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Stringent regulation of peptidoglycan synthesis in Escherichia coli Ramey, William David 1977

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STRINGENT REGULATION OF PEPTIDOGLYCAN SYNTHESIS IN ESCHERICHIA COLI by WILLIAM DAVID RAMEY A THESIS SUBMITTED IN PARTIAL' FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF MICROBIOLOGY We a c c e p t t h i s t h e s i s as 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 UNIVERSITY OF BRITISH COLUMBIA July, 1977 0 W i l l iam D a v i d Ramey, 1977 In presenting this thesis in partial fulfilment of the requ i rement s f o r an advanced degree at the University of B r i t i s h Co lumb ia , I ag ree that the Library shall make it freely available for r e f e r e n c e and s tudy . I further agree that permission for extensive copying o f t h i s t h e s i s for scholarly purposes may be granted by the Head o f my Department or by his representatives. It i s understood that c o p y i n g o r p u b l i c a t i o n o f this thesis for financial gain shall not be allowed without my written permission. Department of Microbiology The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date December 6, 1977 i ABSTRACT D u r i n g amino a c i d d e p r i v a t i o n , t h e amount of m e s o - d i a m i n o p i m e l i c a c i d (Dap) i n c o r p o r a t e d i n t o p e p t i d o g l y c a n by dap l y s amino a c i d a u x o t r o p h s of E s c h e r i c h i a c o l i was found t o be dependent on the a c t i v i t y o f t h e r e l A g e n e . p r o d u c t . I n r e l A + b a c t e r i a , the- i n c o r p o r a t i o n was s u b s t a n t -i a l l y r e d u c e d , whereas t h e i n c o r p o r a t i o n i n r e l A b a c t e r i a was e s s e n t i a l l y e q u a l t o t h a t i n t h e u n s t a r v e d c o n t r o l . The i n h i b i t i o n o f Dap i n c o r p o r a t i o n i n r e l A + b a c t e r i a was r e a d i l y overcome by r e s t o r a t i o n o f the r e q u i r e d amino a c i d o r by a d d i t i o n o f c h l o r a m p h e n i c o l (CAM). Guanosine 5 ' - d i p h o s p h a t e 3 ' - d i p h o s p h a t e (ppGpp) i s t h e p r o d u c t of t h e r e a c t i o n between t h e r e l A gene p r o d u c t and i d l i n g r i b o s o m e s i n s t r i n g e n t c e l l s . J_n v i t r o e x p e r i m e n t s i n d i c a t e d t h a t p h y s i o l o g i c a l l e v e l s of ppGpp i n h i b i t e d a t l e a s t two s t e p s i n p e p t i d o g l y c a n b i o s y n t h e s i s . One was the p h o s p h o - N - a c e t y l m u r a m o y l - p e n t a p e p t i d e t r a n s f e r a s e (EC2.7.1.13) r e a c t i o n and t h e o t h e r i n h i b i t i o n was p r o b a b l y a t t h e t r a n s f e r o f p e p t i d o -g l y c a n p r e c u r s o r s f r o m the g l y c o s y l c a r r i e r l i p i d (GCL) t o t h e n a s c e n t p e p t i d o g l y c a n . Q u a n t i t a t i o n o f t h e p e p t i d o g l y c a n p r e c u r s o r s and t h e n e t p e p t i d o g l y c a n i n r e l A + c o n t r o l and amino a c i d - d e p r i v e d b a c t e r i a i n d i c a t e d t h a t p e p t i d o g l y c a n a c c u m u l a t i o n was i n h i b i t e d . There was as much UDP-MurNAc-pentapeptide and G C L - l i n k e d i n t e r m e d i a t e s i n the amino a c i d - d e p r i v e d b a c t e r i a as i n t h e c o n t r o l b a c t e r i a . T h i s s u g g e s t s t h a t t h e t r a n s f e r o f l i p i d - l i n k e d p r e c u r s o r s t o n a s c e n t a c c e p t o r i s the s i t e o f i n h i b i t i o n o f Dap i n c o r p o r a t i o n . I n a d d i t i o n , t h e p o o l o f s o l u b l e n u c l e o t i d e - l i n k e d p r e c u r s o r s was found t o a c c u m u l a t e when r e l A b a c t e r i a were d e p r i v e d o f r e q u i r e d amino a c i d s . T h i s s u g g e s t s t h a t the s i z e o f the p r e c u r s o r p o o l i s a l s o r e g u l a t e d by the a c t i v i t y o f t h e r e l A gene p r o d u c t . \ i i TABLE OF CONTENTS Page INTRODUCTION I . S t r u c t u r e and f u n c t i o n o f E. c o l i p e p t i d o g l y c a n 1 I I . B i o s y n t h e s i s o f E. c o l i p e p t i d o g l y c a n 4 I I I . R e g u l a t i o n o f f o r m a t i o n o f p e p t i d o g l y c a n and p e p t i d o g l y c a n p r e c u r s o r s 10 IV. S t r i n g e n t r e g u l a t i o n i n E. c O l i 11 V. Purp o s e o f t h i s t h e s i s 13 MATERIALS AND METHODS I . B a c t e r i a l s t r a i n s 14 I I . C u l t u r a l c o n d i t i o n s 14 I I I . Chromatography ^ 14 IV. S c i n t i l l a t i o n c o u n t i n g 16 V. C h e m i c a l s 16 V I . P r e p a r a t i o n o f s u b s t r a t e s , s t a n d a r d s and ppGpp. A. S o l u b l e n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s . a) U D P - M u r N A c - t r i p e p t i d e 16 b) UDP-MurNAc-pentapeptide 17 B. Other s t a n d a r d s a) M u r N A c - p e n t a p e p t i d e 17 b) M u r N A c ( - G l c N A c ) - p e n t a p e p t i d e ( t e t r a p e p t i d e ) 18 c) GCL-P-P-MurNAc-pentapeptide and GCL-P-P-MurNAc(-GlcNAc)-pentapeptide 18 C. ppGpp p r e p a r a t i o n and p u r i f i c a t i o n 18 V I I . I n v i t r o a s s a y s f o r p e p t i d o g l y c a n b i o s y n t h e t i c enzymes. A. Coupled r e a c t i o n f o r p e p t i d o g l y c a n s y n t h e s i s a) P a r t i c u l a t e enzyme p r e p a r a t i o n 19 i i i Page b) C o u p l e d r e a c t i o n a s s a y f o r f o r m a t i o n o f p e p t i d o g l y c a n and G C L - l i n k e d i n t e r m e d i a t e s ..... 20 B. Exchange a s s a y f o r phospho-N-acetylmuramoyl p e n t a p e p t i d e t r a n s f e r a s e 20 C. D - a l a n y l a l a n i n e s y n t h e t a s e and D - a l a n y l a l a n i n e l i g a s e a) Enzyme p r e p a r a t i o n 21 b) A s s a y f o r D - a l a n y l a l a n i n e s y n t h e t a s e 22 c) A s s a y f o r D - a l a n y l a l a n i n e l i g a s e 22 V I I I . In v i v o p e p t i d o g l y c a n s y n t h e s i s A. A s s a y f o r TCA i n s o l u b l e [ ^ j D a p i n c o r p o r a t i o n 22 B. A s s a y f o r the d i s t r i b u t i o n o f i n c o r p o r a t e d [ 3H]Dap a) Q u a n t i t a t i v e a n a l y s i s of t h e l a b e l e d c y t o p l a s m i c , m e m b r a n e - a s s o c i a t e d , and. p e p t i d o g l y c a n components 24 b) Q u a n t i t a t i o n o f U D P - M u r N A c - t r i p e p t i d e and UDP-MurNAc-pentapeptide i n [^JDap l a b e l e d c e l l s 25 c) A n a l y s i s o f l i p i d zone components 1) M i l d a c i d h y d r o l y s i s o f l i p i d zone components 26 2) P r o t e a s e t r e a t m e n t o f l i p i d zone components 27 d) A n a l y s i s of [ 3 H ] l y s i n e c o n t e n t i n [ 3H]Dap l a b e l e d c e l l s 28 e) E x a m i n a t i o n o f s u p e r n a t a n t s f r o m l a b e l e d c u l t u r e s 28 RESULTS I . P e p t i d o g l y c a n - , s y n t h e s i s d u r i n g ' i n h i b i t i o n o f p r o t e i n s y n t h e s i s in v i v o A. E f f e c t o f amino a c i d d e p r i v a t i o n and CAM t r e a t m e n t on [ 3H]Dap i n c o r p o r a t i o n by r e l A b a c t e r i a 29 i v Page B. E f f e c t o f amino a c i d d e p r i v a t i o n of [ 3H]Dap i n c o r p o r a t i o n by r e l A b a c t e r i a 29 C. E f f e c t o f the r e l A gene on u t i l i z a t i o n o f endogenous DAP 38 I I . R e g u l a t i o n o f p e p t i d o g l y c a n s y n t h e s i s I n v i t r o A. C o u p l e d r e a c t i o n 38 B. Exchange r e a c t i o n 54 C. E f f e c t s o f n u c l e o t i d e s on t h e c o u p l e d r e a c t i o n 59 D. Other r e a c t i o n s i n p e p t i d o g l y c a n s y n t h e s i s 59 I I I . A n a l y s i s o f s t r i n g e n t c o n t r o l d u r i n g i n v i v o p e p t i d o g l y c a n s y n t h e s i s A. D i s t r i b u t i o n o f [ ^ j D a p i n c o r p o r a t e d by amino a c i d - d e p r i v e d b a c t e r i a 67 B. E f f e c t of s t r i n g e n t i n h i b i t i o n on the c o m p o s i t i o n of the G C L - l i n k e d i n t e r m e d i a t e s 79 C. S t r i n g e n t c o n t r o l o f s o l u b l e n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s 83 DISCUSSION 91 LITERATURE CITED 101 V LIST OF TABLES Page TABLE I . S t r a i n s of B a c t e r i a Used 15 TABLE I I . E f f e c t o f 2 mM c o n c e n t r a t i o n s o f n u c l e o t i d e s on the a c t i v i t y of the c o u p l e d r e a c t i o n f o r p e p t i d o g l y c a n s y n t h e s i s 60 TABLE I I I . E f f e c t of c o n c e n t r a t i o n o f i n h i b i t i n g n u c l e o t i d e s on i n h i b i t i o n of t h e c o u p l e d r e a c t i o n f o r p e p t i d o g l y c a n s y n t h e s i s 61 TABLE IV. E f f e c t of c o n c e n t r a t i o n o f ppGpp, ATP and EDTA on t h e a c t i v i t y o f the c o u p l e d r e a c t i o n f o r p e p t i d o g l y c a n s y n t h e s i s 62 TABLE V. D i s t r i b u t i o n o f [ 3H]Dap i n c o r p o r a t e d by LD5 ( r e l A + ) d u r i n g l y s i n e d e p r i v a t i o n 70 TABLE V I . A n a l y s i s o f s o l u b l e n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s s y n t h e s i z e d by LD5 ( r e l A ) d u r i n g l y s i n e d e p r i v a t i o n 71 TABLE V I I . D i s t r i b u t i o n o f [ 3H]Dap i n c o r p o r a t e d by LD5456 ( r e l A ) d u r i n g l y s i n e d e p r i v a t i o n 72 TABLE V I I I . D i s t r i b u t i o n o f l a b e l e d m a t e r i a l i n c u l t u r e s u p e r n a t a n t s , e x c l u d i n g f r e e [ 3H]Dap 74 TABLE IX. D i s t r i b u t i o n of [ 3H]Dap i n c o r p o r a t e d by LD52 ( r e l A + ) and LD2 ( r e l A ) d u r i n g 60 m i n u t e s of amino a c i d d e p r i v a t i o n 75 TABLE X. D i s t r i b u t i o n o f [ 3H]Dap i n c o r p o r a t e d by LD5457 ( r e l A ) d u r i n g l y s i n e o r l e u c i n e d e p r i v a t i o n 76 TABLE X I . E f f e c t of l y s i n e d e p r i v a t i o n and CAM t r e a t m e n t on d i s t r i b u t i o n o f [ 3H]Dap i n c o r p o r a t e d by LD5 ( r e l A ) 77 TABLE X I I . E f f e c t of l y s i n e d e p r i v a t i o n and CAM t r e a t m e n t on d i s t r i b u t i o n of [ 3 l l]Dap i n c o r p o r a t e d by LD5456 ( r e l A ) 78 TABLE X I I I . A n a l y s i s o f t h e l a b e l e d components of t h e l i p i d zone of c e l l s i n c u b a t e d w i t h [ 3H]Dap f o r 60 m i n u t e s 80 TABLE XIV. E f f e c t of p r o t e a s e and a c e t i c a c i d h y d r o l y s i s oiji t h e l a b e l e d m a t e r i a l o f t h e l i p i d zone of LD5 ( r e l A ) .. 82 v i Page TABLE XV. Amounts o f [ ^ l y s i n e and r a d i o a c t i v e l i p i d zone p r o t e i n i n whole c e l l s l a b e l e d w i t h [ 3H]Dap 84 TABLE X V I . A n a l y s i s o f s o l u b l e n u c l e o t i d e - l i n k e _ d p e p t i d o g l y c a n p r e c u r s o r s s y n t h e s i z e d by LD5 ( r e l A ) d u r i n g l y s i n e d e p r i v a t i o n and CAM t r e a t m e n t 85 TABLE X V I I . A n a l y s i s of s o l u b l e n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s s y n t h e s i z e d by LD5456 ( r e l A ) d u r i n g l y s i n e d e p r i v a t i o n and CAM t r e a t m e n t 87 TABLE X V I I I . E f f e c t o f D - c y c l o s e r i n e on t h e s i z e o f t h e p o o l o f s o l u b l e n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r i n LD5 ( r e l A ) 88 TABLE XIX. E f f e c t o f D - c y c l o s e r i n e on t h e s i z e of the p o o l o f s o l u b l e n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r i n LD5456 ( r e l A ) 89 v i i L I ST OF FIGURES Page FIGURE 1. S c h e m a t i c d r a w i n g o f the c r o s s - l i n k e d s t r u c t u r e o f a s m a l l p o r t i o n o f t h e p e p t i d o g l y c a n i n E. c o l i 2 FIGURE 2. The major s t e p s i n the b i o s y n t h e s i s o f E. c o l i p e p t i d o g l y c a n 6 FIGURE 3. E f f e c t o f l y s i n e d e p r i v a t i o n and CAM t r e a t m e n t on [ 3H]Dap i n c o r p o r a t i o n by LD5 ( r e l A ) 30 FIGURE 4. E f f e c t of l a t e a d d i t i o n of CAM on [ 3H]Dap + i n c o r p o r a t i o n by l y s i n e - d e p r i v e d LD5 ( r e l A ) 32 FIGURE 5. E f f e c t o f l y s i n e r e s t o r a t i o n on [ 3H]Dap i n c o r p o r a t i o n (A) and c u l t u r e t u r b i d i t y (B) of l y s i n e - d e p r i v e d LD5 ( r e l A ) 34 FIGURE 6. E f f e c t o f amino a c i d d e p r i v a t i o n on f 3H]Dap i n c o r p o r a t i o n by o t h e r r e l s t r a i n s A) s t r a i n LD51, B) s t r a i n LD52, C) s t r a i n LD2 36 FIGURE 7. E f f e c t of l y s i n e d e p r i v a t i o n and CAM t r e a t m e n t on [ 3HjDap i n c o r p o r a t i o n by LD5456 ( r e l A ~ ) 39 FIGURE 8. E f f e c t o f amino a c i d d e p r i v a t i o n on [ 3H]Dap i n c o r p o r a t i o n by o t h e r ( r e l A ) s t r a i n s A) s t r a i n LD5457, B) s t r a i n LD5458 41 FIGURE 9. U t i l i z a t i o n o f i n t r a c e l l u l a r r a d i o a c t i v e p o o l s by LD5 ( r e l A ) (A) and LD5456 ( r e l A ) (B) .; 43 FIGURE 10. I n c o r p o r a t i o n o f { 1 4 C j U D P - M u r N A c - p e n t a p e p t i d e i n t o G C L - l i n k e d i n t e r m e d i a t e s and p e p t i d o g l y c a n by a p a r t i c u l a t e membrane p r e p a r a t i o n i n c u b a t e d i n t h e p r e s e n c e and absence of 2 mM ppGpp 46 FIGURE 11. E f f e c t o f ppGpp c o n c e n t r a t i o n on the amount of p e p t i d o g l y c a n and G C L - l i n k e d i n t e r m e d i a t e s formed by the c o u p l e d r e a c t i o n f o r i n v i t r o p e p t i d o g l y c a n s y n t h e s i s 48 FIGURE 12. E f f e c t of M g C l 2 on t h e amount of p e p t i d o g l y c a n ( A ) and G C L - l i n k e d i n t e r m e d i a t e s ( B ) a c c u m u l a t e d by t h e c o u p l e d r e a c t i o n f o r i n v i t r o p e p t i d o g l y c a n s y n t h e s i s 50 v i i i Page FIGURE 13. I n f l u e n c e o f t h e m o l a r r a t i o o f M g C l ^ t o ppGpp ... on the r e l a t i v e amount of p e p t i d o g l y c a n and G C L - l i n k e d i n t e r m e d i a t e s a c c u m u l a t e d by the c o u p l e d r e a c t i o n f o r i n v i t r o p e p t i d o g l y c a n s y n t h e s i s 52 FIGURE 14. I n c o r p o r a t i o n o f [^ftJMP i n t o UDP-MurNAc-pentapeptide by the exchange r e a c t i o n f o r phospho-N-acetylmuramoyl - p e n t a p e p t i d e t r a n s f e r a s e i n c u b a t e d i n the p r e s e n c e and absence of ppGpp 55 FIGURE 15. (A) E f f e c t of M g C l 2 on t h e a c c u m u l a t i o n o f [ 3H]UMP i n UDP-MurNAc-pentapeptide by t h e exchange a s s a y i n c u b a t e d i n the p r e s e n c e and absence of ppGpp. (B) The e f f e c t of t h e m o l a r r a t i o o f M g C l 2 t o ppGpp on the exchange r e a c t i o n 57 FIGURE 16. E f f e c t of t h e p r e s e n c e or absence o f ppGpp on t h e a c t i v i t y of D - a l a n y l a l a n i n e s y n t h e t a s e (A) and D - a l a n y l a l a n i n e l i g a s e (B) 63 FIGURE 17. (A) E f f e c t o f M n C l 2 on t h e a c c u m u l a t i o n o f [ 3H]UMP i n UDP-MurNAc-pentapeptide by the exchange r e a c t i o n i n c u b a t e d i n t h e p r e s e n c e and absence of ppGpp. (B) The i n f l u e n c e o f t h e m o l a r r a t i o of M n C l 2 t o ppGpp on t h e exchange r e a c t i o n 65 FIGURE 18. D i s t r i b u t i o n of r a d i o a c t i v i t y when c e l l s o f LD5 ( r e l A ) were l a b e l e d w i t h [ H]Dap and t h e n chromatographed i n s o l v e n t s y s t e m I 68 FIGURE 19. S c h e m a t i c d i a g r a m of some of t h e r e g u l a t o r y s i t e s i n t he b i o s y n t h e s i s o f Ej_ c o l i p e p t i d o g l y c a n ....... 98 ACKNOWLEDGEMENT S I a p p r e c i a t e t h e h e l p and u s e f u l s u g g e s t i o n s p r o v i d e d by Dr. E. I s h i g u r o d u r i n g the c o u r s e o f t h e r e s e a r c h . I would l i k e t o than k t h e members of my committee, p a r t i c u l a r l y Dr. E.E. I s h i g u r o , Dr. P.P. D e n n i s and Dr. G. Weeks f o r t h e i r c o n s t r u c t i v e a s s i s t a n c e i n e d i t i n g t he t h e s i s . I would a l s o l i k e t o thank Dr. M. C a s h e l f o r g e n e r o u s l y d o n a t i n g 1 mg of p u r i f i e d ppGpp and J . B a i n f o r d o i n g a l o t of t y p i n g . I am g r a t e f u l f o r the s u p p o r t by an H.R. M a c M i l l a n s c h o l a r s h i p . X ABBREVIATIONS amino a c i d s : s t a n d a r d a b b r e v i a t i o n s recommended by t h e Commission on B i o c h e m i c a l N o m e n c l a t u r e (CBN) CAM : c h l o r a m p h e n i c o l CPM : co u n t s p e r m i n u t e Dap : m e s o - d i a m i n o p i m e l l c a c i d DCS : D - c y c l o s e r i n e GCL : g l y c o s y l c a r r i e r l i p i d ; C ^ ^ - i s o p r e n o i d a l c o h o l GCL-P : GCL-monophosphate GCL-P-P : GCL-diphosphate GlcNAc : N - a c e t y l g l u c o s a m i n e Mur : Muramic a c i d MurNAc : N - a c e t y l m u r a m i c a c i d n u c l e o t i d e s : s t a n d a r d a b b r e v i a t i o n s recommended by the CBN P^ : ph o s p h a t e p e n t a p e p t i d e : L-ala-D-glu-meso-Dap-D-ala-D-ala ppGpp : g u a n o s i n e 5'-diphosphate 3'-diphosphate TCA : t r i c h l o r o a c e t i c a c i d t r i p e p t i d e : L-ala-D-glu-meso-Dap t r i s : t r i s - ( h y d r o x y m e t h y l ) - a m i n o methane h y d r o c h l o r i d e INTRODUCTION I . S t r u c t u r e and f u n c t i o n o f E. c o l i p e p t i d o g l y c a n The n o r m a l p e p t i d o g l y c a n o f E. c o l i i s thought t o be a l a r g e b a g - l i k e m o l e c u l e w h i c h e n c l o s e s t h e c e l l . I n e l e c t r o n m i c r o g r a p h s of s e c t i o n e d c e l l s . i t a p p e a rs as a dense l a y e r , 2-3 nm t h i c k , sandwiched between th e i n n e r and o u t e r membranes. I t seems t o have d i s c r e t e a t t a c h m e n t s t o b o t h of t h e s e membranes, but can be i s o l a t e d by d i s s o l v i n g t h e membranes w i t h b o i l i n g sodium d o d e c y l s u l f a t e . P e p t i d o g l y c a n o b t a i n e d by t h i s method i s an i n t a c t m o l e c u l e , o f t e n r e f e r r e d t o as a s a c c u l u s . I t i s g e n e r a l l y c o n t a m i n a t e d w i t h r e g u l a r l y s p a c e d , bound p r o t e i n . T h i s p r o t e i n c a n be removed by t r e a t m e n t w i t h t r y p s i n o r S t r e p t o m y c e s g r i s e u s p r o t e a s e . The i s o l a t e d p r o t e i n - f r e e s a c c u l u s r e t a i n s the shape of the c e l l f r o m w h i c h i t was d e r i v e d and t h e r e f o r e a p p ears t o p o s s e s s some r i g i d i t y . The g e n e r a l s t r u c t u r e of p e p t i d o g l y c a n i s shown i n f i g u r e 1. I t i s composed of g l y c a n " c h a i n s b r i d g e d by s h o r t p e p t i d e s . The g l y c a n c h a i n s a r e h e t e r o p o l y m e r s of a l t e r n a t i n g N - a c e t y l m u r a m i c a c i d (MurNAc) and N - a c e t y l g l u c o s a m i n e ( G l c N A c ) . There i s an a v e r a g e of 100 hexosamines p e r c h a i n (45, 9 2 ) . T h i s i n d i c a t e s t h a t t h e g l y c a n s s h o u l d be about 0.1 um l o n g i f t h e y a r e u n i f o r m i n l e n g t h . The MurNAc a t the p o t e n t i a l r e d u c i n g end of each g l y c a n i s c o n s i s t e n t l y m o d i f i e d w i t h a l-*'6 a n h y d r o - l i n k a g e (41, 45, 49) and no r e d u c i n g s u g a r s a r e d e t e c t a b l e (41, 4 5 ) . A p p r o x i m a t e l y 94% o f t h e l a c t y l m o i e t i e s a t c a r b o n - 3 of the MurNAc a r e c o v a l e n t l y bonded to t h e L - a l a n i n e i n one of two s h o r t p e p t i d e s ( 1 0 9 ) . The e x a c t p r o p o r t i o n of t h e s e p e p t i d e s v a r i e s w i t h t h e growth c o n d i t i o n s (45, 94, 1 0 9 ) ; g e n e r a l l y , 20% a r e t h e t r i p e p t i d e , L-ala-D-glu-meso-Dap, and t h e r e m a i n d e r a r e t h e t e t r a p e p t i d e , L-ala-D-glu-meso-Dap-D-ala. About 15 t o 25% of t h e D - a l a i n the t e t r a p e p t i d e s i s bonded t o t h e f r e e amino group FIGURE 1. Schematic drawing of the cross-linked structure of a small portion of the peptidoglycan iii E. coli. Note that the D-alanine content is possibly more variable and that the actual relative positions of the cross-linking peptides and the Braun lipoprotein are unknown. After Braun et al. (11). 3 Covalently linked lipoprotein replacing D-Ala 4 of the meso-Dap i n t h e t e t r a p e p t i d e of an a d j a c e n t g l y c a n c h a i n . These form t h e c r o s s - l i n k i n g p e p t i d e s . I n a d d i t i o n , 1 0 % of t h e Dap m o l e c u l e s i n t h e p e p t i d o g l y c a n a r e c o v a l e n t l y bonded t o Braun l i p o p r o t e i n s (8, 1 1 ) . The o r i e n t a t i o n of t h e g l y c a n c h a i n s i s unknown. The l e n g t h p r e c l u d e s the p o s s i b i l i t y t h a t t h e y might r a d i a t e outwards from the l o n g i t u d i n a l a x i s of the c e l l . I n a d d i t i o n , t h e r e a r e o n l y 5 x 10 hexosamines p e r s a c c u l u s ( 1 1 ) . T h i s s u g g e s t s t h a t the s a c c u l u s can o n l y be one r e p e a t i n g u n i t t h i c k i n o r d e r t o a c c o u n t f o r the s u r f a c e a r e a c a l c u l a t e d t o be e n c l o s e d by t h e i n t a c t p e p t i d o g l y c a n ( 1 1 ) . Some of t h e p e p t i d e c h a i n s must be o r i e n t e d outwards i n o r d e r t o accommodate t h e a t t a c h e d B r a u n l i p o p r o t e i n s , w h i c h a r e a l s o p a r t i a l l y embedded i n t h e o u t e r membranes ( 8 ) . The r e l a t i v e p o s i t i o n s of t h e d i f f e r e n t t y p e s of a t t a c h e d p e p t i d e s a r e unknown. There may be l o c a l i z e d s t r u c t u r a l d i f f e r e n c e s (9, 10, 25, 34, 78, 79, 92, 115); e.g.,. t h e p e p t i d o g l y c a n a t t h e s e p t a may be l e s s c r o s s - l i n k e d t h a n e l s e w h e r e (25, 34, 78, 7 9 ) . The f u n c t i o n s of the p e p t i d o g l y c a n a r e not c o m p l e t e l y u n d e r s t o o d . I t i s a n / e s s e n t i a l s t r u c t u r e s i n c e the b a c t e r i a n o r m a l l y l y s e when t r e a t e d w i t h a n t i - m e t a b o l i t e s or enzymes w h i c h d i s r u p t t h e i n t e g r i t y o f the s a c c u l u s . I t may s e r v e as a m e c h a n i c a l s u p p o r t w h i c h a l l o w s t h e b a c t e r i a t o w i t h s t a n d t h e f o r c e s g e n e r a t e d by t h e i n t e r n a l o s m o t i c p r e s s u r e . I t may a l s o be i n v o l v e d i n c o n t r o l l i n g the s i z e and shape of t h e c e l l s . The s t r u c t u r e i s l o o s e enough t h a t p e p t i d o g l y c a n s h o u l d n o t i n t e r f e r e w i t h c e l l p e r m e a b i l i t y ( 1 1 ) , a l t h o u g h i t might b i n d s p e c i f i c m o l e c u l e s . I I . B i o s y n t h e s i s of E. c o l i p e p t i d o g l y c a n Most of the c o n c e p t s of p e p t i d o g l y c a n b i o s y n t h e s i s have been g e n e r a l i z e d f r o m work w i t h a v a r i e t y of b a c t e r i a . These i n c l u d e 5 S t a p h y l o c o c c u s , S t r e p t o c o c c u s , M i c r o c o c c u s , L a c t o b a c i l l u s , B a c i l l u s  s u b t i l u s and Ej_ c o l i . The a r e a s where t h e r e s u l t s o v e r l a p s u g g e s t t h a t t h e g e n e r a l e x t r a p o l a t i o n s f r o m one b a c t e r i a l s p e c i e s t o a n o t h e r a r e v a l i d . However, minor d i f f e r e n c e s do o c c u r and must be c o n s i d e r e d i n v i e w o f t h e v a r i a t i o n i n p e p t i d o g l y c a n s t r u c t u r e between some s p e c i e s or even w i t h i n s p e c i e s grown under d i f f e r e n t c o n d i t i o n s . The b i o s y n t h e t i c s t e p s can be d i v i d e d i n t o t h r e e c a t e g o r i e s : i ) t h e c y t o p l a s m i c r e a c t i o n s i i ) t h e membrane-associated r e a c t i o n s i i i ) t h e w a l l - a s s o c i a t e d r e a c t i o n s . These r e a c t i o n s a r e summarized i n f i g u r e 2. The c y t o p l a s m i c r e a c t i o n s shown i n f i g u r e 2 have been demon-s t r a t e d i n E. c o l i (66, 110, 111). The r e a c t i o n s w h i c h s y n t h e s i z e t h e i n t e r m e d i a t e s i n t h e a l a n i n e pathway ( r e a c t i o n s 6 and 7, f i g u r e 2) and t h e n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e s u r s o r s ( r e a c t i o n s 1, 2, 3, 4 , 5 and 8, f i g u r e 2) a r e c a t a l y z e d by h i g h l y s p e c i f i c s o l u b l e enzymes. The a c t i v i t y o f e ach of t h e a d d i n g enzymes ( r e a c t i o n s 3, 4 , 5 and 8, f i g u r e 2) i s dependent on ATP and r e q u i r e s e i t h e r magnesium o r manganese. The s o u r c e of t h e D - g l u t a m i c a c i d i n r e a c t i o n 4 ( f i g u r e 2) i s unknown. No g l u t a m i c a c i d racemase a c t i v i t y or D - a l a n i n e : 2 - k e t . o g l u t a r a t e t r a n s a m i n a s e a c t i v i t y i s known f o r E. c o l i ( 7 2 ) . The meso-Dap i s an i n t e r m e d i a t e i n L - l y s i n e b i o s y n t h e s i s , b u t i t s i n c o r p o r a t i o n i s c o n f i n e d t o p e p t i d o g l y c a n and p e p t i d o g l y c a n p r e s u r s o r s . The D - a l a n i n e i s e n z y m a t i c a l l y r a c e m i z e d f r o m L - a l a n i n e . I t i s d e s t r o y e d by D - a l a n i n e dehydrogenase (27, 5 8 ) . R e a c t i o n 1 ( f i g u r e 2) i s . i n h i b i t e d by f o s f o m y c i n ( 1 1 0 ) . R e a c t i o n s 6 and 7 ( f i g u r e 2) a r e I n h i b i t e d by D - c y c l o s e r i n e , L - c y c l o s e r i n e , 0 - c a r b a m y l -D - s e r i n e , and c h l o r o - D - a l a n i n e (59, 69, 70, 74). No common a n t i b i o t i c s are.known t o i n t e r f e r e w i t h the o t h e r r e a c t i o n s . 6 FIGURE 2. The major steps in the biosynthesis of E. coli peptidoglycan. Each reaction is denoted by a circled number. Cytoplasmic Reactions UDP NADP NADPH UDP-MurNAc © © ^ L-Ala UDP-MurNAc-L-Ala ©U~D Glu UDP-MurNAc-L-Ala-D-Glu UDP-GlcNAc- - * enoylpyruvate Phosphoenolpyruvate UDP-GlcNAc UMP UDP-MurNAc-L-ala-D-Glu-mesp-Dap-D-Ala-D-Ala (UDP-MurNAc-pentapeptide) UDP-MurNAc-L-Ala-D-Glu-meso-Dap meso Dap © L-Ala D-Ala ^D-Ala-D-Ala © © Membrane-Associated React ions Cel l Wa l l -A s s o c i a t e d Reac t i o n s GCL-P-P-MurNAc -pentapeptide. I ( g ) GlcNAc GCL-P-P-MurNAc-pentapeptide GCL-P GCL-P- ? ' Cell Wall Acceptor Cell Wall Acceptor ' Plus Precursor 8 The m embrane-associated r e a c t i o n s have been most e x t e n s i v e l y examined i n S t a p h y l o c o c c u s , b u t seem t o be s i m i l a r i n E. c o l i (9, 51, 1 0 8 ) ' B o t h of the G C L - l i n k e d p e p t i d o g l y c a n i n t e r m e d i a t e s ( p r o d u c t s of r e a c t i o n s 9 and 10, f i g u r e 2) a r e s y n t h e s i z e d by p a r t i c u l a t e enzymes. These membrane-bound i n t e r m e d i a t e s a r e n o t m o d i f i e d or bonded t o t h e Braun l i p o p r o t e i n u n t i l a f t e r t h e y have been i n c o r p o r a t e d i n t o t h e p e p t i d o g l y c a n ( 9 ) . T h e i r f o r m a t i o n i s s t i m u l a t e d by ATP (2; t h i s t h e s i s ) . T h i s may be due t o c o n v e r s i o n of s m a l l amounts of GCL t o t h e GCL-P (46, 85) r e q u i r e d f o r t h e i n i t i a l t r a n s l o c a s e r e a c t i o n ( r e a c t i o n 9, f i g u r e 2 ) . The de-phos-p h o r y l a t i o n of GCL-P-P i n r e a c t i o n 12 ( f i g u r e 2) i s p r e v e n t e d by b a c i t r a c i n (96, 103). N i s i n and t u n i c a m y c i n m ight i n t e r f e r e w i t h t h e a d d i t i o n of UDP-GlcNAc t o t h e GCL-P-P-MurNAc-pentapeptide i n r e a c t i o n 10 ( f i g u r e 2 ) ( 6 , 63, 112). Vancomycin and many o t h e r a n t i b i o t i c s i n t e r f e r e w i t h r e a c t i o n 11 ( f i g u r e 2 ) ( 3 7 , 63, 7 1 ) . The c e l l w a l l - a s s o c i a t e d r e a c t i o n s a r e not f u l l y u n d e r s t o o d . The p r e c u r s o r s a r e known t o be i n c o r p o r a t e d i n t o t h e p e p t i d o g l y c a n , b u t t h e r e i s n o t much agreement about the p r e c i s e mechanism. Some t h e o r i e s s u g g e s t t h a t each p r e c u r s o r i s t r a n s g l y c o s y l a t e d t o a n a s c e n t g l y c a n c h a i n i n t h e s a c c u l u s . Other t h e o r i e s s u g g e s t t h a t the membrane-bound p r e c u r s o r s a r e e xtended i n t o g l y c a n c h a i n s w h i c h a r e t h e n t r a n s g l y c o s y l a t e d o r t r a n s -p e p t i d a t e d i n t o t h e s a c c u l u s . S t i l l o t h e r t h e o r i e s s u g g e s t t h a t o n l y o c c a s i o n a l p r e c u r s o r s a r e i n i t i a l l y c r o s s - l i n k e d i n t o the p e p t i d o g l y c a n . These p r e c u r s o r s can t h e n be e xtended by subsequent t r a n s g l y c o s y l a t i o n t o f o r m g l y c a n c h a i n s w h i c h a r e s e c u r e l y bonded i n t o t h e s a c c u l u s by con-c u r r e n t o r c o n s e c u t i v e t r a n s p e p t i d a t i o n . I n most models, a u t o l y s i n s a r e t h ought t o open t h e c l o s e d s t r u c t u r e o f the s a c c u l u s t o p e r m i t t h e i n s e r t i o n o f new components and e n l a r g e m e n t . T h i s i s p r o b a b l e , b u t has n o t been 9 p r o v e n . No p o l y m e r i z e d g l y c a n c h a i n s have been d e t e c t e d on t h e Ej_ c o l i GCL ( 9 ) . S i m i l a r l y , M i r e l m a n and Schwartz (78) found no e v i d e n c e f o r t r a n s g l y c o s y l a t i o n o f p e p t i d o g l y c a n p r e c u r s o r s t o t h e g l y c a n s i n t h e s a c c u l u s , when t h e y a s s a y e d p e p t i d o g l y c a n s y n t h e s i s i n e t h e r - p e r m e a b i l i z e d c e l l s . They s u g g e s t e d t h a t t h e t r a n s p e p t i d a t i o n o r c r o s s - l i n k i n g i s the p r i n c i p l e means f o r i n c o r p o r a t i n g the new p e p t i d o g l y c a n i n t o t h e E. c o l i s a c c u l u s ( 7 8 ) . However, most t r a n s p e p t i d a t i o n i s a r e l a t i v e l y d e l a y e d p r o c e s s i n E. c o l i (10, 93; p e r s o n a l o b s e r v a t i o n ) . T h i s would n e c e s s i t a t e t h a t some components a r e i n i t i a l l y i n c o r p o r a t e d by l i m i t e d t r a n s p e p t i d a t i o n and l a t e r s e c u r e d w i t h a s l o w e r , more e x t e n s i v e t r a n s -p e p t i d a t i o n . T h i s m i g h t a g r e e w i t h a*;model f o r the growth o f the E^ c o l i p e p t i d o g l y c a n w h i c h s u g g e s t s t h a t the p e p t i d o g l y c a n p r e c u r s o r s a r e i n i t i a l l y i n c o r p o r a t e d a t the septum, b u t a r e s u b s e q u e n t l y r e l e a s e d and r e d i s t r i b u t e d t o d i v e r s e s i t e s t h r o u g h o u t the s a c c u l u s ( 9 0 ) . However, t h i s model was based on d u b i o u s p u l s e - l a b e l i n g e x p e r i m e n t s and has been q u e s t i o n e d ( 8 , 1 0 ) . A l a r g e sodium d o d e c y l s u l f a t e - s o l u b l e p e p t i d o g l y c a n component has been r e p o r t e d (10, 78, 7 9 ) , b u t was n o t c h a r a c t e r i z e d . I t may be an i n t e r m e d i a t e i n t h e f i n a l a s s e mbly and m o d i f i c a t i o n o f the p e p t i d o g l y c a n . The d i s c r e -pancy between t h e D - a l a n i n e c o n t e n t s o f t h e s a c c u l u s and t h e p e p t i d o g l y c a n p r e c u r s o r s i s a t t r i b u t e d t o t h e t r a n s p e p t i d a s e and c a r b o x y p e p t i d a s e a c t i v i t i e s . The t r a n s p e p t i d a s e s l i b e r a t e t h e t e r m i n a l D - a l a n l n e s d u r i n g t h e f o r m a t i o n o f the bond between the p e n u l t i m a t e D - a l a n i n e i n one g l y c a n c h a i n and the meso-Dap of a n o t h e r . The c a r b o x y p e p t i d a s e a c t i v i t i e s remove t h e D - a l a n i n e s w i t h o u t consequent t r a n s p e p t i d a t i o n . . 10 III. Regulation of formation of peptidoglycan and peptidoglycan precursors Although the sacculus is essential for the normal survival of an E. coll, very l i t t l e is known concerning the regulation of peptidoglycan synthesis. Many of the characterized mutations for enzymes in peptidoglycan synthesis have been mapped at positions near either two minutes or eighty-nine minutes on the revised E. coli K-12 genetic map (3). This clustering suggests that the respective genes may be organized into transcription units (68, 69), but no proof for co-ordinate control of these genes has been demonstrated. The rate of peptidoglycan synthesis increases during cell septation (48, 78, 90). This may be due to changes in the activities of the biosynthetic enzymes or the autolysins. No one has reported on the activities of the individual biosynthetic enzymes during the cell division cycle. The activities of many of the autolysins remains constant (5, 35). However, the activity of the carboxypeptidase II (.5), and possibly an endopeptidase (5, 35), increases prior to septation. The carboxypeptidase II can remove the penultimate D-alanine from some peptidoglycan precursors. The endopeptidase can hydrolyse the peptide cross-links in the sacculus. It may be involved in creating the sites for the incorporation of newly synthesized peptidoglycan. The amount of D-alanine is controlled by feedback inhibition of the D-alanylalanine synthetase (reaction 7, figure 2) (20, 66, 69; personal observation). Further regulation of the amount of D-alanylalanine and D-alanine might be achieved by repressing the alanine racemase (reaction 6, figure 2) (27, 58) or inducing alanine dehydrogenase (27). The amount of UDP-MurNAc-pentapeptide may be regulated by feedback inhibition of the first reaction in the biosynthesis of peptidoglycan 11 p r e c u r s o r s ( r e a c t i o n 1, f i g u r e 2) by UDP-MurNAc-pentapeptide (70, I H , t h i s t h e s i s ) . A c y t o p l a s m i c p e p t i d a s e m i g h t a l s o h y d r o l y s e e x c e s s UDP-MurNAc-pentapeptide ( 3 3 ) . The amount of a v a i l a b l e GCL-P, and c o n s e -q u e n t l y the amount of p o t e n t i a l t r a n s l o c a t i o n ( r e a c t i o n 9, f i g u r e 2 ) , c o u l d be l i m i t e d w i t h s p e c i f i c p h o s p h o r y l a s e s and k i n a s e s (46, 8 5 ) . I n a d d i t i o n , t h e r e a r e c o n t r o l s w h i c h a l l o w the c e l l s t o s e l e c t i v e l y use the GCL f o r t h e s y n t h e s i s o f p e p t i d o g l y c a n , some o u t e r membrane p o l y s a c c h a r i d e s and e x o c e l l u l a r c o l a n i c a c i d (53, 105). The d i f f e r e n t p o l y m e r s can be d i f f e r e n t i a l l y s y n t h e s i z e d (88, 105) and do not compete f o r the GCL-P i n v i v o (89). P e p t i d o g l y c a n s y n t h e s i s i s r e l a t i v e l y i n d e p e n d e n t o f c o n c u r r e n t DNA or RNA s y n t h e s i s . I t a p p a r e n t l y c o n t i n u e s when c e l l s a r e t r e a t e d w i t h n a l i d i x i c a c i d (43, 95) or a c t i n o m y c i n . D ( 1 0 1 ) . I t a l s o c o n t i n u e s a t near n o r m a l r a t e s when p r o t e i n s y n t h e s i s i s i n h i b i t e d by CAM (38, 67, 70, 7 5 ) . However, th e r e l a t i v e r a t e of p e p t i d o g l y c a n s y n t h e s i s i s d e c r e a s e d when p r o t e i n s y n t h e s i s i s i n h i b i t e d by d e p r i v i n g some c e l l s of r e q u i r e d amino a c i d s (E. I s h i g u r o , p e r s o n a l c o m m u n i c a t i o n ; t h i s t h e s i s ) . The d i f f e r e n c e i s shown i n t h i s t h e s i s t o be due t o t h e i n f l u e n c e of the s t r i n g e n t c o n t r o l system on s p e c i f i c r e a c t i o n s of p e p t i d o g l y c a n s y n t h e s i s . IV. S t r i n g e n t r e g u l a t i o n i n Ej_ c o l i D u r i n g amino a c i d d e p r i v a t i o n , some b a c t e r i a s e v e r e l y r e d u c e th e a c c u m u l a t i o n o f s t a b l e RNA. T h i s e f f e c t i s t h e s t r i n g e n t r e s p o n s e . I t i s t h o u g h t t o be caused by ppGpp, a n u c l e o t i d e p r o duced i n l a r g e amounts d u r i n g a s p e c i f i c i n t e r a c t i o n o f t h e r e l A gene p r o d u c t and i d l i n g r i b o s o m e s . The amount of ppGpp a c c u m u l a t e d d u r i n g amino a c i d d e p r i v a t i o n , and the degree of i n h i b i t i o n of RNA a c c u m u l a t i o n , i s dependent on t h e particular amino acid and the bacterial strain (12, 26, 55, 91). The ppGpp is unstable in vivo and rapidly disappears when cultures of amino acid-deprived bacteria are supplemented with the requisite amino acids (12, 14) or treated with CAM (12, 31). The in vivo stability is enhanced by the spoT mutation (14, 55). Bacteria which are mutated in the relA gene are relaxed and do not accumulate ppGpp or stop accumulating stable RNA during amino acid deprivation. Stringent bacteria are phenotypically relaxed by CAM treatment. The activity of the.relA gene product also affects the accumulation or synthesis of: i) specific messenger RNA (23, 60, 81) i i ) proteins involved in translation (22, 23, 29, 30, 62, 84), transcription (62) and probably other functions (30, 57, 98) i i i ) polyamines (14) iv) phosphorylated sugars (50) v) some nucleotides (24, 47, 98) including cyclic-AMP (7) vi) various lipids, including phospholipids (32, 65, 76, 82, 83, 86) and fatty acids (83, 98, 99) such as 8-hydroxymyristic acid (100). Specific reactions, such as some of those involved in phospho-lipid biosynthesis (65, 76), are inhibited in vitro at the high levels of ppGpp (4mM) which occur in amino acid-deprived cells. Other responses, such as the stimulated, accumulation of specific messenger RNA (87, 102), are greatest at the low levels of ppGpp (o.l-0.2 mM) observed in cells growing in minimal media. The basal levels of ppGpp. vary inversely to the growth rate (61, 99). They are reduced when growing cells are treated with CAM (31). They are greatly enhanced by carbon source shiftdown (14, 28, 61), levallorphan treatment (14, 39) or NaCI shock (40), even ih relA cells. The variety of responses has led to the view that ppGpp is a signal molecule, or alarmone (102), which indicates a cellular imbalance and serves to coordinate the cellular activity to correct deficiencies in particular areas of metabolism (87, 102). 13 V. P u r p o s e of t h i s t h e s i s I n t h e r e l A + b a c t e r i a t h e r a t e o f p e p t i d o g l y c a n . s y n t h e s i s d u r i n g amino a c i d d e p r i v a t i o n i s s e v e r e l y r e d u c e d . The r a t e of s y n t h e s i s i s near n o r m a l when t h e c u l t u r e s a r e t r e a t e d w i t h CAM. The d i f f e r e n c e was t h o u g h t t o be due t o t h e I n f l u e n c e of t h e r e l A gene. That p o s s i b i l i t y was i n v e s t i g a t e d i n t h i s t h e s i s , u s i n g b o t h i h v i v o and i n v i t r o t e c h n i q u e s . MATERIALS AND METHODS 14 I . B a c t e r i a l s t r a i n s A l l s t r a i n s u s e d , and t h e i r r e l e v a n t c h a r a c t e r i s t i c s , a r e l i s t e d i n T a b l e 1. I I . C u l t u r a l c o n d i t i o n s S m a l l c u l t u r e s were i n c u b a t e d a e r o b i c a l l y a t 37°C i n a New B r u n s w i c k G y r o t o r y w a t e r b a t h s h a k e r (Model G76) s e t t o 225 rpm. L a r g e b a t c h e s o f c e l l s were i n c u b a t e d a t 37°C i n f o u r l i t e r E r l e n m e y e r f l a s k s on a New B r u n s w i c k G y r o t o r y s h a k e r (Model G10) s e t a t 250 rpm. The s m a l l c u l t u r e s were g e n e r a l l y grown i n s i d e arm f l a s k s . Growth was m o n i t o r e d w i t h a Klett-Summerson C o l o r i m e t e r and, where a p p r o p r i a t e , was c o n v e r t e d t o m i l l i g r a m d r y w e i g h t o f c e l l s w i t h a s t a n d a r d c a l i b r a t i o n c u r v e . B l u e f i l t e r s were used f o r c u l t u r e s i n m i n i m a l media and a r e d f i l t e r f o r t h e c u l t u r e s i n r i c h m e dia. L a r g e c u l t u r e s were m o n i t o r e d a t s p e c i f i c t i m e s by t r a n s f e r r i n g 9 m l a l i q u o t s t o s i d e a r m f l a s k s and m e a s u r i n g c u l t u r e d e n s i t y as above. A l l i n o c u l a were f r e s h o v e r n i g h t s t o c k s grown a t 37°C w i t h t h e wate r b a t h s h a k e r s e t t o 105 rpm. I I I . Chr omatography A l l paper chromatography was done w i t h s h e e t s o f Whatman 3 MM chromatography paper 57 cm l o n g . Chromatographs were d e v e l o p e d w i t h a d e s c e n d i n g s o l v e n t system i n a s e a l e d , g l a s s chromatography chamber. S e v e r a l p a p e r s were g e n e r a l l y d e v e l o p e d a t the same t i m e . S o l v e n t systems were mixed i m m e d i a t e l y p r i o r t o u s e . The s o l v e n t systems were: I . I s o b u t y r i c A c i d - 1 M NH.OH ( 5 : 3 , v / v ) ; I I . E t h a n o l - 1 M NH.Acetate pH 7.2 15 TABLE I . S t r a i n s o f B a c t e r i a Used S t r a i n s R e l e v a n t C h a r a c t e r i s t i c s S o u r c e Bacillus subtilis WB746 B a c i l l u s c e r e u s E s c h e r i c h i a c o l i VC10 E. c o l i LD5 E. c o l i LD2 E. c o l i LD51 E. c o l i LD52 E. c o l i LD5456 E. c o l i LD5457 E. c o l i LD5458 W i l d t y p e W i l d t y p e Spontaneous s t r e p t o m y c i n r e s i s t a n t mutant of W3110 F , t h i , l y s A , dap D, r p s L. C o n j u g a l C r o s s o f AT980 x x l i k e LD5 e x c e p t met. l e u d e r i v a t i v e . o f • LD5 t h r d e r i v a t i v e o f LD5 r e l A d e r i v a t i v e o f LD5 l e u d e r i v a t i v e o f LD5456 t h r d e r i v a t i v e o f LD5456 R.A.J. Warren H. Wiens E.E. I s h i g u r o E.E. I s h i g u r o E.E. I s h i g u r o E.E. I s h i g u r o E.E. I s h i g u r o E.E. I s h i g u r o E.E. I s h i g u r o E.E. I s h i g u r o 16 (7: 3 , v / v ) ; I I I . n - b u t a n o l - a c e t i c a c i d - w a t e r ( 2 : 1 : 1 , v / v ) ; IV. e t h a n o l -NH^OH - w a t e r (180:10:10, v / v ) ; V. p y r i d i n e - a c e t i c a c i d - w a t e r (50:35:15, v/v) . IV. S c i n t i l l a t i o n c o u n t i n g Samples were c o u n t e d i n a N u c l e a r C h i c a g o I s o c a p 300 l i q u i d s c i n -t i l l a t i o n c o u n t e r . The s c i n t i l l a t i o n f l u i d c o n t a i n e d 4g 2 , 5 - d i p h e n y l o x a z o l e (PPO) and 0.5g l , 4 - b i s ( 2 ( 5 - p h e n y l o x a z o l y l ) ) b e n z e n e (POPOP) p e r l i t e r o f t o l -uene. The a p p r o p r i a t e r e g i o n s i n t h e paper chromatographs were c u t i n t o 0.5 t o 1.5 cm l o n g s e r i a l s t r i p s , d e p e n d i n g on t h e r e q u i r e d r e s o l u t i o n . The s e p -a r a t e s t r i p s were t h e n c o u n t e d and t h e i n d i v i d u a l c o u n t s were summed. A l l samples were a i r d r i e d , and t h e n h e a t e d a t 105°C f o r a t l e a s t two h o u r s be-f o r e c o u n t i n g . I n a l l c a s e s , q u e n c h i n g was l o w and c o n s i s t e n t from sample t o sample. V. C h e m i c a l s D - a l a n y l - D - a l a n i n e was o b t a i n e d from C y c l o C h e m i c a l Co. R a d i o -c h e m i c a l s were o b t a i n e d f r o m Amersham/Searle Corp. A n t i b i o t i c s , UDP-GlcNAc, n u c l e o t i d e s and amino a c i d s were o b t a i n e d from Sigma C h e m i c a l Co. A l l o t h e r r e a g e n t s were t h e b e s t a v a i l a b l e g r a d e o f f e r e d by Sigma C h e m i c a l Co. and F i s h e r S c i e n t i f i c Co. V I . P r e p a r a t i o n o f s u b s t r a t e s , s t a n d a r d s , and ppGpp A. S o l u b l e n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s , (a) UDP^-MurNAc-tripeptide T h i s was e x t r a c t e d from B. c e r e u s . The c e l l s were grown i n M9 m i n i m a l s a l t s medium (77) supplemented w i t h 0.5% g l u c o s e and 1% peptone. When t h e c u l t u r e r e a c h e d o n e - t h i r d maximal g r o w t h , D - c y c l o s e r i n e was added t o 50 Ug p e r m l f i n a l c o n c e n t r a t i o n . The c u l t u r e was t r e a t e d 17 f o r 45 m i n u t e s . The c e l l s and c o n t e n t s were th e n p r o c e s s e d up t o t h e l y o p h i l i z a t i o n s t e p i n t h e U D P - M u r N A c - t r i p e p t i d e p u r i f i c a t i o n scheme d e s c r i b e d by B a r n e t t ( 4 ) . A t t h i s p o i n t the sample was a p p l i e d t o Whatman 3MM chromatography paper and s e p a r a t e d i n s o l v e n t s y s t e m I I . The p r e s u m p t i v e U D P - M u r N A c - t r i p e p t i d e band was l o c a t e d as d e s c r i b e d e l s e w h e r e (21, 8 0 ) . T h i s zone was t h e n e l u t e d w i t h w a t e r and q u a n t i f i e d (104) u s i n g UDP-GlcNAc as t h e s t a n d a r d . (b) UDP-MurNAc-pentapeptide Crude U D P - M u r N A c - t r i p e p t i d e was e n z y m a t i c a l l y c o n v e r t e d t o UDP-MurNAc-pentapeptide. The r e a c t i o n was s i m i l a r t o t h a t f o r t h e D - a l a n y l a l a n i n e l i g a s e a s s a y ( s e c t i o n V I I Cc, page 22) w i t h the f o l l o w i n g e x c e p t i o n s : ( i ) 200 mM M g C ^ s u b s t i t u t e d f o r the 20 mM M n C ^ j ( H ) t o l u e n e was added t o p r e v e n t m i c r o b i a l g r o w t h , and ( i i i ) t h e I1hC]D-alanine (36m Ci/mmol) was i n c r e a s e d t o 2.8 mM. T h i s m i x t u r e was i n c u b a t e d a t 30°C. A f t e r 24 h o u r s , i t was b o i l e d f o r 2 m i n u t e s and t h e n c e n t r i f u g e d a t 42,000 x j> f o r 20 m i n u t e s t o remove p r e c i p i t a t e d m a t e r i a l . The s u p e r n a t a n t was d e c a n t e d and l y o p h i l i z e d . The l y o p h i l i z e d m a t e r i a l was resuspended i n a s m a l l volume of d i s t i l l e d w a t e r and s t r e a k e d onto chromatography p a p e r . . The chromatograph was d e v e l o p e d I n s o l v e n t s y s t e m I l o n g enough t o r e s o l v e the UDP-MurNAc-pentapeptide f r o m the U D P - M u r N A c - t r i p e p t i d e . The zone of UDP-MurNAc-pentapeptide was t h e n e l u t e d w i t h w a t e r and a d j u s t e d t o 0.75 mM. N o n - r a d i o a c t i v e UDP-MurNAc-pentapeptide was p r e p a r e d by s u b s t i t u t i n g D - a l a n y l a l a n i n e i n t h e r e a c t i o n . B. Other s t a n d a r d s (a) M u r N A c - p e n t a p e p t i d e T h i s was p r e p a r e d by h e a t i n g UDP-MurNAc-pentapeptide i n 0.1 N HC1 (113). I t was p u r i f i e d by chromatography i n s o l v e n t system I . 18 (b) M u r N A c ( - G l c N A c ) - p e n t a p e p t l d e ( t e t r a p e p t i d e ) T h i s was p r e p a r e d f r o m lysozyme d i g e s t e d s u b t i l i s c e l l w a l l s u s i n g t h e s y s t e m d e v e l o p e d f o r e x a m i n i n g p e p t i d o g l y c a n c r o s s - l i n k i n g i n c o l i (10, 5 4 ) . (c) GCL-P-P-MurNAc-pentapeptide and gCL-P-P-MurNAc(-GlcNAc)-p e n t a p e p t i d e These were n o t p u r i f i e d , b u t t h e r a d i o a c t i v e m a t e r i a l of the l i p i d zone of the i n v i t r o c o u p l e d r e a c t i o n ( s e c t i o n V I I Ab, page 20) were used as t h e s t a n d a r d . T h i s m a t e r i a l was s e n s i t i v e t o m i l d a c i d h y d r o l y s i s . I t had t h e a p p r o p r i a t e i n s o l v e n t system I . I t a l s o was a c c u m u l a t e d i f v a n c o m y c i n was added t o t h e r e a c t i o n and d i m i n i s h e d i f b a c i t r a c i n was added t o the i n v i t r o c o u p l e d r e a c t i o n . T h i s m a t e r i a l was p r o b a b l y t h e G C L - l i n k e d i n t e r m e d i a t e s o f p e p t i d o g l y c a n s y n t h e s i s (9, 5 1 ) . C. ppGpp p r e p a r a t i o n and p u r i f i c a t i o n T h i s was p r e p a r e d by c o m b i n i n g some of t h e p u b l i s h e d p r o c e d u r e s f o r i n v i t r o p r o d u c t i o n o f ppGpp (13, 4 2 ) . An o v e r n i g h t c u l t u r e of VC10 was d i l u t e d one h u n d r e d - f o l d i n t o L - b r o t h (97) and grown t o 150 K l e t t U n i t s ( a p p r o x i m a t e l y o n e - h a l f maximum g r o w t h ) . The c e l l s were h a r v e s t e d and b r o k e n i n a Power L a b o r a t o r y P r e s s (American I n s t r u m e n t Co.) a t 12,000 l b s p e r s q u a r e i n c h . The c e l l d e b r i s was removed by two c e n t r i f u g a t i o n s a t 40,000 x g_ f o r 20 m i n u t e s . The c r u d e r i b o s o m e s were p e l l e t e d i n a S p i n c o M odel L (Beckman I n s t r u m e n t s I n c . ) u s i n g a Beckman a n g l e 40 r o t o r a t 40,000 rpm f o r 2.5 h o u r s . The p e l l e t was r i n s e d , r e s uspended i n t h e b u f f e r d e s c r i b e d by C a s h e l (13) and r e - h a r v e s t e d a t 40,000 rpm f o r 2 h o u r s . These c r u d e r i b o s o m e s seemed t o have more ppGpp-p r o d u c i n g a c t i v i t y t h a n r i b o s o m e s c l e a n e d w i t h 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 (42) o r T r i t o n X-100 washes ( 1 3 ) . They were used t o g e n e r a t e 19 l a r g e q u a n t i t i e s of ppGpp, as d e s c r i b e d e l s e w h e r e ( 1 3 ) . T h i s ppGpp was p u r i f i e d a s d e s c r i b e d by C a s h e l ( 1 3 ) . I t was checked f o r p u r i t y and i d e n t i f i e d by t h e p u b l i s h e d a c t i o n s p e c t r u m (12, 15) and t h e r e l a t i v e m o b i l i t y on p o l y e t h y l e n e i m i n e t h i n l a y e r p l a t e s (Brinkman I n s t r u m e n t s , I n c . ) ( 1 5 ) . An a u t h e n t i c 1 m i l l i g r a m sample of ppGpp was k i n d l y donated by M. C a s h e l . V I I . J_n v i t r o a s s a y s f o r p e p t i d o g l y c a n b i o s y n t h e t i c enzymes A. C o u p l e d r e a c t i o n f o r p e p t i d o g l y c a n s y n t h e s i s (a) P a r t i c u l a t e enzyme p r e p a r a t i o n The p a r t i c u l a t e enzyme was p r e p a r e d by a method s i m i l a r t o t h a t d e s c r i b e d by I z a k i ejt a l . ( 5 1 ) . An o v e r n i g h t c u l t u r e of c o l i VC10 was d i l u t e d one h u n d r e d - f o l d i n M9 m i n i m a l s a l t s media (77) supplemented w i t h 0.5% g l u c o s e and 0.5% casamino a c i d s and grown t o 200 K l e t t u n i t s ( a p p r o x -i m a t e l y t w o - t h i r d s maximum g r o w t h ) . The c u l t u r e was h a r v e s t e d , and the p e l l e t was s t o r e d o v e r n i g h t a t -20°C. The f r o z e n p e l l e t was ground i n a c h i l l e d m o r t a r w i t h t h r e e t i m e s the p e l l e t wet w e i g h t of a l u m i n a Type 305 (Sigma) f o r 5 m i n u t e s . The p a s t e was r e s u s p e n d e d i n a b u f f e r c o n t a i n i n g 5mM t r i s (pH 8.2), 10 mM M g C l 2 and 1 mM m e r c a p t o e t h a n o l . T h i s s u s p e n s i o n was c e n t r i f u g e d two s u c c e s s i v e t i m e s a t 15,000 x j * f o r 5 m i n u t e s to remove t h e a l u m i n a and unbroken c e l l s . The p e l l e t s were d i s c a r d e d . The s u p e r n a t a n t was t h e n c e n t r i f u g e d a t 44,000 x £ f o r 40 m i n u t e s t o p e l l e t t h e p a r t i c u l a t e enzyme. T h i s p e l l e t was resuspended and washed two t i m e s w i t h a b u f f e r c o n t a i n i n g 0.5 mM T r i s (pH 8.2), 0.1 mM M g C l 2 and 1 mM m e r c a p t o e t h a n o l . I t was a s s a y e d f o r p r o t e i n by t h e Lowry p r o c e d u r e (64) w i t h b o v i n e serum a l b u m i n a s the s t a n d a r d , and resuspended t o 15 mg o f p r o t e i n p e r ml. The p r e p a r a t i o n was t h e n d i s p e n s e d i n s m a l l b a t c h e s 20 w h i c h were f r o z e n a t -20 C u n t i l u s e d . The f r o z e n p r e p a r a t i o n s r e t a i n e d f u l l a c t i v i t y f o r s e v e r a l weeks. (b) C o u p l e d r e a c t i o n a s s a y f o r f o r m a t i o n o f p e p t i d o g l y c a n and G C L - l i n k e d i n t e r m e d i a t e s . The i n v i t r o f o r m a t i o n o f p e p t i d o g l y c a n and G C L - l i n k e d i n t e r m e d i a t e s by t h e p a r t i c u l a t e enzyme p r e p a r a t i o n was measured i n an i n c u b a t i o n m i x t u r e w h i c h c o n t a i n e d : ( i ) 5 y l o f a s t o c k s o l u t i o n o f t r i s (pH 8.2, 1M), M g C l 2 (20 mM) and UDP-GlcNAc (2mM), ( i i ) 5 y l o f p a r t i c u l a t e enzyme, ( i i i ) 5 y l o f d i s t i l l e d w a t e r , ( i v ) 5 y l o f p o t a s s i u m p e n i c i l l i n G (1.1 mM). T h i s m i x t u r e was p r e - i n c u b a t e d a t 37°C f o r 10 m i n u t e s . Then t h e r e a c t i o n was i n i t i a t e d by a d d i n g 5 y l o f l l * C - l a b e l e d UDP-MurNAc-p e n t a p e p t i d e (0.75 mM, 18 mCi p e r mmole). A f t e r 10 m i n u t e s o f i n c u b a t i o n , t h e r e a c t i o n s were t e r m i n a t e d by c h i l l i n g them on i c e and a d d i n g 10 y l o f i s o b u t y r i c a c i d . The r a d i o a c t i v e p r o d u c t s were s e p a r a t e d by chromatography i n s o l v e n t s y s t e m I f o r 18 t o 22 h o u r s and c o u n t e d . The o r i g i n r e t a i n e d t h e p e p t i d o g l y c a n , and a f l u o r e s c e n t zone a t 0.8 c o n t a i n e d t h e G C L - l i n k e d i n t e r m e d i a t e s . The e f f e c t s o f ppGpp and o t h e r compounds were checked by s u b s t i -t u t i n g t h e 5 y l o f w a t e r i n t h e r e a c t i o n w i t h t h e a p p r o p r i a t e c o n c e n t r a t i o n s o f t h e m a t e r i a l s t o be t e s t e d . P e n i c i l l i n was added t o i n h i b i t t h e D - a l a n i n e c a r b o x y p e p t i d a s e and p e p t i d o g l y c a n t r a n s p e p t i d a s e a c t i v i t i e s (37, 1 0 6 ) . O m i s s i o n o f t h e p e n i c i l l i n d i d n o t a l t e r t h e g e n e r a l t r e n d o f t h e r e s u l t s , b u t i t d i d d e c r e a s e t h e n e t c o u n t s i n t h e p e p t i d o g l y c a n because o f r 1 k i e n z y m a t i c r e m o v a l o f L C J D - a l a n i n e from t h e i n c o r p o r a t e d p r e c u r s o r s . B. Exchange a s s a y f o r p h o s p h o - N - a c e t y l m u r a m o y l - p e n t a p e p t i d e t r a n s f e r a s e . The p h o s p h o - N - a c e t y l m u r a m o y l - p e n t a p e p t i d e t r a n s f e r a s e r e a c t i o n 21 was measured i n an a s s a y a n a l o g o u s t o t h e exchange a s s a y d e s c r i b e d by Hammes and Neuhaus ( 3 6 ) . The r e a c t i o n used t h e same p a r t i c u l a t e enzyme p r e p a r a t i o n as t h e c o u p l e d r e a c t i o n d e s c r i b e d above. The a s s a y c o n t a i n e d ( i ) 5 Ul o f a s o l u t i o n o f t r i s (pH 8.2, IM) and p o t a s s i u m p e n i c i l l i n G (1.1 mM) , ( i i ) 5 u l of p a r t i c u l a t e enzyme, ( i i i ) 5 u l o f d i s t i l l e d w a t e r , ( i v ) 5 u l o f M g C l 2 o r MnCl^ a t t h e a p p r o p r i a t e c o n c e n t r a t i o n , (v) 5 u l o f a m i x t u r e o f [ 3H]UMP (5mM, 2.5 mCi p e r mmole) and UDP-MurNAc-pentapeptide (1 mM). The s u b s t r a t e s were added a f t e r t h e r e s t o f t h e r e a c t i o n m i x t u r e had been p r e - i n c u b a t e d a t 37°C f o r 10 m i n u t e s . The a s s a y was t h e n i n c u b a t e d f o r 15 m i n u t e s and t e r m i n a t e d by t r a n s f e r r i n g t h e r e a c t i o n v e s s e l s t o an i c e b a t h and a d d i n g 10 U l o f i s o b u t y r i c a c i d . The [^UDP-MurNAc-p e n t a p e p t i d e w h i c h was formed by t h e r e a c t i o n was s e p a r a t e d from [ 3HJUMP by chromatography i n s o l v e n t system I f o r 24 h o u r s and c o u n t e d . The e f f e c t s o f ppGpp and o t h e r n u c l e o t i d e s were examined by s u b s t i t u t i n g them, a t t h e a p p r o p r i a t e c o n c e n t r a t i o n s , f o r t h e 5 U l o f w a t e r added t o t h e a s s a y . KC1 d i d n o t seem t o s t i m u l a t e t h e r e a c t i o n , as i t does when o t h e r b a c t e r i a l membranes a r e used (44) , and was n o t r o u t i n e l y i n c l u d e d . C. D - a l a n y l a l a n i n e s y n t h e t a s e and D - a l a n y l a l a n i n e l i g a s e . (a) Enzyme p r e p a r a t i o n . The enzyme p r e p a r a t i o n was a ' p a r t i a l l y p u r i f i e d m i x t u r e c o n t a i n i n g b o t h a c t i v i t i e s . T h i s p r e p a r a t i o n was made by d i a l y z i n g t h e f i r s t ( N H ^ ^ S O ^ p r e c i p i t a t e o c c u r r i n g i n t h e p u r i f i c a t i o n scheme d e s c r i b e d by Comb ( 2 0 ) . I t was p r e p a r e d from E ^ c o l i VC10 w h i c h had been grown t o t w o - t h i r d s maximal growth i n t h e media used by Comb ( 2 0 ) . The p r e p a r -a t i o n s were a d j u s t e d t o 10 mg o f p r o t e i n p er ml u s i n g t h e Lowry p r o c e d u r e (64) w i t h b o v i n e serum a l b u m i n as t h e s t a n d a r d . S m a l l b a t c h e s were t h e n 22 f r o z e n a t -20°C u n t i l u s e d . (b) A s s a y f o r D - a l a n y l a l a n i n e s y n t h e t a s e . The a s s a y was s i m i l a r t o t h a t d e s c r i b e d e l s e w h e r e (20, 6 6 ) . I t c o n t a i n e d : ( i ) 5 y l o f t r i s (pH 8.7, 400 mM), ( i i ) 10 y l o f ATP (pH 7.2, 125 mM) , ( i i i ) 5 y l o f enzyme p r e p a r a t i o n , ( i v ) 5 y l o f MnCl^ (20mM) and p o t a s s i u m p e n i c i l l i n G (1.4mM), (v) 10 y l o f d i s t i l l e d w a t e r , ( v i ) 5 y l o f [ 1 ^ C ] D - a l a n i n e (0.28 mM, 36 mCi/m m o l e ) . The r e a c t i o n was s t a r t e d by a d d i n g t h e [ 1 ^ C ] D - a l a n i n e and was i n c u b a t e d a t 37°C f o r s p e c i f i e d p e r i o d s o f t i m e . I t was t e r m i n a t e d by b e i n g c h i l l e d i n an i c e b a t h and t h e n h a v i n g 10 y l o f i s o b u t y r i c a c i d added. The e f f e c t o f ppGpp was t e s t e d by sub-s t i t u t i n g 5 y l o f 20 mM ppGpp f o r 5 y l of t h e d i s t i l l e d w a t e r . The p o t a s s i u m p e n i c i l l i n G was added t o i n h i b i t any c a r b o x y p e p t i d a s e a c t i v i t y (106) w h i c h was s t i l l p r e s e n t i n t h e enzyme p r e p a r a t i o n . (c) A s s a y f o r D - a l a n y l a l a n i n e l i g a s e . T h i s a s s a y was e s s e n t i a l l y t h e same as t h e D - a l a n y l a l a n i n e s y n t h e t a s e a s s a y e x c e p t t h a t 5 y l o f 180 mM U D P - M u r N A c - t r i p e p t i d e was s u b s t i t u t e d f o r 5 y l o f t h e d i s t i l l e d w a t e r . As p r e v i o u s l y r e p o r t e d ( 6 6 ) , t h e l i g a s e a c t i v i t y i s s l o w e r t h a n t h e s y n t h e t a s e a c t i v i t y and can be measured as e f f i c i e n t l y w i t h [ 1 "*C]D-alanine as w i t h [ 1 4 C ] D - a l a n y l a l a n i n e . V I I I . I n v i v o p e p t i d o g l y c a n s y n t h e s i s A. A s s a y f o r T C A - i n s o l u b l e [ 3H]Dap i n c o r p o r a t i o n . A l l o f t h e i n v i v o a s s a y s f o r p e p t i d o g l y c a n s y n t h e s i s used dap  l y s a u x o t r o p h s w h i c h would s p e c i f i c a l l y l i m i t t h e [ 3 H ] D a p i n c o r p o r a t i o n t o p e p t i d o g l y c a n components. C e l l s were r o u t i n e l y d i l u t e d i n a 500 m l s i d e -arm f l a s k c o n t a i n i n g 50 ml of-M9 m i n i m a l • s a l t s medium (77) supplemented w i t h 4~mg per ml g l u c o s e , 1 yg per m l t h i a m i n e , 10 yg per ml d i a m i n o p i m e l i c 23 a c i d and 50 yg' p e r ml o f o t h e r r e q u i r e d amino a c i d s . F o r c o n v e n i e n c e , t h i s media i s r e f e r r e d t o as M9. The c u l t u r e was grown f o r 4 t o 5 g e n e r a t i o n s u n t i l i t r e a c h e d 50 t o 60 K l e t t u n i t s . A 40-ml a l i q u o t was t h e n t r a n s f e r r e d t o s t e r i l e , screw-cap c e n t r i f u g e t u b e s and h a r v e s t e d a t 27,000 x £ f o r 3 m i n u t e s i n a S o r v a l RC2B r e f r i g e r a t e d c e n t r i f u g e s e t t o 2°C. The p e l l e t was resuspended and washed once w i t h 40 ml o f 0.9% s t e r i l e s a l i n e . The washed c e l l s were resuspended i n f r e s h N a Cl and d i s p e n s e d t o a f i n a l t u r b i d i t y o f 25 t o 30 K l e t t u n i t s i n f l a s k s c o n t a i n i n g 10 ml o f t h e a p p r o p r i a t e d i a m i n ' o p i m e l i c a c i d - f r e e media. { 3H]Dap; (280-545 mCi/mmol) was t h e n added t o 0.2 ug p e r ml f o r each f l a s k and t h e c u l t u r e s were i n c u b a t e d . Growth resumed i m m e d i a t e l y i n t h e u n t r e a t e d c u l t u r e s . A t d e s i g n a t e d i n t e r v a l s 0.1 ml a l i q u o t s were removed from each c u l t u r e and p i p e t t e d onto 2.4 cm d i a m e t e r d i s c s o f Whatman 3MM f i l t e r p a p e r . When each sample was a b s o r b e d , t h e d i s c was dropped i n t o a f l a s k o f c o l d 5% TCA and l e f t f o r a minimum o f 30 m i n u t e s . The d i s c s were t h e n t r a n s f e r r e d t o f r e s h TCA f o r a n o t h e r 30 m i n u t e s , t h e n soaked f o r two 20-minute p e r i o d s i n c o l d 95% e t h a n o l and f i n a l l y r i n s e d f o r 30 seconds i n d i e t h y l e t h e r . The d i s c s were t h e n d r i e d and c o u n t e d . The r e s u l t s were n o r m a l i z e d t o c e l l mass and e x p r e s s e d as c o u n t s p er minut e p e r m i l l i g r a m d r y w e i g h t o f c e l l s . The u t i l i z a t i o n o f t h e i n t r a c e l l u l a r p o o l s o f Dap was examined by p r e - l o a d i n g t h e Dap p o o l s o f the c e l l s w i t h pHJDap. The c e l l s were grown t o 50-60 K l e t t u n i t s , t h e n washed and t r a n s f e r r e d t o co m p l e t e M9 media c o n t a i n i n g [ 3HjDap as d e s c r i b e d above. A f t e r 30 m i n u t e s , t h e c e l l s were washed f r e e o f exogenous I 3H]Dap and t r a n s f e r r e d t o D a p - f r e e medium. The c u l t u r e s were t h e n m o n i t o r e d . a n d sampled as d e s c r i b e d above. 24 B. Assay f o r the d i s t r i b u t i o n - o f incorporated [ 3H]Dap (a) Q u a n t i t a t i v e a n a l y s i s of the l a b e l e d cytoplasmic, membrane-associated, and peptidoglycan components. The v a r i o u s [ 3HlDap-containing components of the l a b e l e d c e l l s were separated and qu a n t i t a t e d i n a system analogous to that described by Liigtenberg and de Haan (67). The c e l l s were washed and t r a n s f e r r e d to the appropriate [^jDap-containing M9_ media as described above, except that the c e l l s were g e n e r a l l y t r a n s f e r r e d to 18 ml of media. At designated i n t e r v a l s 8-ml samples were p i p e t t e d to c h i l l e d g l a s s Corex c e n t r i f u g e tubes. These were immediately c e n t r i f u g e d at 35,000 £ f o r 3 minutes i n a r e f r i g e r a t e d RC2B c e n t r i f u g e set f o r 2°C. The c e n t r i f u g e tubes were t r a n s f e r r e d to an i c e bath. The supernatants were then removed w i t h a Pasteur p i p e t t e and frozen f o r l a t e r examination. The p e l l e t s were resuspended w i t h 60 y l of c o l d d i s t i l l e d water and a l i q u o t s were a p p l i e d to chromatography paper as 1 cm long s t r e a k s . The v a r i o u s components were simultaneously e x t r a c t e d and f r a c t i o n a t e d by developing the chromatographs i n solvent system I u n t i l the solvent f r o n t was n e a r l y at the end of the paper ( u s u a l l y 19 to 22 hours). The appropriate l a b e l e d components were then l o c a t e d and counted. The peptidoglycan stayed at the o r i g i n i n t h i s solvent system (51, 54, 67), the s o l u b l e n u c l e o t i d e -l i n k e d peptidoglycan precursors migrated w i t h an R^ of 0.1 to 0.2 (51, 67), f r e e [ 3H]Dap migrated w i t h an R f of 0.3 to 0.4, and the GCL-linked intermediates migrated at R^ 0.8 (9, 51, 67). The l a t t e r r e g i o n a l s o contains some p r o t e i n (10, 107), but, f o r convenience, was termed the l i p i d zone. I t f l u o r e s c e d when viewed under u l t r a v i o l e t l i g h t . The samples d r i e d r a p i d l y when streaked onto the paper i n 10 y l p o r t i o n s . They d i d not seem to change when stored i n the i c e bath f o r 25 as much as f i f t e e n m i n u t e s b e f o r e b e i n g u s e d . D u p l i c a t e samples d i f f e r e d by l e s s t h a n 5%. I n a d d i t i o n , t h e r e s u l t s g e n e r a l l y agreed w i t h t h e r o u t i n e , s i m u l t a n e o u s a s s a y s f o r T C A - i n s o l u b l e ['3H]Dap i n c o r p o r a t i o n . I n p r e l i m i n a r y e x p e r i m e n t s , p e l l e t s were resuspended w i t h i s o b u t y r i c a c i d t o i n h i b i t e n z y m a t i c a c t i v i t y and p o s s i b l e a u t o l y s i s . T h i s d i d n o t a l t e r t h e r e l a t i v e p r o p o r t i o n s o f l i p i d zone m a t e r i a l o r s o l u b l e n u c l e o t i d e -l i n k e d i n t e r m e d i a t e s when compared t o t h e amounts i n p e l l e t s r e s uspended w i t h w a t e r , b u t i t d i d cause t h e p e p t i d o g l y c a n t o become clumped and d i f f i c u l t t o h a n d l e . R e s u l t s were n o r m a l i z e d by r e l a t i n g t h e q u a n t i t y o f c e l l s a p p l i e d t o t h e paper t o t h e t u r b i d i t y o f t h e o r i g i n a l c u l t u r e . T h i s was done by d e t e r m i n i n g t h e r a t i o o f T C A - i n s o l u b l e c o u n t s i n t h e c o n c e n t r a t e d sample t o t h e T C A - i n s o l u b l e c o u n t s i n t h e sample b e f o r e c o n c e n t r a t i n g . T h i s c o n c e n t r a t i o n f a c t o r and t h e amount o f m a t e r i a l a p p l i e d t o t h e paper c o u l d t h e n be used t o c a l c u l a t e t h e q u a n t i t y o f c e l l s a p p l i e d . A l t e r n a t i v e l y , t h e t u r b i d i t y o f an a l i q u o t o f the c o n c e n t r a t e d c e l l s was measured a t 410 nm i n a Beckman DB-G s p e c t r o p h o t o m e t e r and c o n v e r t e d t o d r y w e i g h t w i t h a c a l i b r a t i o n c u r v e . B o t h methods gave s i m i l a r r e s u l t s . (b) Q u a n t i t a t i o n o f U D P - M u r N A c - t r i p e p t i d e and UDP-MurNAc-p e n t a p e p t i d e i n J 3 H J D a p - l a b e l e d c e l l s . The p r e c e d i n g p r o c e d u r e d i d n o t ' r e s o l v e the d i f f e r e n t n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s , b u t c o u l d s e p a r a t e them when t h e chromatographs were d e v e l o p e d f o r 62 h o u r s . A d d i t i o n a l l y , t h e p e l l e t o f l a b e l e d c e l l s . w a s r o u t i n e l y r e s u s p e n d e d w i t h c o l d w a t e r c o n t a i n i n g s u f f i c i e n t u n l a b e l e d U D P - M u r N A c - t r i p e p t i d e t o quench when t h e chromato-graphs were s u b s e q u e n t l y examined w i t h u l t r a - v i o l e t l i g h t . The UDP-MurNAc-p e n t a p e p t i d e m i g r a t e d 2 t o 3 cm ahead o f t h e U D P - M u r N A c - t r i p e p t i d e . Any 26 U D P - M u r N A c - t r i p e p t i d e - a l a would m i g r a t e w i t h UDP-MurNAc-pentapeptide, b u t was assumed t o be p r e s e n t i n n e g l i g i b l e amounts. I n s p e c i f i e d i n s t a n c e s , c u l t u r e s were t r e a t e d w i t h D - c y c l o s e r i n e t o a l t e r t h e c o m p o s i t i o n o f t h e p e p t i d o g l y c a n p r e c u r s o r p o o l s o f t h e c e l l s . T h i s was done by a d d i n g D - c y c l o s e r i n e t o 40 Ug per ml f i n a l c o n c e n t r a t i o n . These t r e a t e d Ej_ c o l i c u l t u r e s l y s e d a t 25 t o 30 m i n u t e s and so were o n l y i n c u b a t e d f o r 15 m i n u t e s i n o r d e r t o a v o i d c o m p l i c a t i o n s due t o t h e a u t o l y s i s . (c) A n a l y s i s o f l i p i d zone components. (1) M i l d a c i d h y d r o l y s i s o f l i p i d zone components. The G C L - l i n k e d i n t e r m e d i a t e s do n o t s e p a r a t e r e a d i l y i n s o l v e n t s y s t e m I , b u t t h e y a r e r e a d i l y h y d r o l y s e d i n m i l d a c i d (9) and t h e r e s u l t i n g M u r N A c - p e n t a p e p t i d e and M u r N A c ( - G l c N A c ) - p e n t a p e p t i d e do s e p a r a t e . I n i t i a l a t t e m p t s t o e x t r a c t (108) and t h e n h y d r o l y z e t h e G C L - l i n k e d i n t e r m e d i a t e s from whole c e l l s gave i n c o m p l e t e y i e l d s . T h e r e -f o r e , t h e t e c h n i q u e used by Braun and Bosch t o measure a c i d - h y d r o l y z e d G C L - l i n k e d components i n membrane p r e p a r a t i o n s (9) was m o d i f i e d t o measure G C L - l i n k e d i n t e r m e d i a t e s i n l a b e l e d c e l l s . The c e l l s were p r e -p a r e d and l a b e l e d as d e s c r i b e d e a r l i e r ( s e c t i o n V I I I Ba, page 2 4 ) . The p e l l e t was r e s u s p e n d e d i n a s m a l l volume o f w a t e r and a s m a l l p o r t i o n was put d i r e c t l y o n t o t h e chroma t o g r a p h . The r e m a i n i n g p o r t i o n was t r a n s -f e r r e d t o a g l a s s c e n t r i f u g e tube c o n t a i n i n g 0.35 ml o f b o i l i n g w a t e r and b o i l e d f o r e x a c t l y 4 m i n u t e s . T h i s t r e a t m e n t removed t h e f r e e I 3H]Dap and n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s w h i c h c o u l d i n t e r -f e r e w i t h t h e a s s a y . Four m i n u t e s o f b o i l i n g q u a n t i t a t i v e l y removed t h e s e components w i t h o u t a f f e c t i n g t h e c o u n t s i n t h e l i p i d zone. However, t h e b o i l i n g has t o be p r e c i s e s i n c e s i x m i n u t e s o f b o i l i n g d i d remove some 27 l i p i d zone c o u n t s . A f t e r b o i l i n g , t h e samples were r a p i d l y c o o l e d i n an i c e b a t h and c e n t r i f u g e d a t 36,000 x £ f o r t h r e e m i n u t e s . The p e l l e t was r e s u s p e n d e d i n 0.40 ml o f b o i l i n g w a t e r and r e - c e n t r i f u g e d . I t was t h e n r e s u s p e n d e d w i t h a s m a l l volume o f h o t w a t e r and a s m a l l p o r t i o n was t r a n s f e r r e d t o t h e chromatography p a p e r . The r e m a i n d e r was t r a n s f e r r e d t o a g l a s s ampoule c o n t a i n i n g a f i n a l c o n c e n t r a t i o n o f 10% a c e t i c a c i d . T h i s was s e a l e d and t h e n h e a t e d f o r s i x t y m i n u t e s i n an oven s e t t o 105°C. The c o n t e n t s o f t h e ampoule, and w a t e r from s e v e r a l subsequent r i n s e s , were t r a n s f e r r e d t o t h e chromatography paper. The chromatographs were t h e n d e v e l o p e d i n s o l v e n t system I u n t i l t h e s o l v e n t f r o n t was a l m o s t a t th e end o f t h e paper. The d r i e d paper was t h e n c u t i n t o 0.5 cm l o n g s t r i p s w h i c h were c o u n t e d f o r r a d i o a c t i v i t y ( r e g i o n s where c o u n t s were n o t e x p e c t e d were c u t as 1 cm l o n g s t r i p s ) . The M u r N A c - p e n t a p e p t i d e and MurNAc ( - G l c N A c ) - p e n t a p e p t i d e were l o c a t e d w i t h s t a n d a r d s and equated t o t h e r e s p e c t i v e G C L - l i n k e d i n t e r m e d i a t e s . The b o i l e d b u t n o t h y d r o l y z e d sample s e r v e d as a c o n t r o l t o e n s u r e t h a t a l l o f t h e [ 3 H ] D a p - l a b e l e d s o l u b l e components m i g r a t i n g between t h e o r i g i n and t h e l i p i d zone were removed b e f o r e h y d r o l y s i s . The sample w h i c h was n o t b o i l e d s e r v e d as a s t a n d a r d , t o e n s u r e q u a n t i t a t i v e r e c o v e r y o f t h e l i p i d zone m a t e r i a l a f t e r b o i l i n g and a f t e r h y d r o l y s i s . (2) P r o t e a s e t r e a t m e n t o f l i p i d zone components. The p r o t e i n i n t h e l i p i d zone was s o l u b i l i z e d by d i g e s t i n g t h e b o i l e d membranes i n a p r o t e a s e m i x t u r e a t 37°C f o r 30 m i n u t e s . The i n c u b a t i o n c o n t a i n e d a f i n a l c o n c e n t r a t i o n o f 2 mg per ml o f S t r e p t o m y c e s  g r i s e u s p r o t e a s e (Sigma Type V I p r o t e a s e ) , 5.5 mM p o t a s s i u m p e n i c i l l i n G and 200 mM t r i s ( pH 7) . 28 (d) A n a l y s i s o f [ 3 H ] l y s i n e c o n t e n t i n [ 3 H ] D a p - l a b e l e d c e l l s . Samples o f whole c e l l s were h y d r o l y z e d a t 105°C f o r 20 h o u r s i n s e a l e d ampoules c o n t a i n i n g 6N H C l . The H C l was removed by d r y i n g t h e samples under r e d u c e d p r e s s u r e i n a d e s i c c a t o r c o n t a i n i n g NaOH p e l l e t s and C a C ^ . Samples were t h e n s e p a r a t e d by paper chromatography i n s o l v e n t systems I , I I I , IV and V, and t h e chromatographs were examined f o r r a d i o a c t i v i t y w h i c h was n o t i n t h e [ 3H]Dap s p o t . T h i s m a t e r i a l c o - m i g r a t e d w i t h l y s i n e i n a l l f o u r s o l v e n t systems. (e) E x a m i n a t i o n o f s u p e r n a t a n t s from l a b e l e d c u l t u r e s . The f r o z e n s u p e r n a t a n t s from t h e [ 3H]Dap l a b e l e d c u l t u r e s ( s e c t i o n v i i i Ba, page 24) were l y o p h i l i z e d . The s a l t and much of t h e f r e e [ 3H]Dap were removed on Sephadex G-25. The d e - s a l t e d samples were t h e n l y o p h i l i z e d and f r a c t i o n a t e d by paper chromatography i n s o l v e n t system I f o r 19 t o 22 h o u r s . A f t e r development, l a b e l e d components between t h e o r i g i n .and R f 0.8 were q u a n t i t a t e d . 29 RESULTS I . P e p t i d o g l y c a n s y n t h e s i s d u r i n g i n h i b i t i o n o f p r o t e i n s y n t h e s i s i n v i v o A. E f f e c t o f amino a c i d d e p r i v a t i o n and CAM t r e a t m e n t on [ H]Dap i n c o r p o r a t i o n by r e l A b a c t e r i a . P e p t i d o g l y c a n s y n t h e s i s was m o n i t o r e d i n dap l y s b a c t e r i a by f o l l o w i n g t h e i n c o r p o r a t i o n o f [ 3H]Dap. The e f f e c t o f i n h i b i t i o n o f p r o t e i n s y n t h e s i s on t h e r a t e o f p e p t i d o g l y c a n s y n t h e s i s was d e t e r m i n e d by e i t h e r d e p r i v i n g t h e b a c t e r i a o f r e q u i r e d amino a c i d s o r t r e a t i n g t he b a c t e r i a w i t h CAM. When LD5 ( r e l A + ) was d e p r i v e d o f l y s i n e , t h e r a t e o f [ 3H]Dap i n c o r p o r a t i o n was g r e a t l y d e c r e a s e d r e l a t i v e t o t h e c o n t r o l r a t e ( f i g u r e 3 ) . I n c o n t r a s t , CAM t r e a t m e n t s t i l l p e r m i t t e d a h i g h r a t e o f p e p t i d o g l y c a n s y n t h e s i s . The r a t e o f p e p t i d o g l y c a n s y n t h e s i s i n l y s i n e -d e p r i v e d c u l t u r e s t r e a t e d s i m u l t a n e o u s l y w i t h CAM was s i m i l a r t o t h e r a t e o b s e r v e d w i t h CAM a l o n e . CAM t r e a t m e n t c o u l d a l s o enhance t h e r a t e o f Dap i n c o r p o r a t i o n when added w e l l a f t e r t h e o n s e t o f l y s i n e d e p r i v a t i o n ( f i g u r e 4 ) . The e f f e c t s o f l y s i n e d e p r i v a t i o n on t h e growth o f the c u l t u r e s and t h e r a t e o f Dap i n c o r p o r a t i o n were r e a d i l y r e v e r s e d by r e s t o r i n g t h e l y s i n e ( f i g u r e 5 A, B ) . Oth e r r e l A + s t r a i n s (LD51, LD52 and LD2) had s i m i l a r l o w r a t e s o f Dap i n c o r p o r a t i o n when d e p r i v e d o f one o r more r e q u i r e d amino a c i d s ( f i g u r e 6A, B, C ) . The p r e c e d i n g r e s u l t s suggest- t h a t r e l A . i s • i n v o l v e d I n r e g u l a t i n g p e p t i d o g l y c a n a c c u m u l a t i o n d u r i n g amino a c i d d e p r i v a t i o n . B. E f f e c t o f amino a c i d d e p r i v a t i o n on [ 3H]Dap i n c o r p o r a t i o n by r e l A b a c t e r i a . When LD5456 ( r e l A ) was d e p r i v e d o f l y s i n e , Dap i n c o r p o r a t i o n FIGURE 3. E f f e c t of l y s i n e d e p r i v a t i o n and CAM t r e a t m e n t on [ H]Dap i n c o r p o r a t i o n by LD5 ( r e l A ). C e l l s were h a r v e s t e d , washed once w i t h s a l i n e and t h e n i n o c u l a t e d i n t o M9 ( 0 ) , M9 w i t h o u t l y s i n e ( • ) , M9 p l u s 200 yg CAM per ml ( A ) and M9 w i t h o u t l y s i n e but w i t h 200 yg CAM per ml ( A ). I n c o r p o r a t i o n was m o n i t o r e d as the T C A - i n s o l u b l e r a d i o a c t i v i t y i n 0.1 ml samples. Counts were n o r m a l i z e d t o c e l l d r y w e i g h t i n o r d e r t o compensate f o r d i f f e r e n c e s i n the mass of the samples and t o f a c i l i t a t e c o m p a r i s o n of the v a l u e s . B e f o r e t h e c o r r e c t i o n f o r d i f f e r e n c e s i n the c e l l u l a r mass of t h e samples, a t 60 m i n u t e s , t h e r e s p e c t i v e CPM f o r t h e f o u r l i s t e d c u l t u r e s were: 2,527; 665; 967; and 895. 31 M i n u t e s FIGURE 4. Effect of late addition of CAM on [3H]Dap incorporation by lysine-deprived LD5 (relA +). Washed cells were inoculated into complete M9 ( 0 ), M9 without lysine ( 9 ), and M9 without lysine but with 200 ug per ml CAM ( A ). After 30 minutes (arrow), the lysine-deprived culture without CAM was divided in half and 200 yg per ml CAM was added to one half ( A ). The counts were monitored and normalized as described in figure 3. Before the correction for differences in the cellular mass of the samples, at 60 minutes, the respective CPM for the four listed cultures were: 3,843; 943; 1,255; and 1,134, 33 M i n u t e s 34 FIGURE 5. Effect of lysine restoration on £3HjDap incorporation (A) and culture turbidity (B) of lysine-deprived LD5 (relA +). For the details of the method, see figure 3 . After 30 minutes (arrow) the starved culture was divided in half and lysine was added to one half. Complete M9 ( 0 ), M9 minus lysine ( ® ), M9 with lysine restored ( A ). Before the correction for the differences in the cellular mass of the samples, at 60 minutes, the respective CPM for the three listed cultures were: 4,139; 1,210; and 1,861. / 36 FIGURE 6. E f f e c t of amino a c i d d e p r i v a t i o n on [ 3H]Dap i n c o r p o r a t i o n by other r e l A + amino a c i d auxotrophs of ,E. c o l i . For d e t a i l s of the methods, see f i g u r e 3. A) S t r a i n LD51: Complete M9_ ( 0 ), M9 minus • l y s i n e ( § ), M9 minus l e u c i n e ( A ), M9 minus l y s i n e and l e u c i n e ( A ). B) S t r a i n LD52: Complete M9 ( 0 ), M9 minus l y s i n e ( i ), M9 minus threonine ( A ), M9 minus l y s i n e and threonine ( A ). C) S t r a i n LD2: Complete M9 ( 0 ) , M9_ minus l y s i n e ( 8 ), M9_ minus methionine ( A ), M9 minus l y s i n e and methionine ( A ). Before the c o r r e c t i o n f o r d i f f e r e n c e s i n the c e l l u l a r mass, a t 60 minutes, the r e s p e c t i v e CPM f o r the twelve l i s t e d c u l t u r e s were: 1,265; 660; 694; 713; 1,605; 699; 694; 893; 2,893; 850; 869; and 866. i 1 1 1 1 r M i n u t e s 38 was e q u a l t o or g r e a t e r t h a n the i n c o r p o r a t i o n by t h e c o n t r o l ( f i g u r e 7 ) . I n c o r p o r a t i o n d u r i n g CAM t r e a t m e n t was o n l y s l i g h t l y l o w e r t h a n t h a t i n t h e c o n t r o l . Other r e l A ~ s t r a i n s (LD5457 and LD5458) gave s i m i l a r r e s u l t s when d e p r i v e d o f l y s i n e ( f i g u r e 8A, B ) . When t h e s e s t r a i n s were d e p r i v e d o f o t h e r r e q u i r e d amino a c i d s , t h e Dap i n c o r p o r a t i o n was s l i g h t l y l e s s t h a n i n t h e c o n t r o l b u t was s t i l l r e l a x e d . These r e s u l t s c o n c u r w i t h t h e c o n c l u s i o n t h a t r e l A i s i n v o l v e d i n r e g u l a t i n g p e p t i d o g l y c a n a c c u m u l a t i o n d u r i n g amino a c i d d e p r i v a t i o n . C. E f f e c t o f t h e r e l A gene on u t i l i z a t i o n o f endogenous Dap. Dap a u x o t r o p h s o f E_. c o l i c o n t a i n a l a r g e p o o l o f endogenous Dap w h i c h can be r a d i o a c t i v e l y l a b e l e d by g r o w i n g t h e c e l l s i n [ 3RjDap f o r 30 m i n u t e s . T h i s p o o l i s s u f f i c i e n t t o s u p p o r t p e p t i d o g l y c a n s y n t h e s i s f o r about two d o u b l i n g s i n t h e absence of exogenous Dap. P r e - l a b e l e d l y s i n e - d e p r i v e d c u l t u r e s o f LD5 ( r e l A + ) u t i l i z e d i n t e r n a l Dap a t a l o w e r r a t e t h a n e i t h e r t h e c o n t r o l o r t h e e q u i v a l e n t CAM-t r e a t e d c u l t u r e s ( f i g u r e 9 A ) . I n c o n t r a s t , p r e - l a b e l e d l y s i n e - d e p r i v e d c u l t u r e s o f LD5456 ( r e l A ) u t i l i z e d i n t e r n a l Dap a t n e a r l y t h e same r a t e as t h e c o n t r o l ( f i g u r e 9 B ) . T h i s r a t e was n o t enhanced by CAM t r e a t m e n t . These o b s e r v a t i o n s i n d i c a t e t h a t t h e r e l a t i v e d e c r e a s e i n a c c u m u l a t i o n o f p e p t i d o g l y c a n by amino a c i d - d e p r i v e d s t r i n g e n t b a c t e r i a i s p r o b a b l y n o t due t o d i f f e r e n c e s i n t h e u p t a k e o f t h e exogenous l a b e l e d Dap. The r e s u l t s s u p p o r t t h e c o n c l u s i o n t h a t t h e a c c u m u l a t i o n o f p e p t i d o g l y c a n i n amino a c i d - d e p r i v e d b a c t e r i a i s s t r i n g e n t l y r e g u l a t e d . I I . R e g u l a t i o n o f p e p t i d o g l y c a n s y n t h e s i s i n v i t r o A. C o u p l e d r e a c t i o n . I n i t i a l a t t e m p t s t o l o c a l i z e t h e s i t e o f i n h i b i t i o n o f FIGURE 7. E f f e c t o f l y s i n e d e p r i v a t i o n and CAM t r e a t m e n t on [ 3H]Dap i n c o r p o r a t i o n by LD5456 ( r e l A ) . F o r d e t a i l s o f t h e methods see f i g u r e 3. Complete M9 (.0 ) , M9 w i t h o u t l y s i n e ( © ) , M9 p l u s 200 yg p e r m l CAM ( A ) , and M9_ w i t h o u t l y s i n e b u t w i t h 200 yg p e r m l CAM ( A ) . B e f o r e t h e c o r r e c t i o n f o r d i f f e r e n c e s i n t h e c e l l u l a r mass, a t 60 m i n u t e s , t h e r e s p e c t i v e CPM f o r t h e f o u r l i s t e d c u l t u r e s were: 1,716; 1,137; 705; and 729. 40 M i n u t e s FIGURE 8. E f f e c t o f amino a c i d d e p r i v a t i o n on I 3H]Dap i n c o r p o r a t i o n by o t h e r ( r e l A ) s t r a i n s . F o r d e t a i l s of t h e methods, see f i g u r e 3. A) S t r a i n LD5457: Complete M9_ ( 0 ) , M9 minus l y s i n e ( § ) , M9 minus l e u c i n e ( A ). B) S t r a i n , LD5458: Complete M9_ ( 0 ) , M9 minus l y s i n e (8 ) , M9 minus t h r e o n i n e ( . .A ) . B e f o r e t h e c o r r e c t i o n f o r d i f f e r e n c e s i n t h e c e l l u l a r mass, a t 60 m i n u t e s , t h e r e s p e c t i v e CPM f o r the s i x l i s t e d c u l t u r e s were: 1,806; 1,601; 914; 3,054; 1,522; and 1,399. FIGURE 9. U t i l i z a t i o n of i n t r a c e l l u l a r r a d i o a c t i v e p o o l s by LD5 ( r e l A + ) (A) and LD5456 ( r e l A ~ ) ( B ) . C e l l s were l a b e l e d f o r 30 m i n u t e s , washed w i t h s a l i n e and i n o c u l a t e d i n t o f r e s h D a p - f r e e media w i t h l y s i n e ( 0 ) , w i t h o u t l y s i n e ( 6 ) , and w i t h o u t l y s i n e b u t p l u s 200 yg p e r ml CAM ( k ). I n c o r p o r a t i o n was f o l l o w e d as t h e i n c r e a s e i n T C A - i n s o l u b l e r a d i o a c t i v i t y i n 0.1 m l samples of the c u l t u r e s . To f a c i l i t a t e c o m p a r i s o n t h e v a l u e s were n o r m a l i z e d t o t h e d r y c e l l w e i g h t and z e r o e d by s u b t r a c t i n g t h e c o u n t s w h i c h had been i n c o r p o r a t e d by t h e t i m e t h a t t h e c u l t u r e s were s p l i t (10.8 x 10h CPM p e r mg c e l l d r y w e i g h t f o r LD5, and 9.43 x 1 0 4 CPM pe r mg c e l l d r y w e i g h t f o r LD5456). B e f o r e t h e c o r r e c t i o n f o r d i f f e r e n c e s i n t h e c e l l u l a r mass, a t 60 m i n u t e s a f t e r a d d i n g t h e [ 3H]Dap, t h e r e s p e c t i v e CPM f o r t h e s i x p e r m u t a t i o n s of t h e l i s t e d c u l t u r e s were: 1,802; 865; 1,149; 1,921; 1,288; and 1,024. 45 p e p t i d o g l y c a n s y n t h e s i s d u r i n g s t r i n g e n t c o n t r o l were made by d e t e r m i n i n g t h e e f f e c t o f ppGpp on t h e i n v i t r o a c t i v i t y o f some of t h e enzymes i n v o l v e d i n p e p t i d o g l y c a n s y n t h e s i s . The c o u p l e d r e a c t i o n i n v o l v e s a c r u d e p a r t i c u l a t e c e l l f r a c t i o n w h i c h c a t a l y s e s t h e r e a c t i o n s numbered 9, 10, 11 and 12 i n f i g u r e 2 and a l l o w s b o t h t h e f o r m a t i o n o f G C L - l i n k e d p e p t i d o g l y c a n i n t e r m e d -i a t e s and p e p t i d o g l y c a n t o be m o n i t o r e d . 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 2 mM ppGpp s i g n i f i c a n t l y i n h i b i t e d t h e r a t e o f p e p t i d o g l y c a n s y n t h e s i s b u t d i d n o t i n h i b i t t he a c c u m u l a t i o n o f G C L - l i n k e d i n t e r m e d i a t e s ( f i g u r e 1 0 ) . The d e g r e e o f i n h i b i t i o n o f p e p t i d o g l y c a n f o r m a t i o n i n c r e a s e d w i t h i n c r e a s t -i n g ppGpp c o n c e n t r a t i o n ( f i g u r e 1 1 ) . However, a t h i g h e r c o n c e n t r a t i o n s o f ppGpp t h e a c c u m u l a t i o n o f G C L - l i n k e d i n t e r m e d i a t e s was a l s o r e d u c e d ( f i g u r e 1 1 ) . The c o u p l e d r e a c t i o n was dependent on magnesium ( f i g u r e 1 2 ) . The magnesium dependence was complex and p r o b a b l y r e f l e c t e d d i f f e r e n t magnesium optim a of the v a r i o u s enzymes i n v o l v e d i n t h e a s s a y . The o p t i m a l a c c u m u l a t -i o n o f b o t h t h e G C L - l i n k e d i n t e r m e d i a t e s ( f i g u r e 12B) and t h e p e p t i d o g l y c a n ( f i g u r e 12A) o c c u r r e d a t a p p r o x i m a t e l y 12 mM MgC^- The deg r e e of i n h i b i t i o n o f b o t h r e a c t i o n s by ppGpp d e c r e a s e d w i t h i n c r e a s i n g c o n c e n t r a t i o n o f M g C ^ ( f i g u r e 1 2 ) . However. Lueking. and G o l d f i n e (65) r e p o r t e d t h a t t h e s t r i n g e n t i n h i b i t i o n o f i n v i t r o p h o s p h o l i p i d b i o s y n t h e s i s i s dependent on t h e m o l a r r a t i o o f magnesium t o ppGpp i n t h e r e a c t i o n r a t h e r t h a n t h e a b s o l u t e amount of e i t h e r . T h e r e f o r e , t h e e f f e c t o f t h e m o l a r r a t i o o f magnesium t o ppGpp on t h e c o u p l e d r e a c t i o n was c a l c u l a t e d u s i n g t h e d a t a i n f i g u r e 12. The r e l a t i v e i n h i b i t i o n s o f t h e a c c u m u l a t i o n of p e p t i d o g l y c a n and G C L - l i n k e d i n t e r m e d i a t e s d e c r e a s e d as t h e m o l a r r a t i o o f M g C ^ t o ppGpp i n c r e a s e d ( f i g u r e 1 3 ) . S i m i l a r r e l a t i v e amounts of a c t i v i t y were o b s e r v e d when the. m o l a r r a t i o of M g C l ^ t o ppGpp was v a r i e d by c h a n g i n g t h e ppGpp FIGURE 10. Incorporation of I1^CjUDP-MurNAc-pentapeptide into GCL-linked intermediates ( A, A ) and peptidoglycan ( 0, 8 ) by a particulate membrane preparation incubated in the presence ( A, 81 ) and absence ( A, 0 ) of 2 mM ppGpp. All reactions contained 6 mM MgCl0. P e p t i d o g l y c a n A c c u m u l a t e d (102 C P M ) -p-48 FIGURE 11. E f f e c t of ppGpp concentration on the amount of peptido-glycan ( ® ) and GCL-linked intermediates ( A ) formed by the coupled r e a c t i o n f o r i n v i t r o peptidoglycan synthesis i n ten minutes. Reactions contained 6 mM MgCl_. o FIGURE 12. Effect of MgCl^ on the amount of peptidoglycan ( A ) and GCL-linked intermediates ( B ) accumulated by the coupled reaction for in vitro peptidoglycan synthesis. Reactions were incubated for 10 minutes in the presence ( 8 ) and absence ( 0 ) of 4 mM ppGpp. FIGURE 13. I n f l u e n c e o f t h e m o l a r r a t i o , o f M g C ^ t o ppGpp on t h e r e l a t i v e amount o f p e p t i d o g l y c a n ( © ) and G C L - l i n k e d i n t e r m e d i a t e s ( A )• a c c u m u l a t e d by t h e c o u p l e d r e a c t i o n f o r i n v i t r o p e p t i d o g l y c a n s y n t h e s i s . The v a l u e s were c a l c u l a t e d f r o m t h e r e s u l t s i n f i g u r e 12 and a r e p r e s e n t e d as t h e amounts ac c u m u l a t e d i n t h e p r e s e n c e o f ppGpp r e l a t i v e t o t h e r e s p e c t i v e amounts ac c u m u l a t e d i n t h e absence of ppGpp. 53 54 c o n c e n t r a t i o n i n s t e a d o f t h e M g C l 2 c o n c e n t r a t i o n ( d a t a n o t p r e s e n t e d ) . A g i v e n m o l a r r a t i o of MgCl,> t o ppGpp gave a c o n s i s t e n t l e v e l o f a c c u m u l a t i o n . The p r e c e d i n g d a t a i n d i c a t e t h a t f o r m a t i o n , of p e p t i d o -g l y c a n c o u l d be i n h i b i t e d by ppGpp a t t h e phospho-N-acetylmuramoyl-p e n t a p e p t i d e t r a n s l o c a s e ( r e a c t i o n 9, f i g u r e 2) b u t a l a t e r s t e p i n t h e i n v i t r o p e p t i d o g l y c a n s y n t h e s i s i s even more s e n s i t i v e t o ppGpp. B. Exchange R e a c t i o n . S i n c e t h e r e s u l t s o f t h e c o u p l e d a s s a y f o r p e p t i d o g l y c a n s y n t h e s i s a r e dependent on t h e c o n s e c u t i v e a c t i v i t y o f s e v e r a l enzymes, t h e f i r s t r e a c t i o n c a t a l y z e d by t h e p a r t i c u l a t e system was s p e c i f i c a l l y s t u d i e d w i t h t h e exchange r e a c t i o n . I n t h i s a s s a y , t h e enzyme c a t a l y z e s an exchange r e a c t i o n between I 3H]UMP and UDP-MurNAc-pentapeptide. The r e a c t i o n was l i n e a r f o r a t l e a s t 32 m i n u t e s and was s e n s i t i v e t o ppGpp ( f i g u r e 1 4 ) . The exchange r e a c t i o n was dependent on magnesium ( f i g u r e 15A) . Optimum enzyme a c t i v i t y o c c u r r e d w i t h 6 mM MgCl,,. The dependence on magnesium was not as complex as t h a t o b s e r v e d f o r t h e c o u p l e d r e a c t i o n . The i n h i b i t o r y e f f e c t o f ppGpp on t h e exchange r e a c t i o n s t i l l v a r i e d w i t h t h e c o n c e n t r a t i o n o f t h e M g C l 2 ( f i g u r e 15A). The i n h i b i t i o n was dependent on t h e m o l a r r a t i o o f magnesium t o ppGpp ( f i g u r e 1 5 B ) ; t h e d egree o f i n h i b i t i o n d e c r e a s e d as t h e m o l a r r a t i o i n c r e a s e d . B o t h t h e exchange r e a c t i o n and t h e amount o f G C L - l i n k e d i n t e r m e d i a t e s i n t h e c o u p l e d r e a c t i o n were r e d u c e d by 50% a t a M g C l 2 t o ppGpp m o l a r r a t i o o f a p p r o x -i m a t e l y 1.3. T h i s s u g g e s t s t h a t t h e r e s u l t s from t h e c o u p l e d r e a c t i o n a r e v a l i d and s u p p o r t s t h e c o n c l u s i o n t h a t t h e a c t i v i t y o f t h e phospho-N - a c e t y l m u r a m o y l - p e n t a p e p t i d e t r a n s l o c a s e ( r e a c t i o n 9, f i g u r e 2) can be i n h i b i t e d by ppGpp. FIGURE 14. Incorporation of I3H]UMP into UDP-MurNAc-pentapeptide by the exchange reaction for phospho-N-acetylmuramoyl-pentapeptide transferase (reaction 9, figure 2). Samples were incubated in the presence ( 9 ) and absence ( 0 ) of 2 mM ppGpp. All reactions contained 6 mM MgCl0. 0 8 16 24 32 M i n u t e s FIGURE 15. (A) E f f e c t o f M g C l 2 on t h e a c c u m u l a t i o n of [ 3H]UMP i n UDP-MurNAc-pentapeptide by t h e exchange a s s a y . R e a c t i o n s were i n c u b a t e d f o r 15 m i n u t e s i n t h e absence of ppGpp ( 0 ) and i n th e p r e s e n c e o f 2 mM ppGpp ( 9 ) and 4 mM ppGpp ( A ) a t t h e i n d i c a t e d M g C l 2 c o n c e n t r a t i o n s . (B) The e f f e c t o f t h e m o l a r r a t i o of M g C l 2 t o ppGpp was e x p r e s s e d as t h e p e r c e n t o f t h e c o n t r o l a c t i v i t y o b s e r v e d i n the p r e s e n c e of 2 mM ppGpp ( • ') and 4 mM ppGpp ( A ). 58 C\] oo " ~ ( l A l c D g O O u o ! ; B | n u j n o o v 5 9 C. E f f e c t s o f n u c l e o t i d e s on t h e c o u p l e d r e a c t i o n The i n h i b i t i o n o f t h e c o u p l e d r e a c t i o n by ppGpp was r e l a t i v e l y s p e c i f i c ( T a b l e I I ) . Most o t h e r common n u c l e o t i d e s a t 2 mM c o n c e n t r a t i o n d i d n o t s i g n i f i c a n t l y a f f e c t t h e a c c u m u l a t i o n o f e i t h e r p e p t i d o g l y c a n o r G C L - l i n k e d i n t e r m e d i a t e s . The p r i n c i p l e e x c e p t i o n s were UMP, UDP and UTP. Of t h e s e , o n l y UMP a f f e c t e d t h e r e a c t i o n s a t l o w e r , more p h y s i o -l o g i c a l c o n c e n t r a t i o n s ( T a b l e I I I ) . The i n h i b i t i o n s by UTP and UDP were p r o b a b l y n o t due t o c o n v e r s i o n t o UMP by p h o s p h a t a s e s i n t h e enzyme p r e p a r a t i o n s i n c e UMP was n o t d e t e c t e d i n e i t h e r r e a c t i o n . The e f f e c t s o f ATP were a l s o c o n c e n t r a t i o n - d e p e n d e n t ( T a b l e I V ) . Above 10 mM ATP, a c c u m u l a t i o n o f b o t h t h e p e p t i d o g l y c a n and t h e G C L - l i n k e d i n t e r m e d i a t e s was r e d u c e d . At s l i g h t l y l o w e r c o n c e n t r a t i o n s o n l y t h e a c c u m u l a t i o n o f p e p t i d o g l y c a n was r e d u c e d , and t h e a c c u m u l a t i o n o f G C L - l i n k e d i n t e r m e d i a t e s was g r e a t e r t h a n i n t h e c o n t r o l . C h e l a t i n g a g e n t s , such as EDTA, a l s o i n h i b i t e d t h e c o u p l e d r e a c t i o n ( T a b l e I V ) . D. O t h e r r e a c t i o n s i n p e p t i d o g l y c a n s y n t h e s i s . O t h e r r e a c t i o n s examined i n v i t r o were D - a l a n y l a l a n i n e s y n t h e s i s ( r e a c t i o n 7, f i g u r e 2) and D - a l a n y l a l a n i n e l i g a s e ( r e a c t i o n 8, f i g u r e 2 ) . B o t h enzymes r e q u i r e e i t h e r magnesium o r manganese. At a m o l a r r a t i o o f M n C l 2 t o ppGpp o f one, n e i t h e r r e a c t i o n was s i g n i f i c a n t l y i n h i b i t e d ( f i g u r e 16A, B ) . T h i s l a c k o f i n h i b i t i o n was p r o b a b l y n o t due t o t h e use o f manganese i n t h e r e a c t i o n s s i n c e t h e exchange r e a c t i o n was more s e n s i t i v e t o ppGpp when manganese was s u b s t i t u t e d f o r magnesium ( f i g u r e 17A, B ) . TABLE I I . E f f e c t o f 2 mM c o n c e n t r a t i o n s o f n u c l e o t i d e s on t h e a c t i v i t y o f t h e c o u p l e d r e a c t i o n f o r p e p t i d o g l y c a n s y n t h e s i s 3 . N u c l e o t i d e Added P e p t i d o g l y c a n G C L - l i n k e d I n t e r m e d i a t e s CPM % o f C o n t r o l CPM % of C o n t r o l E x p e r i m e n t 1 None ( c o n t r o l ) 2636 ppGpp .1377 GMP 2650 dGTP 2234 ppppG 2607 c-GMP 2687 CMP 2647 CTP 2378 UMP 643 UDP 892 UTP 1818 ADP 2309 E x p e r i m e n t 2 None ( c o n t r o l ) 1528 GTP 1467 CDP 1591 AMP 1305 E x p e r i m e n t 3 None ( c o n t r o l ) 2756 ppGpp 1461 GDP 2799 ATP 2982 100 763 100 52 779 102 101 633 83 85 752 99 99 583 76 102 776 101 101 693 91 90 783 103 24 580 76 34 252 33 69 638 88 88 761 100 100 2688 100 96 2413 90 104 2752 102 85 2516 94 100 731 100 53 616 84 102 769 ..104 108 809 111 A l l a s s a y s were i n c u b a t e d f o r 10 m i n u t e s and had a f i n a l MgCl c o n c e n t r a t i o n o f 6 mM. 61 TABLE I I I . E f f e c t o f c o n c e n t r a t i o n o f i n h i b i t i n g n u c l e o t i d e s on i n h i b i t i o n o f t h e c o u g l e d r e a c t i o n f o r p e p t i d o g l y c a n s y n t h e s i s . _ , ., . P e p t i d o g l y c a n G C L - l i n k e d I n t e r m e d i a t e s I n h x b x t o r C o n c e n t r a t i o n c p M % q £ C o n t r o l C p M % o f C o n t r o l C o n t r o l (none) 1369 100 203 ' 100 UMP (2.0 mM) 339 25 114 56 (0.4 mM) 739 54 17 8 (0.2 mM) 1114 81 95 46 UDP (2.0 mM) 491 36 82 40 (0.4 mM) 1099 80 162 80 (0.2 mM) 1225 89 235 116 UTP (2.0 mM) 1112 81 169 83 (0.4 mM) 1416 103 209 103 (0.2 mM) 1545 113 259 125 I n c u b a t e d f o r 10 m i n u t e s w i t h a f i n a l MgCl„ c o n c e n t r a t i o n o f 6 mM. 62 TABLE IV. E f f e c t o f c o n c e n t r a t i o n o f ppGpp, ATP and EDTA on t h e a c t i v i t y of t h e c o u p l e d r e a c t i o n f o r p e p t i d o g l y c a n s y n t h e s i s 3 . I n h i b i t o r P e p t i d o g l y c a n G C L - l i n k e d I n t e r m e d i a t e s C o n c e n t r a t i o n CPM % o f C o n t r o l CPM % o f C o n t r o l C o n t r o l (none) 3371 100 1081 100 .ppGpp (2.0 mM) 2088 62 900 .83 (6.0 mM) 466 14 301 28 ATP (0.5 mM) 3825 113 1251 116 (1.0 mM) 3614 107 1128 104 (2.0 mM) 3624 107 1401 130 (4.0 mM) 2933 87 1708 158 (6.0 mM) 1812 54 1477 137 (10.0 mM) 407 12 997 92 (12.0 mM) 168 5 424 39 EDTA (2.0 mM) 2755 82 923 85 (4.0 mM) 1073 32 490 45 (6.0 mM) 137 4 661 61 I n c u b a t e d f o r 10 m i n u t e s w i t h a f i n a l KgCl^ c o n c e n t r a t i o n o f 6 mM. FIGURE 16. E f f e c t of t h e p r e s e n c e ( • ) or absence ( 0 ) of 4 mM ppGpp on t h e a c t i v i t y o f D - a l a n y l a l a n i n e s y n t h e t a s e ( A ) and D - a l a n y l a l a n i n e l i g a s e ( B ) as s a y e d i n t h e p r e s e n c e o f 4 mM MnCl„. Minutes 65 FIGURE 17. (A) E f f e c t o f M n C l 2 on t h e . a c c u m u l a t i o n of I 3RjUMP i n UDP-MurNAc-pentapeptide by t h e exchange a s s a y . R e a c t i o n s were i n c u b a t e d f o r 15 m i n u t e s i n t h e p r e s e n c e ( • ) and absence ( 0 ) of 2 mM ppGpp a t t h e i n d i c a t e d M n C l 2 c o n c e n t r a t i o n s . (B) The i n f l u e n c e o f the m o l a r r a t i o o f M n C l 2 t o ppGpp on t h e exchange v a l u e s was c a l c u l a t e d as t h e p e r c e n t of the a c c u m u l a t i o n i n the p r e s e n c e of ppGpp r e l a t i v e t o t h e a c c u m u l a t i o n i n t h e absence o f ppGpp. 67 I I I . A n a l y s i s o f s t r i n g e n t c o n t r o l d u r i n g i n v i v o p e p t i d o g l y c a n s y n t h e s i s . A. D i s t r i b u t i o n o f [ 3H]Dap i n c o r p o r a t e d by amino a c i d - d e p r i v e d b a c t e r i a . . The s i t e o f s t r i n g e n t r e g u l a t i o n o f p e p t i d o g l y c a n f o r m a t i o n was examined i n v i v o by comparing t h e amount of [ 3H]Dap i n c o r p o r a t e d i n t o t h e p e p t i d o g l y c a n , s o l u b l e n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s and GCL-l i n k e d p e p t i d o g l y c a n i n t e r m e d i a t e s i n t h e p r e s e n c e and absence o f r e q u i r e d amino a c i d s . T h i s was done by l o a d i n g samples of i n t a c t c e l l s t o paper chromatographs and u s i n g t h e d e v e l o p i n g chromatography s o l v e n t t o s i m u l t a n e o u s l y e x t r a c t and s e p a r a t e any l a b e l e d components. A t y p i c a l a n a l y s i s of s t r a i n LD5. ( r e l A + ) i s shown'in f i g u r e 18. The e f f e c t s o f l y s i n e d e p r i v a t i o n on LP5 ( r e l A + ) a r e summarized i n T a b l e V. The amounts of Dap i n c o r p o r a t e d i n t o t h e T C A - i n s o l u b l e and t h e p e p t i d o g l y c a n f r a c t i o n s by l y s i n e - d e p r i v e d c e l l s were a p p r o x i m a t e l y 30% o f t h e amounts i n c o n t r o l c e l l s . The q u a n t i t y of l a b e l e d l i p i d zone m a t e r i a l was c o n s i s t e n t l y 2- t o 3-f o l d h i g h e r i n t h e l y s i n e - d e p r i v e d c u l t u r e s t h a n i n t h e c o n t r o l s . L y s i n e -d e p r i v e d c e l l s a l s o c o n t a i n e d a t l e a s t as much l a b e l e d n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s as c o n t r o l c e l l s . I n b o t h c o n d i t i o n s , UDP-MurNAc-p e n t a p e p t i d e a c c o u n t e d f o r a p p r o x i m a t e l y 95% o f the n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r p o o l ( T a b l e V I ) . These r e s u l t s s u g g e s t t h a t t h e b a s i s f o r s t r i n g e n t c o n t r o l i s n o t l i m i t a t i o n of e i t h e r s o l u b l e n u c l e o t i d e -l i n k e d p r e c u r s o r s o r GCL-P-P-MurNAc-pentapeptide; t h e s i t e o f i n h i b i t i o n i s a l m o s t c e r t a i n l y one ( o r b o t h ) o f t h e enzymes c a t a l y z i n g t h e l a s t two r e a c t i o n s i n p e p t i d o g l y c a n s y n t h e s i s ( r e a c t i o n s 10 and 11, f i g u r e 2).. The, r e l a x e d s t r a i n LD5456 ( r e l A ) c o n t i n u e d t o a c c u m u l a t e p e p t i d o g l y c a n and T C A - i n s o l u b l e m a t e r i a l d u r i n g l y s i n e d e p r i v a t i o n ( T a b l e V I I ) . There was a l s o a r e l a t i v e b u i l d u p o f t h e n u c l e o t i d e - l i n k e d p r e c u r s o r s and t h e l i p i d zone m a t e r i a l . FIGURE 18. D i s t r i b u t i o n o f r a d i o a c t i v i t y when c e l l s o f LD5 + 3  ( r e l A ) were l a b e l e d w i t h [ HjDap and t h e n chromatographed i n s o l v e n t system I . Growing c e l l s were h a r v e s t e d , washed once w i t h 0.9% s a l i n e and t r a n s f e r r e d t o f r e s h D a p - f r e e M9_. [ 3H]Dap was added and a f t e r 60 m i n u t e s a p o r t i o n of t h e c u l t u r e was concen-t r a t e d and a p p l i e d t o Whatman 3 MM chromatography p a p e r . The chromatograph was d e v e l o p e d f o r a p p r o x i m a t e l y 20 h o u r s . Peak A c o r r e s p o n d s to p e p t i d o g l y c a n , Peak B c o r r e s p o n d s t o s o l u b l e n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s , Peak C c o r r e s p o n d s t o f r e e [ 3H]Dap and Peak D i s t h e l i p i d zone. A C c m f r o m O r i g i n TABLE V. D i s t r i b u t i o n o f [ 3H]Dap i n c o r p o r a t e d by LD5 ( r e l A ) d u r i n g l y s i n e d e p r i v a t i o n 3 . 101* CPM per mg C e l l D r y Wt. C u l t u r e C o n d i t i o n s T o t a l TCA C I n s o l u b l e N u c l e o t i d e P r e c u r s o r s - l i n k e d L i p i d Zone P e p t i d o g l y c a n ^ 30 m i n u t e s C o n t r o l 10.44 (100%) 0.62 (100%) 0.08 (100%) 10.74 (100%) Minus L y s i n e 3.12 (30%) 0.90 (145%) 0.19 (238%) 3.18 (30%) 60 m i n u t e s C o n t r o l 20.84 (100%) 0.72 (100%) 0.23 (100%) 21.92 (100%) Minus L y s i n e 6.02 (29%) 1.00 (139%) 0.82 (357%) 4.46 1(20%) C e l l s were p r e p a r e d and grown as d e s c r i b e d i n f i g u r e 3. The c o n t r o l i s t h e u n t r e a t e d c u l t u r e . Net i n c o r p o r a t i o n was m o n i t o r e d as TCA i n s o l u b l e r a d i o a c t i v i t y . D i s t r i b u t i o n o f t h e i n c o r p o r a t e d Dap was d e t e r m i n e d by c h r o m a t o g r a p h i n g c o n c e n t r a t e d samples of whole c e l l s i n s o l v e n t s y s t e m A and t h e n c o u n t i n g t h e r a d i o a c t i v i t y i n t h e r e g i o n s c o r r e s p o n d i n g t o t h e s o l u b l e p r e c u r s o r s , t h e l i p i d zone and t h e p e p t i d o g l y c a n . Counts were n o r m a l i z e d t o c e l l mass i n o r d e r t o compensate f o r d i f f e r e n c e s i n t h e amount o f each sample and t o f a c i l i t a t e c o m p a r i s o n of the v a l u e s . ^ V a l u e s i n b r a c k e t s a r e p e r c e n t of the c o n t r o l , c The r e s p e c t i v e CPM f o r t h i s column, b e f o r e t h e c o r r e c t i o n f o r d i f f e r e n c e s i n mass of t h e s a m p l e s , were: 555; 195; 1,479; and 386. The r e s p e c t i v e CPM f o r t h i s column, b e f o r e t h e c o r r e c t i o n f o r d i f f e r e n c e s i n mass of t h e s a m p l e s , were: 22,379; 5,850; 62,307; and 9,968. 71 TABLE V I . A n a l y s i s o f s o l u b l e n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s s y n t h e s i z e d by LD5 ( r e l A ) d u r i n g l y s i n e d e p r i v a t i o n 3 . 101* CPM per mg C e l l Dry Wt. b n ~ n A ' *-' T o t a l L a b e l e d UDP-MurNAc- C UDP-MurNAc-C u l t u r e C o n d i t i o n s P r e c u r s o r s p e n t a p e p t i d e t r i p e p t i d e 30 m i n u t e s C o n t r o l 0.86 0.83 (97%) 0.03 :(3%) Minus L y s i n e 0.66 0.62 (95%) 0.04 (5%) 60 m i n u t e s C o n t r o l 0.87 0.85 (98%) 0.02 (2%) Minus L y s i n e 0.83 0.78 (94%) 0.05 (6%) The c e l l s were p r e p a r e d and grown as d e s c r i b e d i n f i g u r e 3. The c o n t r o l i s t h e u n t r e a t e d c u l t u r e . The p o o l s o f n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s were s e p a r a t e d by c h r o m a t o g r a p h i n g c o n c e n t r a t e d samples of whole c e l l s . The r a d i o a c t i v i t y i n t h e r e s p e c t i v e zones was n o r m a l i z e d t o t h e c e l l mass i n o r d e r t o f a c i l i t a t e c o m p a r i s o n . ^ V a l u e s i n b r a c k e t s a r e p e r c e n t o f t h e t o t a l r a d i o a c t i v i t y i n t h e p r e c u r s o r p o o l s . The r e s p e c t i v e CPM f o r t h i s column, b e f o r e the c o r r e c t i o n f o r d i f f e r e n c e s i n t h e mass of t h e samples, were: 1,755; 1,615; 2,440; and 2,339. TABLE V I I . D i s t r i b u t i o n o f [ 3H]Dap i n c o r p o r a t e d by LD5456 ( r e l A ) d u r i n g l y s i n e d e p r i v a t i o n . IO1* CPM p e r rag C e l l Dry Wt. b ~ — 0 T o t a l TCA N u c l e o t i d e - l i n k e d L i p i d Zone P e p t i d o g l y c a n ^ C u l t u r e C o n d x t i o n s .. , .. _ v r o j I n s o l u b l e P r e c u r s o r s 30 m i n u t e s C o n t r o l Minus L y s i n e 60 m i n u t e s C o n t r o l Minus L y s i n e 10.04 (100%) 11.85 (118%) 20.01 ;(ioo%) 18.30 (91%) 0.58 (100%) 2.13 (367%) 0.63 • (100%) 3.28 (521%) 0.09 (100%) 0.53 (588%) 0.28 (100%) 2.26 (807%) 8.74 (100%) 8/43 (96%) 17.77 (100%) 12.93 (73%) F o r t h e d e t a i l s o f t h e methods, see T a b l e V. V a l u e s I n b r a c k e t s a r e p e r c e n t of t h e c o n t r o l . c The r e s p e c t i v e CPM f o r t h i s column, b e f o r e the c o r r e c t i o n f o r d i f f e r e n c e s i n t h e mass of t h e s a m p l e s , were: 368; 605; 1,062; and 1,007. ^The r e s p e c t i v e CPM f o r t h i s column, b e f o r e the c o r r e c t i o n f o r d i f f e r e n c e s i n t h e mass of t h e s a m p l e s , were: 18,949; 16,198; 44,267; and 23,387. 73 The d i f f e r e n c e between t h e amount o f p e p t i d o g l y c a n a c c u m u l a t e d by t h e l y s i n e - d e p r i v e d s t r i n g e n t and r e l a x e d c e l l s i s n o t due t o a u t o l y s i s . The s u p e r n a t a n t s from t h e [ 3HJDap l a b e l e d c u l t u r e s o n l y c o n t a i n e d s l i g h t amounts o f p o s s i b l e a u t o l y t i c m a t e r i a l ( T a b l e V I I I ) . These m a t e r i a l s o c c u r r e d i n s i m i l a r amounts i n t h e r e l a x e d and s t r i n g e n t c u l t u r e s , and none o f t h e examined samples c o n t a i n e d more t h a n 1.5% of t h e t o t a l T C A - i n s o l u b l e c o u n t s a s s o c i a t e d w i t h t h e c e l l s . O t h e r r e l A + s t r a i n s (LD52 and LD2) gave t h e same g e n e r a l p a t t e r n o f r e s u l t s as LD5 ( r e l A + ) (see T a b l e i x ) • D u r i n g amino a c i d d e p r i v a t i o n , t h e a c c u m u l a t i o n o f p e p t i d o g l y c a n was s i g n i f i c a n t l y r e d u c e d , whereas the a c c u m u l a t i o n o f s o l u b l e n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s and l i p i d zone m a t e r i a l was n o t . The r e l a x e d s t r a i n LD5457 ( T a b l e X) gave t h e same g e n e r a l p a t t e r n o f r e s u l t s as LD5456 ( r e l A ) . However, i n b o t h t h e s t r i n g e n t and t h e r e l a x e d s t r a i n s t h e e x c e s s i v e a c c u m u l a t i o n o f l a b e l i n t h e l i p i d zone o n l y o c c u r r e d d u r i n g l y s i n e d e p r i v a t i o n and was n o t o b s e r v e d d u r i n g d e p r i v a t i o n f o r t h r e o n i n e , l e u c i n e o r m e t h i o n i n e . CAM t r e a t m e n t r e l a x e d t h e s y n t h e s i s o f p e p t i d o g l y c a n i n LD5 ( r e l A + ) ( T a b l e X I ) . The degree o f r e l a x a t i o n was n o t as h i g h as t h a t o b s e r v e d f o r l y s i n e - d e p r i v e d c e l l s o f LD5456 ( r e l A ) ( T a b l e V i i ) . The d i f f e r e n c e m i ght be due t o s i d e e f f e c t s o f the CAM s i n c e t h e r e l a x e d a c c u m u l a t i o n o f p e p t i d o g l y c a n by l y s i n e - d e p r i v e d c e l l s o f LD5456 ( r e l A ) was l o w e r f o l l o w i n g CAM t r e a t m e n t ( T a b l e X I I ) . CAM t r e a t m e n t a l s o r e s u l t e d i n an a c c u m u l a t i o n o f l a b e l e d n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r i n LD5 ( r e l A + ) ( T a b l e X I ) and p r e v e n t e d the e x c e s s i v e a c c u m u l a t i o n o f l a b e l e d l i p i d zone m a t e r i a l p r e v i o u s l y o b s e r v e d i n b o t h s t r i n g e n t and r e l a x e d c e l l s d e p r i v e d o f l y s i n e . 74 TABLE V I I I . D i s t r i b u t i o n o f l a b e l e d m a t e r i a l i n c u l t u r e 3' Si s u p e r n a t a n t s , e x c l u d i n g f r e e I H]Dap . 1 0 3 CPM p e r mg C e l l Dry Wt. b C u l t u r e C o n d i t i o n s R f 0 . 4 3 C ' 6 R 0.62d ' LD5 ( r e l A + ) C o n t r o l , 60 m i n u t e s 3.12 (0.5%) 0.45 (0.1%) Minus L y s i n e , 60 m i n u t e s 2.60 (1.2%) 0.48 (0.2%) LD5456 ( r e l A ~ ) C o n t r o l , 60 m i n u t e s 5.26 (1.3%) 0.92 (0.2%) Minus L y s i n e , 60 m i n u t e s 3.23 (0.8%) 0.49 (0.1%) " D e s a l t e d and l y p h i l i z e d s u p e r n a t a n t s f r o m c u l t u r e s examined f o r t h e d i s t r i b u t i o n o f i n c o r p o r a t e d [ 3H]Dap (see T a b l e V f o r d e t a i l s ) were chromatographed i n s o l v e n t system I . The chromatographs were t h e n examined f o r r a d i o a c t i v e s p o t s w h i c h d i d not c o r r e s p o n d t o f r e e Dap. The r e s u l t s were n o r m a l i z e d t o t h e d r y w e i g h t o f c e l l s i n t h e o r i g i n a l c u l t u r e s , i n o r d e r t o a l l o w c o m p a r i s o n t o t h e n e t i n c o r p o r a t e d Dap. ^ F i g u r e s i n b r a c k e t s a r e p e r c e n t of t o t a l TCA i n s o l u b l e a c t i v i t y i n t h e c u l t u r e . °Possibly M u r N A c - p e n t a p e p t i d e o r M u r N A c ( - G l c N A c ) - p e n t a p e p t i d e . ^ P o s s i b l y p e n t a p e p t i d e . The r e s p e c t i v e CPM f o r t h i s column, b e f o r e t h e c o r r e c t i o n f o r d i f f e r e n c e s i n t he mass o f t h e o r i g i n a l c u l t u r e s , were: 616; 499; 1,033; and 380. , 4-TABLE IX. D i s t r i b u t i o n o f [ H]Dap i n c o r p o r a t e d by LD52 ( r e l A ^ and LD2 ( r e l A ) d u r i n g 60 m i n u t e s of amino a c i d d e p r i v a t i o n . 10k CPM per mg C e l l Dry Wt. _ — , . . T o t a l TCA N u c l e o t i d e - l i n k e d L i p i d Zone P e p t i d o g l y c a n ^ C u l t u r e C o n d i t i o n s T 1 , 1 c „ r t> J I n s o l u b l e P r e c u r s o r s LD52 ( r e l A ) C o n t r o l 15.94 (100%) 0.48 (100%) 0.12 (100%) 15.09 (100%) Minus L y s i n e 5.20 (33%) 0.44 (92%) 0.35 (292%) 5.06 (34%) Minus T h r e o n i n e 6.41 (40%) 0.89 (185%) 0.11 (92%) 6.64 (44%) Minus L y s i n e and T h r e o n i n e 7.45 (47%) 0.64 (133%) 0.40 (333%) 6.45 (43%) '. ( r e l A + ) C o n t r o l 37.60 . (100%) 0.96 (100%) 0.24 (100%) 37.89 (100%) Minus L y s i n e 12.76 (34%) 1.31 (136%) 0.94 (392%) 8.97 (24%) Minus M e t h i o n i n e 11.45 (30%) 1.40 (146%) 0.22 (92%) 10.05 (27%) Minus L y s i n e and M e t h i o n i n e 10.23 (27%) 1.58 (165%) 0.96 (400%) 10.32 (27%) SL F o r the d e t a i l s of t h e methods, see T a b l e V. ^ V a l u e s i n b r a c k e t s a r e p e r c e n t of the c o n t r o l , c The r e s p e c t i v e CPM f o r t h i s column, b e f o r e t h e c o r r e c t i o n f o r d i f f e r e n c e s i n mass o f t h e s a m p l e s , were: 1,198; 278; 345; 406; 2,734; 691; 741; and 743. ^The r e s p e c t i v e CPM f o r t h i s column, b e f o r e t h e c o r r e c t i o n f o r d i f f e r e n c e s i n mass o f t h e s a m p l e s , were: 34,808; 9,032; 13,212; 11,819;. 72,859; 15,751; 18,695; and 19,527. TABLE X. D i s t r i b u t i o n of [ 3H]Dap i n c o r p o r a t e d by LD5457 ( r e l A ) d u r i n g l y s i n e o r l e u c i n e d e p r i v a t i o n . 10** CPM per mg C e l l Dry Wt. b C u l t u r e C o n d i t i o n s T o t a l TCA I n s o l u b l e N u c l e o t i d e — l i n k e d P r e c u r s o r s L i p i d Zone P e p t i d o g l y c a n 30 m i n u t e s C o n t r o l 8.75 (100%) 0.35 (100%) 0.08 (100%) 7.39 (100%) Minus L y s i n e 12.81 (146%) 1.34 (386%) 0.61 (761%) 10.09 (137%) Minus L e u c i n e 8.91 (94%) 1.26 (363%) 0.10 (125%) 6.22 (84%) 60 m i n u t e s C o n t r o l 16.70 (100%) 0.42 (100%) 0.14 (100%) 15.42 (100%) Minus L y s i n e 18.76 (112%) 1.72 (410%) 0.97 (693%) '13.33 (86%) Minus L e u c i n e 15.49 (93%) 2.35 (560%) 0.10 (71%) 13.42 (87%) a F o r t h e d e t a i l s o f the methods, see T a b l e V. ^ V a l u e s i n b r a c k e t s a r e p e r c e n t of the c o n t r o l . The r e s p e c t i v e CPM f o r t h i s column, b e f o r e the c o r r e c t i o n f o r d i f f e r e n c e s i n mass o f s a m p l e s , were: 583; 648; 401; 1,589; 1,036; and 737. ^The r e s p e c t i v e CPM f o r t h i s column, b e f o r e t h e c o r r e c t i o n f o r d i f f e r e n c e s i n mass o f samples, were: 32,767; 30,108; 19,771; 72,582; 43,904; and 28,744. TABLE X I . E f f e c t of l y s i n e d e p r i v a t i o n and CAM t r e a t m e n t on d i s t r i b u t i o n of [ 3H]Dap i n c o r p o r a t e d by LD5 ( r e l A + ) a . 101* CPM p e r mg C e l l Dry Wt. b C u l t u r e C o n d i t i o n s T o t a l TCA I n s o l u b l e N u c l e o t i d e - l i n k e d P r e c u r s o r s L i p i d Zone P e p t i d o g l y c a n d 30 m i n u t e s C o n t r o l Minus L y s i n e Minus L y s i n e ; P l u s CAM P l u s CAM 26.40 11.58 18.53 18.80 (100%) •(44%) (70%) (71%) 1.54 1.94 6.05 6.36 (100%) (126%) (393%) (413%) 0.53 2.63 0.66 0.39 (100%) (496%) (125%) (74%) 27.00 9.11 18.74 19.15 (100%) (34%) (69%) (71%) 60 m i n u t e s C o n t r o l Minus L y s i n e Minus L y s i n e ; P l u s CAM P l u s CAM 54.80 18.26 29.73 31.20 (100%) (33%) (54%) (57%) 1.67 2.90 8.89 9.51 (100%) (174%) (532%) (569%) 0.65 2,91 0.67 0.48 (100%) (448%) (103%) (74%) 56.53 15.51 30.79 34.03 (100%) (27%) (54%) (60%) F o r t h e d e t a i l s of t h e methods, see T a b l e V. b V a l u e s i n b r a c k e t s a r e p e r c e n t of the c o n t r o l . The r e s p e c t i v e CPM f o r t h i s column, b e f o r e the c o r r e c t i o n f o r d i f f e r e n c e s i n mass o f t h e s a m p l e s , were: 1,559; 758; 932; 892; 4,376; 1,088; 1,487; and 1,467. ^The r e s p e c t i v e CPM f o r t h i s column, b e f o r e the c o r r e c t i o n f o r d i f f e r e n c e s i n mass o f t h e s a m p l e s , were: 32,565; 8,869; 16,601; 17,021; 118,983; 15,138; 37,901; and 35,568. TABLE X I I . E f f e c t o f l y s i n e d e p r i v a t i o n and CAM t r e a t m e n t on d i s t r i b u t i o n of [ 3H]Dap i n c o r p o r a t e d by LD5456 ( r e ! A ~ ) a . 101* CPM p e r mg C e l l . Dry Wt. b C u l t u r e C o n d i t i o n s T o t a l TCA I n s o l u b l e Nu c l eo t i d e-1 i n k e d P r e c u r s o r s L i p i d Zone P e p t i d o g l y c a n * 1 30 m i n u t e s C o n t r o l 17.98 (100%) 1.38 (100%) 0.33 (100%) 17.63 (100%) Minus L y s i n e 22.61 (126%) 4.01 (291%) 1.78 (539%) 16.49 (94%) Minus L y s i n e ; P l u s CAM 14.19 (79%) 4.72 (342%) 0.29 (88%) 13.04 (74%) P l u s CAM 12.73 (71%) 6.55 (475%) 0.33 (100%) 15.62 (89%) 60 m i n u t e s C o n t r o l 35.45 (100%) 1.40 (100%) 0.90 (100%) " 34.53 (100%) Minus L y s i n e 37.10 (105%) 5.71 (408%) 5.30 (589%) 27.42 (79%) Minus L y s i n e ; P l u s CAM 20.41 (58%) 7.31 (522%) 0.46 (51%) 20'. 11 (58%) P l u s CAM 21.36 (60%) 8.21 (586%) 0.40 (44%) 20.44 (59%) 3. For t h e d e t a i l s of the methods , see T a b l e V. V a l u e s i n b r a c k e t s a r e p e r c e n t of t h e c o n t r o l . The r e s p e c t i v e CPM 1,004; 1,090; 709; f o r t h i s column, b e f o r e the 623; 2,888; 1,773; 1,020; and c o r r e c t i o n f o r 1,029. d i f f e r e n c e s i n mass o f t h e sampl e s , were; The r e s p e c t i v e CPM f o r t h i s column, b e f o r e the c o r r e c t i o n f o r d i f f e r e n c e s i n mass o f t h e s a m p l e s , were; 21,846; 19,063; 13,625; 15,446; 74,211; 30,724; 20,144; and 19,592. 79 B. E f f e c t of stringent i n h i b i t i o n on the composition of the GCL-linked intermediates. The preceding r e s u l t s suggest that the^incorporation of Dap into peptidoglycan i s i n h i b i t e d during stringent c o n t r o l at ei t h e r one or both of the following reactions: (i) the addition of UDP-GlcNAc to the GCL-P-P-MurNAc-pentapeptide (reaction 10, figure 2); ( i i ) the subsequent transf e r of the lipid-bound precursor to the nascent acceptor i n the peptidoglycan (reaction 11, figure 2). These p o s s i b i l i t i e s were distinguished by quantitating each GCL-linked intermediate during the stringent response. Representative r e s u l t s are shown i n Table XIII. . Threonine deprivation of LD52 ( r e l A + ) did not s i g n i f i c a n t l y a l t e r the absolute amount of GCL-P-P-MurNAc(-GlcNAc)-pentapeptide. However, the analysis i s p o t e n t i a l l y complicated by two u n i d e n t i f i e d components i n the l i p i d zone. One component was s e n s i t i v e to mild acid h y d r o l y s i s . It constituted about 10% of the labeled l i p i d zone material i n the c e l l s deprived of amino acids and about 15% of the l i p i d zone material i n the c o n t r o l c e l l s . The other u n i d e n t i f i e d component was r e s i s t a n t to mild acid h y d r o l y s i s and constituted 5- to 15% of the t o t a l labeled l i p i d zone material. However, even i f a l l of these u n i d e n t i f i e d materials are at t r i b u t e d to either GCL-P-P-MurNAc-pentapeptide or GCL-P-P-MurNAc(-GlcNAc) -pentapeptide, the r e l a t i v e proportions i n the s t r i n g e n t l y i n h i b i t e d c e l l s are s t i l l s i m i l a r to the proportions i n the growing c e l l s ; there should have been s u f f i c i e n t GCL-P-P-MurNAc(-GlcNAc)-pentapeptide to support a normal rate of peptidoglycan synthesis i n the amino acid-deprived c u l t u r e even though peptidoglycan synthesis was a c t u a l l y i n h i b i t e d by 66%/ These r e s u l t s c l e a r l y indicate that the terminal step i n peptidoglycan synthesis (reaction 11, fig u r e 2) i s i n h i b i t e d during the stringent response. TABLE X I I I . A n a l y s i s of t h e l a b e l e d zone of c e l l s i n c u b a t e d components of t h e l i p i d w i t h [ 3H]Dap f o r 60 m i n u t e s a D i s t r i b u t i o n o f R a d i o a c t i v i t y ( 1 0 3 CPM p e r mg C e l l D r y Wt. ) C u l t u r e C o n d i t i o n s GCL-P-P-•MurNAc-(-GleNAc)-p e n t a p e p t i d GCL-P-P-MurNAc-p e n t a p e p t i d e U n i d e n t i f i e d b R e s i d u a l -L i p i d Zone c Counts Net : Zone L i p i d Counts P e p t i d o g l y c a n * LD52 ( r e l A + ) C o n t r o l 0.71 0.49 0.32 0.25 1.72 (103%) 161.6 Minus T h r e o n i n e 0.78 0.47 0.21 0.15 1 .62 (106%) 70.9 LD5 ( r e l A + ) C o n t r o l 0.80 0.51 0.21 0.32 1.79 (105%) 256.3 Minus L y s i n e 2.03 0.68 N i l 3.44 5.97 (103%) 46.7 a T h e amount of each G C L - l i n k e d p e p t i d o g l y c a n i n t e r m e d i a t e i n the l i p i d zone was d e t e r m i n e d by h y d r o l y z i n g b o i l e d c e l l s i n 10% a c e t i c a c i d a t 105°C f o r 1 h o u r . The b o i l i n g d i d n o t a f f e c t the amount of c o u n t s i n t h e zone, but q u a n t i t a t i v e l y removed the n u c l e o t i d e - l l n k e d p e p t i d o g l y c a n p r e c u r s o r s and t h e endogenous p o o l of Dap. The m i l d a c i d h y d r o l y s i s r e l e a s e d t h e p e p t i d o g l y c a n p r e c u r s o r s f r o m t h e GCL i n forms w h i c h c o u l d be s e p a r a t e d by chromatography i n s o l v e n t system I . The n e t l i p i d zone c o u n t s and p e p t i d o g l y c a n were d e t e r m i n e d w i t h samples of whole c e l l s removed f r o m t h e same c u l t u r e s ( s e e T a b l e V f o r d e t a i l s ) . C ounts were n o r m a l i z e d t o c e l l mass i n o r d e r t o compensate f o r d i f f e r e n c e s i n t h e amount of each sample and f a c i l i t a t e c o m p a r i s o n of t h e v a l u e s . b A f t e r m i l d a c i d h y d r o l y s i s t h i s * m a t e r i a l m i g r a t e d a t Rf 0.53 i n s o l v e n t s y s t e m I . c A f t e r m i l d a c i d h y d r o l y s i s t h i s m a t e r i a l c o n t i n u e s t o m i g r a t e i n t h e l i p i d zone (R^ 0.8) i n s o l v e n t s ystem I . ^The f i g u r e s i n b r a c k e t s a r e t h e p e r c e n t r e c o v e r y of the t o t a l e d o r i g i n a l l i p i d zone components a f t e r m i l d a c i d h y d r o l y s i s and s e p a r a t i o n i n s o l v e n t system I . The r e s p e c t i v e CPM f o r t h i s column, b e f o r e t h e c o o r e c t i o n s f o r d i f f e r e n c e s i n t h e sample s i z e s were: 638; 637; 1,239; and 1,204. ^The r e s p e c t i v e CPM f o r t h i s column,' b e f o r e the c o r r e c t i o n s f o r d i f f e r e n c e s i n t h e mass of t h e samples were: 60,046; 21,537; 62,807; and 10,279. 00 o 81 L y s i n e - d e p r i v e d c u l t u r e s o f LD5 ( r e l A ^ ) gave s i m i l a r r e s u l t s ( T a b l e X I I I ) e x c e p t t h a t t h e u n i d e n t i f i e d component w h i c h was r e s i s t a n t t o m i l d a c i d h y d r o l y s i s c o n s t i t u t e d 50 t o 70% o f t h e l a b e l e d m a t e r i a l i n t h e l i p i d zone. However, t h i s s t i l l a l l o w s more GCL-P-P-MurNAc(-GlcNAc) - p e n t a p e p t i d e i n t h e l y s i n e - d e p r i v e d c e l l s t h a n i n t h e c o n t r o l s s i n c e t h e t o t a l amount o f l a b e l e d l i p i d zone m a t e r i a l was much l a r g e r t h a n i n t h e c o n t r o l s . S i n c e t h e G C L - l i n k e d p e p t i d o g l y c a n i n t e r m e d i a t e s s h o u l d be v e r y s e n s i t i v e t o m i l d a c i d h y d r o l y s i s ( 1 , 9) i t was p o s s i b l e t h a t t h e u n i d e n t i f i e d m a t e r i a l i n t h e l i p i d zone o f l y s i n e - d e p r i v e d LD5 ( r e l A + ) was n o t G C L - l i n k e d p e p t i d o g l y c a n i n t e r m e d i a t e s . T h e r e f o r e , t h e s e n s i t i v i t y o f t h e l i p i d zone m a t e r i a l t o S t r e p t o m y c e s g r i s e u s p r o t e a s e was examined b e c a u s e : ( i ) p r o t e i n s c a n c o - m i g r a t e w i t h t h e G C L - l i n k e d i n t e r m e d i a t e s (10, 1 0 7 ) , ( i i ) CAM, an i n h i b i t o r o f p r o t e i n s y n t h e s i s , p r e v e n t e d t h e e x c e s s a c c u m u l a t i o n o f l a b e l e d l i p i d zone m a t e r i a l d u r i n g l y s i n e d e p r i v a t i o n , ( i i i ) i f t h e l y s i n e a u x o t r p p h s r e t a i n e d any d i a m i n o p i m e l a t e d e c a r b o x y l a s e , Dap c o u l d be c o n v e r t e d t o l y s i n e and would t h e n be r e a d i l y i n c o r p o r a t e d i n t o p r o t e i n i n t h e absence o f exogenous l y s i n e . As shown i n T a b l e XIV l y s i n e d e p r i v a t i o n o f LD5 ( r e l A + ) r e s u l t e d i n n e a r l y a 5 - f o l d i n c r e a s e i n l a b e l e d l i p i d zone m a t e r i a l . O n l y 31% o f t h i s l i p i d zone m a t e r i a l was s e n s i t i v e t o m i l d a c i d h y d r o l y s i s , whereas 80% o f the m a t e r i a l from c o n t r o l c e l l s was h y d r o l y z e d . When t h e l i p i d zone m a t e r i a l was t r e a t e d w i t h p r o t e a s e , t h e r e was no e f f e c t on t h e samples from c o n t r o l c e l l s , b u t 47% o f t h e l i p i d zone m a t e r i a l from t h e l y s i n e - d e p r i v e d c e l l s was removed. F i n a l l y , i f t h e l i p i d zone m a t e r i a l was t r e a t e d w i t h p r o t e a s e and t h e n h y d r o l y z e d by m i l d a c i d , 78% and 87% o f t h e r e s p e c t i v e m a t e r i a l from c o n t r o l c e l l s and l y s i n e - d e p r i v e d c e l l s was r e l e a s e d . 82 TABLE XIV. E f f e c t of p r o t e a s e and a c e t i c a c i d h y d r o l y s i s on t h e l a b e l e d m a t e r i a l o f t h e l i p i d zone of LP5 ( f e l A ) . R a d i o a c t i v i t y i n L i p i d Zone. ( 1 0 2 CPM p e r mg C e l l Dry Wt.) Treatment C o n t r o l Minus L y s i n e None 6.41 (100%) 29.08 (100%) A c e t i c A c i d H y d r o l y s i s 1.28 (20%) 20.07 (69%) P r o t e a s e 6.99 (109%) 15.41 (53%) P r o t e a s e t h e n A c e t i c A c i d H y d r o l y s i s 1.41 (22%) 4.94 (17%) " C e l l s were grown and p r e p a r e d as d e s c r i b e d i n f i g u r e 3. A f t e r s i x t y m i n u t e s of i n c u b a t i o n t h e c e l l s were b o i l e d , t r e a t e d and t h e n a p p l i e d t o Whatman 3 MM f o r chromatography i n s o l v e n t system I . The a c e t i c a c i d h y d r o l y s i s i s d e s c r i b e d i n T a b l e X I I I . The p r o t e a s e t r e a t m e n t was done by i n c u b a t i n g t h e b o i l e d c e l l s a t 37°C f o r 30 m i n u t e s w i t h a 2 mg p e r ml c o n c e n t r a t i o n of S t r e p t o m y c e s g r i s e u s p r o t e a s e . The c o u n t s i n the l i p i d zone were n o r m a l i z e d t o c e l l mass i n o r d e r t o f a c i l i t a t e c o m p a r i s o n of t h e v a l u e s and compensate f o r d i f f e r e n c e s i n t h e mass of t h e samples f r o m t h e two c u l t u r e s . ^ V a l u e s i n b r a c k e t s a r e p e r c e n t r a d i o a c t i v i t y r e l a t i v e t o t h e u n t r e a t e d samples. The r e s p e c t i v e CPM i n the l i p i d zones of the u n t r e a t e d samples of t h e c o n t r o l and minus l y s i n e , b e f o r e t h e n o r m a l i z a t i o n t o a c o n s i s t e n t sample mass, were 618 and 2,551. 83 T h e r e f o r e , much o f t h e a c e t i c a c i d - r e s i s t a n t m a t e r i a l i n t h e l i p i d zone o f l y s i n e - d e p r i v e d c e l l s seemed t o be s e n s i t i v e t o p r o t e a s e d i g e s t i o n and i s p r o b a b l y p r o t e i n . The [ ^ j D a p used i n a l l o f t h e s e e x p e r i m e n t s d i d not c o n t a i n l y s i n e . However, s m a l l amounts o f [ ^ J l y s i n e were d e t e c t e d i n samples o f LD5 ( r e l A + ) and LD5456 ( r e l A ~ ) h y d r o l y z e d w i t h 6N H C l ( T a b l e X V ) . The l y s i n e - d e p r i v e d c e l l s c o n t a i n e d 5- t o 10 t i m e s more [ 3 H j l y s i n e t h a n g r o w i n g c e l l s . Most o f the [ ^ l y s i n e i n t h e l y s i n e - d e p r i v e d c e l l s c o u l d be a c c o u n t e d f o r i f i t was i n t h e l a b e l e d p r o t e i n w h i c h c o - m i g r a t e d w i t h t h e G C L - l i n k e d p e p t i d o g l y c a n i n t e r m e d i a t e s ( T a b l e X V ) . I n s u f f i c i e n t [ d H J l y s i n e o c c u r r e d i n t h e samples o f whole c e l l s t o s i g n i f i c a n t l y a f f e c t t h e measurements o f p e p t i d o g l y c a n s y n t h e s i s o r a l t e r t h e c o n c l u s i o n t h a t p e p t i d o g l y c a n s y n t h e s i s i s s t r i n g e n t l y c o n t r o l l e d d u r i n g l y s i n e d e p r i v a t i o n (compare t h e e f f e c t t h a t s u b t r a c t i n g t h e t o t a l amount o f l y s i n e has on t h e f i n a l amount o f p e p t i d o g l y c a n i n column t h r e e o f T a b l e X V ) . No [ ^ j l y s i n e was r e l e a s e d by b o i l i n g t h e c e l l s o r h y d r o l y z i n g samples o f t h e s o l u b l e n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s from e i t h e r LD5 ( r e l A + ) o r LD5456 ( r e l A ~ ) . C. S t r i n g e n t c o n t r o l o f s o l u b l e n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s . The p r e c e d i n g , r e s u l t s i n d i c a t e t h a t s t r i n g e n t c e l l s i n h i b i t t h e t r a n s f e r o f M u r N A c ( - G l c N A c ) - p e n t a p e p t i d e from GCL t o t h e n a s c e n t a c c e p t o r ( r e a c t i o n 11, f i g u r e 2) d u r i n g amino a c i d d e p r i v a t i o n . However, o t h e r s i t e s i n p e p t i d o g l y c a n s y n t h e s i s a r e a l s o a f f e c t e d by s t r i n g e n t r e g u l a t i o n . The l e v e l o f s o l u b l e p e p t i d o g l y c a n p r e c u r s o r s i n g r o w i n g o r amino a c i d - d e p r i v e d s t r i n g e n t c e l l s remained r e l a t i v e l y c o n s t a n t between 30 and 60 m i n u t e s ( T a b l e X V I ) . However, 60 m i n u t e s o f CAM t r e a t m e n t , 84 TABLE XV. Amounts of I R j l y s i n e and r a d i o a c t i v e l i p i d zone p r o t e i n i n whole c e l l s l a b e l e d w i t h T 3HJDap. a 10k CPM p e r mg C e l l Dry Wt. C u l t u r e I 3 H ] l y s i n e b L i p i d Zone P r o t e i n P e p t i d o -g l y c a n 0 LD5 ( r e l A + ) C o n t r o l 0.035 0.041 21.62 Minus L y s i n e 0.520 0.485 8.28 LD5456 ( r e l A ~ ) C o n t r o l 0.050 0.013 21.92 Minus L y s i n e 0.370 0.245 15.40 C e l l s were p r e p a r e d and grown as d e s c r i b e d i n f i g u r e 3. A f t e r 60 m i n u t e s of i n c u b a t i o n , c o n c e n t r a t e s of whole c e l l s were h y d r o l y z e d i n 6N H C l a t 105°C f o r 20 h o u r s , t o f r e e i n c o r p o r a t e d l y s i n e . The h y d r o l y s a t e s were chromatographed i n s o l v e n t system I I I and t h e r a d i o -a c t i v i t y i n the l y s i n e zone was measured. The r a d i o a c t i v i t y i n t h e l y s i n e zone c o - m i g r a t e d w i t h l y s i n e i n s o l v e n t systems I , I V , and V. L i p i d zone p r o t e i n and p e p t i d o g l y c a n were measured i n a n o t h e r sample, as d e s c r i b e d i n T a b l e s X I I I and XIV. The l i p i d zone p r o t e i n was d e f i n e d as t h e m a t e r i a l c o - m i g r a t i n g w i t h G C L - l i n k e d i n t e r m e d i a t e s w h i c h was s e n s i t i v e t o p r o t e a s e and r e s i s t a n t t o m i l d a c e t i c a c i d h y d r o l y s i s . A l l c o u n t s were n o r m a l i z e d t o the c e l l mass t o compensate f o r d i f f e r e n c e s i n t h e amounts of t h e samples and f a c i l i t a t e c o m p a r i s o n . The r e s p e c t i v e CPM f o r t h i s column b e f o r e t h e n o r m a l i z a t i o n t o a c o n s i s t e n t sample mass, were: 306; 2,368; 344; and 1,671. c The r e s p e c t i v e CPM f o r t h i s column b e f o r e the n o r m a l i z a t i o n t o a c o n s i s t e n t sample mass, were: 96,048; 18,917; 74,850; and 34,850. TABLE XVI. A n a l y s i s of s o l u b l e n u c l e o t i d e - l i n k e d . p e p t i d o g l y c a n p r e c u r s o r s s y n t h e s i z e d by LP5 ( r e l A ) d u r i n g l y s i n e d e p r i v a t i o n and CAM t r e a t m e n t . 3 1 0 4 CPM p e r mg C e l l Dry Wt. C u l t u r e C o n d i t i o n s * ° t a l L a b e l e d UDP-MurNAc- UDP-MurNAc-P r e c u r s o r s p e n t a p e p t i d e t r i p e p t i d e 30 m i n u t e s C o n t r o l 0.86 0.83 (97%) 0.03 (3%) Minus L y s i n e 0.66 0.62 (95%) 0.04 (5%) Minus L y s i n e ; P l u s CAM 2.75 2.64 (96%) 0.11 (4%) P l u s CAM 2.83 , 2.67 (94%) 0.16 (6%) 60 m i n u t e s C o n t r o l 0.87 0.85 (98%) 0.02 (2%) Minus L y s i n e 0.83 0.78 (94%) 0.05 (6%)~ M inus L y s i n e ; P l u s CAM 3.97 3.84 (97%) 0.13 (3%) P l u s CAM 4.88 4.74 (97%) 0.14 (3%) "The n u c l e o t i d e - l i n k e d p r e c u r s o r s were measured as d e s c r i b e d f o r T a b l e V, e x c e p t t h a t the chromatographs were d e v e l o p e d l o n g e r i n o r d e r t o improve r e s o l u t i o n . ^ V a l u e s i n b r a c k e t s a r e p e r c e n t of t h e t o t a l r a d i o a c t i v i t y i n t h e p r e c u r s o r p o o l s . The r e s p e c t i v e CPM f o r t h i s column, b e f o r e t h e c o r r e c t i o n f o r d i f f e r e n c e s i n mass of t h e ^ s a m p l e s , were: 1,755; 1,615; 7,159; 7,295; 2,440; 2,339; 11,597; and 12,463. 86 e i t h e r a l o n e o r i n c o n j u n c t i o n w i t h amino a c i d d e p r i v a t i o n , caused a 4- t o 5 - f o l d a c c u m u l a t i o n o f p r e c u r s o r s . S i m i l a r l y , t h e g r o w i n g r e l a x e d c e l l s had a c o n s t a n t l e v e l o f s o l u b l e n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s ( T a b l e X V I I ) . However, amino a c i d d e p r i v a t i o n , CAM t r e a t m e n t , o r a c o m b i n a t i o n o f amino a c i d d e p r i v a t i o n and CAM t r e a t m e n t , a l l a l l o w e d a r a p i d a c c u m u l a t i o n o f n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s . S i m i l a r r e s u l t s were o b t a i n e d when c e l l s were p r e - l a b e l e d w i t h i 3 H ] D a p f o r 30 m i n u t e s and t h e n examined a f t e r g r o w i n g i n media l a c k i n g any Dap ( d a t a n o t p r e s e n t e d ) . I n a l l c a s e s , t h e r e l a t i v e c o m p o s i t i o n o f t h e p o o l s o f p e p t i d o g l y c a n p r e c u r s o r s was n o t a f f e c t e d . The s i z e o f t h e p o o l s o f n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s i s t h o u g h t t o be governed by f e e d b a c k i n h i b i t i o n by the UDP-MurNAc-pentapeptide (68, 70, 1 1 0 ) . T h e r e f o r e , the e f f e c t of t h e absence o f UDP-MurNAc-pentapeptide on t h e s t r i n g e n t r e g u l a t i o n o f t h e p o o l s i z e was examined. F i f t e e n m i n u t e s a f t e r LD5 ( r e l A + ) c e l l s were t r e a t e d w i t h D - c y c l o s e r i n e (DCS) t o i n h i b i t t h e f o r m a t i o n o f UDP-MurNAc-p e n t a p e p t i d e , t h e p o o l o f n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s had i n c r e a s e d t o 175% o f t h e l e v e l i n u n s t a r v e d c e l l s ( T a b l e X V I I I ) . I n c o n t r a s t , t h e l y s i n e - d e p r i v e d c e l l s w i t h o u t DCS t r e a t m e n t o n l y i n c r e a s e d t h e p o o l t o 111%, The a d d i t i o n o f DCS t o CAM-treated LP5 ( r e l A + ) i n c r e a s e d t h e s i z e o f t h e p o o l t o more t h a n d o u b l e t h e s i z e o f t h e p o o l measured i n CAM-treated c e l l s . I n a c c o r d a n c e w i t h t h e p r e c e d i n g r e s u l t s , t h e p o o l o f p e p t i d o g l y c a n p r e c u r s o r s i n l y s i n e - d e p r i v e d LP5456 ( r e l A ) had i n c r e a s e d t o o v e r 2 . 5 - f o l d t h e l e v e l found i n u n t r e a t e d c o n t r o l c e l l s b y 15 m i n u t e s ( T a b l e X I X ) . The a d d i t i o n o f DCS t o t h e l y s i n e - d e p r i v e d LD5456 ( r e l A ) r e l a x e d t h e r e g u l a t i o n o f t h e p o o l s i z e even more; by 15 m i n u t e s t h e p o o l was n e a r l y 7 . 5 - f o l d t h e c o n t r o l l e v e l . 87 TABLE X V I I . A n a l y s i s of s o l u b l e n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s s y n t h e s i z e d by LD5456 ( r e l A ~ ) d u r i n g l y s i n e d e p r i v a t i o n and CAM t r e a t m e n t . 3 " 1 0 4 CPM pe r mg C e l l Dry Wt . b C u l t u r e C o n d i t i o n s T o t a l L a b e l e d P r e c u r s o r s UDP-MurNAc-. . c p e n t a p e p t i d e UDP-MurNAc-t r i p e p t i d e 30 m i n u t e s C o n t r o l 0.43 0.41 (95%) 0.02 (5%) Minus L y s i n e 1.50 1.33 (89%) 0.17 (11%) Minus L y s i n e ; P l u s CAM 2.32 2.24 (90%) 0.18 (10%) P l u s CAM 2.43 2.24 (87%) 0.19 (13%) 60 m i n u t e s C o n t r o l 0.45 0.40 (89%) 0.05 (11%) Minus L y s i n e 3.55 3.39 (94%) 0.16 (6%) Minus L y s i n e ; P l u s CAM 3.75 3.65 (97%) 0.10 (3%) P l u s CAM 3.70 3.21 (87%) 0.4 9 (13%) " F o r the d e t a i l s o f t h e methods, see T a b l e X V I . ^ V a l u e s i n b r a c k e t s a r e p e r c e n t o f t h e t o t a l r a d i o a c t i v i t y i n t h e p r e c u r s o r p o o l s . The r e s p e c t i v e CPM f o r t h i s column, b e f o r e t h e c o r r e c t i o n f o r d i f f e r e n c e s i n mass of the samples, were: 1,623; 6,087; 6,088; 7,293; 2,601; 9,033; 9,820; and 9,873. 88 TABLE X V I I I . E f f e c t . o f D - c y c l o s e r i n e on t h e s i z e o f the p o o l of s o l u b l e n u c l e o t i d e - l i n k e d p e p t i d o -g l y c a n p r e c u r s o r i n LD5 ( r e l A ) a . C u l t u r e C o n d i t i o n s I O 4 CPM per mg C e l l Dry Wt. T o t a l S o l u b l e P r e c u r s o r s UDP-MurNAc-p e n t a p e p t i d e UDP-MurNAc-t r i p e p t i d e 15 m i n u t e s C o n t r o l 0.95 (100%) Minus L y s i n e 1.05 (111%) Minus L y s i n e ; p l u s D - c y c l o s e r i n e 1.66 (175%) P l u s CAM 2.75 (289%) Minus L y s i n e ; P l u s CAM 2.62 (276%) P l u s CAM; p l u s D - c y c l o s e r i n e 6.63 (698%) Minus L y s i n e ; p l u s CAM; p l u s D - c y c l o s e r i n e 5.14 (541%) P l u s D - c y c l o s e r i n e 6.21 (654%) 30 m i n u t e s C o n t r o l 0.86 (91%) 0.91 0.98 0.03 2.72 2.57 0.02 0.18 0.08 0.78 0.04 0.06 1.63 0.03 0.05 6.61 4.96 6.13 0.08 "The d e t a i l s o f t h e methods a r e t h e same as i n T a b l e X V I , e x c e p t t h a t D - c y c l o s e r i n e was added t o some c u l t u r e s b e f o r e the [ 3H]Dap was added. The D - c y c l o s e r i n e t r e a t e d c e l - l s b e g i n t o l y s e by 30 m i n u t e s so t h e samples were a n a l y s e d a t 15 m i n u t e s i n o r d e r t o a v o i d c o m p l i c a t i o n s . ^ V a l u e s i n b r a c k e t s a r e p e r c e n t of t h e c o n t r o l v a l u e a t 15 m i n u t e s . The r e s p e c t i v e i CPM f o r t h i s column, b e f o r e t h e c o r r e c t i o n f o r d i f f e r e n c e s i n mass of t h e samples, were: 1,771; 1,840; 3,125; 5,735; 6,328;. 10,760; 12,300; 12,128; and 1,668. TABLE X I X . E f f e c t , o f D - c y c l o s e r i n e on t h e s i z e o f t h e p o o l o f s o l u b l e n u c l e o t i d e - l i n k e d g e p t i d o -g l y c a n p r e c u r s o r i n LD5456 ( r e l A ) . C u l t u r e C o n d i t i o n s 101* CPM p e r mg C e l l Dry Wt. T o t a l S o l u b l e P r e c u r s o r UDP-MurNAc-p e n t a p e p t i d e UDP-MurNAc-. t r . i p e p t i d e 15 m i n u t e s C o n t r o l 0.78 Minus L y s i n e 2.07 Minus L y s i n e ; P l u s D - c y c l o s e r i n e 5.02 P l u s D - c y c l o s e r i n e 3.86 30 m i n u t e s C o n t r o l (100%) (265%) (644%) (495%) 0.86 (110%) 0.71 1.90 0.08 0.07 0.78 0.07 0.18 4.94 3.79 0.08 F o r t h e d e t a i l s o f t h e methods r e f e r t o T a b l e s XVI and X V I I I . ^ V a l u e s i n b r a c k e t s a r e p e r c e n t of t h e c o n t r o l v a l u e a t 15 m i n u t e s . The r e s p e c t i v e CPM f o r t h i s column, b e f o r e t h e c o r r e c t i o n f o r d i f f e r e n c e s i n mass o f t h e sa m p l e s , were: 3,558; 9,783; .19,193; 16,482; and 4,142. 90 The above d a t a show t h a t t h e p o o l o f n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s i n c r e a s e d i f r e l a x e d c e l l s were d e p r i v e d o f r e q u i r e d amino a c i d s . The t o t a l p o o l a l s o expands i n t h e amino a c i d -d e p r i v e d s t r i n g e n t c e l l s when t h e l e v e l o f UDP-MurNAc-pentapeptide i n t h e p o o l was d e c r e a s e d . The r e l a t i v e a c c u m u l a t i o n o f n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s was even g r e a t e r when t h e l e v e l o f UDP-MurNAc-p e n t a p e p t i d e was d e c r e a s e d i n r e l a x e d c e l l s . T h i s i n d i c a t e s t h a t t h e s i z e o f t h e p o o l i s r e g u l a t e d by b o t h f e e d b a c k i n h i b i t i o n and by s t r i n g e n t c o n t r o l . 91 DISCUSSION The p r e c e d i n g r e s u l t s i n d i c a t e t h a t p e p t i d o g l y c a n s y n t h e s i s i s s t r i n g e n t l y c o n t r o l l e d i n E_^ _ c o l i . The r a t e o f p e p t i d o g l y c a n s y n t h e s i s i s s i g n i f i c a n t l y r e d u c e d when s t r i n g e n t s t r a i n s a r e d e p r i v e d o f r e q u i r e d amino a c i d s . However, t h e r a t e r e m a i n s s i m i l a r t o t h e r a t e i n t h e u n t r e a t e d c o n t r o l when t h e amino a c i d - d e p r i v e d c e l l s a r e pheno-t y p i c a l l y r e l a x e d by e i t h e r CAM t r e a t m e n t o r by m u t a t i o n s i n t h e r e l A gene. A t t e m p t s were made t o i d e n t i f y t h e s i t e o f s t r i n g e n t i n h i b i t i o n o f p e p t i d o g l y c a n s y n t h e s i s u s i n g i n v i t r o s y s t e m s . E x p e r i m e n t s w i t h c r u d e p a r t i c u l a t e enzyme p r e p a r a t i o n s i n d i c a t e t h a t h i g h l e v e l s o f ppGpp can i n h i b i t p h o s p h o - N - a c e t y l m u r a m o y l - p e n t a p e p t i d e t r a n s l o c a s e a c t i v i t y ( r e a c t i o n 9, f i g u r e 2 ) . A l a t e r s t e p , t h e t r a n s f e r o f t h e G C L - l i n k e d p e p t i d o g l y c a n i n t e r m e d i a t e s t o t h e n a s c e n t a c c e p t o r ( r e a c t i o n 1 1 , f i g u r e 2 ) , i s a l s o i n h i b i t e d . T h i s l a t e r s t e p seems more s e n s i t i v e t o ppGpp t h a n t h e p h o s p h o - N - a c e t y l m u r a m o y l - p e n t a p e p t i d e t r a n s l o c a s e s i n c e G C L - l i n k e d i n t e r m e d i a t e s c a n a c c u m u l a t e t o h i g h l e v e l s under c o n d i t i o n s where p e p t i d o g l y c a n s y n t h e s i s i s i n h i b i t e d by 50%. The a c c u m u l a t e d G C L - l i n k e d i n t e r m e d i a t e s a r e p r o b a b l y n o t GCL-P-P-MurNAc-pentapeptide. The GCL-P-P-MurNAc-pentapeptide i s u n s t a b l e i n t h e i n v i t r o a s s a y s u s i n g membrane p r e p a r a t i o n s (52) and does n o t a c c u m u l a t e when membrane p r e p a r a t i o n s a c t i v e f o r r e a c t i o n s 9, 10 and 11 ( f i g u r e 2) a r e i n c u b a t e d .without UDP-GlcNAc i n t h e r e a c t i o n ( 7 1 ; p e r s o n a l o b s e r v a t i o n ) . However, t h e p h y s i o l o g i c a l s i g n i f i c a n c e o f i n h i b i t i o n by ppGpp i n t h i s s ystem i s n o t c l e a r . S i n c e ppGpp can c h e l a t e and b o t h o f t h e i n h i b i t e d r e a c t i o n s r e q u i r e magnesium,- t h e ppGpp c o u l d be i n h i b i t i n g t h e enzyme r e a c t i o n s by c h e l a t i o n o f magnesium. 92 P h o s p h o l i p i d s y n t h e s i s i s i n h i b i t e d i n v i t r o by t h e same l e v e l s o f ppGpp w h i c h i n h i b i t p e p t i d o g l y c a n s y n t h e s i s ( 6 5 , 7 6 ) . L i k e t h e i n v i t r o p e p t i d o g l y c a n s y n t h e s i s , t h e r e l a t i v e i n h i b i t i o n by ppGpp g e n e r a l l y i n c r e a s e s as t h e m o l a r r a t i o o f M g C ^ t o ppGpp d e c r e a s e s . However, a t m o l a r r a t i o s o f M g C ^ t o ppGpp below 1.0 t h e r e l a t i v e i n h i b i t i o n b e g i n s t o d e c r e a s e . T h i s d e c r e a s e i n i n h i b i t i o n has been used t o argue a g a i n s t t h e p o s s i b i l i t y t h a t ppGpp i n h i b i t s i n v i t r o p h o s p h o l i p i d s y n t h e s i s by c h e l a t i o n s i n c e t h e s m a l l e r amount o f •..'-; , magnesium a v a i l a b l e s h o u l d be c h e l a t e d more r e a d i l y ( 6 5 ) . A s i m i l a r e f f e c t c a n be o b s e r v e d when t h e r e l a t i v e r a t e o f t h e exchange r e a c t i o n f o r p h o s p h o - N - a c e t y l m u r a m o y l - p e n t a p e p t i d e t r a n s l o c a s e i s p l o t t e d a g a i n s t t h e m o l a r r a t i o o f magnesium t o ppGpp i n t h e r e a c t i o n . However, t h e a p p a r e n t d e c r e a s e i n r e l a t i v e i n h i b i t i o n i s an a r t e f a c t w h i c h r e s u l t s when t h e c o n s t a n t ( o r s l i g h t l y d e c r e a s i n g ) r a t e o f a c t i v i t y i n t h e p p G p p - t r e a t e d r e a c t i o n s i s d i v i d e d by the h i g h e r , b u t s t i l l d i m i n i s h i n g r a t e o f a c t i v i t y i n t h e m a g n e s i u m - l i m i t e d c o n t r o l r e a c t i o n s . A l t e r n a t i v e l y , d i f f e r e n t i a l i n h i b i t i o n o f d i f f e r e n t r e a c t i o n s by ppGpp and l a c k o f i n h i b i t i o n by ATP, a n o t h e r n u c l e o t i d e w h i c h c h e l a t e s , have been used t o argue a g a i n s t t h e p o s s i b i l i t y t h a t ppGpp i n h i b i t s i n v i t r o p h o s p h o l i p i d s y n t h e s i s by c h e l a t i o n ( 6 5 ) . S i m i l a r o b s e r v a t i o n s can a g a i n be made w i t h t h e i n v i t r o r e a c t i o n s f o r p e p t i d o g l y c a n s y n t h e s i s . Low l e v e l s o f ATP d i d n o t i n h i b i t t h e c o u p l e d r e a c t i o n f o r p e p t i d o g l y c a n s y n t h e s i s . I n a d d i t i o n , n e i t h e r t h e D - a l a n y l a l a n i n e l i g a s e n o r t h e D - a l a n y l a l a n i n e s y n t h e t a s e a r e i n h i b i t e d by a m o l a r r a t i o o f manganese t o ppGpp a t w h i c h t h e exchange r e a c t i o n f o r p h o s p h o - N - a c e t y l m u r a m o y l - p e n t a p e p t i d e t r a n s l o c a s e i s i n h i b i t e d by 93%. However, t h e r e l a t i v e a f f i n i t i e s o f t h e enzymes, t h e s u b s t r a t e s , and t h e n u c l e o t i d e s f o r t h e magnesium o r manganese i s unknown 93 and can not be readily determined. Inhibition could only be expected when the affinity of the enzyme-substrate complex for the divalent ions is less than the affinity of the nucleotides for the divalent ions. In addition, low concentrations of ATP might stimulate peptidoglycan synthesis (2, 71) sufficiently to mask concurrent inhibition. Both EDTA and higher concen-trations of ATP are inhibitory, probably because of chelation. Therefore, none of the preceding approaches clarify the means by which ppGpp inhibits in vitro peptidoglycan synthesis. However, some of the reactions for in  vitro peptidoglycan synthesis can be inhibited by the levels of ppGpp present in amino acid-deprived cells (4mM) (47, 76) at the average level of magnesium within whole cells (4 mM) (56, 73). The Inhibitions are also specific for ppGpp, and there is no significant inhibition by relatively high concentrations (19, 24) of many other nucleotides, including guanosine 5'-tetraphosphate. In addition, both the in vitro and the in vivo data suggest that the transfer of peptidoglycan precursors to the nascent peptidoglycan could be inhibited during the stringent response. Therefore, at least some of the inhibitory effects of ppGpp on the in vitro reactions may be physiologically significant. The in vivo data indicate that stringent amino acid-deprived cells accumulate proportionally less peptidoglycan than equivalent amounts of growing cells. This effect is probably not due to anomalies in the uptake of the exogenous labeled Dap since a similar effect is observed when the incorporation of Dap from endogenous pools is monitored. There is insufficient autolysis to account for the decreased amount of peptidoglycan accumulated by amino acid-deprived cells, and there is no turnover of E. coli peptidoglycan (17, 18, 88, 90). In addition, the amino acid-94 d e p r i v e d c e l l s do n o t seem t o be d e f i c i e n t i n e i t h e r s o l u b l e or G C L - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s . T h i s s u g g e s t s t h a t t h e s y n t h e s i s o f p e p t i d o -g l y c a n i s i n h i b i t e d a t t h e s t e p i n w h i c h t h e GCL-P-P-MurNAc(-GlcNAc)-p e n t a p e p t i d e i s t r a n s f e r r e d t o t h e n a s c e n t a c c e p t o r ( r e a c t i o n 11, f i g u r e 2 ) . L y s i n e - d e p r i v a t i o n i n s t r i n g e n t c e l l s r e s u l t s i n a s l i g h t a c c u m u l a t i o n o f GCL-P-P-MurNAc(-GlcNAc)-pentapeptide, as w o u l d be e x p e c t e d i f t h e subsequent s t e p i n p e p t i d o g l y c a n s y n t h e s i s i s i n h i b i t e d . However, t h e s l i g h t i n c r e a s e i n the amount o f GCL-P-P-MurNAc(-GlcNAc)-p e n t a p e p t i d e was n o t p r o p o r t i o n a l t o t h e d e c r e a s e i n t h e amount o f p e p t i d o g l y c a n a c c u m u l a t e d and was n o t o b s e r v e d d u r i n g t h r e o n i n e d e p r i v a t i o n . The absence o f e x t e n s i v e a c c u m u l a t i o n o f G C L - l i n k e d p e p t i d o g l y c a n i n t e r m e d i a t e s i s p o s s i b l y due t o a l i m i t i n g amount of GCL-P a v a i l a b l e i n t h e membranes f o r p e p t i d o g l y c a n s y n t h e s i s (46, 8 6 ) . I t i s a l s o p o s s i b l e t h a t t h e p h o s p h o - N - a c e t y l m u r a m o y l - p e n t a p e p t i d e t r a n s l o c a s e ( r e a c t i o n 9, f i g u r e 2) i s s t r i n g e n t l y c o n t r o l l e d , as o b s e r v e d i n v i t r o . I t s h o u l d be n o t e d t h a t t h e p r o p o r t i o n o f GCL-P-P-M u r N A c - p e n t a p e p t i d e t o GCL-P-P-MurNAc(-GlcNAc)-pentapeptide i n b o t h t h e g r o w i n g and t h e t h r e o n i n e - d e p r i v e d c o l i i s s i m i l a r t o t h a t o b s e r v e d i n membrane p r e p a r a t i o n s 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 ( 1 ) . The c o m p o s i t i o n o f t h e p o o l o f n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s i s a p p a r e n t l y not a l t e r e d by amino a c i d d e p r i v a t i o n i n e i t h e r s t r i n g e n t , o r r e l a x e d s t r a i n s o f . b a c t e r i a . The r a t i o of t h e amount of UDP-MurNAc-pentapeptide t o t h e amount of U D P - M u r N A c - t r i p e p t i d e i s r e a s o n a b l y c o n s t a n t and i s s i m i l a r t o the p r o p o r t i o n s o b s e r v e d i n o t h e r E. c o l i ( 7 0 ) . The s i z e o f t h e p o o l o f n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s i s a l s o r e l a t i v e l y c o n s t a n t i n b o t h t h e g r o w i n g c e l l s and t h e 95 amino a c i d - d e p r i v e d s t r i n g e n t c e l l s . T h i s p o o l i s r e l a t i v e l y s m a l l ; Dap a u x o t r o p h s g r o w i n g i n g l u c o s e m i n i m a l medium i n c o r p o r a t e about t h r e e -f i f t h s t h e e q u i v a l e n t o f t h e number o f p r e c u r s o r s i n t h e p o o l each m i n u t e . On t h e o t h e r hand, t h e p o o l o f f r e e Dap w i t h i n t h e dap a u x o t r o p h s i s a p p a r e n t l y l a r g e s i n c e t h e s y n t h e s i s o f p e p t i d o g l y c a n c a n c o n t i n u e f o r up t o two g e n e r a t i o n s i n t h e absence o f exogenous Dap. T h e r e f o r e , t h e c e l l s may have a purpose f o r r e s t r i c t i n g t h e s i z e and c o m p o s i t i o n o f t h e p o o l of n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s . The s i z e o f t h i s p o o l i s g e n e r a l l y t h o u g h t t o be c o n t r o l l e d by f e e d b a c k i n h i b i t i o n ( 7 0 , 1 1 1 ) . However, t h i s p r o p o s a l i s l a r g e l y based on i n d i r e c t e v i d e n c e . L u g t e n b e r g (70) r e p o r t e d t h a t m utants a p p a r e n t l y b l o c k e d i n some of t h e membrane a s s o c i a t e d s t e p s o f p e p t i d o g l y c a n s y n t h e s i s do n o t a c c u m u l a t e UDP-MurNAc-p e n t a p e p t i d e . I n a d d i t i o n , i n v i t r o s t u d i e s i n d i c a t e t h a t UDP-MurNAc-p e n t a p e p t i d e c a n i n h i b i t t h e f i r s t r e a c t i o n i n E. c o l i ( r e a c t i o n 1, f i g u r e 2) (110). However, ^UDP-MurNAc-pentapeptide a l s o i n h i b i t e d t h e e q u i v a l e n t enzyme a c t i v i t y i n e x t r a c t s o f B a c i l l u s c e r e u s (111) even though f e e d b a c k i n h i b i t i o n o f t h i s r e a c t i o n does n o t o c c u r i n v i v o f o r t h i s b a c t e r i u m ( 1 1 1 ) . The d a t a i n T a b l e s X V I I I and XIX i n d i c a t e t h a t d e p l e t i o n o f t h e UDP-MurNAc-pentapeptide p o o l s by t r e a t m e n t w i t h D - c y c l o -s e r i n e r e s u l t e d i n c o n t i n u e d a c c u m u l a t i o n o f U D P - M u r N A c - t r i p e p t i d e . T h i s i s . d i r e c t e v i d e n c e , t h a t UDP-MurNAc-pentapeptide i s p r o b a b l y a p h y s i o l o g i c a l f e e d b a c k i n h i b i t o r , o f an e a r l y r e a c t i o n i n t h e s y n t h e s i s o f n u c l e o t i d e -l i n k e d p e p t i d o g l y c a n p r e c u r s o r s , p o s s i b l y a t t h e f i r s t r e a c t i o n ( r e a c t i o n 1, f i g u r e 2) ( 1 1 1 ) . T h i s s t u d y a l s o i n d i c a t e s t h a t t h e s i z e o f t h e p o o l . : of n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s i s s t r i n g e n t l y c o n t r o l l e d . UDP-MurNAc-pentapeptide c o n t i n u e s t o a c c u m u l a t e when amino a c i d - d e p r i v e d r e l A + c e l l s a r e . r e l a x e d by t r e a t m e n t w i t h CAM. The UDP-MurNAc-pentapeptide 96 a l s o a c c u m u l a t e s when r e l A c e l l s a r e d e p r i v e d of a r e q u i r e d amino a c i d . The f e e d b a c k i n h i b i t i o n and t h e s t r i n g e n t r e g u l a t i o n of t h e s y n t h e s i s o f n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s a r e d i s t i n c t c o n t r o l s . Each c o n t r o l seems t o have some e f f e c t w i t h o u t t h e o t h e r . I f e i t h e r c o n t r o l i s e l i m i n a t e d i n amino a c i d - d e p r i v e d c e l l s , t h e s i z e o f t h e p o o l s i n c r e a s e s but does n o t i n c r e a s e t o t h e e x t e n t o b s e r v e d when b o t h c o n t r o l s a r e e l i m i n a t e d . D u r i n g amino a c i d d e p r i v a t i o n , b o t h c o n t r o l s a r e r e q u i r e d t o m a i n t a i n the p o o l a t the s i z e c h a r a c t e r i s t i c o f t h e g r o w i n g c e l l s . The s t r i n g e n t r e g u l a t i o n o v e r t h e amount of UDP-MurNAc-pentapeptide may be an a u x i l i a r y c o n t r o l w h i c h i s e f f e c t e d when c e l l s a r e s u b j e c t e d t o a s e v e r e s t r e s s s u c h as amino a c i d d e p r i v a t i o n . Feedback i n h i b i t i o n a p p e ars t o be t h e major c o n t r o l i n g r o w i n g c e l l s . I t i s p o s s i b l e t o s p e c u l a t e t h a t b o t h t h e f e e d b a c k i n h i b i t i o n and t h e s t r i n g e n t c o n t r o l a c t a t r e a c t i o n 1 i n f i g u r e 2. However, t h e s i t e of t h e s t r i n g e n t c o n t r o l of t h e p o o l s i z e was n o t d e t e r m i n e d . The r e s u l t s o n l y i n d i c a t e t h a t t h e s i t e o c c u r s a t some p o i n t p r i o r t o t h e f o r m a t i o n of UDP-MurNAc-pentapeptide. The s t r i n g e n t c o n t r o l of t h e p o o l s i z e m i g h t even r e s u l t as a s e c o n d a r y consequence of t h e s t r i n g e n t r e s p o n s e i f t h e amino a c i d - d e p r i v e d c e l l s become l i m i t e d f o r o t h e r e s s e n t i a l m e t a b o l i t e s s u c h as p h o s p h o e n o l p y r u v a t e . A number o f f a c t o r s c o u l d c o n t r i b u t e t o t h e f o r m a t i o n o f s m a l l amounts of [ 3 H ] l y s i n e - l a b e l e d p r o t e i n when l y s i n e - d e p r i v e d c e l l s a r e i n c u b a t e d w i t h [ 3H]Dap. The l y s A m u t a t i o n i n the s t r a i n s w h i c h were used might not c a use c o m p l e t e . i n a c t i v a t i o n of t h e d i a m i n o p i m e l a t e d e c a r b o x y l a s e even though t h e b a c t e r i a a r e dependent on exogenous l y s i n e . The absence of l y s i n e would enhance d e r e p r e s s i o n (16) o f t h e mutated l y s A gene and w o u l d a l s o e l i m i n a t e f e e d b a c k i n h i b i t i o n f o r any r e s i d u a l d i a m i n o p i m e l a t e d e c a r b o x y l a s e ( 1 1 4 ) . I n a d d i t i o n , any [ 3 H ] l y s i n e w h i c h was s y n t h e s i z e d 97 would n o t be d i l u t e d by u n l a b e l e d l y s i n e and would be r e a d i l y u t i l i z e d . The l y s i n e - l a b e l e d p r o t e i n a c c o u n t s f o r some e x c e s s T C A - i n s o l u b l e m a t e r i a l formed d u r i n g t h e i n i t i a l p e r i o d o f l y s i n e d e p r i v a t i o n o f t h e r e l a x e d s t r a i n s . I t a l s o a c c o u n t s f o r some of t h e a c c u m u l a t e d m a t e r i a l w h i c h m i g r a t e s i n t h e l i p i d zone of t h e chromatographs. However, t h e amount o f [ 3 H ] l y s i n e was n o t s u f f i c i e n t t o a f f e c t t h e c o n c l u s i o n t h a t the amino a c i d - d e p r i v e d s t r i n g e n t c e l l s made l e s s p e p t i d o g l y c a n t h a n t h e g r o w i n g c e l l s d e s p i t e t h e p r e s e n c e of an e q u i v a l e n t amount o f p e p t i d o g l y c a n p r e c u r s o r s . S t r i n g e n t c o n t r o l o f p e p t i d o g l y c a n s y n t h e s i s a l l o w s amino a c i d -d e p r i v e d c e l l s t o c o o r d i n a t e t h e s y n t h e s i s o f t h e s a c c u l u s w i t h o t h e r m a c r o m o l e c u l a r syntheses.: T h i s i s c o n s i s t e n t w i t h t h e p r o p o s a l t h a t t h e s t r i n g e n t c o n t r o l s y s t e m c o o r d i n a t e s c e l l u l a r r e s p o n s e t o amino a c i d d e f i c i e n c y (14, 88, 1 0 2 ) . However, t h e p r o p o s e d r e g u l a t i o n can not be g e n e r a l i z e d t o t h e b i o s y n t h e s i s of t h e e n t i r e c e l l w a l l s i n c e t h e s y n t h e s i s o f t h e 0 - a n t i g e n of l i p o p o l y s a c c h a r i d e i s not s t r i n g e n t l y r e g u l a t e d (E. I s h i g u r o , p e r s o n a l c o m m u n i c a t i o n ) . The s t r i n g e n t c o n t r o l of p e p t i d o -g l y c a n s y n t h e s i s s h o u l d be e f f e c t e d when ppGpp a c c u m u l a t e s d u r i n g c a r b o n s o u r c e s t a r v a t i o n (14, 61) o r s h i f t - d o w n (14, 6 1 ) . I t m ight a l s o respond to t h e growth r a t e - d e p e n d e n t changes i n t h e b a s a l l e v e l of ppGpp i n g r o w i n g c e l l s ( 61, 9 9 ) . N e i t h e r o f t h e s e p o s s i b i l i t i e s was i n v e s t i g a t e d . The l a t t e r p o s s i b i l i t y would r e q u i r e t h a t t h e p e p t i d o g l y c a n s y n t h e s i s r e s p o n d t o m i n o r changes i n i n t r a c e l l u l a r c o n c e n t r a t i o n s o f ppGpp. Some o f t h e known and presumed c o n t r o l s o f p e p t i d o g l y c a n s y n t h e s i s a r e summarized i n f i g u r e 19. The few c o n t r o l s shown can a c c o u n t f o r c o o r d i n a t i o n of t h e s y n t h e s i s of p e p t i d o g l y c a n p r e c u r s o r s and p e p t i d o g l y c a n . O ther c o n t r o l s p r o b a b l y e x i s t . I t i s i n t e r e s t i n g t h a t t h e s i t e s of FIGURE 19. S c h e m a t i c d i a g r a m of some of t h e r e g u l a t o r y s i t e s i n t h e b i o s y n t h e s i s o f E. c o l i p e p t i d o g l y c a n . The c i r c l e d numbers c o r r e s p o n d t o t h e d e s i g n a t i o n s f o r t h e same r e a c t i o n s i n f i g u r e 2. The t h i c k b l a c k arrows i n r e a c t i o n s 1 and 7 d e n o t e f e e d b a c k I n h i b i t i o n . The d o t t e d a r r o w i n r e a c t i o n . 6 r e p r e s e n t s a r e a d i l y r e v e r s i b l e r e a c t i o n . The l a r g e h a t c h e d arrows r e p r e s e n t s t r i n g e n t c o n t r o l . N ote t h a t t h e f o r m a t i o n of n u c l e o t i d e - l i n k e d p e p t i d o g l y c a n p r e c u r s o r s i s a f f e c t e d by s t r i n g e n t r e g u l a t i o n , b u t t h e s i t e o f a c t i o n f o r t h i s s t r i n g e n t e f f e c t i s s t i l l unknown. Cytoplasmic Reactions Membrane-Associated Reactions Cell Wall-Assoc iated React ions NADP NADPH UDP-MurNAc v y UDP-GlcNAc (£ ) enoylpyruvate L-Ala Stringent Control UDP-MurNAc-L-Ala Phosphoenolpyruvate UDP-GlcNAc U D PN/ rGCLP-P-MurNAc -pentapeptide. 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