n a t i o n a l E q u i p m e n t Co.  w e r e l a y e r e d on t h e t o p s o f  Ihe 14.2  s p u n f o r 12 h o u r s a t 2 C i n an  Sheet  P r o t e i n e x t r a c t s (0.6  ml g r a d i e n t s and  the  ml)  tubes  IEC m o d e l B60 c e n t r i f u g e u s i n g  t h e SB283 r o t o r , a t 150,000 x g_.  A h o l e was  punched in the  bottom  o f e a c h t u b e u s i n g a 20 g a u g e n e e d l e , and 6 - d r o p f r a c t i o n s w e r e c o l l e c t e d u s i n g t h e LKB 15 ml p e r h o u r .  The f r a c t i o n s were a s s a y e d  a c t i v i t y as d e s c r i b e d  I I.  Iron T r a n s p o r t  1.  f r a c t i o n c o l l e c t o r , at a flow rate of f o r DHB-synthetase  above.  Studies  Iron Uptake The c e l l s were p r e p a r e d ,  incubated with  59  F e , and  assayed 59 f o r i r o n u p t a k e as d e s c r i b e d by P e t e r s and W a r r e n ( 4 6 ) . A Fe  quench-curve  was p r e p a r e d u s i n g  F e o f known s p e c i f i c  From t h i s c u r v e , c o u n t s p e r minute picograms  (pg) o f i r o n .  c e l l s , washing  activity.  (cpm) w e r e c o n v e r t e d t o  E x a c t d e t a i l s a s t o a d d i t i o n s made t o  o f c e l l s and o t h e r t r e a t m e n t s , a r e g i v e n i n  the t e x t . 2.  Iron E f f l u x C u l t u r e s were grown f o r s e v e r a l g e n e r a t i o n s i n t h e p r e s e n c e 59  of  F e . C e l l s w e r e h a r v e s t e d by c e n t r i f u g a t i o n a t room  tempera-  t u r e ( 1 2 , 0 0 0 x g_) i n a S o r v a l l SSI c e n t r i f u g e f o r 5 m i n u t e s , w a s h e d t w i c e i n i r o n - d e f i c i e n t medium w i t h o u t c a r b o n s o u r c e a n d resuspended  i n i r o n - d e f i c i e n t medium t o a c o n s t a n t c e l l d e n s i t y .  F i v e ml s a m p l e s Erlenmeyer  o f t h i s s u s p e n s i o n w e r e d i s t r i b u t e d t o 25 ml  f l a s k s a n d t h e c u l t u r e s i n c u b a t e d a t 37 C a n d 100 rpm  in a M e t a b o l y t e shaking water bath. t i o n s w e r e made a n d 1.0 ml s a m p l e s  A f t e r 10 m i n u t e s , a d d i then removed a t i n t e r v a l s and  f i l t e r e d t h r o u g h 0.45 y M i l l i p o r e f i l t e r s .  The f i l t e r e d  cells  w e r e w a s h e d w i t h 5 ml o f i r o n - d e f i c i e n t medium p r e - w a r m e d t o 37 C, t h e n d r i e d u n d e r a 2 5 0 w a t t h e a t lamp.  The dried  filters  w e r e t h e n p l a c e d i n s c i n t i l l a t i o n v i a l s c o n t a i n i n g 7-5 ml o f L i q u i f l u o r / t o l u e n e (42/958) s c i n t i l l a t i o n f l u i d and t h e r a d i o a c t i v i t y counted as before.  IX.  Chemicals  A l l o r g a n i c media c o n s t i t u e n t s were o f D i f c o brand.  Inorganic  c o n s t i t u e n t s were o b t a i n e d from F i s h e r S c i e n t i f i c Co. C h o r i s m i c a c i d was p u r i f i e d f r o m t h e c u l t u r e s u p e r n a t a n t s o f A. a e r o g e n e s 62-1  b y t h e m e t h o d o f G i b s o n (23) a s d e s c r i b e d b y P e t e r s  (44), a n d c h e c k e d f o r p u r i t y .  DHBG was s y n t h e t i z e d a c c o r d i n g t o  P e t e r s (44) a n d c h e c k e d f o r p u r i t y . F e r r i c h r o m e and F e r r i c h r o m e A were o b t a i n e d from Dr. J . B . Neilands o f the U n i v e r s i t y o f C a l i f o r n i a , Berkeley.  Desferriferri-  c h r o m e a n d D e s f e r r i f e r r i c h r o m e A w e r e p r e p a r e d a c c o r d i n g t o Emery a n d N e i l a n d s (20). A l b o m y c i n was o b t a i n e d f r o m D r . J . T u r k o v a , C z e c h o s l o v a k A c a d e m y of  S c i e n c e , Prague. 5 - h y d r o x y a n t h r a n i l i c a c i d was o b t a i n e d f r o m M r . P a t r i c k  of the Department  Curling  o f M i c r o b i o l o g y , U.B.C.  The f o l l o w i n g c h e m i c a l s were o b t a i n e d from commercial s o u r c e s : 2,3-DHB, 2,4-DHB, 2,5-DHB, 2,6-DHB, 3 - f 1 u o r o b e n z o i c  a c i d , 0-hydroxy-  h i p p u r i c a c i d f r o m A l d r i c h C h e m i c a l C o . ; EDTA f r o m A l l i e d  Chemical  Co.; t h e a r o m a t i c amino a c i d s , c r e s o t i c a c i d , 3 h y d r o x y b e n z o i c  acid,  _  NTA, T i r o n , 1 , 1 0 - p h e n a n t h r o l i n e , s a l i c y l a m i d e ,  N-hydroxymethy1  s a l i c y l a m i d e , m - t o l u i c a c i d , v e r a t r i c acid and v a n i l l i c acid J.T. Baker Chemical Co.; s a l i c y l i c a c i d from The B r i t i s h Houses; hemin, 3 hydroxyanthrani1ic -  from  Drug  a c i d , a-iodoacetamide and p-  c h l o r o m e r c u r i b e n z o a t e f r o m C a l b i o c h e m ; a n t h r a n i l i c and amic a c i d s from Eastman O r g a n i c C h e m i c a l s ; c a t e c h o l , NaN^,  benzohydroxa,a-dipyridy1,  and p - n i t r o p h e n o l f r o m F i s h e r S c i e n t i f i c Co.; b o v i n e s e r u m  albumin o-hydroxyphenylacetate, shikimic a c i d , streptomycin s u l f a t e 59 f r o m Sigma C h e m i c a l Co.;  Fe a s F e C l ^ ( s p e c i f i c a c t i v i t y 2 3 C i / g  Fe) f r o m t h e I n t e r n a t i o n a l C h e m i c a l a n d N u c l e a r C o . ;  lysozyme  and  D N a s e f r o m W o r t h i n g t o n B i o c h e m i c a l C o r p . , F r e e h o l d , New J e r s e y . P o l y e t h y l e n e g l y c o l 6 0 0 0 was o b t a i n e d f r o m J . T . B a k e r Co.  D E A E - c e l l u l o s e a n d S e p h a d e x G10 w e r e p r o d u c t s o f  C h e m i c a l s , U p p s a l a , Sweden.  Chemical  Pharmacia  RESULTS  Growth F a c t o r s , A l b o m y c i n and A l b o m y c i n - A n t a g o n i s m  I ntroduction Peters  (44)  showed t h a t v a r i o u s p h e n o l i c compounds and  h y d r o x a m i c a c i d s h a d d i f f e r e n t e f f e c t s on p h e n o l i c a c i d d u c t i o n by  subt i l l s .  pro-  I t was t h e r e f o r e o f i n t e r e s t t o i n v e s t i -  g a t e t h e c a p a c i t y o f some o f t h e s e c o m p o u n d s t o s u p p o r t  the growth  of b a c t e r i a c u l t i v a t e d under c o n d i t i o n s o f s t r e s s , s i n c e  various  c h e l a t i n g a g e n t s h a v e b e e n shown t o a c t a s " g r o w t h f a c t o r s " f o r a number o f b a c t e r i a l s p e c i e s  (4,  16, 17,  28,  29,  32,  55,  71).  Micrococcus  l y s o d e i k t i c u s was c h o s e n f o r s t u d y b e c a u s e i t r e q u i r e s  ferrichrome  (39)  for growth.  j3. s u b t i l i s was c h o s e n b e c a u s e i t p r o d u c e s DHB(G)  and  (53)  or catechol  and o t h e r  iron-binding  i t was o f i n t e r e s t t o s e e i f DHB(G) o r r e l a t e d c o m p o u n d s  a role in the basic metabolism o f the organism. b e e n shown t h a t f e r r i c h r o m e  were a l s o t e s t e d as p o s s i b l e a n t a g o n i s t s on J3. s u b t i 1 i s a n d M^. l y s o d e i k t i c u s .  Micrococcus  Since  s e r v e s as an a n t a g o n i s t  i n h i b i t o r y a c t i o n o f the a n t i b i o t i c albomycin  1.  compounds  lysodeikticus  (4,  play  i t has  to the  7 6 ) , t h e compounds  to the a c t i o n of  albomycin  (a)  Growth M_. l y s o d e i k t i c u s g r e w v e r y p o o r l y when i n o c u l a t e d  d i r e c t l y f r o m T S Y i n t o S a l t o n ' s m i n i m a l medium.  The length  o f t h e l a g p e r i o d was e v e n g r e a t e r when t h e i n o c u l u m was washed once  i n i r o n - d e f i c i e n t m i n i m a l medium.  No  growth  o c c u r r e d f o r 40 h o u r s when t h e i n o c u l u m was w a s h e d t w i c e , n o r i n 48 h o u r s when t h e i n o c u l u m was w a s h e d t h r e e t i m e s ure 1).  (Fig-  S u b s e q u e n t l y , t h e i n o c u l a were washed t w i c e f o r t h e  e x p e r i m e n t s t e s t i n g t h e v a r i o u s compounds.  The lengths o f  t h e l a g p e r i o d s w e r e i n a l l c a s e s o b t a i n e d by e x t r a p o l a t i o n from growth c u r v e s determined from d u p l i c a t e experiments. (b)  Growth F a c t o r s When f e r r i c h r o m e , a c y c l i c i r o n - c o n t a i n i n g p e p t i d e ,  was a d d e d  t o t h e m e d i u m , g r o w t h was s t i m u l a t e d s i g n i f i c a n t l y  ( F i g u r e 2) a n d t h e l e n g t h o f t h e l a g p e r i o d was f r o m 40 h o u r s t o 10 - 12 h o u r s .  reduced  The length o f t h e l a g p e r i o d  was n o t r e d u c e d s i g n i f i c a n t l y by t h e a d d i t i o n o f EDTA, NTA o r o - h y d r o x y h i p p u r i c a c i d t o t h e g r o w t h medium. when t h e s e c o m p o u n d s w e r e a d d e d  However,  i n t h e p r e s e n c e o f 0.5%  y e a s t e x t r a c t , t h e l e n g t h o f t h e l a g p e r i o d was r e d u c e d a g a i n f r o m 40 h o u r s t o a b o u t  10 - 12 h o u r s  (Figure 2).  e x t r a c t , w h i c h s t i m u l a t e d growth u s i n g unwashed  Yeast inocula,  had no e f f e c t u n d e r t h e e x p e r i m e n t a l c o n d i t i o n s e m p l o y e d . DHBG, 2,4-DHB, a n d t o a l e s s e r e x t e n t DHB a n d c a t e c h o l ,  F i g u r e 1.  E f f e c t o f washing inoculum  o n g r o w t h o f M. l y s o d e i k t i c u s  i n d e f i n e d m e d i u m a t 37 C. Symbols:  to  8, u n t r e a t e d  inoculum;  •  •, t w i c e - w a s h e d i n o c u l u m ;  washed t h r e e  F i g u r e 2.  inoculum;  0 - — 0 , once-washed A  A,  inoculum  times.  E f f e c t o f v a r i o u s c o m p o u n d s o n g r o w t h o f M_. l y s o d e i k t i c u s i n d e f i n e d m e d i u m a t 37 C Symbols: •  I n o c u l u m was w a s h e d  f, no a d d i t i o n s ; 0  p l u s 40 yM NTA o r 0 - H y d r o x y h i p p u r i c A  A, 10 uM  ferrichrome.  0, 0.5%  twice.  yeast extract  a c i d , o r 10 uM EDTA;  um  IDC  in t h e a b s e n c e o f y e a s t e x t r a c t , a l s o reduced t h e l a g period.  S a l i c y l i c a c i d , on t h e o t h e r hand, i n c r e a s e d t h e  length o f the lag period to a s l i g h t extent (c)  ferrichrome,  in Tables  h a n d 5,  i t is evident  l i q u i d a n d s o l i d medium.  In l i q u i d medium, n e i t h e r  had any e f f e c t on g r o w t h , w h e r e a s on  m i n i m a l a g a r p l a t e s , EDTA a n t a g o n i z e d With yeast e x t r a c t  albomycin  i n t h e l i q u i d medium, both  were e f f e c t i v e albomycin a n t a g o n i s t s .  inhibition.  EDTA a n d NTA  F e C l ^ was  s t i m u l a t o r y under both c o n d i t i o n s o f growth.  sligthly  Yeast  extract  had l i t t l e e f f e c t on t h e l e n g t h o f t h e l a g p e r i o d .  Baci11 us (a)  that  DHB a n d DHBG o v e r c a m e a l b o m y c i n a n t a g o n i s m i n  EDTA n o r NTA a l o n e  alone  3).  Growth I n h i b i t i o n and A l b o m y c i n A n t a g o n i s m From t h e d a t a  both  (Table  subt?1is  Growth L i k e iM. l y s o d e i k t i c u s , j$. s u b t i 1 ?s e x h i b i t e d a n e x t e n d e d  l a g p e r i o d when a w a s h e d i n o c u l u m  was u s e d .  Consequently,  u s i n g twice-washed c e l l s as inoculum, t h e e f f e c t s o f v a r i o u s c o m p o u n d s on g r o w t h o f B^. s u b t i 1 i s w e r e e x a m i n e d . extract organism  {0.5%), NTA a n d EDTA a l l o w e d ( F i g u r e 3)-  Ferrichrome  Yeast  r a p i d growth o f t h e  and F e C l ^ f a i l e d t o s h o r t -  en t h e l e n g t h o f t h e l a g p e r i o d , b u t d i d a l l o w a g r e a t e r y i e l d o f c e l l s than DHB(G) , c a t e c h o l  the control  ( F i g . 3).  and o - h y d r o x y h i p p u r i c  Salicylic acid,  a c i d showed no e f f e c t .  T a b l e 3.  T h e e f f e c t o f v a r i o u s i r o n - b i n d i n g c o m p o u n d s on g r o w t h of Micrococcus medium.  l y s o d e ? k t i c u s i n S a l t o n ' s (53) minimal  Results (the average  from t r i p l i c a t e  experiments),  are presented as the length o f the l a g p e r i d ( i n hours) before growth ensued.  "  control  ,  (nil)  Final Concentration (yM)  Length o f l a g P e r i o d (hours) . .  -  42  2.3 dihydroxyberizoate  40  36  2.4- d i h y d r o x y b e n z o a t e  40  30  itoic acid  40  16  catechol  kO  36  sal icy 1ic acid  kO  48  -  T a b l e 4.  E f f e c t o f v a r i o u s compounds as a n t a g o n i s t s t o t h e i n h i b i t t o r y a c t i o n o f a l b o m y c i n on growth o f M i c r o c o c c u s deikticus  i n S a l t o n ' s ( 3 3 ) m i n i m a l medium.  lyso-  Results, (the  average from d u p l i c a t e e x p e r i m e n t s ) , a r e p r e s e n t e d as t h e length o f the l a g p e r i o d ( i n hours) before the onset o f logarithmic growth.  Compound  Final Concentration (uM)  Albomyc " n A l b . * + F e r r i chrome II  +  "  + 1toic acid  2,3-d i h y d r o x y b e n z o a t e Salicylic  acid  o-Hydroxyhi ppurate  Length o f Lag P e r i o d (Hours)  0.1  50  10.0  10  40.0  24  40.0  32  40.0  48  40.0  >50  40.0  >50  1.0  >50  "  + Catechol  "  + FeCl  11  + EDTA  10.0  >50  "  + NTA  10.0  >50  11  + Yeast e x t r a c t  0.5%  48  11  + Y e a s t e x t r a c t * * + NTA  10.0  14  "  + Y e a s t e x t r a c t + EDTA  10.0  13  1 .0  24  3  Yeast e x t r a c t + FeCl,  * A l b o m y c i n was u s e d a t 0.1 yM c o n c e n t r a t i o n t h r o u g h o u t ** Y e a s t e x t r a c t was u s e d a t 0.5% c o n c e n t r a t i o n  T a b l e 5.  E f f e c t o f v a r i o u s compounds as a n t a g o n i s t s o f a l b o m y c i n on g r o w t h o f M_. l y s o d e i k t i c u s  u s i n g t h e paper s t r i p method  o f Z a h n e r e t a l . ( 7 6 ) . E f f e c t i v e compounds p e r m i t t e d growth o f M. l y s o d e i k t i c u s strip.  to within  S l i g h t l y e f f e c t i v e compounds p e r m i t t e d growth  t o 5 mm p a s t t h e n o r m a l albomycin s t r i p . the  l e s s t h a n 1 mm o f t h e a l b o m y c i n up  l i n e o f i n h i b i t i o n around the  I n e f f e c t i v e compounds d i d n o t o v e r c o m e  inhibitory action of albomycin.  Compounds  1  Tested as Albomycin  Effective  SIightly  2  Antagonists  Ineffeet ive  Effeetive  Ferrichrome  NTA  o-hydroxyh i ppurate  2,3 d i hydroxybenzoate  2 ,h-dihydroxybenzoate  o-hydroxyphenylacetate  itoic  2,6-dihydroxybenzoate  S a l i c y l i c acid  catechol  salicylamide  5-hydroxyanthrani1ic  2,5-di hydroxybenzoate  cresotic  Acid  EDTA  hemi n  N-hydroxymethylsalicylamide  FeCK  -  acid  acid  p a p e r s t r i p i m p r e g n a t e d w i t h 1 mM c o n c e n t r a t i o n o f t e s t e d  compound  p a p e r s t r i p i m p r e g n a t e d w i t h 1.0 uM c o n c e n t r a t i o n o f a l b o m y c i n  29  H O U R S  F i g u r e 3-  € f f e c t o f v a r i o u s c o m p o u n d s o n g r o w t h o f B_. s u b t ? 1 i s B1471 a t 37 C i n m i n i m a l medium. t o 10 yM c o n c e n t r a t i o n s a t z e r o  A l l a d d i t i o n s w e r e made time.  S y m b o l s : 0 - — 0 , n o a d d i t i o n s , s a l i c y l i c a c i d , DHB, DHBG, catechol, o-hydroxyhippuric (0.5%); A ferrichrome.  A , EDTA; A  acid; C  A, NTA;  Q  •, y e a s t e x t r a c t Q  FeCl  3  or  (b)  G r o w t h I n h i b i t i o n and A l b o m y c i n  Antagonism  T h e a d d i t i o n o f 0.1 uM a l b o m y c i n t o t h e g r o w t h had  medium  l i t t l e e f f e c t on t h e l e n g t h o f t h e B. s u b t i 1 i s l a g p e r i o d  w h e r e a s 0.4 uM a l b o m y c i n e x t e n d e d t h e l a g t o a b o u t  144  T h e d a t a i n T a b l e s 6 and 7 i n d i c a t e t h a t , a s w i t h M_,  hours. lyso-  d e i k t i c u s , DHBG o v e r c a m e a l b o m y c i n i n h i b i t i o n o f EL s u b t ? 1 i s i n b o t h l i q u i d a n d s o l i d medium. f e r r i c h r o m e and DHB  In c o n t r a s t , h o w e v e r ,  by t h e m s e l v e s w e r e i n e f f e c t i v e , b u t when  t e s t e d i n the presence o f y e a s t e x t r a c t they overcame albomycin antagonism.  Iron, in the presence of yeast e x t r a c t  a l s o overcame the e f f e c t of albomycin.  Yeast extract alone  r e d u c e d t h e l e n g t h o f t h e l a g p e r i o d somewhat.  EDTA a n d  NTA by t h e m s e l v e s s e r v e d a s e f f e c t i v e a l b o m y c i n a n t a g o n i s t s . S a l i c y l a m i d e was e f f e c t i v e i n o v e r c o m i n g a l b o m y c i n  inhibition  i n s o l i d med i urn.  Discussion of Results  Hydroxamic c h e l a t i n g agents  a c i d s ( 4 , 1 7 ) , g l y c i n e p e p t i d e s (16) and o t h e r (28,  29,  32,  71) s e r v e a s e f f e c t i v e g r o w t h  f o r v a r i o u s b a c t e r i a l s p e c i e s c u l t i v a t e d under poor conditions.  The p r e s e n t r e s u l t s  factors  nutritional  indicate that unidentified  growth  f a c t o r s c a r r i e d o v e r from TSY-grown i n o c u l a , or p r e s e n t in y e a s t e x t r a c t , c a n be e f f e c t i v e l y r e p l a c e d by i r o n , f e r r i c h r o m e o r n u m e r o u s  31  T a b l e 6.  E f f e c t o f v a r i o u s compounds a s a n t a g o n i s t s t o t h e i n h i b i t o r y a c t i o n o f a l b o m y c i n o n g r o w t h o f B_. s u b t i 1 i s B1471 a n d WB746.  R e s u l t s , (the average o f d u p l i c a t e experiments),  are presented as the length o f the l a g p e r i o d ( i n hours) before t h e onset o f growth.  Compound  Final Concentration (•••yM)  Albomycin  Length o f Lag P e r i o d (Hours)  0.4  144  10.0  16  Alb." + Ethylenediaminetetraacetic acid "  + N i t r i l o t r i a c e t i c acid  10.0  14  "  + Itoic acid  40.0  15  "  + FeCl  3  1.0  102  "  + Hemin  1.0  124  "  + Ferrichrome  10.0  144  "  + Catechol  10.0  144  11  + Salicylic acid  10.0  144  "  + Yeast e x t r a c t * *  "  + Yeast e x t r a c t + ferrichrome  10.0  12  "  + Yeast e x t r a c t + Itoic acid  10.0  14  "  + Yeast e x t r a c t + 2,3-dihydroxybenzoate 10 yM + Yeast e x t r a c t + F e 20 yM  ."  0.5%  + + +  60  18 32  * A l b o m y c i n was a d d e d a t 0.4 yM f i n a l c o n c e n t r a t i o n ** Y e a s t e x t r a c t was u s e d a t 0 . 5 % c o n c e n t r a t i o n t h r o u g h o u t  T a b l e 7.  E f f e c t o f v a r i o u s compounds as a n t a g o n i s t s t o t h e e f f e c t o f a l b o m y c i n o n g r o w t h o f B_i s u b t i 1 i s B1471  and WB746 u s i n g  t h e p a p e r s t r i p m e t h o d o f Z a h n e r e t a 1. ( 7 6 ) .  Effective  c o m p o u n d s p e r m i t t e d g r o w t h o f B_. s u b t i 1 i s t o w i t h i n l e s s t h a n 1 mm o f t h e a l b o m y c i n s t r i p .  S l i g h t l y e f f e c t i v e com-  p o u n d s p e r m i t t e d g r o w t h up t o 5 mm  p a s t the normal  of i n h i b i t i o n around the albomycin s t r i p .  line  Ineffective  compounds d i d n o t overcome t h e i n h i b i t o r y a c t i o n o f a l b o m y c i n .  Compounds  Effeet ive  1  T e s t e d as A l b o m y c i n  2  Antagonists  Ineffeetive  SIightly Effect ive  Ferrichrome  Catechol  2.5- d i h y d r o x y b e n z o a t e  Itoic'acid  2.3 d i hydroxybenzoate  2.6- d i h y d r o x y b e n z o a t e  Salicylamide  2.4- d i h y d r o x y b e n z o a t e  salicylic  Nitritotriacetic  o-hydroxyhippuric  hemin  aci d  FeCU  -  acid  acid  - also a l l previous compounds e x c e p t F e r r i chrome a t c o n c e n t r a t i o n s o f l e s s t h a n 1 mM  Compounds i m p r e g n a t e d p a p e r s t r i p a t 1 mM c o n c e n t r a t i o n A l b o m y c i n was  u s e d a t 10 uM c o n c e n t r a t i o n on t h e p a p e r  strip  c h e l a t i n g a g e n t s ( i n c l u d i n g p h e n o l i c a c i d s ) f o r J3. s u b t j 1 i s a n d lysodeikticus.  T h e s t i m u l a t i o n o f g r o w t h by c h e l a t i n g  agents  i s u s u a l l y a t t r i b u t e d t o o n e o f two e f f e c t s ( 7 1 ) : ( a ) t h e a g e n t s make a v a i l a b l e t o t h e c e l l s e s s e n t i a l m e t a l  ions that a r e inacces-  s i b l e , o r (b) t h e a g e n t s b i n d t o x i c m e t a l s t h a t m i g h t i n h i b i t growth.  On t h e o t h e r h a n d , t h e h y p o t h e s i s  which has r e c e i v e d  some s u p p o r t  otherwise (39),  o f Neilands  f r o m o t h e r w o r k ( 1 7 , 28),  that  d e g r e e o f s p e c i f i c i t y o f a c h e l a t i n g a g e n t was n e c e s s a r y  some  f o r i t to  s e r v e a s a g r o w t h f a c t o r f o r M^. l y s o d e i k t i c u s w o u l d i m p l y a d i r e c t e f f e c t o f t h e c h e l a t i n g a g e n t on t h e c e l l . Some d e g r e e o f v a r i a t i o n was o b s e r v e d i n t h e c h e l a t i n g  agents  s e r v i n g a s g r o w t h f a c t o r s f o r B_. s u b t i l i s a n d f4. l y s o d e i k t i c u s u n d e r the c o n d i t i o n s employed.  T h e f a c t t h a t NTA a n d EDTA s e r v e d e f f e c t i v e -  l y f o r M_. l y s o d e i k t i c u s i n t h e p r e s e n c e o f y e a s t e x t r a c t b u t n o t i n i t s a b s e n c e , m i g h t i m p l y a mode o f a c t i o n s i m i l a r t o t h a t o f ( a ) above, in which the e s s e n t i a l  i o n ( o r o t h e r f a c t o r ) was  f r o m t h e y e a s t e x t r a c t by t h e EDTA o r t h e NTA.  released  On t h e o t h e r  hand,  EDTA a n d NTA w e r e b o t h e f f e c t i v e f o r B_. s u b t i 1 ?s i n t h e p r e s e n c e or absence o f yeast  extract.  In t h i s c a s e , d i r e c t a c t i o n o f t h e  c h e l a t i n g a g e n t s o n t h e c e l l s may be c o n s i d e r e d  possible.  Alter-  n a t i v e l y , t h e c h e l a t i n g a g e n t s may h a v e r e l i e v e d t h e t o x i c i t y c a u s e d by t h e e x c e s s o f o n e p a r t i c u l a r i o n o v e r a n o t h e r by a l t e r i n g t h e ratios of the various Ferrichrome,  ions  i n t h e medium.  a trihydroxamate containing  i r o n , served  as a  potent  growth f a c t o r f o r both organisms.  w h i c h s e r v e as g r o w t h f a c t o r s a l s o b i n d  The  various  phenolic  iron strongly.  However, f o r B_. s u b t i 1 i s  v a r i a t i o n d i d e x i s t between the phenol i c s which served and  those which served  f o r M.  lysodeikticus.  t h e r e f o r e be t a k e n t o i m p l i c a t e The  r e s u l t s may  a l s o be  These r e s u l t s  t h a t t h e c h e l a t i n g a g e n t s may  by a c t i n g i n one  o f a t l e a s t two,  a g e n t s , s u c h a s EDTA o r NTA, essential  may  i o n s , o r a l t e r n a t i v e l y , but  t o x i c ions.  iron-binding polysaccharide in b a c t e r i a l c e l l s  c h e l a t i n g agent enters i n s u c h a way  Some  s e r v e as " r e l e a s e r s " o f l e s s l i k e l y , as r e m o v e r s o f or  (51).  q u i t e r e a d i l y (40).  A n o t h e r a l t e r n a t i v e may  that i n h i b i t i o n is overcome.  be t h a t  Effectiveness of  unexplained.  the  the  strength,  s h a p e o f t h e c h e l a t e - i o n c o m p l e x , a s w e l l as t h e  roles played  up-  t h e c e l l and a l t e r s t h e n o r m a l m e t a b o l i s m  s i t e s on t h e b a c t e r i a l c e l l The  Even  i s thought r e s p o n s i b l e f o r ion  g r o w t h f a c t o r w o u l d t h e r e f o r e be a m a t t e r o f t h e b i n d i n g s i z e and  the  s e r v e as s p e c i f i c f a c t o r s w h i c h s o l u b i l i z e o r  t r a n s p o r t the e s s e n t i a l ion to the c e l l  take  respect,  s e r v e as g r o w t h f a c t o r s  O t h e r c h e l a t i n g a g e n t s , s u c h as f e r r i c h r o m e  p h e n o l i c a c i d s , may  an  In t h i s  p o s s i b l y t h r e e , ways. simply  involved.  i n d i c a t i n g t h a t some d e g r e e  o f s p e c i f i c i t y f o r the c h e l a t i n g agent does e x i s t . it is evident  may  i r o n as the e s s e n t i a l f a c t o r  i n t e r p r e t e d as  acids  binding  surface.  by a l b o m y c i n and  i t s antagonists  still  remain  At l e a s t t h r e e p o s s i b l e s i t e s of a c t i o n f o r the a n t i -  b i o t i c a c t i v i t y o f a l b o m y c i n have been h y p o t h e s i z e d  (65,  66)  and  each  35  o f t h e s e has r e c e i v e d some e x p e r i m e n t a l w h i c h was  the most p o t e n t a l b o m y c i n  subti1is.  In t h e p r e s e n c e o f y e a s t  an e f f e c t i v e a n t a g o n i s t w i t h B^.  T h i s would i n d i c a t e that albomycin a c t s at d i f f e r e n t  i n t h e s e two o r g a n i s m s , a d i f f e r e n t purpose strains.  Ferrichrome;  a n t a g o n i s t f o r M_. l y s o d e i k t i c u s ,  had a l m o s t no e f f e c t w i t h B_. s u b t i l i s . e x t r a c t , h o w e v e r , f e r r i c h r o m e was  backing.  or a l t e r n a t i v e l y , that ferrichrome serves  in the metabolism  o f e a c h o f t h e s e two  DHBG o v e r c a m e t h e e f f e c t o f a l b o m y c i n  a n d J3. s u b t i 1 i s .  sites  bacterial  f o r b o t h M_. l y s o d e i k t i c u s  NTA and EDTA c o m p l e t e l y a n t a g o n i z e d a l b o m y c i n  with  B_. s u b t i l i s , b u t w i t h M_. l y s o d e i k t i c u s t h e s e c o m p o u n d s w e r e a g a i n e f f e c t i v e o n l y in the presence o f yeast e x t r a c t fore, that albomycin ism.  a c t s on a t l e a s t two p h a s e s o f c e l l u l a r  One o f t h e s e may  The o t h e r e f f e c t a p p e a r s  t i o n of iron w i t h i n the c e l l .  metabol-  t o be a  utiliza-  If such were the c a s e , d i f f e r e n c e s  t o i r o n , t o v a r i o u s c h e l a t o r s and t o h y d r o x a m a t e s w o u l d  be e x p e c t e d .  II.  there-  be an i n t e r f e r e n c e w i t h t r a n s p o r t m e c h a n i s m s  a c r o s s t h e c e l l membrane.  in r e s p o n s e  It appears,  Such d i f f e r e n c e s were, i n f a c t ,  observed.  Some G e n e r a l C h a r a c t e r i s t i c s o f G r o w t h , P h e n o l i c A c i d a n d Coproporphyrin  Production in B a c i l l u s  subtilis.  Introduction Up t o t h i s p o i n t , t h e w o r k had d e a l t m a i n l y w i t h IB. s u b t i l i s s t r a i n B1471.  V a r i o u s o b s e r v a t i o n s had  l e d P e t e r s (hk)  to b e l i e v e  t h a t s t r a i n WB746 was  f r o m B1471 w i t h  distinct  i o n i n g o f t h e TCA c y c l e , and c o n t r o l o f DHB strains  on p h e n o l i c a c i d  In a d d i t i o n , t h e t i m e s  f o r g r o w t h and t h e n  and p o r p h y r i n  of onset  production  of phenolic acid  s t r a i n s were reexamined and c u l t u r e s u p e r n a t a n t s p r o d u c t i o n o f compounds  other  than  to the funct-  production.  B1471 and WB746 w e r e c o m p a r e d c a r e f u l l y .  were t e s t e d as carbon s o u r c e s effects  respect  Therefore,  A number o f  compounds  examined f o r t h e i r by t h e two production analysed  strains. by  these  f o r the  DHB a n d DHBG d u r i n g g r o w t h u n d e r  iron-deficient conditions. 1.  W i l d Type (a)  Stra?ns  E f f e c t o f Carbon  Source  P h e n o l i c a c i d s and p o r p h y r i n were produced o n l y presence of high servations).  l e v e l s of carbon source  The d a t a  i n Table  ( p r e l i m i n a r y ob-  8 show t h a t g l u c o s e  t h e most e f f e c t i v e c a r b o n s o u r c e  used  Acetate  appeared  f o r phenolic acid  by  s t r a i n s , although  both  production  t o be i m p o r t a n t  i f glucose  was  in this  was  respect. production  w i t h s t r a i n WB746 t h e r e was  plus glutamate,  mate, o r g l u t a m a t e a l o n e , were absence o f a c e t a t e .  i n the  succinate plus  included  some gluta-  i n t h e medium i n t h e  In t h e p r e s e n c e o f 0.3%  a c e t a t e , Cop I I I  p r o d u c e d when g l u t a m a t e p l u s ^ s u c c i n a t e o r g l y c i n e p l u s  s u c c i n a t e were  included  i n t h e medium. I I I was  With s t r a i n  produced  B1471  on t h e o t h e r  h a n d , o n l y Cop  i n the absence  of a c e t a t e .  In t h e p r e s e n c e o f a c e t a t e , no p h e n o l i c o r  t a b l e 8.  E f f e c t o f v a r i o u s carbon s o u r c e s on growth, p h e n o l i c a c i d a n d c o p r o p o r p h y r i n p r o d u c t i o n '* by B_. s u b t i 1 i s s t r a i n s B1471 a n d WB746.  Carbon Source (]%  level)  +G1ucose C i trate Acetate Succi nate Lactate G1utamate Glycerol Glucose + C i trate G l u c o s e + Succ i nate Glucose + G1utamate Glucose + Glycine Glutamate + Succinate Glycine + Succinate G l y c i ne + C i t r a t e GIJJC. + G l y . + S u c c . lAcetate +G1ucose *C i t r a t e *G1ucose + Glutamate #Succinate + Glutamate  WB746  B1471 c f ii P h e n o l i c Cop 111 x 1 0 " +  +  -  -  +  -  +  +  -  +  +  -+  +  +  -  -  -  +  +  +  +  -  -  +  3  Phenolic  >15 7.5 >15 7.8 NDI ND ND 3.5 >15 >15 >15 >15 5.8 2.8 >15  " +  >15 12 5.4 >15 >15  + +  -  +  -  + +  -  ±  + ND + +  -  +  -  + +  Cop II 1  cf u x 10'  -  >15 2.8 8.6 7.2 ND 7.3 ND 8 10 ND 14 >15 6.7 > 1 12  -  8.6 12 2.3 12 10  -  ND  -  + +  * Cop I I I p r o d u c t i o n d e t e r m i n e d v i s u a l l y by p r e s e n c e o f p i n k c o l o r i n culture. + T e s t e d w i t h 0.1% A c e t a t e * Tested as sole carbon source I ND = n o t d e t e r m i n e d  Cop  III p r o d u c t i o n o c c u r r e d when g l u t a m a t e  the medium ( T a b l e (b)  to  Growth  T h e pH o f t h e m e d i u m was Although  f r o m one e x p e r i m e n t obtained  added  8).  C h a n g e s i n pH D u r i n g  of c u l t u r e s .  was  f o l l o w e d d u r i n g the growth  t h e r e was  to another,  considerable variation a constant  t r e n d was  always  i n t h e pH p r o f i l e o f t h e g r o w i n g c u l t u r e s .  Ini-  tially  i n t h e r a n g e a r o u n d pH 7, v a l u e s d r o p p e d t o a b o u t  pH 6.2  - 6.5  d u r i n g the f i r s t 7 - 8 hours of growth  then remained constant to  r i s e and c o n t i n u e d  8 was  reached.  f o r 1 - 2 hours.  and  T h e pH t h e n  began  t o do s o u n t i l pH 7 o r a t t i m e s  I t was  pH  not u n t i l the b e g i n n i n g o f the  i n pH t h a t p h e n o l i c a c i d s w e r e f i r s t d e t e c t e d  in c u l t u r e s  g r o w i n g u n d e r i r o n - d e f i c i e n t c o n d i t i o n s ( F i g u r e k). s i m i l a r pH t r e n d was  obtained  i r o n - s u f f i c i e n c y , except  rise  A  f o r c u l t u r e s grown under  t h a t p h e n o l i c a c i d s were not  pro-  duced . (c)  Time of Onset of P h e n o l i c A c i d The  Production  time of onset of p h e n o l i c a c i d production  was  e x a m i n e d u s i n g l a r g e (500 ml) v o l u m e s o f c u l t u r e and e x t r a c t i o n methods p r e v i o u s l y d e s c r i b e d . was  the  W i t h WB746,  DHB  d e t e c t e d as e a r l y as 5 - 6 hours a f t e r i n o c u l a t i o n , o f  t h e f l a s k s , when t h e d e n s i t y had Chromatographic examination  reached  k x 10^ c f u p e r  of c u l t u r e supernatants  ml.  obtained  H O U R S  F i g u r e k.  V a r i a t i o n with time o f growth, phenolic a c i d  production  a n d pH o f t h e c u l t u r e medium o f B_. s u b t i l i s BT471 a n d WB746. Symbols: 0  0, g r o w t h (hO k l e t t u n i t s r e p r e s e n t  forming units per ml); • production K  (O.D._ ) . 5 1 0 nm lri  f , pH; A  10  colony  A, p h e n o l i c a c i d  a f t e r 7, 14 a n d 23 h o u r s i n c u b a t i o n r e v e a l e d t h e p r e s e n c e o f o n l y DHB. S t r a i n B 1 4 7 1 , h o w e v e r , b e h a v e d somewhat d i f f e r e n t l y . O n l y DHBG c o u l d b e d e t e c t e d natants  obtained  hand, although  chromatographically  in super-  a f t e r 13 - 14 h o u r s g r o w t h .  On t h e o t h e r  t h e 6, 7 a n d 8 h o u r s u p e r n a t a n t s  appeared  s i m i l a r t o e a c h o t h e r when c h r o m a t o g r a p h e d , t h e y d i f f e r e d f r o m t h e 13 - 14 h o u r c u l t u r e s i n t h a t s e v e r a l a c i d s were d e t e c t a b l e  i n t h e f o r m e r ( F i g u r e 5).  p h e n o l i c s were d e t e c t e d deficient conditions. compounds p r e s e n t  only  A l l these  i n c u l t u r e s grown u n d e r i r o n -  O f t h e two i r o n - b i n d i n g  in the early supernatants,  ( c , F i g u r e 5) h a s b e e n i d e n t i f i e d a s DHBG.  phenolic  one o f them The other  p o u n d ( a , F i g u r e 5) I s a s y e t u n i d e n t i f i e d . and  phenolic  two-dimensional thin layer chromatography  Acid  hydrolysis  indicated  the p r e s e n c e o f an as y e t u n i d e n t i f i e d amino a c i d . is c u r r e n t l y i n progress  com-  Work  i n an a t t e m p t t o c h a r a c t e r i z e t h i s  unknown c o m p o u n d . 2.  Mutant The  Strains  g r o w t h o f s t r a i n s B1471 a n d WB746 was i n h i b i t e d by 2.0  mM F e C l ^ a n d b y 1.5 mM h e m i n .  Mutants were s e l e c t e d from each  s t r a i n w h i c h w e r e r e s i s t a n t t o 20 mM F e C l ^ o r t o 10 mM h e m i n . The mutants resembled t h e w i l d type s t r a i n s i n t h a t  they  s t a r t e d t o produce p h e n o l i c a c i d s a f t e r about 8 hours growth  F i g u r e 5.  T h i n l a y e r chromatography of c u l t u r e s u p e r n a t a n t  extracts  a f t e r growth of B_. s u b t i 1 i s B1471 f o r 7 o r 14 hours w i t h o r w i t h o u t added i r o n .  Compounds were d e t e c t e d as f l u o r e -  scent s p o t s under u l t r a - v i o l e t Evans reagent ( p h e n o l i c s )  l i g h t and were p o s i t i v e t o  and to F e C l ^  (iron-binding).  The s o l v e n t system used was b e n z e n e : m e t h a n o l : a c e t i c a c i d (10:2:1)  (v/v/v)  (7).  41  I. A B C  origin 2. solvent front growth medium 2,3-dihydroxybenzoate (DHB) 7-hour culture without Fe  D E F G  2,3- dihydroxybenzoylglycine OHBG) 7-hour (s14hour) culture with Fe DHB 14-hour culture without Fe  F i g u r e 5.  ( F i g u r e 6 and 7).  H o w e v e r , t h e F e s t r a i n s o f WB746 p r o d u c e d  a b o u t t w i c e a s much p h e n o l i c a c i d a s t h e w i l d t y p e , b o t h w i t h and w i t h o u t a d d e d i r o n ( F i g u r e 7).  Hem s t r a i n s o f WB746 p r o -  d u c e d t h e same l e v e l s o f p h e n o l i c a c i d s a s t h e w i l d t y p e b u t a g a i n b o t h w i t h and w i t h o u t a d d e d i r o n .  strain,  Both types o f  m u t a n t s grew more s l o w l y than t h e w i l d t y p e s , w i t h o r w i t h o u t added i r o n .  T h e m u t a n t s o f B1471  were s i m i l a r t o those o b t a i n e d  f r o m s t r a i n WB746, e x c e p t t h a t t h e Hem s t r a i n d i d show some response to added i r o n  (Figure 6).  Both t y p e s o f mutants grew  a t t h e same r a t e a s t h e w i l d t y p e u n d e r t h e c o n d i t i o n s t e s t e d .  Discussion of Results  There is l i t t l e understanding of the b a s i c metabolism involved i n p h e n o l i c a c i d p r o d u c t i o n b y B^. s u b t ? 1 i s when g r o w n u n d e r c o n d i t i o n s of iron-deficiency.  In a d d i t i o n t o i r o n d e f i c i e n c y , a s u i t a b l e  carbon s o u r c e i s n e c e s s a r y f o r the p r o d u c t i o n o f s i d e r a m i n e s by Aspergi 1 lus  mel l.eus (14).  I t was  t h e r e f o r e of interest to look  a t p h e n o l i c a c i d p r o d u c t i o n as a f u n c t i o n o f the c a r b o n - s o u r c e f o r g r o w t h o f B_. s u b t ? 1 i s .  used  T h e e v i d e n c e o b t a i n e d shows t h a t a  r e l a t i v e l y high concentration of carbon source is necessary f o r DHB(G) p r o d u c t i o n .  I t i s not s u r p r i s i n g t h a t g l u c o s e proved to be  the most e f f e c t i v e c a r b o n s o u r c e t e s t e d , s i n c e the a r o m a t i c pathway l e a d i n g t o c h o r i s m i c a c i d and u l t i m a t e l y t o DHB p r o d u c t i o n r e q u i r e s a  F i g u r e 6. G r o w t h a n d p h e n o l i c a c i d p r o d u c t i o n b y m u t a n t s t r a i n s o f EL s u b t i 1 i s E31471 i n t h e a b s e n c e a n d p r e s e n c e o f a d d e d i r o n (5.5 y g atoms  Fe  + + +  /1iter).  Symbols: growth i n t h e p r e s e n c e (0 (•  0) a n d a b s e n c e  •) o f a d d e d i r o n (kO K l e t t u n i t s = 1 0 c f u / m l ) , b y 8  R  R  both F e ^and H mutants. P h e n o l i c a c i d p r o d u c t i o n ( O . D . , ) by F e mutant i n t h e p r e s e n c e ( A - — A ) a n d 510 nm absence ( A - — A ) o f added i r o n . P h e n o l i c a c i d p r o d u c t i o n R  (O.D.JJJQ  N  M  )  a b s e n c e (••  by  H  R  mutant  i n the presence ( •  • ) o f added  • ) and  iron.  F i g u r e 7. G r o w t h a n d p h e n o l i c a c i d p r o d u c t i o n b y m u t a n t s t r a i n s o f —• s u b t ? 1 i s WB746 i n t h e a b s e n c e a n d p r e s e n c e o f a d d e d i r o n (5.5 y g atoms Symbols: 0  Fe  + + +  /1iter).  0, g r o w t h o f F e m u t a n t s w i t h o r w i t h o u t R  p  iron; •  g r o w t h o f H" m u t a n t w i t h i r o n ; C — C ,  growth  o f H m u t a n t w i t h o u t a d d e d i r o n ; 1 0 c f u / m l = kO K l e t t 8  R  units.  Phenolic acid production  ( O . D . ^ Q  N  M  )  by F e  R  mutant i n t h e p r e s e n c e ( Q Q ) a n d t h e absence (A A) of added i r o n ; p h e n o l i c a c i d p r o d u c t i o n ( O . D . _ . „ ) by _ 5'0 nm H m u t a n t i n t h e p r e s e n c e ( HS B ) andabsence ( A — A ) of iron.  43  condensation to produce  between e r y t h r o s e - 4 - p h o s p h a t e  sedoheptulose-7~phosphate.  and  phosphoenolypyruvate  S i n c e B_. s u b t i 1 i s c a n  dissi-  m i l a t e g l u c o s e v i a the hexose-monophosphate pathway ( 2 2 ) , t h i s w o u l d be e x p e c t e d  to result  i n t h e p r o d u c t i o n o f an a d e q u a t e  of sedoheptulose-7-phosphate, The clear. carbon  subsequently  r o l e t h a t a c e t a t e p l a y s i n DHB  leading to chorismic a c i d .  p r o d u c t i o n i s not  Except f o r the i n s t a n c e s where glutamate  was  immediately  u s e d as s o l e  source, the absence of a c e t a t e r e s u l t e d in l o s s of p h e n o l i c  a c i d p r o d u c t i o n by B. s u b t i l i s . porphyr in would appear e v e n when a c e t a t e was  However, the p r o d u c t i o n o f  t o i n d i c a t e t h a t t h e TCA absent.  o t h e r t h a n a f u n c t i o n a l TCA  Therefore  c y c l e was  i t appears  ficiency.  converted  t o a - k e t o g l u t a r a t e and t h e n c e The  ment f o r i r o n and c o n s e q u e n t l y  iron-de-  as the s o l e  be e x p l a i n e d on t h e b a s i s o f g l u t a m a t e  o p e r a t i v e heme p a t h w a y .  operating  cycle is necessary for phenolic acid  The p r o d u c t i o n o f p h e n o l i c s w i t h g l u t a m a t e  s o u r c e may  copro-  t h a t some f a c t o r  p r o d u c t i o n by B. s u b t i l i s when grown u n d e r c o n d i t i o n s o f  carbon  supply  being  t o s u c c i n y l Co A a n d  l a t t e r would r e s u l t  an  in a high require-  i r o n - d e f i c i e n c y would soon ensue,  leading u l t i m a t e l y to p h e n o l i c a c i d production. f u n c t i o n i n g o f o t h e r i r o n - r e q u i r i n g enzymes found  Moreover, the in the  citric  a c i d c y c l e and b i o s y n t h e t i c pathways w o u l d a l s o c o n t r i b u t e t o t h e r a p i d u t i l i z a t i o n o f a l l a v a i l a b l e i r o n i n t h e c u l t u r e medium. m e t h o d by w h i c h t h e o n s e t o f p h e n o l i c a c i d p r o d u c t i o n i s a s y e t u n c l e a r , a l t h o u g h a s we  The  commences  s h a l l see l a t e r , the r e s u l t i n g  i r o n - d e f i c i e n c y probably causes a d e r e p r e s s i o n of the DHB(G)-pro-  clucing enzyme  system.  T h e pH c h a n g e s o b s e r v e d d u r i n g g r o w t h do n o t a p p e a r unique to p h e n o l i c a c i d p r o d u c t i o n or the consequences ficiency  i n Eh s u b t i1? s .  t o be  of iron-de-  S i m i l a r p a t t e r n s have been observed  s p o r u l a t i o n s t u d i e s (38).  It appears  in  that d u r i n g the decrease  in  pH, a c e t a t e i s c o n v e r t e d t o CO^ and v a r i o u s o r g a n i c a c i d s v i a t h e cycle.  The a c e t a t e - o x i d i z i n g system  the l a t t e r h a l f of  reaches peak a c t i v i t y d u r i n g  the low pH p e r i o d and d e c r e a s e s  p r o b a b l y t o a l o s s i n a c e t a t e p e r m e a b i l i t y (38). a c t i v i t y o f TCA c y c l e e n z y m e s m i g h t  throughout  r a p i d l y , due Repression of  then o c c u r w i t h d e r e p r e s s i o n  t a k i n g p l a c e when g l u c o s e i s e x h a u s t e d Continuous  TCA  f r o m t h e medium.  p h e n o l i c a c i d p r o d u c t i o n does not appear  t h e e n t i r e g r o w t h c y c l e o f B_. s u b t i l i s .  to occur  Experiments  i n v o l v i n g the e x t r a c t i o n of l a r g e volumes of c u l t u r e s u p e r n a t a n t s , and e x p e r i m e n t s  involving examination  o f mutant s t r a i n s whose c o n t r o l  o v e r p h e n o l i c a c i d p r o d u c t i o n by e x o g e n o u s i r o n a p p e a r s  t o be  lost,  i n d i c a t e t h a t s p e c i f i c c o n d i t i o n s must be r e a c h e d b e f o r e p h e n o l i c a c i d p r o d u c t i o n i s turned on.  These appear  to c o i n c i d e with  the  end o f t h e minimum pH p e r i o d ; p o s s i b l y w i t h t h e d e r e p r e s s i o n o f  TCA  c y c l e enzyme a c t i v i t i e s . I t i s o f i n t e r e s t t h a t w i t h s t r a i n WB746 o n l y DHB was w h e t h e r t h e s u p e r n a t a n t was inoculation 23 h o u r s .  examined as e a r l y as 5 - 6 hours  ( a t a d e n s i t y o f k x 10  produced after  c e l l s p e r ml) o r a s l a t e as  S t r a i n B1471, on t h e o t h e r h a n d , p r o d u c e d  an u n i d e n t i f i e d  • p h e n o l i c compound e a r l y i n i t s g r o w t h c y c l e . l a t e r by t h e p r o d u c t i o n f i e d compound.  T h i s was f o l l o w e d  o f DHBG a n d d i s a p p e a r a n c e  of the unidenti-  S i n c e o t h e r b a c t e r i a have been r e p o r t e d  amino a c i d conjugates  t o produce  o f DHB ( l , 1 2 ) , i t seems t h a t WB746 may be  d e f i c i e n t i n t h e c a p a c i t y t o c o u p l e a n a m i n o a c i d t o DHB.  The i n -  v o l v e m e n t o f a c o u p l i n g enzyme u t i l i z i n g s h o r t c h a i n amino a c i d s p o s s i b l y r e q u i r e s a g r e a t e r o x i d a t i v e f u n c t i o n i n g o f t h e TCA c y c l e i n s t r a i n B1471 t h a n  III.  i n WB746.  E f f e c t o f DHB A n a l o g s a n d C h e l a t i n g A g e n t s o n P h e n o l i c Production  Acid  by B a c i 1 l u s s u b t i 1 i s  Introduction Peters  (44) i n v e s t i g a t e d t h e e f f e c t s o f v a r i o u s analogs  on p h e n o l i c a c i d p r o d u c t i o n was  found  b y g r o w i n g c e l l s o f j3_. s u b t ? 1 i s .  that, in general, meta-substituted  benzoic  It  acids and r e -  l a t e d c o m p o u n d s w e r e t h e m o s t e f f e c t i n h i b i t o r s o f DHB(G) Some a d d i t i o n a l a n a l o g s  o f DHB  production.  h a v e now b e e n i n v e s t i g a t e d a n d t h e e f f e c t s  o f a number o f m e t a l c h e l a t i n g a g e n t s a l s o e x a m i n e d . v e l o p m e n t o f a n i n v i t r o s y s t e m f o r DHB s y n t h e s i s  (see  With the deMaterials  and M e t h o d s ) i t became p o s s i b l e t o e x a m i n e t h e e f f e c t o f v a r i o u s i n h i b i t o r s o n b o t h DHB s y n t h e t a s e eel 1 e x t r a c t s .  a c t i v i t y a n d t h e enzyme l e v e l s i n  47  1.  E f f e c t s o f V a r i o u s A g e n t s on P h e n o l i c A c i d P r o d u c t i o n  by  Growing C e l l s (a)  Analogs  o f DHB  None o f t h e a n a l o g s study caused  in this  s i g n i f i c a n t inhibition of phenolic acid  d u c t i o n by g r o w i n g c e l l s (b)  examined f o r the f i r s t time  pro-  (Table 9 ) .  Chelating Agents T h e p r o d u c t i o n o f p h e n o l i c a c i d s was s t i m u l a t e d  by t h e a d d i t i o n o f v a r i o u s c h e l a t i n g a g e n t s  slightly  to the growth  medium ( T a b l e 1 0 ) . 2.  In V i t r o (a)  Assays  Preliminary  Experiments  Preliminary experiments  indicated that at least a  two h o u r r e a c t i o n t i m e was n e c e s s a r y product  formation  (Figure 8).  to ensure s i g n i f i c a n t  Although  the substrate  appeared r a p i d l y from t h e r e a c t i o n mixture, formed d u r i n g the f i r s t hour ( F i g u r e 8 ) . of excess  dis-  DHB was n o t In t h e  s u b s t r a t e , t h e amount o f p r o d u c t  presence  f o r m e d was  p r o p o r t i o n a l t o t h e amount o f e n z y m e a d d e d ( F i g u r e 9 ) • The  r e a c t i o n p r o d u c t was e x t r a c t e d  s p e c t r a l c h a r a c t e r i s t i c s examined.  i n t o e t h e r and i t s  Its absorption  ( F i g u r e 10) was i d e n t i c a l t o a u t h e n t i c DHB. b e h a v e d l i k e a u t h e n t i c DHB o n c h r o m a t o g r a m s . B. s u b t i 1 i s WB746 c o n v e r t e d  The  spectrum product  Extracts of  some k0% o f t h e s u b s t r a t e ,  T a b l e 9.  Effect of analogs of 2,3-dihydroxybenzoic  a c i d on phenol  a c i d p r o d u c t i o n by B_. s u b t ? 1 i s s t r a i n s B1471 a n d WB746 a f t e r 14 h o u r s i n c u b a t i o n w i t h o u t a d d e d  iron.  ° - - 5 1 0 nm D  Compound  Concentration  B1471  WB746  -  0.25  0.48  M-Toluic acid  0.1  0.26  0.47  V a n i l l i c acid  0.1  0.22  0.44  Veratric acid  0.1  0.20  0.46  1.0  0.24  0.50  (mM) Nil  o-hydroxybenzyl  T a b l e 10.  alcohol  Effect of chelating  agents on p h e n o l i c a c i d p r o d u c t i o n  by f3_. s u b t ? l i s s t r a i n B1471 a n d WB746 a f t e r 14 h o u r s i n c u b a t i o n w i t h o u t added  iron.  , D ,  Compound  Concentration  5 1 0 nm  B1471  WB746  -  0.26  0.54  a,a-dipyridyl  0.1  0.30  0.56  1,10-phenanthroline  0.1  0.35  0.58  Tiron  0.1  0.39  0.58  Ethylenedraminetetraacetate  0.1  0.33  0.58  nitrilotriacetate  0.1  0.28  0.54  (mM) Nil  20  0-3-  -15 E c  CO  Q O  0-2-  -10  01-  -0-5  S L z = _  CM  O O  — i  2 H O U R S  Figure  8.  Enzymatic conversion of chorismic b e n z o i c a c i d , by c e l l - f r e e e x t r a c t s grown under c o n d i t i o n s Symbols: 0-—0,  •  mixture.  #,  2,3 dihydroxy-  o f B_. s u b t i 1 i s WB746  iron-deficiency.  y m o l e s o f DHB  0.075 y m o l e s DHB); from reaction  of  a c i d to  f o r m e d (0.1  O.D.^g  n m  disappearance of chorismic  = acid  50  0-8-  0-6oo io Q  0-4-  o 0-2-  0-95  F i g u r e 9-  1-90 2-85 mg PROTEIN  3-80  Conversion of c h o r i s m i c acid to 2,3-dihydroxybenzoic a c i d as a f u n c t i o n o f . t h e p r o t e i n c o n c e n t r a t i o n .  P r o t e i n was  added as crude, e e l 1-free e x t r a c t s from jB. s u b t i 1 i s WB746 grown under  iron-deficiency.  250  F i g u r e 1.0.  275 300 WAVELENGTH  (nm)  325  350  U l t r a v i o l e t spectra (ether solvent) of substrate, and  intermediate reaction mixture  (b) i n t h e  •conversion of c h o r i s m i c a c i d (c) to acid  product  enzymatic  2,3-dihydroxybenzoic  (a) by c e l l - f r e e e x t r a c t s o f B^. s u b t 11 i s W.B746 g r o w n  under i r o n - d e f i c i e n c y .  chorismic acid  ( a t an i n i t i a l  c o n c e n t r a t i o n o f 1.0 ymole'/ml)  t o DHB a n d t h o s e o f B1471 some 1 5 % . H o w e v e r , y i e l d s somewhat f r o m e x p e r i m e n t t o e x p e r i m e n t (25 and (b)  varied  - 6 5 % f o r WB746  15 - 2 5 % f o r B 1 4 7 1 ) . R e p r e s s i o n o f DHB S y n t h e t a s e F o r m a t i o n Significant  l e v e l s o f a c t i v e enzyme were n o t formed  when t h e c e l l s w e r e g r o w n i n t h e p r e s e n c e o f i r o n o r f e r r i c h r o m e a n d t h e e n z y m e l e v e l was r e d u c e d i n t h e p r e s e n c e of t h e a r o m a t i c amino acids (c)  (Table 11).  or 3 f1uorobehzoic acid _  Enzyme I n h ? b i t i o n In B_. s u b t i 1 i s s t r a i h WB746, 3 - h y d r o x y a n t h r a n i 1 i c a c i d  and  3-hydroxybenzoic a c i d were very e f f e c t i v e  enzyme a c t i o n .  DHB a n d c a t e c h o l  were a l s o  inhibitors of  inhibitory, but  t o a l e s s e r e x t e n t ( T a b l e 1 2 ) . In s t r a i n B1471, i n a d d i t i o n to 3-hydroxyanthrani1ic a c i d and 3-hydroxybenzoic a c i d , both catechol  a n d h e m i n , when t e s t e d  i n the presence o f iron,  p r o v e d t o be 100% e f f e c t i v e a s enzyme i n h i b i t o r s . had  (ron alone  no e f f e c t on enzyme a c t i v i t y w h e r e a s hemin a l o n e  a c t i v i t y o f B1471  extracts  by a b o u t 2 0 % .  3 f1uorobenzoic _  acid  i n h i b i t e d t h e enzyme a c t i v i t y about 50%.  (d)  Enzyme  Reactivation  In a n a t t e m p t t o s e e i f DHB s y n t h e t a s e a c t i v i t y be r e s t o r e d  inhibited  to extracts  1.0 mM F e C l _ ,  extracts  could  o f WB746 g r o w n i n t h e p r e s e n c e o f were d i a l y s e d  overnight at k C  Table  11.  E f f e c t o f v a r i o u s c o m p o u n d s o n l e v e l s o f DHB produced  by growing  synthetase  c e l l s o f B_. s u b t i 1 i s B1471 a n d WB746  % e n z y m e a c t i v i t y was c a l c u l a t e d o n a p e r mg o f p r o t e i n basis.  Results a r e expressed  cate  Final Concentration  -  Nil FeCl,.6H 0 o  Catechol Fe  + + +  *  DHB o-Hydrbxyh i ppurate o-Hydroxyhippurate Dipicolinic  +  Fe  + + +  acid  3-F1uorobenzoic  acid  3-Hydroxyanthrani1ic 3-Hydroxybenzoic  acid  acid  *  Enzyme A c t i v i t y  {%)  WB746  B1471  100  100  0 0  100 uM 50 uM 25 uM 0.25 uM  100  0 0 0 85  1.0 mM  120  100  1 .0 mM  80  175  1.0 mM  1 10  100  10 uM  90  90  10 uM  120  80  100 uM  70  -  1 .0 mM 0.1 mM  50 70  10 40  100 uM  100  100 uM  140  -  2k  Hemi n  1.5 yM  90  Tyrosine  1 .0 mM  10  Tryptophan  1.0 mM  40  Phenylalanine  1.0 mM  80  1 .0 mM  75  Anthranilie * Fe  acid  of 3 repli-  assays.  Compound  Catechol +  as t h e average  was i n c l u d e d a t 0.25 u g a t o m s / m l c o n c e n t r a t i o n  ' T a b l e 12.  Effect of various  c o m p o u n d s o n t h e a c t i v i t y o f DHB  s y n t h e t a s e e n z y m e i n c r u d e e x t r a c t s o f B_. s u b t i 1 i s WB746  a n d B1471 g r o w n u n d e r c o n d i t i o n s  of iron  deficiency.  % e n z y m e a c t i v i t y was c a l c u l a t e d o n a p e r mg o f p r o t e i n basis. cate  Results  a r e expressed as the average o f 3 r e p l i -  assays.  Compound  Final Concentration  Nil  -  1 mM  FeCl .6H 0 2  3  10 yM  F e r r i chrome  1 mM 1 mM  Catechol Catechol  + Fe  + + +  **  5 mM 0.5 mM 1 mM  2,3-dihydroxybenzoic acid Dipicolinic  acid  3 f1uorobenzoic acid  1 mM  3-hydroxyanthrani1ic acid  1 mM  -  3-hydroxyanthrani 1 ic acid + F e  + + +  **  3-hydroxybenzoic acid o-hydroxyhippuric acid  1 mM 1 mM 10 yM  1 yM 1 yM  Enzyme A c t i v i t y  WB746  B1471  100 100  100 100  100  100  60 100 50 100  -  100  100  100 100  100  1 mM  Tryptophan  1 mM  100  Phenylalanine  1 mM 1 mM  100 100  ,;  1-mM  EDTA  Anthranilie  acid  * not determined Fe  + + +  included  a t 0.25 y g a t o m s / m l  concentration  0 100  0  T y r o s i ne  Hemin + F e * * ++  50  0  100 100 100 100  Hemi n  0  0  80 0 100  -  a g a i n s t three changes of v a r i o u s c h e l a t i n g agents M Tris buffer.  The  i n c r e a s e i n e n z y m e a c t i v i t y was  Results  e f f e c t of the analogs  on t h e p r o d u c t i o n o f p h e n o l i c a c i d s  by g r o w i n g c e l l s o f B_. s u b t i 1 i s w e r e n o t u n e x p e c t e d (44)  f i n d i n g s by P e t e r s analogs  observed  13).  (Table  Discussion of  No  0.1  in  that meta-substituted  benzoates  s e r v i n g as the most e f f e c t i v e i n h i b i t o r s .  f o r e , t h a t t h e 3 h y d r o x y g r o u p o f DHB _  may  be  in view of  the  were the  It seems, t h e r e -  involved in shut-off  o f DHB(G) s y n t h e s i s . T h e o b s e r v a t i o n t h a t p h e n o l i c a c i d p r o d u c t i o n was when B_. s u b t i 1 i s was agents which are suggestion  (44)  grown in t h e p r e s e n c e  not a n a l o g s  o f DHB  increased  of strong iron chelating  lent f u r t h e r support  to  the  t h a t t h e p r o d u c t i o n o f DHB(G) i s r e p r e s s e d by i r o n .  The c h e l a t i n g a g e n t s  added t o t h e medium c o u l d have bound r e s i d u a l  i r o n i n the growth medium t h e r e b y making i t u n a v a i l a b l e t o t h e S i n c e t h e m e d i u m was p r o d u c t i o n was produced  low i n i t s i r o n c o n t e n t and p h e n o l i c a c i d  already high,  w o u l d n o t be  cells.  a g r e a t i n c r e a s e i n t h e amount o f p h e n o l i c  expected.  T h e e n z y m e s i n v o l v e d i n DHB(G) s y n t h e s i s a r e r e p r e s s i b l e , a r e s u l t s i m i l a r to that found  f o r t h e E_. c o l i  system  (2)  and  for  • T a b l e 13.  E f f e c t o f c h e l a t i n g a g e n t s on r e s t o r a t i o n o f  DHB-  s y n t h e t a s e a c t i v i t y t o c e l l - f r e e e x t r a c t s o f B_. s u b t i 1 i s s t r a i n WB746 g r o w n i n t h e p r e s e n c e o f 1 mM f o r ]k  F e C l ^ a t 37 C  hours;  Enzyme A c t i v i t y Chelating  Agent  Ethylenediaminetetraacetate  C o n c e n t r a t i on  1  mM  % of s t a r t i n g control Sample  Control  0  70  a,a-dipyridyl  0.1  mM  0  60  T i ron  0.1  mM  0  65  A. a e r o g e n e s ( 7 2 ) . When c e l l s w e r e g r o w n In t h e p r e s e n c e o f i r o n , the e x t r a c t s were devoid  of activity.  Since a c t i v i t y d i d not appear  a f t e r d i a l y s i s of the extracts against various i r o n was n o t s e r v i n g m e r e l y a s a n i n h i b i t o r . supported did  chelating  agents,  This conclusion  was  by t h e f i n d i n g t h a t t h e a d d i t i o n o f i r o n t o a c t i v e  n o t r e s u l t i n a l o s s o f enzyme a c t i v i t y .  Preparations  extracts  from  c e l l s g r o w n i n t h e a b s e n c e o f a d d e d i r o n showed g o o d e n z y m e a c t i v i t y in t h e c o n v e r s i o n  o f c h o r i s m a t e t o DHB.  These r e s u l t s can  be i n t e r p r e t e d a s i n d i c a t i n g t h a t t h e p r e s e n c e o f i r o n the formation  o f the enzyme(s) r e s p o n s i b l e  J3. s u b t i l i s .  Repression  represses  f o r DHB(G) p r o d u c t i o n  control of aromatic  J3. s u b t i 1 ?s h a s b e e n shown o n l y r e c e n t l y  therefore  in  amino a c i d s y n t h e s i s i n  (24, 4 2 ) .  It is of interest  t h a t t h e enzymes i n v o l v e d p r i o r t o p r e p h e n a t e a r e p r i m a r i l y under t h e c o n t r o l o f t y r o s i n e , and t h a t e a c h o f t h e t e r m i n a l with  the possible exception  of the phenylalanine  branches,  pathway, i s under  the r e p r e s s i o n c o n t r o l o f the amino a c i d which i t s y n t h e s i z e s 42).  The aromatic  amino a c i d s , f e r r i c h r o m e  and  (41,  3~fluorobenzoic  a c i d , in addition to iron, a l s o appear to s i g n i f i c a n t l y repress formation contains (40)  i n DHB s y n t h e s i s .  i r o n and a l s o p o s s i b l y s e r v e s  thereby  aromatic ducing  o f t h e enzymes i n v o l v e d  Ferrichrome  as an i r o n t r a n s p o r t  m a k i n g t h e i r o n more a v a i l a b l e t o t h e c e l l .  factor  The  a m i n o a c i d s p o s s i b l y a c t i n d i r e c t l y on t h e s y s t e m b y r e t h e amount o f c h o r i s m i c  acid produced  by l i m i t i n g t h e amount o f DHB(G) f o r m e d .  i n t h e c e l l and  there-  The e f f e c t s o f 3 f 1 u o r o b e h z o -  a c i d a r e unknown. although ' and  n  T h e e f f e c t s o f c a t e c h o l a n d DHB a r e n o t c l e a r ,  i t a p p e a r s t h a t n e i t h e r r e p r e s s e s enzyme  formation.  —• s u b t i l i s , D A H P - s y n t h e t a s e i s i n h i b i t e d b y c h o r i s m i c  acid  by p r e p h e n a t e and p r e p h e n a t e d e h y d r o g e n a s e and d e h y d r a t e a s e a r e  i n h i b i t e d b y t y r o s i n e and p h e n y l a l a n i n e a negative  (41).  Tryptophan acts as  e f f e c t o r of anthranilate synthetase  (18).  3-Hydroxy-  a n t h r a n i l i c a c i d and 3 h y d r o x y b e n z o i c a c i d appear t o e f f e c t i v e l y -  i n h i b i t DHB s y n t h e t a s e  a c t i v i t y , p o s s i b l y by a c t i n g as substrate  analogs.  3-Fluorobenzoate,  w h i c h c a u s e d a 50% r e p r e s s i o n o f e n z y m e  formation  w i t h b o t h s t r a i n s B1471 a n d WB746, a p p e a r s t o i n h i b i t  a c t i v i t y o f t h e B1471 e n z y m e , b u t n o t t h a t o f WB746.  It a l s o appears  t h a t c a t e c h o l a n d DHB a c t a s i n h i b i t o r s o f DHB s y n t h e t a s e I f t h i s i s t r u e , DHB may b e c o n s i d e r e d  a negative  activity.  effector.  the f a c t that these s t u d i e s were c a r r i e d o u t using c e l l - f r e e and  n o t t h e p u r i f i e d e n z y m e may c a u s e d i f f i c u l t y  However, extracts  i n i n t e r p r e t i n g these  results.  IV.  P a r t i a l P u r i f i c a t i o n o f DHB S y n t h e t a s e  Introduct ion  A n a t t e m p t was made t o p u r i f y t h e enzyme c o n v e r t i n g a c i d t o DHB.  chorismic  I t was h o p e d t h a t a k i n e t i c s t u d y o f t h i s r e a c t i o n  w i t h t h e p u r i f i e d e n z y m e w o u l d g i v e some i n d i c a t i o n o f t h e m e c h a n i s m s  involved  i n r e g u l a t i n g DHB  t h i s p h a s e o f t h e w o r k was a e r o g e n e s ( 7 2 , 73, intermediates  74)  synthesis.  However, s h o r t l y a f t e r  s t a r t e d , w o r k w i t h _E. c o l i and  revealed  in the c o n v e r s i o n  the e x i s t e n c e  which would separate ly prevent  of at l e a s t  o f c h o r i s m a t e t o DHB.  mediates are not a v a i l a b l e at present,  A. two  These i n t e r -  s o t h a t any p u r i f i c a t i o n s t e p s  i n d i v i d u a l enzymes of the pathway would e f f e c t i v e -  f u r t h e r assay of the system.  In s p i t e o f t h i s ,  i t was  p o s s i b l e to " p u r i f y " the o v e r a l l system, i . e . that c o n v e r t i n g m a t e t o DHB, g r a p h y and e n z y m e s may  some 9 - f o l d by u s e o f D E A E - c e l l u l o s e sucrose  gradient  centrifugation.  This  column chromatoindicated that  e x i s t as a m u l t i e n z y m e c o m p l e x i n s i d e the  1.  M and  from  0.75  M NaCl  ( F i g u r e 11a)  cell.  b e t w e e n 0.34  and  0.38  eluted  when c r u d e c e l l - f r e e  s u b t i 1 ? s WB746 w e r e s u b j e c t e d  g r a p h y and  between  extracts  to DEAE-cellulose'chromato-  M NaCl  ( F i g u r e l i b ) when p a r t i a l l y  p u r i f i e d e x t r a c t s were used.  2.  Sucrose Gradient  Centrifugation  D H B - s y n t h e s i z i n g p r o t e i n was gradient  the  DEAE - C e l l u l o s e C o l u m n C h r o m a t o g r a p h y  A c t i v e D H B - s y n t h e s i z i n g e n z y m e was 0.40  choris-  found in  sucrose  f r a c t i o n s 25 t o 34 a f t e r c e n t r i f u g a t i o n f o r 12 h o u r s  Figure  TV.  DEAE-cellulose  c h r o m a t o g r a p h y o f DHB s y n t h e t a s e a c t i v i t y  i n an e x t r a c t o f iron.  s u b t i 1 i s WB746 g r o w n w i t h o u t a d d e d  (a) Crude e x t r a c t  protein/ml  (2.0 ml) c o n t a i n i n g  19 mg o f  was a p p l i e d t o t h e c o l u m n a n d e l u t e d b y a  l i n e a r NaCl g r a d i e n t were c o l l e c t e d ,  (0 - 1.0 M ) .  Fractions  (99  (b) P a r t i a l l y p u r i f i e d e x t r a c t  (NH^J^SO^ s a t u r a t i o n )  ( 2 . 0 ml) c o n t a i n i n g  drops) (after  8 mg o f p r o t e i n /  ml was a p p l i e d t o t h e c o l u m n a n d e l u t e d by a l i n e a r NaCl g r a d i e n t  ( 0 . 3 0 - 0.60 M ) .  F r a c t i o n (45  drops)  were c o l l e c t e d . Symbols: 0  0, p r o t e i n ; • — i ,  DHB  synthetase.  F i g u r e 11.  ( F i g u r e 12).  P r o t e i n was  found i n a l l f r a c t i o n s (not shown).  Discussion of Results  A l t h o u g h t h e DHB  s y n t h e t a s e s y s t e m was  not p u r i f i e d e x t e n s i v e l y ,  t h e r e s u l t s i n d i c a t e t h a t t h e e n z y m e s y s t e m may e x i s t i n t h e c e l l as a m u l t i e n z y m e  complex.  Such complexes  a r o m a t i c p a t h w a y o f EL s u b t 11 ?s ( 1 8 , 3 5 ) .  are i n v o l v e d in the That the complex  is  r e l a t i v e l y s t a b l e i s i n d i c a t e d by t h e " p u r i f i c a t i o n " a c h i e v e d on DEAE-cel1ulose chromatography If s u c h a m u l t i e n z y m e  and s u c r o s e g r a d i e n t c e n t r i f u g a t i o n .  c o m p l e x w e r e d i s r u p t e d by t h e t r e a t m e n t s , no  d e m o n s t r a t i o n o f enzyme a c t i v i t y would  V.  be e x p e c t e d .  I ron T r a n s p o r t  Introduction  T h e w o r k on t h e EL s u b t i 1 ? s s y s t e m by P e t e r s and W a r r e n was  (46)  r e p e a t e d and e x t e n d e d t o i n c l u d e an e x a m i n a t i o n o f t h e n a t u r e  o f t h e t r a n s p o r t s y s t e m , t h e r o l e o f i r o n b i n d i n g a g e n t s and s t a t e of the i n t r a c e l l u l a r iron.  In a d d i t i o n , i r o n u p t a k e  S a l m o n e l l a t y p h i m u r i um and by M i c r o c o c c u s l y s o d e i k t i c u s examined  f o r comparative  purposes.  was  by  the  0.20-  0.15-  co  0.10-  Q d  0.05-  Figure  12.  Sucrose gradient  c e n t r i f u g a t i o n o f DHB s y n t h e t a s e  activity  i n a c r u d e e x t r a c t o f B_. s u b t i 1 i s WB746 g r o w n w i t h o u t added i r o n . protein/ml ml).  Crude e x t r a c t  (0.6 ml) c o n t a i n i n g  19.mg  was a p p l i e d t o t h e t o p o f t h e g r a d i e n t  (14.2  C e n t r i f u g a t i o n was p e r f o r m e d a t 2 C f o r 12 h o u r s  a t 150,000 x g .  Fractions  (6 d r o p s ) w e r e c o l 1 e c t e d .  Bacillus subti 1is Strain (a)  B7471 *  P r e l i m i n a r y Experiments R a d i o a c t i v e i r o n was  added  t o c e l l - f r e e medium a n d  i n c u b a t e d as d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s . f r e e s a m p l e s w e r e r e m o v e d p e r i o d i c a l l y o v e r a 30 p e r i o d and p a s s e d t h r o u g h 0.45  When  radioactivity  was  r e t a i n e d on t h e f i l t e r s .  was  o m i t t e d from t h e medium, a h i g h l e v e l o f  was  found to remain a t t a c h e d to the f i l t e r s  H o w e v e r , when c i t r i c  acid  radioactivity and t h i s  i n c r e a s e d o v e r the i n c u b a t i o n p e r i o d .  t h e f i l t e r s w i t h 2.0  minute  y Millipore filters.  t h e m e d i u m u s e d c o n t a i n e d c i t r i c a c i d , no  generally  t o 5.0 ml o f i r o n - d e f i c i e n t  medium radio-  C o n s e q u e n t l y , e x p e r i m e n t s were run w i t h a b a s a l  l e v e l o f c i t r a t e i n t h e medium used t o suspend t h e For  cells.  u p t a k e e x p e r i m e n t s , t h e c e l l s r e t a i n e d by t h e  w e r e w a s h e d w i t h 5 ml o f i r o n - d e f i c i e n t extraneous  level  Washing  lowered, but d i d not e l i m i n a t e , t h i s r e t e n t i o n o f activity.  Cell-  loosely  bound  filters  medium t o r e m o v e  iron.  For c e r t a i n e x p e r i m e n t s ,  i t was  n e c e s s a r y t o wash the  c e l l s several times before adding radioactive iron. e f f e c t s of d i f f e r e n t washing  p r o c e d u r e s on t h e  c a p a c i t y o f B_. s u b t i 1 1 s w e r e e x a m i n e d  The  uptake  with c e l l s which  had b e e n g r o w n w i t h v a r i o u s l e v e l s o f a d d e d  iron.  t h e c e l l s r e d u c e d t h e i r u p t a k e c a p a c i t y by a l m o s t  Washing 50%  fn a l l c a s e s ( T a b l e  14).  A l t h o u g h w a s h i n g by c e n t r i f u g a -  t i o n c a u s e d t h e l e a s t a p p a r e n t damage t o t h e c e l l s ,  the  f i l t e r m e t h o d was p r e f e r r e d b e c a u s e o f i t s s p e e d , (b)  Experiments With I r o n - D e f i c i e n t (i)  Cultures  Iron uptake as a f u n c t i o n o f p h y s i o l o g i c a l age. D u r i n g t h e e a r l y and m i d - l o g phases o f g r o w t h ,  c e l l s o f B. s u b t i l i s t o o k up i r o n a t a c o n s t a n t d u r i n g a 40 m i n u t e i n c u b a t i o n p e r i o d  rate  ( F i g u r e 13).  In l a t e l o g phase h o w e v e r , t h e u p t a k e c a p a c i t y i n creased  2 - 3 - f o l d a n d t h e r a t e o f u p t a k e was  constant  during the incubation period (Figure  It was e v i d e n t simply  not  t h a t t h i s i n c r e a s e d c a p a c i t y was  t h e r e s u l t o f an i n c r e a s e i n c e l l  The i n c r e a s e d increase  13). not  numbers.  r a t e o f u p t a k e was a s s o c i a t e d w i t h an  in phenolic acid production  by t h e g r o w i n g  e e l Is ( F i g u r e 13)• (ii) Effect of metabolic inhibitors T h e u p t a k e o f i r o n f r o m t h e c u l t u r e medium by 'B_. s u b t i 1 i s a p p e a r e d t o i n v o l v e an a c t i v e process (Figure 14). at a concentration  Sodium a z i d e or a - i o d o a c e t a m i d e ,  o f 1 mM,  d e c r e a s e d t h e u p t a k e by  20 - 40% a n d i n c o m b i n a t i o n , by a l m o s t 8 0 % . hydryl-binding  transport  Sulf-  a g e n t s w e r e e v e n more e f f e c t i v e , d e -  c r e a s i n g u p t a k e by 9 0 % ( F i g u r e 1 4 ) .  A l b o m y c i n a t 0.1  65  T a b l e 14.  E f f e c t o f w a s h i n g c e l l s on i r o n u p t a k e c a p a c i t y o f Q. s u b t ? 1 i s B1471 g r o w n w i t h v a r y i n g l e v e l s o f i r o n .  g  cpm/10 Amount o f F e added t o t h e g r o w t h medium  + + +  5.5 y g a t o m s / 1i ter 18.1 y g a t o m s / liter  colony forming units  F i l t e r e d , washed F i l t e r e d , washed and resuspended Control (not washed) and resuspended in extracted i n n o r m a l medium medium  Centrifuged, washed and r e suspended i n e x t r a c t e d medium  512  215  171  266  622  126  299  422  38  72  171  145  66  500-  400tO UJ  ooo  300-  u. o>  200  100"  MINUTES F i g u r e 13- . I r o n u p t a k e by B. s u b t i l i s  B1471  (grown u n d e r . i r o n - d e -  f i c i e n c y ) a s a f u n c t i o n o f p h y s i o l o g i c a l a g e and acid  phenolic  production.  Symbols: 0  0, e a r l y l o g ( 5 i h o u r ) c e l l s , O . D . ^ ^ ^  =  0.02: • •, m i d - l o g p h a s e ( 6 i h o u r ) c e l l s , 0 . D . . = ' ' . 510 nm 0.03; A — - A , ' l a t e r l o g p h a s e ( 7 i h o u r ) c e l l s , O.D. 510 nm 0.12. 59 Fe was a d d e d a t a c o n c e n t r a t i o n o f 100 pg a t o m s r l  p e r ml  67  600 500CO  400"  UJ  ®  Q  o o.  300200100-  10 15 MINUTES F i g u r e 14.  E f f e c t o f . m e t a b o l i c i n h i b i t o r s and s u l f h y d r y l  r e a g e n t s on  i r o n u p t a k e by J3_. s u b t ? 1 i s B1471  iron-deficient  grown under  conditions. Symbols:  0  0, no a d d i t i o n s ;  a i o d o a c e t a m i d e ; A—A, Q  •;  HgCl ; 2  Additions  1 mM  •—  1 mM NaN^  1 mM + 1 mM  NaN^;  sodium  X  arsenite. 59  w e r e ' m a d e 10 m i n u t e s p r i o r t o  A,  1 mM  a-iodoacetamide;  NaN^p-chloromercuribenzoate; , 1 mM  A  X, 50 yM 59  Fe.  a d d e d a t a c o n c e n t r a t i o n o f 100 pg a t o m s / m l .  Fe  was  uM c o n c e n t r a t i o n h a d no e f f e c t o n i r o n u p t a k e c a p a city  ( n o t shown).  ( i i i ) Effect of glucose When c e l l s o f j3. s u b t i 1 i s g r o w i n g i n 1% g l u c o s e were washed and a l l o w e d t o s t a r v e  f o r l i hours i n the  a b s e n c e o f g l u c o s e , t h e i r a b i l i t y t o t a k e up i r o n  from  t h e medium was r e d u c e d more t h a n 80% r e l a t i v e t o a n unstarved control  (Figure  15). If the starved  cells  w e r e s u p p l e m e n t e d w i t h 0.2% g l u c o s e , u p t a k e was i n creased about 3-fold uptake i n the starved  (Figure  15). The rate o f iron  c e l l s g i v e n g l u c o s e was s i g n i -  f i c a n t l y g r e a t e r than that o f t h e unsupplemented c e l l s (Figure 15). (iv) Iron uptake as a f u n c t i o n  o f membrane i n t e g r i t y .  When r e a g e n t s known t o a f f e c t t h e i n t e g r i t y o f t h e 59 c e l l e n v e l o p e were added t o c u l t u r e s  shortly after  Fe,  t h e r e was a d r a m a t i c d e c r e a s e i n t h e a m o u n t o f i r o n bound t o t h e c e l l s ( F i g u r e active  1 6 ) . T h e amount o f r a d i o -  i r o n t a k e n up by t h e c e l l s d r o p p e d r a p i d l y t o  a low a n d a p p a r e n t l y c o n s t a n t l e v e l f o l l o w i n g t h e addition (v)  o f such reagents (Figure 16).  S p e c i f i c i t y o f iron uptake The  uptake o f radioactive  by t h e a d d i t o n o f Go  , Ni  i r o n was n o t a f f e c t e d  , Zn  o r Mn  ions  69  10  F i g u r e 15.  20 MINUTES  30  40  E f f e c t o f g l u c o s e on i r o n u p t a k e c a p a c i t y o f ,B. s u b t i 1 i s B1471  grown under  Symbols: I  iron-deficient conditions.  • , - c e l l s w e r e w a s h e d o n c e and r e s u s p e n d e d  in  m e d i u m w i t h 0.2%  g l u c o s e , then allowed to incubate a t . 59 37 C f o r . 1 . 5 h o u r s b e f o r e Fe a d d i t i o n ; A A, c e l l s w e r e t r e a t e d a s a b o v e e x c e p t t h a t g l u c o s e was o m i t t e d ; 59 0—  0, u n t r e a t e d c e l l s .  ml c o n c e n t r a t i o n .  Fe was  a d d e d a t 100 pg a t o m s /  70  ~l  10  F i g u r e 16.  i  1  MINUTES  20  30  E f f e c t o f l i p o l y t i c c o m p o u n d s o n i r o n u p t a k e by J3_. s u b t ? 1 ?s 59 B1471 g r o w n w i t h o u t a d d e d atomsyml c o n c e n t r a t i o n .  iron.  Fe was "added a t 50 pg  O t h e r a d d i t i o n s w e r e made a t +10  minutes. S y m b o l s : 0 — - 0 , n o a d d i t i o n s ; • — i , 0.1% A-^—A, 0.1% s o d i u m l a u r y l  sulfate.  toluene;  ( F i g u r e 17).  When u n l a b e l led i r o n equal t o the amount  of r a d i o a c t i v e  i r o n , was added t o the f l a s k a t z e r o  t i m e , the amount o f l a b e l  taken up by the c e l l s was  reduced by some 30%.When a 6 - f o l d or a 5 0 - f o l d excess o f u n l a b e l l e d i r o n was added t o the f l a s k a f t e r a d d i t i o n of r a d i o a c t i v e  i r o n , the uptake o f  radioactivity  stopped almost i m m e d i a t e l y , and the amount o f by the c e l l s remained c o n s t a n t  the  label  held  (Figure 17). +++  Preliminary results interfere with  i n d i c a t e t h a t Cr  ions  i r o n uptake in B_. s u b t i 1 i s by r e d u c i n g 59  the amount o f  Fe taken up by the  Experiment With Iron S u f f i c i e n t (?)  cells.  Cultures  E f f e c t on uptake c a p a c i t y When J3.  s u b t i 1 i s was grown in the presence o f 5.5 yg  a t o m s / l i t e r or 18.1 yg a t o m s / l i t e r o f added i r o n and then t e s t e d f o r i t s a b i l i t y t o take up r a d i o a c t i v e  iron  from the growth medium, the amount o f l a b e l taken up was v e r y much l e s s than t h a t  r e t a i n e d by c e l l s grown w i t h o u t  added i r o n (Table 14, F i g u r e 1 8 ) .  A 5-fold  increase  in  59 the amount o f sulted  Fe added t o the i n c u b a t i o n m i x t u r e r e -  in s i g n i f i c a n t uptake by the i r o n - s u f f i c i e n t c e l l s .  However, the amount taken up was c o n s i d e r a b l y l e s s t h a t by the i r o n - d e f i c i e n t c e l l s  than  (Figure 19).  C e l l s grown in the presence of a low l e v e l of Co  700600500-1 to -i _J  LU  4001 300-  x  I  200-1 100-  —A  15 F i g u r e 17.  Pi  MINUTES  "15  35  35  S p e c i f i c i t y o f t h e i r o n u p t a k e s y s t e m o f B. s u b t i 1 i s B1471 Symbols:  0-  -0, no a d d i t i o n s , Co  600 pg a t o m s / m l ; A — A ,  Fe  + + +  ++  Ni  Zn  , 100 pg a t o m s / m l ;  o r Mn I  Fe  + + +  , 600 pg a t o m s / m l  a d d e d a t 7.5 m i n u t e s ( + ) ; k  Fe  + + +  , 5 ng a t o m s / m l  A l l o t h e r a d d i t i o n s w e r e made 3  minutes before  A  at A,  F e , [ a d d e d a t 2.5 m i n u t e s ^ w h i c h was i n e l u d e d a t a c o n c e n t r a t i o n o f 100 p g . a t o m s / m l .  600-  MINUTES F i g u r e 18.  R e p r e s s i o n o f i r o n - u p t a k e c a p a c i t y o f B_. s u b t ? 1 i s B1471 by g r o w t h i n t h e p r e s e n c e o f a d d e d Symbols: 0 — 0 ,  iron.  c e l l s grown u n d e r i r o n - d e f i c i e n c y ; i  c e l l s g r o w n w i t h 5.5 y g a t o m s F e  + + +  c e l l s g r o w n w i t h 18.1 y g a t o m s F e  /liter  + + +  a d d e d a t a c o n c e n t r a t i o n o f 100 pg  added;  /liter. atoms/ml.  "^Fe  A—A. was  MINUTES Figure  19.  E f f e c t o f g r o w t h w i t h a d d e d i r o n on i r o n u p t a k e c a p a c i t y o f B_„ s u b t ! 1 i s B1471 . ' Symbols: 0 0, c e l l s g r o w n w i t h o u t a d d e d i r o n , 500 pg 59 atoms/ml Fe a d d e d ; t t, c e l l s grown w i t h o u t added 59 i r o n , 100 pg a t o m s / m l Fe a d d e d ; A — A , c e l l s g r o w n w i t h 18.1 y g a t o m s / l i t e r F e  + + +  , 500 pg a t o m s / m l  a d d e d ; A — A , c e l l s g r o w n w i t h 18.1 y g a t o m s / l i t e r 59 100 pg a t o m s / m l Fe a d d e d .  "'"Ve Fe  + + +  75  ions appeared t o l y s e immediately  upon w a s h i n g .  Un-  w a s h e d c e l l s , h o w e v e r , showed a n i n c r e a s e d i r o n u p t a k e capacity (personal (d)  Experiments  In C h e m i c a l l y  observations).  with C o n t r o l l e d Levels o f Iron.  Growth  E x t r a c t e d medium .  (i)  E f f e c t o f p h e n o l i c a c i d p r o d u c t i o n on i r o n up-  take  capacity. G r o w t h o f J3. s u b t ? 1 i s  m e d i u m was e x t r e m e l y production  in chemically  slow and v a r i a b l e .  extracted Phenolic acid  b y s u c h c e l l s was a l s o v a r i a b l e .  However,  t h e r e d i d a p p e a r t o be a s t r o n g c o r r e l a t i o n between t h e c a p a c i t i e s o f t h e c e l l s t o t a k e up i r o n a n d t h e i r production of phenolic acids (Figure 20). (e)  Iron Uptake as a F u n c t i o n o f C h e l a t i n g Agents (i)  E f f e c t o f d e s f e r r i f e r r i c h r o m e , DHBG a n d EDTA. When t h e t h r e e i r o n - b i n d i n g a g e n t s d e s f e r r i 59  f e r r i c h r o m e , DHBG a n d EDTA w e r e i n c u b a t e d w i t h  Fe  f o r 15 m i n u t e s a n d t h e n a d d e d t o J3. s u b t i 1 i s B1471 c e l l s grown under i r o n - d e f i c i e n t c o n d i t i o n s , a change i n t h e amount o f l a b e l t a k e n up was 21).  observed  (Figure  D e s f e r r i f e r r i c h r o m e a n d DHBG i n c r e a s e d t h e u p t a k e  by a b o u t h0% a n d 20% r e s p e c t i v e l y , w h e r e a s , EDTA r e d u c e d t h e a m o u n t o f l a b e l t a k e n up by t h e c e l l s b y some 30% ( F i g u r e 2 1 ) .  76  ?  F i g u r e 20.  10  \5 20 MINUTES  iS  I r o n u p t a k e b y _B. s u b t ? 1 i s B1471 g r o w n w i t h levels of iron in extracted  30  different  medium.  Symbols: 0 0, n o a d d i t i o n s , O.D.-.. = 0: • •' .. + 5 1 0 nm 1 y g atoms Fe / l i t e r , 0.D._ ' = 0 . 2 1 ; • — A ; . 5 1 0 nm ' ' +  +  + + +  h y g atoms Fe / l i t e r , 0.D. +++ 8 y g atoms Fe / l i t e r ^ O.D. .  =0.16, Q (S =0.  r l r i  5 1 0  n m  r l  /67  •  5 1 0  nm  .  T~\ ;  F i g u r e 21.  Effect of various  iron-binding,agents  on i r o n u p t a k e b y  B V . . s u b t i l i s * B1471' g r o w n ) u n d e r i r o n - d e f i c i e n t c o n d i t i o n s . " ' 59 Compounds w e r e p r e i n c u b a t e d w i t h Fe b e f o r e a d d i n g t o a f i n a l c o n c e n t r a t i o n o f I yfcl c h e l a t e s a n d 20 pg a t o m s / m l  —  Symbols: 0-6,  no a d d i t i o n s ; • — i ,  ' ferriferrichrome; [ -  5  10  15 MINUTES  DHBG; A — A , d e s -  ; 'EDTA.  20  25  30  (ii) Effect of varying concentrations I t was f o u n d  o f DHBG.  that the increase  59  i n uptake o f  Fe  f r o m t h e medium b y c e l l s g r o w n u n d e r i r o n - d e f i c i e n t c o n d i t i o n s was p r o p o r t i o n a l t o t h e amount o f DHBG 59 added with t h e was  q u i t e small  subti 1 i s Mutant  Fe, although  the r e l a t i v e increase  (Figure 22). Strains  The u p t a k e c a p a c i t i e s o f v a r i o u s mutant s t r a i n s were examined order t o c o r r e l a t e phenolic acid production (a)  Shikimic Acid Requiring The  with  iron uptake.  Mutant  u p t a k e c a p a c i t y o f t h e m u t a n t when g r o w i n g , u n d e r  i r o n l i m i t i n g c o n d i t i o n s was l e s s t h a n t h a t o f t h e w i l d type s t r a i n (Figure 23). stimulated  uptake.  D e s f e r r i f e r r i c h r o m e , DHB a n d DHBG  P h e n o l i c a c i d p r o d u c t i o n was n o t d e t e c t e d  in t h i s s t r a i n . (b)  Streptomycin The  Resistant  u p t a k e c a p a c i t i e s o f two s t r e p t o m y c i n  mutants were c o n s i d e r a b l y ( F i g u r e 2k). slightly. was (c)  Mutants  l e s s than t h a t o f t h e w i l d  S u r p r i s i n g l y , streptomycin Phenolic acid production  l e s s than  i n the w i l d type  Albomycin Resistant  resistant  stimulated  i n both  type uptake  these s t r a i n s  (Table 15).  Strains 59  Although  t h e immediate b i n d i n g o f  F e by a n  r e s i s t a n t m u t a n t was g r e a t e r t h a n t h a t o f t h e w i l d  albomycin type,  79 :  5  1  F i g u r e 22.  10 r  1  1  15 20 MINUTES  - i  25  r  30  E f f e c t o f i t o i c a c i d o n i r o n u p t a k e by B. s u b t ? 1 i s B1471 g r o w n w i t h o u t a d d e d i r o n . DHBG was p r e i n c u b a t e d w i t h 59 59 Fe b e f o r e a d d i n g t o t h e g i v e n c o n c e n t r a t i o n . F e was a d d e d t o 20 pg a t o m s / m l f i n a l c o n c e n t r a t i o n . Symbols: 0 A  0, n o a d d i t i o n s ; • —  A, 1.0 yM DHBG; A  8, 10 yM DHBG;  A, 0.1 yM DHBG.  25020015010050-  10 F i g u r e 23.  15 MINUTES  20  25  30  E f f e c t o f v a r i o u s i r o n - b r i n g i n g a g e n t s on i r o n u p t a k e by B_.' s u b t i 1 i s 1471 y i g r o w n w i t h 10 yM s h i k i m i c a c i d  under  i r o n - d e f i c i e n t condi t ions. Symbols: 0  0, no a d d i t i o n ; A  f e r r i chrome; A — A ,  A,  1.0 yM DHBG; • —  1.0 yM  desferri-  i , 50 yM  DHB.  •c-7--  59 Compounds w e r e p r e i n c u b a t e d w i t h Fe b e f o r e b e i n g added. 59 Fe was p r e s e n t .at 100 pg a t o m s / m l c o n c e n t r a t i o n .  5  F i g u r e 2k.  10  15 MINUTES  20  25  I r o n u p t a k e by two s t r e p t o m y c i n - r e s i s t a n t  30  mutants o f  B. s J b t i 1 i s B1471 g r o w n u n d e r c o n d i t i o n s o f i r o n d e ficiency. Symbols: 0 — 0 ,  A5 w i t h o u t ' S m a d d e d ; 9  100 yM Sm a d d e d ; A — A, A9 w i t h o u t Sm; A 100 yM  Sm.  f, A5  with  A, A9  with  82  T a b l e 15-  U p t a k e o f R a d i o a c t i v e i r o n by m u t a n t s t r a i n s o f B_. s u b t i 1 i s B1471, grown w i t h o r w i t h o u t " a d d e d  cpm x 1 0 / 1 0 3  8  iron.  cells  in 2 minutes  i n 30 m i n u t e s  O.D.  •Fe  +Fe*  -Fe  +Fe  -Fe  A5  5.9  -  57-4  -  .16  A9  4.0  -  40.3  -  .05  A  R 1 2 3  Co H  R  R  Fe  R  Fe H R  R  Bl471  +Fe*  510  rl/%  nm +Fe  6.3  18.5  57.1  29.0  0.25  0.16  13.2  5.3  40.9  38.3  0.40  0.32  5.6  4.6  56.4  25.1  0.19  0.09  8.5  21.4  34.6  75.9  0.28  0.20  4.3  14.5  30.0  58.4  0.24  0.18  8.2  5.6  75.2  54.8  .24  0.12  r e f e r s t o g r o w t h o f B. s u b t i 1 i s i n t h e p r e s e n c e o f 0.1 mM  FeCl^.  t h e t o t a l a m o u n t b o u n d i n t h i r t y m i n u t e s was l e s s 15). was  When g r o w n w i t h 0.1 mM F e C l ^ , i r o n u p t a k e c a p a c i t y repressed about 50%.  P h e n o l i c a c i d p r o d u c t i o n by t h i s  s t r a i n was s l i g h t l y g r e a t e r t h a n by t h e w i l d (d)  (Table  type.  Cobalt R e s i s t a n t Mutant 59 Again  was  the i n i t i a l  g r e a t e r than  binding of  Fe by t h e mutant  i n the wild type, while the total  strain  amount  b o u n d was l e s s ( T a b l e 1 5 ) . T h e c a p a c i t y f o r u p t a k e was u n a f f e c t e d b y g r o w t h w i t h 0.1 mM F e C l ^ . production (e)  by t h i s s t r a i n was g r e a t e r t h a n by t h e w i l d  type.  Hemin R e s i s t a n t M u t a n t The  was  Phenolic acid  i r o n uptake c a p a c i t y o f a hemin r e s i s t a n t mutant  s i m i l a r t o t h a t o f t h e w i l d t y p e when grown w i t h o u t  added  i r o n ( T a b l e 1 5 ) . When g r o w n w i t h 0.1 mM F e C l ^ , i t s u p t a k e c a p a c i t y was r e d u c e d  by more than 50%.  Phenolic acid  d u c t i o n b y t h i s n u t a n t s t r a i n was s i m i l a r t o t h e w i l d (f)  protype.  Iron R e s i s t a n t Mutants Theiron  uptake c a p a c i t y o f an i r o n r e s i s t a n t mutant  grown w i t h o u t  a d d e d i r o n was c o n s i d e r a b l y l e s s t h a n t h a t o f  the w i l d type  ( T a b l e 1 5 ) . H o w e v e r , when g r o w n w i t h 0.1 mM  F e C l ^ , i t s u p t a k e c a p a c i t y more t h a n d o u b l e d .  Phenolic  a c i d p r o d u c t i o n by t h i s m u t a n t s t r a i n was s i m i l a r t o t h a t o f t h e w i l d t y p e , b u t was n o t s i g n i f i c a n t l y r e p r e s s e d when t h e c e l l s were grown i n t h e p r e s e n c e  o f a d d e d i r o n ( F i g u r e 6, T a b l e  (g)  Hemin and  Iron R e s i s t a n t Mutant  T h e u p t a k e by a m u t a n t r e s i s t a n t t o b o t h h e m i n i r o n was (Table  Discussion of  s i m i l a r to t h a t of the i r o n r e s i s t a n t s t r a i n 15).  Results  The work o f P e t e r s r e s u l t s of t h i s study  (44)  c i t y of t h i s organism  and o f P e t e r s and W a r r e n (46)  and  Moreover, i t is evident that the  t o t a k e up  during a p a r t i c u l a r p h y s i o l o g i c a l stage of growth.  significantly  T h i s change in  iron uptake c a p a c i t y is c l o s e l y a s s o c i a t e d with the onset of by t h e c e l l s .  and Wang and Newton (67,  68)  P e t e r s a n d W a r r e n (46)  w i t h E_. c o l i h a v e a l s o shown a c l o s e  M u t a n t s whose c a p a c i t i e s f o r p h e n o l i c a c i d p r o d u c t i o n a r e a l s o show d e c r e a s e d n e c e s s i t y of metabolic  iron uptake a b i l i t y  (46,  the presence  The  production. severely\>  68).  a c t i v i t y f o r iron uptake is evident  f r o m t h e s t u d i e s u s i n g i n h i b i t o r s s u c h a s s o d i u m a z i d e and acetamide.  phenolic  w i t h B_. s u b t ? 1 i s  a s s o c i a t i o n b e t w e e n i r o n u p t a k e c a p a c i t y and p h e n o l i c a c i d  The  capa-  i r o n f r o m t h e g r o w t h medium when  i t i s grown under i r o n - d e f i c i e n t c o n d i t i o n s i n c r e a s e s  acid production  the  i n d i c a t e an e n e r g y - d e p e n d e n t m e c h a n i s m f o r  i r o n u p t a k e i n J3. s u b t ? 1 ? s .  decreased  and  a-iodo-  r e s i d u a l a c t i v i t y found a s s o c i a t e d with the c e l l s in  of the metabolic  i n h i b i t o r s may  an i r o n - b i n d i n g p r o t e i n c l o s e t o t h e c e l l  represent  surface.  iron held  The e f f e c t o f  by  'sulfhydryl or H g  + +  i n h i b i t o r s such as p - c h 1 o r o m e r c u r i b e n z o a t e , sodium  is interesting.  These e f f e c t i v e l y i n h i b i t uptake o f iron  f r o m t h e g r o w t h medium a n d r e d u c e t h e a m o u n t o f r e s i d u a l by t h e c e l l s . iron-binding sulfhydryl  T h i s may be t a k e n t o i n d i c a t e or iron transport  groups.  responsible it available c e l l s starved  arsenite,  protein  that  does e x i s t ,  i r o n bound  i fa specific i t contains  T h e s e In e f f e c t m i g h t s e r v e a s t h e a g e n t s  f o r releasing  iron from a chelated  to the c e l l s .  complex and making  The decrease o f uptake capacity  f o r glucose is further  evidence favoring  in  an e n e r g y  requirement f o r the a c t i v e uptake o f iron. The  l o s s o f l a b e l f o l l o w e d by t h e m a i n t e n a n c e o f a c o n s t a n t  level of r a d i o a c t i v i t y after treatment of the c e l l s with  toluene  or sodium l a u r y l s u l f a t e , both o f which a p p r a r e n t l y d i s r u p t t h e c e l l u l a r membrane w i t h o u t c a u s i n g c e l l that part;  (59), would  indicate  b u t n o t a l l o f t h e i r o n t a k e n up i s r a p i d l y b o u n d  l y by t h e c e l l s . radioactive  lysis  As t h e time i s increased  i r o n and t h e a d d i t i o n  tight-  between t h e a d d i t i o n  of  o f compounds such as t o l u e n e ,  s o d i u m l a u r y l s u l f a t e , f e r r i c h r o m e , DHBG a n d e v e n m e t a b o l i c  inhibi-  tors , l e s s , l a b e l led iron i s l o s t from the b a c t e r i a l  This  istaken  t o mean t h a t  the iron-binding  w i t h r a p i d and a l m o s t q u a n t i t a t i v e  capacity  cells.  of the c e l l s i s great,  u t i l i z a t i o n of the iron  taken  up f r o m t h e medium. The  s p e c i f i c i t y o f t h e u p t a k e s y s t e m f o r i r o n i s e v i d e n c e d by  t h e f a c t t h a t Co  , Ni  , Mn  and Zn  ions d i d n o t i n t e r f e r e  with  the uptake of  Fe.  P r e l i m i n a r y e x p e r i m e n t s , however,  indicate  +++ that Cr  ions i n t e r f e r e with iron uptake, suggesting that the  uptake mechanism i s not c o m p l e t e l y s p e c i f i c f o r i r o n . o f u n l a b e l l e d FeC1^  The  addition  a t any time b e f o r e o r d u r i n g t h e u p t a k e e x p e r i 59  m e n t s c a u s e d an i m m e d i a t e  decrease in  o f t h e l a b e l by n o n - r a d i o a c t i v e Fe  F e u p t a k e , due t o d i l u t i o n ions.  It i s important to  p o i n t out t h a t t h i s d e c r e a s e i n i r o n uptake does not l i k e l y r e p r e s e n t r e p r e s s i o n o f the uptake system f o r i r o n but does  illustrate  the s p e c i f i c i t y of the system f o r the c a t i o n . When J3_. s u b t i 1 i s was g r o w n i n t h e p r e s e n c e o f a d d e d i t s u p t a k e c a p a c i t y a p p e a r e d t o be r e p r e s s e d . h a s b e e n shown f o r i r o n u p t a k e  i n E_. c o l ? ( 6 8 ) .  u p t a k e c a p a c i t y o f s u c h c e l l s was 59 s e e n when a h i g h e r l e v e l o f  A similar  iron, result  However, the  not e l i m i n a t e d c o m p l e t e l y , as  Fe was a d d e d  to the c e l l s .  Since  q u a n t i t a t i v e s t u d i e s on t h e t o t a l amount o f i r o n i n v o l v e d i n s u c h a system were not performed, labelled  i t was  p o s s i b l e t h a t d i l u t i o n o f the  i r o n by b o l d F e C l 3 made i t a p p e a r t h a t u p t a k e was  when i n r e a l i t y  reduced  i t remained c o n s t a n t .  EDTA a p p e a r e d t o make i r o n l e s s a v a i l a b l e t o t h e c e l l s , i n d i c a t i n g t h a t t h e u p t a k e s y s t e m shows some s p e c i f i c i t y f o r c h e l a t ing agent. c h r o m e was  T h e s t i m u l a t i o n o f i r o n u p t a k e by DHBG o r d e s f e r r i f e r r l e s s than t h a t seen p r e v i o u s l y (46). 59  o f DHBG m i x e d w i t h up by t h e c e l l s .  The c o n c e n t r a t i o n  Fe had an e f f e c t on t h e amount o f i r o n  taken  However, the d i f f e r e n c e between t h e s e r e s u l t s  and  t h o s e o f P e t e r s (kk)  and P e t e r s and Warren  explained at present. m e t a b o l i s m may  i n s u c h a way  iron transport  A hydroxamate  readily  B_. s u b t ? 1 ?s i s an u n s t a b l e o r g a n i s m and i t s  have a l t e r e d  a s an e f f e c t i v e  (46) c a n n o t be  t h a t DHBG no l o n g e r s e r v e s  factor.  i s p r o d u c e d by a n o t h e r s t r a i n o f J3. s u b t ? 1 i s a n d  i s t h o u g h t t o p l a y a r o l e i n i r o n t r a n s p o r t by t h i s s t r a i n ( 6 ) . A t t e m p t s t o d e m o n s t r a t e bound h y d r o x y l a m i n e i n S t r a i n B1471 unsuccessful.  However, the i r o n uptake system i n s t r a i n  does respond t o f e r r i c h r o m e , a hydroxamate  were  B1471  o f known b i o l o g i c a l  a c t i v i t y (40).  3.  Micrococcus (a)  lysodeikticus  P r e l i m i n a r y Experiments G r o w t h o f M_. l y s o d e i k t i c u s , u n d e r c o n d i t i o n s o f  d e f i c i e n c y and w i t h o u t c i t r a t e was v a r i a b l e .  i n t h e s u s p e n d i n g medium,  Preliminary results indicated that  p o l y m e r i z e d a n d r e m a i n e d bound  iron-  to the f i l t e r s ,  leading to erroneously high counts of  iron  thereby  radioactivity.  C o n s e q u e n t l y , M_. l y s o d e i k t i c u s was e i t h e r g r o w n i n m i n i mal medium c o n t a i n i n g c i t r a t e o r g r o w n i n S a l t o n ' s m i n i m a l medium, t h e n washed and r e s u s p e n d e d  i n t h e same m e d i u m  c o n t a i n i n g c i t r a t e b e f o r e uptake experiments were run. (b)  E f f e c t o f Growth With or Without  I r o n on U p t a k e C a p a c i t y  C e l l s g r o w n i n c i t r a t e - c o n t a i n i n g medium i n t h e p r e s e n c e  88  o f added i r o n had a g r e a t e r grown w i t h o u t  i r o n uptake c a p a c i t y than  cells  added i r o n ( F i g u r e 2 5 ) .  C e l l s g r o w n i n S a l t o n ' s m i n i m a l medium w i t h o r w i t h o u t a d d e d i r o n a p p e a r e d t o h a v e no a c t i v e u p t a k e m e c h a n i s m f o r iron (Figure 26). (c)  E f f e c t o f Growth W i t h C h e l a t i n g A g e n t s on U p t a k e Growth i n t h e p r e s e n c e o f f e r r i c h r o m e  Capacity  o r benzohydroxamic  a c i d , o r i n t h e p r e s e n c e o f EDTA o r NTA p l u s y e a s t  extract  i n c r e a s e d t h e iron u p t a k e c a p a c i t y o v e r t h a t o f c o n t r o l cultures (Figure 25). (d)  E f f e c t o f C h e l a t i n g Agents on Iron Uptake i n v i t r o EDTA, NTA a n d T i r o n c a u s e d a s i g n i f i c a n t r e d u c t i o n ca  i n t h e amount o f  Fe taken  up b y t h e c e l l s  Desferriferrichrome A a l s o decreased iron taken  up ( F i g u r e 2 8 ) .  (Figure 27).  t h e amount o f  D e s f e r r i f e r r i c h r o m e , on t h e  other  hand, c a u s e d a 1 0 0 % i n c r e a s e , i n t h e amount, o f . i r o n  taken  up, whereas benzohydroxamate and f e r r i c h r o m e A had  a slight stimulatory e f f e c t (Figure 28). Catechol, o-hydroxyhippuric  a c i d , DHB a n d DHBG a l l  a p p e a r e d t o i n c r e a s e t h e amount o f i r o n t a k e n 59 lysodeikticus  (Figure 29).  T h e amount o f  up by M_.  F e t a k e n up  i n t h e p r e s e n c e o f DHBG was a f u n c t i o n o f t h e DHBG c o n centration (Figure 30).  89  F i g u r e 25.  E f f e c t o f g r o w t h w i t h v a r i o u s compounds on i r o n u p t a k e c a p a c i t y i n M^.  lysodeikticus.  Symbols: 0 — 0 ,  0.1% c i t r a t e w i t h o u t added  0.1% c i t r a t e w i t h A  A,  1 yM F e C ^ ; A  1 yM NTA;  1 yM b e n z o h y d r o x a m i c  •  p acid.  A, 1 yM  , 1 yM EDTA;  iron; I  i,  ferrichrome; £| ,  — I  "1  1 1  5  10  1—  I  15  20  1—  —I  25  30  MINUTES •  F i g u r e 26.  I r o n u p t a k e by M_. l y s o d e i k t i c u s grown i n medium  without'  citrate. Symbols: 0  0, no a d d i t i o n s ; •  a-iodoacetamide;  1 mM NaN^ + I mM  At—A, 1 mM s o d i u m a r s e n i t e .  U p t a k e was s i m i l a r - w h e t h e r g r o w n w i t h o u t o r w i t h yg atoms/1 i t e r .  1.2  91  T  1  5  F i g u r e 27.  EffeGt  10  1  15 MINUTES  1  20  a t o m s / l i t e r o f added Symbols: 0 — A,  1—  25  30  o f c h e l a t i n g a g e n t s on i r o n u p t a k e by M_. l y s o -  d e i k t i c u s grown i n c i t r a t e c o n t a i n i n g  A  1  medium w i t h  1.2 y g  iron.  0, no a d d i t i o n s ; 6 —  9,  EDTA; A  A,  NTA;  Tiron. CO  A l l c h e l a t i n g agents were p r e i n c u b a t e d a d d e d t o 1 yM f i n a l  concentration.  with  Fe and  MINUTES F i g u r e 28.  E f f e c t o f h y d r o x a m a t e s on i r o n u p t a k e by M_. grown  i n c i t r a t e - c o n t a i n i n g medium w i t h  l i t e r o f added Symbols: 0 A—A, acid;  lysodeikticus  1.2 y g  atoms/  iron.  0, no a d d i t i o n s ; •  §, f e r r i c h r o m e  A;  d e s f e r r i f e r r i c h r o m e A; A-—-A, b e n z o h y d r o x a m i c • -—Q  , desferriferrichrome. A l l compounds were p r e i n c u b a t e d w i t h 59 Fe a n d a d d e d t o a final concentration  o f I yM.  MINUTES F i g u r e 29.  E f f e c t o f p h e n o l i c c o m p o u n d s o n i r o n u p t a k e b y M. l y s o d e i k t i c u s g r o w n i n c i t r a t e - c o n t a i n i n g medium  with  1.2 y g a t o m s / l i t e r o f a d d e d i r o n . Symbols: 0 DHB; •  0, n o a d d i t i o n s ; •  A, o - H y d r o x y h i p p u r i c  •, catechol; A  acid;  A l l a d d i t i o n s w e r e made t o 1 yM f i n a l after pre-incubation with  Fe.  Q — — • , DHBG. concentration  A,  5  F i g u r e 30.  io~  15 MINUTES  r  20  25  30  E f f e c t o f v a r y ing^ c o n c e n t r a t i o n s o f i t o i c a c i d o n i r o n uptake  ty M_. l y s o d e i k t i c u s g r o w n i n c i t r a t e c o n t a i n i n g  m e d i u m w i t h 1.2 y g a t o m s / l i t e r o f a d d e d Symbols.: 0 10 yM DHBG; A  0, no a d d i t i o n s ; • 4,1,.0 yM DHBG;  •, 50 yM DHBG; A •  DHBG was p r e i n c u b a t e d f o r 15 m i n u t e s addedrto  t h e eel 1s.  iron.  • with  A,  , 0.1 yM DHBG. Fe and then  95  (e)  E f f e c t o f A l b o m y c i n on I r o n U p t a k e A 0.1 yM a l b o m y c i n c o n c e n t r a t i o n a p p e a r e d t o d e c r e a s e  i r o n u p t a k e by a b o u t 50%  (Figure 31).  Discussion of Results  I r o n i s known t o f o r m p o l y m e r s o f h i g h m o l e c u l a r w e i g h t when i n s o l u t i o n s o f n e u t r a l o r b a s i c pH ( 6 1 ) . T h e r e f o r e , n u m e r o u s p r e l i m i n a r y e x p e r i m e n t s were performed u s i n g v a r i o u s media and b u f f e r s t o determine optimal c o n d i t i o n s f o r uptake experiments. I t was f o u n d t h a t a b a s a l l e v e l o f c i t r a t e was n e c e s s a r y i n t h e suspending f l u i d  to prevent the retention of s i g n i f i c a n t levels of  r a d i o a c t i v i t y o n t h e membrane f i l t e r s . M_. l y s o d e i k t i c u s a p p e a r e d t o h a v e a n i r o n u p t a k e s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t o f J3_. s u b t ? l i s .  system  When g r o w n i n  media c o n t a i n i n g no c h e l a t i n g a g e n t o r s o l u b i l i z i n g f a c t o r w i t h o r w i t h o u t added  i r o n , M_. l y s o d e i k t i c u s d i d n o t a p p e a r t o h a v e a n a c t i v e  mechanism f o r the uptake o f i r o n . d i f f u s e d t h r o u g h t h e c e l l membrane. p r e s e n c e o f added  It appeared that iron simply Growth o f t h e o r g a n i s m i n t h e  i r o n when t h e m e d i u m c o n t a i n e d a s o l u b i l i z i n g  f a c t o r such as c i t r a t e , d i d not appear t o r e p r e s s t h e i r o n mechanism.  uptake  On t h e c o n t r a r y , i r o n s e e m e d t o i n d u c e t h e t r a n s p o r t  59  of  Fe i n t o t h e c e l l s .  Growth  under i r o n - d e f i c i e n t c o n d i t i o n s  in t h e p r e s e n c e o f c i t r a t e d i d not appear t o s t i m u l a t e t h e i r o n  96  1  10  F i g u r e 31.  20 30 MINUTES  40  5*0  E f f e c t o f a l b o m y c i n on i r o n u p t a k e by M. l y s o d e i k t i c u s -8 grown w i t h y e a s t e x t r a c t added  p l u s 10  M EDTA a n d w i t h o u t '  iron.  S y m b o l s : 0 - — 0 , no a d d i t i o n s ; 1  •  i , a l b o m y c i n (0.1 yM  c o n c e n t r a t i o n ) added 3 minutes p r i o r t o  CO  Fe.  u p t a k e c a p a c i t y o f M_. l y s o d e i k t i c u s .  However, growth i n t h e  p r e s e n c e o f v a r i o u s o t h e r c h e l a t i n g a g e n t s d i d seem t o s t i m u l a t e uptake.  T h e d e g r e e o f t h i s s t i m u l a t i o n d e p e n d e d t o some  on t h e t y p e o f c h e l a t i n g a g e n t .  Consequently, one might  extent suspect  that a degree of s p e c i f i c i t y e x i s t s in this microorganism f o r a c h e l a t i n g agent which "induces"  iron uptake.  Albomycin appears to i n h i b i t iron uptake t o a s l i g h t extent. This supports,  but does not d i s t i n g u i s h between, the  suggestions  t h a t a l b o m y c i n a c t s e i t h e r by i n h i b i t i n g t r a n s p o r t o f an e s s e n t i a l metabolyte across with  the cell  membrane (65), o r t h a t  the internal u t i l i z a t i o n of F e  the c e l l  k.  + + +  i t interferes  ions once they a r e i n s i d e  (75).  Salmonella (a)  typhimurium s t r a i n LT-2  Iron  Uptake  (i)  Growth Under I r o n - D e f i c i e n t a. E f f e c t o f M e t a b o l i c  Conditions  I n h i b i t o r s on I r o n U p t a k e  When s o d i u m a z i d e a n d a - i o d o a c e t a m i d e w e r e a d d e d t o w a s h e d c e l l s o f S_. t y p h i m u r i u m ,  three  59 minutes p r i o r to the a d d i t i o n o f  F e , t h e amount  o f i r o n t a k e n up was r e d u c e d t o o n e - t h i r d of the c o n t r o l .  The a d d i t i o n o f  p-nitrophenol  a t 10 u m o l e s / m l a l s o c a u s e d a r e d u c t i o n take  (Figure 32).  that  i n up-  5  F i g u r e 32.  10  15 MINUTES  Effect of metabolic  20  25  i nh i b i t o r s • on i r o n u p t a k e by S_.  t y p h Imur ium L T - 2 g r o w n w i t h o u t  added i r o n . 59 t i o n s w e r e made 10 m i n u t e s p r i o r t o Fe. Symbols: 0 ' — 0 ,  30  A l l addi-.  no a d d i t i o n s ; • — p - n i t r o p h e n o l  (10 i i m o l e s / m l ) ; A — - A , 59 mM) . - ^ F e was p r e s e n t  s o d i u m a z i d e + a - i o d o a c e t a m i d e (1 a t 40 pg a t o m s / m l  concentration.  99  b. E f f e c t o f I r o n - C h e l a t i n g  Agents  When d e s f e r r i f e r r i c h r o m e , DHB, DHBG o r - E D T A 59  were p r e i n c u b a t e d  with  Fe and t h e m i x t u r e s  a d d e d t o S_. t y p h i m u r i u m , c h a n g e s i n u p t a k e were observed  ( F i g u r e 33)-  r e d u c t i o n w h e r e a s DHBG c a u s e d increase both The  iii uptake.  EDTA c a u s e d  creased  a 50%  an a p p r o x i m a t e  50%  DHB a n d d e s f e r r i f e r r i c h r o m e  increased uptake about 3"fold initial  patterns  ( F i g u r e 33).  b i n d i n g o f i r o n by t h e c e l l s was i n -  c o n s i d e r a b l y by d e s f e r r i f e r r i c h r o m e , b u t  n o t by t h e o t h e r  i r o n - b i n d i n g a g e n t s ( F i g u r e 33).  c. S p e c i f i c i t y o f t h e System When e x c e s s u n l a b e l l e d i r o n was a d d e d t o t h e i n c u b a t i o n m i x t u r e , t h e u p t a k e o f 59 F e was r e d u c e d (/n o t shown).  Preliminary results indicate a  dramatic 59  i n c r e a s e f o l l o w e d by a s l o w r e l e a s e i n i n i t i a l l y b y t h e c e l l s when C r after  59  Fe. The additbn of Cr  Fe bound  ions a r e added +++  ions t e n minutes  before the a d d i t i o n o f r a d i o a c t i v e iron, appears to stimulate Further  i r o n u p t a k e c a p a c i t y more than  i n v e s t i g a t i o n s i n t o t h e r o l e p l a y e d by  +++ Cr  100%.  i o n s i n t h e i r o n u p t a k e s y s t e m o f S_.  typhimuriurn  a r e underway.  100  —  -r  -i  5  10  i  1  15  20  r  r  25  30  MINUTES *  F i g u r e 33.  *  E f f e c t o f c h e l a t i n g a g e n t s o n i r o n u p t a k e by S .'• t y p h i m u r ium g r o w n u n d e r c o n d i t i o n s o f i r o n - d e f i c i e n c y ' . S y m b o l s : 0 — 0 , no a d d i t o n s ;  •—  A  •  J  A, d e s f e r r i f e r r i c h r o m e ;  A l l compounds were p r e i n c u b a t e d with  5 9  Fe.  i , DHB; A " — A , • a t 1 mM  DHBG;  EDTA. concentration  101  ( i i ) Growth Under I r o n - S u f f ? c i e n t C o n d i t i o n s . , E f f e c t on U p t a k e  Capacity.  C e l l s g r o w n i n t h e p r e s e n c e o f 0.1 mM F e C l ^ h a d about o n e - t e n t h t h e c a p a c i t y f o r i r o n uptake as c e l l s g r o w n w i t h o u t a d d e d i r o n ( F i g u r e 34). o f DHBG i n c r e a s e d  The addition  t h i s uptake c a p a c i t y about  3 fold -  ( F i g u r e 34). (b)  Iron E f f l u x When c e l l s , grown f o r s e v e r a l g e n e r a t i o n s i n t h e p r e s e n c e  o f r a d i o a c t i v e i r o n , were washed and resuspended  in iron-  d e f i c i e n t m e d i u m , a s l i g h t l o s s o f r a d i o a c t i v i t y was o b s e r v e d (Table 16).  The addition o f excess unlabelled  iron or  d e s f e r r i f e r r i c h r o m e t o t h e i n c u b a t i o n medium c a u s e d n o additional  loss o f l a b e l (Table 16).  Toluene and sodium  59 a r s e n i t e caused f u r t h e r loss o f  Fe from t h e c e l l s ,  a - i o d o a c e t a m i d e appeared t o c a u s e an i n c r e a s e amount o f r a d i o a c t i v i t y b o u n d t o t h e c e l l s 5.  -S_. t y p h i m u r ? u r n M u t a n t (a)  Preliminary  |n t h e  (Table 16).  Strains  Experiments  P r e l i m i n a r y e x a m i n a t i o n o f i r o n u p t a k e showed  that  s t r a i n s f e r l a n d fer3 w e r e s i m i l a r , a s w e r e s t r a i n s f e r D 9 and f e r D 1 9 -  C o n s e q u e n t l y , s t r a i n s f e r l and ferD19 were  used i n a l l  subsequent  experiments  MINUTES Figure.34.  R e p r e s s i o n o f i r o n - u p t a k e c a p a c i t y o f S. t y p h i m u r i u m  by  g r o w t h i n t h e p r e s e n c e o f 0.1 mM F e C l ^ a n d t h e e f f e c t o f . DHBG o n i r o n Symbols: 0 • 59  uptake. 0, c e l l s grown w i t h o u t  c e l l s grown w i t h o u t  i r o n ; no a d d i t o n s ;  i r o n , 1 yM DHBG a d d e d w i t h  Fe; A A, c e l l s g r o w n w i t h i r o n ; n o a d d i t i o n s ; A A, c e l l s g r o w n w i t h i r o n , 1 yM DHBG a d d e d w i t h ^ F e .  T a b l e 16.  E f f e c t o f v a r i o u s compounds o n  F e e f f l u x f r o m S_.  t y p h imur i um.  Compound a d d e d  Concentration  cpm/10  cells  after  (mM)  0 Time  10 M i n u t e s  -  2160  2270  1928  0 .1  2230  2300  2050  0 .1  2163  2108  2062  0 .1  2294  2222  2240  0 .1  2190  2250  2150  0. 1  2260  2210  1930  2342  2214  1616  1 .0  2498  2080  2445  a lodoacetamide + F e C l ^  1 .0  1815  1953  2090  Sodium a r s e n i t e  1 .0  2895  2840  2607  Sodium a r s e n i t e + F e C l ^  1 .0  1967  2012  1926  Sodium a z i d e  1 .0  2112  2279  2206  Sodium a z i d e + F e C l ,  1 .0  2033  1966  1612  Nil FeCl  3  Desferriferrichrome 1 toic  1  acid  FeCl^ + Itoic  acid  FeCl^ + Ferrichrome Toluene a  lodoacetamide  (0  .n)  30 M i n u t e s  104  (b)  Comparison with Wild  Type  59 Uptake o f but o t h e r w i s e  Fe i n both  f e r l a n d f e r D 1 9 was l e s s  s i m i l a r t o that o f the parent  than,  s t r a i n LT-2  ( F i g u r e 35) • (c)  E f f e c t o f C h e l a t i n g A g e n t s on U p t a k e With the f e r l  s t r a i n , DHB a n d DHBG a p p e a r e d t o s t i m u -  l a t e i r o n u p t a k e s l i g h t l y , w h e r e a s EDTA c a u s e d a s i g n i f i c a n t decrease.  D e s f e r r i f e r r i c h r o m e , on t h e o t h e r hand, caused  59 about a 5 f o l d increase _  i n t h e amount o f  the mutant c u l t u r e ( F i g u r e  Fe taken  up b y  36).  W i t h m u t a n t f e r D 1 9 , DHB, DHBG a n d EDTA a l l r e s u l t e d in a d e c r e a s e  i n iron uptake,  whereas-desferriferrichrome 59  caused a 10-fold Discussion of Results  The  increase  e f f e c t s o f metabolic  in  Fe uptake ( F i g u r e  37).  i n h i b i t o r s show t h a t i r o n i s t a k e n  up by .S. t y p h i m u r i u m v i a a n a c t i v e t r a n s p o r t s y s t e m .  This  system,  l i k e t h a t shown i n B_. s u b t i l i s , h a s a s p e c i f i c i t y o f r e s p o n s e t o certain  iron binding agents,  the c o n c e n t r a t i o n ferrichrome  a n d a l s o a p p e a r s t o be i n f l u e n c e d by  o f i r o n i n t h e medium.  stimulate  DHB, DHBG a n d d e s f e r r i -  i r o n u p t a k e more i n S^. t y p h i m u r ? u r n t h a n  subti1is , with desferriferrichrome again giving the greatest tion.  i n JJ. stimula-  105  T  i  10  5 F i g u r e 35.  T  15 MINUTES  r  20  r  25  T  30  I r o n u p t a k e c a p a c i t y o f S_. t y p h i m u r i u m w i l d t y p e a n d +++  mutants.  M u t a n t s t r a i n s w e r e g r o w n w i t h k y g a t o m s Fe ''/ml  Symbols: 0 — 0 ,  w i l d t y p e ; f — t , f e r 1; A  A, f e r D19.  MINUTES F i g u r e 36.  E f f e c t b f c h e l a t i n g a g e n t s on i r o n u p t a k e by S_. t y p h i m u r i u m mutant f e r 1 grown w i t h k y g atoms o f F e Symbols: 0 — A—-A,  /ml.'  9, DHB; A — A ,  0, no a d d i t i o n s ; © —  desferriferrichrome; Q  + + +  a  ,EDTA.  A l l c o m p o u n d s w e r e p r e i n c u b a t e d w i t h " ^ F e a t 1 mM centration.  DHBG; con-  107  250^  hi  200  O  00  o  150  l2  2 loo  10  F i g u r e 37.  r 30  r  T  20  MINUTES  E f f e c t o f c h e l a t i n g a g e n t s on i r o n u p t a k e by S_. t y p h i m u r i u m m u t a n t f e r D19 g r o w n w i t h k y g a t o m s o f F e S y m b o l s : 0 - — 0 , no a d d i t i o n s ; • — • A—A,  desferriferrichrome,  •  , DHB; A •  A l l compounds were p r e i n c u b a t e d w i t h centration.  + + +  /ml. A, DHBG,  , EDTA. 59 F e a t 1 mM  con-  As w i t h J3_. s u b t i 1 i s a n d E_. c o l i , g r o w t h o f S_. t y p h imur ?um w i t h 0.1 mM F e C l ^  reduced i t s iron uptake capacity  H o w e v e r , DHBG s t i m u l a t e d  u p t a k e by i r o n - s u f f i c i e n t c e l l s .  f o r e , DHBG a p p e a r e d t o s e r v e a s a n i r o n t r a n s p o r t a l l conditions  considerably.  factor  Thereunder  o f growth.  Iron remained t i g h t l y bound t o t h e c e l l s a f t e r up b y S_. t y p h i m u r i u m , s i n c e  i t was t a k e n  t h e r e was no l o s s o f l a b e l f r o m  cells  59 grown w i t h  Fe and then i n c u b a t e d w i t h e x c e s s u n l a b e l l e d  However, d e s f e r r i f e r r i c h r o m e t h e same c o n d i t i o n s .  appeared t o cause loss o f label  o f t h e c e l l membrane t h e r e f o r e the c e l l .  The i n t e g r i t y  appeared important i n retaining When t h e c e l l s w e r e t r e a t e d  azide i n the presence o f excess unlabelled  c l e a r how t h e s e i n h i b i t o r s c o u l d  The  with sodium  i r o n , o r sodium  w i t h o u t a d d e d i r o n , some l o s s o f l a b e l was a l s o o b s e r v e d .  the b a c t e r i a l  under  L o s s o f l a b e l was a l s o o b s e r v e d i f t o l u e n e ,  w i t h o r w i t h o u t e x c e s s F e C l ^ was a d d e d t o t h e c e l l s .  bound i r o n w i t h i n  iron.  arsenite It i s not  a c t t o c a u s e i r o n t o be l o s t f r o m  e e l 1.  m u t a n t s t r a i n s o f S_. t y p h i m u r i u m d i f f e r e d f r o m t h e w i l d  type in t h e i r response t o chelating DHBG a n d d e s f e r r i f e r r i c h r o m e in t h e w i l d  agents, but not otherwise.  stimulate  iron uptake s i g n i f i c a n t l y  type s t r a i n , but only desferriferrichrome  with both types o f mutant.  DHB,  is effective  The exact roles o f ferrichrome i n iron  m e t a b o l i s m remain t o be e x p l a i n e d .  109  VI.  Effect of 3-Fluorobenzoic Baci1lus  subti1 is S t r a i n  A c i d on G e n e r a l  Properties of  B1471.  Introduct ion  Among t h e i n h i b i t o r s o f p h e n o l i c a c i d 3 f 1 u o r o b e n z o i c a c i d was  particularly effective.  _  compound was  i n B.  most e f f e c t i v e  iron uptake.  subtilis,  Therefore,  e x a m i n e d i n some d e t a i l f o r i t s e f f e c t on  p h e n o l i c a c i d p r o d u c t i o n and  1.  production  this  growth,  S i n c e t h e compound  i n s t r a i n B1471, t h i s o r g a n i s m  was  chosen  was  f o r study.  Growth Although  t h e r a t e o f g r o w t h was  s l i g h t decrease benzoate tion.  was  in the f i n a l  u n a f f e c t e d , t h e r e was  c e l l y i e l d when 1 mM  3 fluoro_  added t o t h e growth medium a t the time o f i n o c u l a -  T h e r e was  a s i m i l a r , but s m a l l e r , e f f e c t  was  a d d e d a f t e r 7 o r 11 h o u r s  2.  Phenolic Acid  i f the  of growth ( F i g u r e  Production -  d i d not c o m p l e t e l y  inhibitor  38).  The a d d i t i o n o f 3 f 1 u o r o b e n z o a t e a t z e r o time p r e v e n t , DHBG f o r m a t i o n  l a t e r t h e t i m e a t w h i c h t h e i n h i b i t o r was was  i t s e f f e c t on p h e n o l i c a c i d p r o d u c t i o n  3.  Iron The  reduced,  ( F i g u r e 39).  c a p a c i t y o f B1471 decreased  (Figure  39).  t o t a k e up i r o n f r o m t h e g r o w t h  by m o r e t h a n 50%  but The  added, the s m a l l e r  Uptake  m e d i u m was  a  when t h e c e l l s w e r e  110  500  400  300-  200-  !00-  9 HOURS F i g u r e 38.  12  E f f e c t o f 3 f l u o r b e n z o i c a c i d on g r o w t h o f IB. s u b t i 1 i s -  Symbols:  0~—0,  a c i d added added  no a d d i t i o n ;  ®  a t z e r o t i m e ; 6-—Q,  C, 1 mM  7 h o u r s a f t e r i n o c u l a t i o n ; A-  z o i c a c i d added  11 h o u r s a f t e r  1 mM  B1471  3 f1uorobenzoic _  3~f1uorobenzoic A, 1 mM  inoculation.  acid  3 fluoroben_  Ill  F i g u r e 39.  E f f e c t of growth with 3 f 1 u o r o b e n z o i c _  a c i d on p h e n o l i c a c i d  p r o d u c t i o n by B_. s u b t i 1 i s 6 1 4 7 1 . Symbols': 0 — 0 ,  no a d d i t i o n s ; ©  a c i d added a t ' z e r o time; A  S, 1 mM  A, 1 mM  added 7 hours a f t e r i n o c u l a t i o n ; A z o i c a c i d a d d e d 11 h o u r s a f t e r  3"fl uorobenzoic  f1uorobenzoic A,  1 mM  inoculation.  acid  3 fluoroben_  grown under i r o n - d e f i c i e n t c o n d i t i o n s i n the p r e s e n c e 3 f1uorobenzoate (Figure _  Discussion of  Peters effectively JL*  40).  Results  (44)  found  t h a t t h e DHB  analog,  3 fluorobenzoic -  acid,  i n h i b i t e d p h e n o l i c a c i d p r o d u c t i o n by g r o w i n g c e l l s  s u b t ? l i s . T h i s i n h i b i t i o n i s t h e o n l y known r o l e o f  b e n z o i c a c i d as a m e t a b o l i c  inhibitor.  t o i n v e s t i g a t e i t s e f f e c t s on g r o w t h and w e l l as on p h e n o l i c a c i d Any  o f 1 mM  I t was  3 fluoro_  therefore of i n t e r e s t  i r o n u p t a k e c a p a c i t y as  production.  e f f e c t of 3 fluorobenzoate _  on p h e n o l i c a c i d  production  o r t h e i r o n u p t a k e c a p a c i t y o f B. s u b t i 1 i s i s n o t a d i r e c t quence of i n h i b i t i o n of general that 3 fluorobenzoate -  of  c e l l metabolism.  conse-  It i s u n l i k e l y  a c t s i n d i r e c t l y by i n h i b i t i n g p a r t o f t h e  TCA  c y c l e s i n c e m a l o n a t e does not cause a r e d u c t i o n in p h e n o l i c a c i d production  (personal observations).  The  reduction in iron uptake  c a p a c i t y as a c o n s e q u e n c e o f the r e p r e s s i o n o f p h e n o l i c a c i d p r o d u c t i o n by 3 ~ f l u o r o b e n z o a t e p h e n o l i c a c i d s and  again p o i n t s to the r e l a t i o n s h i p between  i r o n t r a n s p o r t i n j3_. s u b t i l i s .  113  5 •  F i g u r e kO.  10  15 20 MINUTES  25  30  »  E f f e c t o f g r o w t h w i t h 1 mM take capacity  3 f 1 u o r o b e n z o i c a c i d on _  o f B_. s u b t i 1 i s B1471  grown u n d e r i r o n  iron  deficient  cond i t ions. Symbols: 0-—0,  no a d d i t i o n s ;  a c i d added at the  time of  8—6,  1 mM  inoculation.  up-  3-fluorobenzoic  GENERAL D I S C U S S I O N  It a p p e a r s t h a t c h e l a t i n g a g e n t s a c t e i t h e r d i r e c t l y o r i n d i r e c t l y on t h e metabolism o f t h e b a c t e r i a l c e l l , depending the c h e l a t i n g agent i t s e l f .  M_. l y s o d e i k t i c u s shows s e l e c t i v i t y  f o r t h e c h e l a t i n g agent which supports i t s growth. e a r l i e r that t h i s might'be t h e case (39)specificity complex  lies  I t was s u g g e s t e d  Whether t h i s degree o f  i n t h e s i z e , shape o r s t a b i l i t y o f t h e c h e l a t e  i s unclear.  in t h e i n i t i a l  upon  P o s s i b l y s i z e and shape p l a y an i m p o r t a n t r o l e  state of contact  the m i c r o o r g a n i s m .  o f t h e m e t a l - c h e l a t e complex  with  I t m i g h t be e x p e c t e d , t h e n , t h a t t h e mode o f  ,j„ | |  r e l e a s e o f t h e Fe  i o n from t h e complex would d i f f e r  depending  on t h e c h e l a t i n g a g e n t a n d h e n e a g a i n a d e g r e e o f s p e c i f i c i t y w o u l d exi s t . In M_. l y s o d e i k t i c u s , EDTA a n d p o s s i b l y NTA a p p e a r t o a c t i n d i r e c t l y on t h e c e l l  by r e m o v i n g t o x i c c a t i o n s , s i n c e EDTA d o e s  n o t s e r v e a s an i r o n t r a n s p o r t  factor.  However,  desferriferrichrome  a n d DHBG b o t h s e r v e e f f e c t i v e l y a s i r o n t r a n s p o r t f a c t o r s .  The  m e c h a n i s m o f i r o n u p t a k e b y M_. l y s o d e i k t i c u s s t i l l  unclear.  remains  It w i l l be n e c e s s a r y t o u s e l a b e l l e d c h e l a t i n g a g e n t s t o d e t e r m i n e whether o r not iron enters ion.  t h e c e l l as a complex, o r as a " f r e e  In B_. s u b t i 1 i s a n d S_. t y p h i m u r i u r n a l s o , DHBG a n d d e s f e r r i -  ferrichrome  appear t o serve as iron transport  EDTA makes i r o n u n a v a i l a b l e  to the cells.  factors,  whereas  A l b o m y c i n , an i r o n -  c o n t a i n i n g a n t i b i o t i c which  i n h i b i t s g r o w t h o f b o t h M^. l y s o d e i k t i -  c u s a n d B_. s u b t ? 1 i s a n d r e d u c e s t h e q u a n t i t y o f i r o n t a k e n up b y t h e former organism, has a s t r u c t u r e s i m i l a r t o that o f f e r r i c h r o m e ( F i g u r e kl).  The Fe(DHBG)^ and t h e Fe(EDTA) complexes  i l l u s t r a t e d f o r comparative purposes (Figure  are also  kl).  T h e m e c h a n i s m o f i r o n u p t a k e i s s i m i l a r i n B^. s u b t i 1 i s a n d S_. t y p h i m u r i u m , b u t d i f f e r e n t  i n M. l y s o d e i k t i c u s .  In t h e t w o  f o r m e r o r g a n i s m s , t h e u p t a k e s y s t e m i s r e p r e s s e d by g r o w t h presence o f high levels o f iron.  :  in the  M_. l y s o d e i k t i c u s h a s a n i n d u c i b l e  u p t a k e mechanism, w i t h e i t h e r a c h e l a t i n g agent o r an i ron.-chelate complex s e r v i n g as the inducer.  E_. c o l ? h a s a r e p r e s s i b l e  s y s t e m s i m i l a r t o t h o s e o f B_. s u b t i 1 i s a n d S_. t y p h i m u r i u m  uptake (68) .  H o w e v e r , t h e s e s y s t e m s hsow a d i f f e r e n t r e s p o n s e t o c h r o m i u m  ions.  The s y s t e m s examined h e r e a l l i n v o l v e a t l e a s t two s t e p s : a r a p i d , energy-independent b i n d i n g o f iron t o t h e c e l l and a s l o w e r , energydependent  uptake  cf i r o n .  T h e s e c o n d s t e p i s i n h i b i t e d by s u l f -  h y d r y l r e a g e n t s , s u g g e s t i n g t h a t p r o t e i n ( s ) may b e i n v o l v e d a t this state, similar t o the sulfate-binding protein involved in s u l f a t e u p t a k e i n S^. t y p h i m u r i u m ( 1 9 ) . In S_. t y p h i m u r i u m , DHBG s t i m u l a t e s i r o n u p t a k e by b o t h t h e w i l d type s t r a i n and one o f t h e mutants.  DHBS s u p p o r t s g r o w t h o f  t h e s e m u t a n t s o n medium c o n t a i n i n g c i t r a t e ( P o l l a c k , " p e r s o n a l communication).  C i t r a t e appeared t o i n h i b i t growth o f t h e mutants.  J.R. P o l l a c k , D e p t . o f B i o c h e m i s t r y , U n i v e r s i t y o f C a l i f o r n i a , Berkeley.  S i n c e b o t h c i t r a t e and  DHBS complex? i r o n , i t a p p e a r s t h a t i n t h e  Fe c i t r a t e c o m p l e x t h e  i r o n becomes u n a v a i l a b l e , whereas in the  FeCDHBS)^ complex, the  i r o n i s r e a d i l y taken  C o n t r o l o f DHB(G) f o r m a t i o n at  by  i n t o the  cells.  iron involves repression  l e a s t one o f t h e e n z y m e s t a k i n g p a r t i n i t s b i o s y n t h e s i s  u r e 43).  S i n c e DHB  of (Fig-  s y n t h e s i s appears to involve a multienzyme  complex, i t i s p o s s i b l e t h a t none o f the enzymes in t h i s pathway a r e f o r m e d when an o r g a n i s m i s g r o w n i n t h e p r e s e n c e o f i r o n . Iron  i s t h o u g h t t o a c t as a c o - r e p r e s s o r  o:f p r o t e i n s y n t h e s i s . volved  o r e v e n as an  (43)  inducer  However, i r o n i s not the o n l y f a c t o r i n -  i n t h e r e g u l a t i o n o f DHB  synthesis.  a c i d c y c l e a l s o a p p e a r s t o be n e c e s s a r y  An o p e r a t i v e  citric  f o r the s y n t h e s i s of t h i s  compound. —•  subt1 l i s p r o b a b l y  production, with The  possesses  an o p e r o n c o n t r o l l i n g (DHB(G)  i r o n s e r v i n g as a c o - r e p r e s s o r  e x i s t e n c e o f an o p e r o n i n t h e DHBS s y s t e m o f SL  i s s o o n t o be p u b l i s h e d  ( P o l l a c k , p e r s . comm.).  t h a t work w i t h d e f i n e d c o n t r o l mutants w i l l t h a t DHB(G) and iron  of t h i s system.  related phenolics  play  typhimurium  I t i s t o be  soon c l a r i f y the  hoped role  in the a c t i v e t r a n s p o r t  of  (and p o s s i b l y o t h e r m e t a l s ) i n t o the b a c t e r i a l c e l l .  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