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11(1):167. DOI: 10.1128/AAC.11.1.167.

1977,Antimicrob. Agents Chemother.Lee Chao

Escherichia coliAlterations in

Nalidixic Acid-Induced Protein

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A N T I M I C R O B I A L A G E N T AND C H E M O T H E R A P Y , J a n . 1 9 7 7 , p . 1 6 7 - 1 7 0C o p y r i g h t 1 9 7 7 A m e r i c a n S o c i e t y f o r M i c r o b i o l o g y V o l . 1 1 , N o . 1P r i n t e d i n U . S . A .

NOTESN a l i d i x i c A c i d - I n d u c e d P r o t e i n A l t e r a t i o n s i nE s c h e r i c h i a c o l i

LEE CHAOD e p a r t m e n t o f B i o c h e m i s t r y , M e d i c a l U n i v e r s i t y o f S o u t h C a r o l i n a , C h a r l e s t o n , S o u t h C a r o l i n a 2 9 4 0 1

R e c e i v e d f o r p u b l i c a t i o n 1 5 J u l y 1 9 7 6T h e e f f e c t o f n a l i d i x i c a c i d o n p u l s e - l a b e l e d p r o t e i n p a t t e r n s was a n a l y z e d b ys o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d e s l a b g e l e l e c t r o p h o r e s i s . T h e d r u g wasf o u n d t o a l t e r p r o t e i n p a t t e r n s i n a d r u g - s e n s i t i v e s t r a i n b u t n o t i n t h e i s o g e n i cd r u g - r e s i s t a n t s t r a i n o f E s c h e r i c h i a c o l i . T h e d r u g - i n d u c e d a l t e r a t i o n o f p r o t e i np a t t e r n s w a s d e m o n s t r a t e d b y t h e a p p e a r a n c e o f s o m e a n d t h e d i s a p p e a r a n c e o fo t h e r p u l s e - l a b e l e d p e p t i d e s e l i c i t e d b y t h e d r u g .

N a l i d i x i c a c i d i s a n e x t e n s i v e l y u s e d a n t i b a c -t e r i a l d r u g w h o s e m o d e o f a c t i o n i s p o o r l y u n -d e r s t o o d . I t i s known t h a t n a l i d i x i c a c i d s p e c i f i -c a l l y i n h i b i t s b a c t e r i a l d e o x y r i b o n u c l e i c a c i d( D N A ) s y n t h e s i s b u t o n l y p a r t i a l l y i n h i b i t s t h es y n t h e s i s o f r i b o n u c l e i c a c i d ( R N A ) a n d p r o -t e i n s ( 2 , 1 2 ) . T h e p r i m a r y t a r g e t o f i n h i b i t i o n i st h e s e m i c o n s e r v a t i v e DNA r e p l i c a t i o n ( 2 1 ) ,w i t h a p o s s i b l e e f f e c t o n t h e r e p a i r s y n t h e s i s o fDNA ( 1 0 ) . T h e i n h i b i t i o n o f DNA s y n t h e s i s i sn o t a t t r i b u t a b l e t o a n y i n t e r a c t i o n b e t w e e n n a l -i d i x i c a c i d a n d t h e DNA t e m p l a t e ( 3 , 4 ) . I t h a sb e e n s h o w n ( 1 8 ) t h a t t h e t h e r m a l s u s c e p t i b i l i t yo f a DNA l i g a s e t e m p e r a t u r e - s e n s i t i v e m u t a n ti s e n h a n c e d b y n a l i d i x i c a c i d , b u t t h e s e a r c h f o ra d i r e c t i n t e r f e r e n c e o f t h e d r u g w i t h e n z y m e si n v o l v e d i n DNA m e t a b o l i s m h a s b e e n s o f a ru n s u c c e s s f u l ( 4 , 1 9 ) . A r e c e n t r e p o r t b y C r u m -p l i n a n d S m i t h ( 8 ) i n d i c a t e s t h a t , i n a d d i t i o n t oi n h i b i t i n g DNA s y n t h e s i s , n a l i d i x i c a c i d ma yi n h i b i t RNA s y n t h e s i s a s a s e c o n d t a r g e t s i t e .H o w e v e r , t h e i n h i b i t i o n o f b a c t e r i a l RNA s y n -t h e s i s i s o n l y p a r t i a l ( 1 2 ) a n d ma y o r may n o ta f f e c t b a c t e r i a l g r o w t h s i g n i f i c a n t l y , s i n c e t h er a t e o f b a c t e r i a l RNA s y n t h e s i s c a n a l s o v a r yw i d e l y d e p e n d i n g o n t h e g r o w t h c o n d i t i o n s .S t u d i e s h a v e t h e r e f o r e b e e n c o n d u c t e d t o i n v e s -t i g a t e t h e p o s s i b i l i t y t h a t n a l i d i x i c a c i d ma ya l t e r t h e e x p r e s s i o n o f many g e n e p r o d u c t s i n aq u a l i t a t i v e m a n n e r . R e s u l t s o f t h e s e s t u d i e s ,r e p o r t e d h e r e , i n d i c a t e t h a t t h e p r o t e i n p a t t e r ni n b a c t e r i a i s i n d e e d a l t e r e d b y n a l i d i x i c a c i dt r e a t m e n t . T h e a l t e r a t i o n i s q u i t e c o m p l e x i nt h a t t h e s y n t h e s i s o f c e r t a i n p r o t e i n s i s d e -p r e s s e d w h e r e a s t h a t o f o t h e r p r o t e i n s i s i n -d u c e d b y t h e d r u g .

T h e b a c t e r i a l s t r a i n s u s e d w e r e E s c h e r i c h i ac o l i K L 1 6 1 a n d K L 1 6 6 . T h e s e t w o s t r a i n s a r ei s o g e n i c e x c e p t t h a t KL166 i s a NalA d e r i v a t i v eo f K L 1 6 1 ( 1 ) . T h e g r o w t h o f K L 1 6 1 i s t o t a l l yi n h i b i t e d b y n a l i d i x i c a c i d a t a c o n c e n t r a t i o n o f1 0 , g / m l i n m in im um me dium M9 s u p p l e -m e n t e d w i t h 1 0 0 g g o f t h y m i n e p e r m l . ( 5 ) . T h eg r o w t h o f K L 1 6 6 i s u n a f f e c t e d b y n a l i d i x i c a c i da t 5 0 0 g g / m l i n t h e same m e d i u m . A d i r e c ta n a l y s i s o f t h e e f f e c t o f n a l i d i x i c a c i d o n t h ep r o f i l e s o f p u l s e - l a b e l e d RNA i s c l e a r l y n o t f e a -s i b l e . An i n d i r e c t a p p r o a c h o f a n a l y z i n g p u l s e -l a b e l e d p r o t e i n s o n s o d i u m d o d e c y l s u l f a t e -p o l y a c r y l a m i d e g e l was u s e d . T h i s a p p r o a c h i sb a s e d o n t h e a s s u m p t i o n t h a t t h e p r o f i l e o fp u l s e - l a b e l e d p r o t e i n s r e f l e c t s t h a t o f t h e RNAa n d t h a t n a l i d i x i c a c i d d o e s n o t a c t o n t r a n s l a -t i o n ( 8 ) .C e l l s a t a d e n s i t y o f 3 x 1 0 8 p e r m l i n M9medium p l u s 1 0 0 ,g o f t h y m i n e p e r m l w e r et r e a t e d w i t h n a l i d i x i c a c i d ( S i g m a C h e m i c a lC o . ) a t 1 0 0 g g / m l . N a l i d i x i c a c i d - t r e a t e d a n du n t r e a t e d s a m p l e s w e r e t h e n d i v i d e d a n d w e r es e p a r a t e l y p u l s e l a b e l e d w i t h [ 1 4 C ] t h y m i d i n e( 0 . 5 , u C i / m l ) , [ 3 H ] u r i d i n e ( 1 , u C i / m l ) , a n d 1 4 C -l a b e l e d a m i n o a c i d m i x t u r e s ( 2 , C i / m l ) a t p r e -d e t e r m i n e d t i m e i n t e r v a l s . A l l l a b e l e d c h e m i -c a l s w e r e p u r c h a s e d f r o m Ne w E n g l a n d N u -c l e a r . [ ' 4 C ] t h y m i d i n e - a n d [ 3 H ] u r i d i n e - l a b e l e ds a m p l e s o f 1 ml e a c h w e r e s t o p p e d w i t h 10 %t r i c h l o r o a c e t i c a c i d a n d c o u n t e d . S a m p l e s ( 1 mle a c h ) l a b e l e d w i t h ' 4 C - l a b e l e d a m i n o a c i d s w e r es t o p p e d b y a d d i n g 2 m l o f i c e - c o l d 2% u n l a b e l e da m i n o a c i d m i x t u r e a n d 1 0 % g l y c e r o l . L a b e l e dc e l l s w e r e p e l l e t e d a n d l y s e d i n t h e e x t r a c t i o nb u f f e r ( 1 5 ) . P o r t i o n s o f t h e l y s a t e w e r e p r e c i p i -1 6 7

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1 6 8 NOTESt a t e d w i t h 5% t r i c h l o r o a c e t i c a c i cA n o t h e r p o r t i o n o f e a c h s a m p l es o d i u m d o d e c y l s u l f a t e - p o l y a c r y l et r o p h o r e s i s a c c o r d i n g t o t h e s t a w( 1 6 ) .T h e r e s u l t s o f t h e p u l s e - l a b e l i ra re s h o w n i n F i g . 1 . T h e r a t e o f ps i s i n K L 1 6 1 a n d KL166 was red i x i c a c i d t r e a t m e n t b y a b o u t 2 5s p e c t i v e l y . T h e r a t e o f RNA s y n tt w o s t r a i n s was a l s o r e d u c e d proFr e p o r t e d b y o t h e r s ( 1 2 ) . T h y m i dt i o n was i n h i b i t e d i n K L 1 6 1 b u t( d a t a n o t s h o w n ) . T h u s , t h e b a c to f n a l i d i x i c a c i d d o e s n o t seems p e c i f i c a l l y t o i t s a c t i o n on b a c tp r o t e i n s y n t h e s i s . H o w e v e r , a ct i o n o f i n d i v i d u a l p e p t i d e s b y s (s u l f a t e - p o l y a c r y l a m i d e g e l a n a l ,

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F I G . 1 . E f f e c t o f n a l i d i x i c a c i d onand p r o t e i n s y n t h e s i s . E . c o l i KL161grown i n M9 m i n i m a l medium s u p1 0 0 pg o f t h y m i n e pe r ml a t 30c e l l s l m l . N a l i d i x i c a c i d was added tzero t i m e t o g i v e a f i n a l c o n c e n t r a t i cS a m p l e s o f 1 ml e a c h w e r e t a k e n lt i m e a nd p u l s e l a b e l e d w i t h e i t h e r, u C i / m l ) f o r 1 min o r 1 4 C - l a b e l e d ami( 2 u C i / m l ) f o r 3 m i n . L a b e l e d R Bw e r e d e t e r m i n e d b y s c i n t i l l a t i o n c o uc i p i t a b l e c o u n t s w e r e p l o t t e d a s t h ec o n t r o l w h i c h was p u l s e l a b e l e d b e f o rt i o n . [ 3 H ] u r i d i n e i n c o r p o r a t i o n : ( IK L 1 6 6 . " 4 C - l a b e l e d a m i n o a c i d i n cK L 1 6 1 ; ( E ) K L 1 6 6 .

A N T I M I C R O B . A G E N T S C H E M O T H E R .I a n d c o u n t e d . o t h e r w i s e . C e r t a i n m a j o r q u a l i t a t i v e d i f f e r -! w a s u s e d f o r e n c e s i n p r o t e i n p a t t e r n s b e t w e e n K L 1 6 1 a n du m i d e g e l e l e c - K L 1 6 6 e l i c i t e d b y n a l i d i x i c a c i d a r e s h o w n i nn d a r d m e t h o d F i g . 2 . T h e p a t t e r n o f p u l s e - l a b e l e d p r o t e i n s

w a s d r a s t i c a l l y a l t e r e d b y n a l i d i x i c a c i d i na g e x p e r i m e n t K L 1 6 1 b u t n o t i n K L 1 6 6 . F u r t h e r m o r e , i nr o t e i n s y n t h e - K L 1 6 1 t h e s y n t h e s i s o f many p r o t e i n s was d e -d u c e d b y n a l i - p r e s s e d w h e r e a s t h a t o f o t h e r s wa s i n d u c e d b yi a n d 1 0 % , r e - n a l i d i x i c a c i d . T h e m o s t p r o m i n e n t p r o t e i nt h e s i s i n t h e s e b a n d s w h o s e s y n t h e s i s wa s d e p r e s s e d b y n a l i -) o r t i o n a t e l y a s d i x i c a c i d w e r e t h e 1 6 5 - K ( 1 6 5 , 0 0 0 d a l t o n s ) , 1 5 5 -i n e i n c o r p o r a - K , 9 5 - K a n d 3 0 - K p e p t i d e s . O f t h e s e p e p t i d e s ,n o t i n K L 1 6 6 o n l y t h e 1 6 5 - a n d 1 5 5 - K p e p t i d e s h a v e b e e ne r i c i d a l a c t i o n i d e n t i f i e d . T h e s e a r e t h e ( 3 ' a n d ( 8 s u b u n i t s o ft o b e r e l a t e d RNA p o l y m e r a s e , r e s p e c t i v e l y ( 1 7 ; m y u n p u b -t e r i a l RNA o r l i s h e d d a t a ) . T h e s y n t h e s i s o f t h e s e t w o s u b -l o s e e x a m i n a - u n i t s wa s d e p r e s s e d b y a b o u t 1 0 - f o l d i n K L 1 6 1x d i u m d o d e c y l b u t o n l y b y a b o u t 3 0 % i n K L 1 6 6 b y n a l i d i x i cL y s i s i n d i c a t e s a c i d a s r e v e a l e d b y d i r e c t s c a n n i n g o f t h e b a n d so n r a d i o a u t o g r a m s ( d a t a n o t s h o w n ) . I n K L 1 6 1t h e f o l l o w i n g p e p t i d e s w e r e i n d u c e d b y n a l i -d i x i c a c i d : 1 3 5 , 1 1 0 , 9 0 , 5 9 , 5 5 , 5 2 , 4 0 , a n d 1 8 K .T h e 1 3 5 - K p e p t i d e h a s b e e n i d e n t i f i e d a s ( 3 -g a l a c t o s i d a s e ( u n p u b l i s h e d d a t a ) . Shuman a n dS c h w a r t z h a v e s h o w n ( 2 0 ) t h a t n a l i d i x i c a c i du - - - - - - - - o r e d u c e s t h e l e v e l o f i n d u c e d f - g a l a c t o s i d a s e i nE . c o l i s t r a i n K - 1 2 . U n f o r t u n a t e l y , t h e y d i d n o t* - - - - - - - . m e n t i o n t h e e f f e c t o f n a l i d i x i c a c i d on t h e l e v e l0 o f 8 - g a l a c t o s i d a s e i n u n i n d u c e d c u l t u r e s . T h er e l a t i o n s h i p b e t w e e n t h e a c t i o n o f n a l i d i x i c

a c i d a n d t h e i n d u c t i o n o f 8 3 - g a l a c t o s i d a s e , a n dp o s s i b l y t h e i n d u c t i o n o f o t h e r c a t a b o l i t e - r e -p r e s s i b l e o p e r o n s , a p p e a r s t o b e c o m p l e x i n t h a tf 3 - g a l a c t o s i d a s e i s o n l y p o o r l y i n d u c e d b y i s o -p r o p y l - , 3 - r - t h i o g a l a c t o s i d e i n K L 1 6 6 a s c o m -p a r e d w i t h K L 1 6 1 e v e n i n t h e a b s e n c e o f n a l i -d i x i c a c i d ( L . C h a o , m a n u s c r i p t i n p r e p a r a -t i o n ) . T h e o t h e r r e a d i l y i d e n t i f i a b l e p e p t i d e i st h e 4 0 - K p e p t i d e , w h i c h c o r r e s p o n d s t o p r o t e i nX p r e v i o u s l y r e p o r t e d b y I n o u y e a n d P a r d e e( 1 4 ) . T h i s p r o t e i n h a s b e e n i m p l i e d t o f u n c t i o nI i n DNA r e p a i r ( 1 3 ) . N o t e t h a t c e r t a i n p e p t i d e s ,

3 0 4 0 s u c h a s t h e 5 9 - a n d 5 2 - K p e p t i d e s , a r e o n l yi n d u c e d t r a n s i e n t l y i n K L 1 6 1 b y n a l i d i x i c a c i d .T h e i n d u c t i o n o f o t h e r p e p t i d e s i n K L 1 6 1 p e r -t h e r a t e o f RNA s i s t s u n t i l t h e v i a b i l i t y o f t h e c e l l s b e g i n t o d r o p? n p d K L l 6 d w e i t r h e s i g n i f i c a n t l y ( d a t a n o t s h o w n ) .

D o C t o 3 x 1 0 8 I t i s k n o w n t h a t n a l i d i x i c a c i d t r e a t m e n t c o n -o e a c h c u l t u r e a t v e r t s c e l l u l a r DNA i n t o a c i d - s o l u b l e f r a g m e n t sn o f 1 0 0 g / I m l . a n d t h a t t h i s s o l u b i l i z a t i o n i s a n t a g o n i z e d b ya t t h e i n d i c a t e d c o n d i t i o n s r e s t r i c t i n g RNA a n d p r o t e i n s y n t h e -[ 3 H ] u r i d i n e ( 1 s i s ( 6 ) . I n a d d i t i o n , n a l i d i x i c a c i d , l i k e o t h e ri n o a c i d m i x t u r e a g e n t s t h a t c a u s e l e s i o n s i n DNA, i n d u c e s p r o -J A a n d p r o t e i n s p h a g e s ( 7 , 1 1 , 2 2 ) . I t i s t h e r e f o r e v e r y a t t r a c t i v e, n t i n g . A c i d - p r e - t o i m p l i c a t e n a l i d i x i c a c i d i n i n d u c i n g a DNAu e r c e n t a g e o f t h e l e s i o n o r i n h i b i t i n g DNA r e p a i r , o r b o t h . T h er e t h e d r u g a d d i - r e s u l t s p r e s e n t e d h e r e a r e v e r y s u g g e s t i v e o f0 ) K L 6 1 ; ( a ) s u c h a c t i o n s b y n a l i d i x i c a c i d . F i r s t , n a l i d i x i c, o r p o r a t i o n : ( 0 ) a c i d d r a s t i c a l l y a l t e r s t h e p a t t e r n o f n e w l y s y n -

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8/2/2019 Nalidixic Acid Paper

4/5

V O L . 1 1 , 1 9 7 7

KLI61

11

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2 3 4 12 3 4F I G . 2 . E f f e c t o f n a l i d i x i c a c i d on p u l s e - l a b e l e d p r o t e i n p a t t e r n s . KL16 1 a nd KL166 were t r e a t e d w i t hn a l i d i x i c a c i d and p u l s e l a b e l e d w i t h ' 4 C - l a b e l e d amino a c i d m i x t u r e as d e s c r i b e d i n t h e l e g e n d t o F i g . 1 .S a m p l e s c o r r e s p o n d i n g t o 5 x 1 0 7 c e l l s were l y s e d a n d a n a l y z e d on a 7 . 5 t o 1 2 . 5 % g r a d i e n t s o d i u m d o d e c y ls u l f a t e - p o l y a c r y l a m i d e s l a b g e l . P u l s e - l a b e l e d p r o t e i n p a t t e r n s were o b t a i n e d b y a u t o r a d i o g r a p h y . Th ef o l l o w i n g p r o t e i n s were e l e c t r o p h o r e s e d on t h e same g e l f o r m o l e c u l a r w e i g h t d e t e r m i n a t i o n : E . c o l i RNAp o l y m e r a s e s u b u n i t s ( 1 6 5 , 1 5 5 , 9 0 and 3 9 K ) , b o v i n e serum a l b u m i n ( 6 7 K ), o v a l b u m i n ( 4 5 K ) , u r i c a s e ( 3 3 K )and m y o g l o b i n ( 1 8 K ) . ( 1 ) C o n t r o l ; ( 2 ) 1 0 m i n , ( 3 ) 20 m i n , and ( 4 ) 30 m i n a f t e r d r u g a d d i t i o n . Th e m o l e c u l a rw e i g h t s ( i n k i l o d a l t o n s ) o f r e l e v a n t p e p t i d e s a re i n d i c a t e d .

t h e s i z e d p r o t e i n s i n s e n s i t i v e b u t n o t i n r e s i s t -a n t s t r a i n s o f b a c t e r i a . T h i s a l t e r a t i o n i s l i k e l yd u e t o a d i r e c t o r i n d i r e c t e f f e c t o f n a l i d i x i c a c i don g e n e e x p r e s s i o n a t t h e RNA l e v e l a n d n o t a tt h e l e v e l o f t r a n s l a t i o n ( 8 ) . H o w e v e r , a m o d i f i -c a t i o n o f t h e t r a n s l a t i o n a l m a c h i n e r y b y t h ed r u g c a n n o t b e t o t a l l y e x c l u d e d a t t h e p r e s e n tt i m e . S e c o n d , n a l i d i x i c a c i d e l i c i t s d i f f e r e n t e f -f e c t s on d i f f e r e n t g e n e p r o d u c t s , a s s h o w n b yt h e i n d u c t i o n o f c e r t a i n p r o t e i n s a n d t h e d e -p r e s s i o n o f o t h e r s . Some o f t h e i n d u c e d p r o -t e i n s , s u c h a s 8 - g a l a c t o s i d a s e , a re a p p a r e n t l yn o t r e l a t e d t o t h e b a c t e r i c i d a l e f f e c t o f t h e d r u g .O t h e r i n d u c e d o r d e p r e s s e d p r o t e i n s may p l a y ar o l e i n t h e d r u g a c t i o n . On e o f t h e p o s s i b i l i t i e s

i s t h a t some o f t h e d r u g - d e p r e s s e d p r o t e i n s mayb e i n v o l v e d i n c e l l u l a r DNA metabolism i n away s i m i l a r t o DNA r e p a i r e n z y m e s o r n u-c l e a s e s . R e c e n t l y C r u m p l i n an d Smith r e p o r t e d( 9 ) t h a t t h e s t e p b l o c k e d b y n a l i d i x i c a c i d i nDNA s y n t h e s i s i s t h e c o n v e r s i o n o f 3 8 S s i n g l e -s t r a n d e d DNA f r a g m e n t s i n t o h i g h - m o l e c u l a r -w e i g h t DNA i n s e n s i t i v e c e l l s . The n a l i d i x i ca c i d - i n d u c e d p r o t e i n s may t h e r e f o r e d i r e c t l y i n -t e r f e r e w i t h t h e c o n v e r s i o n o f t h e 3 8 S f r a g -ments i n t o f u l l - s i z e d DNA o r r a p i d l y d e g r a d et h e 388 i n t e r m e d i a t e f r a g m e n t s , and t h u s e x e r tt h e b a c t e r i c i d a l a c t i o n o f t h e d r u g . T h i s n o t i o ni s s u p p o r t e d b y t h e o b s e r v a t i o n t h a t c o m p e t e n tRNA a n d p r o t e i n s y n t h e s e s a re e s s e n t i a l p r e -

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1 7 0 NOTESr e q u i s i t e s f o r b o t h e x o n u c l e a s e a c t i o n a n d t h eb a c t e r i c i d a l a c t i o n o f n a l i d i x i c a c i d ( 6 , 8 ) .

I a m g r a t e f u l t o J . D . Karam f o r h i s g e n e r o s i t y i n p r o v i d -i n g e q u i p m e n t a n d h i s p a t i e n c e i n i n s t r u c t i n g i n t h e u s e o fs l a b g e l t e c h n i q u e s . I a l s o w i s h t o t h a n k J . D . Karam a n d B .B a g g e t t f o r t h e i r c r i t i c a l r e a d i n g o f t h e m a n u s c r i p t . T h eb a c t e r i a l s t r a i n s w e r e k i n d l y p r o v i d e d b y B a r b a r a B a c h -m a n .T h i s w o r k w a s s u p p o r t e d b y P u b l i c H e a l t h S e r v i c e G e n -e r a l R e s e a r c h G r a n t R R 0 5 4 2 0 ( f r o m t h e D i v i s i o n o f R e -s e a r ch R e s o u r ce s ) t o t h e M e d i c a l U n i v e r s i t y a n d b y t h eS o u t h C a r o l i n a S t a t e A p p r o p r i a t i o n f o r B i o m e d i c a l R e -s e a r c h .LITERATURE CITED

1 . B a c h m a n , B . J . 1 9 7 2 . P e d i g r e e s o f s o m e m u t a n t s t r a i n so 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 . 3 6 : 5 2 5 - 5 5 7 .2 . B a u e r n f e i n d , A . , a n d G . G r u m m e r . 1 9 6 5 . B i o c h e m i c a le f f e c t s o f n a l i d i x i c a c i d o n E s c h e r i c h i a c o l i . C h e m o t h -e r a p i a 1 0 : 9 5 - 1 0 2 .3 . B o u r g u i g n o n , G . J . , M . L e v i t t , a n d R . S t e r n g l a n z .1 9 7 3 . S t u d i e s o n t h e m e c h a n i s m o f a c t i o n o f n a l i d i x i ca c i d . A n t i m i c r o b . A g e n t s C h e m o t h e r . 4 : 4 7 9 - 4 8 6 .4 . B o y l e , J . V . , T . M . C o o k , a n d W . A . G o s s . 1 9 6 9 . M e c h a -n i s m o f a c t i o n o f n a l i d i x i c a c i d o n E s c h e r i c h i a c o l i .V I . C e l l - f r e e s t u d i e s . J . B a c t e r i o l . 9 7 : 2 3 0 - 2 3 6 .5 . C h a m p e , S . P . , a n d S . B e n z e r . 1 9 6 2 . R e v e r s a l o f m u t a n tp h e n o t y p e s b y 5 - f l u o r o u r a c i l : a n a p p r o a c h t o n u c l e o -t i d e s e q u e n c e s i n m e s i e n g e r - R N A . P r o c . N a t l . A c a d .S c i . U . S . A . 4 8 : 5 3 2 - 5 4 6 .6 . C o o k , T . M . , W. H . D e i t z , a n d W. A . G r o s s . 1 9 6 6 .M e c h a n i s m o f a c t i o n o f n a l i d i x i c a c i d o n E s c h e r i c h i ac o l i . I V . E f f e c t o n t h e s t a b i l i t y o f c e l l u l a r c o n s t i t u -e n t s . J . B a c t e r i o l . 9 1 : 7 7 4 - 7 7 9 .7 . C o w l i s h a w , J . , a n d W . G i n o z a . 1 9 7 0 . I n d u c t i o n o f Ap r o p h a g e b y n a l i d i x i c a c i d . V i r o l o g y 4 1 : 2 4 4 - 2 5 5 .8 . C r u m p l i n , G . C . , a n d J . T . S m i t h . 1 9 7 5 . N a l i d i x i c a c i d :a n a n t i b a c t e r i a l p a r a d o x . A n t i m i c r o b . A g e n t s C h e m -o t h e r . 8 : 2 5 1 - 2 6 1 .9 . C r u m p l i n , G . C . , a n d J . T . S m i t h . 1 9 7 6 . N a l i d i x i c a c i da n d b a c t e r i a l c h r o m o s o m e r e p l i c a t i o n . N a t u r e ( L o n -d o n ) 2 6 0 : 6 4 3 - 6 4 5 .1 0 . E b e r l e , H . , a n d W. M a s k e r . 1 9 7 1 . E f f e c t o f n a l i d i x i ca c i d o n s e m i c o n s e r v a t i v e r e p l i c a t i o n a n d r e p a i r s y n -

A N T I M I C R O B . A G E N T S C H E M O T H E R .t h e s i s a f t e r u l t r a v i o l e t i r r a d i a t i o n i n 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 . 1 0 5 : 9 0 8 - 9 1 2 .1 1 . G e i s s l e r , E . 1 9 6 7 . U n t e r s u c h u n g e n u b e r d e n M e c h a n i s -mus d e r I n d u k t i o n l y s o g e n e r B a k t e r i e n . X I . D e r E i n -f l u s s v o n D N A - S y n t h e s e h e m m e r n a u f l y s o g e n e B a k -t e r i e n . B i o l . Z e n t r a l b l . 8 6 ( S u p p l . ) : 5 5 - 6 7 .1 2 . G r o s s , W. A . , W. H . D e i t z , a n d T . M . C o o k . 1 9 6 5 .M e c h a n i s m o f a c t i o n o f n a l i d i x i c a c i d o n E s c h e r i c h i ac o l i . I I . I n h i b i t i o n o f d e o x y r i b o n u c l e i c a c i d s y n t h e s i s .J . B a c t e r i o l . 8 9 : 1 0 6 8 - 1 0 7 4 .1 3 . G u d a s , L . J . , a n d A . B . P a r d e e . 1 9 7 5 . M o d e l f o r r e g u l a -t i o n o f E s c h e r i c h i a c o l i DNA r e p a i r f u n c t i o n s . P r o c .N a t l . A c a d . S c i . U . S . A . 7 2 : 2 3 3 0 - 2 3 3 4 .1 4 . I n o u y e , M . a n d A . B . P a r d e e . 1 9 7 0 . C h a n g e s o f mem-b r a n e p r o t e i n s a n d t h e i r r e l a t i o n t o d e o x y r i b o n u c l e i ca c i d s y n t h e s i s a n d c e l l d i v i s i o n i n E s c h e r i c h i a c o l i . J .B i o l . C h e m . 2 4 5 : 5 8 1 3 - 5 8 1 9 .1 5 . K a r a m , J . D . , a n d M . G . B o w l e s . 1 9 7 4 . M u t a t i o n t oo v e r p r o d u c t i o n o f b a c t e r i o p h a g e T 4 g e n e p r o d u c t s . J .V i r o l . 1 3 : 4 2 8 - 4 3 8 .1 6 . L a e m m l i , U . K . 1 9 7 0 . C l e a v a g e o f s t r u c t u r a l p r o t e i n sd u r i n g a s s e m b l y o f t h e h e a d b a c t e r i o p h a g e T 4 . N a -t u r e ( L o n d o n ) 2 2 7 : 6 8 0 - 6 8 5 .1 7 . M a t z u r a , H . , S . M o l i n , a n d 0 . M a a l o e . 1 9 7 1 . S e q u e n -t i a l b i o s y n t h e s i s o f t h e P a n d , 3 ' s u b u n i t s o f t h e DNA-d e p e n d e n t RN A p o l y m e r a s e f r o m E s c h e r i c h i a c o l i . J .M o l . B i o l . 5 9 : 1 7 - 2 5 .1 8 . N i s h i d a , M . , Y . M i s h i m a , J . K a w a d a , a n d K . L . Y i e l d -i n g . 1 9 7 5 . E n h a n c e m e n t b y n a l i d i x i c a c i d o f t h e t h e r -m a l s u s c e p t i b i l i t y o f t h e T s - 7 m u t a n t o f E s c h e r i c h i ac o l i T A U - b a r . A n t i m i c r o b . A g e n t s C h e m o t h e r . 8 : 3 8 4 -3 8 6 .1 9 . P e d r i n i , A . M . , D . G e r o l d i , A . S i c c a r d i , a n d A . F a l a s h i .1 9 7 2 . S t u d i e s o n t h e m o d e o f a c t i o n o f n a l i d i x i c a c i d .E u r . J . B i o c h e m . 2 5 : 3 5 9 - 3 6 5 .2 0 . S h u m a n , H . , a n d M . S c h w a r t z . 1 9 7 5 . T h e e f f e c t o f n a l i -d i x i c a c i d o n t h e e x p r e s s i o n o f s o m e g e n e s i n E s c h e -r i c h i a c o l i K - 1 2 . B i o c h e m . B i o p h y s . R e s . Commun.6 4 : 2 0 4 - 2 0 9 .2 1 . S i m o n , T . J . , W. E . M a s k e r , a n d P . C . H a n a w a l t . 1 9 7 4 .S e l e c t i v e i n h i b i t i o n o f s e m i c o n s e r v a t i v e DNA s y n t h e -s i s b y n a l i d i x i c a c i d i n p e r m e a b i l i z e d b a c t e r i a .B i o c h i m . B i o p h y s . A c t a 3 4 9 : 2 7 1 - 2 7 4 .2 2 . T a k e t o , A . , a n d H . W a t a n b e . 1 9 6 7 . E f f e c t o f n a l i d i x i ca c i d o n t h e g r o w t h o f b a c t e r i a l v i r u s e s . J . B i o c h e m .6 1 : 5 2 0 - 5 2 2 .

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