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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 I N ESCHERICHIA C O L I  by W I L L I A M DAVID RAMEY  A T H E S I S SUBMITTED I N PARTIAL' FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY I N THE DEPARTMENT OF MICROBIOLOGY  We a c c e p t t h i s to  t h e s i s as  the required  conforming  standard  THE U N I V E R S I T Y OF B R I T I S H COLUMBIA  July, 1977  0  W i l l i a m D a v i d Ramey, 1977  OF  In presenting this thesis in partial fulfilment of an  advanced degree at the University of  British  the Library shall make it freely available for  the  Columbia,  for scholarly purposes may be granted by the Head It  is  understood that  I  agree  r e f e r e n c e and  I further agree that permission for extensive copying  by his representatives.  requirements  of  of  this  or  thesis  Department of  Microbiology  The University of B r i t i s h Columbia  2075 Wesbrook Place Vancouver, Canada V6T 1W5  Date  D e c e m b e r 6, 1977  or  publication  o f this thesis for financial gain shall not be allowed without my written permission.  that  study.  my D e p a r t m e n t  copying  for  i  ABSTRACT  D u r i n g amino a c i d d e p r i v a t i o n , acid  (Dap) i n c o r p o r a t e d  t h e amount o f m e s o - d i a m i n o p i m e l i c  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  of E s c h e r i c h i a  c o l i was f o u n d t o b e d e p e n d e n t o n t h e a c t i v i t y  gene.product.  In relA  ially  +  bacteria,  the- i n c o r p o r a t i o n  reduced, whereas t h e i n c o r p o r a t i o n  equal to that in relA  +  i n the unstarved control.  i n relA  b a c t e r i a was e s s e n t i a l l y incorporation  b a c t e r i a was r e a d i l y o v e r c o m e b y r e s t o r a t i o n o f t h e r e q u i r e d (CAM).  Guanosine 5'-diphosphate 3'-diphosphate  (ppGpp) i s t h e p r o d u c t  t h e r e a c t i o n b e t w e e n t h e r e l A gene p r o d u c t and i d l i n g  stringent  of the relA  substant-  The i n h i b i t i o n o f Dap  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  of  was  auxotrophs  cells.  J_n v i t r o  experiments i n d i c a t e d  that  ribosomes i n  physiological  levels  o f 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 . was 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 f e r a s e reaction  peptidoglycan.  Quantitation  peptidoglycan i n r e l A  indicated  that  +  acid-deprived  of  (GCL) t o t h e n a s c e n t  c o n t r o l and amino a c i d - d e p r i v e d  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 .  bacteria T h e r e was a s  i n t e r m e d i a t e s i n t h e amino  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 .  This suggests  that  t r a n s f e r of l i p i d - l i n k e d precursors to nascent acceptor i s the s i t e 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 .  nucleotide-linked  precursor pool i s also  product.  In addition,  the pool of  soluble  p r e c u r s o r s was f o u n d t o a c c u m u l a t e when r e l A  were d e p r i v e d o f r e q u i r e d the  lipid  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  much U D P - M u r N A c - p e n t a p e p t i d e a n d G C L - l i n k e d  the  (EC2.7.1.13)  a n d 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 -  glycan precursors from the g l y c o s y l c a r r i e r  net  One  amino a c i d s . regulated  This suggests that  by t h e a c t i v i t y  bacteria  the size of  of the r e l A  gene  \  ii  T A B L E OF CONTENTS Page INTRODUCTION I.  S t r u c t u r e a n d 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  II.  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  III.  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 peptidoglycan precursors  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.  Purpose of t h i s  13  thesis  MATERIALS AND METHODS I.  Bacterial strains  14  II.  Cultural  14  III.  Chromatography  IV.  Scintillation  V.  Chemicals  VI.  Preparation of substrates, standards A.  B.  conditions ^  14  counting  16 16 a n d ppGpp.  Soluble nucleotide-linked peptidoglycan  precursors.  a)  UDP-MurNAc-tripeptide  16  b)  UDP-MurNAc-pentapeptide  17  Other  standards  a)  MurNAc-pentapeptide  17  b)  MurNAc(-GlcNAc)-pentapeptide(tetrapeptide)  18  c)  GCL-P-P-MurNAc-pentapeptide and GCL-P-P-MurNAc(-GlcNAc)-pentapeptide  C. VII.  ppGpp p r e p a r a t i o n a n d p u r i f i c a t i o n  I n v i t r o assays A.  18  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.  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 a)  18  P a r t i c u l a t e enzyme p r e p a r a t i o n  synthesis 19  i i i  Page b)  B.  C.  VIII.  Coupled r e a c t i o n assay f o r formation 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  .....  Exchange assay f o r phospho-N-acetylmuramoyl pentapeptide transferase D-alanylalanine D-alanylalanine  synthetase ligase  20  20  and  a)  Enzyme p r e p a r a t i o n  b)  Assay f o r D-alanylalanine  synthetase  22  c)  Assay f o r D-alanylalanine  ligase  22  In v i v o p e p t i d o g l y c a n  21  synthesis  A.  A s s a y f o r TCA i n s o l u b l e [ ^ j D a p  B.  Assay f o r the d i s t r i b u t i o n of incorporated a)  b)  incorporation [ H]Dap 3  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 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  25  Analysis of l i p i d 1)  zone components  M i l d a c i d h y d r o l y s i s of l i p i d  zone  components 2) d) e)  24  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 cells  c)  22  Protease  26  treatment  of l i p i d  zone components  A n a l y s i s of [ H ] l y s i n e content i n [ H]Dap labeled cells Examination of supernatants from labeled cultures 3  27  3  28 28  RESULTS I.  Peptidoglycan-,synthesis s y n t h e s i s in v i v o A.  during ' i n h i b i t i o n of p r o t e i n  E f f e c t o f a m i n o a c i d d e p r i v a t i o n a n d CAM t r e a t m e n t on [ H ] D a p i n c o r p o r a t i o n b y r e l A b a c t e r i a 3  29  iv  Page B.  C.  E f f e c t o f amino a c i d d e p r i v a t i o n 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  of [ H]Dap 3  29  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 e n d o g e n o u s DAP  II.  III.  Regulation  38  of peptidoglycan  synthesis  In vitro  A.  Coupled 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 of nucleotides  D.  Other r e a c t i o n s  on t h e c o u p l e d r e a c t i o n  i n peptidoglycan  A n a l y s i s of s t r i n g e n t c o n t r o l during peptidoglycan synthesis A.  B.  C.  synthesis  59 59  i n vivo  D i s t r i b u t i o n of [^jDap incorporated acid-deprived bacteria  by amino 67  E f f e c t o f s t r i n g e n t i n h i b i t i o n on t h e 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  Stringent c o n t r o l of s o l u b l e peptidoglycan precursors  83  DISCUSSION LITERATURE CITED  nucleotide-linked  91 101  V  L I S T OF TABLES Page TABLE I .  S t r a i n s of B a c t e r i a Used  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 the a c t i v i t y of the coupled r e a c t i o n f o r peptidoglycan synthesis  TABLE I I I .  TABLE I V .  TABLE V.  TABLE V I .  TABLE V I I .  TABLE V I I I .  15 on 60  E f f e c t of c o n c e n t r a t i o n of 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 p l e d r e a c t i o n f o r peptidoglycan synthesis  61  E f f e c t o f c o n c e n t r a t i o n o f p p G p p , ATP a n d EDTA o n the a c t i v i t y of the coupled r e a c t i o n f o r peptidoglycan synthesis  62  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 during lysine deprivation 3  b y LD5  (relA ) +  70  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 peptidoglycan p r e c u r s o r s s y n t h e s i z e d b y LD5 ( r e l A ) d u r i n g l y s i n e deprivation  71  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 (relA ) during lysine deprivation  72  3  by LD5456  D i s t r i b u t i o n of labeled material i n culture 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 [ H]Dap  74  3  TABLE I X .  TABLE X.  TABLE X I .  D i s t r i b u t i o n of [ H ] D a p i n c o r p o r a t e d b y 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 o f a m i n o a c i d deprivation  75  D i s t r i b u t i o n o f [ H ] D a p i n c o r p o r a t e d b y LD5457 (relA ) during l y s i n e or l e u c i n e d e p r i v a t i o n  76  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 a n d CAM t r e a t m e n t o n d i s t r i b u t i o n o f [ H ] D a p i n c o r p o r a t e d b y LD5 ( r e l A )  77  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 a n d CAM t r e a t m e n t o n d i s t r i b u t i o n o f [ ll]Dap i n c o r p o r a t e d by LD5456 (relA )  78  A n a l y s i s o f t h e l a b e l e d components o f t h e l i p i d o f c e l l s i n c u b a t e d w i t h [ H ] D a p f o r 60 m i n u t e s  80  3  +  3  3  TABLE X I I .  3  TABLE X I I I .  zone  3  TABLE X I V .  E f f e c t o f p r o t e a s e a n d 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 z o n e o f LD5 ( r e l A ) ..  82  vi  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 w h o l e c e l l s l a b e l e d w i t h [ H]Dap  84  Analysis of soluble nucleotide-linke_d peptidoglycan p r e c u r s o r s s y n t h e s i z e d b y 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 a n d CAM t r e a t m e n t  85  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 a n d CAM t r e a t m e n t  87  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 soluble nucleotide-linked peptidoglycan precursor i n LD5 ( r e l A )  88  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 soluble nucleotide-linked peptidoglycan precursor i n LD5456 ( r e l A )  89  3  TABLE X V I .  TABLE X V I I .  TABLE X V I I I .  TABLE X I X .  vii  L I S T OF FIGURES Page FIGURE 1.  FIGURE 2.  FIGURE 3.  Schematic drawing of the c r o s s - l i n k e d s t r u c t u r e of a s m a l l p o r t i o n of t h e peptidoglycan i n E. c o l i  2  The m a j o r s t e p s i n t h e b i o s y n t h e s i s peptidoglycan  6  o f E.  coli  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 a n d CAM t r e a t m e n t o n [ H ] D a p i n c o r p o r a t i o n b y LD5 ( r e l A )  30  E f f e c t o f l a t e a d d i t i o n o f CAM o n [ H ] D a p i n c o r p o r a t i o n b y l y s i n e - d e p r i v e d LD5 ( r e l A )  32  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 [ H]Dap i n c o r p o r a t i o n (A) a n d c u l t u r e t u r b i d i t y (B) o f l y s i n e - d e p r i v e d LD5 ( r e l A )  34  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 H ] D a p 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 L D 5 2 , C ) s t r a i n LD2  36  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 a n d CAM t r e a t m e n t on [ H j D a p i n c o r p o r a t i o n by LD5456 ( r e l A ~ )  39  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 [ H]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 L D 5 4 5 7 , B) s t r a i n L D 5 4 5 8  41  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) a n d LD5456 ( r e l A ) (B) .;  43  Incorporation of { CjUDP-MurNAc-pentapeptide 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 a n d a b s e n c e o f 2 mM ppGpp  46  E f f e c t o f ppGpp c o n c e n t r a t i o n on t h 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 formed 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 peptidoglycan synthesis  48  E f f e c t o f M g C l o n t h e amount o f 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 ) accumulated by the coupled r e a c t i o n f o r i n v i t r o peptidoglycan synthesis  50  3  FIGURE 4.  FIGURE 5.  FIGURE 6.  FIGURE 7.  3  +  3  3  3  FIGURE 8.  FIGURE 9.  FIGURE 10.  FIGURE 1 1 .  FIGURE 1 2 .  3  1 4  2  viii  Page FIGURE 1 3 .  FIGURE 14.  FIGURE 1 5 .  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 ... o n 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 a n d G C L - l i n k e d i n t e r m e d i a t e s accumulated by the coupled reaction f o r i n v i t r o peptidoglycan synthesis  52  I n c o r p o r a t i o n o f [^ftJMP i n t o U D P - M u r N A c - p e n t a p e p t i d e by t h e e x c h a n g e 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 -pentapeptide transferase incubated i n the presence a n d a b s e n c e o f ppGpp  55  ( A ) E f f e c t o f M g C l o n t h e a c c u m u l a t i o n o f [ H]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 t h e p r e s e n c e a n d a b s e n c e o f ppGpp. (B) 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 M g C l t o ppGpp o n t h e e x c h a n g e r e a c t i o n  57  E f f e c t o f t h e p r e s e n c e o r a b s e n c e o f ppGpp o n 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) a n d D - a l a n y l a l a n i n e l i g a s e (B)  63  ( A ) E f f e c t o f M n C l o n t h e a c c u m u l a t i o n o f [ H]UMP i n UDP-MurNAc-pentapeptide by t h e 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 a n d a b s e n c e o f 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 o f M n C l t o ppGpp o n t h e e x c h a n g e r e a c t i o n  65  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 ( r e l A ) w e r e l a b e l e d w i t h [ H]Dap a n d t h e n chromatographed i n s o l v e n t system I  68  S c h e m a t i c d i a g r a m o f some o f 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 Ej_ c o l i p e p t i d o g l y c a n .......  98  3  2  2  FIGURE 16.  FIGURE 17.  3  2  2  FIGURE 18.  FIGURE 19.  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 by D r . E. I s h i g u r o d u r i n g t h e c o u r s e I would l i k e  to thank  provided  of the research. t h e members o f my  committee,  p a r t i c u l a r l y D r . E.E. I s h i g u r o , D r . P.P. D e n n i s a n d D r . G. Weeks f o r their  constructive assistance i n editing I would a l s o l i k e  the thesis.  t o t h a n k D r . 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 o f p u r i f i e d ppGpp a n d J . B a i n f o r d o i n g a l o t o f t y p i n g . I am g r a t e f u l f o r t h e s u p p o r t scholarship.  by a n H.R.  MacMillan  X  ABBREVIATIONS amino a c i d s  :  standard  a b b r e v i a t i o n s recommended b y t h e C o m m i s s i o n  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  :  chloramphenicol  CPM  :  counts  Dap  :  meso-diaminopimellc acid  DCS  :  D-cycloserine  GCL  :  glycosyl carrier  GCL-P  :  GCL-monophosphate  GCL-P-P  :  GCL-diphosphate  GlcNAc  :  Mur  :  Muramic  MurNAc  :  N-acetylmuramic  nucleotides  :  standard  P^  :  phosphate  pentapeptide  :  L-ala-D-glu-meso-Dap-D-ala-D-ala  ppGpp  :  guanosine 5'-diphosphate  TCA  :  trichloroacetic  tripeptide  :  tris  :  per minute  lipid;  C^^-isoprenoid  alcohol  N-acetylglucosamine acid acid  a b b r e v i a t i o n s recommended b y t h e CBN  3'-diphosphate  acid  L-ala-D-glu-meso-Dap tris-(hydroxymethyl)-amino  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 The  f u n c t i o n o f E.  coli  peptidoglycan  n o r m a l p e p t i d o g l y c a n o f E.  bag-like molecule  which encloses  coli  the c e l l .  s e c t i o n e d c e l l s . i t a p p e a r s as a dense l a y e r , t h e i n n e r and both  o u t e r membranes.  o f t h e s e membranes, b u t  with boiling i s an  sodium d o d e c y l  i n t a c t molecule,  contaminated removed by  was  c a n be sulfate.  is  (45, 9 2 ) .  94% to  between to  t h e membranes this  method  It i s generally  This p r o t e i n can  be  Streptomyces g r i s e u s protease. the shape of the c e l l some  The  from which  rigidity.  short peptides.  (GlcNAc).  and  This i n d i c a t e s t h a t the glycans  of the l a c t y l  no  The  i n length.  The  reducing sugars  glycan  (MurNAc)  T h e r e i s a n a v e r a g e o f 100  and  hexosamines  MurNAc a t t h e p o t e n t i a l  It  chains  s h o u l d be a b o u t 0.1  o f e a c h 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 49)  sandwiched  dissolving  of a l t e r n a t i n g N-acetylmuramic a c i d  long i f they are uniform  45,  of  g e n e r a l s t r u c t u r e o f 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.  N-acetylglucosamine  (41,  thick,  bound p r o t e i n .  t h e r e f o r e appears to possess  are heteropolymers  end  nm  to as a s a c c u l u s .  composed o f g l y c a n " c h a i n s b r i d g e d b y  chain  2-3  P e p t i d o g l y c a n o b t a i n e d by  often referred  treatment w i t h t r y p s i n or  The  In e l e c t r o n micrographs  i s o l a t e d by  w i t h r e g u l a r l y spaced,  d e r i v e d and  t o be a l a r g e  I t seems t o h a v e d i s c r e t e a t t a c h m e n t s  isolated protein-free sacculus retains it  i s thought  per um  reducing  anhydro-linkage  are d e t e c t a b l e (41, 45).  Approximately  m o i e t i e s a t c a r b o n - 3 o f t h e MurNAc a r e c o v a l e n t l y b o n d e d  t h e L - a l a n i n e i n one  o f two  short peptides  (109).  The  exact  proportion  o f 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 g r o w t h c o n d i t i o n s ( 4 5 , 94,  109);  generally,  the  20% a r e t h e t r i p e p t i d e ,  L - a l a - D - g l u - m e s o - D a p , and  remainder are the t e t r a p e p t i d e , L-ala-D-glu-meso-Dap-D-ala.  A b o u t 15  to  25% of the 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. c o l i .  Note that the  D-alanine content i s possibly more variable and that the actual relative positions of the cross-linking peptides and the Braun lipoprotein are unknown. After Braun et a l . (11).  3  Covalently linked lipoprotein replacing D-Ala  4  o f t h e meso-Dap i n t h e t e t r a p e p t i d e o f a n a d j a c e n t form the c r o s s - l i n k i n g p e p t i d e s .  glycan chain.  I n a d d i t i o n , 1 0 % o f t h e Dap  These  molecules  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 The precludes  o r i e n t a t i o n o f t h e g l y c a n c h a i n s i s unknown.  the p o s s i b i l i t y  In addition,  hexosamines per  This suggests  be  one  repeating unit  calculated  t o be  (11).  t h i c k i n order  e n c l o s e d by  p e p t i d e c h a i n s m u s t be  (8).  f o r the s u r f a c e  the i n t a c t peptidoglycan  o r i e n t e d outwards i n order  The  (11).  T h e r e may  differences  79,  (9, 10,  t h e s e p t a may The It  be  25,  34,  78,  92,  be 115);  area  t o accommodate  types  (25,  the p e p t i d o g l y c a n a r e not  to w i t h s t a n d may  a l s o be  s e r v e as a m e c h a n i c a l  the f o r c e s generated  by  involved i n controlling  34,  78,  completely  understood.  l y s e when t r e a t e d the  support which a l l o w s the b a c t e r i a  the i n t e r n a l osmotic t h e s i z e and  pressure.  c e l l p e r m e a b i l i t y (11), although  molecules.  coli  It  shape of the c e l l s . interfere  B i o s y n t h e s i s o f E.  at  79).  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  II.  of  e.g.,. t h e p e p t i d o g l y c a n  w i t h a n t i - m e t a b o l i t e s o r 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 I t may  the  structural  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  sacculus.  the  embedded i n t h e  localized  l e s s c r o s s - l i n k e d than elsewhere  f u n c t i o n s of  only  Some o f  r e l a t i v e p o s i t i o n s of the d i f f e r e n t  a t t a c h e d p e p t i d e s a r e unknown.  10  t h a t the s a c c u l u s can  attached Braun l i p o p r o t e i n s , which are a l s o p a r t i a l l y o u t e r membranes  the  there are only 5 x  to account  11).  length  t h a t they might r a d i a t e outwards from  l o n g i t u d i n a l a x i s of the c e l l . sacculus  The  (8,  i t might bind s p e c i f i c  with  peptidoglycan  Most of the concepts  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 from 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  The  5  Staphylococcus, s u b t i l u s and  Streptococcus,  Ej_ c o l i .  The  Micrococcus,  areas  b a c t e r i a l species to another  H o w e v e r , m i n o r d i f f e r e n c e s do  occur  view of the v a r i a t i o n i n p e p t i d o g l y c a n  c a n be d i v i d e d i n t o  reactions  three categories:  i n E.  coli  (66,  110,  111).  The  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 f i g u r e 2) a r e c a t a l y z e d b y  activity  of each of the adding  i s d e p e n d e n t o n ATP source  and  glutamic acid  i s known f o r E.  peptidoglycan  from L-alanine. Reaction 7  coli  or  The b i o s y n t h e t i c  1  2 h a v e b e e n demon-  reactions which synthesize  presursors  7,  f i g u r e 2)  ( r e a c t i o n s 1,  highly specific  2,  or D-alanine  (72).  The  The  I t i s destroyed  and 4,  3,  4,  5 and  8,  The  figure  by D - a l a n i n e  to  No  transaminase  fosfomycin  (110).  in  peptidoglycan  i s enzymatically dehydrogenase  racemized  (27,  58).  Reactions  6  and  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 -  chloro-D-alanine  (59,  69,  70,  74).  are.known to i n t e r f e r e w i t h the other r e a c t i o n s .  No  2)  The  ( f i g u r e 2) i s unknown. : 2-ket.oglutarate  5  meso-Dap i s a n i n t e r m e d i a t e  D-alanine  by  the  s o l u b l e enzymes.  ( r e a c t i o n s 3,  in reaction 4  ( f i g u r e 2) i s . i n h i b i t e d  and  2.  i t s incorporation i s confined  presursors.  ( f i g u r e 2) a r e I n h i b i t e d  D-serine,  in  cytoplasmic  ( r e a c t i o n s 6 and  enzymes  racemase a c t i v i t y  L - l y s i n e b i o s y n t h e s i s , but and  considered  r e q u i r e s e i t h e r magnesium o r manganese.  of the D - g l u t a m i c a c i d  activity  i ) 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  i n t e r m e d i a t e s i n the a l a n i n e pathway  8,  conditions.  These r e a c t i o n s a r e summarized i n f i g u r e The  and  m u s t be  are  i i ) the membrane-associated r e a c t i o n s i i i ) the w a l l - a s s o c i a t e d  reactions.  strated  and  that  s t r u c t u r e b e t w e e n some s p e c i e s  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 steps  Bacillus  where the r e s u l t s o v e r l a p suggest  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 valid.  Lactobacillus,  common a n t i b i o t i c s  6  FIGURE 2.  The major steps i n the biosynthesis of E. c o l i  peptidoglycan.  Each reaction i s denoted by a circled number.  Cytoplasmic  Membrane-Associated  Reactions UDP  NADP UDP-MurNAc  ©^  Phosphoenolpyruvate  NADPH  GCL-P-P-MurNAc -pentapeptide.  I  (g)  GlcNAc Cell Wall Acceptor  UDP-GlcNAc  UDP-GlcNAc- - * enoylpyruvate  ©  Reactions  Cell W a l l Associated React ions  L-Ala UMP  GCL-P-P-MurNAc-pentapeptide Cell Wall Acceptor ' Plus Precursor  UDP-MurNAc-L-Ala  ©U~  D  UDP-MurNAc-L-ala-D-Glu-mesp-Dap-D-Ala-D-Ala  Glu  (UDP-MurNAc-pentapeptide)  UDP-MurNAc-L-Ala-D-Glu  UDP-MurNAc-L-Ala-D-Glu-meso-Dap  ©  meso Dap L-Ala  ©  D-Ala  ©  ^D-Ala-D-Ala  GCL-P  GCL-P- ? '  8  The  m e m b r a n e - a s s o c i a t e d 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 , Both of the GCL-linked 9 and  10,  but  seem t o be  peptidoglycan  intermediates  until after  coli  (products  i s s t i m u l a t e d b y ATP  the i n i t i a l  (9, 51,  T h e s e membrane-  or bonded t o the Braun  (2; t h i s  thesis).  t o t h e GCL-P  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,  p h o r y l a t i o n o f GCL-P-P i n r e a c t i o n 12 (96,  103).  N i s i n and  T h i s may ( 4 6 , 85)  108)'  of r e a c t i o n s  lipoprotein  they have been i n c o r p o r a t e d i n t o the p e p t i d o g l y c a n  c o n v e r s i o n o f s m a l l a m o u n t s o f GCL  bacitracin  i n E.  f i g u r e 2) a r e s y n t h e s i z e d b y p a r t i c u l a t e e n z y m e s .  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  formation  similar  (9).  be due  Their  to  required for  figure 2).  The  ( f i g u r e 2) i s p r e v e n t e d  de-phosby  tunicamycin might i n t e r f e r e w i t h  the  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 (figure  2 ) ( 6 , 63,  w i t h r e a c t i o n 11 The The  112).  V a n c o m y c i n and  (figure 2)(37,  63,  many o t h e r a n t i b i o t i c s  fully  in  the sacculus.  i n t o the s a c c u l u s .  These p r e c u r s o r s  can  Still  are i n i t i a l l y t h e n be  then  other  t h e o r i e s suggest that  c r o s s - l i n k e d i n t o the  e x t e n d e d by  c u r r e n t or consecutive  c o m p o n e n t s and  enlargement.  This  trans-  only  peptidoglycan.  subsequent t r a n s g l y c o s y l a t i o n to  transpeptidation.  thought t o open the c l o s e d s t r u c t u r e of  chain  precursors  t r a n s g l y c o s y l a t e d or  form 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  o f new  Some t h e o r i e s  O t h e r t h e o r i e s s u g g e s t t h a t t h e membrane-bound  occasional precursors  but  i s t r a n s g l y c o s y l a t e d to a nascent glycan  are extended i n t o g l y c a n chains which are peptidated  understood.  i n c o r p o r a t e d i n t o the p e p t i d o g l y c a n ,  t h e r e i s n o t much a g r e e m e n t a b o u t t h e p r e c i s e m e c h a n i s m . suggest t h a t each p r e c u r s o r  interfere  71).  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 are not  p r e c u r s o r s a r e known t o be  10  t h e s a c c u l u s by  con-  I n most m o d e l s , a u t o l y s i n s a r e  the  sacculus  i s probable,  to permit but has  the not  insertion been  9  proven.  No p o l y m e r i z e d  GCL ( 9 ) .  g l y c a n c h a i n s have been d e t e c t e d  S i m i l a r l y , M i r e l m a n and Schwartz  transglycosylation of peptidoglycan s a c c u l u s , when t h e y cells.  They s u g g e s t e d  ( 7 8 ) f o u n d no e v i d e n c e f o r  precursors  assayed peptidoglycan  o n t h e Ej_ c o l i  to the glycans  synthesis i n ether-permeabilized  that the transpeptidation or c r o s s - l i n k i n g i s  t h e 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 t h e new p e p t i d o g l y c a n E.  coli  delayed  sacculus process  (78).  i n E. c o l i  t r a n s p e p t i d a t i o n and l a t e r  ( 1 0 , 93; p e r s o n a l o b s e r v a t i o n ) .  secured  This  would  i n c o r p o r a t e d by l i m i t 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 -  T h i s m i g h t a g r e e w i t h a*;model f o r t h e g r o w t h o f t h e E ^ c o l i  peptidoglycan which suggests  that the peptidoglycan precursors are i n i t i a l l y  i n c o r p o r a t e d a t t h e septum, b u t a r e s u b s e q u e n t l y to  into the  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  n e c e s s i t a t e t h a t some c o m p o n e n t s a r e i n i t i a l l y  peptidation.  i nthe  diverse sites  throughout the sacculus  (90).  r e l e a s e d and r e d i s t r i b u t e d H o w e v e r , t h i s m o d e l was  b a s e d 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 h a s b e e n q u e s t i o n e d A l a r g e sodium dodecyl reported in  sulfate-solublepeptidoglycan  ( 1 0 , 7 8 , 7 9 ) , b u t was n o t c h a r a c t e r i z e d .  the f i n a l  a s s e m b l y and m o d i f i c a t i o n  pancy between t h e D-alanine precursors activities.  i s attributed  contents  of the peptidoglycan.  D-alanines  without  carboxypeptidase  the terminal D-alanlnes  t h e f o r m a t i o n o f t h e bond between t h e p e n u l t i m a t e  the  The d i s c r e -  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  liberate  c h a i n a n d t h e meso-Dap o f a n o t h e r .  component has been  I t may b e a n i n t e r m e d i a t e  t o t h e t r a n s p e p t i d a s e and  The t r a n s p e p t i d a s e s  (8, 10).  D-alanine  The c a r b o x y p e p t i d a s e  consequent t r a n s p e p t i d a t i o n . .  during  i n one g l y c a n  activities  remove  10 III.  Regulation of formation of peptidoglycan and peptidoglycan precursors Although the sacculus is essential for the normal survival of an  E. c o l l , 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 eightynine minutes on the revised E. c o l i 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 c e l l 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 c e l l 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 f i r s t reaction in the biosynthesis of peptidoglycan  11  precursors this  ( r e a c t i o n 1,  thesis).  A cytoplasmic  UDP-MurNAc-pentapeptide quently could  the  be  addition,  f i g u r e 2) by  peptidase might a l s o hydrolyse  (33).  limited with there  are  controls which allow  synthesis  and  exocellular colanic acid  differentially  of p e p t i d o g l y c a n ,  synthesized  (53,  (88,  o r RNA  nalidixic  105).  105)  synthesis. acid  However, the  (43,  synthesis  I t apparently  shown i n t h i s  95)  or actinomycin. D  personal  Stringent  do  conse-  figure (46,  2),  85).  In  to s e l e c t i v e l y use  polysaccharides  d i f f e r e n t p o l y m e r s can not  the  compete f o r the  be  GCL-P  i s t h o u g h t t o be  ribosomes.  The  independent of  (101).  due  treated  with  I t a l s o continues at  near  i s i n h i b i t e d by  depriving  to the  CAM  synthesis some c e l l s  of  are  (38,  67,  70,  75).  i s d e c r e a s e d when of r e q u i r e d  thesis).  influence  of p e p t i d o g l y c a n  concurrent  the  The  amino  difference  stringent  control  synthesis.  r e g u l a t i o n i n Ej_ c o l i  accumulation of  the  cells  communication; t h i s  t h e s i s t o be  specific reactions  amounts d u r i n g  and  kinases  c o n t i n u e s when c e l l s  D u r i n g amino a c i d d e p r i v a t i o n ,  It  ( r e a c t i o n 9,  is relatively  i s i n h i b i t e d by  (E. I s h i g u r o ,  s y s t e m on  the  excess  some o u t e r membrane  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  protein synthesis  IV.  the  The  and  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  is  IH,  (89). Peptidoglycan  acids  (70,  amount o f a v a i l a b l e GCL-P, a n d  s p e c i f i c p h o s p h o r y l a s e s and  f o r the  DNA  The  amount o f p o t e n t i a l t r a n s l o c a t i o n  GCL  in vivo  UDP-MurNAc-pentapeptide  s t a b l e RNA. c a u s e d by  This  some b a c t e r i a s e v e r e l y  e f f e c t i s the  ppGpp, a n u c l e o t i d e  a s p e c i f i c i n t e r a c t i o n of  the  i n h i b i t i o n o f RNA  produced i n  response. large  r e l A g e n e p r o d u c t and  amount o f ppGpp a c c u m u l a t e d d u r i n g  degree of  stringent  reduce  amino a c i d  idling  deprivation,  a c c u m u l a t i o n , i s d e p e n d e n t on  the  particular amino acid and the bacterial strain (12, 26, 55, 91).  The  ppGpp i s 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). by the spoT mutation (14, 55).  The in vivo stability i s enhanced  Bacteria which are mutated i n the relA gene  are relaxed and do not accumulate ppGpp or stop accumulating stable RNA during amino acid deprivation. relaxed by CAM  Stringent bacteria are phenotypically  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-  l i p i d 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 i n cells growing in minimal media. rate (61, 99). (31).  The basal levels of ppGpp. vary inversely to the growth  They are reduced when growing cells are treated with CAM  They are greatly enhanced by carbon source shiftdown (14, 28, 61),  levallorphan treatment (14, 39) or NaCI shock (40), even i h relA  cells.  The variety of responses has led to the view that ppGpp i s 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.  Purpose of t h i s  thesis  In the r e l A during  +  b a c t e r i a the r a t e  amino a c i d d e p r i v a t i o n  i s severely  of peptidoglycan reduced.  i s n e a r 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 was  t h o u g h t t o b e due  was  investigated  techniques.  to the Influence  i n this  thesis, using  .synthesis  The r a t e o f CAM.  of t h e r e l A gene. both i h vivo  and  The  synthesis  difference  That  possibility  in vitro  14 MATERIALS AND METHODS  I.  Bacterial All  in  strains s t r a i n s used,  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  T a b l e 1.  II.  Cultural  conditions  S m a l l c u l t u r e s w e r e 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 Brunswick Gyrotory water bath shaker  ( M o d e l G 7 6 ) s e t t o 225 r p m .  b a t c h e s o f c e l l s w e r e i n c u b a t e d a t 37°C i n f o u r l i t e r o n 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  ( M o d e l G10) s e t a t 2 5 0 r p m .  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 . w i t h a Klett-Summerson to  Erlenmeyer  Large flasks  The s m a l l  Growth was m o n i t o r e d  C o l o r i m e t e r a n d , w h e r e a p p r o p r i a t e , was c o n v e r t e d  milligram d r y weight of c e l l s w i t h a standard c a l i b r a t i o n curve.  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 m e d i a and a r e d f i l t e r  c u l t u r e s i n r i c h media. transferring as above.  9 ml aliquots  bath shaker  III.  Chr omatography All  chromatography  Acid  at specific  t o sidearm f l a s k s and measuring  culture  times by density  s e t t o 105 rpm.  paper paper  chromatography 57 cm l o n g .  s o l v e n t system  immediately p r i o r  w a s d o n e w i t h s h e e t s o f Whatman 3 MM Chromatographs were developed w i t h a  i n a s e a l e d , g l a s s chromatography  papers were g e n e r a l l y developed mixed  for the  A l l i n o c u l a w e r e f r e s h o v e r n i g h t s t o c k s g r o w n a t 37°C w i t h t h e  water  descending  Large c u l t u r e s were monitored  Blue  a t t h e same t i m e .  t o use.  - 1 M NH.OH ( 5 : 3 , v / v ) ; I I .  chamber.  S o l v e n t systems  The s o l v e n t systems were:  I.  Several  were  Isobutyric  E t h a n o l - 1 M N H . A c e t a t e pH 7.2  15  TABLE I .  Strains  Bacillus subtilis Bacillus  WB746  cereus  S t r a i n s o f B a c t e r i a Used  Relevant C h a r a c t e r i s t i c s  Source  Wild  type  R.A.J.  Wild  type  H.  Warren  Wiens  Escherichia c o l i  VC10  Spontaneous s t r e p t o m y c i n r e s i s t a n t m u t a n t o f W3110  E.E.  Ishiguro  E. c o l i  LD5  F , t h i , l y s A , d a p D, r p s L. C o n j u g a l C r o s s o f AT980 x x  E.E.  Ishiguro  E. c o l i  LD2  l i k e LD5 e x c e p t met.  E.E.  Ishiguro  E. c o l i  LD51  leu  d e r i v a t i v e . o f • LD5  E.E.  Ishiguro  E. c o l i  LD52  thr  d e r i v a t i v e o f LD5  E.E.  Ishiguro  E. c o l i  LD5456  relA  E.E.  Ishiguro  E. c o l i  LD5457  leu  d e r i v a t i v e o f LD5456  E.E.  Ishiguro  E.  LD5458 t h r  d e r i v a t i v e o f LD5456  E.E.  Ishiguro  coli  d e r i v a t i v e o f LD5  16 (7:3,  v / v ) ; I I I . n-butanol-acetic acid-water (2:1:1, v / v ) ; IV.  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 acid - water  ethanol(50:35:15,  v/v) .  IV.  S c i n t i l l a t i o n counting S a m p l e s w e r e 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  t i l l a t i o n counter. (PPO)  fluid  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  a n d 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  uene. to  The s c i n t i l l a t i o n  The a p p r o p r i a t e r e g i o n s i n t h e p a p e r c h r o m a t o g r a p h s  1.5 cm l o n g  serial  s t r i p s , depending  scin-  of  tol-  were c u t i n t o  on t h e r e q u i r e d r e s o l u t i o n .  0.5  The s e p -  a r a t e s t r i p s w e r e t h e n c o u n t e d a n d t h e i n d i v i d u a l c o u n t s w e r e summed. A l l s a m p l e s w e r e a i r d r i e d , a n d t h e n h e a t e d a t 105°C f o r a t l e a s t fore counting.  two h o u r s b e -  I n a l l c a s e s , q u e n c h i n g was l o w a n d c o n s i s t e n t f r o m s a m p l e t o  sample.  V.  Chemicals D - a l a n y l - D - a l a n i n e was o b t a i n e d f r o m C y c l o C h e m i c a l Co.  c h e m i c a l s were o b t a i n e d from Amersham/Searle Corp.  Antibiotics,  RadioUDP-GlcNAc,  n u c l e o t i d e s a n d a m i n o a c i d s w e r e o b t a i n e d f r o m S i g m a C h e m i c a l Co.  A l l other  r e a g e n t s w e r e 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 b y S i g m a C h e m i c a l Co. a n d Fisher  VI.  Scientific  Co.  Preparation of substrates, A.  s t a n d a r d s , a n d ppGpp  Soluble nucleotide-linked peptidoglycan precursors, (a)  UDP^-MurNAc-tripeptide  T h i s was e x t r a c t e d f r o m B. c e r e u s . m i n i m a l s a l t s medium ( 7 7 ) s u p p l e m e n t e d  w i t h 0.5% g l u c o s e a n d 1 % p e p t o n e .  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 m a x i m a l a d d e d t o 50 Ug p e r m l f i n a l  The c e l l s w e r e g r o w n i n M9  concentration.  g r o w t h , D - c y c l o s e r i n e was The c u l t u r e was  treated  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 w e r e t h e n p r o c e s s e d up  l y o p h i l i z a t i o n step i n the UDP-MurNAc-tripeptide p u r i f i c a t i o n d e s c r i b e d by B a r n e t t ( 4 ) . 3MM  chromatography  A t t h i s p o i n t t h e s a m p l e was  scheme  applied  t o Whatman  p a p e r 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  U D P - M u r N A c - t r i p e p t i d e b a n d was T h i s z o n e was  to the  l o c a t e d as d e s c r i b e d  t h e n e l u t e d w i t h w a t e r and  elsewhere  quantified  presumptive  (21, 80).  (104) u s i n g U D P - G l c N A c  as the standard. (b)  UDP-MurNAc-pentapeptide  C r u d e U D P - M u r N A c - t r i p e p t i d e was UDP-MurNAc-pentapeptide.  The  D - a l a n y l a l a n i n e l i g a s e assay exceptions: was  ( i ) 200 mM  r e a c t i o n was  After  MgC^  x j> f o r 20 m i n u t e s d e c a n t e d and  mM.  the  T h i s m i x t u r e was  The  was  MnC^j  following (H)  toluene  I C]D-alanine  (36m  1h  i n c u b a t e d a t 30°C.  t h e n c e n t r i f u g e d a t 42,000  t o remove p r e c i p i t a t e d m a t e r i a l .  The  l y o p h i l i z e d m a t e r i a l was  s m a l l v o l u m e o f d i s t i l l e d w a t e r and chromatograph  (iii)  b o i l e d f o r 2 m i n u t e s and  lyophilized.  to that f o r the  s u b s t i t u t e d f o r t h e 20 mM  i n c r e a s e d t o 2.8  24 h o u r s , i t was  similar  ( s e c t i o n V I I C c , p a g e 22) w i t h t h e  a d d e d 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  C i / m m o l ) was  The  enzymatically converted to  supernatant resuspended  s t r e a k e d onto chromatography  was in a  paper.  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  from the UDP-MurNAc-tripeptide.  UDP-MurNAc-pentapeptide  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  was  N o n - r a d i o a c t i v e UDP-MurNAc-pentapeptide D-alanylalanine i n the B.  T h i s was HC1  (113).  was  p r e p a r e d by  The  zone  of  substituting  reaction.  Other standards (a)  I t was  .  MurNAc-pentapeptide p r e p a r e d by h e a t i n g U D P - M u r N A c - p e n t a p e p t i d e p u r i f i e d by chromatography  i n 0.1  i n s o l v e n t system I .  N  mM.  18  (b)  MurNAc(-GlcNAc)-pentapeptlde  T h i s was  prepared  u s i n g the system developed coli  (10,  (tetrapeptide)  from lysozyme d i g e s t e d  subtilis  f o r examining peptidoglycan  cell  walls  cross-linking  in  54). (c)  G C L - P - P - M u r N A c - p e n t a p e p t i d e and  gCL-P-P-MurNAc(-GlcNAc)-  pentapeptide These were not p u r i f i e d , but lipid  zone of  the i n v i t r o  were used as  the standard.  hydrolysis.  I t had  accumulated  was  ppGpp p r e p a r a t i o n and  diluted  Klett Units harvested at  prepared  one  by  centrifugations pelleted angle  (13, 42).  square inch.  An  The  Model L  40 r o t o r a t 4 0 , 0 0 0 rpm  cell  Press  producing fugation  f o r 2 hours. activity (42)  (9,  was 51).  hours.  The  The  procedures  c u l t u r e of grown t o  The  cells  150 were  (American Instrument  d e b r i s was  r e s u s p e n d e d 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 4 0 , 0 0 0 rpm  was  This material synthesis  (97) and  (Beckman I n s t r u m e n t s f o r 2.5  It also  the p u b l i s h e d  maximum g r o w t h ) .  a t 4 0 , 0 0 0 x g_ f o r 20 m i n u t e s .  i n a Spinco  acid  diminished i f  overnight  into L-broth  one-half  20)  purification  b r o k e n i n a Power L a b o r a t o r y  12,000 l b s p e r  to m i l d  reaction.  c o m b i n i n g some o f  hundred-fold  (approximately  and  coupled  the  page  i n s o l v e n t system I .  intermediates of p e p t i d o g l y c a n  i n v i t r o p r o d u c t i o n o f ppGpp  VC10  sensitive  a d d e d t o t h e r e a c t i o n and  added t o the i n v i t r o  T h i s was for  the a p p r o p r i a t e  the GCL-linked C.  r e a c t i o n ( s e c t i o n V I I Ab,  T h i s m a t e r i a l was  i f v a n c o m y c i n was  b a c i t r a c i n was probably  coupled  the r a d i o a c t i v e m a t e r i a l of  removed by  Co.)  two  crude ribosomes were I n c . ) u s i n g a Beckman  p e l l e t was  (13)  and  rinsed,  re-harvested  at  T h e s e c r u d e r i b o s o m e s seemed t o h a v e more p p G p p -  than ribosomes cleaned w i t h sucrose  o r T r i t o n X-100  washes  (13).  gradient  They were used t o  centrigenerate  19  l a r g e q u a n t i t i e s o f ppGpp, a s d e s c r i b e d e l s e w h e r e  (13).  was  checked  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  identified  by  (13).  I t was  the p u b l i s h e d a c t i o n spectrum  ( 1 2 , 15)  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  and  (15).  An a u t h e n t i c 1 m i l l i g r a m s a m p l e o f ppGpp was  b y M.  Cashel.  VII.  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 Coupled (a) The  d e s c r i b e d by diluted 0.5%  imately  I z a k i ejt a l . ( 5 1 ) .  0.5%  An  minimal  s a l t s media  c a s a m i n o a c i d s and  donated  enzymes  The  (Sigma) f o r 5 m i n u t e s . 5mM  tris  (pH 8 . 2 ) ,  was  c e n t r i f u g e d two  The  10 mM  p a s t e was  MgCl  2  and  1 mM  weight  unbroken c e l l s .  T h i s p e l l e t was  w i t h a b u f f e r c o n t a i n i n g 0.5 I t was  mM  assayed  w i t h b o v i n e serum a l b u m i n as  Tris  in a  Type  resuspended  (pH 8 . 2 ) ,  t h e s t a n d a r d , and  305  i n a buffer containing This  f o r 40 m i n u t e s  0.1  f o r p r o t e i n by  p r e p a r a t i o n was  the  suspension to  p e l l e t s were d i s c a r d e d .  t h e n c e n t r i f u g e d a t 44,000 x £  t h e p a r t i c u l a t e enzyme.  (approx-  ground  of alumina  mercaptoethanol.  The  with  h a r v e s t e d , and  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  r e m o v e t h e a l u m i n a and  The  units  f r o z e n p e l l e t was  resuspended  was  (77) s u p p l e m e n t e d  c u l t u r e was  The  to t h a t  c o l i VC10  g r o w n t o 200 K l e t t  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 t h e p e l l e t wet  p r o t e i n per ml.  kindly  o v e r n i g h t c u l t u r e of  s t o r e d o v e r n i g h t a t -20°C.  mercaptoethanol.  Instruments,  p r e p a r e d by a method s i m i l a r  t w o - t h i r d s maximum g r o w t h ) .  s u p e r n a t a n t was  relative  P a r t i c u l a t e enzyme p r e p a r a t i o n  one h u n d r e d - f o l d i n M9  p e l l e t was  the  and  reaction for peptidoglycan synthesis  p a r t i c u l a t e enzyme was  g l u c o s e and  for purity  (Brinkman  Inc.)  A.  T h i s ppGpp  mM  to  The  pellet  a n d w a s h e d two MgCl  2  and  1  times mM  the Lowry procedure  resuspended  t o 15 mg  then dispensed i n small  (64) of  batches  20  w h i c h w e r e f r o z e n a t -20 C u n t i l u s e d . full  activity  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 o r s e v e r a l weeks. (b)  Coupled r e a c t i o n assay and  The i n v i t r o  f o r formation  GCL-linked  intermediates.  formation  of peptidoglycan  and  of  peptidoglycan  GCL-linked  i n t e r m e d i a t e s b y 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 m e a s u r e d i n a n incubation mixture  which contained:  (pH 8.2, 1 M ) , M g C l enzyme,  (iii)  ( 1 . 1 mM).  (20 mM)  2  ( 0 . 7 5 mM,  by adding  in  acid.  (2mM),  l l  G  Then  * C - l a b e l e d UDP-MurNAc-  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 ,  by c h i l l i n g  t h e m o n i c e and a d d i n g  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  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 a n d c o u n t e d .  the p e p t i d o g l y c a n ,  penicillin  a t 37°C f o r 10 m i n u t e s .  5 y l of  tris  ( i i ) 5 y l of p a r t i c u l a t e  ( i v ) 5 y l of potassium  18 m C i p e r m m o l e ) .  the r e a c t i o n s were t e r m i n a t e d isobutyric  water,  was p r e - i n c u b a t e d  t h e r e a c t i o n was i n i t i a t e d pentapeptide  and UDP-GlcNAc  5 y l of d i s t i l l e d  This mixture  ( i ) 5 y l of a stock s o l u t i o n of  and a f l u o r e s c e n t zone a t  10 y l o f  by chromatography  The o r i g i n r e t a i n e d  0.8 c o n t a i n e d  the  GCL-linked  intermediates. The e f f e c t s o f ppGpp a n d o t h e r  compounds w e r e c h e c k e d b y  t u t i n g the 5 y l of water i n the r e a c t i o n with the appropriate o f t h e m a t e r i a l s t o be t e s t e d . carboxypeptidase  transpeptidase a c t i v i t i e s  Omission of the p e n i c i l l i n d i d not a l t e r but  i t d i d decrease the net counts r 1 k  enzymatic  removal of B.  L  concentrations  P e n i c i l l i n was a d d e d t o i n h i b i t  and p e p t i d o g l y c a n  substi-  the  D-alanine  (37, 106).  the general trend of the r e s u l t s ,  i n the peptidoglycan because of  i  CJD-alanine  Exchange assay  from the i n c o r p o r a t e d  precursors.  f o r phospho-N-acetylmuramoyl-pentapeptide  transferase. 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 assay analogous t o t h e exchange assay d e s c r i b e d by  Hammes a n d N e u h a u s ( 3 6 ) . preparation (i)  T h e r e a c t i o n u s e d t h e same p a r t i c u l a t e  as t h e coupled  reaction described  above.  enzyme  The a s s a y  contained  5 Ul o f a s o l u t i o n o f t r i s ( p H 8 . 2 , IM) a n d 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 o f p a r t i c u l a t e e n z y m e , ( i i i ) 5 u l o f d i s t i l l e d w a t e r , 5 u l of MgCl mixture  The s u b s t r a t e s were added a f t e r  been p r e - i n c u b a t e d  an i c e b a t h  and a d d i n g  substituting  them, a t t h e a p p r o p r i a t e  d o e s when o t h e r  routinely  The  3  n u c l e o t i d e s were examined by  concentrations,  b a c t e r i a l membranes a r e u s e d  for the 5 Ulof  (44) , a n d w a s n o t  synthetase  and D - a l a n y l a l a n i n e  enzyme p r e p a r a t i o n was a ' p a r t i a l l y This  purified  I t was p r e p a r e d  scheme d e s c r i b e d  f r o m E ^ c o l i VC10 w h i c h h a d b e e n g r o w n t o  t w o - t h i r d s m a x i m a l g r o w t h i n t h e m e d i a u s e d b y Comb ( 2 0 ) . a t i o n s were a d j u s t e d  mixture  p r e p a r a t i o n was made b y d i a l y z i n g t h e  (NH^^SO^ p r e c i p i t a t e occurring i n the p u r i f i c a t i o n  Comb ( 2 0 ) .  ligase.  Enzyme p r e p a r a t i o n .  containing both a c t i v i t i e s .  (64)  f r o m [ HJUMP  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 , a s  D-alanylalanine (a)  by  [^UDP-MurNAc-  included. C.  first  The  s y s t e m I f o r 24 h o u r s a n d c o u n t e d .  o f ppGpp a n d o t h e r  w a t e r added t o t h e a s s a y . it  acid.  the reaction vessels  w h i c h w a s f o r m e d b y t h e r e a c t i o n was s e p a r a t e d  effects  mixture  T h e a s s a y was t h e n  by t r a n s f e r r i n g  10 U l o f i s o b u t y r i c  chromatography i n s o l v e n t The  the rest of the reaction  a t 37°C f o r 10 m i n u t e s .  f o r 15 m i n u t e s a n d t e r m i n a t e d  pentapeptide by  (v) 5 u l o f a  3  incubated to  concentration,  o f [ H ] U M P (5mM, 2.5 m C i p e r mmole) a n d U D P - M u r N A c - p e n t a p e p t i d e  (1 mM). had  o r MnCl^ a t t h e a p p r o p r i a t e  2  (iv)  The  t o 10 mg o f p r o t e i n p e r m l u s i n g t h e L o w r y  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 .  Small  preparprocedure  b a t c h e s were  then  22  f r o z e n a t -20°C u n t i l (b) The contained: 125  used.  Assay f o r D-alanylalanine  a s s a y was s i m i l a r ( i ) 5 y l of t r i s  synthetase.  to that described  (pH 8.7, 4 0 0 mM),  mM) , ( i i i ) 5 y l o f enzyme p r e p a r a t i o n ,  elsewhere  ( i i ) 10 y l o f ATP (pH 7.2,  ( i v ) 5 y l of MnCl^  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 of  [ ^C]D-alanine 1  ( 0 . 2 8 mM,  adding t h e [ ^C]D-alanine 1  of time.  36 mCi/m m o l e ) .  by being  10 y l o f i s o b u t y r i c a c i d a d d e d . stituting  chilled  i n an i c e bath  (c)  present  periods  and t h e n  having  The e f f e c t o f ppGpp was t e s t e d b y s u b -  5 y l o f 20 mM ppGpp f o r 5 y l o f t h e d i s t i l l e d  w h i c h was s t i l l  (vi) 5 y l  a t 37°C f o r s p e c i f i e d  p o t a s s i u m p e n i c i l l i n G was a d d e d t o i n h i b i t (106)  water,  (20mM) a n d  The r e a c t i o n was s t a r t e d b y  a n d was i n c u b a t e d  I t was t e r m i n a t e d  (20, 66). I t  water.  any carboxypeptidase  The activity  i n t h e enzyme p r e p a r a t i o n .  Assay f o r D-alanylalanine  ligase.  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 a s t h e D - a l a n y l a l a n i n e synthetase  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  substituted the  f o r 5 y l of the d i s t i l l e d water.  ligase activity  i s slower  than the synthetase  measured as e f f i c i e n t l y w i t h  [ "*C]D-alanine  VIII.  synthesis  In vivo peptidoglycan A. All  lys to  1  Assay f o r TCA-insoluble  as w i t h  components.  1 4  3  limit  s y n t h e s i s used dap  the[ H]Dap incorporation 3  C e l l s were r o u t i n e l y d i l u t e d  f l a s k c o n t a i n i n g 50 m l of-M9 m i n i m a l • s a l t s medium per ml glucose,  and c a n be  [ C]D-alanylalanine.  of the i n vivo assays f o r peptidoglycan  peptidoglycan  4~mg  activity  [ H]Dap i n c o r p o r a t i o n .  auxotrophs which would s p e c i f i c a l l y  arm  As p r e v i o u s l y r e p o r t e d ( 6 6 ) ,  1 yg per ml thiamine,  i n a 500 m l s i d e -  (77) supplemented  10 y g p e r m l  diaminopimelic  with  23  a c i d a n d 50 yg' p e r m l o f o t h e r r e q u i r e d t h i s media i s r e f e r r e d generations u n t i l was at  t o a s M9.  to sterile,  27,000 x £ f o r 3 m i n u t e s  saline.  screw-cap  A 40-ml  centrifuge  aliquot  t u b e s and h a r v e s t e d set to  a n d w a s h e d o n c e w i t h 40 m l o f 0.9%  The washed c e l l s were r e s u s p e n d e d  and d i s p e n s e d t o a f i n a l containing  T h e c u l t u r e was g r o w n f o r 4 t o 5  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  T h e p e l l e t was r e s u s p e n d e d  sterile  For convenience,  i t r e a c h e d 50 t o 60 K l e t t u n i t s .  then transferred  2°C.  amino a c i d s .  i n fresh  NaCl  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  10 m l 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.  { H ] D a p ; ( 2 8 0 - 5 4 5 mCi/mmol) was t h e n a d d e d t o 0.2 u g p e r m l f o r e a c h  flask  3  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 m l a l i q u o t s w e r e r e m o v e d from each c u l t u r e filter  paper.  flask of cold  and p i p e t t e d  When e a c h  f o r two 20-minute 30 s e c o n d s  s a m p l e was a b s o r b e d , t h e d i s c was d r o p p e d  5% TCA a n d l e f t  were then t r a n s f e r r e d  o n t o 2.4 cm d i a m e t e r d i s c s o f Whatman 3MM  f o r a minimum o f 30 m i n u t e s .  The d i s c s  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  periods i n cold  i n diethyl ether.  into a  soaked  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  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 w e r e n o r m a l i z e d t o c e l l mass a n d e x p r e s s e d a s c o u n t s p e r m i n u t e per m i l l i g r a m The  d r yweight utilization  of c e l l s . of the i n t r a c e l l u l a r  p o o l s o f Dap was e x a m i n e d  b y p r e - l o a d i n g t h e Dap p o o l s o f t h e c e l l s w i t h p H J D a p .  The c e l l s  were  g r o w n t o 50-60 K l e t t u n i t s , t h e n w a s h e d a n d t r a n s f e r r e d  t o complete  M9  media c o n t a i n i n g  [ HjDap as d e s c r i b e d above. 3  After  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 H]Dap and t r a n s f e r r e d 3  medium.  The c u l t u r e s w e r e t h e n m o n i t o r e d . a n d  sampled  t o Dap-free  a s 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 i n c o r p o r a t e d  [ H]Dap  (a)  cytoplasmic,  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 membrane-associated, and  The  various  were s e p a r a t e d and  3  components.  [ H l D a p - c o n t a i n i n g components of the l a b e l e d i n a system analogous t o t h a t  by Liigtenberg and de Haan ( 6 7 ) .  The  c e l l s were washed and  [ ^ j D a p - c o n t a i n i n g M9_ media as d e s c r i b e d  i n t e r v a l s 8-ml  c e n t r i f u g e tubes.  described  transferred  except t h a t 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 t o 18 ml At d e s i g n a t e d  cells  3  quantitated  t o the a p p r o p r i a t e  peptidoglycan  above, of media.  samples were p i p e t t e d t o c h i l l e d g l a s s Corex  These were i m m e d i a t e l y c e n t r i f u g e d at 35,000 £ f o r  3 m i n u t e s 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. tubes were t r a n s f e r r e d t o an i c e b a t h . removed w i t h a P a s t e u r p i p e t t e and  The  The  centrifuge  s u p e r n a t a n t s were then  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  aliquots  were a p p l i e d t o chromatography paper as 1 cm l o n g s t r e a k s .  various  components were s i m u l t a n e o u s l y  e x t r a c t e d and  The  f r a c t i o n a t e d by  developing  t h e chromatographs i n s o l v e n t system I u n t i l the s o l v e n t f r o n t n e a r l y at the end  of the paper ( u s u a l l y 19 t o 22 h o u r s ) .  l a b e l e d components were then l o c a t e d and  counted.  The  The  was appropriate  peptidoglycan  stayed  at the o r i g i n i n t h i s s o l v e n t system (51, 54, 6 7 ) , the s o l u b l e n u c l e o t i d e linked peptidoglycan 67), free  [ H]Dap m i g r a t e d w i t h an R 3  intermediates contains  m i g r a t e d at R^ 0.8  f  of 0.3  (9, 51, 67).  t o 0.4, The  and  l a t t e r region  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  The  samples d r i e d r a p i d l y when s t r e a k e d  They d i d not  t o 0.2  (51,  the G C L - l i n k e d  some p r o t e i n (10, 107), b u t , f o r c o n v e n i e n c e , was  l i p i d zone.  portions.  p r e c u r s o r s m i g r a t e d w i t h an R^ of 0.1  termed  also the  light.  onto the paper i n 10 y l  seem t o change when s t o r e d i n the i c e b a t h f o r  25  a s much a s 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 . 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  r o u t i n e , simultaneous  assays  preliminary experiments, to i n h i b i t  enzymatic  f o r TCA-insoluble  difficult  with the  [' H]Dap i n c o r p o r a t i o n .  In  3  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 .  acid  This d i d not a l t e r  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 -  i n t e r m e d i a t e s when c o m p a r e d t o t h e a m o u n t s i n p e l l e t s  w i t h water,  differed  p e l l e t s were resuspended w i t h i s o b u t y r i c  the r e l a t i v e proportions of l i p i d linked  D u p l i c a t e samples  resuspended  b u t i t d i d c a u s e t h e p e p t i d o g l y c a n t o become c l u m p e d a n d  t o handle. 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 the q u a n t i t y of c e l l s  a p p l i e d t o t h e paper t o the 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 . done by d e t e r m i n i n g  the ratio  sample t o t h e T C A - i n s o l u b l e  of TCA-insoluble  counts  counts  T h i s was  i n the concentrated  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 a n d 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 then 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 . Alternatively,  t h e t u r b i d i t y o f an a l i q u o t o f t h e c o n c e n t r a t e d  m e a s u r e d a t 4 1 0 nm i n a Beckman DB-G to d r y weight  with a c a l i b r a t i o n curve. (b)  preceding  nucleotide-linked  was  and c o n v e r t e d  Both methods gave s i m i l a r  results.  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 a n d UDP-MurNAcpentapeptide  The  spectrophotometer  cells  i n J HJDap-labeled  procedure  cells.  3  d i d not'resolve the d i f f e r e n t  peptidoglycan precursors, but could separate  the chromatographs were developed  f o r 62 h o u r s .  t h e m when  A d d i t i o n a l l y , the p e l l e t  of l a b e l e d c e l l s . w a s r o u t i n e l y resuspended w i t h c o l d water 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 q u e n c h when t h e c h r o m a t o graphs were s u b s e q u e n t l y pentapeptide  migrated  examined w i t h u l t r a - v i o l e t  light.  T h e UDP-MurNAc-  2 t o 3 cm a h e a d 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  UDP-MurNAc-tripeptide-ala  would migrate  w i t h UDP-MurNAc-pentapeptide, but  w a s assumed t o be p r e s e n t  i n negligible  amounts.  In to  alter  specified  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  the composition  T h i s was d o n e b y a d d i n g  of the peptidoglycan D-cycloserine  D-cycloserine  precursor pools of the c e l l s .  t o 40 Ug p e r m l f i n a l  concentration.  T h e s e t r e a t e d E j _ 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 a n d s o w e r e only incubated  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  autolysis. (c)  Analysis of l i p i d (1)  The  zone components.  Mild acid hydrolysis of l i p i d  GCL-linked  zone components.  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  system I , but they a r e r e a d i l y hydrolysed  i n solvent  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 a n d 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 separate.  Initial  a t t e m p t s t o e x t r a c t (108) and then h y d r o l y z e t h e  GCL-linked  intermediates  f o r e , the technique GCL-linked  gave i n c o m p l e t e  used by Braun and Bosch t o measure  c o m p o n e n t s i n membrane p r e p a r a t i o n s  measure G C L - l i n k e d pared  from whole c e l l s  intermediates  There-  acid-hydrolyzed  ( 9 ) was m o d i f i e d t o  i n labeled cells.  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  yields.  The c e l l s were  pre-  ( s e c t i o n V I I I B a , p a g e 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 v o l u m e o f w a t e r a n d a s m a l l p o r t i o n was put d i r e c t l y onto t h e chromatograph. ferred and  The r e m a i n i n g  p o r t i o n was t r a n s -  t o a g l a s s c e n t r i f u g e t u b e c o n t a i n i n g 0.35 m l o f b o i l i n g  b o i l e d f o r e x a c t l y 4 minutes.  This treatment  I H]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 3  fere w i t h t h e assay. components w i t h o u t  water  removed t h e f r e e  precursors which could  inter-  F o u r 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  a f f e c t i n g the counts  i n the l i p i d  zone.  these  However, t h e  b o i l i n g h a s t o b e 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 r e m o v e some  27  lipid ice  zone c o u n t s .  After boiling,  t h e samples were r a p i d l y c o o l e d i n an  b a t h a n d 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 .  resuspended  i n 0.40  m l o f b o i l i n g w a t e r and  The  re-centrifuged.  pellet  I t was  r e s u s p e n d e d w i t h a s m a l l v o l u m e o f h o t w a t e r and a s m a l l p o r t i o n transferred  to the chromatography  paper.  The  r e m a i n d e r 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 o v e n  The  c o n t e n t s o f t h e a m p o u l e , and w a t e r  were t r a n s f e r r e d  to the chromatography  then developed i n s o l v e n t system I u n t i l the  end o f t h e p a p e r .  The  dried  The  This  rinses,  chromatographs  t h e n c u t i n t o 0.5  were  almost at  cm  long  s t r i p s which were counted 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 w e r e c u t a s 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 w e r e l o c a t e d w i t h s t a n d a r d s and e q u a t e d the  respective GCL-linked intermediates.  sample  [ H]Dap-labeled 3  s o l u b l e c o m p o n e n t s m i g r a t i n g b e t w e e n t h e o r i g i n and t h e l i p i d The  s a m p l e w h i c h was  not b o i l e d  standard, to ensure q u a n t i t a t i v e r e c o v e r y of the l i p i d a f t e r b o i l i n g and a f t e r (2) The  zone  zone  material  hydrolysis. Protease treatment of l i p i d  p r o t e i n i n the l i p i d  incubation contained a f i n a l  z o n e was  zone  solubilized  c o n c e n t r a t i o n o f 2 mg  g r i s e u s p r o t e a s e ( S i g m a T y p e V I p r o t e a s e ) , 5.5 tris  ( H p  7) .  were  s e r v e d as a  components. by d i g e s t i n g  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 .  a n d 200 mM  to  The b o i l e d b u t n o t h y d r o l y z e d  s e r v e d as a c o n t r o l t o ensure t h a t a l l of the  removed b e f o r e h y d r o l y s i s .  to  s e t t o 105°C.  t h e s o l v e n t f r o n t was  p a p e r was  was  acid.  from s e v e r a l subsequent paper.  then  transferred  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 was  was  mM  per ml of  the  The  Streptomyces  potassium p e n i c i l l i n  G  28 (d) Samples i n sealed the  ampoules  A n a l y s i s of [ H ] l y s i n e content i n [ H]Dap-labeled c e l l s . 3  3  o f whole c e l l s were h y d r o l y z e d containing  6N H C l .  a t 105°C f o r 20 h o u r s  The H C l was r e m o v e d  s a m p l e s u n d e r 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 ^ . i n solvent  Samples w e r e t h e n s e p a r a t e d by paper  This  3  co-migrated w i t h l y s i n e i n a l l four (e)  f  systems.  3  o n Sephadex  G-25.  T h e s a l t a n d much o f t h e f r e e [ H ] D a p 3  The d e - s a l t e d  a n d f r a c t i o n a t e d by p a p e r c h r o m a t o g r a p h y hours.  samples were t h e n  i n solvent  quantitated.  lyophilized  s y s t e m I f o r 19 t o 22  A f t e r development, l a b e l e d components between  0.8 w e r e  material  supernatants from t h e [ H]Dap l a b e l e d c u l t u r e s ( s e c t i o n  B a , p a g e 24) w e r e l y o p h i l i z e d .  w e r e removed  solvent  examined  Examination o f supernatants from l a b e l e d c u l t u r e s .  The f r o z e n viii  chromatography  s y s t e m s I , I I I , I V a n d V, a n d t h e c h r o m a t o g r a p h s w e r e  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 [ H ] D a p s p o t .  R  by d r y i n g  t h e o r i g i n .and  29  RESULTS  I.  Peptidoglycan A.  synthesis  during  i n h i b i t i o n of protein synthesis  E f f e c t o f a m i n o a c i d d e p r i v a t i o n a n d CAM t r e a t m e n t o n [ H]Dap i n c o r p o r a t i o n b y r e l A  Peptidoglycan by  synthesis  on t h e r a t e o f p e p t i d o g l y c a n  either depriving  bacteria with  CAM.  i n dap  the bacteria of required  s y n t h e s i s was d e t e r m i n e d amino a c i d s  When LD5 ( r e l A ) was d e p r i v e d +  3  peptidoglycan deprived  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 synthesis.  cultures treated  o b s e r v e d w i t h CAM a l o n e . Dap  to the control  permitted  The r a t e o f p e p t i d o g l y c a n simultaneously  or t r e a t i n g the  of l y s i n e , the rate of  [ H ] D a p 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 (figure 3).  l y s bacteria  The e f f e c t o f i n h i b i t i o n o f  3  protein synthesis  bacteria.  was m o n i t o r e d  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 [ H]Dap.  by  i n vivo  a high  synthesis  rate  rate of i n lysine-  w i t h CAM was s i m i l a r t o t h e r a t 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  i n c o r p o r a t i o n when a d d e d 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  (figure 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 o n t h e growth o f t h e  c u l t u r e s a n d t h e r a t e o f Dap i n c o r p o r a t i o n w e r e 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 ) . Other r e l A  +  s t r a i n s ( L D 5 1 , LD52 a n d L D 2 ) h a d 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 o n e o r more r e q u i r e d  amino  acids  ( 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 s u g g e s t - t h a t r e l A . i s • i n v o l v e d I n regulating peptidoglycan B.  accumulation during  amino a c i d  deprivation.  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 [ H]Dap i n c o r p o r a t i o n 3  by  relA  When LD5456  bacteria. ( 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 o f l y s i n e d e p r i v a t i o n and  [ H]Dap i n c o r p o r a t i o n by LD5 washed once w i t h  s a l i n e and  w i t h o u t l y s i n e ( • ) , M9 w i t h o u t l y s i n e but was  m o n i t o r e d as  samples.  with  then inoculated 200  yg  yg  CAM  CAM  per  the T C A - i n s o l u b l e  Counts were n o r m a l i z e d  ml  c o m p a r i s o n of  f o r d i f f e r e n c e s i n the minutes, the  radioactivity  2,527; 665;  967;  and  f o r the  the  ) and  M9 M9  Incorporation i n 0.1  ml  s a m p l e s and  B e f o r e the  c e l l u l a r mass o f  895.  ( A  ( 0 ),  to c e l l dry weight i n order  the v a l u e s .  r e s p e c t i v e CPM  ( A ).  on  harvested,  i n t o M9  per ml  c o m p e n s a t e f o r d i f f e r e n c e s i n t h e mass o f facilitate  treatment  ( r e l A ). C e l l s were  plus 200  CAM  four  the  to  correction  samples, at  listed  to  60  c u l t u r e s were:  31  Minutes  FIGURE 4. Effect of late addition of CAM on [ H]Dap incorporation 3  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 i n 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  Minutes  34  FIGURE 5. Effect of lysine restoration on £ HjDap incorporation (A) 3  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 i n 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 i n 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 o f amino a c i d d e p r i v a t i o n  by o t h e r r e l A  +  amino a c i d auxotrophs o f ,E. c o l i .  methods, see f i g u r e 3.  A)  S t r a i n LD51:  • l y s i n e ( § ) , M9 minus l e u c i n e B)  on [ H]Dap  S t r a i n LD52:  3  incorporation  F o r d e t a i l s o f the  Complete M9_ ( 0 ) , M9 minus  ( A ), M9 minus l y s i n e and l e u c i n e  ( A ).  Complete M9 ( 0 ) , M9 minus l y s i n e ( i ) , M9 minus  t h r e o n i n e ( A ), M9 minus l y s i n e and t h r e o n i n e ( A ).  C)  S t r a i n LD2:  Complete M9 ( 0 ) , M9_ minus l y s i n e ( 8 ) , M9_ minus m e t h i o n i n e ( A ), M9 minus l y s i n e and m e t h i o n i n e ( A ).  Before the c o r r e c t i o n f o r differences  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 l i s t e d c u l t u r e s were: 850; 869; and 866.  CPM f o r t h e t w e l v e  1,265; 660; 694; 713; 1,605; 699; 694; 893; 2,893;  i  1  1  1  Minutes  1  r  38  was e q u a l t o o r g r e a t e r  than the i n c o r p o r a t i o n  during  the  O t h e r r e l A ~ s t r a i n s (LD5457 a n d L D 5 4 5 8 ) g a v e s i m i l a r  r e s u l t s when d e p r i v e d deprived  t r e a t m e n t was o n l y  slightly  (figure 7).  Incorporation control.  CAM  by t h e c o n t r o l  o f l y s i n e ( f i g u r e 8A, B ) .  of other required  a m i n o a c i d s , t h e Dap  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 concur with  the conclusion  peptidoglycan  that  relA i s involved  accumulation during  C.  amino a c i d  lower than that i n  When t h e s e s t r a i n s w e r e incorporation relaxed.  These r e s u l t s  i n regulating  deprivation.  E f f e c t o f t h e r e l A g e n e o n u t i l i z a t i o n o f e n d o g e n o u s Dap.  Dap a u x o t r o p h s o f E_. c o l i  contain  a large pool  o f endogenous  Dap w h i c h c a n b e r a d i o a c t i v e l y l a b e l e d b y g r o w i n g t h e c e l l s f o r 30 m i n u t e s . synthesis  This  pool  i s sufficient  f o r a b o u t two d o u b l i n g s  Pre-labeled  was  lysine-deprived  t o support  i n [ RjDap 3  peptidoglycan  i n t h e a b s e n c e o f e x o g e n o u s Dap.  c u l t u r e s o f LD5  (relA ) utilized +  internal  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 treated  cultures  ( f i g u r e 9A).  In contrast,  c u l t u r e s o f LD5456 ( r e l A ) u t i l i z e d as  the control  ( f i g u r e 9B).  These o b s e r v a t i o n s peptidoglycan  indicate that  support the conclusion  Regulation A.  t h e same r a t e treatment.  the r e l a t i v e decrease i n accumulation of stringent b a c t e r i a i s probably not  Coupled  that  the accumulation of  bacteria i s stringently  of peptidoglycan  Initial  i n t e r n a l Dap a t n e a r l y  i n t h e u p t a k e o f t h e e x o g e n o u s l a b e l e d Dap.  i n amino a c i d - d e p r i v e d  II.  lysine-deprived  r a t e was n o t e n h a n c e d b y CAM  by amino a c i d - d e p r i v e d  due t o d i f f e r e n c e s results  This  pre-labeled  CAM-  synthesis  peptidoglycan  regulated.  i n vitro  reaction.  attempts to l o c a l i z e  The  the s i t e of i n h i b i t i o n of  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 a n d CAM t r e a t m e n t  [ H ] D a p i n c o r p o r a t i o n by LD5456 3  s e e f i g u r e 3.  C o m p l e t e M9  p l u s 200 y g p e r m l CAM 200 y g p e r m l CAM  (relA ).  F o r d e t a i l s o f t h e methods  (.0 ) , M9 w i t h o u t l y s i n e ( © ) , M9  ( A ) , a n d M9_ w i t h o u t l y s i n e b u t w i t h  ( A ).  Before the c o r r e c t i o n f o r differences  i n t h e c e l l u l a r m a s s , a t 60 m i n u t e s , t h e r e s p e c t i v e four  listed  on  CPM f o r t h e  c u l t u r e s w e r e : 1,716; 1,137; 7 0 5 ; a n d 7 2 9 .  40  Mi  nutes  FIGURE 8. by 3.  other 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 on I H ] D a p 3  (relA ) strains. S t r a i n LD5457:  M9 m i n u s l e u c i n e  ( A ).  M9 m i n u s l y s i n e (8 for differences  F o r d e t a i l s of t h e methods, see f i g u r e  C o m p l e t e M9_ ( 0 ) , M9 m i n u s l y s i n e ( § ) , B)  S t r a i n , LD5458:  ) , M9 m i n u s t h r e o n i n e  (..A ) .  C o m p l e t e M9_ ( 0 ) , Before the correction  i n t h e c e l l u l a r m a s s , a t 60 m i n u t e s , t h e  CPM f o r t h e s i x l i s t e d 1,522; a n d  incorporation  1,399.  c u l t u r e s were:  1,806; 1 , 6 0 1 ; 914;  respective 3,054;  FIGURE 9. (relA )  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  (A) and L D 5 4 5 6  +  m i n u t e s , washed w i t h media w i t h but  plus  (relA~)  ( B ) . C e l l s were l a b e l e d  s a l i n e and i n o c u l a t e d  l y s i n e ( 0 ), without l y s i n e  200 y g p e r m l CAM  as  the increase  of  the c u l t u r e s .  r a d i o a c t i v e p o o l s b y LD5  ( k ).  into fresh  Incorporation  per  mg  x 10  h  cell  differences  was  lysine  followed  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 s a m p l e s To f a c i l i t a t e  been incorporated  (10.8  Dap-free  ( 6 ) , and w i t h o u t  comparison 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 b y s u b t r a c t i n g had  f o r 30  by t h e time t h a t  CPM p e r mg  the counts which  the c u l t u r e s were  split  c e l l d r y w e i g h t f o r L D 5 , and 9.43 x 1 0  d r y weight f o r LD5456).  4  CPM  Before the c o r r e c t i o n f o r  i n t h e c e l l u l a r m a s s , a t 60 m i n u t e s a f t e r a d d i n g t h e  [ H]Dap, t h e r e s p e c t i v e 3  c u l t u r e s were:  CPM f o r t h e s i x p e r m u t a t i o n s o f t h e  1,802; 8 6 5 ; 1,149; 1 , 9 2 1 ; 1,288; and 1,024.  listed  45 peptidoglycan  synthesis during  s t r i n g e n t c o n t r o l w e r e made by d e t e r m i n i n g  e f f e c t o f ppGpp o n t h e i n v i t r o a c t i v i t y peptidoglycan cell  synthesis.  The c o u p l e d  o f some o f t h e e n z y m e s i n v o l v e d i n  r e a c t i o n involves a crude  particulate  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, 1 0 , 1 1 a n d 12 i n  f i g u r e 2 and a l l o w s b o t h i a t e s and p e p t i d o g l y c a n t h a t 2 mM  ppGpp  the formation  of GCL-linked  t o be m o n i t o r e d .  significantly  but d i d not i n h i b i t  inhibited  the accumulation  the r a t e of peptidoglycan  of GCL-linked  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 ) . the accumulation  of GCL-linked  peptidoglycan  intermed-  P r e l i m i n a r y experiments i n d i c a t e d  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  ppGpp  the  intermediates  formation  synthesis (figure 10).  increased w i t h increast-  However, a t h i g h e r  concentrations of  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  (figure 11). The c o u p l e d  r e a c t i o n was d e p e n d e n t o n m a g n e s i u m ( f i g u r e 1 2 ) .  m a g n e s i u m d e p e n d e n c e was c o m p l e x a n d 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  o p t i m a o f t h e 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 . ion of both  the GCL-linked  ( f i g u r e 12A) o c c u r r e d  intermediates  a t approximately  o f b o t h r e a c t i o n s b y ppGpp d e c r e a s e d (figure 12).  ratio  o f magnesium  of e i t h e r . on  the coupled  ( f i g u r e 12B) a n d t h e p e p t i d o g l y c a n  12 mM M g C ^ -  The d e g r e e o f  i n the r e a c t i o n r a t h e r than  MgC^  that the stringent  the absolute  amount  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 m a g n e s i u m t o ppGpp  r e l a t i v e i n h i b i t i o n s of the accumulation  the data  i n f i g u r e 12.  of p e p t i d o g l y c a n and  as the molar r a t i o of MgC^  t o ppGpp  o f M g C l ^ t o ppGpp was v a r i e d b y c h a n g i n g  The  GCL-linked increased  S i m i l a r r e l a t i v e amounts o f a c t i v i t y were o b s e r v e d  the. m o l a r r a t i o  inhibition  with increasing concentration of  r e a c t i o n was c a l c u l a t e d u s i n g  intermediates decreased  accumulat-  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 molar  t o ppGpp  Therefore,  (figure 13).  The o p t i m a l  However. Lueking. and G o l d f i n e (65) r e p o r t e d  i n h i b i t i o n of i n v i t r o  The  when  t h e ppGpp  FIGURE 10. Incorporation of I ^CjUDP-MurNAc-pentapeptide into 1  GCL-linked intermediates ( A, A ) and peptidoglycan ( 0, 8 ) by a particulate membrane preparation incubated i n the presence ( A, 81 ) and absence ( A, 0 ) of 2 mM ppGpp. A l l reactions contained 6 mM MgCl . 0  Peptidoglycan  Accumulated  (10  2  CPM)  -p-  48  FIGURE 11. glycan  Effect  of 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 -  ( ® ) 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 p e p t i d o g l y c a n minutes.  Reactions  contained  6 mM  MgCl_.  synthesis i n ten  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 1 3 .  Influence of the molar r a t i o , o f MgC^  t o ppGpp o n  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 ( © ) a n d G C L - l i n k e d intermediates vitro  ( A )• a c c u m u l a t e d b y t h e c o u p l e d r e a c t i o n f o r i n  peptidoglycan synthesis.  The v a l u e s w e r e c a l c u l a t e d  from  t h e r e s u l t s i n f i g u r e 12 a n d a r e p r e s e n t e d a s t h e a m o u n t s a c 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 accumulated  t o t h e r e s p e c t i v e amounts  i n t h e a b s e n c e o f ppGpp.  53  54  concentration  i n s t e a d of the M g C l  concentration  2  (data not  presented).  A g i v e n m o l a r r a t i o o f MgCl,> t o ppGpp g a v e a c o n s i s t e n t l e v e l accumulation.  The  g l y c a n c o u l d be pentapeptide in vitro  preceding  i n h i b i t e d by  translocase  peptidoglycan B.  Exchange  data  i n d i c a t e t h a t formation, of  ppGpp a t t h e  ( r e a c t i o n 9,  f i g u r e 2) b u t  an  the consecutive  the exchange r e a c t i o n .  ( f i g u r e 14). 15A) .  The  e x c h a n g e r e a c t i o n was  not  peptidoglycan  specifically  t h e enzyme c a t a l y z e s  UDP-MurNAc-pentapeptide.  f o r a t l e a s t 32 m i n u t e s and  was  The  s e n s i t i v e t o ppGpp  d e p e n d e n t on m a g n e s i u m ( f i g u r e  Optimum enzyme a c t i v i t y o c c u r r e d  on m a g n e s i u m was  the  a c t i v i t y o f s e v e r a l enzymes,  In t h i s assay,  3  linear  assay f o r  t h e p a r t i c u l a t e s y s t e m was  e x c h a n g e r e a c t i o n b e t w e e n I H]UMP and  r e a c t i o n was  step i n  Reaction.  r e a c t i o n c a t a l y z e d by  studied with  a later  s y n t h e s i s i s e v e n more s e n s i t i v e t o ppGpp.  s y n t h e s i s a r e d e p e n d e n t on first  peptido-  phospho-N-acetylmuramoyl-  Since the r e s u l t s of the coupled  the  of  w i t h 6 mM  MgCl,,.  The  dependence  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  reaction.  The  i n h i b i t o r y e f f e c t o f ppGpp on t h e e x c h a n g e r e a c t i o n s t i l l  varied  the  c o n c e n t r a t i o n of the M g C l  was  d e p e n d e n t on  2  ( f i g u r e 15A).  The  t h e m o l a r r a t i o o f m a g n e s i u m t o ppGpp ( f i g u r e 1 5 B ) ;  degree o f i n h i b i t i o n d e c r e a s e d as the m o l a r r a t i o e x c h a n g e r e a c t i o n and  t h e amount o f G C L - l i n k e d  r e a c t i o n were r e d u c e d by i m a t e l y 1.3.  This  are v a l i d  supports  and  50%  at a M g C l  2  ppGpp.  increased.  intermediates  suggests t h a t the r e s u l t s  the  i n the  from the coupled  the c o n c l u s i o n t h a t the a c t i v i t y of the ( r e a c t i o n 9,  the  Both  t o ppGpp m o l a r r a t i o o f  N-acetylmuramoyl-pentapeptide translocase i n h i b i t e d by  inhibition  with  coupled  approx-  reaction phospho-  f i g u r e 2)  can  be  FIGURE 14.  Incorporation of I H]UMP into UDP-MurNAc-pentapeptide 3  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. A l l reactions contained 6 mM MgCl . 0  0  8  16  Minutes  24  32  FIGURE 1 5 .  (A)  Effect  of MgCl  on t h e a c c u m u l a t i o n o f [ H]UMP 3  2  i n U D P - M u r N A c - p e n t a p e p t i d e by t h e e x c h a n g e a s s a y . i n c u b a t e d f o r 15 m i n u t e s the presence indicated ratio  activity ppGpp  o f 2 mM ppGpp  MgCl  of M g C l  2  2  ( 9 ) and 4 mM ppGpp  concentrations.  (B)  t o ppGpp was e x p r e s s e d  observed  ( A ).  i n t h e a b s e n c e o f ppGpp  i n the presence  R e a c t i o n s were ( 0 ) and i n  ( A ) at the  The e f f e c t o f t h e m o l a r as t h e percent o f t h e c o n t r o l  o f 2 mM ppGpp  ( • ') and 4 mM  58  C\]  oo  "~  (lAlcDgOO  uo!;B|nujnoov  59  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  The i n h i b i t i o n o f t h e c o u p l e d specific  (Table I I ) .  UTP.  intermediates.  Of t h e s e , o n l y UMP  l o g i c a l concentrations were p r o b a b l y  the accumulation  was r e d u c e d .  (Table  of either peptidoglycan or  t o UMP  the peptidoglycan lower  i n the c o n t r o l .  the coupled D.  and  physioUDP  b y p h o s p h a t a s e s i n t h e enzyme  i n either reaction. (Table  The  effects  I V ) . A b o v e 10 mM  and t h e G C L - l i n k e d  ATP,  intermediates  concentrations only the accumulation  p e p t i d o g l y c a n was r e d u c e d , a n d t h e a c c u m u l a t i o n  inhibited  UDP  I I I ) . The i n h i b i t i o n s b y UTP a n d  was n o t d e t e c t e d  At s l i g h t l y  was g r e a t e r t h a n  concentration  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  o f ATP w e r e 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 of both  relatively  The p r i n c i p l e e x c e p t i o n s w e r e UMP,  n o t due t o c o n v e r s i o n  p r e p a r a t i o n s i n c e UMP  accumulation  r e a c t i o n b y ppGpp was  M o s t o t h e r common n u c l e o t i d e s a t 2 mM  did not s i g n i f i c a n t l y a f f e c t GCL-linked  reaction  of GCL-linked  C h e l a t i n g agents,  of  intermediates  s u c h a s EDTA, a l s o  r e a c t i o n (Table I V ) .  Other r e a c t i o n s i n peptidoglycan  synthesis.  Other 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 synthesis  ( r e a c t i o n 7, f i g u r e 2) a n d D - a l a n y l a l a n i n e l i g a s e  8, f i g u r e 2 ) . a molar r a t i o inhibited  (reaction  B o t h enzymes r e q u i r e e i t h e r magnesium o r manganese. of MnCl  2  t o ppGpp o f o n e , n e i t h e r r e a c t i o n was  ( f i g u r e 16A, B ) .  t o t h e u s e o f manganese  At  significantly  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  i n t h e r e a c t i o n s s i n c e t h e e x c h a n g e r e a c t i o n was  more s e n s i t i v e t o ppGpp when m a n g a n e s e was s u b s t i t u t e d f o r m a g n e s i u m ( 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 peptidoglycan synthesis . 3  N u c l e o t i d e Added  Peptidoglycan CPM  Experiment  Intermediates  CPM  % of C o n t r o l  1  None ( c o n t r o l ) ppGpp GMP dGTP ppppG c-GMP CMP CTP UMP UDP UTP ADP Experiment  % of Control  GCL-linked  2636 .1377 2650 2234 2607 2687 2647 2378 643 892 1818 2309  100 52 101 85 99 102 101 90 24 34 69 88  763 779 633 752 583 776 693 783 580 252 638 761  100 102 83 99 76 101 91 103 76 33 88 100  1528 1467 1591 1305  100 96 104 85  2688 2413 2752 2516  100 90 102 94  2756 1461 2799 2982  100 53 102 108  731 616 769 809  100 84 ..104 111  2  None ( c o n t r o l ) GTP CDP AMP Experiment 3 None ( c o n t r o l ) ppGpp GDP ATP  A l l assays were incubated c o n c e n t r a t i o n o f 6 mM.  f o r 10 m i n u t e s a n d h a d a f i n a l M g C l  61  TABLE I I I .  _ , ., . Inhxbxtor Concentration  Peptidoglycan c  Control  (none)  UMP  (2.0  UDP  UTP  Effect of concentration of 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 the cougled reaction f o rpeptidoglycan synthesis .  p  M  %  q  £  C  o  n  t  r  o  l  GCL-linked C  p  M  %  '  o  Intermediates f  C  o  n  t  r  o  l  100  1369  100  203  mM)  339  25  114  56  (0.4  mM)  739  54  17  8  (0.2  mM)  1114  81  95  46  (2.0  mM)  491  36  82  40  (0.4  mM)  1099  80  162  80  (0.2  mM)  1225  89  235  116  (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 I V .  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 o n t h e a c t i v i t y o f t h e c o u p l e d reaction f o rpeptidoglycan synthesis . 3  Inhibitor Concentration  Peptidoglycan CPM  % of Control  GCL-linked CPM  Intermediates % of Control  Control  (none)  3371  100  1081  100  .ppGpp  ( 2 . 0 mM)  2088  62  900  .83  ( 6 . 0 mM)  466  14  301  28  ( 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  ( 1 0 . 0 mM)  407  12  997  92  ( 1 2 . 0 mM)  168  5  424  39  ( 2 . 0 mM)  2755  82  923  85  ( 4 . 0 mM)  1073  32  490  45  ( 6 . 0 mM)  137  4  661  61  ATP  EDTA  I n c u b a t e d f o r 1 0 m i n u t e s w i t h a f i n a l KgCl^  concentration of 6  mM.  FIGURE 1 6 .  Effect  of the presence  ppGpp on t h e a c t i v i t y D-alanylalanine MnCl„.  ligase  ( • ) or absence  of D-alanylalanine ( B ) assayed  ( 0 ) o f 4 mM  s y n t h e t a s e ( A ) and  i n t h e p r e s e n c e o f 4 mM  Minutes  65  FIGURE 17.  (A)  E f f e c t of M n C l  i n UDP-MurNAc-pentapeptide  on t h e . a c c u m u l a t i o n o f I RjUMP 3  2  by t h e exchange a s s a y .  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 2 mM ppGpp a t t h e i n d i c a t e d M n C l of  the molar r a t i o  calculated  of MnCl  2  2  R e a c t i o n s were  ( • ) and a b s e n c e  concentrations.  (B)  The  ( 0 ) of influence  t o ppGpp o n t h e e x c h a n g e v a l u e s was  as t h e percent of the accumulation i n the presence 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 a b s e n c e o f ppGpp.  67 III.  Analysis A.  of  stringent control during  D i s t r i b u t i o n of  i n vivo peptidoglycan  [ H]Dap incorporated  by  3  amino  synthesis.  acid-deprived  bacteria.. The  s i t e of  e x a m i n e d i n v i v o by peptidoglycan, linked  s t r i n g e n t r e g u l a t i o n of peptidoglycan  comparing  soluble nucleotide-linked  peptidoglycan  amino a c i d s .  This  c h r o m a t o g r a p h s and simultaneously a n a l y s i s of  intermediates was  done by  using  s t r a i n LD5.  precursors  p r e s e n c e and  absence of  samples of  the d e v e l o p i n g chromatography s o l v e n t  to  l a b e l e d components.  ( r e l A ) are  s u m m a r i z e d i n T a b l e V.  The  amounts of  +  the peptidoglycan  labeled  deprived  i n the  lipid  lysine-deprived  precursors  cultures  as c o n t r o l c e l l s .  basis linked  precursor  pool  f o r s t r i n g e n t c o n t r o l i s not precursors  reactions  (or both) of  i n peptidoglycan  The, r e l a x e d  c o n s i s t e n t l y 2-  than i n the  of  the  the  synthesis  (reactions  TCA-insoluble material  was  also a r e l a t i v e buildup zone m a t e r i a l .  during of  3Lysine-  UDP-MurNAc-  nucleotide-linked  either soluble  10  cells.  nucleotide-linked  s i t e of  enzymes c a t a l y z i n g t h e  s t r a i n LD5456 ( r e l A ) c o n t i n u e d  and  to  These r e s u l t s suggest t h a t  l i m i t a t i o n of  Dap  by  controls.  In both conditions,  or GCL-P-P-MurNAc-pentapeptide; the  a l m o s t c e r t a i n l y one  lipid  (Table V I ) .  fractions  lysine  t h e amounts i n c o n t r o l  a t l e a s t a s much l a b e l e d  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% peptidoglycan  of  z o n e m a t e r i a l was  c e l l s also contained  peptidoglycan  typical  e f f e c t s of  The  higher  paper  The  c e l l s were a p p r o x i m a t e l y 30%  fold  required  i s s h o w n ' i n f i g u r e 18.  +  i n t o t h e T C A - i n s o l u b l e and  of  GCL-  (relA )  lysine-deprived quantity  A  the  and  to  s e p a r a t e any  was  into  3  peptidoglycan  i n the  loading  [ H]Dap incorporated  intact cells  e x t r a c t and  d e p r i v a t i o n on LP5 incorporated  t h e amount o f  formation  and  11,  to accumulate  lysine deprivation  the n u c l e o t i d e - l i n k e d  the  nucleotide-  inhibition last  two  f i g u r e 2).. peptidoglycan  (Table V I I ) . precursors  and  There the  is  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  of  LD5  + ( r e l A ) w e r e l a b e l e d w i t h [ H j D a p and t h e n c h r o m a t o g r a p h e d 3  solvent system I . 0.9%  Growing c e l l s were h a r v e s t e d , washed once  s a l i n e and t r a n s f e r r e d  added and a f t e r  t o f r e s h D a p - f r e e M9_.  c h r o m a t o g r a p h was  t o Whatman 3 MM  with  [ H ] D a p was 3  60 m i n u t e s a p o r t i o n o f t h e c u l t u r e was  t r a t e d and a p p l i e d  in  chromatography paper.  concenThe  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 to s o l u b l e nucleotide-linked  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 [ H ] D a p and P e a k D i s t h e l i p i d 3  zone.  A  C  cm  from  Origin  TABLE V.  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 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  3  10 * CPM 1  Culture  Conditions  T o t a l TCA Insoluble  C  p e r mg  Nucleotide-linked Precursors  C e l l Dry Lipid  Wt. Zone  Peptidoglycan^  30 m i n u t e s Control Minus  Lysine  10.44  (100%)  0.62  (100%)  0.08  (100%)  10.74  (100%)  3.12  (30%)  0.90  (145%)  0.19  (238%)  3.18  (30%)  20.84  (100%)  0.72  (100%)  0.23  (100%)  21.92  (100%)  6.02  (29%)  1.00  (139%)  0.82  (357%)  4.46  1(20%)  60 m i n u t e s Control Minus  Lysine  C e l l s w e r e p r e p a r e d and g r o w n a s 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 a s 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 s a m p l e s o f w h o l e c e l l s i n s o l v e n t s y s t e m A a n d 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 peptidoglycan. C o u n t s w e r e 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 c o m p e n s a t e f o r d i f f e r e n c e s i n t h e amount o f e a c h sample and t o f a c i l i t a t e c o m p a r i s o n o f t h e v a l u e s . ^Values  i n brackets  are percent  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 c o l u m n , b e f o r e 555; 1 9 5 ; 1,479; and 386.  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 , w e r e :  T h e r e s p e c t i v e CPM f o r t h i s c o l u m n , 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 , w e r e : 2 2 , 3 7 9 ; 5,850; 62,307; and 9,968.  71  TABLE V I .  A n a l y s i s of s o l u b l e nucleotide-linked peptidoglycan p r e c u r s o r s s y n t h e s i z e d b y LD5 ( r e l A ) d u r i n g l y s i n e deprivation . 3  10 * CPM p e r mg C e l l D r y 1  n ~ n A ' *-' Culture Conditions  T o t a l Labeled Precursors  UDP-MurNAcpentapeptide  C  Wt.  b  UDP-MurNActripeptide  30 m i n u t e s Control  0.86  0.83  (97%)  0.03  :(3%)  Minus  0.66  0.62  (95%)  0.04  (5%)  Control  0.87  0.85  (98%)  0.02  (2%)  Minus  0.83  0.78  (94%)  0.05  (6%)  Lysine  60 m i n u t e s  Lysine  The c e l l s w e r e p r e p a r e d a n d g r o w n a s 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 z o n e s 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 . ^Values i n brackets precursor pools.  are percent  of the t o t a l r a d i o a c t i v i t y i n the  The r e s p e c t i v e CPM f o r t h i s c o l u m n , 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 mass o f t h e s a m p l e s , w e r e : 1,755; 1,615; 2,440; a n d 2,339.  TABLE V I I .  D i s t r i b u t i o n of [ H]Dap i n c o r p o r a t e d (relA )during lysine deprivation . 3  IO * CPM 1  ~ — Culture  0  Condxtions  T o t a l TCA .. , .. Insoluble  p e r rag C e l l D r y  Nucleotide-linked _ Precursors  by LD5456  Wt.  b  L i p i d Zone v  Peptidoglycan^ r o j  30 m i n u t e s Control  10.04  (100%)  0.58  (100%)  0.09  (100%)  8.74  (100%)  Minus  11.85  (118%)  2.13  (367%)  0.53  (588%)  8/43  (96%)  Control  20.01  ;(ioo%)  0.63 •  (100%)  0.28  (100%)  17.77  (100%)  Minus  18.30  (91%)  3.28  (521%)  2.26  (807%)  12.93  (73%)  see Table  V.  Lysine  60 m i n u t e s  For  Lysine  t h e d e t a i l s of t h e methods,  Values In brackets  are percent of the  control.  c The r e s p e c t i v e CPM f o r t h i s c o l u m n , b e f o r e were: 3 6 8 ; 605; 1,062; and 1,007.  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 mass o f t h e s a m p l e s ,  ^The r e s p e c t i v e CPM f o r t h i s c o l u m n , b e f o r e were: 1 8 , 9 4 9 ; 1 6 , 1 9 8 ; 4 4 , 2 6 7 ; and 23,387.  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 mass o f t h e s a m p l e s ,  73  The d i f f e r e n c e b e t w e e n t h e amount o f p e p t i d o g l y c a n by t h e l y s i n e - d e p r i v e d autolysis. contained  s t r i n g e n t and r e l a x e d  cells  i s n o t due t o  The s u p e r n a t a n t s f r o m t h e [ H J D a p l a b e l e d  These m a t e r i a l s  of possible  occurred  autolytic material  i n s i m i l a r amounts  t o t a l TCA-insoluble counts associated Other r e l A  pattern  with  s t r a i n s (LD52 a n d LD2)  +  o f r e s u l t s a s LD5  (relA ) +  g a v e t h e same  ( s e e T a b l e ix) •  z o n e m a t e r i a l was n o t .  ( T a b l e X) g a v e t h e same g e n e r a l However,  pattern  was  deprivation leucine  and was n o t o b s e r v e d d u r i n g  +  observed  treatment relaxed  (Table X I ) .  for lysine-deprived  l o w e r f o l l o w i n g CAM  precursor  strain  LD5457  (relA ). excessive  occurred during  lysine  deprivation  for  threonine,  the synthesis  of peptidoglycan  The d e g r e e o f r e l a x a t i o n was n o t a s h i g h  accumulation of peptidoglycan  resulted  peptidoglycan  s t r a i n s the  cells  o f LD5456  by l y s i n e - d e p r i v e d  treatment  (Table X I I ) .  since  (relA ) +  accumulation of labeled  CAM  s t r i n g e n t and r e l a x e d  zone m a t e r i a l  c e l l s deprived  previously  of l y s i n e .  the  treatment  ( T a b l e X I ) and p r e v e n t e d t h e lipid  as  that The  relaxed  c e l l s o f LD5456 ( r e l A )  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 i n LD5  i n LD5  ( r e l A ) (Table V i i ) .  d i f f e r e n c e m i g h t b e due t o s i d e e f f e c t s o f t h e CAM  was  reduced,  or methionine. CAM  (relA )  acid  significantly  o f r e s u l t s a s LD5456  zone o n l y  of  general  D u r i n g amino  The r e l a x e d  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  accumulation of l a b e l i n the l i p i d  stringent  more t h a n 1.5%  whereas the a c c u m u l a t i o n of s o l u b l e n u c l e o t i d e - l i n k e d and l i p i d  and  the c e l l s .  deprivation, the accumulation of peptidoglycan  precursors  only  (Table V I I I ) .  i n the relaxed  c u l t u r e s , and n o n e o f t h e e x a m i n e d s a m p l e s c o n t a i n e d the  cultures  3  s l i g h t amounts  accumulated  also  peptidoglycan  excessive observed  i n both  74  TABLE V I I I .  D i s t r i b u t i o n of labeled 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 . 10 Culture Conditions  LD5  CPM p e r mg C e l l D r y  3  .43 ' C  R  f  0  6  R  Wt.  b  0.62 ' d  (relA ) +  Control,  60 m i n u t e s  Minus L y s i n e , LD5456  60 m i n u t e s  3.12  (0.5%)  0.45  (0.1%)  2.60  (1.2%)  0.48  (0.2%)  5.26  (1.3%)  0.92  (0.2%)  3.23  (0.8%)  0.49  (0.1%)  (relA~)  Control,  60 m i n u t e s  Minus L y s i n e ,  60 m i n u t e s  " 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 [ H]Dap (see T a b l e V f o r d e t a i l s ) were c h r o m a t o g r a p h e d i n s o l v e n t s y s t e m I . The c h r o m a t o g r a p h s w e r e t h e n e x a m i n e d 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 n o t 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 the d r y weight o f c e l l s i n the 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. 3  ^ 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 o f t o t a l TCA i n s o l u b l e a c t i v i t y culture.  i n the  °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 . ^Possibly pentapeptide. T h e r e s p e c t i v e CPM f o r t h i s c o l u m n , 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 mass o f t h e o r i g i n a l c u l t u r e s , w e r e : 616; 4 9 9 ; 1,033; a n d 3 8 0 .  TABLE I X .  4-  ,  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 L D 5 2 ( r e l A ^ a n d ( r e l A ) d u r i n g 60 m i n u t e s o f a m i n o a c i d d e p r i v a t i o n . 10  k  _ — ,. . Culture Conditions  T  T o t a l TCA , Insoluble 1  1  c  „  CPM  p e r mg  Nucleotide-linked Precursors  C e l l Dry Lipid  LD2  Wt. Zone  Peptidoglycan^ r t> J  LD52 ( r e l A ) 15.94  Control  (100%)  0.48  (100%)  0.12  (100%)  15.09  (100%)  Minus  Lysine  5.20  (33%)  0.44  (92%)  0.35  (292%)  5.06  (34%)  Minus  Threonine  6.41  (40%)  0.89  (185%)  0.11  (92%)  6.64  (44%)  7.45  (47%)  0.64  (133%)  0.40  (333%)  6.45  (43%)  (100%)  0.96  (100%)  0.24  (100%)  37.89  (100%)  Minus L y s i n e Threonine  and  '. ( r e l A ) +  Control  37.60  Minus  Lysine  12.76  (34%)  1.31  (136%)  0.94  (392%)  8.97  (24%)  Minus  Methionine  11.45  (30%)  1.40  (146%)  0.22  (92%)  10.05  (27%)  10.23  (27%)  1.58  (165%)  0.96  (400%)  10.32  (27%)  Minus L y s i n e Methionine SL  For  .  and  the d e t a i l s of t h e methods,  ^Values i n brackets  s e e T a b l e V.  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 c o l u m n , 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 , w e r e : 1,198; 278; 3 4 5 ; 406; 2,734; 6 9 1 ; 741; and 7 4 3 . ^The r e s p e c t i v e CPM f o r t h i s c o l u m n , 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 , w e r e : 3 4 , 8 0 8 ; 9,032; 1 3 , 2 1 2 ; 11,819;. 72,859; 1 5 , 7 5 1 ; 1 8 , 6 9 5 ; a n d 1 9 , 5 2 7 .  TABLE X.  D i s t r i b u t i o n o f [ H ] D a p i n c o r p o r a t e d b y LD5457 (relA ) during l y s i n e or leucine d e p r i v a t i o n . 3  10** CPM  Culture  Conditions  T o t a l TCA Insoluble  p e r mg  Nucleotide—linked Precursors  C e l l Dry Lipid  Wt.  b  Zone  Peptidoglycan  30 m i n u t e s Control  8.75  (100%)  0.35  (100%)  0.08  (100%)  7.39  (100%)  12.81  (146%)  1.34  (386%)  0.61  (761%)  10.09  (137%)  8.91  (94%)  1.26  (363%)  0.10  (125%)  6.22  (84%)  Control  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 Leucine  15.49  (93%)  2.35  (560%)  0.10  (71%)  13.42  (87%)  Minus L y s i n e Minus L e u c i n e 60 m i n u t e s  a  For  t h e d e t a i l s of the methods, see T a b l e  ^Values i n brackets  V.  a r e percent 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 c o l u m n , b e f o r e 5 8 3 ; 648; 4 0 1 ; 1,589; 1,036; a n d 737.  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 s a m p l e s , w e r e :  ^The r e s p e c t i v e CPM f o r t h i s c o l u m n , 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 s a m p l e s , w e r e : 3 2 , 7 6 7 ; 3 0 , 1 0 8 ; 1 9 , 7 7 1 ; 7 2 , 5 8 2 ; 4 3 , 9 0 4 ; and 28,744.  TABLE X 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 a n d CAM t r e a t m e n t o n d i s t r i b u t i o n o f [ H ] D a p i n c o r p o r a t e d b y LD5 (relA ) . 3  +  a  10 * CPM 1  Culture  Conditions  T o t a l TCA Insoluble  p e r mg  Nucleotide-linked Precursors  C e l l Dry Lipid  Wt.  b  Zone  Peptidoglycan  d  30 m i n u t e s Control  26.40  (100%)  1.54  (100%)  0.53  (100%)  27.00  (100%)  Minus  11.58  •(44%)  1.94  (126%)  2.63  (496%)  9.11  (34%)  Minus L y s i n e ; P l u s CAM  18.53  (70%)  6.05  (393%)  0.66  (125%)  18.74  (69%)  Plus  18.80  (71%)  6.36  (413%)  0.39  (74%)  19.15  (71%)  Control  54.80  (100%)  1.67  (100%)  0.65  (100%)  56.53  (100%)  Minus  18.26  (33%)  2.90  (174%)  2,91  (448%)  15.51  (27%)  Minus L y s i n e ; P l u s CAM  29.73  (54%)  8.89  (532%)  0.67  (103%)  30.79  (54%)  Plus  31.20  (57%)  9.51  (569%)  0.48  (74%)  34.03  (60%)  see Table  V.  Lysine  CAM  60 m i n u t e s  For b  Lysine  CAM  the d e t a i l s  of t h e methods,  Values i n brackets  a r e percent 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 c o l u m n , 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 m a s s o f t h e s a m p l e s , w e r e : 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 c o l u m n , 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 m a s s o f t h e s a m p l e s , w e r e : 3 2 , 5 6 5 ; 8,869; 1 6 , 6 0 1 ; 1 7 , 0 2 1 ; 1 1 8 , 9 8 3 ; 1 5 , 1 3 8 ; 3 7 , 9 0 1 ; and 3 5 , 5 6 8 .  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 a n d CAM t r e a t m e n t o n d i s t r i b u t i o n of [ H]Dap i n c o r p o r a t e d by LD5456 (re!A~) . 3  a  10 * CPM p e r 1  Culture  T o t a l TCA Insoluble  Conditions  Nu c l eo t i d e-1 i n k e d Precursors  mg C e l l . D r y Lipid  Wt.  b  Zone  Peptidoglycan*  1  30 m i n u t e s Control  17.98  (100%)  1.38  (100%)  0.33  (100%)  17.63  (100%)  Minus  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%)  Plus  12.73  (71%)  6.55  (475%)  0.33  (100%)  15.62  (89%)  Control  35.45  (100%)  1.40  (100%)  0.90  (100%)  Minus  37.10  (105%)  5.71  (408%)  5.30  Minus L y s i n e ; P l u s CAM  20.41  (58%)  7.31  (522%)  Plus  21.36  (60%)  8.21  (586%)  Lysine  CAM  60 m i n u t e s  3.  For  Lysine  CAM  the d e t a i l s  "  34.53  (100%)  (589%)  27.42  (79%)  0.46  (51%)  20'. 11  (58%)  0.40  (44%)  20.44  (59%)  o f t h e m e t h o d s , s e e T a b l e V.  Values i n brackets are  percent of t h e 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 c o l u m n , 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 1,004; 1,090; 709; 623; 2,888; 1,773; 1,020; and 1,029.  i n mass o f t h e s a m p l e s ,  were;  The r e s p e c t i v e CPM f o r t h i s c o l u m n , 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 , w e r e ; 2 1 , 8 4 6 ; 1 9 , 0 6 3 ; 1 3 , 6 2 5 ; 1 5 , 4 4 6 ; 7 4 , 2 1 1 ; 3 0 , 7 2 4 ; 20,144; and 1 9 , 5 9 2 .  79  B.  E f f e c t of s t r i n g e n t GCL-linked  i n h i b i t i o n on the composition of the  intermediates.  The p r e c e d i n g r e s u l t s suggest that  the^incorporation  into peptidoglycan i s i n h i b i t e d during stringent or both o f the f o l l o w i n g the  reactions:  GCL-P-P-MurNAc-pentapeptide  stringent  response.  o f UDP-GlcNAc to  p r e c u r s o r t o the nascent  each GCL-linked i n t e r m e d i a t e d u r i n g the  o f LD52 ( r e l A ) +  lipid  XIII.  d i d not s i g n i f i c a n t l y a l t e r the  a b s o l u t e amount of GCL-P-P-MurNAc(-GlcNAc)-pentapeptide. analysis  acceptor  These p o s s i b i l i t i e s were  R e p r e s e n t a t i v e r e s u l t s a r e shown i n T a b l e  . Threonine d e p r i v a t i o n  one  ( r e a c t i o n 10, f i g u r e 2 ) ; ( i i ) the  i n the p e p t i d o g l y c a n ( r e a c t i o n 11, f i g u r e 2 ) . by q u a n t i t a t i n g  control at either  ( i ) the a d d i t i o n  subsequent t r a n s f e r of the l i p i d - b o u n d  distinguished  of Dap  However, the  i s p o t e n t i a l l y c o m p l i c a t e d by two u n i d e n t i f i e d components  zone.  constituted  One component was  s e n s i t i v e to mild  about 10% o f the l a b e l e d  d e p r i v e d o f amino a c i d s  lipid  acid hydrolysis.  zone m a t e r i a l  and about 15% of the l i p i d  The o t h e r u n i d e n t i f i e d component was  acid hydrolysis  and c o n s t i t u t e d  It  i n the c e l l s  zone m a t e r i a l  control cells.  i n the  i n the  r e s i s t a n t to mild  5- to 15% of the t o t a l l a b e l e d  lipid  material.  However, even i f a l l of these u n i d e n t i f i e d m a t e r i a l s  attributed  t o e i t h e r GCL-P-P-MurNAc-pentapeptide  zone  are  or GCL-P-P-MurNAc(-GlcNAc)  - p e n t a p e p t i d e , the r e l a t i v e p r o p o r t i o n s i n the s t r i n g e n t l y i n h i b i t e d are  still  s i m i l a r to the p r o p o r t i o n s i n the growing c e l l s ;  have been s u f f i c i e n t  GCL-P-P-MurNAc(-GlcNAc)-pentapeptide  a normal 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 n the amino  c u l t u r e even though p e p t i d o g l y c a n s y n t h e s i s These r e s u l t s c l e a r l y i n d i c a t e synthesis  that  was  the t e r m i n a l  cells  t h e r e should t o support  acid-deprived  a c t u a l l y i n h i b i t e d by  66%/  step i n peptidoglycan  ( r e a c t i o n 11, f i g u r e 2) i s i n h i b i t e d d u r i n g the s t r i n g e n t  response.  TABLE X I I I .  A n a l y s i s of the l a b e l e d components of t h e l i p i d a zone of c e l l s i n c u b a t e d w i t h [ H ] D a p f o r 60 m i n u t e s 3  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 (10 GCL-P-P•MurNAc(-GleNAc)pentapeptid  Culture Conditions LD52  GCL-P-PMurNAcpentapeptide  Unidentified  b  3  CPM  p e r mg  ResidualL i p i d Zone c Counts  C e l l Dry  Wt. )  N e t :L i p i d Zone Counts  Peptidoglycan*  (relA ) +  Control  0.71  0.49  0.32  0.25  1.72  (103%)  161.6  Minus Threonine  0.78  0.47  0.21  0.15  1.62  (106%)  70.9  Control  0.80  0.51  0.21  0.32  1.79  (105%)  256.3  Minus  2.03  0.68  Nil  3.44  5.97  (103%)  46.7  LD5  (relA ) +  Lysine  T h e amount o f e a c h 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 t h e l i p i d z o n e was d e t e r m i n e d b y h y d r o l y z i n g b o i l e d c e l l s i n 1 0 % 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 t h e amount o f c o u n t s i n t h e z o n e , b u t q u a n t i t a t i v e l y removed t h e 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 e n d o g e n o u s p o o l o f 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 f o r m s w h i c h c o u l d be s e p a r a t e d by c h r o m a t o g r a p h y i n s o l v e n t s y s t e m I . T h e n e t l i p i d z o n e c o u n t s and peptidoglycan w e r e d e t e r m i n e d w i t h s a m p l e s o f w h o l e c e l l s r e m o v e d 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 o u n t s w e r e 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 c o m p e n s a t e f o r d i f f e r e n c e s i n t h e amount o f e a c h s a m p l e and f a c i l i t a t e comparison of the v a l u e s . a  A f t e r mild c After mild system I .  b  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 R f 0.53 a c i d h y d r o l y s i s t h i s material continues  i n solvent  to migrate i n the l i p i d  ^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 o f t h e t o t a l e d o r i g i n a l 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 s y s t e m I . The r e s p e c t i v e CPM f o r t h i s 637; 1,239; and 1,204.  column, b e f o r 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 w e r e : 60,046; 21,537; 6 2 , 8 0 7 ; a n d 10,279.  system I .  the coorections  zone  lipid  (R^ 0.8)  i n solvent  zone components a f t e r m i l d  f o r d i f f e r e n c e s i n the sample s i z e s were:  638; 00  t h e 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 o f t h e  samples  o  81  Lysine-deprived (Table  X I I I ) except that  to mild the  zone.  However, t h i s  - p e n t a p e p t i d e i n the  ( r e l A ^ ) gave s i m i l a r r e s u l t s  t h e u n i d e n t i f i e d c o m p o n e n t w h i c h was  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  lipid  total  c u l t u r e s o f LD5  still  lysine-deprived  amount o f l a b e l e d l i p i d  t o 70%  of the  resistant  labeled material  in  a l l o w s more G C L - P - P - M u r N A c ( - G l c N A c ) c e l l s than i n the  z o n e m a t e r i a l was  controls since  much l a r g e r t h a n i n  the  the  controls. Since  the  s e n s i t i v e to mild  GCL-linked peptidoglycan  acid hydrolysis  unidentified material was of  not the  lipid  GCL-linked peptidoglycan lipid  because: (10,  i n the  zone m a t e r i a l  (i) proteins  107),  ( i i ) CAM,  intermediates.  co-migrate with  inhibitor  ( i i i ) i f the  d e c a r b o x y l a s e , Dap incorporated T a b l e XIV increase  was  c e l l s was  s e n s i t i v e to mild  47%  protease,  of the  removed.  (relA ) +  sensitivity  Finally,  then hydrolyzed  from c o n t r o l c e l l s  would  t h e n be  +  hydrolyzed. no  i f the by m i l d  When t h e  e f f e c t on  lipid  from the  the  lipid  lysine-deprived  and  a  shown i n 5-fold zone  the  zone  material  samples from c o n t r o l  lysine-deprived  z o n e m a t e r i a l was  a c i d , 78%  of  readily  As  of t h i s l i p i d  a c i d h y d r o l y s i s , whereas 80%  zone m a t e r i a l  and  diaminopimelate  (relA ) resulted in nearly Only 31%  the  lysine  absence of exogenous l y s i n e .  t h e r e was  lipid  examined  intermediates  zone m a t e r i a l d u r i n g  zone m a t e r i a l .  very  the  the  GCL-linked  c o n v e r t e d t o l y s i n e and  f r o m c o n t r o l c e l l s was  p r o t e a s e and material  be  in labeled l i p i d  c e l l s , but  the  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  i n t o p r o t e i n i n the  treated with  Therefore,  be  of p r o t e i n s y n t h e s i s , prevented  l y s i n e d e p r i v a t i o n o f LD5  m a t e r i a l was material  could  possible that  z o n e o f l y s i n e - d e p r i v e d LD5  excess accumulation of l a b e l e d l i p i d deprivation,  i t was  should  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  can  an  ( 1 , 9)  intermediates  87%  treated  of the  c e l l s was  with  respective  released.  82  TABLE X I V .  E f f e c t o f 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 t h e l a b e l e d m a t e r i a l of the l i p i d zone of LP5  Radioactivity in Lipid ( 1 0 CPM p e r mg C e l l D r y 2  on (felA ) .  Zone. Wt.)  Treatment Control None Acetic Acid  Hydrolysis  Protease Protease then A c e t i c Hydrolysis  Minus  Lysine  6.41  (100%)  29.08  (100%)  1.28  (20%)  20.07  (69%)  6.99  (109%)  15.41  (53%)  1.41  (22%)  4.94  (17%)  Acid  " C e l l s w e r e g r o w n and p r e p a r e d a s d e s c r i b e d i n f i g u r e 3. After sixty m i n u t e s o f i n c u b a t i o n t h e c e l l s w e r e 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 c h r o m a t o g r a p h y i n s o l v e n t s y s t e m I . The a c e t i c acid hydrolysis i s described i n Table XIII. The p r o t e a s e t r e a t m e n t was d o n e 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 m l c o n c e n t r a t i o n 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 . The c o u n t s i n the l i p i d zone w e r e 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 o f t h e v a l u e s and c o m p e n s a t e f o r d i f f e r e n c e s i n t h e mass o f t h e s a m p l e s f r o m t h e two c u l t u r e s . ^Values i n brackets untreated samples.  are percent  radioactivity  r e l a t i v e to  the  The r e s p e c t i v e CPM i n t h e l i p i d z o n e s o f t h e u n t r e a t e d s a m p l e s o f c o n t r o l and m i n u s 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 s a m p l e m a s s , w e r e 618 and 2,551.  the  83  T h e r e f o r e , much o f  the  of  cells  lysine-deprived  and  i s probably The  lysine. o f LD5 The  ( r e l A ) and  the  amounts of  accounted  cells  contained  Most of  the  f o r i f i t was  [ HJlys ine occurred d  i n the  deprivation l y s i n e has XV).  No  the  final  [ ^ j l y s i n e was  samples of the e i t h e r LD5  6N  HCl  t i m e s more i n the  synthesis  effect that  (Table  XV).  [ Hjlysine 3  lysine-deprived  (Table XV).  or a l t e r  subtracting  the  the  Insufficient  t o t a l amount  c e l l s or  +  the  peptidoglycan  that  lysine  released  boiling  affect  conclusion  i n column three  by  cells  co-migrated  amount o f p e p t i d o g l y c a n  soluble nucleotide-linked  Stringent  i n samples  labeled p r o t e i n which  intermediates  contain  samples of whole c e l l s to s i g n i f i c a n t l y  ( r e l A ) o r LD5456 C.  with  i s s t r i n g e n t l y controlled during  (compare the on  t o 10  i n the  the measurements of p e p t i d o g l y c a n synthesis  5-  [^lysine  GCL-linked peptidoglycan  peptidoglycan  zone  digestion  [ ^ J l y s i n e were d e t e c t e d  LD5456 ( r e l A ~ ) h y d r o l y z e d  +  than growing c e l l s .  with  s e n s i t i v e to protease  lipid  [ ^ j D a p used i n a l l of t h e s e experiments d i d not  However, s m a l l  be  seemed t o be  i n the  protein.  lysine-deprived  could  acetic acid-resistant material  of  of  Table  hydrolyzing  precursors  from  (relA~).  c o n t r o l of  soluble nucleotide-linked  peptidoglycan  precursors. The  preceding,  r e s u l t s indicate that  stringent cells  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 f r o m GCL ( r e a c t i o n 11, sites  f i g u r e 2)  i n peptidoglycan  regulation.  The  and  60 m i n u t e s  amino a c i d d e p r i v a t i o n .  synthesis  l e v e l of  amino a c i d - d e p r i v e d 30  during  are  soluble  a l s o a f f e c t e d by  peptidoglycan  stringent cells (Table XVI).  to the  inhibit  nascent  the  acceptor  However, o t h e r stringent  precursors  i n growing  remained r e l a t i v e l y constant  H o w e v e r , 60 m i n u t e s o f CAM  or  between  treatment,  84  TABLE XV.  A m o u n t s o f 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 HJDap. 3  a  10  k  I H]lysine 3  Culture  LD5  CPM b  p e r mg C e l l D r y L i p i d Zone Protein  Wt.  Peptidoglycan 0  (relA ) +  Control  0.035  0.041  21.62  Minus  0.520  0.485  8.28  Control  0.050  0.013  21.92  Minus  0.370  0.245  15.40  LD5456  Lysine  (relA~)  Lysine  C e l l s w e r e p r e p a r e d a n d g r o w n a s 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 w e r e c h r o m a t o g r a p h e d i n s o l v e n t s y s t e m I I I and t h e r a d i o a c t i v i t y i n t h e l y s i n e z o n e was m e a s u r e d . 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 z o n e 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 s y s t e m s I , I V , and V. L i p i d z o n e p r o t e i n and p e p t i d o g l y c a n w e r e m e a s u r e d i n a n o t h e r s a m p l e , a s d e s c r i b e d i n T a b l e s X I I I and X I V . The l i p i d z o n e 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 intermediates 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 a n d 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 hydrolysis. A l l c o u n t s w e r e n o r m a l i z e d t o t h e c e l l mass t o c o m p e n s a t e f o r d i f f e r e n c e s i n t h e a m o u n t s o f t h e s a m p l e s and f a c i l i t a t e c o m p a r i s o n . T h e r e s p e c t i v e CPM f o r t h i s c o l u m n b e f o r e t h e n o r m a l i z a t i o n c o n s i s t e n t s a m p l e m a s s , w e r e : 3 0 6 ; 2,368; 344; a n d 1,671.  to a  c The r e s p e c t i v e CPM f o r t h i s c o l u m n 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 s a m p l e m a s s , w e r e : 9 6 , 0 4 8 ; 1 8 , 9 1 7 ; 7 4 , 8 5 0 ; and 3 4 , 8 5 0 .  TABLE X V 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 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 treatment. 3  10  Culture Conditions  30  t  a  l  L  a  b  e  l  e  p e r mg  C e l l Dry  UDP-MurNAcpentapeptide  d  Wt.  UDP-MurNActripeptide  minutes Control  0.86  0.83  (97%)  0.03  (3%)  Minus L y s i n e  0.66  0.62  (95%)  0.04  (5%)  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%)  Minus  60  * ° Precursors  CPM  4  Lysine;  ,  minutes Control  0.87  0.85  (98%)  0.02  (2%)  Minus L y s i n e  0.83  0.78  (94%)  0.05  (6%)~  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%)  Minus  Lysine;  "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 w e r e m e a s u r e d a s 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 t h e c h r o m a t o g r a p h s w e r e d e v e l o p e d l o n g e r i n order to improve r e s o l u t i o n . ^Values i n brackets precursor pools.  are percent  of the  total radioactivity  in  The r e s p e c t i v e CPM f o r t h i s c o l u m n , 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 m a s s o f t h e ^ s a m p l e s , w e r e : 1,755; 1,615; 7,159; 7,295; 2,440; 2,339; 1 1 , 5 9 7 ; and 1 2 , 4 6 3 .  the  86  e i t h e r alone 4-  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  to 5 - f o l d accumulation  c e l l s had  a constant  precursors  of p r e c u r s o r s .  level  (Table X V I I ) .  or a combination  S i m i l a r l y , the  H o w e v e r , a m i n o a c i d d e p r i v a t i o n , CAM CAM  treatment,  of n u c l e o t i d e - l i n k e d peptidoglycan  30 m i n u t e s and  (data not  then examined a f t e r  presented).  t o be  UDP-MurNAc-pentapeptide  (68, 70,  examined.  feedback  110).  Therefore,  (DCS)  of the l e v e l  to 111%,  The  to i n h i b i t  i n unstarved  a d d i t i o n o f DCS  by  the e f f e c t of  increased to over 15 m i n u t e s  the  (relA ) cells  the were  +  cells DCS  (Table X V I I I ) .  treatment  t o C A M - t r e a t e d LP5 the  only  (Table XIX).  The  found  In  increased +  s i z e of the  In accordance w i t h the preceding  2.5-fold the l e v e l  pool  results,  (relA  i n untreated control  a d d i t i o n o f DCS  n e a r l y 7.5-fold the c o n t r o l  ) cells  to the 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 e v e n m o r e ; b y m i n u t e s t h e p o o l was  had  (relA )  the 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  by  the  t h e f o r m a t i o n o f UDP-MurNAc-  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  measured i n CAM-treated c e l l s .  had  the  the s t r i n g e n t r e g u l a t i o n of  c o n t r a s t , the l y s i n e - d e p r i v e d c e l l s without  the  of  Dap  the pool of n u c l e o t i d e - l i n k e d peptidoglycan precursors  i n c r e a s e d t o 175%  increased  inhibition  F i f t e e n m i n u t e s a f t e r LD5  treated with D-cycloserine  the pool  3  not a f f e c t e d .  governed by  a b s e n c e o f U D P - M u r N A c - p e n t a p e p t i d e on  pentapeptide,  i H]Dap  s i z e of the pools 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 thought  p o o l s i z e was  a l l allowed  i n m e d i a l a c k i n g any  In a l l cases, the r e l a t i v e composition  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 The  growing  treatment,  precursors.  S i m i l a r r e s u l t s w e r e o b t a i n e d when c e l l s w e r e p r e - l a b e l e d w i t h for  relaxed  of s o l u b l e n u c l e o t i d e - l i n k e d peptidoglycan  o f a m i n o a c i d d e p r i v a t i o n and  a rapid accumulation  growing  a  level.  15  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 peptidoglycan 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 treatment. " 3  10  Culture  Conditions  4  CPM p e r  Total Labeled Precursors  . b mg C e l l D r y Wt  UDP-MurNAc.. c pentapeptide  UDP-MurNActripeptide  30 m i n u t e s Control  0.43  0.41  (95%)  0.02  (5%)  Minus  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%)  Plus  2.43  2.24  (87%)  0.19  (13%)  Control  0.45  0.40  (89%)  0.05  (11%)  Minus  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%)  Plus  3.70  3.21  (87%)  0.4 9  (13%)  Lysine  CAM  60 m i n u t e s  "For  Lysine  CAM  the d e t a i l s  of the methods, see Table XVI.  ^Values i n brackets precursor pools.  are percent  of the t o t a l r a d i o a c t i v i t y  i n the  The r e s p e c t i v e CPM f o r t h i s c o l u m n , 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 , w e r e : 1,623; 6,087; 6,088; 7,293; 2 , 6 0 1 ; 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 t h e 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  IO  Culture  4  CPM  Total Soluble Precursors  Conditions  p e r mg  C e l l Dry  Wt.  UDP-MurNAcpentapeptide  UDP-MurNActripeptide  15 m i n u t e s Control  0.95  (100%)  0.91  0.04  Minus L y s i n e  1.05  (111%)  0.98  0.06  plus D-cycloserine  1.66  (175%)  0.03  1.63  P l u s CAM  2.75  (289%)  2.72  0.03  2.57  0.05  2.62  (276%)  6.63  (698%)  0.02  6.61  D-cycloserine  5.14  (541%)  0.18  4.96  Plus D-cycloserine  6.21  (654%)  0.08  6.13  0.86  (91%)  0.78  0.08  Minus L y s i n e ;  Minus L y s i n e ; P l u s CAM P l u s CAM; p l u s D-cycloserine Minus L y s i n e ; p l u s CAM; p l u s  30 m i n u t e s Control  "The d e t a i l s o f t h e m e t h o d s a r e t h e same a s 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 a d d e d t o some c u l t u r e s b e f o r e t h e [ H ] D a p was a d d e d . T h e 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 b y 30 m i n u t e s s o t h e s a m p l e s w e r e 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 . 3  ^Values i n brackets  are percent  o f 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 iCPM 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 , w e r e : 1,771; 1,840; 3,125; 5,735; 6,328;. 1 0 , 7 6 0 ; 1 2 , 3 0 0 ; 1 2 , 1 2 8 ; a n d 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 pool of s o l u b l e n u c l e o t i d e - l i n k e d geptidog l y c a n p r e c u r s o r i n LD5456 ( r e l A ) .  10 * CPM p e r mg C e l l D r y Wt. 1  Culture  Total Soluble Precursor  Conditions  UDP-MurNAcpentapeptide  UDP-MurNAc. tr.ipeptide  15 m i n u t e s Control  0.78  (100%)  0.71  0.07  Minus L y s i n e  2.07  (265%)  1.90  0.18  (644%)  0.08  4.94  (495%)  0.07  3.79  (110%)  0.78  0.08  Minus Plus  Lysine;  D-cycloserine  5.02  Plus D-cycloserine  3.86  30 m i n u t e s  0.86  Control  For  the d e t a i l s  o f t h e methods r e f e r  ^Values i n brackets  are percent  t o T a b l e s X V I and X V I I I .  o f 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 .  T h e r e s p e c t i v e CPM f o r t h i s c o l u m n , 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 , w e r e : 3,558; 9,783; .19,193; 1 6 , 4 8 2 ; a n d 4,142.  90  The a b o v e d a t a peptidoglycan precursors r e q u i r e d amino a c i d s . deprived  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 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  The t o t a l p o o l a l s o e x p a n d s i n t h e a m i n o  s t r i n g e n t c e l l s when t h e l e v e l  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  o f UDP-MurNAc-pentapeptide i n  accumulation  of nucleotide-linked  p e p t i d o g l y c a n p r e c u r s o r s was e v e n g r e a t e r when t h e l e v e l pentapeptide size  was d e c r e a s e d  acid-  i n relaxed c e l l s .  This  o f UDP-MurNAc-  indicates that the  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 b y s t r i n g e n t  control.  91  DISCUSSION The p r e c e d i n g  results  is stringently controlled  indicate that peptidoglycan  i n E_^_ c o l i .  The r a t e o f  synthesis  peptidoglycan  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 r e q u i r e d amino a c i d s . the untreated  However, t h e r a t e r e m a i n s s i m i l a r  to the rate i n  c o n t r o l when t h e a m i n o a c i d - d e p r i v e d c e l l s  t y p i c a l l y r e l a x e d b y e i t h e r CAM  treatment  of  a r e pheno-  o r by mutations  i n the r e l A  gene. A t t e m p t s w e r e 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 of peptidoglycan  synthesis using i n v i t r o  systems.  inhibition  Experiments  with  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  inhibit  phospho-N-acetylmuramoyl-pentapeptide 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 ) . peptidoglycan is  A later  step, the t r a n s f e r of the  intermediates to the nascent acceptor  also inhibited.  This later  activity  GCL-linked  (reaction 11, figure 2 ) ,  s t e p seems more s e n s i t i v e t o ppGpp  the phospho-N-acetylmuramoyl-pentapeptide t r a n s l o c a s e since  than  GCL-linked  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 peptidoglycan  s y n t h e s i s i s i n h i b i t e d by 50%.  intermediates are probably  not GCL-P-P-MurNAc-pentapeptide.  P-P-MurNAc-pentapeptide i s u n s t a b l e preparations  The a c c u m u l a t e d  i n the i n v i t r o  assays  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 ) .  clear.  The  GCL-  u s i n g membrane  ( 5 2 ) a n d d o e s 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 a n d 11 ( f i g u r e 2) a r e i n c u b a t e d  physiological  GCL-linked  However, t h e  s i g n i f i c a n c e o f i n h i b i t i o n b y ppGpp i n t h i s  S i n c e ppGpp  magnesium,- t h e ppGpp  can c h e l a t e and b o t h c o u l d be i n h i b i t i n g  c h e l a t i o n o f magnesium.  .without  of the i n h i b i t e d  system i s not reactions require  t h e enzyme r e a c t i o n s by  92  Phospholipid  synthesis  o f ppGpp w h i c h i n h i b i t  i s i n h i b i t e d i n v i t r o b y t h e same l e v e l s  peptidoglycan  synthesis  v i t r o peptidoglycan  synthesis, the r e l a t i v e  generally  as the molar r a t i o  increases  However, a t m o l a r r a t i o s o f M g C ^ i n h i b i t i o n begins to decrease. used t o argue a g a i n s t phospholipid  i n h i b i t i o n b y ppGpp  of MgC^  t o ppGpp d e c r e a s e s .  This  decrease i n i n h i b i t i o n has been t h a t ppGpp i n h i b i t 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  magnesium a v a i l a b l e s h o u l d  Like the i n  t o ppGpp b e l o w 1.0 t h e r e l a t i v e  the p o s s i b i l i t y  synthesis  (65, 76).  be c h e l a t e d  i n vitro  amount o f  more r e a d i l y ( 6 5 ) .  A similar  e f f e c t c a n b e 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 e x c h a n g e for phospho-N-acetylmuramoyl-pentapeptide translocase the molar r a t i o  apparent decrease i n r e l a t i v e when t h e c o n s t a n t  differential  but s t i l l  i n the magnesium-limited c o n t r o l reactions.  i n the diminishing Alternatively,  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 b y ppGpp a n d l a c k o f  argue against  the  i s an a r t e f a c t which r e s u l t s  i s d i v i d e d by the h i g h e r ,  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  synthesis  against  However, t h e  (or s l i g h t l y decreasing) rate of a c t i v i t y  ppGpp-treated r e a c t i o n s rate of a c t i v i t y  inhibition  reaction  i s plotted  o f m a g n e s i u m t o ppGpp i n t h e r e a c t i o n .  •..'-; ,  the p o s s i b i l i t y  by c h e l a t i o n  i n vitro reactions  did not i n h i b i t  (65).  which c h e l a t e s , have been used t o  t h a t ppGpp i n h i b i t s  i n vitro  Similar observations  f o r peptidoglycan  can again  synthesis.  the D-alanylalanine  b e made w i t h  Low l e v e l s o f ATP  the coupled r e a c t i o n f o r peptidoglycan  addition, neither  phospholipid  l i g a s e nor the  synthesis.  In  D-alanylalanine  s y n t h e t a s e a r e i n h i b i t e d b y a m o l a r r a t i o o f m a n g a n e s e t o ppGpp a t w h i c h the is  exchange r e a c t i o n f o r phospho-N-acetylmuramoyl-pentapeptide i n h i b i t e d by 93%.  substrates,  However, t h e r e l a t i v e a f f i n i t i e s  and t h e n u c l e o t i d e s  translocase  o f t h e enzymes, t h e  f o r t h e m a g n e s i u m o r m a n g a n e s e 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 i s 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 i n 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 i n vivo data indicate that stringent amino acid-deprived cells accumulate proportionally less peptidoglycan than equivalent amounts of growing cells.  This effect i s probably not due to anomalies in the uptake  of the exogenous labeled Dap since a similar effect i s 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 i s no turnover of E. c o l i peptidoglycan (17, 18, 88, 90). In addition, the amino acid-  94  deprived  c e l l s do n o t seem t o b e d e f i c i e n t  peptidoglycan  precursors.  glycan i s inhibited pentapeptide  This suggests  i n e i t h e r s o l u b l e or  GCL-linked  that the synthesis of peptido-  a t t h e s t e p i n which t h e GCL-P-P-MurNAc(-GlcNAc)-  i s t r a n s f e r r e d to the nascent acceptor  ( r e a c t i o n 11,  figure 2). Lysine-deprivation i n stringent cells results accumulation expected  slight  o f G C L - P - P - M u r N A c ( - G l c N A c ) - p e n t a p e p t i d e , a s w o u l d be  i f the subsequent step i n p e p t i d o g l y c a n  However, t h e s l i g h t pentapeptide  synthesis i s inhibited.  i n c r e a s e i n t h e amount o f G C L - P - P - M u r N A c ( - G l c N A c ) -  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  peptidoglycan  a c c u m u l a t e d a n d was n o t o b s e r v e d  deprivation.  The a b s e n c e o f e x t e n s i v e a c c u m u l a t i o n  peptidoglycan  intermediates  during  threonine  of  GCL-linked  i s p o s s i b l y due t o a l i m i t i n g  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 It  in a  amount o f  synthesis  (46, 8 6 ) .  i s a l s o p o s s i b l e that the phospho-N-acetylmuramoyl-pentapeptide  translocase observed  ( 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 ,  i n vitro.  I t should  be noted  as  t h a t t h e p r o p o r t i o n o f GCL-P-P-  MurNAc-pentapeptide t o GCL-P-P-MurNAc(-GlcNAc)-pentapeptide i n both the 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  coli  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 The c o m p o s i t i o n precursors in  either  i s apparently  i s similar  to that  observed  lysodeikticus (1).  of the pool of n u c l e o t i d e - l i n k e d peptidoglycan n o t a l t e r e d by amino a c i d d e p r i v a t i o n  stringent, or relaxed strains o f . b a c t e r i a .  The r a t i o o f  t h e amount o f U D P - M u r N A c - p e n t a p e p t i d e t o t h e amount o f U D P - M u r N A c - t r i p e p t i d e i s reasonably E. c o l i  constant  and i s s i m i l a r  t o the p r o p o r t i o n s observed  i n other  ( 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  precursors  i s also relatively  constant  i n both  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 . Dap a u x o t r o p h s fifths  growing  This pool i s r e l a t i v e l y  i n glucose minimal  small;  medium i n c o r p o r a t e a b o u t t h r e e -  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 e a c h m i n u t e .  On t h e o t h e r h a n d , 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 apparently large since the synthesis of peptidoglycan can continue f o r up  t o two g e n e r a t i o n s  i n t h e a b s e n c e o f e x o g e n o u s Dap.  c e l l s may h a v e a p u r p o s e 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  pool of n u c l e o t i d e - l i n k e d peptidoglycan p r e c u r s o r s . is  g e n e r a l l y thought  Therefore, the  t o be c o n t r o l l e d by f e e d b a c k  The s i z e o f t h i s  inhibition  (70, 111).  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 . (70)  reported that mutants apparently blocked  associated  pentapeptide  In addition, can i n h i b i t  f i g u r e 2) (110).  though feedback this bacterium  i nv i t r o  the f i r s t  i n some o f t h e membrane  s t u d i e s i n d i c a t e t h a t UDP-MurNAc-  r e a c t i o n i n E. c o l i  ( r e a c t i o n 1,  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 of B a c i l l u s cereus  (111) even  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 occur (111).  The d a t a i n T a b l e s  i nvivo for  X V I I I and X I X i n d i c a t e  d e p l e t i o n o f the UDP-MurNAc-pentapeptide p o o l s by treatment serine resulted  feedback linked 1,  i n continued accumulation  with  D-cycloThis  a physiological  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 the s y n t h e s i s of n u c l e o t i d e -  peptidoglycan precursors, possibly a t the f i r s t  reaction (reaction  This study a l s o i n d i c a t e s that the s i z e of the pool  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 UDP-MurNAc-pentapeptide continues +  that  of UDP-MurNAc-tripeptide.  evidence, t h a t UDP-MurNAc-pentapeptide i s probably  f i g u r e 2) ( 1 1 1 ) .  relA  Lugtenberg  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-  pentapeptide.  is.direct  pool  cells  . :  controlled.  t o a c c u m u l a t e when a m i n o a c i d - d e p r i v e d  a r e . r e l a x e d by treatment  w i t h CAM.  The U D P - M u r N A c - p e n t a p e p t i d e  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 are deprived  The  the  f e e d b a c k i n h i b i t i o n and  of n u c l e o t i d e - l i n k e d  stringent  peptidoglycan  of a r e q u i r e d  r e g u l a t i o n of  precursors  are  the  distinct  E a c h c o n t r o l seems t o h a v e some e f f e c t w i t h o u t t h e o t h e r . control i s eliminated increases are  but  does not  eliminated.  t o m a i n t a i n the The be  i n amino a c i d - d e p r i v e d increase  pool  at  the  t o be  amino a c i d d e p r i v a t i o n .  f e e d b a c k i n h i b i t i o n and  r e a c t i o n 1 i n f i g u r e 2. s i z e was  not  stringent  consequence of  c o n t r o l of the  the  However, the  determined.  s i t e o c c u r s a t some p o i n t The  might not  growing  the  pool  The  the  3  [ H]Dap. 3  The  d e c a r b o x y l a s e (114).  to  a  appears  speculate at of  i n d i c a t e that  formation  UDP-MurNAc-pentapeptide.  of  of the  d e p e n d e n t on (16)  s u c h as to the  secondary  formation cells  cells  of  small  are  s t r a i n s which were used  diaminopimelate  decarboxylase The  absence  the mutated l y s A gene  f e e d b a c k i n h i b i t i o n f o r any  and  r e s i d u a l diaminopimelate  [ H ] l y s i n e w h i c h was 3  the  phosphoenolpyruvate.  exogenous l y s i n e . of  a  amino a c i d - d e p r i v e d  l y s A m u t a t i o n i n the  I n a d d i t i o n , any  may  stringent control  p r o t e i n when l y s i n e - d e p r i v e d  cause c o m p l e t e . i n a c t i v a t i o n  eliminate  required  r e s u l t s only  contribute  l y s i n e would enhance d e r e p r e s s i o n  would a l s o  controls  subjected  s i z e m i g h t e v e n r e s u l t as  factors could  [ H]lysine-labeled  pools  cells.  stringent c o n t r o l act  s i t e of  p r i o r to the  even though the b a c t e r i a are of  the  I t i s possible to  s t r i n g e n t response i f the  A number o f  incubated with  the  Feedback i n h i b i t i o n  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  amounts of  either  amount o f U D P - M u r N A c - p e n t a p e p t i d e  the major c o n t r o l i n growing c e l l s .  pool  If  s i z e of  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  that both the  controls.  e x t e n t o b s e r v e d when b o t h  s i z e c h a r a c t e r i s t i c of  s t r i n g e n t r e g u l a t i o n over the an  the  synthesis  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  s e v e r e s t r e s s s u c h as  the  to the  cells,  amino a c i d .  synthesized  97  w o u l d n o t be d i l u t e d by The  unlabeled  l y s i n e and  l y s i n e - l a b e l e d p r o t e i n accounts  w o u l d be r e a d i l y  f o r some e x c e s s  TCA-insoluble  formed d u r i n g the i n i t i a l p e r i o d of l y s i n e d e p r i v a t i o n of the strains. migrates of  I t a l s o accounts i n the l i p i d  [ H ] l y s i n e was 3  not  utilized. material  relaxed  f o r some o f 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  zone of t h e chromatographs. sufficient  However, t h e  amount  to a f f e c t the c o n c l u s i o n t h a t the  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  than  c e l l s d e s p i t e t h e p r e s e n c e o f an  peptidoglycan  e q u i v a l e n t amount o f  the  amino  growing  precursors. 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 deprived  cells  to coordinate the s y n t h e s i s of the sacculus w i t h  m a c r o m o l e c u l a r syntheses.:  This  i s c o n s i s t e n t w i t h the proposal  s t r i n g e n t c o n t r o l system coordinates deficiency  ( 1 4 , 88,  generalized  102).  c e l l u l a r r e s p o n s e t o amino  that  I s h i g u r o , personal communication).  The  stringently  s t r i n g e n t c o n t r o l of  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  source  or shift-down  to  61).  synthesis  peptidocarbon  I t might a l s o respond  t h e g r o w t h r a t e - d e p e n d e n t c h a n g e s i n t h e b a s a l l e v e l o f ppGpp i n  growing c e l l s The  (14,  be  regulated  g l y c a n s y n t h e s i s s h o u l d be 61)  the  acid  However, t h e p r o p o s e d r e g u l a t i o n can not  of l i p o p o l y s a c c h a r i d e i s not  s t a r v a t i o n (14,  acid-  other  to the b i o s y n t h e s i s of the e n t i r e c e l l w a l l s i n c e the  of the 0-antigen (E.  s y n t h e s i s a l l o w s amino  latter  (61, 99).  N e i t h e r of  t h e s e p o s s i b i l i t i e s was  p o s s i b i l i t y would r e q u i r e that the p e p t i d o g l y c a n  respond t o minor changes i n i n t r a c e l l u l a r Some o f t h e known and a r e s u m m a r i z e d i n f i g u r e 19.  c o n c e n t r a t i o n s of  investigated. synthesis ppGpp.  presumed c o n t r o l s of p e p t i d o g l y c a n  The  few  c o n t r o l s shown c a n a c c o u n t f o r  c o o r d i n a t i o n o f 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 p r e c u r s o r s and Other c o n t r o l s probably  exist.  synthesis  It i s interesting  peptidoglycan.  that the s i t e s  of  FIGURE 1 9 .  S c h e m a t i c d i a g r a m o f some o f 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 . correspond  The c i r c l e 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 a r r o w s i n r e a c t i o n s 1 a n d 7 d e n o t e Inhibition.  precursors  feedback  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  reversible reaction. control.  numbers  a readily  The l a r g e h a t c h e d a r r o w s r e p r e s e n t  stringent  Note that the formation of n u c l e o t i d e - l i n k e d peptidoglycan 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  action forthis  stringent effect  i s still  unknown.  Cytoplasmic Reactions  Membrane-Associated Reactions  UDP NADP UDP-MurNAc  NADPH  v  Cell WallAssoc iated React i o n s  Phosphoenolpyruvate  N/ r  GCLP-P-MurNAc -pentapeptide.  I  GlcNAc Cell Wall Acceptor  UDP-GlcNAc  y UDP-GlcNAc enoylpyruvate  (£) L-Ala  ,  . ®Stringpnt  M U U 4 U U U  GCL-P-P-MurNAc-pentapeptide  Stringent Control  Cell Wall Acceptor Plus Precursor  UDP-MurNAc-L-Ala  UDP-MurNAc-L-ala-D-Glu-meso-Dap -D-Ala-D-Ala  -Glu  Control  \«GCL-P -  ^  GCLPP'  (UDP-MurNAc-pentapeptide) UDP-MurNAc-LAIa-D-Glu  UDP-MurNAc-L-Ala-D-Glu-meso-Dap  y©  meso Dap L-Ala' — = ^ 5 r D-Ala _T ©  • D-Ala-C  © vo VO  100  regulation are also the sites of action of many c e l l wall antibiotics. This may mean that some of the antibiotics work by interfering with the normal regulation of peptidoglycan  synthesis.  101  LITERATURE CITED  1.  A n d e r s o n , J . S . , M. M a t s u h a s h i , M.A. fiaskin a n d J . L . S t r o m i n g e r . 1967. " B i o s y n t h e s i s o f t h e p e p t i d o g l y c a n o f b a c t e r i a l c e l l w a l l s . I I . P h o s p h o l i p i d c a r r i e r s i n t h e r e a c t i o n sequence." J . B i o l . 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