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 ASPECTS OF PHENOLIC A C I D AND IRON METABOLISM IN SELECTED B A C T E R I A L STRAINS by BARRY L. WALSH B . S c . ( A g r . ) i n M i c r o b i o l o g y M c G i l l U n i v e r s i t y , 1967 A T H E S I S SUBMITTED IN P A R T I A L FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE i n t h e D e p a r t m e n t o f M i c r o b i o l o g y We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE U N I V E R S I T Y OF B R I T I S H COLUMBIA S e p t e m b e r , 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r 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 a t 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 and s t u d y . I f u r t h e r a g r e e t h a p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada i i ABSTRACT B a c i l l u s s u b t i l i s and M i c r o c o c c u s l y s o d e i k t i c u s grew p o o r l y when s u b j e c t e d t o c o n d i t i o n s o f l i m i t i n g i r o n n u t r i t i o n a f t e r i n o c u l a w e re w a s h e d . Gro w t h o f b o t h o r g a n i s m s was s t i m u l a t e d by f e r r i -c hrome and v a r i o u s p h e n o l i c a c i d s . The c h e l a t i n g a g e n t s 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 and n i t r i l o t r i a c e t i c a c i d s t i m u l a t e d g r o w t h o f JB; s u b t i 1 i s b u t n o t o f -M.. l y s o d e i k t i c u s . The e f f e c t o f t h e s e compounds 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 p e p t i d e 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 c o n d i t i o n s . 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 j l i s requ? r e d r e l a t i v e l y h i g h l e v e l s o f a c a r b o n s o u r c e , w i t h g l u c o s e s e r v i n g a s t h e most e f f e c t i v e s u b s t r a t e o f t h o s e t e s t e d . B_. s u b t i 1 i s WB7*t6 p r o d u c e d o n l y 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 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 a c i d e a r l y i n l o g p h a s e and 2 , 3~dihydroxy-b e n z o y l g l y c i n e i n t h e l a t e l o g and e a r l y s t a t i o n a r y p h a s e s o f g r o w t h , 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 ! M u t a n t s t r a i n s o f B_. s u b t ? 1 ?s p r o d u c e d p h e n o l i c a c i d s i n t h e a b s e n c e o r p r e s e n c e o f i r o n i n t h e g r o w t h medium. DHB s y n t h e s i z i n g enzymes were 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 i n t h e p r e s e n c e o f i r o n , f e r r i c h r o m e o r t h e a r o m a t i c a m i n o a c i d s . A c t i v e 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 n o t a f f e c t e d by t h e s e compounds. T h e DHB 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 a r t i a l l y 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 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 ; t h e r e s u l t s u g g e s t e d t h e e x i s t e n c e o f a m u l t i e n z y m e c o m p l e x . I r o n u p t a k e by B_. s u b t i 1 i s and t y p h i m u r i um was shown t o be e n e r g y - d e p e n d e n t and r e p r e s s i b l e by g r o w t h i n t h e p r e s e n c e o f a d e q u a t e i r o n . T he i r o n 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 a p p e a r e d 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 . F e r r i c h r o m e and d i h y d r o x y b e n z o i c a c i d a p p e a r e d 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 o r g a n i s m s , w h e r e a s 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 | d i n h i b i t e d i r o n u p t a k e . 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 l i t t l e e f f e c t on g r o w t h , b u t d i d r e d u c e b o t h p h e n o l i c a c i d p r o d u c t i o n b y , and 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 s u b t i l i s . TABLE OF CONTENTS Page INTRODUCTION 1 MATERIALS AND METHODS . . . . 5 I. B a c t e r i a l S t r a i n s 5 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 m u t a n t s 6 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. W i l d t y p e ' S_. t y p h i m u r i u m 6 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 . . . . . . . . 6 I I . M e d i a and C u l t u r e C o n d i t i o n s 8 1 . G e n e r a l 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 I I I . D e t e r m i n a t i o n 10 1. Growt h 10 2. P h e n o l i c a c i d s 10 a) DHB(G) 1^0 b) O t h e r p h e n o l i c s 10 c) C h r o m a t o g r a p h y 11 i ) P a p e r c h r o m a t o g r a p h y 11 V T a b l e o f C o n t e n t s ( C o n t i n u e d ) Page i i ) T h i n l a y e r c h r o m a t o g r a p h y . . . . . . .11 3. C o p r o p o r p h y r i n I I I 12 k. P r o t e i n \k IV. A n t a g o n i s m S t u d i e s . . 1 . L i qu i d med i um \k 2. S o l i d medi um \k V. P r e p a r a t i o n s o f C e l l s and C e l l - f r e e E x t r a c t s . . . . .15 VI . E n z y m e ' A s s a y . S y n t h e s i s o f DHB 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 c o l u m n c h r o m a t o g r a p h y . . . . . .17 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 18 V I I I . I r o n T r a n s p o r t ' S t u d i e s 18 1 . I r o n u p t a k e 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 22 I. G r o w t h 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 . . .22 I n t r o d u c t i o n 22 1. M. l y s o d e i k t i c u s .22 v i T a b l e o f C o n t e n t s ( C o n t i n u e d ) Page a) Growth 23 b) Growth 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 and a l b o m y c i n a n t a g o n i s m . . . 30 D i s c u s s i o n o f R e s u l t s 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 I n t r o d u c t i o n 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) Changes i n pH d u r i n g g r o w t h 38 c) T i m e 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 D i s c u s s i o n o f R e s u l t s 2^ I I I . E f f e c t o f A n a l o g s and 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 k(> I n t r o d u c t i o n . . 6^ v i i T a b l e o f C o n t e n t s ( C o n t i n u e d ) 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 . . . . p r o d u c t i o n by g r o w i n g c e l l s 47 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 2. 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 D i s c u s s i o n o f R e s u l t s 55 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 I n t r o d u c t i o n 58 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 . . . . . 59 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 . 5 9 D i s c u s s i o n o f R e s u l t s 61 V. I r o n T r a n s p o r t 61 I n t r o d u c t i o n 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 ) I r o n u p t a k e 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 a g e 64 v i i i T a b l e o f C o n t e n t s ( C o n t i nued) Page i i ) 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 6k i i i ) E f f e c t o f g l u c o s e 68 i v ) I r o n u p t a k e as a f u n c t i o n o f membrane. i n t e g r i t y 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 68 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 i ) E f f e c t on u p t a k e c a p a c i t y 71 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 l e v e l s o f . 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 o n . i r o n u p t a k e c a p a c i t y 75 d) I r o n u p t a k e as a f u n c t i o n o f c h e l a t i n g a g e n t s . . 75 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 and EDTA 75 i i ) E f f e c t o f v a r y i n g c o n c e n t r a t i o n s o f DHBG 78 2. B. s u b t i 1 i s m u t a n t s t r a i n s 78 a) S h i k i m i c a c i d r e q u i r i n g m u t a n t 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 i x T a b l e o f C o n t e n t s ( C o n t i n u e d ) 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 ) I r o n r e s i s t a n t m u t a n t s 83 g) Hemin and I r o n r e s i s t a n t m u t a n t 84 D i s c u s s i o n o f R e s u l t s 84 3. M_. l y s o d e i k t l c u s 87 a) P r e l i m i n a r y e x p e r i m e n t s 87 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 . . . . on u p t a k e c a p a c i t y 87 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 a g e n t s . . . . on u p t a k e c a p a c i t y . 88 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 i r o n u p t a k e \n_ v i t r o 88 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 95 D i s c u s s i o n o f R e s u l t s 95 4. S_. t y p h i m u r i u r n s t r a i n LT-2 97 a) I r o n u p t a k e 97 i ) G r o w t h 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 . . 97 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 . . . i r o n u p t a k e 97 b) E f f e c t o f i r o n - c h e l a t i n g a g e n t s . . . . 99 c) S p e c i f i c i t y o f t h e s y s t e m 99 X T a b l e o f C o n t e n t s ( C o n t i n u e d ) Page i i ) G r o w t h u n d e r 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 . 101 b) I r o n e f f l u x 101 5. 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 on u p t a k e 104 D i s c u s s i o n o f R e s u l t s 104 V I . 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 109 I n t r o d u c t i o n . 1 0 9 1. G r o w t h . . 109 2. P h e n o l i c a c i d p r o d u c t i o n 109 3. I r o n u p t a k e 109 D i s c u s s i o n o f R e s u l t s . 112 GENERAL DISCUSSION 114 L I S T OF REFERENCES 120 X I L I S T OF FIGURES Page F i g u r e 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_. l y s b d e i k t i c u s 2k F i g u r e 2. E f f e c t o f v a r i o u s compounds on g r o w t h o f M_. l y s o d e i k t i c u s 2k F i g u r e 3. E f f e c t o f v a r i o u s compounds on g r o w t h o f B_. s u b t ? 1 i s 31 F i g u r e 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 and 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 39 F i g u r e 5. T h i n l a y e r c h r o m a t o g r a p h y o f e x t r a c t s o f c u l t u r e s u p e r n a t a n t s f r o m B. s u b t i1 i s kl F i g u r e 6. G r o w t h and p h e n o l i c a c i d p r o d u c t i o n by mut a n t s t r a i n s o f B. s u b t i 1 i s BU71 k3 F i g u r e 7. G r o w t h and p h e n o l i c a c i d p r o d u c t i o n by mut a n t s t r a i n s o f B. s u b t i 1 i s WB746 k3 F i g u r e 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 t o 2 , 3 - d i h y d r o x y 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 o f B_. s u b t ? l i s WB746 kS F i g u r e 9. 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 2,3-d 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 p r o t e i n c o n c e n t r a t i o n 50 F i g u r e 10. U l t r a v i o l e t s p e c t r a o f s u b s t r a t e , p r o d u c t 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 o f c h o r i s m i c a c i d t o 2 , 3 - d i h y d r o x y 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 o f B_. s u b t i 1 i s WB746 51 F i g u r e 11. 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 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 B. s u b t i l i s WB746 60 x i i L i s t o f F i g u r e s ( C o n t i n u e d ) Page F i g u r e 12. 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 DHB-s y n t h e t a s e a c t i v i t y i n a c r u d e e x t r a c t o f i - s u b t i l i s WB746 62 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 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 a ge and p h e n o l i c a c i d p r o d u c t i o n 66 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 B_. s u b t i 1 i s B1471 67 F i g u r e 15. 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 F i g u r e 16. E f f e c t o f l i p o l y t i c compounds on i r o n u p t a k e by 13. s u b t i 1 i s B1471 70 F i g u r e 17. 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 l i s B1471 72 F i g u r e 18. R e p r e s s i o n o f 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 B1471 by g r o w t h w i t h a d d e d i r o n 73 F i g u r e 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 by B_. s u b t i 1 i s B1471 74 F i g u r e 20. I r o n u p t a k e by B_. s u b t i 1 i s B1471 grown w i t h d i f f e r e n t l e v e l s o f i r o n i n e x t r a c t e d medium 76 F i g u r e 21. 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 F i g u r e 22. 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 F i g u r e 23. 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 EL s u b t i 1 ? s l471y 80 F i g u r e 24. 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 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 o f H. 1 y s b d e i k t ? c u s 89 F i g u r e 26. Iron 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 w i t h o u t c i t r a t e 90 F i g u r e 27. 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 M_. l y s o d e i k t i c u s 91 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 on i r o n u p t a k e by M_. l y s b d e i k t i c u s 92 F i g u r e 29. E f f e c t o f p h e n o l i c compounds 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 93 F i g u r e 30. E f f e c t o f v a r y i n g 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 on i r o n u p t a k e by M. l y s b d e i k t i c u s Sk F i g u r e 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 F i g u r e 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 i r o n u p t a k e by S_. t y p h i murium LT-2 98 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 on i r o n u p t a k e by S_. t y p h i r i i u r i urn LT-2 100 F i g u r e 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 murium LT-2 by 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 i r o n u p t a k e 102 F i g u r e 35. 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 LT-2 a n d m u t a n t s t r a i n s 105 F i g u r e 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 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 - 1 10.6. F i g u r e 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 u p t a k e by S_. t y p h i m u r i u m mutant f e r D19 107 x i v L i s t o f F i g u r e s ( C o n t i n u e d ) F i g u r e 38. F i g u r e 39-F i g u r e 40. F i g u r e 41. F i g u r e 42. F i g u r e 43. 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 o f B_. s u b t i l i s B1471 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 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 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 a l b o m y c i n and f e r r i c h r o m e 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 F e ( E D T A ) c o m p l e x e s I n t e r m e d i a t e s i n t h e b i o s y n t h e s i s o f 2 , 3 " d i h y d r o x y b e n z o i c a c i d Page 110 111 113 116 117 119 XV L I S T OF TABLES T a b l e I. T a b l e I I . T a b l e I I I . T a b l e IV. T a b l e V. T a b l e V I . T a b l e V I I . T a b l e V I I I . T a b l e IX. T a b l e X. T a b l e X I . T a b l e X I I . T a b l e X I I I . Page D e s c r i p t i o n o f b a c t e r i a l s t r a i n s 7 R f 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 g r o w t h o f M_. l y s b d e i k t i c u s 26 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 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 t h e p a p e r s t r i p method 28 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 B_. s u b t i 1 i s 31 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 B_. s u b t i 1 i s 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 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 IB. s u b t i 1 i s 37 E f f e c t o f DHB a n a l o g 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 48 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 by 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 compounds 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 f r o m B_. s u b t i 1 ?s by v a r i o u s compounds 54 A t t e m p t e d r e s t o r a t i o n o f D H B - s y n t h e t a s e 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 grown i n t h e p r e s e n c e o f i r o n 56 x v i L i s t o f T a b l e s ( C o n t i n u e d ) Page T a b l e X I V . 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 B. s u b t i 1 i s B1471 65 T a b l e XV. 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 8 2 T a b l e X V I . 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 x v i i GLOSSARY OF ABBREVIATIONS C o p r o p o r p h y r i n I I I Cop I I I 2 , 3 - d i h y d r o x y b e n z o i c a c i d DHB 2 , 3 - d i h y d r o x y b e n z o y l g l y c i n e ( i t o i c a c i d ) DHBG 2 , 3 - d i h y d r o x y b e n z o y l s e r i n e DHBS 2 - N , 6 - N - D i - ( 2 , 3 - d i h y d r o x y b e n z o y l ) - L - l y s i n e DHBL 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 n i t r i l o t r i a c e t i c a c i d 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 r o n A l b o m y c i n A l b F e r r i c h r o m e F c 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 and a d e q u a t e a e r a t i o n , B a c i l l u s s u b t i l i s a l t e r s i t s m e t a b o l i s m i n s u c h a way 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 (45). The f o r m e r h a v e been 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 com-pounds ( D H B ( G ) ) , d e p e n d i n g 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 c o n s i d e r a t i o n . U n d e r 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 p r o d u c e s (DHBS) (1), A e r o b a c t e r a e r o g e n e s p r o d u c e s DHB (49, 72) and A z o t o - b a c t e r v i n e l a n d i i p r o d u c e s b o t h DHB and DHBL. (12). The p o r p h y r i n 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 I I I (33). In B_. s u b t i l i s , an 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 tween 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 and t h e l e v e l o f DHB(G) p r o d u c e d by g r o w i n g c e l l s (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 normal 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 i n t h e a c c u m u l a t i o n i n 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 III w h i c h i s r a p i d l y o x i d i z e d t o Cop III (33). Compounds o t h e r t h a n i r o n ( o r t h o s e c o n t a i n i n g i r o n , s u c h as f e r r i c h r o m e ) a f f e c t DHB(G) 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 . Of t h e s e , t h e most p o t e n t i n h i b i t o r s o f 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 m e t a - s u b s t i t u t e d d e r i v a t i v e s o f b e n z o i c a c i d (44). Compounds w h i c h i n h i b i t n ormal f u n c t i o n i n g o f t h e TCA c y c l e c a u s e d e c r e a s e d Cop 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 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 (44). 2 T h e m e t a b o l i c a l t e r a t i o n ( s ) i n t h e a r o m a t i c pathway c a u s e d by 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 have n o t been f u l l y c h a r a c t e r i z e d . W o r k i n g w i t h A. a e r o g e n e s , Young and c o - w o r k e r s (72) h a v e shown t h a t DHB i s s y n t h e s i z e d by way o f a t l e a s t two i n t e r m e d i a t e s (73, 74) 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 b i o -s y n t h e t i c p a t h way o f a r o m a t i c compounds. B r o t , G o o d w i n and F a l e s (1) 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 ? grown 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 a r e c a p a b l e o f c o n v e r t i n g DHB t o DHBS. P e t e r s (44) p r e s e n t e d p r e l i m i n a r y e v i d e n c e f o r t h e i n v i t r o f o r m a t i o n o f DHB(G) by e x t r a c t s o f EL s u b t i l i s . 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 t h e i n v i t r o f o r m a t i o n o f DHB(G) was c a r r i e d o u t i n t h e p r e s e n t s t u d y . T h e e v i d e n c e t h a t i r o n a p p e a r s t o c o n t r o l p h e n o l i c a c i d p r o -d u c t i o n (2, 45) and t h e h y p o t h e s i s t h a t p h e n o l i c a c i d s , w h i c h a r e s t r o n g i r o n - b i n d i n g a g e n t s , 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 a g e n t s f o r t h e b e n e f i t o f t h e o r g a n i s m s p r o d u c i n g them (39), l e d t o a s t u d y o f i r o n u p t a k e , and t h e r o l e p l a y e d by i r o n - b i n d i n g a g e n t s i n i r o n u p t a k e , w i t h Q_. s u b t i 1 i s. F u r t h e r m o r e , s i n c e v a r i o u s c h e l a t i n g compounds 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 l y s o d e i k t i c u s , i r o n u p t a k e was i n v e s t i g a t e d i n t h i s o r g a n i s m . In a d d i t i o n , s i n c e t h e p e p t i d e a n t i b i o t i c a l b o m y c i n i s t h o u g h t t o a c t by b l o c k i n g t h e i n s e r t i o n o f i r o n i n t o t h e p r o t o p o r p h y r i n r i n g (75) o r by i n t e r -f e r i n g w i t h t h e t r a n s f e r o f m e t a b o l i c p r e c u r s o r s t h r o u g h b a c t e r i a l membranes (65, 66) , i t was 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 r o l e o f 3 v a r i o u s i r o n - s e q u e s t e r i n g a g e n t s 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 -m y c i n , and a s a g e n t s i n v o l v e d 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 and M_. l y s o d e i k t i c u s . As t h e work on i r o n u p t a k e w i t h t h e two o r g a n i s m s m e n t i o n e d 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  t y p h i m u r i u m r e q u i r i n g i r o n a n d / o r DHB as g r o w t h f a c t o r s was b r o u g h t t o my a t t e n t i o n ( 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 ) . As a c o n -s e q u e n c e o f t h i s , i r o n u p t a k e s t u d i e s w ere p e r f o r m e d w i t h t y p h i m u r i u m and a r e i n c l u d e d i n t h i s t h e s i s . T h e l i t e r a t u r e c o n t a i n s few r e p o r t s on c a t i o n u p t a k e by m i c r o o r g a n i s m s and u n t i l v e r y r e c e n t l y t h e m a j o r i t y o f t h o s e p u b l i s h -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 by 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 by Conway and c o - w o r k e r s (9, 10, 11) and by t h e R o t h s t e i n g r o u p (21, 52), i n d i c a t e d t h a t M g + + , C o + + , Z n + + , M n + + a n d ! N i + + 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 s y s t e m s . The 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 an e q u i v a -l e n t s e c r e t i o n o f H + i o n s i n y e a s t (9), i n S t r e p t o c o c c u s f a e c a l i s (77), and i n E^ . c o l i (54) l e d t o t h e h y p o t h e s i s t h a t i o n a c c u m u l a t i o n 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). W h e t h e r t h i s e x c h a n g e i s 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 e n e r g y d e p e n d e n t d e p e n d s l a r g e l y 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 and on t h e whim o f t h e i n v e s t i g a t o r . T h e r e i s e v i d e n c e 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 enzyme 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). T h e u p t a k e o f M n + + and M g + + was i n h i b i t e d by o t h e r c a t i o n s i n Gram-p o s i t i v e b u t n o t i n G r a m - n e g a t i v e s p e c i e s (Webb, 69, 70). T u n g s t a t e 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 1i'but d i d n o t i n h i b i t i t s i n t r a c e l l u l a r f u n c t i o n ; F e + + a l s o i n h i b i t e d M o + + u p t a k e (3, 30, 31). T h r e e v e r y r e c e n t p a p e r s 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 s e p -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 + + (58) and f o r M g + + (37, 57). In a d d i t i o n , e v i d e n c e f o r an a c t i v e i r o n u p t a k 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 + + + i o n s i n t e r f e r e has j u s t been p u b l i s h e d (67, 68). P r e l i m i n a r y e v i d e n c e s u g g e s t e d t h e e x i s t e n c e o f an 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). T h i s work has been e x t e n d e d i n t h e p r e s e n t s t u d y . In a d d i t i o n , i r o n u p t a k e has been e x a m i n e d i n o t h e r b a c t e r i a l s p e c i e s i n an a t t e m p t t o g a i n an u n d e r s t a n d i n g o f i r o n u p t a k e p r o c e s s e s i n b a c t e r i a . 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 f r o m Dr. J . B . N e i l a n d s , U n i v e r s i t y o f C a l i f o r n i a a t B e r k e l e y . S t r a i n WB746 (a d e r i v a t i v e o f t h e M a r b u r g s t r a i n ) was o b t a i n e d f r o m Dr. E.W. N e s t e r , U n i v e r s i t y o f W a s h i n g t o n , S e a t t l e . 2. M u t a n t S t r a i n s o f B_. s u b t i 1 i s M u t a n t s t r a i n s w ere s e l e c t e d as s p o n t a n e o u s 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 d e r i v a t i v e s , (a) S p o n t a n e o u s M u t a n t s S t r a i n s r e s i s t a n t t o low (2 mM) o r h i g h (100 mM) l e v e l s o f F e + + + , C o + + , h e m i n , a l b o m y c i n o r s t r e p t o m y c i n were o b t a i n e d by s e r i a l t r a n s f e r o f t h e w i l d t y p e s t r a i n i n m i n i m a l medium c o n t a i n i n g i n c r e a s i n g c o n c e n t r a t i o n s o f t h e compounds. The f i n a l c u l t u r e s w e r e d i l u t e d and s p r e a d on m i n i m a l a g a r p l a t e s and s i n g l e c o l o n i e s w ere p i c k e d and t e s t e d f o r r e s i s t a n c e t o t h e d e s i r e d compound. R e s i s t a n t c o l o n i e s w ere s t r e a k e d on m i n i m a l medium and s i n g l e c o l o n i e s p i c k e d as r e s i s t a n t s t r a i n s . 6 (b) NNG-Induced M u t a n t s The method o f L o r e i i c e and N e s t e r (35) was f o l l o w e d t o o b t a i n N N G - i n d u c e d m u t a n t s a u x o t r o p h i c f o r a r o m a t i c a m i n o a c i d s o r t h e i r b i o s y n t h e t i c p r e c u r s o r s . A l l 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 i n c u b a t i o n a t 37 C. 3. 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 f r o m Dr. M.R.J. S a l t o n , 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 . I t was s t o r e d a t k C on T S Y - a g a r s l a n t s and t r a n s f e r r e d m o n t h l y . h. 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 f r o m Dr. B.N. Ames, U n i v e r s i t y o f C a l i f o r n i a a t B e r k e l e y . I t was s t o r e d a t k C on T S Y - a g a r s l a n t s and t r a n s f e r r e d m o n t h l y . 5. 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 o b t a i n e d f r o m Dr. Ames. The p r o p e r t i e s o f a l l b a c t e r i a l s t r a i n s u s e d a r e o u t l i n e d i n T a b l e 1. 7 T a b l e 1. D e s c r i p t i o n o f B a c t e r i a l S t r a i n s P a r e n t O r g a n i s m M u t a n t S t r a i n C h a r a c t e r i s t i c s S o u r c e j3. s u b t ? 1 i s B1471 B_. s u b t i 1 i s WB746 I471y H R1 Fe J i— R UR Fe H Co 123 R A 5 & A 9 Fe R R H r e q u i r e s s h i k i m i c a c i d f o r g r o w t h r e s i s t a n t t o 1 mM hemin r e s i s t a n t t o 100 mM F e C l 3 r e s i s t a n t t o 1 mM hemin and 100 mM F e C l 3 r e s i s t a n t t o 100 mM A l b o m y c i n r e s i s t a n t t o 100 mM C o C l 2 r e s i s t a n t t o 50 ug/ml S t r e p t o m y c i n r e s i s t a n t t o 100 mM F e C l 3 r e s i s t a n t t o 1 mM hemi n p r e s e n t r e s e a r c h t y p h i m u r i u m LT-2 f e r - 1 r e q u i r e s Fe f o r g r o w t h f e r - 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 DHB f o r g r o w t h f e r D9 r e q u i r e s Fe o r DHB f o r g r o w t h Dr. 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 . G e n e r a l B a c t e r i a w e r e grown 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 l a s k s f i t t e d w i t h s i d e a r m s . Medium ( 1 0 % by v o l u m e o f f l a s k c a p a c i t y ) r e c e i v e d a 0.5% 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 h o u r i n t h e c a s e o f Mu 1 y s o d e ? k t i c u s ) g r o w i n g i n T r y p t i c a s e - S o y -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 . F l a s k s were s h a k e n a t 250 rpm i n a model G77 M e t a b o l y t e W a t e r B a t h (New B r u n s w i c k S c i e n t i f i c Co., New B r u n s w i c k , New J e r s e y ) . A l l g l a s s w are was a u t o c l a v e d t w i c e i n g l a s s - d i s t i l l e d w a t e r p r i o r t o u s e t o e l i m i n a t e r e s i d u a l i r o n . A l l m e d i a were made up w i t h g l a s s - d i s t i l l e d w a t e r . S u g a r s o l u t i o n s were s t e r i l i z e d s e p -a r a t e l y as c o n c e n t r a t e d s o l u t i o n s (20 - 50%) i n g l a s s - d i s t i l l e d w a t e r . T h e f o l l o w i n g compounds were s t e r i l i z e d as s o l u t i o n s by p a s s a g e 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 ( M i l l i p o r e C o r p . , Bed-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, DHBG, f e r r i c h r o m e , hemin a n t h r a n i l i c a c i d , 3 ~ h y d r o x y a n t h r a n i 1 i c a c i d , 3 - h y d r o x y b e n z o i c a c i d , s h i k i m i c a c i d , s a l i c y l a m i d e , N - h y d r o x y m e t h y l s a l i c y l a m i d e . 2. Bac?11 us s u b t i 1 i s T h e m e d i a u s e d f o r g r o w t h 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 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 i n i m a l medium 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; N e u r o -s p o r a t r a c e e l e m e n t s o l u t i o n o f V o g e l (63), 0.01% by v o l u m e . T h e medium was a d j u s t e d t o pH 7-4 w i t h 5N NaOH, u s i n g a F i s h e r A c c u m e t Model 220 pH m e t e r ( F i s h e r S c i e n t i f i c Co.) and s t e r i l i z e d 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 . S t e r i l e 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 t h e d e s i r e d c o n c e n t r a t i o n ( r o u t i n e l y \ % ) . 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 ( s e e b e l o w ) were p r e p a r e d a c c o r d i n g t o S p i z i z e n ( 6 2 ) . T h e medium c o n t a i n e d , i n g / l i t e r ; ( N H ^ S O ^ , 2.0; I^HPO^, 14.0; K H 2P0 / (, 6.0; s o d i u m c i t r a t e . 2 H 2 0 , 1.0; M g S O ^ H ^ , 0.2; and g l u c o s e , 5.0. The g l u c o s e was a d d e d s e p a r a t e l y as a s t e r i l e s o l -u t i o n . F u r t h e r a d d i t i o n s w ere made i m m e d i a t e l y p r i o r t o t h e p o u r i n g o f p l a t e s . C h e m i c a l l y e x t r a c t e d medium was p r e p a r e d by t h e method o f V o g e l (63) u s i n g 8 - h y d r o x y q u i n o l i n e . 3. M i c r o c o c c u s l y s o d e i k t i c u s 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 t h a t o f S a l t o n (53) and c o n t a i n e d , i n g / l i t e r : K H^O^, 6.8; M g S O ^ H ^ , 0.1; N H ^ C l , 1.0; s o d i u m g l u t a m a t e , 10.0; NH^ a c e t a t e , 10,0; M n C l 2 . 4 H 2 0 , 0.002; and B i o t i n , 0.0005. T h e medium was a d j u s t e d t o pH 7-5 p r i o r t o a u t o c l a v i n g . A g a r (1.5%) was a d d e d t o p r e p a r e m i n i m a l a g a r p l a t e s w here i n d i c a t e d . 4. S a l m o n e l l a t y p h i m u r i u m S_. t y p h i m u r i u m was grown i n t h e b a s a l medium o f V o g e l and B o n n e r (64) w i t h o u t a d d e d t r a c e e l e m e n t s . T h e medium c o n t a i n e d , i n g / l i t e r : ^ H P O ^ , 10.0; 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. The medium was pH 7-0 b e f o r e a u t o c l a v i n g . S t e r i l e 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.. D e t e r m i n a t i o n s 1. G r o w t h 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 w i t h a g r e e n (540 nm) f i l t e r . K l e t t r e a d i n g s were c o n v e r t e d t o v i a b l e c e l l s p e r ml by u s e o f p r e p a r e d s t a n d a r d g c u r v e s . A v i a b l e c e l l d e n s i t y o f 10 c o l o n y f o r m i n g u n i t s ( c f u ) 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 and 32 u n i t s f o r S_. t y p h i m u r i u m . 2. P h e n o l i c A c i d s (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 P e t e r s and W a r r e n ( 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 p r o d u c e d i n c u l t u r e s u p e r n a t a n t s , 500 ml v o l u m e s o f c u l t u r e s grown i n 2 - l i t e r E r l e n m e y e r f l a s k s w i t h o u t f i t t e d s i d e a r m s 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-28 r o t o r o f an IEC model 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 Co., Needham H t s . , M a s s . ) . T h e s u p e r n a t a n t s w e r e a d -j u s t e d t o pH 2 w i t h 6N HC1 and c l a r i f i e d by f u r t h e r c e n t r i -f u g a t i o n . 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 vacuum 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 (Chem-Q u i p Co., 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 , and e x t r a c t e d w i t h 3 v o l u m e s o f e t h y l a c e t a t e . T h e e t h y l a c e t a t e f r a c t i o n was c o n c e n t r a t e d on t h e e v a p o r a t o r t o a b o u t 3 m l . The a q u e o u s 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 , and t h e 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 a b o u t 3 m l . Th e 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 w e r e e x a m i n e d by p a p e r 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 s c a n n e d f o r a b s o r p t i o n o f u l t r a v i o l e t 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 s p e c t r o p h o t o m e t e r ( U n i c a m I n s t r u m e n t s L t d . , C a m b r i d g e , E n g l a n d ) . (c) C h r o m a t o g r a p h y ( i ) P a p e r C h r o m a t o g r a p h y T h e f o l l o w i n g s o l v e n t s y s t e m s were u s e d : 1. n - b u t a n o l : a c e t i c a c i d : w a t e r (k:1:5)(v/v/v). (27). 2. t - b u t a n o l : m e t h y l e t h y l k e t o n e : w a t e r : d i e t h y l a m i n e ( I 0 : 1 0 : 5:l)(v/v/v/v). (27). ( i i ) T h i n L a y e r C h r o m a t o g r a p h y P l a t e s (20 cm x 20 cm) were p r e p a r e d w i t h S i l i c a g e l G ( E . M e r c k and C o . ) . T h e f o l l o w i n g s o l v e n t s y s t e m s were u s e d : 3. 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 ) . 4. same as #2 a b o v e . ( 2 7 ) . 5. b e n z e n e : a c e t i c a c i d ( 2 0 : 5 ) ( v / v ) s a t u r a t e d w i t h w a t e r . ( 1 2 ) . ( i i i ) D e t e c t i o n S y s t e m s T h e f o l l o w i n g d e t e c t i o n s y s t e m s were u s e d f o r b o t h p a p e r and t h i n l a y e r c h r o m a t o g r a p h y : 1. E v a n s r e a g e n t , u s e d t o d e t e c t o - d i h y d r i c p h e n o l s (13). 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 ( 6 0 ) . 3> P o t a s s i u m 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 p o l y h y d r i c -p h e n o l s ( 6 0 ) . 5. F e r r i c c h l o r i d e i n w a t e r : a c e t o n e ( 4 : 1 ) ( v / v ) , t o d e t e c t i r o n b i n d i n g compounds ( 4 8 ) . 6. C h r o m a t b - v v e c a b i n e t ( U l t r a - v i o l e t P r o d u c t s , I n c . , San G a b r i e l , C a l . ) w h i c h a b s o r b u l t r a v i o l e t l i g h t . The R.p v a l u e s and r e a c t i o n s w i t h some o f t h e r e a g e n t s a r e g i v e n i n T a b l e 2 f o r DHB, DHBG and some common a n a l o g s run r o u t i n e l y a s s t a n d a r d s . C o p r o p o r p h y r i n I I I Cop I I I was d e t e r m i n e d by t h e method o f L e s s i e S S i s t r o m 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 ( 5 0 ) . T a b l e 2. R^ . 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 common p h e n o l i c a c i d s i n p a p e r and t h i n - l a y e r c h r o m a t o g r a p h y . ^ R e a c t i o n w i t h : Compound P a p e r * f T L C * * Evan s R e a g e n t F e C l 2 , 3 - d i hyd r o x y b e h z o y 1 g 1 y c i ne 0.42 0 .31 p i n k + 2 , 3 - d i h y d r o x y b e n z o i c a c i d 0.64 0 .48 p i n k + 2 , 4 - d i h y d r o x y b e n z o i c a c i d 0 .49 p i nk + 2 , 5 _ d i h y d r o x y b e n z o i c a c i d 0 .44 p i nk + 2 , 6 - d i h y d r o x y b e h z o i c a c i d 0 .18 p i n k + m - h y d r o x y b e n z o a t e 0 .57 ye 11ow -p - h y d r o x y b e n z o a t e 0 .63 ye 11ow -o - h y d r o x y p h e n y 1 a c e t a t e 0 .55 ye 11ow -o - h y d r o x y h i p p u r a t e 0 .51 p i nk + c a t e c h o l 0 .60 p i n k + s a l i c y l i c a c i d 0 .75 g r e e n + a n t h r a n i 1 i c a c i d 0 .75 ye 11ow -* S o l v e n t s y s t e m : t - b u t a n o l : m e t h y l e t h y 1 k e t o n e : w a t e r : d i e t h y l a m i n e (10 :10 :5 :1)(v/v/v/v) (27) ** S o l v e n t s y s t e m : 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) 14 0. 48 u m o l e s o f Cop I I I p e r ml = 0 . 1 O.D. ( E j JJ M (400 nm) = 0.1) 4. P r o t e i n D e t e r m i n a t i o n P r o t e i n was a s s a y e d a c c o r d i n g t o t h e method o f G r o v e s , D a v i s a n d S e l l s ( 2 6 ) . C r y s t a l l i n e b e e f serum a l b u m i n was u s e d a s a s t a n d a r d . The method gave 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 ( 3 6 ) , u s i n g s t a n d a r d BSA and t h e o r g a n i s m s u n d e r s t u d y and was more e a s i l y a n d r a p i d l y r u n u n d e r r o u t i n e c o n d i t i o n s . I V . A n t a g o n i s m S t u d i e s 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 a n t i -b i o t i c a l b o m y c i n a r e s a i d t o a n t a g o n i z e t h e d r u g , o r t o s e r v e as a l b o m y c i n a n t a g o n i s t s . 1. 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 ere 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 . A t t h e t i m e o f 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 compounds t o be t e s t e d w ere made t o t h e f l a s k s a t t h e d e s i r e d c o n c e n t r a t i o n s . Growth was f o l l o w e d as d e s c r i b e d a b o v e . 2. 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 d e t e r m i n e d e s s e n t i a l l y a c c o r d -i n g t o t h e method o f Z a h n e r , Bachmann, 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 g r o w i n g i n m i n i m a l 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 . S t r i p s o f s t e r i l e 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 , w ere 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 f o r m o f a c r o s s . The p l a t e s w ere t h e n i n c u b a t e d a t 37 C and e x a m i n e d a t i n t e r v a l s . Z o n e s o f g r o w t h and i n h i b i t i o n w ere r e c o r d e d . V. P r e p a r a t i o n s o f C e l l s and 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 were 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 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 SS4 c e n t r i f u g e ( I v a n S o r v a l l I n c . , N o r w a l k , C o n n e c t i c u t ) . T h e c e l l s w e re 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 and 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 wet 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. Ly s o z y m e (100 u g / m l ) , DNase (10 ug/ml) and M g C l 2 (5 x 10 M) were a d d e d and 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. The 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 SSI c e n t r i -f u g e . 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 enzyme a s s a y s . V I . Enzyme A s s a y . S y n t h e s i s o f DHB. T h e f o r m a t i o n o f 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 t h e method o f Y o u n g , Cox a n d G i b s o n (72), w i t h t h e f o l l o w i n g m o d i f i c a t i o n s (a) 0.1 ml o f IN HC1 was u s e d t o t e r m i n a t e t h e enzyme r e a c t i o n . (b) T h e 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 i n t o 2.0 ml o f e t h e r . ( c ) The e t h e r e a l l a y e r was 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 (200 x g_) f o r 3 m i n u t e s i n a GLC-1 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 S o r v a l l I n c . , N o r w a l k , C o n n . ) , t h e n s c a n n e d i m m e d i a t e l y f r o m 300 nm t o 350 nm 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 -p h o t o m e t e r . C a r e was t a k e n t o a d j u s t t h e e t h e r l a y e r t o t h e o r i g i n a l v o l u m e . (d) A r e a c t i o n b l a n k was run f o r e a c h i n d i v i d u a l a s s a y . F o r i d e n t i f i c a t i o n p u r p o s e s , i n a d d i t i o n t o t h e u l t r a v i o l e t s c a n , a p o r t i o n o f t h e e x t r a c t was c h r o m a t o g r a p h e d and compared w i t h a u t h e n t i c DHB ( s e e C h r o m a t o g r a p h y , a b o v e ) . DHB p r o d u c t i o n was q u a n t i t a t e d r o u t i n e l y f r o m a s t a n d a r d c u r v e p r e p a r e d u s i n g c o m m e r c i a l DHB. U n d e r t h e s e c o n d i t i o n s , 0.1 O.D. (318 nm) r e p r e s e n t s 0.075 y m o l e s o f DHB p e r ml ( E ^ 5 y H (3l8 nm) = 0.1). 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 S y n t h e t a s e 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. subt? 1 i s WB746 were 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 M T r i s / H C l b u f f e r , pH 8.1, c o n t a i n i n g 1 mM 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 mM m e r c a p t o -e t h a n o l . T h e s e were 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 a p e r i o d o f s e v e r a l d a y s d u r i n g c o l u m n c h r o m a t o g r a p h y . The c r u d e e x t r a c t was t r e a t e d i n i t i a l l y i n one o f t h r e e ways: e i t h e r (a) a l t e r -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 20,000 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 i c e - b a t h 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 d e s c r i b e d a b o v e ; 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 ( N H ^ ^ S O ^ a t 4 C, w i t h t h e f r a c t i o n p r e c i p i t a t i n g between 30 and 60% 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 a s d e s c r i b e d . In e a c h c a s e , t h e enzyme p r e p a r a t i o n was t h e n d e s a l t e d by p a s s a g e t h r o u g h a 1.0 x 10 cm c o l u m n o f S e p h a d e x G-10. The r e s u l t -i n g 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 P o l y e t h y l e h e g l y c o l 6000, 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 c h r o m a t o g r a p h y o r 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 . 1. D E A E - C e l l u l o s e C h r o m a t o g r a p h y A l l o p e r a t i o n s were p e r f o r m e d a t 4 C. D E A E - c e l 1 u l o s e was p r o c e s s e d and c o l u m n s (2 x 20 cm) w ere p a c k e d a c c o r d i n g t o P e t e r s o n and S o b e r ( 4 7 ) . A maximum o f 2.5 ml o f c r u d e e x t r a c t , o r 45 mg o f p r o t e i n , was a p p l i e d t o t h e column and t h e p r o t e i n was 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 d r o p s w e re c o l l e c t e d i n 16 x 150 mm t e s t t u b e s u s i n g an LKB f r a c t i o n c o l l e c t o r (LKB P r o d u k t e r AB, S t o c k h o l m , Sweden). A f l o w r a t e o f 15 ml p e r h o u r was m a i n t a i n e d . T h e f r a c t i o n s were a s s a y e d f o r enzyme 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 o f e a c h r u n . T h e e f f l u e n t was m o n i t o r e d f o r a b s o r b a n c e a t 280 nm u s i n g a U n i c a m SP800 r e c o r d i n g s p e c t r o p h o t o m e t e r . 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 E x p o n e n t i a l 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 p r e p a r e d 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 S h e e t #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. P r o t e i n e x t r a c t s (0.6 ml) w e re l a y e r e d on t h e t o p s o f Ihe 14.2 ml g r a d i e n t s and t h e t u b e s s p u n f o r 12 h o u r s a t 2 C i n an IEC model 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 p u n c h e d i n t h e b o t t o m o f e a c h t u b e u s i n g a 20 gauge n e e d l e , and 6 - d r o p f r a c t i o n s w ere c o l l e c t e d u s i n g t h e LKB f r a c t i o n c o l l e c t o r , a t a f l o w r a t e o f 15 ml p e r h o u r . The f r a c t i o n s w ere a s s a y e d f o r D H B - s y n t h e t a s e a c t i v i t y as d e s c r i b e d a b o v e . I I. I r o n T r a n s p o r t S t u d i e s 1. I r o n U p t a k e 59 T h e c e l l s w e r e p r e p a r e d , i n c u b a t e d w i t h F e , and a s s a y e d 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 q u e n c h - c u r v e was p r e p a r e d u s i n g Fe o f known s p e c i f i c a c t i v i t y . From t h i s c u r v e , c o u n t s p e r m i n u t e (cpm) w e r e c o n v e r t e d t o p i c o g r a m s (pg) o f i r o n . 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 c e l l s , w a s h i n g 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 t h e t e x t . 2. I r o n E f f l u x C u l t u r e s w ere 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 o f F e . C e l l s w ere 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 t e m p e r a -t u r e (12,000 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 , washed 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 and r e s u s p e n d e d 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 o f t h i s s u s p e n s i o n were d i s t r i b u t e d t o 25 ml E r l e n m e y e r f l a s k s and t h e c u l t u r e s i n c u b a t e d a t 37 C and 100 rpm i n a M e t a b o l y t e s h a k i n g w a t e r b a t h . A f t e r 10 m i n u t e s , a d d i -t i o n s w ere made and 1.0 ml s a m p l e s t h e n 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 . T h e f i l t e r e d c e l l s w e r e washed w i t h 5 ml o f i r o n - d e f i c i e n t medium pre-warmed t o 37 C, t h e n d r i e d u n d e r a 250 w a t t h e a t lamp. T h e d r i e d f i l t e r s 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 c o u n t e d a s b e f o r e . IX. C h e m i c a l s A l l o r g a n i c m e d i a c o n s t i t u e n t s were o f D i f c o b r a n d . I n o r g a n i c c o n s t i t u e n t s w ere o b t a i n e d f r o m 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 by t h e method o f G i b s o n (23) as d e s c r i b e d by P e t e r s (44), and 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) and 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 f r o m Dr. J . B . N e i l a n d s o f t h e U n i v e r s i t y o f C a l i f o r n i a , B e r k e l e y . D e s f e r r i f e r r i -c hrome and 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 and 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 Dr. J . T u r k o v a , C z e c h o s l o v a k Academy o f S c i e n c e , P r a g u e . 5-hydroxy 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 Mr. P a t r i c k C u r l i n g o f t h e D e p a r t m e n t o f M i c r o b i o l o g y , U.B.C. T h e 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 f r o m c o m m e r c i a l 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 Co.; EDTA f r o m A l l i e d C h e m i c a l Co.; t h e a r o m a t i c a m i n o 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 a c i d , 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-hydroxymethy 1 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 a c i d and v a n i l l i c a c i d f r o m J . T . B a k e r C h e m i c a l Co.; s a l i c y l i c a c i d f r o m T h e B r i t i s h Drug H o u s e s ; h e m i n , 3 - h y d r o x y a n t h r a n i 1 i c a c i d , a - i o d o a c e t a m i d e 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 b e n z o h y d r o x -a m i c a c i d s f r o m E a s t m a n O r g a n i c C h e m i c a l s ; c a t e c h o l , a , a - d i p y r i d y 1 , NaN^, 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 a l b u m i n o - h y d r o x y p h e n y l a c e t a t e , s h i k i m i c a c i d , s t r e p t o m y c i n s u l f a t e 59 f r o m Sigma C h e m i c a l Co.; Fe as 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 and N u c l e a r Co.; l y s o z y m e and DNase 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 6000 was o b t a i n e d f r o m J . T . B a k e r C h e m i c a l Co. D E A E - c e l l u l o s e and S e p h a d e x G10 w e r e p r o d u c t s o f P h a r m a c i a C h e m i c a l s , U p p s a l a , Sweden. RESULTS Grow t h 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 n t r o d u c t i o n P e t e r s (44) showed t h a t v a r i o u s p h e n o l i c compounds a n d h y d r o x a m i c a c i d s had 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 p r o -d u c t i o n by s u b t i l l s . 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 compounds t o s u p p o r t t h e g r o w t h o f b a c t e r i a c u l t i v a t e d u n d e r c o n d i t i o n s o f s t r e s s , s i n c e v a r i o u s c h e l a t i n g a g e n t s h a v e been shown t o a c t as " 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, 7 1 ) . M i c r o c o c c u s 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 f e r r i c h r o m e (39) o r c a t e c h o l (53) and o t h e r i r o n - b i n d i n g compounds f o r g r o w t h . 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 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 compounds p l a y a r o l e i n t h e b a s i c m e t a b o l i s m o f t h e o r g a n i s m . S i n c e i t has been shown t h a t f e r r i c h r o m e s e r v e s a s an a n t a g o n i s t t o t h e i n h i b i t o r y a c t i o n o f t h e a n t i b i o t i c a l b o m y c i n (4, 7 6 ) , t h e compounds were a l s o t e s t e d a s p o s s i b l e a n t a g o n i s t s t o t h e a c t i o n o f a l b o m y c i n on J3. s u b t i 1 i s and M^ . l y s o d e i k t i c u s . 1. M i c r o c o c c u s l y s o d e i k t i c u s (a) Growth M_. l y s o d e i k t i c u s grew 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 TSY i n t o S a l t o n ' s m i n i m a l medium. T h e l e n g t h 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 o n c e i n i r o n - d e f i c i e n t m i n i m a l medium. No g r o w t h 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 washed 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 washed t h r e e t i m e s ( F i g -u r e 1 ) . S u b s e q u e n t l y , t h e i n o c u l a w ere 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 l e n g t h s o f t h e l a g p e r i o d s were 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 f r o m g r o w t h c u r v e s d e t e r m i n e d f r o m d u p l i c a t e e x p e r i m e n t s . (b) G r o w t h 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 medium, 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) and 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 f r o m 40 h o u r s t o 10 - 12 h o u r s . T he l e n g t h 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. However, when t h e s e compounds w e r e a d d e d 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 ( F i g u r e 2 ) . Y e a s t e x t r a c t , w h i c h s t i m u l a t e d g r o w t h u s i n g unwashed i n o c u l a , 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, and t o a l e s s e r e x t e n t DHB and c a t e c h o l , F i g u r e 1. E f f e c t o f w a s h i n g i n o c u l u m on 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 medium a t 37 C. S y m b o l s : to 8, u n t r e a t e d i n o c u l u m ; 0 - — 0 , o n c e - w a s h e d i n o c u l u m ; • •, t w i c e - w a s h e d i n o c u l u m ; A A, i n o c u l u m washed t h r e e t i m e s . F i g u r e 2. E f f e c t o f v a r i o u s compounds on 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 medium a t 37 C I n o c u l u m was washed t w i c e . S y m b o l s : • f, no a d d i t i o n s ; 0 0, 0.5% y e a s t e x t r a c t 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 c i d , o r 10 uM EDTA; A A, 10 uM f e r r i c h r o m e . u m I D C i n 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 r e d u c e d t h e l a g p e r i o d . S a l i c y l i c a c i d , on t h e o t h e r h a n d , i n c r e a s 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 t o a s l i g h t e x t e n t ( T a b l e 3). ( 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 From t h e d a t a i n T a b l e s h and 5, i t i s e v i d e n t t h a t f e r r i c h r o m e , DHB and 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 b o t h l i q u i d and s o l i d medium. In l i q u i d medium, n e i t h e r EDTA n o r NTA a l o n e 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 a l b o m y c i n i n h i b i t i o n . W i t h y e a s t e x t r a c t i n t h e l i q u i d medium, b o t h EDTA and NTA w e r e 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 . F e C l ^ was s l i g t h l y s t i m u l a t o r y u n d e r b o t h c o n d i t i o n s o f g r o w t h . Y e a s t e x t r a c t a l o n e 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 s u b t ? 1 i s (a) 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 an e x t e n d e d l a g p e r i o d when a washed i n o c u l u m was u s e d . C o n s e q u e n t l y , u s i n g t w i c e - w a s h e d c e l l s as i n o c u l u m , t h e e f f e c t s o f v a r i o u s compounds 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 . Y e a s t e x t r a c t {0.5%), NTA and EDTA a l l o w e d r a p i d g r o w t h o f t h e o r g a n i s m ( F i g u r e 3)- F e r r i c h r o m 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 t h a n t h e c o n t r o l ( F i g . 3). S a l i c y l i c a c i d , DHB(G) , c a t e c h o l and o - h y d r o x y h i p p u r i c a c i d showed no e f f e c t . T a b l e 3. The 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 compounds on g r o w t h o f M i c r o c o c c u s l y s o d e ? k t i c u s i n S a l t o n ' s (53) m i n i m a l medium. R e s u l t s ( t h e a v e r a g e f r o m t r i 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 a s t h e l e n g t h o f t h e l a g p e r i d ( i n h o u r s ) b e f o r e g r o w t h e n s u e d . , F i n a l L e n g t h o f l a g " C o n c e n t r a t i o n P e r i o d ( h o u r s ) (yM) . . c o n t r o l ( n i l ) - 42 2 . 3 - d i h y d r o x y b e r i z o a t e 40 36 2.4- d i h y d r o x y b e n z o a t e 40 30 i t o i c a c i d 40 16 c a t e c h o l kO 36 s a l i c y 1 i c a c i d 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 g r o w t h o f M i c r o c o c c u s l y s o - d e i k t i c u s i n S a l t o n ' s (33) m i n i m a l medium. R e s u l t s , ( t h e a v e r a g e f r o m 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 l e n g t h o f t h e l a g p e r i o d ( i n h o u r s ) b e f o r e t h e o n s e t o f l o g a r i t h m i c g r o w t h . Compound F i n a l C o n c e n t r a t i o n L e n g t h o f L a g P e r i o d (uM) ( H o u r s )  A l b o m y c " n 0.1 50 A l b . * + F e r r i chrome 10.0 10 II + 2,3-d i h y d r o x y b e n z o a t e 40.0 24 " + 1 t o i c a c i d 40.0 32 S a l i c y l i c a c i d 40.0 48 o - H y d r o x y h i p p u r a t e 40.0 >50 " + C a t e c h o l 40.0 >50 " + F e C l 3 1.0 >50 11 + EDTA 10.0 >50 " + NTA 10.0 >50 11 + Y e a s t 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 Y e a s t e x t r a c t + F e C l , 1 .0 24 * 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 p a p e r 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 g r o w t h o f M. l y s o d e i k t i c u 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 compounds p e r m i t t e d g r o w t h up t o 5 mm p a s t t h e n o r m a l l i n e o f i n h i b i t i o n a r o u n d t h e a l b o m y c i n s t r i p . 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 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 . 1 2 Compounds T e s t e d a s A l b o m y c i n A n t a g o n i s t s E f f e c t i v e S I i g h t l y E f f e e t i v e I n e f f e e t i v e F e r r i c h r o m e 2 , 3 - d i h y d r o x y b e n z o a t e i t o i c a c i d c a t e c h o l 2 , 5 - d i h y d r o x y b e n z o a t e EDTA N - h y d r o x y m e t h y l s a l i c y l a m i d e NTA o - h y d r o x y h i p p u r a t e 2 ,h-dihydroxybenzoate o - h y d r o x y p h e n y l a c e t a t e 2 , 6 - d i h y d r o x y b e n z o a t e S a l i c y l i c a c i d s a l i c y l a m i d e c r e s o t i c a c i d hemi n F e C K 5 - h y d r o x y a n t h r a n i 1 i c A c i d 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 compounds on 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. A l l a d d i t i o n s were made t o 10 yM c o n c e n t r a t i o n s a t z e r o t i m e . S y m b o l s : 0 - — 0 , no a d d i t i o n s , s a l i c y l i c a c i d , DHB, DHBG, c a t e c h o l , o - h y d r o x y h i p p u r i c a c i d ; C •, y e a s t e x t r a c t (0.5%); A A, EDTA; A A, NTA; Q Q F e C l 3 o r f e r r i c h r o m e . (b) 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 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 medium 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 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 h o u r s . The 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_, l y s o - 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 and s o l i d medium. In c o n t r a s t , h o w e v e r , f e r r i c h r o m e and DHB by t h e m s e l v e s were i n e f f e c t i v e , b u t when t e s t e d 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 t h e y 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 r o n , 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 a l s o 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 . Y e a s t e x t r a c t a l o n e 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 and NTA by t h e m s e l v e s s e r v e d as 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 i n h i b i t i o n i n s o l i d med i urn. D i s c u s s i o n o f R e s u l t s H y d r o x a m i c 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 c h e l a t i n g a g e n t s (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 a c t o r s 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 u n d e r p o o r n u t r i t i o n a l c o n d i t i o n s . The p r e s e n t r e s u l t s i n d i c a t e t h a t u n i d e n t i f i e d g r o w t h f a c t o r s c a r r i e d o v e r f r o m TSY-grown i n o c u l a , o r p r e s e n t i n 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 numerous 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 and WB746. R e s u l t s , ( t h e a v e r a g e o f 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 a s t h e l e n g t h o f t h e l a g p e r i o d ( i n h o u r s ) b e f o r e t h e o n s e t o f g r o w t h . Compound F i n a l C o n c e n t r a t i o n L e n g t h o f Lag P e r i o d (•••yM) ( H o u r s ) A l b o m y c i n 0.4 144 A l b . " + 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 10.0 16 " + N i t r i l o t r i a c e t i c a c i d 10.0 14 " + I t o i c a c i d 40.0 15 " + F e C l 3 1.0 102 " + Hemin 1.0 124 " + F e r r i c h r o m e 10.0 144 " + C a t e c h o l 10.0 144 11 + S a l i c y l i c a c i d 10.0 144 " + Y e a s t e x t r a c t * * 0.5% 60 " + Y e a s t e x t r a c t + f e r r i c h r o m e 10.0 12 " + Y e a s t e x t r a c t + I t o i c a c i d 10.0 14 " + Y e a s t e x t r a c t + 2 , 3 - d i h y d r o -x y b e n z o a t e 10 yM 18 . " + Y e a s t e x t r a c t + F e + + + 20 yM 32 * A l b o m y c i n was ad 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 a s 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 on 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 method o f Z a h n e r e t a 1. ( 7 6 ) . E f f e c t i v e compounds 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-pounds p e r m i t t e d g r o w t h up t o 5 mm p a s t t h e n o r m a l l i n e o f i n h i b i t i o n a r o u n d t h e a l b o m y c i n s t r i p . 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 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 . 1 2 Compounds T e s t e d a s A l b o m y c i n A n t a g o n i s t s E f f e e t i v e S I i g h t l y E f f e c t i v e I n e f f e e t i v e F e r r i c h r o m e I t o i c ' a c i d S a l i c y l a m i d e N i t r i t o t r i a c e t i c a c i d C a t e c h o l 2 . 3 - d i h y d r o x y b e n z o a t e 2.4- d i h y d r o x y b e n z o a t e o - h y d r o x y h i p p u r i c a c i d F e C U 2.5- d i h y d r o x y b e n z o a t e 2.6- d i h y d r o x y b e n z o a t e s a l i c y l i c a c i d hemin - a l s o a l l p r e v i o u s 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 s t r i p 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 and l y s o d e i k t i c u s . 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 a g e n t s i s u s u a l l y a t t r i b u t e d t o one 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 i o n s t h a t a r e i n a c c e s -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 o t h e r w i s e i n h i b i t g r o w t h . 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 o f N e i l a n d s (39), w h i c h has r e c e i v e d some s u p p o r t f r o m o t h e r work ( 1 7 , 28), t h a t some 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 f o r i t t o 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 a g e n t s 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 t h e c o n d i t i o n s e m p l o y e d . T h e f a c t t h a t NTA and 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) a b o v e , i n w h i c h t h e 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 r e l e a s e d 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. On t h e o t h e r h a n d , EDTA and NTA were 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 o r a b s e n c e o f y e a s t e x t r a c t . 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 on t h e c e l l s may be c o n s i d e r e d p o s s i b l e . A l t e r -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 one 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 r a t i o s o f t h e v a r i o u s i o n s i n t h e medium. F e r r i c h r o m e , a t r i h y d r o x a m a t e c o n t a i n i n g i r o n , s e r v e d a s a p o t e n t g r o w t h f a c t o r f o r b o t h o r g a n i s m s . T h e v a r i o u s p h e n o l i c a c i d s 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 i r o n s t r o n g l y . However, v a r i a t i o n d i d e x i s t b e tween t h e p h e n o l i c s w h i c h s e r v e d f o r B_. s u b t i 1 i s and t h o s e w h i c h s e r v e d f o r M. l y s o d e i k t i c u s . T h e s e r e s u l t s may 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 i r o n a s t h e e s s e n t i a l f a c t o r i n v o l v e d . T h e r e s u l t s may a l s o 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 some d e g r e e o f s p e c i f i c i t y f o r t h e c h e l a t i n g a g e n t d o e s e x i s t . In t h i s r e s p e c t , i t i s e v i d e n t t h a t t h e c h e l a t i n g a g e n t s may s e r v e as g r o w t h f a c t o r s by a c t i n g i n one o f a t l e a s t two, p o s s i b l y t h r e e , ways. Some a g e n t s , s u c h a s EDTA o r NTA, may s i m p l y s e r v e a s " r e l e a s e r s " o f e s s e n t i a l i o n s , o r a l t e r n a t i v e l y , b u t l e s s l i k e l y , as r e m o v e r s o f t o x i c i o n 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 a s f e r r i c h r o m e o r t h e p h e n o l i c a c i d s , may 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 t h e e s s e n t i a l i o n t o t h e c e l l q u i t e r e a d i l y ( 4 0 ) . E ven an i r o n - b i n d i n g p o l y s a c c h a r i d e i s t h o u g h t r e s p o n s i b l e f o r i o n up-t a k e i n b a c t e r i a l c e l l s (51). A n o t h e r a l t e r n a t i v e may be t h a t t h e c h e l a t i n g a g e n t e n t e r s 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 i n s u c h a way t h a t i n h i b i t i o n i s o v e r c o m e . E f f e c t i v e n e s s o f t h e 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 t r e n g t h , s i z e and 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 , as w e l l as t h e b i n d i n g s i t e s on t h e b a c t e r i a l c e l l s u r f a c e . T h e r o l e s p l a y e d by a l b o m y c i n and i t s a n t a g o n i s t s s t i l l r e m a i n u n e x p l a i n e d . A t l e a s t t h r e e p o s s i b l e s i t e s o f a c t i o n f o r t h e 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 e a c h 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 b a c k i n g . F e r r i c h r o m e ; w h i c h was t h e most p o t e n t a l b o m y c i n 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 . In 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 , h o w e v e r , f e r r i c h r o m e was 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^ . s u b t i 1 i s . T h i s w o u l d i n d i c a t e t h a t a l b o m y c i n a c t s a t d i f f e r e n t s i t e s i n t h e s e two o r g a n i s m s , o r a l t e r n a t i v e l y , t h a t f e r r i c h r o m e s e r v e s a d i f f e r e n t p u r p o s e i n t h e m e t a b o l i s m o f e a c h o f t h e s e two b a c t e r i a l s t r a i n s . 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 f o r b o t h M_. l y s o d e i k t i c u s a n d J3. s u b t i 1 i 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 w i t h 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 compounds were a g a i n e f f e c t i v e o n l y 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 - I t a p p e a r s , t h e r e -f o r e , t h a t a l b o m y c i n 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 m e t a b o l -ism. One o f t h e s e may 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 mechanisms a c r o s s t h e c e l l membrane. The o t h e r e f f e c t a p p e a r s t o be a u t i l i z a -t i o n o f i r o n w i t h i n t h e c e l l . I f s u c h were t h e c a s e , d i f f e r e n c e s i n r e s p o n s e 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 . Such d i f f e r e n c e s w e r e , i n f a c t , o b s e r v e d . 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 a c i l l u s s u b t i l i s . I n t r o d u c t i o n Up t o t h i s p o i n t , t h e work 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) t o b e l i e v e t h a t s t r a i n WB746 was d i s t i n c t from B1471 w i t h r e s p e c t t o t h e f u n c t -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 p r o d u c t i o n . T h e r e f o r e , s t r a i n s B1471 and WB746 were compared c a r e f u l l y . A number o f compounds were t e s t e d as ca r b o n s o u r c e s f o r growth and then examined f o r t h e i r e f f e c t s on p h e n o l i c a c i d and p o r p h y r i n p r o d u c t i o n by the two s t r a i n s . In a d d i t i o n , the t i m e s 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 by t h e s e 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 a n a l y s e d f o r the p r o d u c t i o n o f compounds o t h e r than DHB and DHBG d u r i n g 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 . 1. W i l d Type S t r a ? n s (a) 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 i n the p r e s e n c e o f h i g h l e v e l s o f c a r b o n s o u r c e ( p r e l i m i n a r y ob-s e r v a t i o n s ) . The d a t a i n T a b l e 8 show t h a t g l u c o s e was the most e f f e c t i v e c a r b o n s o u r c e used i n t h i s r e s p e c t . A c e t a t e appeared t o be i m p o r t a n t 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 by both s t r a i n s , a l t h o u g h w i t h s t r a i n WB746 t h e r e was some p r o d u c t i o n i f g l u c o s e p l u s g l u t a m a t e , s u c c i n a t e p l u s g l u t a -mate, o r g l u t a m a t e a l o n e , were i n c l u d e d i n t h e medium i n t h e absence o f a c e t a t e . In the p r e s e n c e o f 0.3% a c e t a t e , Cop I I I was produced 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 i n c l u d e d i n t h e medium. With s t r a i n B1471 on t h e o t h e r hand, o n l y Cop I I I was produced i n the absence o f 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 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 B_. s u b t i 1 i s s t r a i n s B1471 and WB746. B 1 4 7 1 WB746 C a r b o n S o u r c e c f i i c f u ( ] % l e v e l ) P h e n o l i c Cop 111 x 1 0 " 3 P h e n o l i c Cop II 1 x 10' +G1ucose + + >15 " + >15 C i t r a t e - - 7.5 - - 2.8 A c e t a t e + + >15 + - 8.6 S u c c i n a t e - - 7.8 - - 7.2 L a c t a t e - NDI + - ND G 1 u t a m a t e - + ND + - 7.3 G l y c e r o l + - ND - - ND G l u c o s e + C i t r a t e - - 3.5 ± - 8 G l u c o s e + S u c c i n a t e + + >15 + - 10 G l u c o s e + G 1 u t a m a t e - - >15 ND ND ND G l u c o s e + G l y c i n e + + >15 + - 14 G l u t a m a t e + S u c c i n a t e - - >15 + + >15 G l y c i n e + S u c c i n a t e - - 5.8 - + 6.7 G l y c i ne + C i t r a t e - - 2.8 - - > 1 GIJJC. + G l y . + S u c c . + + >15 + - 12 l A c e t a t e + + >15 + 8.6 +G1ucose - - 12 + - 12 *C i t r a t e - - 5.4 - - 2.3 * G 1 u c o s e + G l u t a m a t e - - >15 + - 12 # S u c c i n a t e + G l u t a m a t e - + >15 + - 10 * 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 c u l t u r e . + T e s t e d w i t h 0.1% A c e t a t e * T e s t e d a s s o l e c a r b o n s o u r c e 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 was a d d e d t o t h e medium ( T a b l e 8). (b) C hanges i n pH D u r i n g Growth T h e pH o f t h e medium was f o l l o w e d d u r i n g t h e g r o w t h o f c u l t u r e s . A l t h o u g h t h e r e was c o n s i d e r a b l e v a r i a t i o n f r o m one e x p e r i m e n t t o a n o t h e r , a c o n s t a n t t r e n d was a l w a y s o b t a i n e d 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 . I n i -t i a l l y 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 t h e f i r s t 7 - 8 h o u r s o f g r o w t h and t h e n r e m a i n e d c o n s t a n t f o r 1 - 2 h o u r s . The pH t h e n began t o r i s e and c o n t i n u e d t o do s o u n t i l pH 7 o r a t t i m e s pH 8 was r e a c h e d . I t was n o t u n t i l t h e b e g i n n i n g o f t h e r i s e i n pH t h a t p h e n o l i c a c i d s w ere f i r s t d e t e c t e d i n 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). A s i m i l a r pH t r e n d was o b t a i n e d f o r c u l t u r e s grown u n d e r i r o n - s u f f i c i e n c y , e x c e p t t h a t p h e n o l i c a c i d s were n o t p r o -d u c e d . ( c ) T i m e 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 T h e t i m e 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 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 t h e 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 . W i t h WB746, DHB was d e t e c t e d a s e a r l y a s 5 - 6 h o u r s 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 r e a c h e d k x 10^ c f u p e r m l . C h r o m a t o g r a p h i c e x a m i n a t i o n o f c u l t u r e s u p e r n a t a n t s o b t a i n e d H O U R S F i g u r e 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 and 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 and WB746. S y m b o l s : 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 10 c o l o n y f o r m i n g u n i t s p e r m l ) ; • f, pH; A A, p h e n o l i c a c i d p r o d u c t i o n (O.D._ l r i ) . K 510 nm a f t e r 7, 14 and 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 B1471, 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 be d e t e c t e d c h r o m a t o g r a p h i c a l l y i n s u p e r -n a t a n t s o b t a i n e d 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 h a n d , a l t h o u g h t h e 6, 7 and 8 h o u r s u p e r n a t a n t s a p p e a r e d 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 p h e n o l i c a c i d s w ere 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). A l l t h e s e p h e n o l i c s w ere d e t e c t e d o n l y i n c u l t u r e s grown 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 . Of t h e two i r o n - b i n d i n g p h e n o l i c compounds p r e s e n t i n t h e e a r l y s u p e r n a t a n t s , one o f them ( c , F i g u r e 5) has been i d e n t i f i e d a s DHBG. T h e o t h e r com-pound ( a , F i g u r e 5) Is a s y e t u n i d e n t i f i e d . A c i d h y d r o l y s i s and t w o - d i m e n s i o n a l t h i n l a y e r c h r o m a t o g r a p h y i n d i c a t e d t h e 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 . Work i s c u r r e n t l y i n p r o g r e s s 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 compound. 2. M u t a n t S t r a i n s T h e g r o w t h o f s t r a i n s B1471 and WB746 was i n h i b i t e d by 2.0 mM F e C l ^ and by 1.5 mM hemin. M u t a n t s w e r e s e l e c t e d f r o m e a c h s t r a i n w h i c h were r e s i s t a n t t o 20 mM F e C l ^ o r t o 10 mM hemi n . T h e m u t a n t s r e s e m b l e d t h e w i l d t y p e s t r a i n s i n t h a t t h e y s t a r t e d t o p r o d u c e p h e n o l i c a c i d s a f t e r a b o u t 8 h o u r s g r o w t h F igure 5. Th in l aye r chromatography of c u l t u r e supernatant e x t r a c t s a f t e r growth of B_. subt i 1 is B1471 f o r 7 or 14 hours w i th or wi thout added i r o n . Compounds were detected as f l u o r e -scent spots under u l t r a - v i o l e t l i g h t and were p o s i t i v e to Evans reagent (phenol i cs ) and to FeCl^ ( i r o n - b i n d i n g ) . The so l ven t system used was benzene :methano l :ace t i c a c i d (10:2:1) (v/v/v) (7 ) . 41 I. origin 2. solvent front A growth medium B 2,3-dihydroxybenzoate (DHB) C 7-hour culture without Fe D 2,3- dihydroxybenzoylglycine OHBG) E 7-hour (s14hour) culture with Fe F DHB G 14-hour culture without Fe F i g u r e 5. ( F i g u r e 6 and 7). However, t h e Fe 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 as much p h e n o l i c a c i d as 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 as t h e w i l d t y p e s t r a i n , 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 . B o t h t y p e s o f m u t a n t s grew more s l o w l y t h a n 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 a d d e d 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 t h o s e 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 r e s p o n s e t o a d d e d i r o n ( F i g u r e 6 ) . B o t h t y p e s o f m u t a n t s 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 . D i s c u s s i o n o f R e s u l t s T h e r e i s l i t t l e u n d e r s t a n d i n g o f t h e b a s i c m e t a b o l i s m i n v o l v e d i 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 ? 1 i s 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 . 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 c a r b o n s o u r c e i s n e c e s s a r y f o r t h e p r o d u c t i o n o f s i d e r a m i n e s by A s p e r g i 1 l u s mel l.eus (14). I t was t h e r e f o r e o f i n t e r e s t t o l o o k a t p h e n o l i c a c i d p r o d u c t i o n a s a f u n c t i o n o f t h e c a r b o n - s o u r c e u s e d f o r g r o w t h o f B_. s u b t ? 1 i s . 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 h i g h c o n c e n t r a t i o n o f c a r b o n s o u r c e i s n e c e s s a r y f o r DHB(G) p r o d u c t i o n . I t i s n o t s u r p r i s i n g t h a t g l u c o s e p r o v e d t o be 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 t e s t e d , s i n c e t h e 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. 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 mut 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 and p r e s e n c e o f a d d e d i r o n (5.5 y g atoms F e + + + / 1 i t e r ) . S y m b o l s : g r o w t h i n t h e p r e s e n c e (0 0) and a b s e n c e (• •) o f add e d i r o n (kO K l e t t u n i t s = 1 0 8 c f u / m l ) , by R R b o t h F e ^ and H m u t a n t s . 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 R mutant i n t h e p r e s e n c e ( A - — A ) and 510 nm a b s e n c e ( A - — A ) o f add e d 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 . J J J Q N M ) by H R m u t a n t i n t h e p r e s e n c e ( • • ) and a b s e n c e (•• • ) o f add e d i r o n . F i g u r e 7. Growth 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 by mut 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 and p r e s e n c e o f add e d i r o n (5.5 y g atoms F e + + + / 1 i t e r ) . S y m b o l s : 0 0, g r o w t h o f F e R m u t a n t s w i t h o r w i t h o u t p i r o n ; • g r o w t h o f H" muta n t w i t h i r o n ; C — C , g r o w t h o f H R m u t a n t w i t h o u t a d d e d i r o n ; 1 0 8 c f u / m l = kO K l e t t u n i t s . P h e n o l i c a c i d p r o d u c t i o n ( O . D . ^ Q N M ) by F e R m u t a n t i n t h e p r e s e n c e ( Q Q ) and t h e a b s e n c e (A A) o f a d d e d 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 ) and a b s e n c e ( A — A ) o f i r o n . 43 c o n d e n s a t i o n between e r y t h r o s e - 4 - p h o s p h a t e and p h o s p h o e n o l y p y r u v a t e t o p r o d u c e s e d o h e p t u l o s e - 7 ~ p h o s p h a t e . S i n c e B_. s u b t i 1 i s c a n d i s s i -m i l a t e g l u c o s e v i a t h e h e x o s e - m o n o p h o s p h a t e pathway ( 2 2 ) , t h i s w o u l d be e x p e c t e d t o r e s u l t 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 s u p p l y o f s e d o h e p t u l o s e - 7 - p h o s p h a t e , s u b s e q u e n t l y l e a d i n g t o c h o r i s m i c a c i d . The r o l e t h a t a c e t a t e p l a y s i n DHB p r o d u c t i o n i s n o t i m m e d i a t e l y c l e a r . E x c e p t f o r t h e i n s t a n c e s w here g l u t a m a t e was u s e d as s o l e c a r b o n s o u r c e , t h e a b s e n c e o f a c e t a t e r e s u l t e d i n l o s s 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 B. s u b t i l i s . However, t h e p r o d u c t i o n o f c o p r o -p o r p h y r i n w o u l d a p p e a r t o i n d i c a t e t h a t t h e TCA c y c l e was o p e r a t i n g e v e n when a c e t a t e was a b s e n t . T h e r e f o r e i t a p p e a r s t h a t some f a c t o r o t h e r t h a n a f u n c t i o n a l TCA c y c l e i s n e c e s s a r y 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 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 i r o n - d e -f i c i e n c y . T he 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 a s t h e s o l e c a r b o n s o u r c e may 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 b e i n g c o n v e r t e d t o a - k e t o g l u t a r a t e and t h e n c e t o s u c c i n y l Co A and an o p e r a t i v e heme pa t h w a y . T h e l a t t e r w o u l d r e s u l t i n a h i g h r e q u i r e -ment f o r i r o n and c o n s e q u e n t l y i r o n - d e f i c i e n c y w o u l d s o o n e n s u e , l e a d i n g u l t i m a t e l y t o p h e n o l i c a c i d p r o d u c t i o n . M o r e o v e r , t h e 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 f o u n d i n t h e c i t r i c a c i d c y c l e and b i o s y n t h e t i c p a t h w a y s 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. The method 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 commences 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 s h a l l s e e l a t e r , t h e r e s u l t i n g i r o n - d e f i c i e n c y p r o b a b l y c a u s e s a d e r e p r e s s i o n o f t h e D H B ( G ) - p r o -c l u c i n g enzyme s y s t e m . 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 t o be u n i q u e t o p h e n o l i c a c i d p r o d u c t i o n o r t h e c o n s e q u e n c e s o f i r o n - d e -f i c i e n c y i n Eh s u b t i1? s. S i m i l a r p a t t e r n s have been o b s e r v e d i n s p o r u l a t i o n s t u d i e s (38). I t a p p e a r s t h a t d u r i n g t h e d e c r e a s e i n 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 TCA c y c l e . T h e a c e t a t e - o x i d i z i n g s y s t e m r e a c h e s peak a c t i v i t y d u r i n g t h e l a t t e r h a l f o f the low pH p e r i o d and d e c r e a s e s r a p i d l y , due 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). R e p r e s s i o n o f a c t i v i t y o f TCA c y c l e enzymes m i g h t t h e n 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 f r o m t h e medium. C o n t i n u o u s p h e n o l i c a c i d p r o d u c t i o n d o e s n o t a p p e a r t o o c c u r t h r o u g h o u t 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 . E x p e r i m e n t s i n v o l v i n g t h e e x t r a c t i o n o f l a r g e v o l u m e s o f 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 i n v o l v i n g e x a m i n a t i o n o f m u t a n t 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 l o s t , 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 t u r n e d o n . T h e s e a p p e a r t o c o i n c i d e w i t h t h e 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 p r o d u c e d w h e t h e r t h e s u p e r n a t a n t was e x a m i n e d a s e a r l y a s 5 - 6 h o u r s a f t e r i n o c u l a t i o n ( a t a d e n s i t y o f k x 10 c e l l s p e r ml) o r a s l a t e as 23 h o u r s . 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 . T h i s was f o l l o w e d l a t e r by t h e p r o d u c t i o n o f DHBG and d i s a p p e a r a n c e o f t h e u n i d e n t i -f i e d compound. 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 t o p r o d u c e a m i n o a c i d c o n j u g a t e s 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 an amino a c i d t o DHB. T h e 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 a m i n o 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 i n WB746. I I I . E f f e c t o f DHB A n a l o g s and 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 a c i 1 l u s s u b t i 1 i s I n t r o d u c t i o n P e t e r s (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 a n a l o g s o f DHB on 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 o f j3_. s u b t ? 1 i s . I t was f o u n d t h a t , i n g e n e r a l , m e t a - s u b s t i t u t e d b e n z o i c a c i d s and r e -l a t e d compounds were t h e most e f f e c t i n h i b i t o r s o f DHB(G) p r o d u c t i o n . Some a d d i t i o n a l a n a l o g s have now been i n v e s t i g a t e d and 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 . W i t h t h e d e -v e l o p m e n t o f an 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 ( s e e M a t e r i a l s 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 on b o t h DHB s y n t h e t a s e a c t i v i t y and t h e enzyme l e v e l s i n e e l 1 e x t r a c t s . 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  G r o w i n g C e l l s (a) A n a l o g s o f DHB None o f t h e a n a l o g s e x a m i n e d f o r t h e f i r s t t i m e i n t h i s s t u d y c a u s e d s i g n i f i c a n t i n h i b i t 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 g r o w i n g c e l l s ( T a b l e 9 ) . (b) C h e l a t i n g A g e n t s The 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 s l i g h t l y 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 t o t h e g r o w t h medium ( T a b l e 1 0 ) . 2. In V i t r o A s s a y s (a) P r e l i m i n a r y E x p e r i m e n t s P r e l i m i n a r y e x p e r i m e n t s i n d i c a t e d t h a t a t l e a s t 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 t o e n s u r e s i g n i f i c a n t p r o d u c t f o r m a t i o n ( F i g u r e 8 ) . A l t h o u g h t h e s u b s t r a t e d i s -a p p e a r e d r a p i d l y f r o m t h e r e a c t i o n m i x t u r e , DHB was n o t f o r m e d d u r i n g t h e f i r s t h o u r ( F i g u r e 8 ) . In t h e p r e s e n c e o f e x c e s s s u b s t r a t e , t h e amount o f p r o d u c t 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 enzyme 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 i n t o e t h e r and i t s s p e c t r a l c h a r a c t e r i s t i c s e x a m i n e d . I t s a b s o r p t i o n s p e c t r u m ( 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. The p r o d u c t b e h a v e d l i k e a u t h e n t i c DHB on c h r o m a t o g r a m s . E x t r a c t s o f B. s u b t i 1 i s WB746 c o n v e r t e d some k0% o f t h e s u b s t r a t e , T a b l e 9. E f f e c t o f a n a l o g s o f 2 , 3 - d i h y d r o x y b e n z o i c a c i d on p h e n o l 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 and 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 i r o n . ° - D - 5 1 0 nm Compound C o n c e n t r a t i o n B1471 WB746 (mM) N i l - 0.25 0.48 M - T o l u i c a c i d 0.1 0.26 0.47 V a n i l l i c a c i d 0.1 0.22 0.44 V e r a t r i c a c i d 0.1 0.20 0.46 o - h y d r o x y b e n z y l a l c o h o l 1.0 0.24 0.50 T a b l e 10. 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 by f3_. s u b t ? l i s s t r a i n B1471 and 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 i r o n . , D , 5 1 0 nm Compound C o n c e n t r a t i o n B1471 WB746 (mM) N i l - 0.26 0.54 a , a - d i p y r i d y l 0.1 0.30 0.56 1 , 1 0 - p h e n a n t h r o l i n e 0.1 0.35 0.58 T i r o n 0.1 0.39 0.58 E t h y l e n e d r a m i n e t e t r a a c e t a t e 0.1 0.33 0.58 n i t r i l o t r i a c e t a t e 0.1 0.28 0.54 0-3-Q O 0-2-01-S L z = _ — i 2 H O U R S 20 -15 -10 E c CO CM O O -0-5 F i g u r e 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 t o 2 , 3 - d i h y d r o x y -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 o f B_. s u b t i 1 i s WB746 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 . S y m b o l s : 0 - — 0 , y m o l e s o f DHB f o r m e d (0.1 O . D . ^ g n m = 0.075 y m o l e s DHB); • #, d i s a p p e a r a n c e o f c h o r i s m i c a c i d f r o m r e a c t i o n m i x t u r e . 50 0-8-0-6-oo io Q o 0 -4-0-2-0-95 1-90 2-85 mg PROTEIN 3-80 F igure 9- Convers ion of cho r i sm ic ac id 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 ro t e i n was added as crude, eel 1-free e x t r a c t s from jB. subt i 1 is WB746 grown under i r o n - d e f i c i e n c y . 250 275 300 325 350 WAVELENGTH (nm) F i g u r e 1.0. U l t r a v i o l e t s p e c t r a ( e t h e r s o l v e n t ) o f s u b s t r a t e , p r o d u c t and i n t e r m e d i a t e r e a c t i o n m i x t u r e (b) i n t h e 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 ( c ) t o 2 , 3 - d i h y d r o x y b e n z o i c a c i d (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 grown u n d e r i r o n - d e f i c i e n c y . c h o r i s m i c a c i d ( 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 and t h o s e o f B1471 some 15%. However, y i e l d s v a r i e d 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 - 65% f o r WB746 and 15 - 25% f o r B 1 4 7 1 ) . (b) 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 S i g n i f i c a n t l e v e l s o f a c t i v e enzyme were n o t f o r m e d when t h e c e l l s w e re grown 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 -chrome and t h e enzyme 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 o f t h e a r o m a t i c a m i n o a c i d s o r 3 _ f 1 u o r o b e h z o i c a c i d ( T a b l e 1 1 ) . ( c ) 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 - h y d r o x y b e n z o i c a c i d w ere v e r y e f f e c t i v e i n h i b i t o r s o f enzyme a c t i o n . DHB and c a t e c h o l w ere a l s o i n h i b i t o r y , b u t 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 t o 3 - h y d r o x y a n t h r a n i 1 i c a c i d a n d 3 - h y d r o x y b e n z o i c a c i d , b o t h c a t e c h o l and h e m i n , when t e s t e d i n t h e p r e s e n c e o f i r o n , p r o v e d t o be 100% e f f e c t i v e as enzyme i n h i b i t o r s . ( r o n a l o n e had 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 i n h i b i t e d a c t i v i t y o f B1471 e x t r a c t s by a b o u t 20%. 3 _ f 1 u o r o b e n z o i c a c i d i n h i b i t e d t h e enzyme a c t i v i t y a b o u t 50%. (d) Enzyme R e a c t i v a t i o n In an 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 c o u l d be r e s t o r e d t o e x t r a c t s o f WB746 grown i n t h e p r e s e n c e o f 1.0 mM F e C l _ , e x t r a c t s w ere d i a l y s e d o v e r n i g h t a t k C T a b l e 11. E f f e c t o f v a r i o u s compounds on l e v e l s o f DHB s y n t h e t a s e p r o d u c e d by g r o w i n g c e l l s o f B_. s u b t i 1 i s B1471 and WB746 % enzyme a c t i v i t y was c a l c u l a t e d on a p e r mg o f p r o t e i n b a s i s . R e s u l t s a r e e x p r e s s e d as t h e a v e r a g e o f 3 r e p l i -c a t e a s s a y s . Compound F i n a l C o n c e n t r a t i o n Enzyme A c t i v i t y {%) WB746 B1471 N i l - 100 100 F e C l , . 6 H o 0 100 uM 0 0 50 uM 0 0 25 uM 2k 0 0.25 uM 100 85 C a t e c h o l 1.0 mM 120 100 C a t e c h o l + F e + + + * 1 .0 mM 80 175 DHB 1.0 mM 1 10 100 o - H y d r b x y h i p p u r a t e 10 uM 90 90 o - H y d r o x y h i p p u r a t e + F e + + + * 10 uM 120 80 D i p i c o l i n i c a c i d 100 uM 70 -3 - F 1 u o r o b e n z o i c a c i d 1 .0 mM 50 10 0.1 mM 70 40 3 - H y d r o x y a n t h r a n i 1 i c a c i d 100 uM 100 -3 - H y d r o x y b e n z o i c a c i d 100 uM 140 -Hemi n 1.5 yM 90 -T y r o s i n e 1 .0 mM 10 -T r y p t o p h a n 1.0 mM 40 -P h e n y l a l a n i n e 1.0 mM 80 -A n t h r a n i l i e a c i d 1 .0 mM 75 -* Fe was i n c l u d e d a t 0.25 ug atoms/ml c o n c e n t r a t i o n ' T a b l e 12. E f f e c t o f v a r i o u s compounds on t h e a c t i v i t y o f DHB s y n t h e t a s e enzyme 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 and B1471 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 . % enzyme a c t i v i t y was c a l c u l a t e d on a p e r mg o f p r o t e i n b a s i s . R e s u l t s a r e e x p r e s s e d a s t h e a v e r a g e o f 3 r e p l i -c a t e a s s a y s . Compound F i n a l C o n c e n t r a t i o n Enzyme A c t i v i t y WB746 B1471 N i l - 100 100 F e C l 3 . 6 H 2 0 1 mM 100 100 F e r r i chrome 10 yM 100 100 C a t e c h o l 1 mM 60 0 C a t e c h o l + F e + + + * * 1 mM 100 2 , 3 - d i h y d r o x y b e n z o i c a c i d 5 mM 0.5 mM 50 100 -D i p i c o l i n i c a c i d 1 mM 100 100 3 - f 1 u o r o b e n z o i c a c i d 1 mM 100 50 3 - h y d r o x y a n t h r a n i 1 i c a c i d 1 mM 0 0 3 - h y d r o x y a n t h r a n i 1 i c a c i d + F e + + + * * 1 mM 100 100 3 - h y d r o x y b e n z o i c a c i d 1 mM 0 0 o - h y d r o x y h i p p u r i c a c i d 10 yM 100 -Hemi n 1 yM 100 80 Hemin + F e + + * * - , ; 1 yM 100 0 EDTA 1-mM 100 100 T y r o s i ne 1 mM 100 -T r y p t o p h a n 1 mM 100 -P h e n y l a l a n i n e 1 mM 100 -A n t h r a n i l i e a c i d 1 mM 100 -* n o t d e t e r m i n e d F e + + + i n c l u d e d a t 0.25 y g atoms/ml c o n c e n t r a t i o n a g a i n s t t h r e e c h a n g e s o f v a r i o u s c h e l a t i n g a g e n t s i n 0.1 M T r i s b u f f e r . No i n c r e a s e i n enzyme a c t i v i t y was o b s e r v e d ( T a b l e 13). D i s c u s s i o n o f R e s u l t s The e f f e c t o f t h e a n a l o g s 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 i n v i e w o f t h e f i n d i n g s by P e t e r s (44) t h a t m e t a - s u b s t i t u t e d b e n z o a t e s w ere t h e a n a l o g s s e r v i n g a s t h e most e f f e c t i v e i n h i b i t o r s . I t seems, t h e r e -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 i n v o l v e d i n s h u t - o f f 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 i n c r e a s e d when B_. s u b t i 1 i s was grown i n t h e p r e s e n c e o f s t r o n g i r o n c h e l a t i n g a g e n t s w h i c h a r e not a n a l o g s o f DHB l e n t f u r t h e r s u p p o r t t o t h e s u g g e s t i o n (44) 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 a d d e d t o t h e medium c o u l d h a v e bound r e s i d u a l i r o n i n t h e g r o w t h medium t h e r e b y m a k i n g i t u n a v a i l a b l e t o t h e c e l l s . S i n c e t h e medium was 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 p r o d u c t i o n was a l r e a d y h i g h , 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 p r o d u c e d w o u l d n o t be e x p e c t e d . The enzymes 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 t o t h a t f o u n d f o r t h e E_. c o l i s y s t e m (2) and f o r •Table 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 grown i n t h e p r e s e n c e o f 1 mM F e C l ^ a t 37 C f o r ]k h o u r s ; C h e l a t i n g A g e n t C o n c e n t r a t i on Enzyme A c t i v i t y  % o f s t a r t i n g c o n t r o l Sample C o n t r o l E t h y l e n e d i a m i n e t e t r a a c e t a t e 1 mM a , a - d i p y r i d y l 0.1 mM T i r o n 0.1 mM 0 0 0 70 60 65 A. a e r o g e n e s ( 7 2 ) . When c e l l s were grown In t h e p r e s e n c e o f i r o n , t h e e x t r a c t s w ere d e v o i d o f a c t i v i t y . S i n c e a c t i v i t y d i d n o t a p p e a r a f t e r d i a l y s i s o f t h e e x t r a c t s a g a i n s t v a r i o u s c h e l a t i n g a g e n t s , i r o n was n o t s e r v i n g m e r e l y a s an i n h i b i t o r . T h i s c o n c l u s i o n was s u p p o r t e d 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 e x t r a c t s d i d 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 . P r e p a r a t i o n s f r o m c e l l s grown i n t h e a b s e n c e o f added i r o n showed good enzyme a c t i v i t y i n 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. T h e s e r e s u l t s c a n t h e r e f o r e 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 r e p r e s s e s t h e f o r m a t i o n o f t h e e n z y m e ( s ) r e s p o n s i b l e f o r DHB(G) p r o d u c t i o n i n J3. s u b t i l i s . R e p r e s s i o n c o n t r o l o f a r o m a t i c a m i n o a c i d s y n t h e s i s i n J3. s u b t i 1 ?s has been shown o n l y r e c e n t l y ( 2 4 , 4 2 ) . I t i s o f i n t e r e s t 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 u n d e r 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 b r a n c h e s , w i t h t h e p o s s i b l e e x c e p t i o n o f t h e p h e n y l a l a n i n e p a t h w a y , i s u n d e r t h e r e p r e s s i o n c o n t r o l o f t h e a m i n o a c i d w h i c h i t s y n t h e s i z e s ( 4 1 , 4 2 ) . T h e a r o m a t i c a m i n o a c i d s , f e r r i c h r o m e and 3 ~ f l u o r o b e n z o i c a c i d , i n a d d i t i o n t o i r o n , a l s o a p p e a r t o s i g n i f i c a n t l y r e p r e s s f o r m a t i o n o f t h e enzymes i n v o l v e d i n DHB s y n t h e s i s . F e r r i c h r o m e c o n t a i n s i r o n and a l s o p o s s i b l y s e r v e s as an i r o n t r a n s p o r t f a c t o r (40) t h e r e b y 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 . T h e a r o m a t i c 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 by r e -d u c i n g t h e amount o f c h o r i s m i c a c i d p r o d u c e d i n t h e c e l l and t h e r e -by l i m i t i n g t h e amount o f DHB(G) f o r m e d . 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. T h e e f f e c t s o f c a t e c h o l and DHB a r e n o t c l e a r , a l t h o u g h 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 f o r m a t i o n . ' n —• 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 by c h o r i s m i c a c i d and 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 by t y r o s i n e and p h e n y l a l a n i n e (41). T r y p t o p h a n a c t s a s a n e g a t i v e e f f e c t o r o f a n t h r a n i l a t e s y n t h e t a s e (18). 3 - H y d r o x y -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 a p p e a r 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 s u b s t r a t e a n a l o g s . 3 - F l u o r o b e n z o a t e , 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 enzyme f o r m a t i o n w i t h b o t h s t r a i n s B1471 and 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 enzyme, b u t n o t t h a t o f WB746. I t a l s o a p p e a r s 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 a c t i v i t y . I f t h i s i s t r u e , DHB may be c o n s i d e r e d a n e g a t i v e e f f e c t o r . However, t h e f a c t t h a t t h e s e s t u d i e s w ere c a r r i e d o u t u s i n g c e l l - f r e e e x t r a c t s and n o t t h e p u r i f i e d enzyme may c a u s e d i f f i c u l t y i n i n t e r p r e t i n g t h e s e r e s u l t s . 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 I n t r o d u c t i o n An 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 c h o r i s m i c a c i d t o DHB. I t was hoped 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 enzyme 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 i n v o l v e d i n r e g u l a t i n g DHB s y n t h e s i s . However, s h o r t l y a f t e r t h i s p h a s e o f t h e work was s t a r t e d , work w i t h _E. c o l i and A. a e r o g e n e s (7 2 , 73, 74) r e v e a l e d t h e e x i s t e n c e o f a t l e a s t two i n t e r m e d i a t e s i n 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. T h e s e i n t e r -m e d i a t e s a r e n o t a v a i l a b l e a t p r e s e n t , s o t h a t any p u r i f i c a t i o n s t e p s w h i c h w o u l d s e p a r a t e i n d i v i d u a l enzymes o f t h e pathway w o u l d e f f e c t i v e -l y p r e v e n t f u r t h e r a s s a y o f t h e s y s t e m . In s p i t e o f t h i s , i t was p o s s i b l e t o " p u r i f y " t h e o v e r a l l s y s t e m , i . e . t h a t c o n v e r t i n g c h o r i s -mate t o DHB, 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 c o l u m n c h r o m a t o -g r a p h y 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 . T h i s i n d i c a t e d t h a t t h e enzymes may e x i s t a s 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 t h e c e l l . 1. DEAE - C e l l u l o s e Column 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 enzyme was e l u t e d between 0.40 M and 0.75 M NaCl ( F i g u r e 11a) when c r u d e c e l l - f r e e e x t r a c t s f r o m s u b t i 1 ? s WB746 were s u b j e c t e d t o D E A E - c e l l u l o s e ' c h r o m a t o -g r a p h y and b e tween 0.34 and 0.38 M N a C l ( 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 w ere u s e d . 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 D H B - s y n t h e s i z i n g p r o t e i n was f o u n d i n s u c r o s e g r a d i e n t 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 F i g u r e TV. D E A E - c e l l u l o s e 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 s u b t i 1 i s WB746 grown w i t h o u t a d d e d i r o n . (a) C r u d e e x t r a c t (2.0 ml) c o n t a i n i n g 19 mg o f p r o t e i n / m l was a p p l i e d t o t h e c o l u m n and e l u t e d by a l i n e a r NaCl g r a d i e n t (0 - 1.0 M). F r a c t i o n s (99 d r o p s ) w ere c o l l e c t e d , (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 ( a f t e r (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 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 and e l u t e d by a l i n e a r NaCl g r a d i e n t (0.30 - 0.60 M). F r a c t i o n (45 d r o p s ) w ere c o l l e c t e d . S y m b o l s : 0 0, p r o t e i n ; • — i , DHB s y n t h e t a s e . F i g u r e 11. ( F i g u r e 1 2 ) . P r o t e i n was f o u n d i n a l l f r a c t i o n s ( n o t s h o w n ) . D i s c u s s i o n o f R e s u l t s 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 n o t 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 enzyme 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 c o m p l e x . Such c o m p l e x e s a r e i n v o l v e d i n t h e a r o m a t i c pathway o f EL s u b t 11 ?s ( 1 8 , 3 5 ) . T h a t t h e c o m p l e x i s 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 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 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 . I f s u c h a m u l t i e n z y m e c o m p l e x were 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 w o u l d be e x p e c t e d . V. I r o n T r a n s p o r t I n t r o d u c t i o n T h e work 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 (46) was 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 t h e s t a t e o f t h e i n t r a c e l l u l a r i r o n . In a d d i t i o n , i r o n u p t a k e by 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 was e x a m i n e d f o r c o m p a r a t i v e p u r p o s e s . 0.20-0.15-co 0.10-Q d 0.05-F i g u r e 12. 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 DHB s y n t h e t a s e a c t i v i t y 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 grown w i t h o u t a d d e d i r o n . C r u d e e x t r a c t (0.6 ml) c o n t a i n i n g 19.mg p r o t e i n / m l 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 m l ) . 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. F r a c t i o n s (6 d r o p s ) were c o l 1 e c t e d . B a c i l l u s s u b t i 1 i s S t r a i n B 7 4 7 1 * (a) P r e l i m i n a r y E x p e r i m e n t s R a d i o a c t i v e i r o n was a d d e d t o c e l l - f r e e medium an 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 and M e t h o d s . C e l l -f r e e s a m p l e s were removed p e r i o d i c a l l y o v e r a 30 m i n u t e p e r i o d and p a s s 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 . When t h e medium u s e d c o n t a i n e d c i t r i c a c i d , no r a d i o a c t i v i t y was r e t a i n e d on t h e f i l t e r s . However, when c i t r i c a c i d was o m i t t e d f r o m t h e medium, a h i g h l e v e l o f r a d i o a c t i v i t y was f o u n d t o r e m a i n a t t a c h e d t o t h e f i l t e r s and t h i s l e v e l g e n e r a l l y i n c r e a s e d o v e r t h e i n c u b a t i o n p e r i o d . W a s h i n g t h e f i l t e r s w i t h 2.0 t o 5.0 ml o f i r o n - d e f i c i e n t medium l o w e r e d , b u t d i d n o t e l i m i n a t e , t h i s r e t e n t i o n o f r a d i o -a c t i v i t y . C o n s e q u e n t l y , e x p e r i m e n t s were r u n 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 u s e d t o s u s p e n d t h e c e l l s . F o r 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 f i l t e r s w e r e washed w i t h 5 ml o f i r o n - d e f i c i e n t medium t o remove e x t r a n e o u s l o o s e l y bound i r o n . F o r 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 t h e c e l l s s e v e r a l t i m e s b e f o r e a d d i n g r a d i o a c t i v e i r o n . T he e f f e c t s o f d i f f e r e n t w a s h i n g p r o c e d u r e s on t h e u p t a k e c a p a c i t y o f B_. s u b t i 1 1 s w ere e x a m i n e d w i t h c e l l s w h i c h had been grown w i t h v a r i o u s l e v e l s o f a d d e d i r o n . W a s h i n g 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 50% f n a l l c a s e s ( T a b l e 1 4 ) . 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 , t h e f i l t e r method 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) E x p e r i m e n t s W i t h I r o n - D e f i c i e n t C u l t u r e s ( i ) I r o n u p t a k e 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 . D u r i n g t h e e a r l y and m i d - l o g p h a s e s 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 r a t e 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 ( F i g u r e 1 3 ) . 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 -c r e a s e d 2 - 3 - f o l d and t h e r a t e o f u p t a k e was n o t c o n s t a n t d u r i n g t h e i n c u b a t i o n p e r i o d ( F i g u r e 1 3 ) . I t was e v i d e n t 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 n o t s i m p l y 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 numbers. T h e i n c r e a s e d 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 i n c r e a s e i n 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 e g r o w i n g e e l Is ( F i g u r e 1 3 ) • ( i i ) 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 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 t r a n s p o r t p r o c e s s ( F i g u r e 1 4 ) . Sodium a z i d e o r a - i o d o a c e t a m i d e , a t a c o n c e n t r a t i o n 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% and i n c o m b i n a t i o n , by a l m o s t 80%. S u l f -h y d r y l - b i n d i n g a g e n t s were 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 90% ( 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 grown 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 c o l o n y f o r m i n g u n i t s Amount o f F e + + + C o n t r o l F i l t e r e d , washed C e n t r i f u g e d , F i l t e r e d , washed and r e s u s p e n d e d washed and r e -ad d e d t o t h e ( n o t washed) and r e s u s p e n d e d i n e x t r a c t e d s u s p e n d e d i n g r o w t h medium i n normal medium medium e x t r a c t e d medium 512 215 171 266 5.5 y g a t o m s / 1 i t e r 622 126 299 422 18.1 y g a t o m s / l i t e r 38 72 171 145 66 500-400-tO UJ o 300-oo 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 age and p h e n o l i c a c i d p r o d u c t i o n . S y m b o l s : 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 . r l . = ' ' . 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. 59 0.12. p e r ml 510 nm 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 atoms 67 CO UJ o 600 500-400" ® Q 300-o. 200-100-10 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 grown 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 . S y m b o l s : 0 0, no a d d i t i o n s ; • — 1 mM NaN^; A A, 1 mM a i o d o a c e t a m i d e ; A — A , 1 mM NaN^ + 1 mM a - i o d o a c e t a m i d e ; Q •; 1 mM N a N ^ p - c h l o r o m e r c u r i b e n z o a t e ; X X, 50 yM H g C l 2 ; , 1 mM s o d i u m a r s e n i t e . 59 59 A d d i t i o n s were'made 10 m i n u t e s p r i o r t o F e . 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 / m l . uM c o n c e n t r a t i o n had no e f f e c t on i r o n u p t a k e c a p a -c i t y ( n o t shown). ( i i i ) E f f e c t o f g l u c o s e 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 w e r e washed and a l l o w e d t o s t a r v e f o r l i h o u r s i n t h e 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 f r o m 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 an u n s t a r v e d c o n t r o l ( F i g u r e 1 5 ) . I f t h e s t a r v e d c e l l s 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 -c r e a s e d a b o u t 3 - f o l d ( F i g u r e 1 5 ) . T h e r a t e o f i r o n u p t a k e i n t h e s t a r v e d 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 t h a n t h a t o f t h e u n s u p p l e m e n t e d c e l l s ( F i g u r e 1 5 ) . ( i v ) I r o n u p t a k e a s 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 s h o r t l y a f t e r F e , 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 amount o f i r o n bound t o t h e c e l l s ( F i g u r e 1 6 ) . T h e amount o f r a d i o -a c t i v e 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 and 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 a d d i t i o n o f s u c h r e a g e n t s ( F i g u r e 1 6 ) . (v) S p e c i f i c i t y o f i r o n u p t a k e The u p t a k e o f r a d i o a c t i v e i r o n was n o t a f f e c t e d by t h e a d d i t o n o f Go , Ni , Zn o r Mn i o n s 69 10 20 30 40 MINUTES F i g u r e 15. 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 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 . S y m b o l s : I • , - c e l l s w e re washed o n c e and r e s u s p e n d e d i n medium w i t h 0.2% g l u c o s e , t h e n a l l o w e d t o i n c u b a t e 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 ere 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 . Fe was added a t 100 pg a t o m s / ml c o n c e n t r a t i o n . 70 ~l 1 i 10 20 30 MINUTES F i g u r e 16. E f f e c t o f l i p o l y t i c compounds on i r o n u p t a k e by J3_. s u b t ? 1 ?s 59 B1471 grown w i t h o u t a d d e d i r o n . Fe was "added a t 50 pg atomsyml c o n c e n t r a t i o n . O t h e r a d d i t i o n s w ere made a t +10 m i n u t e s . S y m b o l s : 0 — - 0 , no a d d i t i o n s ; • — i , 0.1% t o l u e n e ; A-^—A, 0.1% s o d i u m l a u r y l s u l f a t e . (F igure 17). When un labe l led i ron equal to the amount of r a d i o a c t i v e i r o n , was added to the f l a s k at zero t ime , the amount of l abe l taken up by the c e l l s was reduced by some 30%.When a 6-fo ld or a 50- fo ld excess of un labe l led i ron was added to the f l a s k a f t e r the 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 of r a d i o a c t i v i t y stopped almost immediate ly , and the amount of l abe l held by the c e l l s remained constant (F igure 17) . +++ P r e l i m i n a r y r e s u l t s i n d i c a t e that Cr ions i n t e r f e r e w i th i ron uptake in B_. sub t i 1 is by reducing 59 the amount of Fe taken up by the c e l l s . Experiment With Iron S u f f i c i e n t Cu l tu res (?) E f f e c t on uptake c apac i t y When J3. sub t i 1 is was grown in the presence of 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 ron and then tes ted f o r i t s a b i l i t y to take up r a d i o a c t i v e i ron from the growth medium, the amount of l abe l taken up was very much less than that re ta ined by c e l l s grown without added i ron (Table 14, F igure 18). A 5-fo ld increase in 59 the amount of Fe added to the incubat ion mixture r e -su l t ed 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 cons ide rab l y l ess than that by the i r o n - d e f i c i e n t c e l l s (F igure 19). C e l l s grown in the presence of a low l eve l of Co 700-600-500-1 to - i _J L U 4001 300-x I 200-1 100-— A 15 Pi 35 "15 35 MINUTES F i g u r e 17. 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 ++ S y m b o l s : 0- Ni Zn o r Mn a t -0, no a d d i t i o n s , Co 600 pg a t o m s / m l ; A — A , F e + + + , 100 pg a t o m s / m l ; I F e + + + , 600 pg atoms/ml a d d e d a t 7.5 m i n u t e s ( + ) ; k A , F e + + + , 5 ng a t o m s / m l A A l l o t h e r a d d i t i o n s w ere made 3 m i n u t e s b e f o r e F e , [ a d d e d a t 2.5 m i n u t e s ^ w h i c h 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 . was i n -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 added i r o n . S y m b o l s : 0 — 0 , 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 grown w i t h 5.5 y g atoms F e + + + / l i t e r a d d e d ; A — A . c e l l s grown w i t h 18.1 y g atoms F e + + + / l i t e r . " ^ F e 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 / m l . MINUTES F i g u r e 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 . ' S y m b o l s : 0 0, c e l l s grown w i t h o u t added 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 a d d e d 59 i r o n , 100 pg atoms/ml Fe a d d e d ; A — A , c e l l s grown w i t h 18.1 y g a t o m s / l i t e r F e + + + , 500 pg atoms/ml "'"Ve a d d e d ; A — A , c e l l s grown w i t h 18.1 y g a t o m s / l i t e r F e + + + 59 100 pg atoms/ml Fe a d d e d . 75 i o n s a p p e a r e d t o l y s e i m m e d i a t e l y upon w a s h i n g . Un-washed c e l l s , h o w e v e r , showed an i n c r e a s e d i r o n u p t a k e c a p a c i t y ( p e r s o n a l o b s e r v a t i o n s ) . (d) 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 L e v e l s o f I r o n . G r o w t h  In C h e m i c a l l y 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-t a k e c a p a c i t y . G rowth o f J 3 . s u b t ? 1 i s i n c h e m i c a l l y e x t r a c t e d medium was e x t r e m e l y s l o w and v a r i a b l e . P h e n o l i c a c i d p r o d u c t i o n by 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 b e tween 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 and t h e i r 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 ( F i g u r e 2 0 ) . (e) I r o n U p t a k e a s a F u n c t i o n o f C h e l a t i n g A g e n t s ( 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 and 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 and EDTA were 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 and t h e n a d d e d t o J 3 . s u b t i 1 i s B1471 c e l l s grown 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 , a c h a n g e i n t h e amount o f l a b e l t a k e n up was o b s e r v e d ( F i g u r e 2 1 ) . D e s f e r r i f e r r i c h r o m e and 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% and 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 amount o f l a b e l t a k e n up by t h e c e l l s by some 30% ( F i g u r e 2 1 ) . 76 ? 10 \5 20 iS 30 MINUTES F i g u r e 20. I r o n u p t a k e by _B. s u b t ? 1 i s B1471 grown w i t h d i f f e r e n t l e v e l s o f i r o n i n e x t r a c t e d medium. S y m b o l s : 0 0, no a d d i t i o n s , O.D.-.. = 0: • • ' .. + + + 510 nm 1 y g atoms Fe / l i t e r , 0.D._ ' = 0 . 2 1 ; • — A ; + + + . 510 nm ' ' h y g atoms Fe / l i t e r , 0 . D. r l r i = 0 . 1 6 , Q T~\ ; +++ 5 1 0 n m (S 8 y g atoms Fe / l i t e r ^ O .D. r l. = 0 . /67 • 5 1 0 n m. F i g u r e 21. 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 V . . s u b t i l i s * B1471' 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 . — " ' 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 atoms/ml S y m b o l s : 0 - 6 , no a d d i t i o n s ; • — i , DHBG; A — A , d e s -' f e r r i f e r r i c h r o m e ; [ - ; 'EDTA. 5 10 15 20 25 30 MINUTES ( i i ) E f f e c t o f v a r y i n g c o n c e n t r a t i o n s o f DHBG. 59 I t was f o u n d t h a t t h e i n c r e a s e i n u p t a k e o f Fe f r o m t h e medium by c e l l s grown 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 a d d e d w i t h t h e F e , a l t h o u g h t h e r e l a t i v e i n c r e a s e was q u i t e s m a l l ( F i g u r e 2 2 ) . s u b t i 1 i s M u t a n t S t r a i n s 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 m u t a n t s t r a i n s w ere e x a m i n e d o r d e r t o c o r r e l a t e p h e n o l i c a c i d p r o d u c t i o n w i t h i r o n u p t a k e . (a) S h i k i m i c A c i d R e q u i r i n g M u t a n t T h e 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 t y p e s t r a i n ( F i g u r e 2 3 ) . D e s f e r r i f e r r i c h r o m e , DHB and DHBG s t i m u l a t e d u p t a k e . 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 i n t h i s s t r a i n . (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 The 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 r e s i s t a n t m u t a n t s were 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 t h e w i l d t y p e ( F i g u r e 2k). S u r p r i s i n g l y , s t r e p t o m y c i n s t i m u l a t e d u p t a k e s l i g h t l y . P h e n o l i c a c i d p r o d u c t i o n i n b o t h t h e s e s t r a i n s was l e s s t h a n i n t h e w i l d t y p e ( T a b l e 1 5 ) . (c) A l b o m y c i n R e s i s t a n t S t r a i n s 59 A l t h o u g h t h e i m m e d i a t e b i n d i n g o f Fe by an a l b o m y c i 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 t y p e , 79 : 1 r 1 1 - i r 5 10 15 20 25 30 MINUTES F i g u r e 22. 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 ? 1 i s B1471 grown 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 . Fe was a d d e d t o 20 pg atoms/ml f i n a l c o n c e n t r a t i o n . S y m b o l s : 0 0, no a d d i t i o n s ; • — 8, 10 yM DHBG; A A, 1.0 yM DHBG; A A, 0.1 yM DHBG. 250-200-150-100-50-10 15 20 MINUTES 25 30 F i g u r e 23. 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 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 . S y m b o l s : 0 0, no a d d i t i o n ; A A, 1.0 yM d e s f e r r i -f e r r i c hrome; A — A , 1.0 yM DHBG; • — i , 50 yM DHB. •c-7--59 Compounds were 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 a d d e d . 59 Fe was p r e s e n t .at 100 pg atoms/ml c o n c e n t r a t i o n . 5 10 15 20 25 30 MINUTES F i g u r e 2k. 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 J b t i 1 i s B1471 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 . S y m b o l s : 0 — 0 , A5 w i t h o u t ' S m a d d e d ; 9 f, A5 w i t h 100 yM Sm a d d e d ; A — A, A9 w i t h o u t Sm; A A, A9 w i t h 100 yM Sm. 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 i r o n . cpm x 1 0 3 / 1 0 8 c e l l s i n 2 m i n u t e s i n 30 m i n u t e s O.D. r l / % 510 nm •Fe +Fe* -Fe +Fe -Fe +Fe A5 5.9 - 57-4 - .16 A9 4.0 - 40.3 - .05 A R 1 2 3 6.3 18.5 57.1 29.0 0.25 0.16 C o R 13.2 5.3 40.9 38.3 0.40 0.32 H R 5.6 4.6 56.4 25.1 0.19 0.09 F e R 8.5 21.4 34.6 75.9 0.28 0.20 F e R H R 4.3 14.5 30.0 58.4 0.24 0.18 B l 4 7 1 8.2 5.6 75.2 54.8 .24 0.12 +Fe* 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 F e C l ^ . t h e t o t a l amount bound i n t h i r t y m i n u t e s was l e s s ( T a b l e 1 5 ) . When grown 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 was r e p r e s s e d a b o u t 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 t y p e . (d) C o b a l t R e s i s t a n t M u t a n t 59 A g a i n t h e i n i t i a l b i n d i n g o f Fe by t h e mu t a n t s t r a i n was g r e a t e r t h a n i n t h e w i l d t y p e , w h i l e t h e t o t a l amount bound 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 by g r o w t h w i t h 0.1 mM F e C l ^ . 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 g r e a t e r t h a n by t h e w i l d t y p e . (e) Hemin R e s i s t a n t M u t a n t T h e i r o n u p t a k e c a p a c i t y o f a hemin r e s i s t a n t m utant 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 when grown w i t h o u t added i r o n ( T a b l e 1 5 ) . When grown 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 t h a n 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 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 t y p e . ( f ) I r o n R e s i s t a n t M u t a n t s T h e i r o n u p t a k e c a p a c i t y o f an i r o n r e s i s t a n t m u t a n t 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 t h e w i l d t y p e ( T a b l e 1 5 ) . However, when grown 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 . 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 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 w e re 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 I r o n R e s i s t a n t M u t a n t The 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 hemin and i r o n was s i m i l a r t o t h a t o f t h e i r o n r e s i s t a n t s t r a i n ( T a b l e 15). D i s c u s s i o n o f R e s u l t s T h e work o f P e t e r s (44) and o f P e t e r s and W a r r e n (46) and t h e r e s u l t s o f t h i s s t u d y i n d i c a t e an e n e r g y - d e p e n d e n t mechanism f o r i r o n u p t a k e i n J3. s u b t ? 1 ? s . M o r e o v e r , i t i s e v i d e n t t h a t t h e c a p a -c i t y o f t h i s o r g a n i s m t o t a k e up i r o n f r o m t h e g r o w t h medium when i t i s grown 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 i n c r e a s e s s i g n i f i c a n t l y d u r i n g a p a r t i c u l a r p h y s i o l o g i c a l s t a g e o f g r o w t h . T h i s c h a n g e i n i r o n u p t a k e c a p a c i t y i s c l o s e l y a s s o c i a t e d w i t 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 by t h e c e l l s . P e t e r s a n d W a r r e n (46) w i t h B_. s u b t ? 1 i s and Wang and Newton (67, 68) w i t h E_. c o l i h ave a l s o shown a c l o s e a s s o c i a t i o n between 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 p r o d u c t i o n . 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 s e v e r e l y \ > d e c r e a s e d a l s o show d e c r e a s e d i r o n u p t a k e a b i l i t y (46, 68). T h e n e c e s s i t y o f m e t a b o l i c a c t i v i t y f o r i r o n u p t a k e i s e v i d e n t 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 a - i o d o -a c e t a m i d e . T h e r e s i d u a l a c t i v i t y f o u n d a s s o c i a t e d w i t h t h e c e l l s i n t h e p r e s e n c e o f t h e m e t a b o l i c i n h i b i t o r s may r e p r e s e n t i r o n h e l d by 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 s u r f a c e . T h e e f f e c t o f ' s u l f h y d r y l i n h i b i t o r s s u c h as p - c h 1 o r o m e r c u r i b e n z o a t e , s o d i u m a r s e n i t e , o r H g + + i s i n t e r e s t i n g . T h e s e e f f e c t i v e l y i n h i b i t u p t a k e o f i r o n f r o m t h e g r o w t h medium and r e d u c e t h e amount o f r e s i d u a l i r o n bound by t h e c e l l s . T h i s may be t a k e n t o i n d i c a t e t h a t i f a s p e c i f i c i r o n - b i n d i n g o r i r o n t r a n s p o r t p r o t e i n d o e s e x i s t , i t c o n t a i n s s u l f h y d r y l g r o u p s . 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 r e s p o n s i b l e f o r r e l e a s i n g i r o n f r o m a c h e l a t e d c o m p l e x and m a k i n g i t a v a i l a b l e t o t h e c e l l s . T h e d e c r e a s e o f u p t a k e c a p a c i t y i n c e l l s s t a r v e d f o r g l u c o s e i s f u r t h e r e v i d e n c e f a v o r i n g an e n e r g y r e q u i r e m e n t f o r t h e a c t i v e u p t a k e o f i r o n . T h e 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 l e v e l o f r a d i o a c t i v i t y a f t e r t r e a t m e n t o f t h e c e l l s w i t h t o l u e n e o r s o d i u m l a u r y l s u l f a t e , b o t h o f w h i c h 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 l y s i s ( 5 9 ) , w o u l d i n d i c a t e t h a t p a r t ; 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 bound t i g h t -l y by t h e c e l l s . As t h e t i m e i s i n c r e a s e d b e t ween t h e a d d i t i o n o f r a d i o a c t i v e i r o n and t h e a d d i t i o n o f compounds s u c h 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 and e v e n m e t a b o l i c i n h i b i -t o r s , l e s s , l a b e l l e d i r o n i s l o s t f r o m t h e b a c t e r i a l c e l l s . T h i s i s t a k e n t o mean t h a t t h e i r o n - b i n d i n g c a p a c i t y o f t h e c e l l s i s g r e a t , 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 u t i l i z a t i o n o f t h e i r o n t a k e n up f r o m t h e medium. T h e 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 i o n s d i d n o t i n t e r f e r e w i t h t h e u p t a k e o f F e . P r e l i m i n a r y e x p e r i m e n t s , h o w e v e r , i n d i c a t e +++ t h a t C r i o n s i n t e r f e r e w i t h i r o n u p t a k e , s u g g e s t i n g t h a t t h e u p t a k e m e c h a n i s m i s n o t 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 . T h e a d d i t i o n o f u n l a b e l l e d FeC1^ a t any t i m e 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 ments c a u s e d an i m m e d i a t e d e c r e a s e i n Fe u p t a k e , due t o d i l u t i o n 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 i o n s . I t i s i m p o r t a n t t o p o i n t o u t t h a t t h i s d e c r e a s e i n i r o n u p t a k e d o e s n o t 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 t h e u p t a k e s y s t e m f o r i r o n b u t d o e s i l l u s t r a t e t h e s p e c i f i c i t y o f t h e s y s t e m f o r t h e c a t i o n . When J3_. s u b t i 1 i s was 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 , 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 . A s i m i l a r r e s u l t has been shown f o r i r o n u p t a k e i n E_. c o l ? ( 6 8 ) . However, t h e 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 n o t e l i m i n a t e d c o m p l e t e l y , as 59 s e e n when a h i g h e r l e v e l o f Fe was a d d e d t o t h e c e l l s . S i n c e 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 s y s t e m were n o t p e r f o r m e d , 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 t h e l a b e l l e d 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 r e d u c e d when i n r e a l i t y i t r e m a i n e d 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 -i n g a g e n t . 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 -chrome was l e s s t h a n t h a t s e e n p r e v i o u s l y ( 4 6 ) . The c o n c e n t r a t i o n 59 o f DHBG m i x e d w i t h Fe had an e f f e c t on t h e amount o f i r o n t a k e n up by t h e c e l l s . However, t h e d i f f e r e n c e b e tween 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 W a r r e n (46) c a n n o t be r e a d i l y e x p l a i n e d a t p r e s e n t . 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 m e t a b o l i s m may h a v e a l t e r e d i n s u c h a way t h a t DHBG no l o n g e r s e r v e s a s an e f f e c t i v e i r o n t r a n s p o r t f a c t o r . A h y d r o x a m a t e 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 and 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 were u n s u c c e s s f u l . However, t h e i r o n u p t a k e s y s t e m i n s t r a i n B1471 d o e s r e s p o n d t o f e r r i c h r o m e , a h y d r o x a m a t e o f known b i o l o g i c a l a c t i v i t y ( 4 0 ) . 3. M i c r o c o c c u s l y s o d e i k t i c u s (a) P r e l i m i n a r y E x p e r i m e n t s 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 i r o n -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 i n t h e s u s p e n d i n g medium, was v a r i a b l e . P r e l i m i n a r y r e s u l t s i n d i c a t e d t h a t i r o n p o l y m e r i z e d and r e m a i n e d bound t o t h e f i l t e r s , t h e r e b y l e a d i n g t o e r r o n e o u s l y h i g h c o u n t s o f r a d i o a c t i v i t y . 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 grown 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 grown 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 medium c o n t a i n i n g c i t r a t e b e f o r e u p t a k e e x p e r i m e n t s were r u n . (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 on U p t a k e C a p a c i t y C e l l s grown 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 a d d e d i r o n had a g r e a t e r i r o n u p t a k e c a p a c i t y t h a n c e l l s grown w i t h o u t a d d e d i r o n ( F i g u r e 2 5 ) . C e l l s grown 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 have no a c t i v e u p t a k e m echanism f o r i r o n ( F i g u r e 2 6 ) . ( 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 C a p a c i t y G rowth 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 o r b e n z o h y d r o x a m i c 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 e x t r a c t 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 c u l t u r e s ( F i g u r e 2 5 ) . (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 I r o n U p t a k e i n v i t r o EDTA, NTA and 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 t a k e n up by t h e c e l l s ( F i g u r e 2 7 ) . D e s f e r r i f e r r i c h r o m e A a l s o d e c r e a s e d t h e amount o f i r o n t a k e n up ( F i g u r e 2 8 ) . 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, c a u s e d a 100% i n c r e a s e , i n t h e amount, o f . i r o n t a k e n up, w h e r e a s b e n z o h y d r o x a m a t e and f e r r i c h r o m e A had a s l i g h t s t i m u l a t o r y e f f e c t ( F i g u r e 2 8 ) . C a t e c h o l , o - h y d r o x y h i p p u r i c a c i d , DHB and 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 up by M_. 59 l y s o d e i k t i c u s ( F i g u r e 2 9 ) . T h e amount o f Fe 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 -c e n t r a t i o n ( F i g u r e 3 0 ) . 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^ . l y s o d e i k t i c u s . S y m b o l s : 0 — 0 , 0.1% c i t r a t e w i t h o u t added i r o n ; I i , 0.1% c i t r a t e w i t h 1 yM F e C ^ ; A A, 1 yM f e r r i c h r o m e ; A A, 1 yM NTA; • p , 1 yM EDTA; £ | , 1 yM b e n z o h y d r o x a m i c a c i d . — I 1 "1 1 — I — I 1— 1 5 10 15 20 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 w i t h o u t ' c i t r a t e . S y m b o l s : 0 0, no a d d i t i o n s ; • 1 mM NaN^ + I mM a - i o d o a c e t a m i d e ; 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 grown w i t h o u t o r w i t h 1.2 y g atoms/1 i t e r . 9 1 T 1 1 1 1 1— 5 10 15 20 25 30 MINUTES F i g u r e 27. E f f e G 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 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 medium 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 i r o n . S y m b o l s : 0 — 0, no a d d i t i o n s ; 6 — 9, EDTA; A A, NTA; A A, T i r o n . CO A l l c h e l a t i n g a g e n t s were p r e i n c u b a t e d w i t h Fe and a d d e d t o 1 yM f i n a l c o n c e n t r a t i o n . 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_. 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 medium 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 i r o n . S y m b o l s : 0 0, no a d d i t i o n s ; • §, f e r r i c h r o m e A; A — 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 a c i d ; • -—Q , 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 w i t h a f i n a l c o n c e n t r a t i o n o f I yM. 59 Fe and a d d e d t o MINUTES F i g u r e 29. E f f e c t o f p h e n o l i c compounds on 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 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 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 . S y m b o l s : 0 0, no a d d i t i o n s ; • • , c a t e c h o l ; A A, DHB; • A, o - H y d r o x y h i p p u r i c a c i d ; Q — — • , DHBG. A l l a d d i t i o n s w ere made t o 1 yM f i n a l c o n c e n t r a t i o n a f t e r p r e - i n c u b a t i o n w i t h F e . 5 io~ 15 20 MINUTES r 25 30 F i g u r e 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 on i r o n u p t a k e ty 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 medium 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 i r o n . Symbols.: 0 0, no a d d i t i o n s ; • •, 50 yM DHBG; A A, 10 yM DHBG; A 4,1,.0 yM DHBG; • • , 0.1 yM DHBG. 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 w i t h Fe and t h e n a d d e d r t o t h e e e l 1s. 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% ( F i g u r e 3 1 ) . D i s c u s s i o n o f R e s u l t s 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 , numerous p r e l i m i n a r y e x p e r i m e n t s were p e r f o r m e d u s i n g v a r i o u s m e d i a and b u f f e r s t o d e t e r m i n e o p t i m a l c o n d i t i o n s f o r u p t a k e e x p e r i m e n t s . 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 s u s p e n d i n g f l u i d t o p r e v e n t t h e r e t e n t i o n o f s i g n i f i c a n t l e v e l s o f r a d i o a c t i v i t y on 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 an i r o n u p t a k e s y s t e m 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 . When grown i n m e d i a 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 have an a c t i v e m e c h a n i s m f o r t h e u p t a k e o f i r o n . I t a p p e a r e d t h a t i r o n s i m p l y d i f f u s e d t h r o u g h t h e c e l l membrane. Growth o f t h e o r g a n i s m i n t h e p r e s e n c e o f a d d e d i r o n when t h e medium 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 s u c h as c i t r a t e , d i d n o t a p p e a r t o r e p r e s s t h e i r o n u p t a k e m e c h a n i s m . On t h e c o n t r a r y , i r o n seemed t o i n d u c e t h e t r a n s p o r t 59 o f Fe i n t o t h e c e l l s . Growth 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 i n t h e p r e s e n c e o f c i t r a t e d i d n o t a p p e a r t o s t i m u l a t e t h e i r o n 96 1 10 20 30 MINUTES 40 5*0 F i g u r e 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 -8 grown w i t h y e a s t e x t r a c t p l u s 10 M EDTA and w i t h o u t ' a d d e d i r o n . S y m b o l s : 0 - 1 — 0 , no a d d i t i o n s ; • i , a l b o m y c i n (0.1 yM CO c o n c e n t r a t i o n ) a d d e d 3 m i n u t e s p r i o r t o F e . 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, g r o w t h 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 u p t a k e . T he 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 e x t e n t on t h e t y p e o f c h e l a t i n g a g e n t . C o n s e q u e n t l y , one m i g h t s u s p e c t t h a t a d e g r e e o f s p e c i f i c i t y e x i s t s i n t h i s m i c r o o r g a n i s m f o r a c h e l a t i n g a g e n t w h i c h " i n d u c e s " i r o n u p t a k e . A l b o m y c i n a p p e a r s t o i n h i b i t i r o n u p t a k e t o a s l i g h t e x t e n t . T h i s s u p p o r t s , b u t d o e s n o t d i s t i n g u i s h b e t w e e n , t h e s u g g e s t i o n s 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 m e t a b o l y t e a c r o s s t h e c e l l membrane (65), o r t h a t i t i n t e r f e r e s w i t h t h e i n t e r n a l u t i l i z a t i o n o f F e + + + i o n s o n c e t h e y a r e i n s i d e t h e c e l l (75). k. S a l m o n e l l a t y p h i m u r i u m s t r a i n LT-2 (a) I r o n U p t a k e ( i ) G rowth 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 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 I r o n U p t a k e When s o d i u m a z i d e and 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 washed c e l l s o f S_. t y p h i m u r i u m , t h r e e 59 m i n u t e s p r i o r t o t h e 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 t h a t o f t h e c o n t r o l . T h e a d d i t i o n o f p - n i t r o p h e n o l 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 i n up-t a k e ( F i g u r e 3 2 ) . 5 10 15 20 25 30 MINUTES F i g u r e 32. E f f e c t o f m e t a b o l i c 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 LT-2 grown w i t h o u t a d d e d i r o n . A l l a d d i - . 59 t i o n s were made 10 m i n u t e s p r i o r t o F e . S y m b o l s : 0 ' — 0 , 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 , 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 59 mM) . - ^ F e was p r e s e n t a t 40 pg atoms/ml c o n c e n t r a t i o n . 99 b. E f f e c t o f I r o n - C h e l a t i n g A g e n t s 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 w i t h Fe and t h e m i x t u r e s a dded 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 p a t t e r n s w e r e o b s e r v e d ( F i g u r e 33)- EDTA c a u s e d a 50% r e d u c t i o n w h e r e a s DHBG c a u s e d an a p p r o x i m a t e 50% i n c r e a s e i i i u p t a k e . DHB and d e s f e r r i f e r r i c h r o m e b o t h i n c r e a s e d u p t a k e a b o u t 3 " f o l d ( F i g u r e 33). Th e i n i t i a l 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 r e a s e d 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 no 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 S y s t e m 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 -59 / b a t i o n m i x t u r e , t h e u p t a k e o f Fe was r e d u c e d ( n o t sh o w n ) . P r e l i m i n a r y r e s u l t s i n d i c a t e a d r a m a t i c 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 Fe bound i n i t i a l l y by t h e c e l l s when C r i o n s a r e ad d e d 59 +++ a f t e r F e . T h e a d d i t b n o f C r i o n s t e n m i n u t e s b e f o r e t h e a d d i t i o n o f r a d i o a c t i v e i r o n , a p p e a r s t o s t i m u l a t e i r o n u p t a k e c a p a c i t y more t h a n 100%. F u r t h e r 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 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_. t y p h i m u r i u r n a r e un d e r w a y . 100 — -r -i 1 i r r 5 10 15 20 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 on i r o n u p t a k e by S .'• t y p h i mur- ium 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 ' . S y m b o l s : 0 J — 0 , no a d d i t o n s ; • — i , DHB; A " — A , DHBG; A A, d e s f e r r i f e r r i c h r o m e ; • • EDTA. A l l compounds were p r e i n c u b a t e d a t 1 mM c o n c e n t r a t i o n w i t h 5 9 F e . 101 ( i i ) G r o w t h U n d e r 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 C a p a c i t y . C e l l s grown i n t h e p r e s e n c e o f 0.1 mM F e C l ^ had a b o u t 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 u p t a k e as c e l l s grown w i t h o u t added i r o n ( F i g u r e 34). T h e a d d i t i o n o f DHBG i n c r e a s e d t h i s u p t a k e c a p a c i t y a b o u t 3 - f o l d ( F i g u r e 34). (b) I r o n 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 , w ere washed and r e s u s p e n d e d i n i r o n -d e f i c i e n t medium, 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 ( T a b l e 1 6 ) . T h e a d d i t i o n o f e x c e s s u n l a b e l l e d i r o n o r 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 no a d d i t i o n a l l o s s o f l a b e l ( T a b l e 1 6 ) . T o l u e n e and s o d i u m 59 a r s e n i t e c a u s e d f u r t h e r l o s s o f Fe f r o m t h e c e l l s , a - i o d o a c e t a m i d e a p p e a r e d t o c a u s e an i n c r e a s e |n t h e amount o f r a d i o a c t i v i t y bound t o t h e c e l l s ( T a b l e 1 6 ) . 5. -S_. t y p h i m u r ? u r n M u t a n t S t r a i n s (a) P r e l i m i n a r y E x p e r i m e n t s 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 t h a t s t r a i n s f e r l and fer3 were s i m i l a r , a s were s t r a i n s f e r D 9 and ferD19- 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 u s e d i n a l l s u b s e q u e n t e x p e r i m e n t s MINUTES F i g u r e . 3 4 . 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 ^ and t h e e f f e c t o f . DHBG on i r o n u p t a k e . S y m b o l s : 0 0, 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 ; • c e l l s grown w i t h o u t i r o n , 1 yM DHBG ad d e d w i t h 59 F e ; A A, c e l l s grown w i t h i r o n ; no a d d i t i o n s ; A A, c e l l s grown w i t h i r o n , 1 yM DHBG added 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 on Fe e f f l u x f r o m S_. t y p h imur i um. Compound a d d e d C o n c e n t r a t i o n cpm/10 c e l l s a f t e r (mM) 0 T i m e 10 M i n u t e s 30 M i n u t e s N i l - 2160 2270 1928 F e C l 3 0 .1 2230 2300 2050 D e s f e r r i f e r r i c h r o m e 1 0 .1 2163 2108 2062 1 t o i c a c i d 0 .1 2294 2222 2240 F e C l ^ + I t o i c a c i d 0 .1 2190 2250 2150 F e C l ^ + F e r r i c h r o m e 0 . 1 2260 2210 1930 T o l u e n e (0 .n) 2342 2214 1616 a l o d o a c e t a m i d e 1 .0 2498 2080 2445 a l o d o a c e t a m i d e + F e C l ^ 1 .0 1815 1953 2090 Sod i u m a r s e n i t e 1 .0 2895 2840 2607 Sod i u m a r s e n i t e + F e C l ^ 1 .0 1967 2012 1926 Sod i u m a z i d e 1 .0 2112 2279 2206 S o d i u m a z i d e + F e C l , 1 .0 2033 1966 1612 104 (b) C o m p a r i s o n w i t h W i l d T y p e 59 U p t a k e o f Fe i n b o t h f e r l a nd f e r D 1 9 was l e s s t h a n , b u t o t h e r w i s e s i m i l a r t o t h a t o f t h e p a r e n t 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 W i t h t h e f e r l s t r a i n , DHB and 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 d e c r e a s e . 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 h a n d , c a u s e d 59 a b o u t a 5 _ f o l d i n c r e a s e i n t h e amount o f Fe t a k e n up by t h e m u t a n t c u l t u r e ( F i g u r e 36). W i t h m u t a n t f e r D 1 9 , DHB, DHBG and EDTA a l l r e s u l t e d i n a d e c r e a s e i n i r o n u p t a k e , w h e r e a s - d e s f e r r i f e r r i c h r o m e 59 c a u s e d a 1 0 - f o l d i n c r e a s e i n Fe u p t a k e ( F i g u r e 37). D i s c u s s i o n o f R e s u l t s T h e e f f e c t s o f m e t a b o l i c 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 an a c t i v e t r a n s p o r t s y s t e m . T h i s s y s t e m , 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 c e r t a i n i r o n b i n d i n g a g e n t s , and 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 t h e c o n c e n t r a t i o n o f i r o n i n t h e medium. DHB, DHBG and d e s f e r r i -f e r r i c h r o m e s t i m u l a t e 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 i n JJ. s u b t i 1 i s , w i t h d e s f e r r i f e r r i c h r o m e a g a i n g i v i n g t h e g r e a t e s t s t i m u l a -t i o n . 105 T 5 i 10 T r 15 20 MINUTES r 25 T 30 F i g u r e 35. 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 and +++ m u t a n t s . M u t a n t s t r a i n s w ere grown w i t h k y g atoms Fe ''/ml S y m b o l s : 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 m u t a n t f e r 1 grown w i t h k y g atoms o f F e + + + / m l . ' S y m b o l s : 0 — 0, no a d d i t i o n s ; © — 9, DHB; A — A , DHBG; A — - A , d e s f e r r i f e r r i c h r o m e ; Q a ,EDTA. A l l compounds were p r e i n c u b a t e d w i t h " ^ F e a t 1 mM c o n -c e n t r a t i o n . 107 250-^ 200 hi O 00 l2 2 loo o 150 T r 10 20 MINUTES r 30 F i g u r e 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 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 grown w i t h k y g atoms o f F e + + + / m l . S y m b o l s : 0 - — 0 , no a d d i t i o n s ; • — • , DHB; A A, DHBG, A — A , d e s f e r r i f e r r i c h r o m e , • • , EDTA. 59 A l l compounds were p r e i n c u b a t e d w i t h Fe a t 1 mM c o n -c e n t r a t i o n . As w i t h J3_. s u b t i 1 i s and 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 ^ r e d u c e d i t s i r o n u p t a k e c a p a c i t y c o n s i d e r a b l y . However, 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 . T h e r e -f o r e , DHBG a p p e a r e d t o s e r v e a s an i r o n t r a n s p o r t f a c t o r u n d e r a l l c o n d i t i o n s o f g r o w t h . I r o n r e m a i n e d t i g h t l y bound t o t h e c e l l s a f t e r i t was t a k e n up by S_. t y p h i m u r i u m , s i n c e t h e r e was no l o s s o f l a b e l f r o m c e l l s 59 grown w i t h Fe and t h e n 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 i r o n . However, d e s f e r r i f e r r i c h r o m e a p p e a r e d t o c a u s e l o s s o f l a b e l u n d e r t h e same c o n d i t i o n s . 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 . T h e i n t e g r i t y o f t h e c e l l membrane t h e r e f o r e a p p e a r e d i m p o r t a n t i n r e t a i n i n g bound i r o n w i t h i n t h e c e l l . When t h e c e l l s w e re t r e a t e d w i t h s o d i u m a z i d e i n t h e p r e s e n c e o f e x c e s s u n l a b e l l e d i r o n , o r s o d i u m a r s e n i t e 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 . I t i s n o t 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 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 t h e b a c t e r i a l e e l 1. The 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 t y p e i n t h e i r r e s p o n s e t o c h e l a t i n g a g e n t s , b u t n o t o t h e r w i s e . DHB, DHBG and d e s f e r r i f e r r i c h r o m e s t i m u l a t e i r o n u p t a k e s i g n i f i c a n t l y i n t h e w i l d t y p e s t r a i n , b u t o n l y d e s f e r r i f e r r i c h r o m e i s e f f e c t i v e w i t h b o t h t y p e s o f m u t a n t . T h e e x a c t r o l e s o f f e r r i c h r o m e i n i r o n m e t a b o l i s m r e m a i n t o be e x p l a i n e d . 109 VI. 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 e n e r a l P r o p e r t i e s o f B a c i 1 l u s s u b t i 1 i s S t r a i n B1471. I n t r o d u c t i o n 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 p r o d u c t i o n i n B. s u b t i l i s , 3 _ f 1 u o r o b e n z o i c a c i d was p a r t i c u l a r l y e f f e c t i v e . T h e r e f o r e , t h i s compound was 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 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 and i r o n u p t a k e . S i n c e t h e compound was most e f f e c t i v e i n s t r a i n B1471, t h i s o r g a n i s m was c h o s e n f o r s t u d y . 1. Growth A l t h o u g h t h e r a t e o f g r o w t h was u n a f f e c t e d , t h e r e was a s l i g h t d e c r e a s e i n t h e f i n a l c e l l y i e l d when 1 mM 3 _ f l u o r o -b e n z o a t e was a d d e d t o t h e g r o w t h medium a t t h e t i m e o f i n o c u l a -t i o n . T h e r e was a s i m i l a r , b u t s m a l l e r , e f f e c t i f t h e i n h i b i t o r was a d d e d a f t e r 7 o r 11 h o u r s o f g r o w t h ( F i g u r e 38). 2. P h e n o l i c A c i d P r o d u c t i o n 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 t i m e r e d u c e d , b u t d i d n o t c o m p l e t e l y p r e v e n t , DHBG f o r m a t i o n ( F i g u r e 39). The 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 a d d e d , t h e s m a l l e r 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 ( F i g u r e 39). 3. Iron U p t a k e T h e c a p a c i t y o f B1471 t o t a k e up i r o n f r o m t h e g r o w t h medium was d e c r e a s e d by more t h a n 50% when t h e c e l l s w ere 110 500 400 300-200-!00-9 12 HOURS F i g u r e 38. 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 B1471 S y m b o l s : 0 ~ — 0 , no a d d i t i o n ; ® C, 1 mM 3 _ f 1 u o r o b e n z o i c a c i d a d d e d a t z e r o t i m e ; 6-—Q, 1 mM 3 ~ f 1 u o r o b e n z o i c a c i d a d d e d 7 h o u r s a f t e r i n o c u l a t i o n ; A- A, 1 mM 3 _ f l u o r o b e n -z o i c a c i d a d d e d 11 h o u r s a f t e r i n o c u l a t i o n . I l l F i g u r e 39. E f f e c t o f g r o w t h w i t h 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 61471. Symbols': 0 — 0 , no a d d i t i o n s ; © S, 1 mM 3 " f l u o r o b e n z o i c a c i d a d d e d a t ' z e r o t i m e ; A A, 1 mM f 1 u o r o b e n z o i c a c i d a d d e d 7 h o u r s a f t e r i n o c u l a t i o n ; A A , 1 mM 3 _ f l u o r o b e n -z o i c a c i d a d d e d 11 h o u r s a f t e r i n o c u l a t i o n . grown 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 i n t h e p r e s e n c e o f 1 mM 3 _ f 1 u o r o b e n z o a t e ( F i g u r e 40). D i s c u s s i o n o f R e s u l t s P e t e r s (44) f o u n d t h a t t h e DHB a n a l o g , 3 - f l u o r o b e n z o i c a c i d , e f f e c t i v e l y 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 o f JL* 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 3 _ f l u o r o -b e n z o i c a c i d as a m e t a b o l i c i n h i b i t o r . 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 i t s e f f e c t s on g r o w t h and i r o n u p t a k e c a p a c i t y as w e l l as on p h e n o l i c a c i d p r o d u c t i o n . Any e f f e c t o f 3 _ f l u o r o b e n z o a t e on p h e n o l i c a c i d p r o d u c t i o n 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 c o n s e -q u e n c e o f i n h i b i t i o n o f g e n e r a l c e l l m e t a b o l i s m . I t i s u n l i k e l y t h a t 3 - f l u o r o b e n z o a t e 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 d o e s n o t c a u s e a r e d u c t i o n i n p h e n o l i c a c i d p r o d u c t i o n ( p e r s o n a l o b s e r v a t i o n s ) . The r e d u c t i o n i n i r o n u p t a k e c a p a c i t y a s a c o n s e q u e n c e o f t h e 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 a g a i n p o i n t s t o t h e r e l a t i o n s h i p b e tween p h e n o l i c a c i d s and 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 10 15 20 25 30 MINUTES • » F i g u r e kO. E f f e c t o f g r o w t h w i t h 1 mM 3 _ f 1 u o r o b e n z o i c a c i d on i r o n up-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 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 . S y m b o l s : 0 - — 0 , no a d d i t i o n s ; 8—6, 1 mM 3 - f l u o r o b e n z o i c a c i d a d d e d a t t h e t i m e o f i n o c u l a t i o n . GENERAL DISCUSSION I t 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 m e t a b o l i s m o f t h e b a c t e r i a l c e l l , d e p e n d i n g upon t h e c h e l a t i n g a g e n t 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 a g e n t w h i c h s u p p o r t s i t s g r o w t h . I t was s u g g e s t e d e a r l i e r t h a t t h i s m i g h t ' b e t h e c a s e ( 3 9 ) - W h e t h e r t h i s d e g r e e o f s p e c i f i c i t y l i e s i n t h e s i z e , s h a p e o r s t a b i l i t y o f t h e c h e l a t e c o m p l e x i s u n c l e a r . P o s s i b l y s i z e and s h a p e p l a y an i m p o r t a n t r o l e i n t h e i n i t i a l s t a t e o f c o n t a c t o f t h e m e t a l - c h e l a t e c o m p l e x w i t h t h e m i c r o o r g a n i s m . It 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 f r o m t h e c o m p l e x w o u l d d i f f e r d e p e n d i n g on t h e c h e l a t i n g a g e n t and hene 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 e x i s t . In M_. l y s o d e i k t i c u s , EDTA and 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 do 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 f a c t o r . However, d e s f e r r i f e r r i c h r o m e and DHBG b o t h s e r v e e f f e c t i v e l y as i r o n t r a n s p o r t f a c t o r s . T h e me c h a n i s m o f 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 s t i l l r e m a i n s u n c l e a r . I t 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 w h e t h e r o r n o t i r o n e n t e r s t h e c e l l as a c o m p l e x , o r as a " f r e e i o n . In B_. s u b t i 1 i s and S_. t y p h i m u r i u r n a l s o , DHBG and d e s f e r r i -f e r r i c h r o m e a p p e a r t o s e r v e a s i r o n t r a n s p o r t f a c t o r s , w h e r e a s EDTA makes i r o n u n a v a i l a b l e t o t h e c e l l s . 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 w h i c h 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 and B_. s u b t ? 1 i s and 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 by t h e f o r m e r o r g a n i s m , has a s t r u c t u r e s i m i l a r t o t h a t o f f e r r i c h r o m e ( F i g u r e kl). T h e F e ( D H B G ) ^ and t h e Fe ( E D T A ) c o m p l e x e s a r e a l s o i l l u s t r a t e d f o r c o m p a r a t i v e p u r p o s e s ( F i g u r e 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 and 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 i n t h e p r e s e n c e o f h i g h l e v e l s o f i r o n . M_. l y s o d e i k t i c u s has an i n d u c i b l e u p t a k e m e c h a n i s m , w i t h e i t h e r a c h e l a t i n g a g e n t o r an i r o n . - c h e l a t e c o m p l e x s e r v i n g a s t h e i n d u c e r . E_. c o l ? has a r e p r e s s i b l e u p t a k 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 and S_. t y p h i m u r i u m (68) . However, 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 chr o m i u m i o n s . T h e s y s t e m s e x a m i n e d 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 , e n e r g y - i n d e p e n d e n t b i n d i n g o f i r o n t o t h e c e l l and a s l o w e r , e n e r g y -d e p e n d e n t u p t a k e 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 be i n v o l v e d a t t h i s s t a t e , s i m i l a r t o t h e s u l f a t e - b i n d i n g p r o t e i n i n v o l v e d i n 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 t y p e s t r a i n and one o f t h e m u t a n t s . 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 on 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 c o m m u n i c a t i o n ) . C i t r a t e a p p e a r e d t o i n h i b i t g r o w t h o f t h e m u t a n t s . 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 , B e r k e l e y . 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 , w h e r e a s i n t h e FeCDHBS)^ c o m p l e x , t h e i r o n i s r e a d i l y t a k e n i n t o t h e c e l l s . C o n t r o l o f DHB(G) f o r m a t i o n by i r o n i n v o l v e s r e p r e s s i o n o f a t l e a s t one o f t h e enzymes 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 ( F i g -u r e 43 ) . S i n c e DHB s y n t h e s i s a p p e a r s t o i n v o l v e a m u l t i e n z y m e c o m p l e x , i t i s p o s s i b l e t h a t none o f t h e enzymes i n 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 grown i n t h e p r e s e n c e o f i r o n . I r o n 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 r e v e n as an i n d u c e r (43) o:f p r o t e i n s y n t h e s i s . However, i r o n i s n o t t h e o n l y f a c t o r i n -v o l v e d i n t h e r e g u l a t i o n o f DHB s y n t h e s i s . An o p e r a t i v e c i t r i c 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 f o r t h e s y n t h e s i s o f t h i s compound. —• subt1 l i s p r o b a b l y p o s s e s s e s an o p e r o n c o n t r o l l i n g (DHB(G) p r o d u c t i o n , w i t h i r o n s e r v i n g as a c o - r e p r e s s o r o f t h i s s y s t e m . T h e 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 t y p h i m u r i u m 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.). I t i s t o be hoped t h a t work w i t h d e f i n e d c o n t r o l m u t a n t s w i l l s o o n c l a r i f y t h e r o l e t h a t DHB(G) and r e l a t e d p h e n o l i c s p l a y i n t h e a c t i v e t r a n s p o r t o f i r o n (and p o s s i b l y o t h e r m e t a l s ) i n t o t h e b a c t e r i a l c e l l . T h i s has r e c e n t l y been c o n f i r m e d f o r t h e A. a e r o g e n e s and t h e E_. c o l i s y s t e m s by I.G. Young and F. G i b s o n , B i o c h i m . B i o p h y s . A c t a 177:401-411 (1969)-119 HZ HE F i g u r e 43. I n t e r m e d i a t e s i n t h e B i o s y n t h e s i s o f 2 , 3 - d i h y d r o x y b e n z o i c a c i d . C h o r i s m i c a c i d ( l ) , i s o c h o r i s m i c a c i d ( I I ) , 2 , 3 - d i h y d r o - 2 , 3 - d i h y d r o x y b e n z o i c a c i d ( I I I ) a n d 2 , 3 - d i h y d r o x y b a c i d ( I V ) . e n z o i c REFERENCES B r o t , N., J . Goodwin and H. F a l e s . 1966. In v i v o and i n v i t r o f o r m a t i o n o f 2 , 3 - d i h y d r o x y b e n z o y l s e r i n e by E s c h e r i c h i a c o l i K ^ -Bio c h e m . B i o p h y s . R es. Commun. 25:454-461 . B r o t , N., and J . Goodwin. 1968. R e g u l a t i o n o f 2 , 3 - d i h y d r o x y -b e n z o y l s e r i n e s y n t h e t a s e by i r o n . J . B i o l . Chem. 243:510-513. B u l e n , W.A. 1961. E f f e c t o f t u n g s t a t e on t h e u p t a k e and f u n c t i o n o f m o l y b d a t e i n A z o t o b a c t e r a g i l i s . J . B a c t e r i o l . 82:130-134. Burnham, B.F. 1963. I n v e s t i g a t i o n s o n t h e a c t i o n o f t h e i r o n -c o n t a i n i n g g r o w t h f a c t o r s , s i d e r a m i n e s ; and t h e i r o n - c o n t a i n i n g a n t i b i o t i c s , s i d e r o m y c i n s . J . Gen: M i c r o b i o l . 32:117-121. Burnham, B.F., and J . B . N e i l a n d s . 1961. S t u d i e s on t h e meta-b o l i c f u n c t i o n o f t h e f e r r i c h r o m e compounds. J . B i o l . Chem. 236:554-559. B y e r s , B.R., and C.E. L a n k f o r d . 1968. R e g u l a t i o n o f s y n t h e s i s o f 2 , 3 - d i h y d r o x y b e n z o i c a c i d i n Baci11 us s u b t i 1 i s by i r o n a nd a b i o l o g i c a l s e c o n d a r y h y d r o x a m a t e . B i o c h i m . B i o p h y s . A c t a 165: 563-566. C a t l i n , E.R., C H . H a s s a l and B.C. P r a t t . 1968. B i o s y n t h e s i s o f p h e n o l s . XIV. I s o l a t i o n o f some s h i k i m i c a c i d - d e r i v e d m e ta-b o l i t e s f r o m m u t a n t s t r a i n s o f S t r e p t o m y c e s r i m o s u s u n a b l e t o p r o d u c e o x y t e t r a c y c l i n e . B i o c h i m . B i o p h y s . A c t a 156:109~118. C a t r o u x , G., G.C. F o u r i n e r and J . R i v i e r e . 1969. M i s i en 121 e v i d e n c e r a p i d e de 1 1 u t i 1 i z a t i o n des a c i d e s p h e n o l s p a r d e s s o u c h e s b a c t e r i e n n e s i s o l e e s du s o l . Ann. L ' l n s t . P a s t . 116: 99-102. 9. Conway, E . J . and T.G. B r a d y . 1949- B i o l o g i c a l p r o d u c t i o n o f a c i d and a l k a l i . 1. Q u a n t i t a t i v e r e l a t i o n s o f s u c c i n i c and c a r b o n i c a c i d s t o t h e p o t a s s i u m a n d h y d r o g e n i o n e x c h a n g e i n f e r m e n t i n g y e a s t . B i o c h e m . J . 47:360-369. 10. Conway, E . J . and M.E. B e a r y . 1958. A c t i v e t r a n s p o r t o f magne-s i u m a c r o s s t h e y e a s t c e l l membrane. B i o c h e m . J . 69:275~280. 11. Conway, E . J . and F. Duggan. 1958. A c a t i o n c a r r i e r i n t h e y e a s t c e l l w a l l . B i o c h e m . J . 69:265-274. 12. C o r b i n , J . L . and W.A. B u l e n . 1969- The i s o l a t i o n and i d e n t i f i -c a t i o n o f 2 , 3 - d i h y d r o x y b e n z o i c a c i d and 2 - N ^ 6 - N - D i - ( 2 , 3 d i h y d r o x y -b e n z o y l ) - L - l y s i n e f o r m e d by i r o n - d e f i c i e n t A z o t o b a c t e r v i n e l a n d i i . B i o c h e m . 8:757-762. 13. C o u l s o n , C B . and W.C. E v a n s . 1958. P a p e r c h r o m a t o g r a p h y and p a p e r e l e c t r o p h o r e s i s o f p h e n o l s and g l y c o s i d e s . J . C h r o m a t o g . 1:374-379. 14. C r u e g e r , W. and H. Z a h n e r . 1968. S t o f f w e c h s e l p r o d u k t e v o n m i k r o o r g a n i s m e n 70. M i t t e i l u n g . U l s e r den e i n f l u B d e r k o h l e n -s t o f f q u e l l a a u f d i e s i d e r a m i n b i I d u n g v o n A s p e r g i l l u s m e l l e u s yukawa. A r c h i v . f u r M i k r o b i o l . 63 :376-384. 15. Damadian, R. 1968. I r o n m e t a b o l i s m i n a p o t a s s i u m a c c u m u l a -t i o n m utant o f E s c h e r i c h i a c o l i B. 1. P o t a s s i u m m e t a b o l i s m . 122 J . B a c t e r i o l . 95:113-122. 16. Demain, A . L . and D. H e n d l i n . 1958. Growth s t i m u l a t i o n o f a s t r a i n o f B a c i l l u s s u b t i l i s by g l y c i n e p e p t i d e s . J . B a c t e r i o l . 75:46-50. 17- Demain, A . L . and D. H e n d l i n . 1959. I r o n T r a n s p o r t compounds a s gr o w t h s t i m u l a t o r s f o r M i c r o b a c t e r i u m s p . J . Gen. M i c r o b i o l . 21: 72-79. 18. Doy, C H . 1968. C o n t r o l o f a r o m a t i c b i o s y n t h e s i s p a r t i c u l a r l y w i t h r e g a r d t o t h e common pathway and t h e a l l o s t e r i c enzyme, 3 - d e o x y - D - a r a b i n o h e p t u l o s o n a t e 7 - p h o s p h a t e s y n t h e t a s e . Rev. Pu r e and A p p l . Chem. 18:41-78. 19- D r e y f u s s , J . 1964. C h a r a c t e r i z a t i o n o f s u l f a t e - and t h i o s u l -f a t e - t r a n s p o r t i n g s y s t e m i n S a l m o n e l l a t y p h i m u r i u m . J . B i o l . Chem. 239:2292-2297-20. Emery, T., and J . B . N e i l a n d s . i960. C o n t r i b u t i o n t o t h e s t r u c t -u r e o f t h e f e r r i c h r o m e compounds; c h a r a c t e r i z a t i o n o f t h e a c y l m o i t i e s o f t h e h y d r o x a m a t e f u n c t i o n s . J . Am. Chem. S o c . 82:3658-3662. 2+ 21. Fuhrmann, G.F. and A. R o t h s t e i n . 1968. The t r a n s p o r t o f Zn , 2+ 2+ Co and Ni i n t o y e a s t c e l l s . B i o c h i m . B i o p h y s . A c t a 163:325-330. 22. G a r y , N.D. and R . C B a r d . 1952. E f f e c t o f n u t r i t i o n on t h e g r o w t h and m e t a b o l i s m o f B a c i l l u s s u b t i l i s . J . B a c t e r i o l . 64:501-512. 23. G i b s o n , F. 1968. C h o r i s m i c a c i d . B i o c h e m . p r e p s . 12:94-97. 24. G i b s o n , F. and J . P i t t a r d . I968. P a t h w a y s o f b i o s y n t h e s i s o f 123 a r o m a t i c amino a c i d s and v i t a m i n s and t h e i r c o n t r o l i n m i c r o -o r g a n i s m s . B a c t e r i o l . Rev. 32:465-492. 25. Goldman, D., S.G. S c h u l t z and W. E p s t e i n . 1966. R e p r e s s i v e c o n t r o l o f p o t a s s i u m t r a n s p o r t i n E s c h e r i c h ? a c o l ? . B i o c h i m . B i o p h y s . A c t a . 130:546-548. 26. G r o v e s , W.E., F.C. D a v i s , J r . and B.H. S e l l s . 1968. S p e c t r o -p h o t o m e t r y d e t e r m i n a t i o n o f m i c r o g r a m q u a n t i t i e s o f p r o t e i n w i t h o u t n u c l e i c a c i d i n t e r f e r e n c e . A n a l . B i o c h e m . 22:195-210. 27. I t o , T. and J . B . N e i l a n d s . 1958. P r o d u c t s o f " l o w - i r o n f e r m e n -t a t i o n " w i t h B a c ? 1 l u s s u b t ? l i s : i s o l a t i o n , c h a r a c t e r i z a t i o n and s y n t h e s i s o f 2 , 3 - d i h y d r o x y b e n z o y l g l y c i n e . J . Am. Chem. S o c . 80: 4645-4647. 28. J o n e s , G.E. 1964. E f f e c t o f c h e l a t i n g a g e n t s on t h e g r o w t h o f E s c h e r i c h i a c o l i i n s e a - w a t e r . J . B a c t e r i o l . 87:483~499. 29. J o r d e n , D.C. 1959. C h e l a t i o n and t h e g r o w t h - i n i t i a t i o n o f R h i z o b i u m m e l i l o t i . B a c t e r i o l . P r o c . 1959:41. 30. K e e l e r , R.F. 1957- I n f l u e n c e o f c u l t u r e c o n d i t i o n s on u p t a k e and d i s t r i b u t i o n o f molybdenum i n A z o t o b a c t e r v i n e l a n d i i . J . B a c t e r i o l . 73:582-583. 31. K e e l e r , R.F., W.A. B u l e n and J . E . V a r n e r . 1956. D i s t r i b u t i o n 99 o f molybdenum i n c e l l - f r e e p r e p a r a t i o n s o f A z o t o b a c t e r v i n e l a n d i i . J . B a c t e r i o l . 72=395-396. 32. L a n k f o r d , C.E., T.Y. K u s t o f f and T.P. S e r g e a n t . 1957. C h e l a t i n g a g e n t s i n g r o w t h i n i t i a t i o n o f B a c i 1 l u s g l o b i g i i . J . B a c t e r i o l . 72:737-748. 124 3 3 . L a s c e l l e s , J . 1 9 6 4 . T e t r a p y r r o l e b i o s y n t h e s i s and i t s r e g u l a -t i o n . W.A. B e n j a m i n I n c . , New Y o r k . 3k. L e s s i e , T.G. and W.R. S i s t r o m . 1 9 6 4 . C o n t r o l o f p o r p h y r i n s y n t h e s i s i n Rhodopseudomonas s p h e r o i d e s . B i o c h i m . B i o p h y s . A c t a 86^ :250-259-3 5 . L o r e n c e , J.H. and E.W. N e s t e r . 1967- M u l t i p l e m o l e c u l a r f o r m s o f c h o r i s m a t e m u t a s e i n B a c i l l u s s u b t i l i s . B i o c h e m . 6_: 1541 - 1 5 5 3 . 3 6 . L o w r y , 0 . , M. R o s b r o u g h , L. F a r r and R. R a n d a l l . 1 9 5 1 . P r o t e i n m e asurement w i t h t h e F o l i n - p h e n o l r e a g e n t . J . B i o l . Chem. 1 9 3 : 2 6 5 - 2 7 5 . 3 7 . L u s k , J . E . and E.P. Kennedy. 1969- Magnesium t r a n s p o r t i n E s c h e r i c h i a c o l i . . . J . B i o l . Chem. 2 4 4 : 1 6 5 3 - 1 6 5 5 . 3 8 . M u r r e l l , W.G. 1 9 6 7 . T h e b i o c h e m i s t r y o f t h e b a c t e r i a l e n d o s p o r e . _l_n A d v a n c e s i n M i c r o b i a l P h y s i o l o g y J _ : 1 3 3 _ 2 5 1 . A.H. Rose and J . F . W i l k i n s o n , e d i t o r s . A c a d e m i c P r e s s , L o n d o n . 3 9 . N e i l a n d s , J . B . 1 9 5 7 . Some a s p e c t s o f m i c r o b i a l i r o n m e t a b o l i s m . B a c t e r i o l . Rev. 21 : 1 0 1 - 1 1 1 . 4 0 . N e i l a n d s , J . B . 1 9 6 7 . H y d r o x a m i c a c i d s i n n a t u r e . S c i e n c e 1 5 6 : 1 4 4 3 - 1 4 4 7 . 4 1 . N e s t e r , E.W. and R.A. J e n s e n . 1 9 6 6 . C o n t r o l o f a r o m a t i c a c i d b i o s y n t h e s i s i n B a c i 11 us s u b t i l i s : s e q u e n t i a l f e e d b a c k i n h i b i t i o n . J . B a c t e r i o l . 91=1594-1598. 4 2 . N e s t e r , E.W., R.A. J e n s e n and D.S. N a s s e r . 1969- R e g u l a t i o n o f enzyme s y n t h e s i s i n t h e a r o m a t i c amino a c i d pathway o f B a c i 1 1 us 125 s u b t i l i s . J . B a c t e r i o l . 97_:83-90. 43. Padmanaban, G., S. M u t h u k r i s h n a n and P.S. Sarma. 1967. The r o l e o f i r o n i n heme, s y n t h e s i s i n N e u r o s p o r a c r a s s a . B i o c h i m . B i o -p h y s . A c t a 141 :187-190• 44. P e t e r s , W.J. I968. C o n s e q u e n c e s o f i r o n d e f i c i e n c y i n B a c i 1 l u s  s u b t ? l i s . Ph.D. T h e s i s , 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 . 45. P e t e r s , W.J. and R.A.J. W a r r e n . 1968. I t o i c a c i d s y n t h e s i s i n B a c i 1 l u s s u b t i 1 i s . J . . B a c t e r i o l . 95:360-366. 46. P e t e r s , W.J. and R.A.J. Warren., 1968. P h e n o l i c a c i d s and i r o n t r a n s p o r t i n B a c i 1 l u s s u b t i l i s . B i o c h i m . B i o p h y s . A c t a 165:225-232. 47. P e t e r s o n , E.A. and H.A. S o b e r . 79&3• Column c h r o m a t o g r a p h y o f p r o t e i n s ; s u b s t i t u t e d c e l l u l o s e s . _l_n_ M e t h o d s i n E n z y m o l o g y . 5_: 3-27. 48. P i t t a r d , A . J . , F. G i b s o n and C H . Doy. 1961. P h e n o l i c compounds a c c u m u l a t e d by washed c e l l s u s p e n s i o n s o f a t r y p t o p h a n a u x o t r o p h o f A e r o b a c t e r a e r o g e n e s . B i o c h i m . B i o p h y s . A c t a 49:485~494. 49. R a t l e d g e , C. 1967. T h e p r o d u c t i o n o f an N - a c y l a n t h r a n ? 1 i c a c i d f r o m s h i k i m i c a c i d and t h e e f f e c t o f i r o n d e f i c i e n c y on t h e b i o s y n t h e s i s o f o t h e r a r o m a t i c compounds by A e r o b a c t e r a e r o g e n e s . B i o c h i m . B i o p h y s . A c t a 141:55~63. 50. R i m i n g t o n , C. i960. S p e c t r a l - a b s o r p t i o n c o e f f i c i e n t s o f some p o r p h y r i n s i n t h e s o r e t - b a n d r e g i o n . B i o c h e m . J . 75:620-623. 51. Rorem, E.S. 1955. U p t a k e o f r u b i d i u m and p h o s p h a t e i o n s by p o l y s a c c h a r i d e - p r o d u c i n g b a c t e r i a . J . B a c t e r i o l . 70:691~701• 126 52. R o t h s t e i n , A., A. H a y e s , D. J e n n i n g s and D. Ho o p e r . 1958. T h e a c t i v e t r a n s p o r t o f M g + + and M n + + i n t o t h e y e a s t c e l l . J . Gen. ! P h y s i o l . 4l_:585-59^. 53. S a l t o n , M.R.J. 1964. R e q u i r e m e n t o f d i h y d r o x y p h e n o l s f o r t h e g r o w t h o f M i c r o c o c c u s l y s o d e i k t i c u s i n s y n t h e t i c medium. B i o c h i m . B i o p h y s . A c t a 86:421-422. 54. S c h u l t z , S.G., W. E p s t e i n and A.K. So l o m o n . 1963. C a t i o n t r a n s -p o r t i n E s c h e r i c h i a c o l i IV. K i n e t i c s o f n e t K u p t a k e . J . Gen. P h y s i o l . 47:329-346. ; 55. S e r g e a n t , T.P., C.E. L a n k f o r d and R.W. T r a x i e r . 1957. A u t o c l a v e d g l u c o s e and p h o s p h a t e i n g r o w t h i n i t i a t i o n o f B a c i 1 l u s s p p . B a c t e r i o l . P r o c . 1957:55. , 56. S h a p i r o , J . 1967- I n d u c e d r a p i d r e l e a s e and u p t a k e o f p h o s p h a t e by m i c r o o r g a n i s m s . S c i e n c e 155:1269-1271. SI- S i l v e r , S. 1969- A c t i v e t r a n s p o r t o f magnesium i n E s c h e r i c h i a c o l i : P r o c . N a t . A c a d . S c i . Wash. 62:764-771. 58. S i l v e r , S. and M.L. K r a l o v i c . 1969- Manganese a c c u m u l a t i o n by E s c h e r i c h i a c o l i ; e v i d e n c e f o r a s p e c i f i c t r a n s p o r t s y s t e m . B i o c h e m . B i o p h y s . R e s . Commun. 34:640-645. 59- S i l v e r , S. and L. Wendt. 1967• M e c h a n i s m o f a c t i o n o f phe n -e t h y l a l c o h o l : breakdown o f c e l l u l a r p e r m e a b i l i t y b a r r i e r . J . B a c t e r i o l . 93:560-566. 60. S m i t h , I. 1958. P h e n o l i c a c i d s . In C h r o m a t o g r a p h i c T e c h n i q u e s . Heinemann, L o n d o n , pp. 291-307-127 6 1 . S p i r o , T.G., S.E. A l l e r t o n , J . R e n n e r , A. T e r z i s , R. B i l s and P. S a l t m a n . 1 9 6 6 . T h e h y d r o l y t i c p o l y m e r i z a t i o n o f i r o n ( I I I ) . J . Am. Chem. S o c . 8 8 : 2 7 2 1 - 2 7 2 6 . 6 2 . S p i z i z e n , J . 1 9 5 8 . T r a n s f o r m a t i o n o f b i o - c h e m i c a l l y d e f i c i e n t s t r a i n s o f B a c i l l u s s u b t i l i s , b y d e o x y r i b o n u c l e a s e . P r o c . N a t . A c a d . S c i . Wash. 4 4 : 1 0 7 2 - 1 0 7 8 . 6 3 . V o g e l , H. J . 1 9 5 6 . A c o n v e n i e n t g r o w t h medium f o r N e u r o s p o r a (Medium N ) . M i c r o b i o l . Gen. B u l l . ]V.k2. 6 4 . V o g e l , H.J. a nd D.M. B o n n e r . 1 9 5 6 . A s i m p l e medium f o r E s c h e r i c h i a c o l i . M i c r o b i o l . Gen. B u l l . 1 3 : 4 3 . 65. V o r i s e k , J . and D. G r u n b e r g e r . 1 9 6 7 . E f f e c t o f a l b o m y c i n ( G r i s e i n ) on s y n t h e s i s o f n u c l e i c a c i d s and p r o t e i n s i n B a c i 1 l u s  c e r e u s an d E s c h e r i c h i a c o l i . C o l l . C z e c h o s l o v . Chem. Commun. 3 2 : 1 0 6 4 - 1 0 7 7 . 6 6 . V o r i s e k , J . and D. G r u n b e r g e r . 1 9 6 6 . T h e i n h i b i t o r y e f f e c t o f a l b o m y c i n ( G r i s e i n ) on g r o w t h o f B a c i l l u s c e r e u s an d E s c h e r i c h i a  c o l ? . F o l i a M i c r o b i o l o g i c a 1 1 : 4 6 5 ~ 4 7 1 • 67. Wang, C.C. and A. Newton. 1969- R e l a t i o n s h i p b e t w e e n c h r o m i u m s e n s i t i v i t y and h i g h i r o n r e q u i r e m e n t i n m u t a n t s o f E s c h e r i c h i a  c o l i . J . B a c t e r i o l . 98.: 1 1 3 5 - 1 1 4 1 . 6 8 . Wang, C.C. and A. Newton. 1 9 6 9 . I r o n t r a n s p o r t i n E s c h e r i c h i a  c o l i : r o l e s o f e n e r g y - d e p e n d e n t u p t a k e and 2 , 3 - d i h y d r o x y b e n z o y l -s e r i n e . J . B a c t e r i o l . 9 8 : 1 1 4 2 - 1 1 5 0 . 6 9 . Webb, M. 1 9 6 6 . T h e u t i l i z a t i o n o f magnesium by c e r t a i n Gram-128 p o s i t i v e a n d G r a m - n e g a t i v e b a c t e r i a . J . Gen. M i c r o b i o l . 43: 401-409. 70. Webb, M. 1968. The i n f l u e n c e o f c e r t a i n t r a c e m e t a l s on b a c t e r i a l g r o w t h and magnesium u t i l i z a t i o n . J . Gen. M i c r o b i o l . 51 :325~335. 71. W e i n b e r g , E.D. 1957- C h e l a t i n g a g e n t s a s b a c t e r i a l g r o w t h f a c t o r s . B a c t e r i o l . P r o c . 1957:54. 72. Y o u n g , I.G., G.B. Cox and F. G i b s o n . 1967- 2 , 3 ~ d i h y d r o x y -b e n z o a t e a s a b a c t e r i a l g r o w t h f a c t o r and i t s r o u t e o f b i o -s y n t h e s i s . B i o c h i m . B i o p h y s . A c t a 141:319~331. 73. Y o u n g , I.G., L.M. Jackman and F. G i b s o n . 1967. 2 , 3 - d i h y d r o -2 , 3 - d i h y d r o x y b e n z o i c a c i d : an i n t e r m e d i a t e i n t h e b i o s y n t h e s i s o f 2 , 3 - d i h y d r o x y b e n z o i c a c i d . B i o c h i m . B i o p h y s . A c t a 148:313~315. 74. Y o u n g , I.G., T. B a t t e r h a m and F. G i b s o n . 1968. I s o c h o r i s m i c a c i d : a new i n t e r m e d i a t e i n t h e b i o s y n t h e s i s o f 2 , 3 ~ d i h y d r o x y -b e n z o i c a c i d . B i o c h i m . B i o p h y s . A c t a 16$:567~568. 75. Z a h n e r , H. 1964. A n t i b i o t i c a i n d e r m i k r o b i o l o g i e . N a t u r w . R u n d s c h a u 17:391-399. 76. Z a h n e r , H., E . Bachmann, R. H u t t e r and J . N u e s c h . 1962. S i d e r -a m i n e , e i s e n h a l t i g e w a c h s t u m s f a k t o r e n a u s m i k r o o r g a n i s m e n . P a t h . M i c r o b i o l . 25:708-736. 77. Z a r l e n g o , M.H. and S.G. S c h u l t z . 1966. C a t i o n t r a n s p o r t and m e t a b o l i s m i n S t r e p t o c o c c u s f a e c a l i s . B i o c h i m . B i o p h y s . A c t a 126:308-320. 

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