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Structural and functional studies on the role of the outer membrane of Pseudomonas aeruginosa in resistance… Angus, Barbara Lee 1986

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STRUCTURAL AND MEMBRANE OF  FUNCTIONAL STUDIES ON THE ROLE OF THE PSEUDOMONAS AERUGINOSA IN RESISTANCE PERMEABILITY TO ANTIBIOTICS  OUTER AND  by Barbara B.Sc.  A  Lee  University  Angus  of B r i t i s h  1980  Columbia,  THESIS SUBMITTED IN PARTIAL FULFILLMENT OF REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  THE  in THE  FACULTY  OF  (Department  We  accept this standard  THE  of  thesis  UNIVERSITY  as  OF  September (ci)  Barbara  GRADUATE  Lee  STUDIES  Microbiology)  conforming  BRITISH  to  COLUMBIA  1986  Angus,  the  1986  required  In p r e s e n t i n g requirements  this thesis f o r an  of  British  it  freely available  agree that for  Library  s h a l l make  for reference  and  study.  I  for extensive copying of  h i s or  be  her  g r a n t e d by  shall  not  be  Hi CjZ-&l£ J 0L-O  The U n i v e r s i t y o f B r i t i s h 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3  Date  /an  OCT-  /ZA/  Columbia  /?&  of  further this  thesis  head o f  this  my  It is thesis  a l l o w e d w i t h o u t my  permission.  Department o f  the  representatives.  copying or p u b l i c a t i o n  f i n a n c i a l gain  University  the  s c h o l a r l y p u r p o s e s may  understood that  the  the  I agree that  permission by  f u l f i l m e n t of  advanced degree a t  Columbia,  department or for  in partial  written  ABSTRACT  The  intrinsic  examined This  using  outer to  mutant  structure.  a l l antibiotics  mutant  strain  carbenicillin  were  characterised, resistance two  conjugated  that  in  hypersusceptible  permeability  lactamase, permeable wild  type  permeable  had  responsible  a wild  type  One o f t h e s e  the  to B-lactam  other and  methods.  Using  t h e mutant  an a s s a y  strain  to the chromogenic parent  WT1.  N-naphthylamine  t h a n was  ii  and  f o r which antibiotic separately  genetic  background,  transconjugants  (P2-6)  t o a wide r a n g e o f  (P2-8)  was  partially antibiotics  Outer  membrane  was c h a r a c t e r i s e d by  three  involving periplasmic  beta-  M1 was f o u n d  t o be f i v e - f o l d  cephalosporin  nitrocefin  type  more  than i t s  mutant M1 was much more  f l u o r e s c e n t compound  wild  the  been  aminoglycoside  In a d d i t i o n ,  to the hydrophobic  and  partial  for  agent, trimethoprim.  o f t h e above s t r a i n s  agent,  strains,  had  to  using  recovered  Two o t h e r  with  not to the hydrophobic  different  these  of  P1-6),  selective  t o be p a r t i a l l y h y p e r s u s c e p t i b l e whereas  respect  hypersusceptible  and  t h e mutant M1)  into a strain  antibiotics,  but  the  (apparently  were a l s o c h a r a c t e r i s e d . found  as  was  strain.  permeability  t o be  (P1-4  to a l l a n t i b i o t i c s . loci  mutant  Two s p o n t a n e o u s r e v e r t a n t s  isolated  revealing  genetic  membrane  I t was f o u n d  gentamicin  hypersusceptibility  was  hypersusceptible  outer  tested.  or  aeruginosa  (M1) was c h a r a c t e r i s e d w i t h  susceptibility,  membrane  r e s i s t a n c e of  an a n t i b i o t i c  multiple  antibiotic  antibiotic  strain  WT1.  1-N-phenylIn c o n t r a s t ,  mutant the or  o u t e r membrane  to lysozyme  used  to  concluded  from  antibiotic more  confirm  that  susceptibility of  Partially  and c l a r i f y  these data  the o u t e r  to permeabilisation  by a m i n o g l y c o s i d e  ethylenediaminetetraacetate.  were  or  M1 was l e s s s u s c e p t i b l e  strain  these  antibiotics  r e s i s t a n t mutants results.  the mutations  It  responsible  i n t h e mutant were a c t i n g  membrane  of  constituents,  was for  upon  one  protein  and  lipopolysaccharide. Characterisation polyacrylamide  gel  of outer  electrophoresis  differences  between o u t e r membrane  and  strains.  mutant  lipopolysaccharide respect gels  to  Further  in  i t s composition,  that  at l e a s t three  mutant w h i c h c o n t r i b u t e d these  while  characterised.  was  to  partial  l o s s o f an o u t e r  on  porin  the  no  SDS-  observable  of the w i l d showed  type  that  M1 was a l t e r e d  on  the with  SDS-polyacrylamide From t h e s e d a t a ,  i t  mutations existed  i n the  susceptibility.  Two o f  f o r separate third that  susceptibility  by  alterations  mutation the  could  chief  i n strain  membrane-stabilising,  not  to be  alteration M1  involved  Mg -binding + +  lipopolysaccharide.  In three  separate  proposed  leading  site  strain  to a n t i b i o t i c  lipopolysaccharide,  antibiotic  proteins  mobility  m u t a t i o n s were r e s p o n s i b l e  It  showed  properties.  + +  proteins  examination  t h e mutant  and i n i t s M g - b i n d i n g  appeared  membrane  P_j_  aeruginosa  were  technique.  another  portion  o u t e r membrane  demonstrated P_^  of the study, n a t i v e  aeruginosa  using protein iii  proteins  a  and one E_;_  chemical F,  oligomers of coli  crosslinking  t h e major c o n s t i t u t i v e  outer  membrane  porin,  membranes and whole c e l l preparations  of  P_^_  starvation-induced) crosslinked  was c r o s s l i n k e d crosslinking  aeruginosa and  to r e v e a l  coli  protein  d i m e r s and t r i m e r s  SDS, native  F was a b o l i s h e d  indicating  that  associations  i n the outer  iv  F, PhoE  upon  P  in  Purified (phosphate also  two-dimensional Crosslinking  o f the p r o t e i n  p r o d u c t s were  membrane.  outer  ( I c ) were  analysis.  by p r e t r e a t m e n t  the c r o s s l i n k e d  dimers  experiments.  proteins  SDS-polyacrylamide g e l e l e c t r o p h o r e t i c of p r o t e i n  to  due  with to  TABLE  OF  CONTENTS  Page ABSTRACT  i i  TABLE OF CONTENTS  v  List  of Tables  x  List  of Figures  List  of Abbreviations  x i i xiv  ACKNOWLEDGEMENTS  xv  INTRODUCTION  1  1.  A n t i b i o t i c r e s i s t a n c e and m e d i c a l o f Pseudomonas a e r u g i n o s a  2.  Classes of a n t i b i o t i c s aeruginosa  3.  Mechanisms o f a n t i b i o t i c  4.  Structure  used  importance  against  Pseudomonas  resistance  o f t h e gram n e g a t i v e  outer  1 4 8  membrane  10  a.  Proteins  12  b.  Lipopolysaccharide  14  5.  Permeability  of the outer  membrane  6.  Role of the o u t e r membrane o f Pseudomonas aeruginosa i n permeation of a n t i b i o t i c s  20  7.  Antibiotic  21  8.  Aim o f t h e s t u d y  hypersusceptible  mutants  18  23  METHODS  24 1.  Medium  and c u l t u r e c o n d i t i o n s  2.  Bacterial  3.  Antibiotic  strains susceptibility  v  24 24  testing  25  Page  26  4.  Antibiotics  5.  Isolation  6.  Bacteriophage  7.  Beta-lactamase  8.  NPN u p t a k e  9.  Permeabilisation  o f whole c e l l s  Dansyl  binding  10.  and c h e m i c a l s  of spontaneous  revertants  sensitivity assay  o f M1  28  testing  28  and c a l c u l a t i o n s  assay  polymyxin  to  lysozyme  Preparation  of outer  13.  Preparation  of c e l l  14.  Isolation  15.  Preparation  16.  Chemical  of  30 31  32  assay  33  11 . Phase p a r t i t i o n i n g o f c e l l s 12.  27  34  membranes  34  envelopes  34  lipopolysaccharide  36  of L i p i d A  36  assays  a.  Protein  assay  b.  3-deoxy-D-mannooctulosonic a c i d assay  c.  Phosphate  (KDO)  assay  17.  SDS-polyacrylamide  18.  E l e c t r o p h o r e t i c b l o t t i n g o f p r o t e i n s and lipopolysaccharide  38  19.  Immunoenzymatic s t a i n i n g o f p r o t e i n s or l i p o p o l y s a c c h a r i d e on b l o t s  38  20.  Gel  39  staining a. b.  gel electrophoresis  procedures  37  Protein Lipopolysaccharide  21 . P r o t e a s e d i g e s t i o n o f o u t e r membranes  40  22 .  Assay  41  23.  Gas  f o r LPS c a r b o h y d r a t e  liquid  chromatography vi  o f whole  cell  41  fatty 24.  CHAPTER  Page.  acids  Crosslinking proteins  of outer  membranes and p u r i f i e d  42  o f an A n t i b i o t i c Mutant o f Pseudomonas a e r u g i n o s a  44  ONE  Characterisation Hypersusceptible INTRODUCTION  44  RESULTS  47  PART I .  A n t i b i o t i c s u s c e p t i b i l i t y o f w i l d type and mutant s t r a i n s o f Pseudomonas a e r u g i n o s a  47  1.  Derivation of b a c t e r i a l s t r a i n s used in 47 characterisation of a n t i b i o t i c s u s c e p t i b i l i t y and o u t e r membrane alterations  2.  S u s c e p t i b i l i t y o f w i l d t y p e and mutant s t r a i n s to a v a r i e t y o f a n t i b i o t i c s w i t h d i f f e r e n t mechanisms o f a c t i o n  51  3.  A n t i b i o t i c s u s c e p t i b i l i t y of transconjugant and t r a n s d u c t a n t s t r a i n s d e r i v e d from mutant s t r a i n M1  56  4.  Bacteriophage s e n s i t i v i t y of wild mutant s t r a i n s  t y p e and  59  5.  Other p r o p e r t i e s o f the a n t i b i o t i c s u s c e p t i b l e mutant s t r a i n  hyper-  62  6. PART  a.  Frequency  of reversion  b.  Growth r a t e  c.  Colony  o f t h e mutant  morphology  Summary II.  62 63 63 64  C h a r a c t e r i s a t i o n of a l t e r a t i o n s i n outer membrane p e r m e a b i l i t y o f mutant s t r a i n s  1.  Hydrophilic whole c e l l s antibiotic  2.  Induction  (porin-mediated) permeability of to a chromogenic cephalosporin  properties vii  o f chromosomal b e t a -  66 67  74  Page lactamase  from  wild  t y p e and mutant  strains  3.  O u t e r membrane p e r m e a b i l i t y t o h y d r o p h o b i c compounds  77  4.  S u r f a c e charge mutant c e l l s  81  5.  S e l f - p r o m o t e d o u t e r membrane p e r m e a b i l i t y : 83 Aminoglycoside enhancement o f o u t e r membrane p e r m e a b i l i t y t o a h y d r o p h o b i c compound  6.  S e l f - p r o m o t e d o u t e r membrane p e r m e a b i l i t y : 86 EDTA, a m i n o g l y c o s i d e and p o l y m y x i n B enhancement o f l y s o z y m e - m e d i a t e d cell lysis  7.  Summary  PART I I I .  properties of wild  type  and  90  C h a r a c t e r i s a t i o n of a l t e r a t i o n s i n outer membrane components i n v o l v e d i n a n t i b i o t i c susceptibility membrane  protein  patterns  92  1.  Outer  92  2.  Densitometry scans of e l e c t r o p h o r e s e d outer membrane samples s t a i n e d f o r p r o t e i n o r carbohydrate  95  3.  Protease antibody  98  4.  Self-promoted o u t e r membrane p e r m e a b i l i t y : i n t e r a c t i o n o f p o l y m y x i n B w i t h w i l d t y p e and mutant c e l l s u r f a c e s  101  5.  Interaction saccharide:  102  6.  I n t e r a c t i o n o f polymyxin B with l i p o p o l y s a c c h a r i d e : b i n d i n g of d a n s y l polymyxin t o the i s o l a t e d L i p i d A f r a c t i o n o f LPS  104  7.  C o m p e t i t i o n by M g binding to i s o l a t e d Lipid A  105  8.  Whole c e l l  9.  L i p o p o l y s a c c h a r i d e rough composition  d i g e s t i o n p a t t e r n s and m o n o c l o n a l r e a c t i v i t y o f o u t e r membrane p r o t e i n s  o f polymyxin B with l i p o p o l y binding of d a n s y l polymyxin  + +  fatty  of d a n s y l polymyxin l i p o p o l y s a c c h a r i d e and  acid  viii  composition core n e u t r a l  109 sugar  111  Page 10.  SDS-PAGE b a n d i n g p a t t e r n o f r o u g h c o r e l i p o p o l y s a c c h a r i d e i n w i l d t y p e and mutant strains  115  11.  SDS-PAGE b a n d i n g p a t t e r n o f major l i p o p o l y saccharide O-antigen-containing species  117  12.  I m m u n o r e a c t i v i t y o f a l t e r e d o u t e r membrane components w i t h m o n o c l o n a l a n t i b o d i e s directed against lipopolysaccharide  119  13.  LPS p h o s p h a t e  121  14.  Summary  content  124  DISCUSSION  CHAPTER  126  TWO  Chemical Crosslinking o f Pseudomonas a e r u g i n o s a and E s c h e r i c h i a c o l i O u t e r Membrane P o r i n P r o t e i n s to Reveal N a t i v e Oligomers  147  INTRODUCTION  147  RESULTS  151  1.  C r o s s l i n k i n g o f E_j_ c o l i p r o t e i n Pho E  2.  Crosslinking  o f P_j_ a e r u g i n o s a  protein  P  155  3.  Crosslinking  o f P_j_ a e r u g i n o s a  protein  F  159  4.  C r o s s l i n k i n g o f p r o t e i n s i n P_j_ a e r u g i n o s a o u t e r membranes w i t h v a r i o u s concentrations of c r o s s l i n k e r  DISCUSSION  outer  membrane  151  161  170  LITERATURE CITED  177  ix  LIST OF TABLES Page Table I  P_;_ a e r u g i n o s a  strains  50  II  Minimal inhibitory c o n c e n t r a t i o n s (MIC) o f a v a r i e t y o f a n t i b i o t i c s f o r p a r e n t and a n t i b i o t i c hypersusceptible strains.  52  Ill  M i n i m a l i n h i b i t o r y c o n c e n t r a t i o n s (MIC) o f t h r e e a n t i b i o t i c s i n spontaneous r e v e r t a n t s , t r a n s c o n j u g a n t s and t r a n s d u c t a n t s o f M l .  55  S u s c e p t i b i l i t y to various a n t i b i o t i c s a e r u g i n o s a s t r a i n s by d i s c i n h i b i t i o n assay  57  IV  V  VI  o f a l l P_^ diameter  Bacteriophage s e n s i t i v i t y o f w i l d type a n t i b i o t i c hypersusceptible strains.  and  61  Chromosomal b e t a - l a c t a m a s e a c t i v i t y and n i t r o cefin p e r m e a t i o n t h r o u g h t h e o u t e r membrane o f w i l d t y p e and a n t i b i o t i c h y p e r s u s c e p t i b l e P. aeruginosa strains.  70  VII  Outer membrane n i t r o c e f i n using  72  VIII  Induction o f chromosomal b e t a - l a c t a m a s e i n P_^ a e r u g i n o s a w i l d type and a n t i b i o t i c hypersusceptible strains.  75  Fluorescence increase whole c e l l s .  78  IX X XI  p e r m e a b i l i t y o f whole c e l l s t o TEM-2 beta-lactamase  Phase p a r t i t i o n i n g of aeruginosa.  due t o u p t a k e  of wild  type  o f NPN i n  and mutant c e l l s  82  Rate o f f l u o r e s c e n c e i n c r e a s e due t o uptake o f NPN by whole c e l l s a f t e r a d d i t i o n o f 4 ug/ml o f gentamicin.  84  XII  P e r m e a b i l i s a t i o n o f whole c e l l s t o lysozyme by EDTA, g e n t a m i c i n o r p o l y m y x i n B.  88  XIII  B i n d i n g o f d a n s y l p o l y m y x i n B t o whole c e l l s , L P S and L i p i d A, and c o m p e t i t i o n o f b i n d i n g by Mg++  103  XIV  Fatty acid aeruginosa  110  c o m p o s i t i o n o f whole c e l l s o f ?^ s t r a i n s WT1 , P1 — 1 , P1-4 and M1.  x  Page XV  Analysis  of l i p o p o l y s a c c h a r i d e  neutral  XVI  Phosphate c o n t e n t L i p i d A from w i l d  XVII  Summary o f t h e c r o s s l i n k e d m u l t i m e r s p r o t e i n s observed i n t h i s study.  sugars.  o f l i p o p o l y s a c c h a r i d e and t y p e and mutant s t r a i n s  xi  of p o r i n  114 122 171  LIST OF FIGURES Page  Figure 1.  Structure  of  beta-lactam  2.  Gentamicin cells.  3.  P e r m e a b i l i s a t i o n o f whole c e l l s t o l y s o z y m e by EDTA, g e n t a m i c i n o r p o l y m y x i n B.  87  4.  SDS-PAGE o f o u t e r membranes and c e l l e n v e l o p e s from w i l d t y p e and a n t i b i o t i c h y p e r s u s c e p t i b l e mutant s t r a i n s o f P_;_ a e r u g i n o s a .  93  5.  Densitometry scans o f SDS-PAGE of a e r u g i n o s a o u t e r membranes s t a i n e d f o r r a t e and/or p r o t e i n .  P_^ carbohyd-  96  6.  SDS-PAGE of protease digestion and immunor e a c t i v i t y o f o u t e r membrane samples from s t r a i n s WT1 and M1.  99  7.  Binding o f d a n s y l polymyxin c o m p e t i t i o n o f b i n d i n g by M g  enhancement  antibiotics  o f NPN u p t a k e  gentamicin A.  to + +  5  by whole  Lipid  A  79  and  106  d a n s y l polymyxin  108  .  8.  Inhibition by b i n d i n g to L i p i d  9.  S p e c t r o p h o t o m e t r i c scans of c a r b o h y d r a t e assay on p u r i f i e d LPS from P_;_ a e r u g i n o s a s t r a i n s WT1 , WT3 and M1.  112  10.  SDS-PAGE o f LPS from susceptible strains.  116  11.  Immunoreactivity of e l e c t r o p h o r e t i c a l l y LPS from w i l d t y p e and mutant s t r a i n s .  12.  Model suggesting how LPS a l t e r a t i o n s i n an a n t i b i o t i c h y p e r s u s c e p t i b l e mutant o f Pseudomonas a e r u g i n o s a a f f e c t o u t e r membrane p e r m e a b i l i t y  146  13-  One- and t w o - d i m e n s i o n a l c r o s s l i n k i n g of p u r i f i e d E_;_ c o l i p r o t e i n PhoE.  analysis  152  14.  One- and t w o - d i m e n s i o n a l c r o s s l i n k i n g of p u r i f i e d a e r u g i n o s a p r o t e i n P.  analysis  156  15.  Two-dimensional c r o s s l i n k i n g P. a e r u g i n o s a p r o t e i n F.  of p u r i f i e d  160  wild  xii  of  type  and a n t i b i o t i c  analysis  blotted  120  16.  One-dimensional SDS-PAGE of o u t e r membrane samples treated with various concentrations DSP f o r two m i n u t e s .  162 of  17.  Two-dimensional c r o s s l i n k i n g u s i n g 40 ug DSP/mg p r o t e i n .  of  outer  membranes  164  18.  Two-dimensional c r o s s l i n k i n g of u s i n g 200 ug DSP/mg p r o t e i n .  outer  membranes  165  19.  T w o - d i m e n s i o n a l SDS-PAGE of membrane s a m p l e s s t a i n e d f o r  20.  T w o - d i m e n s i o n a l SDS-PAGE o f o u t e r membrane s a m p l e s .  xiii  c r o s s l i n k e d outer carbohydrate. uncrosslinked  control  167 168  LIST  proteose sodium  OF  peptone  dodecyl  ABBREVIATIONS  no.2  growth  medium  sulphate  ethylenediaminetetraacetate dansyl  polymyxin  B  N-phenyl-1-naphthy1amine lipopolysaccharide minimal  inhibitory  Absorbance  a t 600  concentration nm  dithio-bis-(succinimidyl polyacrylamide phosphate nanoSiemen  propionate)  crosslinker  gel electrophoresis  buffered (unit  saline of e l e c t r i c a l  xiv  conductance,  ohm"  ACKNOWLEDGEMENTS  I the  would  Canadian  during thank life  t o acknowledge t h e f i n a n c i a l  Cystic Fibrosis  my t e r m  o f Ph.  D.  Foundation  support  candidacy.  Poole,  Chan,  T h a l i a Nicas,  Kazuhiro  Nakajima,  Andrew W i e c z o r e k , Crockford,  Vasington Richard  Moore,  Siehnel,  Martin,  Janet  Rich  I would  Alice  Rich Darveau,  Bernadette  Loh,  Niamh K e l l y , Sawyer,  Wendy  also like who have  C a r e y , Lucy  Val Raffle,  Moore, Tom P a r r ,  o f both  and D r . R.E.W. Hancock  t h e f o l l o w i n g members o f t h e l a b o r a t o r y , more e n j o y a b l e :  Keith  Gord  like  Janet  C h r i s Grant, Betty  Woodruff  and  Lydia  Mouat, Joe Lam,  Battershill,  Gus B e l l ,  made  Mutharia,  Lynn D e n t ,  Pixie  to  Lynn  P a t r i c k Wong, Worobec, Nancy especially  Bob  Hancock.  "When remains,  you  have  eliminated  however i m p r o b a b l e ,  the  must be t h e t r u t h . " (A.  xv  impossible,  C. D o y l e )  whatever  INTRODUCTION  1. A n t i b i o t i c  resistance  and m e d i c a l  importance  o f Pseudomonas  aeruginosa. The  emergence o f r e s i s t a n c e  organisms  became  clinical first  use  microorganisms recent  higher  during to  therapy  to a n t i m i c r o b i a l  This  rapid  of b a c t e r i a l  resistant  to  infections (and  possession  target  of  1960's,  Antibiotic  resistance  a  strains G  were  w i t h wounds. in  these  altered  of  whereby  consequently  at  has  led  hospitals.  Staphylococcus  able  much  non-random  resistance  of  to  Resistance to  to For  aureus  cause  organisms  A  so-called  be t r a n s f e r r e d  within  of  frequency.  advent  serious  penicillins  was  due  to  beta-lactam-inactivating b i n d i n g p r o t e i n s , the  (Brunton,  1984).  and p e n i c i l l i n a s e  inhibitors  i n dealing  such  with  Semisynthetic as  these  clavulanic two  types  resistance. Pseudomonas a e r u g i n o s a more ^ r e c e n t l y  one  low  ability  penicillin  of p e n i c i l l i n s  successful  the  resistance,  may  plasmid-carried,  or m u l t i p l y  were l a r g e l y  widespread  the  infections  penicillin  of  sites  with  i n pathogenic  after  to  of a n t i b i o t i c  antibiotics)  beta-lactams acid  type  i n patients  other  enzymes,  the  is  agents  and  occurrence.  in  due  plasmid-mediated  frequencies  example,  began.  of concern  or  spread  shortly  mutate a t a r e l a t i v e l y  source  "infectious" resistance  concern  of a n t i b i o t i c s  emerged  more  a medical  to a n t i b i o t i c s  of  the  major c a u s e s  of  nosocomial 1  joined  S_;_  aureus  as  (hospital-acquired)  infections pathogen  and,  individuals, patients their of  although it  who  are  injuries  the  eye,  tracts  surrounding  toxemic)  can  (cystic  serious  such  may  infections  i n rare cases,  tissue  to the n a t u r e  the  also  damage c a u s e d  in  tends  cause  by  in of  treatment of  local  intestinal  conjunctiva to i n f e c t  v e s s e l s i n each  aeruginosa  healthy  infections  wounds, u r i n a r y ,  organism  small blood  to  i s used  fibrosis),  The  opportunistic  v a r i o u s types  case  of  tissue  (Lowbury,  generalised (septicemic and  because of i t s h i g h r e s i s t a n c e  extensive  due  as  cause  i n burn  i s an danger  either  therapy  ear.  no  cause  immunosuppressed,  or i n the  P_;_  poses  to  f o r example,  respiratory  certain  it  able  or to drug  or  1975 ) .  P_;_ a e r u g i n o s a  aeruginosa  infections,  or  is  cancer.  areas  1975).  (Lowbury,  the  death  is  almost  to a n t i b i o t i c s  and  organism  to  prior  the onset  of s e p t i c e m i a . P. to  aeruginosa  virulence  large  variety  gram  factors  pathogens  are  various  collagenase), hemolysin),  (Fox  and  i n the  not.  proteases  The  Although  value  1953),  i n combatting 2  a  Included  amongst  these  and  C  elastase  (lecithinase  exotoxins  t o P_;_  do  produces  other  not  A  aeruginosa  significant  these  respect  whereas  and  Human immunity  organism  (including  A  with  factors,  serum o f p a t i e n t s i n f e c t e d  Lowbury,  protective  do  hyaluronidase,  understood.  appear  immunity.  phospholipases  (Lowbury , 1 975 ) . well  and  medically significant  of excreted v i r u l e n c e  negative  factors  i s also  levels with  and  i s not  of P_;_  The  and S yet  antibody aeruginosa  always appear  infection.  and  t o be  of  prevailing  theories  are  inaccessible by  either  antibodies  in  In  the  when serum a n t i b o d y so  that  lung  of i n f e c t i o n  the a n t i b o d i e s the types  are  could  that  are  degraded  alveolar  from  phagocytic  where s e p t i c e m i a  be p r o t e c t i v e ,  extensive  somehow  for  i s protected  at the stage  is  of anti-Pseudomonal  non-opsonic  the organism  addition,  usually  that  proteases,  macrophages, or t h a t  is  the s i t e  to a n t i b o d i e s ,  P_;_ a e r u g i n o s a  attack.  that  occurs,  prior tissue  immunity  damage  i s no l o n g e r  of  any  consequence. The  aforementioned  w h i c h were s u c c e s s f u l limited the  i n dealing  success with  most p a r t ,  alteration,  approaches  on  al.,  on i n a c c e s s i b i l i t y 1981).  resistance)  i s mediated  bacteria,  which  forms  molecules  above  a  reaching  their  sites  cell.  This  accounts  aeruginosa varying  to  sites  membrane  of action for  are  of action within  other  limit  (Leive,  1974;  thus  prevented  general  g r o u p o f b a c t e r i a , s u c h as E s c h e r i c h i a  3  P_j_  from  or w i t h i n  resistance  the c e l l .  t o most a n t i b i o t i c s  negative  and  i n the p e r i p l a s m the  e_t  intrinsic  i n gram  a wide v a r i e t y o f a n t i b i o t i c s ,  much l e s s s u s c e p t i b l e  (Costerton  b a r r i e r to a n t i b i o t i c s  Antibiotics  agents  1982) and p r o b a b l y  of s o - c a l l e d  exclusion  for  or t a r g e t  antimicrobial  i n vivo  (one t y p e  defined  1980).  therapy  only  was,  deactivation  to  by t h e o u t e r a  resistance  and N i k a i d o ,  to drug  Impermeability  Zimmermann,  since  impermeability  therapy,  a u r e u s , have had  on e n z y m i c  (Angus e_t a_l. , 1982; Y o s h i m u r a also  with  aeruginosa  not dependent  but  to a n t i m i c r o b i a l  despite  the  of  P.  their  aeruginosa  is  than are the e n t e r i c  coli  and S_j_ t y p h i m u r i u m  1975;  (Brown, exceptions  to t h i s  which w i l l have  Zimmermann rule  been  aeruginosa,  developed  and t o w h i c h  susceptible  than  improved  antibiotics, useful  toxicity,  difficult  the bacterium  for  levels  means t h a t  Several throughout  of these  of  i n the  different this  study  used  types of a n t i b i o t i c s for  the  The  pertinent  lend  themselves  t o each  Beta-lactams  inhibiting  the c e l l  penicillin  cytoplasmic  purpose This  without remain  membrane  of  illustrating  P_;_  of  antibiotics The s i t e  which  acts  affinities  by  of action i s of  the  1974),  and each  for  different  o f some o f t h e major  a r e d e s c r i b e d by F i g u r e 1.  4  aeruginosa.  antibiotics  and S t r o m i n g e r ,  The s t r u c t u r e s  beta-lactam  specific  at the outer s u r f a c e  has c h a r a c t e r i s t i c  antibiotics.  used  a r e t h e r e f o r e d e s c r i b e d below.  process.  (Blumberg  been  demonstrating  of these  group  division  of beta-lactams  manipulation  properties  binding proteins  proteins  beta-lactam classes  physical  for  have of  a c r o s s t h e o u t e r membrane o f  o f uptake  and  purposes  types  these  body,  a g a i n s t P. a e r u g i n o s a .  resistance  of  more  achieving  aeruginosa i n f e c t i o n s  susceptibility/  on  P.  D e s p i t e the advent of  difficulty  P_^  against  to t r e a t .  2. C l a s s e s o f a n t i b i o t i c s  a)  use  B,  antibiotics  i s proportionally  bacteria. the  Notable  and p o l y m y x i n  Newer b e t a - l a c t a m  specifically  are enteric  therapeutically  1977).  Rosselet,  are aminoglycosides  be d i s c u s s e d l a t e r .  also  causing  and  antibiotics  to  Structural yield  many  variations influence cells,  of  these  absorption  to  binding  susceptible  (eg.  carbenicillin,  The  in  active  vary the  ring,  above  chemical  to  In  many  these  types  and  "third  (Fig.  ( F i g . 1,  (c-e))  as b e t a - l a c t a m a s e  possesses  a new  compounds a r e r e f e r r e d  5  which  In  can  be  the second  i n the 1 p o s i t i o n , and  such  and i s 1983).  Vasudevan, b u t have  a  substitute bond  sixoxygen  in  the  compounds  are  as c l a v u l a n i c  compounds a l s o  exists  the b e t a - l a c t a m  t o as "monobactams"  Sykes e t a l . , 1 9 8 1 ) .  role  antibiotic.  Some o f t h e s e  structure,  1(a))  figure  o r have a d o u b l e  of these  and  G, f o r example,  similar,  inhibitors,  class  one r i n g  the  Newer d e r i v a t i v e s atom  1(a),  influence  (Rao  are  the  to f u r t h e r  sulphur  1(b))  f o r the s u l p h u r  ring  of  is  an i m p o r t a n t  o f R-groups  In p e n i c i l l i n with  plays  positions  as a "penam" r i n g  addition,  only  -  are  "cepham" r i n g .  carbon  useful  for  antibiotics  molecule  (2 i n  ring  and t h e r e f o r e  a t the d e s i g n a t e d  Cephalosporins  second  bacterial  P_;_ a e r u g i n o s a i s  1 in figure  i s numbered  The a d j a c e n t  i s five-membered,  membered  to  affinity  ticarcillin)  o f the b e t a - l a c t a m  which  properties.  referred  and  penicillin  and  mentioned p r o p e r t i e s  there  substituted  or  portion  i n structure  addition,  ring  piperacillin  into  1982).  (Abraham,  t o t h e more r e c e n t  does n o t v a r y .  does  penetration  designed  cephalosporins.  beta-lactam which  was  beta-lactamase,  proteins  more  generation"  classes  i n t h e body,  stability  penicillin  different  ring,  (Figure  acid. which and 1 (f)  Figure 1. Structure of (3-lactam antibiotics a)  b)  i 0  -N-  •N  ^COOH  0  COOH Penicillin  R  Cephalosporin  0  d)  R  R>  N0  COOH COOH Oxycephalosporin  Oxypenicillin e)  f)  R-  R 'COOH Penem  R« -»si  R  Monobactam  b) A m i n o g l y c o s i d e s aminoglycosides free  amino  g r o u p s , making  event  uptake  into  been  discussed  in  large,  (Bryan  at  length  destabilising  effect  Hancock,1980).  Some t h e r a p e u t i c a l l y  are  streptomycin,  limited  the  outer  some  against  combination  with  oxypenicillin  antibiotics  Polymyxin structure  is  Polymyxin  B  B  tail  Polymyxin  B also  membrane  integrity,  membrane  than  newer  which  that  as g e n t a m i c i n , used in  cephalosporin  or  antibiotic of  but i t s  aminoglycosides.  methyloctanoyl  via a tripeptide  to  a  as w e l l  (Weinstein,  s y n t h e s i s and d i s r u p t s  the  only  usually  of diaminobutyric acid  although  have  1983).  of a hydrophobic  protein  a and  which are c u r r e n t l y  and D - p h e n y l a l a n i n e  inhibits  exert  aminoglycosides  as w e l l  a polycationic  attached  mostly  L-leucine  the  (Maxwell,  i s also  i s composed  composed  threonine,  of  (1981).  (Nicas  aeruginosa.  much d i f f e r e n t  methylheptanoyl peptide  one  to  membrane  n e o m y c i n and k a n a m y c i n ,  success  energised  Hancock  important  s i s o m y c i n and n e t i l m i c i n ,  actual  1977) > and has  reported  a c t i v i t y a g a i n s t P^ a e r u g i n o s a ,  tobramycin, with  on  inhibit  the  the a c t of  by  been  molecules.  subunit to  and van den E l z e n ,  also  three to s i x  although  to i n v o l v e  some  have  with  polycationic  prokaryotes,  i s thought  the c e l l  Aminoglycosides  these  rings  t o t h e 30S r i b o s o m a l  bind  synthesis  bactericidal  The g e n e r a l s t r u c t u r e o f  i s three carbohydrate  Aminoglycosides protein  and p o l y m y x i n  main  effect  may  or  cyclic as  L-  1975). outer be  on  energisation.  c) Others  Tetracyclines  are large  7  molecules  composed  of  four  six-membered  synthesis Unlike  rings.  aminoglycosides,  50S  ribosomal  synthesis.  ring  one o f w h i c h  to i n h i b i t  free  amino g r o u p s .  bacterial  cell  to i n h i b i t  Miller,  1984), w h i c h  acids  and c e r t a i n  and  aeruginosa  of  dichloroacetic  composed  i s a benzyl  of  derivative  Three generally a)  types  of  and two  (Hamilton-  i n the s y n t h e s i s o f n u c l e i c  I t i s also  i s consequently  two  a c t s w i t h i n the  d i h y d r o f o l a t e reductase  amino a c i d s .  to  i n nature.  This a n t i b i o t i c  i s important  3• Mechanisms o f a n t i b i o t i c  not  protein  one w h i c h c o n t a i n s two n i t r o g e n atoms i n t h e r i n g  associated  fairly  very  resistant  hydrophobic, to i t .  resistance.  antibiotic  resistance  mechanisms  are  recognised: specific  lactamases,  for  inactivating penicillins  phosphorylating  or  aminoglycosides  (Bryan,  Acquisition these  derivative  diaminopyridine  structures,  bacteriocidal.  agent which b i n d s  q u i t e hydrophobic a  protein  i n t h e same f a s h i o n .  subunit of prokaryotes  is  inhibit  r a t h e r than  i s a bacteriostatic  and i s c o n s e q u e n t l y  resonant  to  a r e n o t p o l y c a t i o n s and do  I t i s a nitrobenzyl  Trimethoprim  and  they  t h e o u t e r membrane  Chloramphenicol  acid  act  but are b a c t e r i o s t a t i c ,  destabilise  the  These  enzymes  concentration  by  a  enzymes, and  bacterium  and  of a plasmid  can d r a s t i c a l l y  enzymes,  8  antibiotics.  and for  chloramphenicol. specifying  i n c r e a s e the minimal  values f o r these  beta-  cephalosporins,  ADP-ribosylating 1984)  including  one  of  inhibitory  Derepression  of a  chromosomally  specified  of  or aminoglycoside  E_j_  coli,  aeruginosa b)  not for  alterations,  ribosomes  affect  such  or s p e c i f i c  function  such  3'-phosphotransferase  e_t ajl. , 1983) may a l s o  (Okii target  proteins,  gene f o r b e t a - l a c t a m a s e ,  increase  as ampC I I o f P.  resistance.  as i n p e n i c i l l i n  biosynthetic  binding  enzymes, w h i c h do  o f the p r o t e i n s but lower  their  affinity  binding of a n t i b i o t i c s . c)  lie  permeability,  interior  cell.  t o t h e o u t e r membrane  A decrease  mechanism  which  antibiotics  s i n c e the t a r g e t s f o r the  with  and  many a r e w i t h i n  i n p e r m e a b i l i t y to a n t i b i o t i c s can  explain  varying  resistance  physical  antibiotics  to  the  i s the  only  range  of  a  p r o p e r t i e s and  sites  of  action. It two  should  of these  membrane  be m e n t i o n e d  parameters,  permeability,  resistance  level  that  synergistic  f o r example b e t a - l a c t a m a s e may be v e r y  (Vu and N i k a i d o ,  a c t t o slow  important 1985).  barrier  may  targets  i n the p e r i p l a s m s u f f i c i e n t l y  beta-lactamase  can  1974).  The  necessary  to allow i n d u c t i o n  beta-lactamase amount might a  of  which  beta-lactam  be c r i t i c a l  small  change  o f them  between  the  that  (Richmond amount  by t h e  which g a i n s access  i n determining  cell  (up o r down) i n any o f t h e s e  9  and  Curtis,  beta-lactam t h e amount o f  the  survival.  their  low l e v e l s o f  cell,  to  membrane  with  of  of beta-lactamase,  c a n be p r o d u c e d  and o u t e r  The o u t e r  such  between  f o r the observed  contact of beta-lactams  dispose  balance  action  and  the  periplasm,  F o r example,  could upset  the  balance  such  that  large  changes  in  susceptibility  are  manifested. In  P_^_  aeruginosa,  the t h i r d  permeability)  appears  explaining  intrinsic  high  4. S t r u c t u r e Gram  outside  membrane. protein and  The  of  cells  of t h e i r outer  certain  to allow  organised  such  phopholipid  that  outer  contains  remaining  cell  diffusion The  surface  surface  X-ray  diffraction  studies  (Yamada  The be  as a s e l e c t i v e  bacteria  phospholipid, conformation,  of molecules  below  appears  t o be r i g i d l y  o f E_^ c o l i and  associated  and  have a much l e s s  barrier.  complex  10  be the the an  1975).  The  have  a  stability  and  appears,  from  into a  1980;  hexagonal  p r o t e i n molecules i n  lipopolysaccharide.  o f t h e gram n e g a t i v e permeability  to  most o f  Mizushima,  organised  a  occupying  et a l . ,  membrane  on t h e p o s i t i o n i n g o f p o r i n  purpose  of  cytoplasmic  components a r e p r o t e i n s w h i c h  The c e l l  a t r i m e r i c f a s h i o n with  in  bilayer  ethanolamine)  (Smit  diffusion.  based  and  lipopolysaccharide  passive  1974),  wall  bilayer  s i g n i f i c a n c e i n both outer  Rosenbusch,  additional  i n an o r d e r e d  phosphatidyl  45$ o f t h e c e l l  array,  an  i s composed  limit.  estimated  functional  importance  the i n n e r monolayer c o n t a i n s  surface  membrane  membrane.  peptidoglycan  passive  (primarily  monolayer  greatest  possess  membrane  size-exclusion  (outer  r e s i s t a n c e to a n t i b i o t i c s .  and l i p o p o l y s a c c h a r i d e ,  functions  outer  be  o f t h e gram n e g a t i v e negative  envelope  to  mechanism  outer  membrane  Since  peptidoglycan  gram wall,  seems t o negative thinner  and  without  membrane  may  crosslinked and  be  Nikaido,  agents,  size  addition,  cells.  which  the exist  nutrient  nutrients  across  specifically  bind  from and  solution, thus  outer  of  excluded 1974), are  (which from and  excluded  its  to  are  as  cell  enzymes proteins  break  down  may  well  i n the  be  as  w h i c h removes  protection  to them  periplasm,  peptidoglycan)  by  the  certain  membrane  w h i c h make  1 1  is  (Nikaido,  these  below.  the  protein  effectively  (Sanderson  of  several  the  and h y d r o p h o b i c  properties  in detail  against  For example,  lipopolysaccharide  discussed  to  outer  the  which  membrane,  concentration  non-ionic detergents due  an  The  used  gram  transport.  destroys target  are  and,  to  catabolic  components  molecules.  membrane components  possible  their  cells  of  environment.  as  destructive  unique  presence  and s u g a r s ,  membrane p r o v i d e s  membrane m o l e c u l e , outer  amino a c i d s  antagonistic  lysozyme  into  active  from  (Nakae  such  which a l l o w s  space  highly  functions,  secreted  cytoplasmic  allowing  facilitate  The types  periplasmic  the  the  outer  cells  gram p o s i t i v e  space,  and  the  thick,  some a s p e c t s  i n a protected  transferred  some  of  instance,  macromolecules  the  and p r o t e c t i o n  possible  i n the  1979),  gram p o s i t i v e  performs  a periplasmic  cell  to  of  peptidoglycan  For  concentrate  outside  layer  exclusion to  (Mirelman,  alternative  It  makes  membrane c r e a t e s to  an  1975).  similarly  negative  acids  peptidoglycan  rudimentary  in  teichoic  e_t  §_1. ,  antibiotics unique 1979).  essential  outer The  functions  a)  Proteins  The  outer  membrane  contains  a small  in  large  copy number.  are  the s o - c a l l e d p o r i n  integral  Other  analogues OmpA  other  native  sensitivity 1978). gels,  proteins  so f a r examined  as  oligomers  by d e t e r g e n t s  proteins).  the range  reported  distinguishing Porins  to contain  (DiRienzo  i n t e g r a l membrane  proteins,  alpha-helical structure Although  rather  than  (DiRienzo respect  a l l porin active  e_t al^. ,  high  transport  on SDS-PAGE,  which u s u a l l y  proteins  operate  example,  their  e_t  a_l. ,  of  outer weights  and a r e  beta-structure  have l a r g e  areas  1974). by p a s s i v e d i f f u s i o n ,  as i n t h e c y t o p l a s m i c  membrane  1 9 7 8 ) , t h e y may be e i t h e r n o n - s p e c i f i c  to substrate  or s e l e c t i v e f o r c e r t a i n  12  in  temperatures of  amounts o f  (Rosenbusch,  as  on S D S - p o l y a c r y l a m i d e  have monomeric m o l e c u l a r  unusually  such  in  characteristic  o f 30,000 - 50,000 d a l t o n s  have  to e x i s t  vary  including h e a t - m o d i f i a b i l i t y at varying (a  1983).  negative  For  appear  which  c a u s e s anomalous b e h a v i o u r  passive  gram  characteristics.  to s o l u b i l i s a t i o n  This  membrane  of  are s t r u c t u r a l proteins  some b a s i c  these  (and w h i c h o f t e n  seem t o be common t o a l l  forms  solubilisation  for  coli  proteins  of the p o r i n  of  (Lugtenberg,  Porin  and s h a r e  present  size-limited,  membrane in  species)  t o be  characterised  membrane j j r o t e i n ) and l i p o p r o t e i n s .  their  in  found  so f a r  w h i c h a r e membrane-spanning, for  outer  tend  (outer  species all  the  of proteins in  proteins,  examined  which  The most w e l l  responsible  through  types  a l l species  variety of proteins  proteins  diffusion  of  substrates  with on  the  basis  protein, only  of structure OmpF,  weakly  strongly size  or charge.  i s an example  cation-selective  prefer  appears  1 976 ; Benz ejt a_l. , 1 9 7 9 ) , This  substituted,lipid by  measuring  coli  also  relative forces IV  respectively.  porins  results  synthesising which  in  and  (with  apparently  an  3000  daltons  proteins  influenzae, Porins toward  than limit  porin,  bacteria  1976). and t h e  and  of the  proteins  generalised f o r mutants  limit  1981),  of  1978),  Chai,  function with  aeruginosa  larger exclusion  ompB ( T o l  and E a r h a r t ,  o f both  (Foulds  studied  loci,  pressure  include  typhimurium,  OmpC,  (Lundrigan  selective  Well  radiolabelled  a r e c o n t r o l l e d by o s m o t i c  by m u t a t i o n  proteins  porin-  1976) and  (Nakae,  the r e g u l a t o r y  - Benz and Hancock,  from  to  Nikaido,  and N i k a i d o ,  i n d i c a t e s the importance  i n other  other  from  of  (Decad  1975) and envY  strong  functions  found  and  obtained assays  two p o r i n s  Loss  OmpC p o r i n s .  i t is  exclusion  (Decad  permeation  through  new membrane  therefore  OmpF  was  has a n o t h e r g e n e r a l i s e d  1984),  since  been  I t s size  i n t o whole c e l l s  and R e e v e s ,  not  porin  by any c r i t e r i o n  vesicle exclusion  and t e m p e r a t u r e  porin,  coli  above w h i c h d i f f u s i o n i s d r a s t i c a l l y  measurement  amounts o f t h e s e  - Davies  has  600 d a l t o n s  differential  oligosaccharides E.  and  nature).  t o be a p p r o x i m a t e l y  lowered.  of a general  certain substrates  (and h y d r o p h i l i c  The major  of  similar  protein  F  approximately  as w e l l  as  porin  N. g o n o r r h o e a e , C h l a m y d i a e , H.  Y. p e s t i s , and R_^ c a p s u l a t a . have  been i d e n t i f i e d  different substrates.  which  The b e s t  13  display  selectivity  known f r o m amongst  this  class  i s the  which  is relatively  oligomers Another P•  lambda phage r e c e p t o r p r o t e i n (LamB) o f E_^  and  synthesis  which  (0.25nS)  Some porins  is  i s unusual  and  for  proteins  are  high  have n o t  a l s o of note.  OmpA o f  and  o f 7^  H1  i s thought  t o be  outer  aeruginosa  membrane  lipoprotein covalently  o f E_j_ linked  r e s i d u e and  which  is  covalently  transport b) The membrane  coli  glucose.  and  channel phosphate  o f P^  and  (Braun  and  i n the  outer  aeruginosa,  important  (Skurray  Rehn,  outer  to  displays  t o the  be heat  recipient  et a l . ,  growth  1974).  i n low Mg++  effect  Hancock,  of M g  1980).  + +  in  Murein  1969 ) i s ,  in  part,  at i t s carboxy-terminal  a c i d s at  i t s amino  membrane.  The  p r o t e i n I , was  to p e p t i d o g l y c a n  minor  coli  peptidoglycan fatty  attached  been d e m o n s t r a t e d  stabilising  to three  inserted  Numerous identified  by  for i t s small  i s i n d u c i b l e by  (Nicas  t o the  lysine  lipoprotein  dextrose  p o r i n i s p r o t e i n P from  anion  conjugation  i s p o s t u l a t e d t o r e p l a c e the  the  higher  repressed  both  which  i n F pilus-mediated  Protein  its  and  (Hancock e_t a_l. , 1982).  modification cell  f o r maltose  extensively studied selective  selectivity  and  whose  aeruginosa,  size  specific  coli,  not  terminus, equivalent  shown to  be  (Hancock e_t a_l. , 1 9 8 1 a ) .  membrane p r o t e i n s have  w h i c h have some i n v o l v e m e n t  in iron  also  been  or v i t a m i n  B12  (Hancock e_t a_l. , 1 9 7 7 ) .  Lipopolysaccharide other is  major a  lipopolysaccharide  component  molecule (LPS).  unique LPS, 14  as  of the to the  gram the  negative cell  name s u g g e s t s ,  outer  envelope, contains  a  lipid  layer  portion  of  (core  therefore  from  interacts  with  especially  hydrophobic  for  cells,  addition,  basic  the  fatty  acids  similar  ( R o s n e r e_t a_l. ,  Lipid  A fatty  (C14)  acid  the  and  bile with  phagocytes  resulting  In  tissue  body.  portion  of  LPS,  The  coli  and  also  however,  relative strains.  1 983 ),  oxygen  tetradecanoic,  acids.  Lipid  the  major  3-hydroxytetradecanoic  to glucosamine  15  nature of  and  v a r y between  6 -  between  S a l m o n e l l a typhimurium  seem t o be  (C16)  with  number and  Takeyama ejb §JL_. ,  of  beta-1,  o f two  are s u b s t i t u t e d  may  amounts  hexadecanoic  and  pathogens.  the t o x i c  acids.  acid  species  smaller  A,  between s p e c i e s  (both e s t e r i f i e d  with and  acid  which  1 979 ;  agents,  interaction  for plant  I t i s composed  fatty  positive  amongst a l l gram n e g a t i v e b a c t e r i a  fatty  varies  may  compound, known as e n d o t o x i n ,  of L i p i d  o f b o t h E_j_  From s t u d i e s  (C12),  and  detergents  amounts a r e p r e s e n t e d t o t h e  chain  o f each  cells  toward  t o many  influences  a n a p h y l a x i s and  1983).  s i x long  proportions  linked)  extends  adhesion s i t e s  a toxic  glucosamine u n i t s  and  also  outer  polysaccharide  environment  s u c h as  plant  structure  e_t a_l. ,  four  A  and  eliciting  remarkably  linked  the  lipopolysaccharide  for resistance  tissue  is itself  damage i f s u f f i c i e n t  (Qureshi  LPS  including  LPS of  appears  a  The  cell's  ones  1976).  animal pathogens  The  A.  the  i s responsible  (Nikaido,  capable  and  into  a n a l o g o u s l y t o t h e c a p s u l e s e e n i n some Gram LPS  other  i s inserted  O - a n t i g e n ) which  the L i p i d  bacteria.  salts  which  sugars plus  environment  function  A),  t h e o u t e r membrane b i l a y e r ,  component the  (Lipid  The  enteric  and  amide  dodecanoic group  of  bacteria  seem  whereas  P_;_  to  have 3 - 0 H - t e t r a d e c a n o i c  aeruginosa,  3-OH-dodecanoic  tetradecanoic  acid  1973).  Lipid  and  4' p o s i t i o n s  be  monophosphoryl  modified  or  by l i n k a g e t o e t h a n o l a m i n e  Lipid  diglucosamine. molecule  or  There  seem  to  phosphorylethanolamine et  al.,  common  are  be  1984).  Next  heptose  situation  at  two  to three  substituted  groups  Beyond  heptose,  may  be b r a n c h e d  species  than  t h e two p r e v i o u s l y  glucose,  Typical  acetylfucosamine. heptose  acid of  KDO  residues  per  species,  and t h e s e  phosphate 1 975 ;  There  or  Nurminen  found  usually  16  to the  have no  net  more  between  components  (KDO and  i n this  region  N-acetylglucosamine  The above components,  due t o t h e r o u g h  to  a short chain of various  mentioned  sugars,  also  may be two  similarly  and v a r y s u b s t a n t i a l l y  rhamnose,  and n e u t r a l  c o r e LPS,  (in  position  by  These s u g a r s  carbohydrates  galactose,  further  t o KDO i n t h e LPS c h a i n i s h e p t o s e ,  charge,  heptose).  be  3'  (Drewry e_t al.,  i s attached.  rough  the  r e s i d u e s p e r LPS m o l e c u l e ,  w i t h KDO.  may  may  1979).  on t h e b a c t e r i a l  carbohydrates  KDO,  and  t o most gram n e g a t i v e b a c t e r i a .  three  These g r o u p s  and 4 - a m i n o a r a b i n o s e  (Osborn,  KDO)  o f LPS d e p e n d i n g  e_t a l . ,  3-deoxy-D-mannooctulosonic  A i s attached to  (ketodeoxyoctonate  unit.  pyrophosphoryl,  3-0H-  at the 1 (reducing)  groups  o f the diglucosamine  common,  for  1969 ; C h e s t e r  (Hancock and Meadow,  S a l m o n e l l a ) o r o t h e r compounds  also  3-OH-decanoic  and  A c o n t a i n s phosphate  in  substitutes 2-0H-  f o r example,  dodecanoic,  acid  including  include and  Lipid  are together r e f e r r e d appearance  of colonies  NA,  t o as from  Salmonella  and  substitution  of their  0-  somatic)  (or  describe antigen of  Escherichia "core"  mutants  was a term o r i g i n a l l y  t h e major n o n - f l a g e l l a r , species.  heat-stable,  I t comprises  t h e LPS and i s c h a r a c t e r i s e d by r e p e a t i n g  five  carbohydrate  carry  substituted found  moieties.  more p o t e n t i a l l y to  In c o n t r a s t  charged  acid,  2-amino-2-deoxy-galacturonic 1975;  Kropinski  high  variability  immunological difficult bacteria  in  and with  causes  to  long  sugars  et, al.  of  LPS  also  complement,  since  more  sensitive  serum  Goldman,  e_t a_l. ,  function  for  acts  and  1 984).  the outer  I t i s the  which  serotypes  makes bacteria seen  in and  1975). LPS c a n p l a y  an i m p o r t a n t  o f an o r g a n i s m .  r e c o g n i t i o n by  being  or pyocins  to protect  smooth  cells  strains  LPS a l s o p r o v i d e s membrane.  17  and  Welbourn,  , 1983).  r o u g h mutants o f many b a c t e r i a l than  acid  (smooth) LPS (Fensom  t a r g e t f o r phages and c o l i c i n s  1976).  N-acetylfucosamine,  many gram n e g a t i v e  and p a t h o g e n i c i t y surface  these  Some examples o f  components  chain  and Meadow,  cell  sugars,  may be  multitude  p r e v i o u s l y mentioned,  contributes  al. ,  the  relatively  immunogenicity  receptor  0-antigen  protection against  Meadow, 1970; K o v a l As  of  region  u n i t s of three to  (Wilkinson  e t a l . , 1982; K n i r e l  surface  the outer  acetimidoguluronic acid  to  although,they  o f "smooth" LPS a r e  N-acetylimidomannuronic  used  cell  to core  residues,  n e u t r a l i s e charge.  i n the 0-antigen  O-antigen  LPS b a c k b o n e .  antigen  of Salmonella  lacking  Whereas  I t also a  major  (Hancock e_t from  serum  species are  (Taylor, a strong a  role  1983; barrier  phospholipid  bilayer it  i s an e f f e c t i v e  permits  the uptake  barrier  of hydrophobic  Lipopolysaccharide,  being  by  and b i n d i n g  divalent cations  of  hydrophobic membrane  Hancock,  1984).  5.  Permeability As  barrier of  nutrients  though  expected.  routes made  porin  phospholipid i)  size  exclusion  al.,  membrane  or  (1983)  negative  efficient 1979;  al. ,  cell, by  i s an  effective  however  permeation  passive  diffusion  or  of a n t i b i o t i c  i n t o the c e l l  might  membrane, one o f two p o s s i b l e through w a t e r - f i l l e d i i ) uptake  across  channels the  membrane  s e t by t h e c h a n n e l  by N i k a i d o  showed  intact  that  and R o s e n b e r g  beta-lactams  coli  ( t o some e x t e n t charge  v i a porins,  a  w a t e r s o l u b l e and a l s o s m a l l e r  limit(s)  Studies  hydrophobicity  i ) passage  the outer  relatively  of  membrane  largely  the outer  molecules  To c r o s s  be  et  e_t  uptake  LPS-  bilayer.  must  proteins.  the outer  one pathway o f e n t r y  To c r o s s  stabilised  a more  Because o f the l a r g e d i v e r s i t y  must be t a k e n : by  and  prevents  Stan-Lotter  t h e gram n e g a t i v e  more t h a n  molecules.  membrane.  can be a c h i e v e d  porins.  structures, be  above,  surrounding  to proteins,  1974;  o f the outer  discussed  i n nature  and i s t h e r e f o r e  (Leive,  molecules,  or amphiphilic  quite polar  substances  barrier  t o many h y d r o p h i l i c  cells  - Yoshimura  18  than  of the  (1981) and  penetrated  via  o f the beta-lactams  size  molecule  porins,  porin Nikaido  the but  and N i k a i d o , adversely  the  outer that 1982)  affected  their  permeation.  through et  E_^ c o l i  al. ,  1977;  1982),  ii)  deficient  this  hydrophobic  less  from  possess  crossing  and  showed  susceptible  susceptible  tetracyclines  lower  MIC  outer  Evidence  to  a r e more s u s c e p t i b l e  than  their  to t e t r a c y c l i n e whereas  the  core-  a) w i l d  hydrophobicity,  smooth  and  to hydrophobic  ( N i k a i d o , 1976). that  very  (0-antigen  deep r o u g h  several  Experiments  of Leive  t y p e E_j_  type  of  cells  of  mutant  regardless  than w i l d  coli  derivatives  b) a deep r o u g h  agents  1973 ) and  ( G u s t a f s s o n e_t al_. ,  antibiotics  to  which  suspected of  mutants w h i c h  (1984)  are not  the L P S / p h o s p h o l i p i d b i l a y e r .  comes  LPS  (100-fold  confirmed  antibiotics  gram n e g a t i v e b a c t e r i a  as G e n t i a n v i o l e t  al. ,  (Kobayashi  ( N i k a i d o e_t a l . ,  N i k a i d o e t a l . , 1983)  therefore  through  hydrophobic  liposomes  mutants  t y p e c o u n t e r p a r t s w i t h smooth LPS  such  et  into  permeation  results.  are  deficient) wild  of porin  against  membrane  of c e p h a l o s p o r i n  reconstituted  Because c e r t a i n  they  suggest  and  these  effective LPS,  porins  studies  e t a l . , 1982;  Jaffe  extended  Other  became  increasing was  equally  hydrophobicity  f o r t h e most  hydrophobic  derivative). iii)  Another  aminoglycoside demonstrated Nicas  and  utilise  antibiotics, f o r P^  Hancock, this  pathway has  Osborn,  1980),  pathway  1979;  and  aeruginosa  gram n e g a t i v e b a c t e r i a and  been p o s t u l a t e d has  (Zimelis  so f a r  and  although polymyxin  to c r o s s  and  19  and  B also  Teuber,  Viljanen,  only  Jackson,  t h e o u t e r membranes  (Schindler  Vaara  which  f o r uptake  1983;  1975;  of been 1973;  appears of  to  other  Schindler  Hancock,  1984).  This of  so-called outer  self-promoted  membrane  stabilising  specific  binding sites  membrane  integrity.  aminoglycoside  increase 1968),  6.  Mg  on LPS, This  molecules  i n v o l v e s the  or  by  + +  aminoglycosides  of  i n entry of any  1 981 b ).  p r e s u m a b l y by r e m o v i n g M g  o f t h e P.  aeruginosa  + +  outer  either  more  impermeable  EDTA was a l s o  from  this  at  of  other,  o u t e r membrane p e r m e a b i l i t y i n E_^ c o l i  Role  replacement  causing destruction  results  (Hancock e_t a_l. ,  molecules  uptake  shown  cells  to  (Leive,  LPS b i n d i n g  site.  o u t e r membrane i n p e r m e a t i o n o f  antibiotics.  The  o u t e r membrane o f P_^  particularly high to  intrinsic  hydrophilic  workers lower  barrier  antibiotic beta-lactam  that  observed  antibiotics cell  i n E_j_ c o l i  protein enteric  F actually bacteria  Nikaido,  estimated porin,  that  based  1979) b u t t h a t  on  (this  swelling  exclusion limit 1978;  1 0 0 - f o l d lower  than  lipid  bilayer  experiments  in  vivo  outer  membrane  assays  and  Hancock,  ( N i k a i d o and R o s e n b e r g , 20  I t was porin  than  Hancock,  o f these  be  (Brown,  e_t a j l , 1 9 7 9 ) .  the a c t i v i t y  Nicas  several  p e r m e a b i l i t y must  black  thesis;  led  a e r u g i n o s a major  (Hancock and N i k a i d o ,  1981),  experiments  size  a  on i t s  and S_^ t y p h i m u r i u m  t h e P_^  has a l a r g e r  t o be a p p r o x i m a t e l y  Hancock,  liposome  discovered  be  Its resistance  originally  envelope  to  based  profile.  S y k e s and Matthew, 1976; S u g i n a k a  subsequently  appears  to a n t i b i o t i c s ,  resistance  to p o s t u l a t e that  than  1975;  and  effective  aeruginosa  do  Decad  p o r i n s was  E_j_ c o l i (Benz  OmpF and  permeability 1983), 1983).  and  Permeability aeruginosa and  S_^  appears  based  self-promoted  high  have  i n P_^  u p t a k e pathway.  (aminoglycosides been  used  of outer  and p o l y m y x i n  therapeutically  LPS  w h i c h makes t h e o u t e r  challenged  with  self-promoted  antibiotic  A mutants species. degree,  have  aeruginosa  so  stable  destabilise In  by N i c a s  of  P. to  i t  when  effect,  the  and  Hancock  "chink  i n t h e armour" i n  antibiotic  hypersusceptible  been d e s c r i b e d i n t h e  Although  literature,  of  in  several  n o t a l l have been c h a r a c t e r i s e d t o an e q u a l  n e v e r t h e l e s s show a few g e n e r a l  susceptibility,  trends.  w e l l c h a r a c t e r i s e d mutations  the  m a j o r i t y were mutant  o u t e r membrane, r a t h e r t h a n to p o r i n d e f i c i e n c y  hydrophilic  P_;_  organism.  of i n t r i n s i c a l l y  Firstly,  Mutation  the  h y p e r s u s c e p t i b l e mutants  number  these  permeation  w h i c h use t h i s  membrane  pathway p r o p o s e d  of this  It is  The p r o p e r t y  act to  t o be t h e p r o v e r b i a l  treatment  7. A n t i b i o t i c  of success.  may a l s o  ooli  i s a f f o r d e d by  the a p p r o p r i a t e molecule.  uptake  (1980) may p r o v e  MIC d a t a .  against  aeruginosa  agents  P.  B) i n c l u d e a number w h i c h  some d e g r e e  of  of  t h a t o f E_;_  The a n t i b i o t i c s  with  types  bilayer  membrane  aeruginosa  infections  many  than  on a n t i b i o t i c  t h a t the only type  i s relatively  route  the LPS-phospholipid  t o be as low o r l o w e r  typhimurium,  interesting which  through  antibiotic  to  antibiotic  i n t h e LPS  of  i n p o r i n p r o t e i n s (Hancock, was a s s o c i a t e d w i t h  resistance  21  in  the  1984).  c o n v e r s i o n to  coli  and  S^_  typhimurium plausible  hypothesis  enteric  bacteria,  therefore outer  porin  1977;  Jaffe  to e x p l a i n t h i s porin  of  observation  molecules  aeruginosa  to v a r i o u s  may  are  already  be  considered  mutant  not  to a measurable decrease  (T.I. Nicas, Most  and  compounds t h a n  personal  to  be  noteworthy.  The  (R-factor)  series  best  0-antigen  did  periplasmic  enzymes  Reithmeier,  coli,  porin  P.  activity-  antibiotic  resistance  LPS  some  LPS  property mutants are  were  and  are  the  Rfa  defects,  RfaA, where o n l y  from  (heptoseless) not  and  far characterised  These  are LPS  appear  to  mutants  missing, mutant.  to  whereas  susceptibility EDTA  a  100-fold  typhimurium.  appreciably,  detergents,  RfaE  hydrophobic  phospholipases, (Sanderson  e_t  and  jal. ,  the  to  RfaE,  Less  severe  alter  the  have  antibiotic mutant  had  antibiotics, tended  1974;  to Singh  leak and  1975).  Similar strains  in  p o r t i o n s o f LPS  i s a deep r o u g h  enhanced  in  c h a r a c t e r i s e d LPS  i n S_^  repeating  susceptibility  as  m u t a n t s so  altered  s e v e r i t y of  mutations  t h a t o f E_;_  outer  communication).  increasing  which  -  in  t h e r e f o r e p o r i n p r o t e i n d e f i c i e n c y would  hypersusceptible  determined  the  10  in  active,  decrease  Since  to be  A  i s that,  relatively  i s estimated  deficient lead  1982).  d e f i c i e n c y causes a s i g n i f i c a n t  P_^  permeable  aeruginosa  LPS  e_t a_l. ,  membrane p e r m e a b i l i t y t o a n t i b i o t i c s .  membrane less  ( B a v o i l ejt a_l. ,  p r o p e r t i e s were f o u n d  (Meadow and  carbenicillin  Wells,  1985)  susceptibility  22  and  in certain w h i c h had  P_;_  aeruginosa  been s e l e c t e d f o r  sensitivity  to  rough  LPS-  specific  bacteriophages.  increasing rhamnose  defects and  of  S_^  Other  the  been  with  respect  to  of confusion the  LPS  8.  molecule  mutants  0-antigen core  In c o n t r a s t ,  t h e envA  permeability  to  the outer  both  increased mutant  hydrophobic  b u t has no o b v i o u s  defects  described either  which  their  but a c a u s a l  have  the  been  susceptibility  relationship  between  Consequently,  a  e x i s t s as t o t h e r o l e o f e a c h and  with  plus  had  interrelation  great portion  between  and between LPS and p r o t e i n m o l e c u l e s  in  LPS  stability  membrane.  Aim o f t h e s t u d y . This  study  antibiotic of  still  of  e_t a l . , 1 9 8 0 ) .  have  o r LPS chemotype,  molecules of  (Grundstrom  the  two p h e n o t y p e s was n o t d e r i v e d .  deal of  from  violet,  mutants  characterised  series  those with  shows h i g h e r  s u c h as G e n t i a n  LPS s t r u c t u r e  pattern  missing  a  to a n t i b i o t i c s .  typhimurium  substances in  i n LPS, o n l y  glucose  susceptibility  Of  the  describes  r e s i s t a n c e o f P_^  study,  characterised  the r o l e of the outer aeruginosa.  an a n t i b i o t i c to  identify  In the f i r s t  hypersusceptible  outer  membrane  membrane  in  portion  mutant  components  was which  m i g h t be r e s p o n s i b l e  for antibiotic resistance/susceptibility  in  The s e c o n d  the  P_^_  aeruginosa.  native  aeruginosa technique  configuration  of  and one f r o m E . c o l i to determine  possible  23  portion two using  of t h i s  porin  proteins  a chemical  trimeric  study  examines from  P.  crosslinking  organisation.  METHODS  1. Medium  and c u l t u r e  Proteose  peptone  used  as a r i c h  al.,  1974),  at  grown 37°C  were  source  was used  with  grown  0.5 mM MgSO^  (glucose,  succinate,  as a d e f i n e d  i n volumes o f from  under  ( D i f c o 0121-01)  and BM2 m i n i m a l medium  supplemented  citrate)  were  #2 (1$ [w/v])  medium,  20 mM o f c a r b o n or  conditions  conditions  f  (PP2)  (Gilleland  10 uM FeSO^, and  Liquid  cultures  10 ml t o 10 1 i n f l a s k s aeration.  on t h e same media w i t h  e_t  pyruvate, g l y c e r o l  medium.  o f good  was  Plate  the a d d i t i o n  shaken cultures  of  2% a g a r .  2. B a c t e r i a l S t r a i n s  P. W.  aeruginosa  s t r a i n s WT1  Zimmermann o f C i b a  had  been  mutagenised  methanesulphonate, nitrosoguanidine were  Geigy,  selected  cephalosporin  by C.  replica  and  AK  Strains were a k i n d Strain  were  a  Kingston, WT2, gift  four  steps  with  from  gift  with  M1.  rich  i n a separate Dr.  ethyl  N-methyl-N'-nitro-N-  p l a t i n g on  from  Dr.  S t r a i n WT1  Single agar  section.  A.  colonies  containing  I s o l a t i o n o f spontaneous r e v e r t a n t  and P1-6 i s d e s c r i b e d  University,  once  Switzerland.  t o p r o d u c e mutant s t r a i n  P1-4  43  Basel, in  then  and M1 were a g i f t  strains  S t r a i n s WT3  Kropinski,  Queen's  Ontario.  P2-6, from D r .  WT2 was a m u l t i p l y  P 2 - 6 t , WT2-6, P2-8, P2-8t and P2-6,8 J . Fyfe  of Univ. o f Edinburgh,  auxotrophic  24  PAO d e r i v a t i v e  U.K.  (PA0222).  Strains as  P2-6  donor  and  P2-8  strain,  were c o n s t r u c t e d  M1  were s e l e c t e d by  subsequently  tested  and  from  recipient  F116C  given  on  p.  P2-8.  performing strain  wild  type  a  onto  coli  Recombinant  transduction  as  P2-6,8 was  0.5  both  strains  K-12  the  same  used  for  constructed  from  strain  these  ml  and  were  (JC3272) and  P2-6  strains  are  constructed  by  donor  concentration  RP1-carrying  0.1  ml  f o r three  medium.  aeruginosa  recipient  strain.  above i n n u t r i e n t b r o t h  plus  shaken  RP1-containing  e a c h P_^  hypersusceptible  described  selective  high  recipient  hours.  to  were  Cells  placed  were  R e c i p i e n t s were s e l e c t e d of c a r b e n i c i l l i n coli  and  (0.5  mid-  then using  mg/ml)  non-plasmid-carrying  to P.  were s u s c e p t i b l e .  Antibiotic Minimal  obtained  markers  and  Strains  using  e x p e r i m e n t s between an  or a n t i b i o t i c  n u t r i e n t broth  aeruginosa  3.  o f E_^  phase, then  which  respectively,  RP1-containing  were grown as  very  prototrophy  phage F 1 1 6 C  D e t a i l e d genotypes of  conjugation  donor  plated  recipient.  susceptibility.  prototrophic  using,  50.  Plasmid  in  and  by  as  to  t r a n s d u c t i o n o f pro+ p r o t o t r o p h y  strain  log  P2-8,  experiments.  into  Cells  for antibiotic  and  strain  conjugation by  P2-6  WT2  conversion  P 2 - 8 t were c o n s t r u c t e d  strains  conjugation  l e u " (R68.45) and  Recombinants  P2-6t  by  by  incorporated  Susceptibility inhibitory plate  Testing  concentration  dilution  at v a r i o u s  method.  concentrations  25  (MIC)  into  values  were  Antibiotics  were  PP2-agar  plates.  Bacteria ml  of  were grown o v e r n i g h t , bacterial  Depending was  on t h e number  done  either  applicator 37°C.  of  antibiotic  MIC  Single  were  For  disc 1/100  diluted sterile  swab.  aliquots  of  culture 10  lower  than  inhibition then  spread  Results antibiotic  tested.  diameter of  itself,  4. A n t i b i o t i c s  and  from  P l a t e s were grown as t h e l o w e s t  placed  around  thienamycin  overnight  concentration  of growth.  were o b t a i n e d  by  diluting  s p r e a d - p l a t i n g 0.1  used  ml  onto  Concentrations  f o r MIC  used  determinations.  b a c t e r i a grown o v e r n i g h t onto  obtained  onto  were  PP2-agar p l a t e s u s i n g  s o l u t i o n s onto  zone was  sterile  filter  expressed  the d i s c ,  0.01  by s p o t t i n g  t h e lawn o f e a c h  ampicillin, streptomycin  chloramphenicol  Sigma C h e m i c a l  formyl  were  a  strain  the  ml  discs  as t h e  including  a  to  total  diameter  Chemicals  sulphate,  hydrochloride,  multisyringe  using  w h i c h measured 6mm.  Benzylpenicillin, gentamicin  evenly  The i n h i b i t i o n  of i n h i b i t i o n  the d i s c  assays,  plates.  by  antibiotic. those  the  the s p o t t i n g  and t h e n  - 6  onto  0.01  t o be t e s t e d ,  resistance levels  w h i c h were i m m e d i a t e l y be  spotted  inhibition  plates containing  somewhat  or  were d e f i n e d  which produced  overnight  PP2-agar  of s t r a i n s  manually  values  cell  was  e_t a_l. , 1 9 6 0 ) .  (Zierdt  at  an  suspension  1 0 0 - f o l d and t h e n  diluted  Co.  polymyxin sulphate,  and t r i m e t h o p r i m  ( S t . L o u i s , USA).  (Imipenem)  26  were o b t a i n e d  B  sulphate,  tetracycline were  purchased  N o r f l o x a c i n and Nf r o m Merck, S h a r p  and  Dohme  research  l a b (New  m o x a l a c t a m were a g i f t USA).  Cefsulodin  Switzerland).  was  Azthreonam  Carbenicillin  (Montreal,  were a g i f t  (DSP) USA).  of Glaxo  purchased  Disulphide  gift  Ciba  was  a gift  a gift  purchased  from  reducing  A c r y l a m i d e and  (Rochester, dodecyl obtained LKB  Chemicals  from  BDH  (Stockholm,  purchased The  from  was  lysozyme  specially  Chemicals.  Sweden). Schleicher  fluorescent were  Ayerst a gift  Chemical  USA),  purchased  Schuell  from  Sigma  p r e p a r e d as d e s c r i b e d  chemicals  used  were  o f the  propionate)  Co.,  (Rockford  Inc.  highest  2-  purchased Co.  Sodium  and  was  were p u r c h a s e d  from  membrane s h e e t s were (Keene,  Chemical i n Methods. standard  Revertants of  27  and  Laboratories  quality  available.  of Spontaneous  nitrocefin  N.H.,  1-N-phenylnapthylamine  B was  Isolation  Beecham  Eastman Kodak  purified  polymyxin  5.  from  (Toronto, Canada).  Nitrocellulose  probe,  Sons,  Laboratories  Bio-Rad  Ampholines  and  Inc.  and  b i s - a c r y l a m i d e were  USA)  BDH  (Basel,  L t d . , Greenford, England.  from  (Indiana,  sulphate  from  Squibb  C e f t a z i d i m e and  Laboratories and  AG  agents d i t h i o e r y t h r i t o l  obtained  and  (Indianapolis,  Geigy  o f E.R.  the P i e r c e  (Richmond, Miles  Co.  dithio-b_is-(succinimidyl  were  from  Tobramycin  o f R o u s s e l Canada  was  Canada).  mercaptoethanol USA).  from  Group R e s e a r c h  crosslinker,  was  and  Ticarcillin  (Pt. C l a i r e ,  USA).  E l i Lilly  was  was  Canada).  Laboratories  The  a  Cefotaxime  (Montreal). Inc.  from  Jersey,  M1  USA).  (NPN),and Co.  Dansyl A l l other  commercially  Cells were  with  wild  isolated  as  supersusceptible 1$  PP2  cells  6.  cell  large  K.  variety An  sensitivity  of  P_^  Ph.D.  tested.  Reeves  strains  The  using  phage s t o c k s  PFU/ml, t h e n  spotted  Beta-lactamase  cephalosporin, Cultures in  PP2  sodium  onto  was  broth  and  phosphate  then or  were t e s t e d  nm  the  of  a  medium  M1  same  1.0.  to  The  100-fold  containing  between  and  in  the  WT1.  for sensitivity  phages  (Nicas  to  a  and  Hancock,  r o u g h - s p e c i f i c phage, D8  (0PLS27 -  Queen's U n i v . ,  Ontario,  described  multisyringe  by  phage  to g i v e  1980),  was  Hancock  and  applicator.  approximately  lawn on  10^  PP2-agar.  calculations assayed  with  the  (O'Callaghan  optical  suspended 5 mM  the  testing  nitrocefin  were grown to an  on  for strains  a bacterial  and  of  concentrations  were d i l u t e d  assay  Beta-lactamase  a  ml  600  plated  a s s a y s were done as  (1975),  Briefly,  at  aeruginosa  Thesis,  at  of  grown o v e r n i g h t  resuspended  and  gentamicin  was  i n t o 10  density  o f PP2  a d d i t i o n a l deep  Jarrell,  also  ml  bacterial  1980).  optical  antibiotics  revertants  subcultured  resistance levels  Bacteriophage All  7.  or  r e s i s t a n c e to  S t r a i n M1  centrifuged,  i n 0.1  carbenicillin single  then  then  concentration  partial  spontaneous  grown t o an  were  or  mutant M1.  medium and  medium and  type  density  t o one  at  a^.,  600  quarter  Na-HEPES b u f f e r 28  £t  (both  chromogenic  nm  of  1972). 0.5-0.8  volume i n 50 pH  7.0).  mM One  hundred  mierolitres  nitrocefin  solution  semimicro  cuvette,  were  monitored  of  A  hydrolysis lactamase and  broken  the  strains.  pressure for EDTA  i  n  g  to c o n t r o l  a  P  e  r  kin  Elmer  ml  of  hydrolysis  dual  chart  f o rpossible Assays  i n buffer  recorder.  periplasmic  whole  leakage  of  were p e r f o r m e d  were b r o k e n  which  beam  by s i m u l t a n e o u s l y p r e p a r e d  t o g i v e a measure o f r e l a t i v e  2  releasing  0.65  of n i t r o c e f i n  to a Sargent-Welch  a t 20,000 l b / i n ,  proved  beta-lactamase  cell beta-  on whole  crypticity  once  by  in  French  t o be more  efficient  t h a n were  MgCl^  o  r  treatments.  lactamase ratio  calculated the r a t e  was  calculated  activity  nitrocefin  of  s  of n i t r o c e f i n  Cells  Crypticity  The  u  the periplasm.  cells  to  s u p e r n a t a n t s were s u b t r a c t e d f r o m  values from  M g 5  coupled  culture  added  and t h e k i n e t i c s  Values f o r h y d r o l y s i s cell-free  were  (0.1 - 0.25 mg/ml i n t h e same b u f f e r ) i n a  at  spectrophotometer  cells  of  the  ( S ) over  as t h e r a t i o  o f broken  cells  to that  apparent  periplasmic  beta-  whole  cells.  concentration  of  Q  using the Michaelis-Menten  of hydrolysis  concentrations  of  the  t h e o u t s i d e medium c o n c e n t r a t i o n ( S ) was  g  hydrolysis  of  in  i n intact broken  cells cells  equation,  substituting  ( v ) f o r V and t h e at  high  rate  nitrocefin  as V max • V  ^m  w  a  s  " max V  x  determined  s  e ^ m K  from  +  s  e  assays performed  on  broken  cells. The  permeability  parameter  29  "C" was  calculated  from  the  hydrolysis  rate  concentration using  "v" o f  "S n f o  Fick's  Q  nitrocefin  nitrocefin  first  law  Zimmermann and R o s s e l e t  of  total the  area  distance  calculated For  across  across  partitioning by T s u j i  and  5mM  quantified  by  unhydrolysed reference ratio  0  i  assaying  n  (1977),  the  diffuses  and  S  w  cells  and a s s a y e d was  benzyl  as a b o v e .  performed  equal  7.0.  as  volumes o f 1-  Nitrocefin  absorbance  (390 nm) u s i n g  s  were  0.2 t o 200 ug o f  pH  a  equation.  using  peak  d i v i d e d by  was  value  for  the a p p r o p r i a t e  blank  were p r e s e n t e d  as t h e  The r e s u l t s phases.  assay  were s u s p e n d e d  in  mM  t o an o p t i c a l  Na-HEPES b u f f e r pH  phenylnaphthylamine concentration  by  ( p r o p o r t i o n a l to the  nitrocefin  the top / bottom  Cells 5  medium,  expressed  membrane)  broken  Na-HEPES b u f f e r ,  f o r each phase.  8. NPN u p t a k e  nm  of  e_t a_l. ,  nitrocefin  of A^g  as  beta-lactamase,  containing  p e r ml as an i n d u c e r ,  Phase  octanol  the  of induced  grown f o r 2 h i n PP2 b r o t h  described  coefficient  t h e above M i c h a e l i s - M e n t e n  measurement  penicillin  diffusion,  which n i t r o c e f i n  travelled  from  i n the surrounding  the  se)'  C i s the p e r m e a b i l i t y  surface  over  (1977):  v = C (S o wherein  i n whole c e l l s  (NPN)  o f 0.5 mM  was  7.0  - 5mM  dissolved  and u s e d ,  30  density  o f 0.5 a t  Na-azide. in  as d e s c r i b e d  acetone  600 1-N-  at  a  by Loh e t a l .  (1984), mM.  at a f i n a l  Fluorescence  650-10S  concentration (in c e l l  suspension)  intensity  with  water bath  at  temperature.  5  a steady NPN  were 350  nm.  Total  Gentamicin cell  and  was  fluorescence  by  nm,  each  to a f i n a l  and  had  cells  equipped  and  Perkin-Elmer with a  emission  respectively,  then  i n c r e a s e were  alone  caused  Excitation  420  added  a  0.01  Haake  t o m a i n t a i n t h e c u v e t t e h o l d i n g chamber  f l u o r e s c e n c e of c e l l  suspension  MgCl^  measured  f l u o r e s c e n c e spectrophotometer  circulating  for  was  of  suspensions  slit  widths  was  measured.  c o n c e n t r a t i o n o f 4 ug/ml  rates  of  monitored.  negligible  a l o n e was  with  wavelengths  NPN,  and  in  gentamicin-induced gentamicin  fluorescence.  minimal  of  was  and  Fluorescence  subtracted  from  total fluorescence.  9.  P e r m e a b i l i s a t i o n o f whole c e l l s  Cells medium  were grown t o m i d l o g  then  centrifuged  and  to  phase i n p r o t e o s e  resuspended  inhibit  respiration),  5  density  at  approximately  Chemical  Co.,  ug/ml,  then  polymyxin Lysis  600nm St.  L o u i s ) was  B were added  of  observation  cells  was  to l y s i s ,  the presence  mM  in  Na-Hepes pH  added  5mM 7-0  0.5.  No.2  Na-azide  (to  an  followed the  used and  of lysozyme  in  decrease  optical  Lysozyme  a in  ^ftoo*  that  decrease  controls  were p e r f o r m e d  50  gentamicin (Fig.  Perkin-Elmer  t o check  or 3).  OV/VIS  Microscopic ^^QQ  was  to ensure  that  in  or p e r m e a b i l i s e r a l o n e d i d not  31  (Sigma  to a c o n c e n t r a t i o n of  a t the g i v e n c o n c e n t r a t i o n s  was as  peptone  to  t h e p e r m e a b i l i s i n g compounds EDTA,  spectrophotometer  related  of  lysozyme  cause  lysis.  10.  Dansyl  polymyxin  Dansylated as  polymyxin  d e s c r i b e d by  the  dansyl  650-10S  (DPX)  S c h i n d l e r and  group  wavelengths  b i n d i n g assay  was  o f 340  and  was  Teuber  measured 485  fluorescence  prepared  nm,  at  by Dr.  (1975).  spectrophotometer  Moore  F l u o r e s c e n c e of  excitation  respectively,  R.  and  in a  emission  Perkin-Elmer  (Norwalk,  Conn,  U.S.A.). The  assay  ^ 600=0.5  mixture  prior  A  (4.0  ug/ml)]  azide  (to i n h i b i t  DPX  using  bound  sodium  to the p o i n t  DPX  calculate  compared in  recorded  in  aliquots  or L i p i d  added  to determine  Lipid  -5mM  A  sodium of  Fluorescence  arbitrary  units  Industrial).  o f LPS  DPX  cells  pmole a l i q u o t s  was  A,  were bound  to g i v e a f l u o r e s c e n c e  bound.  what f r a c t i o n  which c o u l d  o f DPX  These  t o the f l u o r e s c e n c e v a l u e a t s a t u r a t i o n  excess,  actually  all  7.0  saturation.  t o a g i v e n amount o f LPS,  when  pH  t h e amount o f d a n s y l p o l y m y x i n  to a 1 0 0 - f o l d excess value  of  of  ug/ml) o r  t o w h i c h 500  b i n d i n g was  (Beckman  (3-0  Hepes b u f f e r  respiration),  a voltmeter To  be  i n 5mM  after  of a suspension  t o a d d i t i o n ) [ o r LPS  were added up  increase  consisted  values when DPX  o f t h e added  DPX  were was was  bound.  Inhibition examined containing  by  of  adding cells,  DPX  b i n d i n g by M g  aliquots LPS  gentamicin  to  assay  as w e l l  as DPX  of i n h i b i t o r  or L i p i d  32  or  + +  A,  was  mixtures at  near  saturating measured  conditions.  Decrease  as d e s c r i b e d  i n observed  and e x p r e s s e d  f l u o r e s c e n c e was  as p e r c e n t i n h i b i t i o n  of  fluorescence.  11.  Phase p a r t i t i o n i n g  Cells  were  of  grown  cells  t o mid  phase,  concentrated  ten-fold  phase p o l y m e r  system  described  prepared  adding  volumes o f 20$(w/w)  (PEG)  by  6000  (Sigma  (Pharmacia,  6.2%,  a s s a y was  each  X-100 were  two-  (1977)  polyethylene  Co.) and 20$(w/w)  was  glycol  Dextran  T500  c o n c e n t r a t i o n s o f 4.4$  i n 30mM T r i s - H C l  the cells  performed  buffer,  hours  and  pH 7.0. T h i s  to allow separation  i n glass  o f t h e t o p and bottom  and 0.1 then  minutes  ml o f 1 0 - f o l d  inverted  phases  top was  and  expressed  t u b e s by a d d i n g as w e l l  concentrated  20 t i m e s and l e f t  a t room t e m p e r a t u r e . bottom  Aliquots  cells.  was into  u  e  to the  0 0  i  n  t  h  e  ml  Triton  The  tubes for  45  both of  Lambda  The r e s u l t s  top/bottom  ml  presence  i n a P e r k i n - E l m e r UV/VIS  of A g  1.0  were removed f r o m  using appropriate blanks.  as t h e r a t i o  as 0.1  to e q u i l i b r a t e  and A ^ Q Q ( j  phases  r e c o r d e d f o r each  spectrophotometer,  12.  The aqueous  by Magnusson e_t a_l.  a t 4°C f o r s e v e r a l  and  phases. The  of  Chemical  respectively,  centrifuged  t o the assay.  Sweden) t o g i v e f i n a l  equilibrated two  prior  log  3  were  phases.  P r e p a r a t i o n o f o u t e r membranes Cells  were h a r v e s t e d by c e n t r i f u g a t i o n  33  and, t o p l a s m o l y s e  the  cells,  were  sucrose-1OmM Hancock aid  resuspended  and N i k a i d o  breakage  and  15,000  supernatant  gradient  with  treated  was  Cell  decanted  T0% s u c r o s e  at  l e a s t 12 h i n a Beckman  layer.  This  g  i n a French debris  was c e n t r i f u g e d SW41  for onto  pancreatic cell  removed  by  10 min  and  a  two-step sucrose  a t 183,000 X g f o r  o r SW27Ti r o t o r .  by  to  Pressure  was  of  frozen  l a y e r and 60%  band was c o l l e c t e d f r o m  i n a Beckman  procedure  mg/ml  and l a y e r e d  b o t t o m d r i p method, c o n c e n t r a t e d X  0.5  i n t h e bottom  the upper  membrane  the  3000 rpm i n an SS34 r o t o r  in  outer  with  twice  inch.  at  following  C e l l s were t y p i c a l l y  and b r o k e n  lb/sq.  centrifugation the  (1978). then  deoxyribonuclease at  pH 8.0,  Tris-HCl  i n 20%  t o 100X c o n c e n t r a t i o n  The  the gradient  lower  by  the  c e n t r i f u g a t i o n a t 160,000  Type 50 o r 70 r o t o r  and k e p t  frozen  at  20°C .  13.  Preparation To  were  of c e l l  prepare  cell  centrifuged  diluted  in  envelopes,  and b r o k e n  10 mM  14.  deionized  as  mid-logarithmic described  phosphate b u f f e r  160,000 X g f o r 1 h o u r . in  envelopes phase  cells  previously,  pH 7.4 and c e n t r i f u g e d  The e n v e l o p e  pellet  was  then at  resuspended  water.  I s o l a t i o n of lipopolysaccharide The  Hancock described  method used  (1983). above  f o r LPS i s o l a t i o n Mid-log  and  phase  resuspended  34  was t h a t  cells to  were  o f D a r v e a u and harvested  approximately  as  70-fold  concentration mg/ml  i n 10 mM  T r i s - H C l b u f f e r pH 8.0 - 2mM M g C l ^  deoxyribonuclease  ribonuclease. through  a  cell  French  resulting DNase  The  cell  and  suspension  pressure  lysate  cell  and  0.05 mg/ml, r e s p e c t i v e l y ,  at  37°C  final (final  pH  solubility remove  the  added  sample  added,  was Two  mixed  incubated  and c o o l e d  The  pellet  2%  (w/v) SDS i n 10mM  LPS  was  (w/v),  to  Pronase  mg/ml was  The  bursts. 0.2  incubated to  respectively,  vortexed  supernatant  was  to  ensure  decanted  overnight  at 2  37°C i  with  g5eg  n  e  thanol .  was r e - a d d e d  of  with  h a t 15°C i n t h e p r e s e n c e  any  to a f i n a l  and t h e sample was i n c u b a t e d  c e n t r i f u g e d to c o l l e c t  were  The sample -k ° C .  0  i n t h e same volume o f 0.1 M EDTA  ensure d e n a t u r a t i o n  resuspended  and  constant  T r i s - H C l pH 8 and was i n c u b a t e d  precipitated  and  c o n c e n t r a t i o n o f 0.2 mg/ml  t o 0°C i n an i c e b a t h  was r e s u s p e n d e d  min.  described, and  sec  c e n t r i f u g e d a t 12,000 X g f o r 15 min a t  proteins. 0.025  lb/sq.in.  - EDTA and SDS were added  volumes o f 0 .375 M M g C l  then  20  The  to a f i n a l  was  for  twice  concentrations of  2%  1  and c e n t r i f u g e d a t 50 ,000 X g f o r 30 min a t 20<>c t o  was  shaking.  passed  f o r two 30  The sample was  peptidoglycan.  Pronase  then  15,000  <  pancreatic  and t h e s u s p e n s i o n  o f 0.1 M and  9.5).  was  at  to f i n a l  Tetrasodium  concentrations  mg/ml  was  was s o n i c a t e d  and RNase were r e - a d d e d  f o r 2 h.  0.025  Q  + +  85°C  SDS-resistant  concentration  overnight  Mg -ethanol  at  as  at  of  37°C.  previously  i n t h e same volume o f 10 mM T r i s - H C l t h e LPS p e l l e t o f 25 mM  35  MgCl . 9  a t 200,000 X g f o r 2  15.  Preparation Lipid  of  A was  Rietschel  for  KDO  of L i p i d  A  prepared using  e_t al..  (1977).  a modification  Equimolar  content) of l y o p h i l i s e d  LPS  were  resuspended  t o 15 ml  i n 50  3.0.  The  buffered  a t 3.0  chance  pH  was  of removing  labile  resuspended  LPS  thawed  times to  four  was  were c e n t r i f u g e d minutes washed  twice  more  acetate  prior  removal  o f LPS  each  acetate  groups  from  f o r 1.5  once  glass  h,  with deionised  for loss  strain  buffer  the  Lipid  The  A.  then f r o z e n  acetate  water  pH  to l e s s e n  A.  to  for 5  buffer, remove  and  Samples  t u b e s a t 9500 rpm  w i t h sodium  then sodium  Samples were r e s u s p e n d e d  o f KDO  c a r b o h y d r a t e from  content to ensure  Lipid  in  complete  A.  assays  Protein and  p r e p a r e d from sodium  method  (standardised  promote a g g r e g a t i o n o f L i p i d  to l y o p h i l i s a t i o n .  assayed  1 6 . Chemical  a t 100°C  in thick-walled  washed  amounts  i n an a t t e m p t  phosphate  heated  and  and  mM  o f the  a s s a y was  Strominger  (1972)  performed using  as d e s c r i b e d  1?  (w/v)  SDS  by  Sandermann  to  solubilise  proteins. 3-Deoxy-D-mannooctulosonic the  colourimetric  al.  (1978) on  samples  min  at 1 0 0 ° ^  The  acid  thiobarbituric o f LPS  (KDO)  acid  was  assay of  h y d r o l y s e d i n 0.2  ammonium s a l t  o f KDO  determine  by  Karkhanis  e_t  N H^so^  (Sigma) was  f o r 30 used  as  the s t a n d a r d . Phosphate  was  assayed  using 36  the  ascorbate/molybdate  method  17.  o f Ames and  Dubin  SDS-Polyaorylamide  The  (1960).  gel  Laemmli g e l method,  (1975),  was  used  proteins.  used  a constant r a t i o  at  44:0.8.  A l l stacking  0.4$  (w/v)  routinely  SDS  10$  EDTA,  samples  added from  were  behaviour.  Gels weight  97,400  ),  bovine  wt.  (mol.  (see  to  ), r a b b i t  were  pH  Samples  6.8.  LPS  were of  which  were 0.1  M  their  4$  were  MgCl  a g g r e g a t i o n , or  In c e r t a i n  pH  contained  presence  agents  mix  2 >  40  circumstances, heat-unmodified  at a constant v o l t a g e of  150  V.  were E . c o l i b e t a - g a l a c t o s i d a s e  muscle  phosphorylase  ( mol.  wt.  ), c a r b o n i c a n h y d r a s e  Coomassie  of  i n 0.375 M T r i s - H C l and  analyse  albumen  inhibitor  with  acrylamide  acrylamide  promoting  ( mol.  wt.  1 4 , 3 0 0 ) , a l l o b t a i n e d from  were s t a i n e d  outer  bis-acrylamide  Other  banding.  heated  45,000  trypsin wt.  LPS  serum  :  solubilisation  s t a n d a r d s used  116,000  of  e_t a l .  of  h e a t i n g t o 88°C i n t h e  were r u n  wt.  soybean  by  M Tris-HCl  t h e g e l by  not  (mol.  ( mol.  SDS  were 3% (w/v)  t o the  t o improve  Molecular  (w/v)  (v/v) 2-mercaptoethanol.  remove LPS  mM  of acrylamide  i n 0.125  solubilised  occasionally to  gels  Lugtenberg  gel electrophoresis  Various percentages  G e l s c o n t a i n e d 0.4?  8.8.  and  as m o d i f i e d by  for a l l routine  membrane  SDS  electrophoresis  Brilliant  66,000 ( mol.  20,100  Sigma Blue  B  ( mol.  ), ovalbumen wt.  ) and  Chemical dye  wt.  29,000  ),  lysozyme Co.  Gels  or s i l v e r  stain  below). Gels  for  the e x a m i n a t i o n  37  of l i p o p o l y s a c c h a r i d e  were  as  above but  with  15$  Frasch,  1982)  length  O-antigen  added  to  improve  18.  to  the  LPS  acrylamide,  improve  gels.  or  The  blotting  LPS  gels  SDS  at  of  1 mA  composed  of  192  methanol  pH  8.3.  antigens  (see  19.  for  in  bovine 30  sites.  of  (Tsai  heterogeneous (40  mM)  and  chain  was  solubilisation  and  also  mix  to  from  or  i n contact  These were t h e n either  f o r 2 h at  400  glycine,  25  mM  Blots  polyacrylamide  with  been  0.45  The  blots  into  overnight  at  Transfer Tris-HCl  pore in  a Bio a  Rad  constant  buffer  and  were i m m u n o - s t a i n e d  staining  Mutharia 3$  37°C t o  diluted at  um  pre-soaked  placed  mA. mM  gels  Towbin e_t a_l. (1979) f o r  membranes w h i c h had  blotted  LPS  was  20$  for  of  proteins  e_t_ aJL.  (v/v)  or  LPS  (1982) was  foetal calf  on  (v/v)  specific  4°C.  saturate  serum  non-specific  were t h e n r i n s e d in  3$ The  FCS-PBS antibody  38  briefly was  blots  used.  Blots  (FCS)  serum albumen i n p h o s p h a t e - b u f f e r e d at  overnight  method o f  were p l a c e d  either  min  antibody  added  below).  method  incubated (w/v)  the  60°C.  Immunoenzymatic The  the  proteins  were t r a n s f e r r e d  a p p a r a t u s and  current  of  mentioned  b l o t t i n g of  nitrocellulose (w/v)  u r e a was  Tetrasodium-EDTA  previously  to n i t r o c e l l u l o s e u s i n g  0.1$  resolution  bands.  Electrophoretic  size  4M  resolution.  Proteins  SDS  and  or  saline  protein  i n PBS  added  d i l u t i o n used  and  were 3$ (PBS)  binding 10  ml  of  and  incubated  was  sufficient  t o g i v e an ELISA extensively IgG  Fab  added,  1  titre  o f between  washed w i t h fragments  in  10  PBS and t h e n  conjugated  - 10 *.  2  The b l o t s  1  10 ml o f g o a t  to a l k a l i n e  3$ FCS/PBS and i n c u b a t e d  anti-mouse  phosphatase  2 h a t 37°C.  phosphoric  salt  [Sigma]  development  20. a)  acid,  i n 50 mM  usually  Gel s t a i n i n g  disodium Tris-HCl  took  salt  were  The  were a g a i n washed e x t e n s i v e l y and s u b s t r a t e (1 mg/ml AS-MX  were  blots  Naphthol  + 2 mg/ml F a s t Red  pH 8.5)  was  added.  TR  Colour  p l a c e w i t h i n 5 min.  procedures  Protein Coomassie  R-250  dye  acetic  acid.  al.  was composed  were s t a i n e d  (1981).  G e l s were i n c u b a t e d  (w/v)  stained  NaOH  extensively (v/v)  o f Coomassie B r i l l i a n t  a t 0.5 mg/ml i n 20$ ( v / v )  Proteins  methanol,  50$  stain  +  with  1.4$  with  and d e v e l o p e d  formaldehyde  silver  silver  (v/v)  methanol  solution ammonium  i n 0.005$  in distilled  - 7.5$ ( v / v )  by t h e method  overnight with  water.  o f Wray e_t  shaking  i n 50%  [0.5$ (w/v) i n hydroxide],  (w/v) c i t r i c  Blue  0.75$ rinsed  acid,  0.019$  G e l s were s t o r e d  in  methanol.  b) L i p o p o l y s a c c h a r i d e Silver method to  stain  of Tsai  the p r o t e i n  step  f o r LPS employed  and F r a s c h stain  modification  (1982) and was p e r f o r m e d  above, but w i t h  to allow s t a i n i n g  a  a periodate  o f the sugars.  39  of the similarly  pre-treatment  The g e l s were  soaked  i n 40$  overnight  5 min i n 0 . 7 $  soaked this  (v/v) methanol with  point  periodate  the  staining  Staining  agitated  f o r 10 min,  (also  as  halted  with  reagent  f o r protein  t h e method  stain)  scanning  of S c h i f f  or f o r p r o t e i n  Quick  rinsed  changed  o f f under  well.  was added  r i n s e d and  and  running  stain)  At the  deionised  was added and  Developing and t h e  solution  staining  was  50$ m e t h a n o l .  Densitometer  1969),  was  (as f o r p r o t e i n  then  then  i n 40$ m e t h a n o l .  acid  container  and m e t h a n o l were washed  water.  by  (w/v) p e r i o d i c  agitation  Scan  Jr.  of gels  stained  (as m o d i f i e d  scanner  carbohydrate,  by Z a c h a r i u s  by C o o m a s s i e b l u e ,  gel  for  (Helena  e_t a _ l .  was p e r f o r m e d  with a  Laboratories  Corp.,  resuspended  to  Beaumont, T e x . ) .  21 . P r o t e a s e  digestion  Outer  membrane  (w/v)  and c o o l e d  added the Each  6.8,  pH  Bromphenol  min.  in  samples  0 . 5 $ SDS, blue  dye.  t o 37°C  membrane  sample  was t h e n  divided  SDS, a t a f i n a l  to  was  other  added  identify  2-ME-modifiable  further  heated  at  ml sample  This  digestion.  concentrations  88°c  into  SDS and 2-ME  fragments and t h e n  40  indicated  two p o r t i o n s ,  concentration (10$  of protein loaded  for  Protease  for 1 h  at to  o f 2$  onto  a  2  was and  37°C.  one  of  (w/v), and  - v/v) F.  M  0.0001$  a t 100°C  was h e a t e d  a  0.125  buffer:  10$ ( v / v ) g l y c e r o l and  s a m p l e s were d i g e s t e d  w h i c h was added the  were  f o r protease  1 u l amounts a t t h e  outer  membranes  o f 0 . 5 mg/ml i n 0 . 1  concentration Tris-HCl  of outer  to  help  These  were  14$  (w/v)  acrylamide g e l . was  found  The  initial  to g r e a t l y  aid digestion  resistant  o u t e r membrane  22.  f o r LPS  Assay  LPS Wright A  0.1  Rebers  ml  [KDO]  and  ml  warmed was  to  left  to  scanned and  was  assayed  of  15  M H^Q^  then heated develop  LPS  w  a  and  s  ug/ml) was a  d  d  e  d  ml  t o 100°C f o r 20  nm  f o r coloured products  used  heptose  as s t a n d a r d s  Gas  liquid  For  f o r LPS  .  T  h  min.  e  s a m  The  were  fatty  premixed acid)  analysis,  internal  was  samples  Samples were h y d r o l y s e d i n s c r e w - c a p p e d 1 ml  (Instant  o f 2M  methanolic  Methanolic  State College,  HC1  were  fatty  10 mg  600  nm  sugars. were  of  acids lyophilised  (0.103  mg  of  with  methanolic  tubes  i n the  presence  acetyl  chloride  prepared  from  Kit;  Applied  Science  H y d r o l y s i s was  41  then  respectively.  HC1,  Pa.).  were  L-rhamnose)  standard  before hydrolysis  4°c  cysteine  and  hexose,  o f whole c e l l  acid  with  and  keto-  ple  of c e r t a i n  (and  assay.  for  o f 3% (w/v)  typical  D-glucose  chromatography  whole c e l l  pentadecanoic  Inc.,  acid  by  c o o l e d to  c o l o u r f o r 3 h i n the dark  and  of  modification  s p e c t r o p h o t o m e t r i c a l l y between t h e w a v e l e n g t h s  350  cells  protease-  (standardised  0.02  D-glycero-L-mannoheptose  23.  using a  c o n t e n t o f 4.5  room t e m p e r a t u r e  added,  fairly  SDS  proteins.  of p u r i f i e d  deoxyoctonate 0.9  of the  (1972) o f t h e c y s t e i n e - s u l p h u r i c  sample  then  s t e p i n 0.5%  carbohydrate  carbohydrate  and  solubilisation  HC1.  Laboratories  carried  out  under  nitrogen were  f o r 16  tube, salts  and  t h e s e were t h e n  o r Dowex.  a glass D0H  2°C/min;  100-  a t 18  Cells  The  by  2 mm  Internal  added  to  insoluble  supernatant  initial  ramp  (helium)  Sp-  Inc.,  used:  200°C;  gas  were  equipped  (Supelco  programme was  Carrier  each  c o n t a i n i n g 3%  ID)  temperature,  3 min.  silver  chromatograph  Supelcoport  following  final  purified  medium p l u s g l u c o s e .  L.  Chan from  cells  P were i s o l a t e d  from  cells  limiting  minimal  previous  publications  medium.  rate,  flow  was  membrane i n 0.015  following  grown  on  the  on  HEPES-based  and  PhoE - Benz e_t a l . , purified  protein  v o l u m e s o f 0.2M specifications  42  were  protein  PP2  D a r v e a u and  P r o t e i n s were p u r i f i e d  and ml  experiments  grown  (by R.  proteins  Purified  (F - Hancock, Decad  - Hancock e t al_. , 1982;  8.5,  by  with  t o remove the  o f o u t e r membranes and  PhoE and  respectively)  pH  cm  samples  ml/min.  minimal  obtained  crosslinked  305  the  g).  was  f o r o u t e r membrane c r o s s l i n k i n g  grown i n BM2  Outer  ug)  Sigma 3 gas  200-mesh  time,  Crosslinking  Proteins  to  160°C;  final  maintained  (ca.  Pa.).  temperature,  829  0.031  P o r t i o n s (1 t o 2 u l ) o f t h e  column  on  period,  neutralised  centrifuged  i n a Perkin-Elmer  Bellefonte,  was  and  (methyltetradecanoate;  analysed  24.  this  o r Dowex AG1-X8 (HCC^-) ( c a .  standard  2100  After  c o o l e d t o room t e m p e r a t u r e  carbonate  with  h a t lOOOrj.  F  medium. K.  Poole,  phosphateas  stated  N i k a i d o , 1979;  in P  1984). preparations  were  triethanolamine buffer of Reithmeier  and  Bragg  (1977).  The  crosslinker,  dimethylsulphoxide dilution  to  indicated time,  give  excess  1M  and  reaction  20$  0.8$  atop  8.5 was  (w/v)  10$  added  4$ (w/v) SDS,  described  DSP  as  reaction  without  to  0.5M T r i s - H C l  reducing  above.  and  Two-dimensional  gel  by s o a k i n g t h e f i r s t  and  dimension  (v/v)  f o r 30 min a t room t e m p e r a t u r e , dimension  pH  agent  2-mercaptoethanol  agarose  of  minutes  1/15  1:1  containing  the second  a  and t h e p r e p a r a t i o n s were d i l u t e d  was p e r f o r m e d  strips  two  as  the  electrophoresis  dithioerythritol  samples  in  stop  as  in  dissolved  concentration  After  (v/v) g l y c e r o l  strips  was  the  final  pH  electrophoresed  gel  to  experiment. Tris-HCl  sample b u f f e r  6.8  added  t h e optimum  f o r each  crosslinking into  and  DSP,  or  10mM  s e a l i n g the  SDS-polyacrylamide g e l  electrophoresing  as  with  described  previously. Whole  cell  described  by  logarithmic 10^0  crosslinking Palva  phase  cells/ml  and  cells  whole  Cells  no  reaction membranes by  by  appreciable  reagent,  reacted  for  was t e r m i n a t e d w i t h e x c e s s were i s o l a t e d  (by t h e  two-dimensional  two  43  criterion  were cell  minutes,  previously  gel electrophoresis.  X  motile, surface.  o f 0.01 - 0.1 mg/ml o f  1M T r i s - H C l  as d e s c r i b e d  1  centrifugation,  d i s t u r b a n c e of the  by t h e a d d i t i o n  as  concentrating  approximately  After  was n o t a l t e r e d  performed  b e t a - l a c t a m a s e a s s a y ) and c e l l s  were c r o s s l i n k e d  crosslinking  were  (1976)  i n a volume o f one m l .  cell  suggesting  Randall  100-fold to give  o u t e r membrane p e r m e a b i l i t y of  experiments  then  pH 8.5. and  the Outer  analysed  CHAPTER CHARACTERISATION  OF  A  ONE  HYPERSUSCEPTIBLE MUTANT  OF  PSEUDOMONAS  AERUGINOSA  INTRODUCTION  The aeruginosa highly  high  has  intrinsic  been  impermeable  (Benz  activity  those  of Escherichia  was f o u n d  (6000 d a l t o n  exclusion  major  o f E_;_ c o l i  the  total  porin  conductance In  other  outer  major  lipopolysaccharide  coli,  molecules  organisms spp.  chain  Bryan,  length,  c e r t a i n types  passage  as  membrane aeruginosa  both with  compared  aeruginosa  limit),  Hancock,  1981).  be a f f e c t e d  by  some  which  i s the  constituent.  The different  to average  1984) and  were f o u n d  t o be much more  and d e t e r g e n t s ,  length of  sugars.  w h i c h p o s s e s s LPS  44  major  bilayer  1984),  of a n t i b i o t i c s  to  lipid  of constituent  (Hitchcock,  to  molecules  exclusion  i s considerably  respect  a  1979) t h a n t h e  by b l a c k  also  P.  of  i s due  of larger  (Benz and  could  and i n t h e t y p e s  and  t h e P_j_  as measured  s u c h as N e i s s e r i a  (Parr  average to  of  channels  the l i p o p o l y s a c c h a r i d e ,  o f P_^  of  suggested  impermeability  (OmpF - 600 d a l t o n  permeability  been  - Hancock e_t aJL. ,  was much l o w e r  of  LPS  to permit  limit  characteristic(s)  that  this  Although  activity,  assay,  addition,  from  I t has  o f Pseudomonas p o r i n  protein  porin  membrane.  coli.  resistance  t o be due t o p o s s e s s i o n  1981) t h a t  poor  porin  proposed outer  and Hancock,  antibiotic  Other  Hemophilus of  short  susceptible  e s p e c i a l l y to  hydrophobic such  agents  as  P_;_  aeruginosa  susceptibility structure(s) antibiotic could  be  i n the  M1 ,  outer  from  mutant  a wild  as  this  M1  type  1979).  study  of  P1-4  In a d d i t i o n , two  for  aeruginosa  were f o u n d  to the Royle  One proA  Dr.  strain  et a l . ,  a l l antibiotics. linked caused  antibiotics  of these (40  t o t h e met-28  other (30  hypersusceptibility only.  min  min)  £artially  Fyfe  strains  (Univ.  of  prepared  by  a change  Two to  - PAO  to a l l a n t i b i o t i c s i t was  more  45  a  genetic antibiotic  transconjugant  partial  and  with  P2-6),  chromosomal  hypersusceptibility  transconjugant t_rp_C,D,E and  (35  the  to t h a t o f  susceptible  loci,  aminoglycoside  (P2-6,8) e x h i b i t e d similarly  P2-8),  min)  A transductant constructed using  susceptibility  that  J.  to B-lactam  susceptible strains  isolated  partial resistance  t h e FP2  ( i n the  partially  except  on  and  antibiotics  for  recipient.  ( i n the  1981), c a u s e d The  WT1 ,  o f t h e mutant M1  a s s o c i a t e d with  locus  mutagenesis  of  P1-6,  transconjugant  R68.45-carrying  susceptibility.  P2-6,  responsible  multiple  recovered  t o be  two  the  aeruginosa,  and  loci  and  that  variety  m u l t i p l y auxotrophic  was  hoped  s p o n t a n e o u s r e v e r t a n t s were  (designated  isolated an  P_;_  PA0222-derived,  to  mutant,  significant  i n Pseudomonas  to a  p h e n o t y p e ) w h i c h had  Edinburgh)  map;  i t was  d e r i v e d by  strain  Two  a l l antibiotics.  crossing  was  hypersusceptible  susceptible  linked  with  membrane w h i c h a r e  resistance/susceptibility  (Zimmermann,  to  strain  studying a  pinpointed.  selected  in  By  to s e v e r a l a n t i b i o t i c s  The steps  1979).  (Nikaido,  to  above  partial strain  gentamicin,  similarly  t o P2-8 ( T a b l e Due  to  II).  t h e wide s p e c t r u m  mutant M1 and a l l i t s d e r i v a t i v e investigative such  proteins, 1980)  which  and  possible The  were  on  i) the  from  i n M1  the  outer  antibiotic  t h r e e major  the p a t t e r n  permeability iii) outer  - Zimmermann,  to  the  permeability  which  and,  a  hypersusceptibility. aeruginosa  revertants,  relationship  antibiotics  and  between  alterations  in  membrane,  characterisation  membrane  as  o f s u s c e p t i b i l i t y t o a n t i b i o t i c s and  certain  o f the outer  to  areas:  establishing  susceptibility  binding  membrane  i s o l a t i o n of a n t i b i o t i c p a r t i a l l y r e s i s t a n t ii)  our  mechanisms  penicillin  of a n t i b i o t i c s u s c e p t i b i l i t y i n the  mutant M1 c o v e r e d  which  (eg, beta-lactamase),  of  for this  (eg.  unaltered  enzymes  permeability  mechanism  study  i n fact  to  were s u s c e p t i b l e ,  diverted  alterations  destructive  concentrate  strains  e f f o r t s were l a r g e l y  as s p e c i f i c t a r g e t  of a n t i b i o t i c s  of the a l t e r e d  caused  increased  consequently,  structures outer  antibiotic  i n the membrane hyper-  susceptibility. These separately  sections  i n Chapter  are  One.  46  presented  and  summarised  RESULTS  PART  I.  ANTIBIOTIC STRAINS OF  SUSCEPTIBILITY OF  Since  determination  of  the  study,  antibiotic  isolated  M1  as  cycle  group was  Dr. and  of  (EMS)  antibiotic  the u l t i m a t e g o a l  used.  of  Pae  had  followed  in this  showed  differences  penicillins. susceptibility  might  be  antibiotics,  by  study  necessary  into  separate  strains  selected  of a n t i b i o t i c  resistance.  1980) and  that wild  results  suggested  due  o u t e r membrane  to attempt  a  was  t h e r e were type  no  parent  in  binding proteins for a variety  t o an  t o t h e a p p a r e n t l y complex  was  mutant  therefore i t  between mutant  t h e number o f m u t a g e n e s i s  one (NTG)  this to  of  and  (Zimmermann,  of p e n i c i l l i n These  shown t h a t  more s u s c e p t i b l e  beta-lactam  been  using 4 cycles  mutagenesis  had  this  K799/61,  This strain  h y p e r s u s c e p t i b l e by  10,000-fold  he  affinity  Due  was  Zimmermann  of g r e a t i n t e r e s t  In a d d i t i o n ,  the  mechanism  N-methyl-N'-nitro-N-nitrosoguanidine  of d i f f e r e n t  discernible  study)  sulphonate  mutagenesis. 8-  was  of  alterations.  hypersusceptible strain  in this  of  between  aeruginosa  antibiotic  ethylmethane  MUTANT  in characterisation  o u t e r membrane  in  (designated  used  and  resistance an  strains  susceptibility  P_^  AND  PSEUDOMONAS AERUGINOSA  1. D e r i v a t i o n o f b a c t e r i a l antibiotic  WILD TYPE  phenotype  that  antibiotic alteration.  o f mutant M1  steps involved i n i t s i s o l a t i o n , to segregate  individual  for characterisation.  47  To  of  and it  mutations  move w i l d  type  genes  into  M1,  the  were a t t e m p t e d . frequency lower  of  than  transfer into  by  so  wild  that  on  they  had  those  Of  eleven  the  The  antibiotic resulted of  a  fifteen  were Tables  23  antibiotic  and  by  M1  later  decided  that  would  as  in  be this  I I ) and  resistance  nine  of  e i t h e r gentamicin  or  selected  WT1  mutant  these  its  as  not  and  Tables  I  and  revertant. were  as  presumably  confirmed  fully by  its  shown).  carbenicillin  (represented  between  isolated M1,  that  antiDiocic-  c l a s s of  strains  (.represented had  (see  in  Detailed characterisation  four  (data  48  revertants  and  subsequently was  for gentamicin  intermediate  pnenotypic  selected  revertants  cells  partial  reversions.  from  plating  S t r a i n P1-6  this  mutant M1  revertants  partial I  the  M1.  of  susceptible  with  Apparently,  of mutations  patterns  strain  revertants  representative  Of  type  mutant,  four  revertants  resistance  from u n s t a b l e  identity  was  resistance  nature  were shown t o be  representative  other  antibiotic  selected directly  wild  hypersusceptible I I ) was  method  (see  therefore  n u t r i e n t medium c o n t a i n i n g  antibiotic  of  the  from mutant  inhibited  I t was  number and  were  carbenicillin. resistance,  either  because  determined.  Revertants Ml  transduction  (10-7/CFU).  equally  below). type  the  by  d i r e c t i o n (that i s , not  J. Fyfe,  m i g h t be  strain  was  M1  frequency  opposite  to  isolated,  into strain  reversion  type)  revertants  strain  transfer  i n the  experiments  and  These a t t e m p t s were u n s u c c e s s f u l  the  wild  methods o f c o n j u g a t i o n  by  resistant,  strain  reverted strain  to  14  P1-4,  see  wild  type  WT1-1).  A M1  series  was  M1  auxotrophy  and  strain  recipient selected  by  UK;.  strain  This  transconjugants  prepared  Edinburgh, of  of  w h i c h had  mated  having  chromosomal map.  altered  along  in  mutant M1  to  were f o u n d ,  P2-6  and  and  wild  prepared  type  by  +  retained parents,  the  Dr.  from  virtue  antibiotic  into  Tables  antibiotic  t h e r e f o r e the  the  I  wild  III).  III).  susceptibility  of  PA0222  and  M1  Another  Dr. y  way,  PAO map  to (see  strain,  strain  P2-6 Fyfe  but then  transconjugants, (see  strains  The  transductants  their  transconjugant  genes r e s p o n s i b l e f o r t h e s e  49  this  susceptibility type  to  an  been  separate  o f PA0222  created  the  susceptibility  a PA0222 b a c k g r o u n d and  had In  genes at  susceptibility. newly  the  of p o s s e s s i n g  which  similarly  the  of  well characterised  i n the  I and  of  on  receipt  to i n c r e a s e  those  Tables  of  the  were  a t one  positions  pattern  Fyfe,  PA0222  transconjugants  arising  transferred  prototrophy  phage F 1 1 6 C , a g a i n P2-8t,  R68.45.  nutrient prototrophy.  between  transductants  and  plasmic  auxotrophic  various  trimethoprim  P2-8,  received pro  retained  P2-6t  Two  derivative leucine  to p r o t o t r o p h y  into  strain  to  responsible for antibiotic  intermediate  WT2-6,  using  the  a  mutated  multiply  at  were t r a n s f e r r e d  earbenicillin  strains  a  of  University,  using  conjugative  resistance  background.  values  the  were s e l e c t e d by  with  genes a p p a r e n t l y  locations  performed  Transconjugants  the  genetic  was  (Edinburgh  Putative  sites  antibiotic  transferred  Fyfe  been c o n v e r t e d  g e n e s f r o m M1  desired  with  (WT2).  marker  transductants  spontaneously  which c a r r i e d  was  auxotrophic  J.  Conjugation  strain as  Dr.  and  phenotypes  Table  I.  Strain  Pseudomonas a e r u g i n o s a  strains.  Derivation  WT1  Pae K799/WT  M1  mutant 61 i s o l a t e d by r e p e a t e d m u t a g e n e s i s o f WT1 u s i n g EMS and NTG ; p r o t o t r o p h  WT1-1  Spontaneous f u l l r e v e r t a n t o f M1 isolated 0.5 ug/ml c a r b e n i c i l l i n j p r o t o t r o p h  P1-4  Spontaneous p a r t i a l r e v e r t a n t o f M1 i s o l a t e d on 0.25 ug/ml c a r b e n i c i l l i n j p r o t o t r o p h  P1-6  Spontaneous p a r t i a l r e v e r t a n t o f M1 on 0.4 ug/ml g e n t a m i c i n ; p r o t o t r o p h  WT3  P.aeruginosa  AK  43  (ATCC  12055);  prototroph  on  isolated  PA01 ; p r o t o t r o p h  arg_C54, c h l - 2 , phage E 7 9  r  rough  LPS p h e n o t y p e  WT2  met-28. t r p - 6 . l y s - 1 2 , h i s - 4 . pro-82 . i l v - 2 2 6 ;wild type a n t i b i o t i c r e s i s t a n c e , a u x o t r o p h (PA0222)  P2-6  PAZ3 ; pro+ r e c o m b i n a n t f r o m M1 l e u R68.45 X PA0222 ; a n t i b i o t i c p a r t i a l l y h y p e r s u s c e p t i b l e phenotype  WT2-6  as above, but a n t i b i o t i c resistance phenotype remained w i l d type  P2-6t  pro+ t r a n s d u c t a n t o f PA0222 u s i n g F 1 1 6 C grown on P2-6; p a r t i a l l y hypersusceptible to antibiotics  P2-8  PAZ1 ; met*, t r p _ r e c o m b i n a n t from M1 l e u R68.45 X PA0222 ; B - l a c t a m s u s c e p t i b l e  P2-8t  met+ t r a n s d u c t a n t on P2-8  P2-6,8  met+, p r o t r a n s d u c t a n t prepared from s t r a i n s P2-6 and P2-8 ; p a r t i a l l y h y p e r s u s c e p t i b l e t o antibiotics  +  o f PA0222 u s i n g  F116C  grown  +  S t r a i n WT1 and i t s a n t i b i o t i c - s u p e r s u s c e p t i b l e mutant M1 were a g i f t f r o m W. Zimmermann. S t r a i n s P2-6 , P 2 - 6 t , WT2-6, P2-8, P2-8t and P2-6.8 were a g i f t from J . F y f e . S t r a i n s WT3 and AK 43 were a g i f t f r o m A. K r o p i n s k i . O t h e r s t r a i n s were isolated as d e s c r i b e d in Methods. EMS, ethylmethane s u l p h o n a t e ; NTG, N-methyl-N'-nitro-N-nitrosoguanidine. 50  appeared 8)  t o be c l o s e l y (P2-6)  o r pro-82  prepared into  loci.  by t r a n s d u c i n g  s t r a i n P2-6,  antibiotic  2.  associated  forming  MICs  were  phase,  volumes  PP2-agar  to  were  incorporated  MICs  were  plates  at  for a  organism.  F o r example,  including  moderately inhibits are  phopholipid MICs .type  and p o l y m y x i n inhibition  II).  varying  variety  The  of  of and  virtue  hydrophobic sites i n the bind  Agents  of their  polycationic  synthesis,  following  t o and  of effects  a n t i b i o t i c trimethoprim reductase.  plates.  enzymes on t h e o u t e r  a variety  protein  u l  antibiotics  membrane,  have  10  antibiotics  the agar  target  into  i n  beta-lactams  synthetic  t o as "hydrophobic" by  to a  applied  polycationic  cytoplasmic  dihydrofolate  (1976),  putative  i n PP2 b r o t h  variety  hydrophilic  hydrophobic  referred  Nikaido  wide  specific peptidoglycan  aminoglycosides  both  P2-8  t h e chromosome.  dilutions into  a n t i b i o t i c s with  the  was  strain  strains  grown  (see Table  various  and  cell  and mutant  1/100 a n d t h e n  hydrophilic,  of  from  s i t e s from  f o rcells  diluted  compounds,  surface  (P2-6,8)  strain  (P2-  d i f f e r e n t mechanisms o f a c t i o n .  determined  representing  type  determined  stationary  t h e met-28  a s t r a i n possessing  susceptibility-encoding  a n t i b i o t i c s with  inhibit  Another  either  t h e met p r o t o t r o p h y  Susceptibility of wild of  with  used  on t h e  and  the  (Table I I I ) i n this  study  the nomenclature of  tendency  to  permeate  bilayers. were d e t e r m i n e d  parent  o f M1 ) ,  M1  f o r WT3  (PA01 w i l d  type),  ( a n t i b i o t i c hypersusceptible  51  WT1  (wild  mutant)  Table II. Minimal a n t i b i o t i c s f o r parent  inhibitory concentrations (MIC) o f a and a n t i b i o t i c h y p e r s u s c e p t i b l e s t r a i n s .  M I C  Strain B-lactam  variety  of  (ug/ml)  Aminoglycoside  BP  AP  TC  CB  CF  MX  GM  SM  WT1  >2000  >500  20  25  2  20  10  WT1-1  >2000  >500  20  25  2  20  WT3  >2000  >500  20  25  2  TM  Other CM  PX  TET  50  >200  20  100  10  50  >200  15  1 00  20  8  20  0.8  >200  10  1 00  P1-4  10  5  0.5  0.5  0.3  5  1  5  0.5  >200  10  5  P1-6  10  5  0.5  0.5  0.3  0.2  2  5  0.5  50  5  50  1.0  0.5  0.05  0.05 0.1  0.2  0. 1  2  0.5  25  5  2  M1  M i n i m a l i n h i b i t o r y c o n c e n t r a t i o n s were p e r f o r m e d as d e s c r i b e d i n M e t h o d s . BP, benzylpenicillin; AP, a m p i c i l l i n ; TC, t i c a r c i l l i n ; CB, c a r b e n i c i l l i n ; CF, cefsulodin; MX, moxalactam; GM, gentamicin; SM, s t r e p t o m y c i n ; TM, t o b r a m y c i n ; CM, c h l o r a m p h e n i c o l ; PX, p o l y m y x i n B; TET, t e t r a c y c l i n e .  and and  spontaneous P1-6)  strains  r e v e r t a n t s to f u l l  antibiotic of  P_;_  resistance.  aeruginosa  conventional  penicillins,  ampicillin,  was  that  found  high.  in  well and  as the  lower,  f o r the  lactamases  due  of these  mutant  partial  not  of w i l d  WT1-1)  to  the and  more t h a n 4 0 - f o l d  shown). and  For  newer  carbenicillin,  cephalosporin,  increased  type  benzylpenicillin was  (0'Callaghan,  M1  was  antibiotics  type  resistance  stability 1979;  as  cefsulodin,  to  was  beta-  Murakami  P1-6  between w i l d  beta-lactams  tested  i t s parent  (selected  (selected levels  much more s u s c e p t i b l e  than  P1-4  and  strain  for  f o r gentamicin  and  Both  resistance)  mutant.  were i d e n t i c a l  WT1.  a l l  carbenicillin  f o r a l l beta-lactams, type  to  which  Resistances  f o r the  two  types  had were to of  revertants.  Susceptibility streptomycin although  and  less  because  the  tobramycin  o f the on  particular,  was  also  to  drastically  antibiotics  There  by  ticarcillin  the  agents  resistance  intermediate  part  as  to  revertants  r e s i s t a n c e ) and  partial  (data  generation  strain  beta-lactam-type  most  and  (P1-4  1981).  The  similar  WT1  o x y c e p h a l o s p o r i n , moxalactam, w i l d mostly  Yoshida,  (WT3,  partial  resistance  This resistance  such  third  The  represented  E.coli  carboxypenicillins,  (WT1-1) and  wild very  appeared  aminoglycosides was  than  increased  P_j_  effective  t o be  the  even  on  mutant,  perhaps  in  class  of  of t h i s  aeruginosa.  Tobramycin,  wild  a minor d i f f e r e n c e  53  in  for beta-lactams,  greater effectiveness type  gentamicin,  type  in  strains.  in resistance  to  aminoglycosides although its  the  full  parent.  specific  and  could  of  susceptibility distinguish For  mutant The  differences however.  in The  was  apparently  not  susceptible  to  antibiotic  was  dramatic  the  antibiotic  and  WT3  was  to  of  used.  In  never g r e a t e r  full  enough  which  protein  partial  to  these  two  antibiotics,  nalidixic  B than  the  mutant,  f r o m 2-  to  contrast,  P1-6.  wild  agents  novobiocin  four-fold  more  between WT1  and  for a l l  >2000-fold, the  and  type.  resistance M1,  and  to  but  Subsequent  i s between 50-  acid  two-  than 2 . 5 - f o l d .  54  resistance  chloramphenicol  strain  only  than  displayed  a d d i t i o n a l hydrophobic  was  are  synthesis  revertants  mutant M1  in antibiotic  ranged  had  except  a l s o more s u s c e p t i b l e  to  the  violet,  susceptible  and  P1-6  between  large  1976)  was  the  S t r a i n M1  difference  not  more r e s i s t a n t t o  polymyxin  its  M1  shown t h a t  shown).  margin  isolated for carbenicillin  more s u s c e p t i b l e  ribosomal  and  tetracycline,  t e t r a c y c l i n e than  crystal  P1-4  WT1,  strain-  aminoglycosides,  was  and  classes  400-fold  SV,  or  WT1,  level.  strain  strain  to  presumably  surface  where the  (Nikaido,  have a l s o  rifamycin  to  strain  identical  revertants  resistance  studies  The  partial  parent  was  to minor c e l l  resistance  two  the  therefore  susceptibility  r e s i s t a n t to  (data  WT1-1  intermediate  type.  less  due  hydrophobic  inhibitors,  (P1-4)  strain  tobramycin and  an  and  chloramphenicol  moderately  wild  be  l e v e l s of  case  WT3  d i f f e r e n c e was  Once a g a i n ,  intermediate the  revertant  This  differences.  in  between s t r a i n  antibiotics  depending  on  d i f f e r e n c e between  WT1  The  fact  that  M1  was  Table  III. Minimal i n h i b i t o r y c o n c e n t r a t i o n s (MIC) of three antibiotics in spontaneous revertants, t r a n s c o n j u g a n t s and t r a n s d u c t a n t s o f M1.  Strain  Phenotype  M I C  ( u g / m l  )  CB  GM  WT1  20  5  100  WT1-1  20  5  1 00  TP  P1-4  AbsA,  ?  ,AbsC  0.5  0.5  10  P1-6  AbsA,  ?  ,AbsC  0.5  1 .0  10  M1  AbsA,AbsB,AbsC  <0 .25  0 .05  20  5  WT2 P2-6  AbsA  0.25  WT2-6 P2-6t  20 AbsA  0.5  2 100 20  5  200  0.5  1  100  P2-8  AbsB  1  0 .1  200  P2-8t  AbsB  1  0 .1  200  0.25  0 .1  20  P2-6 ,8  AbsA,AbsB  Overnight c u l t u r e s were d i l u t e d 1/100 t h e n 10 ul spots (approximately 2 X 1 0 c e l l s ) were a p p l i e d t o PP2-agar p l a t e s containing various concentrations of a n t i b i o t i c . Minimal inhibitory c o n c e n t r a t i o n s were t h e l o w e s t concentrations of a n t i b i o t i c a t w h i c h g r o w t h was i n h i b i t e d . CB, c a r b e n i c i l l i n ; GM, g e n t a m i c i n ; TP, t r i m e t h o p r i m . A l l data in this table were taken from a single experiment f o r comparative purposes, a l t h o u g h v e r y s i m i l a r numbers have been obtained i n many t r i a l s . The p h e n o t y p i c designations Abs ( a n t i b i o t i c s u s c e p t i b i l i t y ) A, B, and C a r e d i s c u s s e d i n t h e text. 5  55  more  susceptible  increase  in  to a l l a n t i b i o t i c s  for i t santibiotic  alternative  explanations  enzyme-mediated  susceptibility  transductant  gentamicin  strains  hydrophobic parallel  in  on  f o r wild  susceptibility  resistance  in  antibiotics  that  to  to a l l tested  type  by t h e i r  three a n t i b i o t i c s .  the  (WT2-6), w h i l e  resistance  and  including  alterations  revertants  antibiotic  Fyfe  pro , +  P2-8  to trimethoprim.  56  but had  two P2-8)  p a t t e r n s on  s u s c e p t i b l e to  than  the w i l d  type  transconjugant  (met+ t r p + ) was more  and g e n t a m i c i n  showed  (P2-6 and  (pro+) was more  non-susceptible  three  I I ) . The  resistance  and t r i m e t h o p r i m  the  aminoglycosides  Table  by J .  in  involved a l l  beta-lactams, (see a l s o  P2-6  gentamicin  carbenicillin  and  carbenicillin,  The r e s u l t s ,  the p a r t i a l  be i d e n t i f i e d  or  M1.  aminoglycosides  p a t t e r n s always  could  carbenicillin,  and  transconjugant  polycationic  of transconjugants separated  "sibling"  or  c o n s i d e r e d t o be r e p r e s e n t a t i v e  types  (WT2)  alterations  antibiotics,  For r e v e r t a n t s ,  intermediate  parent  the  transconjugant  for  respectively.  III.  tested,  these  were  t y p e , mutant and r e v e r t a n t s t r a i n s , a r e  antibiotics  other  of  made  three  beta-lactams,  Table  antibiotic  and  were  antibiotics,  data  target  an  probable  than  d e r i v e d from mutant s t r a i n  and t r i m e t h o p r i m ,  of h y d r o p h i l i c  shown  strains  determinations  transductant  a more  susceptibility  of s p e c i f i c  that  resistance.  Antibiotic  MIC  indicated  o u t e r membrane p e r m e a b i l i t y was  explanation  3.  tested  susceptible  approximately  These r e s u l t s  showed  wild that  Table  IV.  S u s c e p t i b i l i t y to v a r i o u s a n t i b i o t i c s of a l l P. a e r u g i n o s a s t r a i n s by d i s c i n h i b i t i o n d i a m e t e r a s s a y .  Strain  Disk  diameter  (mm)  B-lactams  Other Hifi  CB 500  CF 50  CX 100  -  10  7  48  32  40  30  WT1-1  7  10  -  -  P1-4  35  26  29  P1-6  38  34  WT2  1 1  P2-6  BP 5000  CZ 2  AZ 5  NT 5  -  -  -  -  50  21  38  -  -  17  43  30  30  16  -  -  36  30  19  WT2-6  9  9  P2-6t  36  P2-8 P2-8t  WT 1 M1  MX 50  -  GM 5  TC 200  Hifo NR 5  TP 200  CM 200  -  -  -  -  14  -  25  18  28  28  -  -  -  -  -  -  10  24  14  -  23  -  -  39  18  33  10  -  1 1  10  17  -  -  -  14  29  -  -  -  -  28  -  -  -  -  -  -  -  28  44  29  7  13  26  -  26  15  14  9  -  -  1 1  -  -  -  22  15  13  9  7  -  1 1  -  -  -  12  -  8  -  8  7 9  -  9  -  18  -  -  Antibiotics were a p p l i e d i n 10 u l volumes o f the indicated concentrations ( i n ug/ml) t o s t e r i l e f i l t e r d i s c s on PP2-agar plates onto which a lawn o f b a c t e r i a had been swabbed. -, r e s i s t a n t ( i n h i b i t i o n zone d i a m e t e r _< 6 mm); CB, carbenicillin; CF, cefsulodin; CX, cefotaxime; MX, moxalactam; BP, benzylpenicillin; CZ, ceftazidime; AZ, azthreonam; NT, Nformimidoylthienamycin (Imipenem); GM, gentamicin; TC, tetracycline; NR, norfloxacin; TP, trimethoprim; CM, chloramphenicol; H i f i , hydrophilic; Hifo, hydrophobic. The above data were t a k e n from a single experiment but separate trials performed on a l l strains provided similar numbers.  57  the  two  produced By  s e p a r a t e l y mapping m u t a t i o n s different  antibiotic  surveying  a greater variety  disc  assay  the  inhibition  zone  from  ( f o r most  this  comparitive  type  the d i s c of  raised  disc  two-  as w e l l as o t h e r s .  assay  this  of  assay  as  evident  mutant M1 and  both  using  certain  the  from  penicillin  strains  appeared  was  less  (cefotaxime  monobactam  Strain  was  also and  N-  less the  subsequently  zone d i a m e t e r s results and  MIC  and  the not  by  the  s u s c e p t i b l e to the  58  observed  t h a t the  susceptible  third  ceftazidime),  - see  generation  the  greater  two than  agents  In p a r t i c u l a r ,  antibiotic  third  from  were  determinations.  IV was  N-formimidoylthienamycin  (a  moxalactam,  affected  outward  trimethoprim, were  less  to  the  r e v e r t a n t s to h y d r o p h o b i c  beta-lactams.  azthreonam P2-8  i n Table  due  though  P2-6t was  plate  t o be  f o r other  diffuse  example, even  and  been i n the  or p a r t i a l  G,  not  Consequently,  o f P2-6  used  evident.  antibiotics,  inhibition  the r e s u l t s  to s e l e c t e d  cephalosporins  For  partially  were  be  o n l y t o compare s t r a i n s ,  i t had  strains  do  the  P2-8  c o u l d not  norfloxacin the  susceptibility  transconjugant  general  and  increased.  were used  and  chloramphenicol,  or f o u r - f o l d ,  substantially  What was  the  because  gentamicin,  trimethoprim in  P2-6  of assay  purposes  formimidoylthienamycin  not  of a n t i b i o t i c s  h y d r o p h o b i c i t y or l a r g e s i z e ,  levels  mutations)  phenotypes.  antibiotics),  phenotypes of s t r a i n s  Unfortunately,  either  susceptibility  of  ( T a b l e IV) , f o r which r e s i s t a n c e i s p r o p o r t i o n a l to  susceptible  than  (or s e t s  in  growth  generation as  well  (Imipenem) also  Fig.  as and 1).  oxycephalosporin, cephalosporin,  cefsulodin. and  S t r a i n P 2 - 6 t was l e s s s u s c e p t i b l e  trimethoprim  this  (Table  transductant  parent,  P2-6.  were c h a r a c t e r i s e d  bind  to  their  of  either  molecule 1976;  or  phages  of  All  the  for their  protein  of  cell  surfaces  ability  must before  cell.  The  membrane,  lipopolysaccharide one  type  of  (Hancock and R e e v e s , To c r u d e l y  o f a l l the w i l d each  type  lineage,  membrane  assess and  several  receptor  t o l y s e b a c t e r i a on t h e  sites surface  (Table V ) . t h e phages used  i n this  (Mutharia  study  had  and  determine  whether  t h e mutant  phenotype  Kropinski  as s t r a i n 59  unspecified  e_t al., 1982) e x c e p t  Phage D8 was i s o l a t e d as a r o u g h  Jarrell  LPS  assay f o r  outer  1980).  s t r a i n s from  by  rough  of the  o f more t h a n  Bradley,  strains  on t h e b a c t e r i u m  a specific  composed  1977;  o r LPS r e c e p t o r s  and pRD1.  to i t s  Bacteriophages  c a n be i n j e c t e d i n t o t h e  as p i l i n ,  susceptible  an a g a r p l a t e  phage  site  known t o have d i f f e r e n t o u t e r  were t e s t e d  D8  structures.  receptor  a receptor  Datta et a l , ,  antibiotic  of  such  and mutant  testing i s a valuable  i n a c h a r a c t e r i s t i c conformation  relatedness  identical  that  s t r a i n s P2-6 and P2-8  type  must be a c o n s t i t u e n t  a protein  structure  surface  material  site  i t was p o s s i b l e  only  of wild  sensitivity  surface  genetic  reason,  cefotaxime  further.  cell  a cell  receptor  this  sensitivity  Bacteriophage comparison  therefore  was n o t p h e n o t y p i c a l l y  For  4. B a c t e r i o p h a g e  III)  to  (1981)  strain  M1  and  LPS-specific was  displayed  AK 43 (on w h i c h  phages  used the  t h e phage  to same had  been  isolated).  carrying  the a n t i b i o t i c  negative strains  control  and  wild  full to  type  comparisons mutant  were  typing  strain),  strain the not  o f phage  strain,  typing  were  type  to s t r a i n  WT3 (a  phage,  was r e s i s t a n t t o phage  indicated  o f WT1,  binding  the p o s s i b i l i t y  but simply  phage  i n contrast  and P1-6 were  strain  patterns identical  60  AK 43.  to i t s  additional  t h e mutant  cell  t o have more  However,  D8 and t h e r e f o r e  demonstrated that  sensitivity  that  and seemed  sites.  that  each  and L P S - s p e c i f i c  WT1 , w h i c h was r e s i s t a n t t o t h e f o u r  bacteriophage  the  Further  made between  to a l l protein  This  and  strain.  f o r one p i l u s - s p e c i f i c  phages.  to a l l  receptors.  therefore  parent  was d i f f e r e n t t o t h a t  P1-4  their  hand, were r e s i s t a n t  protein  u n d e t e r m i n e d ) was e i t h e r n o t p r e s e n t  strains  these  (Nicas  S t r a i n WT1  WT1-1, on t h e o t h e r  and i t s own w i l d  eliminate  The  of  a  aeruginosa  related  same r o u g h LPS p h e n o t y p e as s t r a i n  mutant,  none  isolated for  o f P_;_  and LPS r e c e p t o r s .  strain  protein-specific  exposed  since  by v i r t u e o f i t s s e n s i t i v i t y  o f t h e phages w i t h  phages e x c e p t  surface  RP1) was used as  phages  strain  Mutant M1 was s u s c e p t i b l e  parent  cells  A l l o f t h e phages u s e d , w i t h t h e  (PA0222) was c l e a r l y  protein  revertant several  for  1980). WT2  with  plasmid  resistance,  t o grow on a PA01 t y p e  Strain  phages  D8,  ( w h i c h was s p e c i f i c  resistance  f o r phage  o f phage  Hancock,  PA01  pRD1  c a r r i e d the plasmid.  exception ability  Phage  the  mutant  d i d not d i s p l a y This  result did  M1 was a r o u g h  t h e D8 r e c e p t o r  LPS  (as y e t  or not exposed. of  the  to that  partial  revertant  of  mutant.  the  Table  V.  Bacteriophage s e n s i t i v i t y of w i l d hypersusceptible strains.  Strain  Bacteriophage Protein  type  and  receptor  Pilus  LPS  and  antibiotic  type R-LPS  RP1  A2  A4  A8  B9A  B9B  C3  D3  C9  A7  B2  B4  D8  pRD1  WT1  R  S  S  R  R  R  S  R  S  S  S  R  R  WT1-1  R  S  S  R  R  R  S  R  S  S  S  R  R  P1-4  S  S  S  S  S  S  S  R  S  S  S  R  R  P1-6  S  S  S  S  S  S  S  R  S  S  S  R  R  M1  S  S  S  S  S  S  S  R  S  S  S  R  R  WT3  S  S  S  S  S  S  S  S  S  S  S  R  R  WT2  S  S  S  S  S  S  S  S  S  S  S  R  R  WT2-6  S  S  S  S  S  S  S  S  S  S  S  R  R  P2-6  S  S  s  s  s  s  S  S  S  S  S  R  R  P2-8  S  S  s  s  s  s  s  s  s  S  S  R  R  Bacteriophage suspensions were s p o t t e d o n t o b a c t e r i a l lawns using a multisyringe i n o c u l a t o r . P o s i t i v e r e s u l t s for s e n s i t i v i t y (a c l e a r e d zone o f b a c t e r i a l l y s i s due to phage) was determined a f t e r i n c u b a t i o n a t 37°C f o r 20 h. R, r e s i s t a n t ; S, s e n s i t i v e . Phages a r e grouped a c c o r d i n g to p u t a t i v e c e l l s u r f a c e receptor. Protein, undetermined outer membrane protein receptor; LPS, undetermined lipopolysaccharide receptor; R-LPS, rough LPSs p e c i f i c ; RP1, s p e c i f i c f o r c e l l s c a r r y i n g RP1 plasmid.  61  The  transconjugant  phages t h e r e f o r e in  these  strain to  surface  the  conclusions  Other  could  be  WT1-1  wild  changes  antibiotic  5.  no  strains  revertant similar  d e r i v a t i v e s o f WT2 as  made.  the  cell The  displayed  type parent  in  to  were s e n s i t i v e t o  a  WT1  surface a l t e r a t i o n s  fact  that  phage  were  the  typing  indicated  mutant  all  that  somehow  full  pattern the  cell  related  to  susceptibility.  properties  of  the  antibiotic  hypersusceptible  mutant  strain. a)  Frequency  As  discussed  derivation  of  or  than  frequency  the  transfer  the  full  of w i l d  strain  M1.  conjugation  antibiotic  reversion earlier  strains,  partial  mutant  of  type  susceptibility  M1,  s i n g l e step  phenotypically  one  identical  i n mutant M1.  reversion mutation  mutation  may  i s that  causing  should  suggested One there  62  into  that  the to  (see  there  into  to  was  wild  from  mutant were  sections,  a s i n g l e major for  i n mutant M1,  Another  the  type  which  other  explanation  susceptibility,  for  segregate  isolate  revertants  t y p e WT1  exists,  another.  greater  obtained  Fyfe  the  either  distinguished  J.  used  full  possible  antibiotic  stabilise  by  procedure  to w i l d  be  from M1  s e l e c t i o n of  chapter) strongly  mutation step  complex  to  p h e n o t y p e was  transduction  performed loci  with  r e s i s t a n c e f r o m WT1  frequency  experiments  the  or  dealing  of r e v e r s i o n  resistance  antibiotic  This  section  frequency  of conjugation  Despite  this  the  antibiotic  strain. the  i n the  but  explanation  single  more  than  that  one  is  that  the o r i g i n a l additional original  mutation  may  mutation,  mutation,  not  have r e v e r t e d ,  which c o u l d d i s g u i s e  may  have o c c u r r e d  but  the e f f e c t  of r e v e r t a n t s u s i n g c a r b e n i c i l l i n ,  one  major m u t a t i o n  another  of  i n t h e M1  b)  strain  loci  transferred  Growth r a t e  Mutant WT1  opposed  medium  carbon  (50  source  response  that  cellular to  sensitive  low  slower  - d a t a not  min). M1  than  of  separate were  of  shown) and  was  min  as  maintained  the  not  assumed  from  sole these  s u s t a i n e d e x t e n s i v e damage  challenge.  temperature  50  type  medium w i t h g l u c o s e as  It had  the w i l d  f u n c t i o n s which c o u l d perhaps  antibiotic  to  two  of  background.  ( g e n e r a t i o n time  i n minimal  strain  reversion  mutant  rich  i n PP2  the  g e n e t i c background  the PA0222  in  min  of  reversion  However,  t h e M1  grew o n l y s l i g h t l y  as WT1  gross  into  o f the  same g r o w t h r a t e  to  from  M1  t o 45  observations  have f a v o u r e d  g e n e t i c background.  mutation-containing successfully  may  an  (phenotypic suppression).  Upon s e l e c t i o n type  instead,  Mutant  killing,  influence i t s M1  was  more  however  (B.  Angus,  P2-6  P2-6,8 were  unpublished observation). c) Colony  morphology  Colonies  o f mutant M1,  noticeably strains  and  glossier also  i n appearance  had  aeruginosa  strain  refractive  surface.  to  an  alteration  P1-4,  P1-6, than  a smooth, rounded  used  i n LPS  in this The  study  mutant  (see l a t e r  63  P2-8 edge. had  a  and  and  the w i l d  The  type  P.  characteristically  c o l o n y t y p e was sections,  wild  type  probably  this chapter).  due  6.  all  SUMMARY  The  major c o n c l u s i o n s  a)  Mutant  classes  M1 was f o u n d  of a n t i b i o i c s  sensitivity b)  strain  tested,  one, p a r t  one were:  t o be h y p e r s u s c e p t i b l e  and p o s s e s s e d  a mutant  to  phage  pattern.  Spontaneous  resistance tested,  from c h a p t e r  between  yet  partial that  retained  revertants  o f M1  and WT1  the i d e n t i c a l  had  for  intermediate  a l l  phage t y p i n g  antibiotics pattern  of  mutant M1 .  c)  Spontaneous  resistance d) respective  not  the  other,  transconjugants  resistant  phenotypes,  same p h e n o t y p e  mutations From  as  mapping could the  be  outer  target  site  parent  strain  and  to wild  and  as  auxotrophic  had  their  partially evidently  revertants  or  each  to c e r t a i n  beta-  trimethoprim, markers  WT1.  (and  t h e s e were  as e i t h e r p a r t i a l  well  type  P2-8  P2-8t)  however  antibiotic  and  with  on  which  the the  mobilised.  wide  hypersusceptibility enhanced  patterns  on d i f f e r e n c e s i n s u s c e p t i b i l i t y  antibiotics  separately  had i d e n t i c a l  P2-6  P2-6t  transductants  based  lactam  revertants  and phage s e n s i t i v i t y Both  antibiotic  full  range  of  i n strain  membrane  alteration  antibiotic  M1 was s u g g e s t e d  permeability  rather  or plasmid-mediated  WT1. 6k  types  involved,  t o be c a u s e d by  t h a n by  antibiotic  resistance  in  the  Based assigned of  on to  separate  the  M1 .  involved  are  phenotypic attempt  was  represents to  represents partial t o be  later  responsible  linked found  (part three  the  the  of  in  the  associated  phenotypically  chapter  strains  P1-4  with  LPS  an  65  The  The  and  P2-6. a  AbsC  P1-6.  structural (see  This  structural phenotype phenotype phenotype  phenotype AbsC was  three,  of also  alteration,  part  of  antibiotic  AbsB  susceptible  f r o m AbsA  one) .  with  of  three  phenotype  susceptible  P2-8.  number  least  partially  one).  antibiotic  strain  distinct  at  mutant  associated  partially antibiotic  revertant  that  f o r the  the  were  susceptibility  t o pro-82 i n s t r a i n  t o be  partially  met-28  to d e s c r i b e  It i s postulated  phenotype  designations  in antibiotic  AbsA p h e n o t y p e r e p r e s e n t s  change i n LPS  was  i n an  mutations  phenotype  found  each s t r a i n  M1.  susceptible  the  data,  strain  The  linked  MIC  mutations  mutant  separate  the  but  chapter  PART  I I . CHARACTERISATION PERMEABILITY The  results  increase  in  responsible the  P_^  OF MUTANT  of the previous  outer  membrane  f o r the increase  aeruginosa  explained  by  degrading  these  antibiotics  affect  mutant  s t r a i n s had i n c r e a s e d Therefore,  designed  to s p e c i f i c a l l y  in  strain.  examine  uptake.  aeruginosa assess  type  the  M1 .  the  In  hydrophobic addition,  strain.  66  and a l l  experiments  membrane  were  permeability to  properties  contribution of this  for these  separately  and s e l f - p r o m o t e d  the  be  t o a wide r a n g e o f  I d chromosomal b e t a - l a c t a m a s e  potential  since  antibiotics  of  not  responsible  following  measure o u t e r  primarily  could  sites,  susceptibility  an  susceptibility  The d a t a  groups o f  that  was  D i f f e r e n t methods were used  porin-mediated,  membrane  permeability  or i n t a r g e t  specific  MEMBRANE  section indicated  a l t e r a t i o n s e i t h e r i n enzymes  only  antibiotics.  IN OUTER  STRAINS  in antibiotic  mutant s t r a i n  would  each  OF ALTERATIONS  of  outer the  P.  were examined t o enzyme  in  each  1 . Hydrophilic a  (porin-mediated)  ohromogenic All  P_j_  Abraham, whole  encoded  1964).  cells  1984),  periplasm  that  In  the to  view  properties  and  The  were  the  by  measured  by  between  6.2  (Table  VI),  lactamase  type,  mutant  nitrocefin The  and  activity  9.6  structural  section  Hancock and  Wong,  represented  uptake  in  wild  in  membrane  the  the a  type  the r e l a t i o n s h i p  of  and  P.  between barrier  chromosomal  t o be  revertant  of  beta-  c o u l d be  beta-lactamase  Permeation  will  d e r i v e d from  production  the  same and  produced  be  was  French-pressed  the n o n - i n d u c e d approximately  enzyme  strains  hydrolysed/min-mg.  gene f o r b e t a - l a c t a m a s e .  of t h i s  beta-lactamase  of a l l s t r a i n s  nmol n i t r o c e f i n  therefore  appeared  chromosomal  membrane  membrane  outer  assay  the AbsA, B o r C p h e n o t y p e s the  outer  of and  located  amount o f chromosomal  wild  suspensions.  EDTA  enzyme was  both  and  activity  i n c r e a s e d by the  and  measured.  total  produced  be  outer  inducibility  (Sabath  beta-lactamase  this  of t h i s ,  susceptibility  inducible,  beta-lactamase  antibiotic  antibiotic  lactamase  an  (Hancock ejt aJL. , 1 981 b ;  barrier  aeruginosa.  contain  disrupt  postulated that  and  significant  Id  which  barrier  i t was  type  a e r u g i n o s a can  agents  permeability  strains  S i n c e the a p p a r e n t  of  gentamicin,  to  cephalosporin a n t i b i o t i c .  aeruginosa  chromosomally  p e r m e a b i l i t y o f whole c e l l s  The  by  of  cell WT1  was cells beta-  so none  of  a mutation  in  inducibility  discussed  in  of  another  chapter. of  nitrocefin  67  was  measured  as  a  functional  demonstration  of the d i f f e r e n c e i n outer  between  strains  location  of  WT1  and  aeruginosa  therefore  forms  substrates  and  the  Zimmermann  and  Rosselet  membrane the  the  will  across  sufficiently the  high  periplasm.  will for  level  a  spectrophotometrically  was e q u a l  measure  technique  periplasm rate  of beta-lactamase  to outer  of  product  measured.  The  t o 1.0  (legend,  hydrophobic  Table  i n comparison  is of  that whole  that  a  activity  exists  in  by whole  cells  permeability nitrocefin  was  which  could  be  partition  ratio  for  system  VI),  outer  beta-lactams  membrane  hydrolysis  of  assuming  cephalosporin  i n a phase-partitioning  was s l i g h t l y  technique  to  barrier,  The c h r o m o g e n i c  provide  nitrocefin  i n the  membrane  beta-lactam  The  of t h i s  by t h e d i f f u s i o n  be p r o p o r t i o n a l  each s t r a i n . to  between  (1977) was used  membrane  periplasmic  the outer  enzyme.  The b a s i s  permeability  the  The e q u i l i b r i u m h y d r o l y s i s r a t e  therefore  used  barrier  hydrolysis rate  outer  of  beta-lactamase,  hydrolytic  be l i m i t e d  the  Because  major  permeability.  beta-lactam  cells  M1.  membrane  of  1-octanol/water  therefore with  this  other  compound  beta-lactam  molecules. The whole  activity  cells  calculate  The =  was  a  Zimmermann  ( S  q  nitrocefin  measured  value  _ s ), e  in  and  f o r the  and R o s s e l e t  permeability 0  o f chromosomal b e t a - l a c t a m a s e the  results  permeability  were  parameter  (1977) and f o r c r y p t i c i t y  p a r a m e t e r was c a l c u l a t e d f r o m where v r e p r e s e n t s whole  i n b r o k e n and  cells, 68  S  Q  the ±S  to  "C"  of  (Table V I ) .  the equation  hydrolysis  the  used  v  rate  of  concentration  of  nitrocefin  added  nitrocefin  S  and  G  i  s  concentration.  hydrolysis  rate  therefore  is sufficiently  be  negligible  proportional  The  so  substrate  permeability  10~3  mi  full  revertant  4.5-fold  type  WT1.  higher The  approximately  two-fold  P1-4  be  could  not  Crypticity resistance Table the  II  beta-lactamase  cells  and,  the  also  activity for  in cells.  possession  strains. enzyme  takes  levels,  high  enzyme  cells  contributes  variable  levels  revertant  G  by  to v a r y by  a  y  P1-6  the  MIC  was  over  however  antibiotic  defined  that  of  the  where b e t a - l a c t a m a s e  s u b s t a n t i a l l y to 69  the  as  an  beta-lactamase  provide  a control between  important level that  in  whole  C provided  amounts o f b e t a - l a c t a m a s e  approach  also  permeability,  total can  i t s wild  determinations  membrane  therefore  an  criterion.  parameter  the  X the  had  was  the  Crypticity cells  1.6  and  type  this  with  i s u s u a l l y only m  to  WT1  than w i l d  f r o m WT1  outer  Crypticity  1.1  than  permeability of  between  value  i n t o account  where S  permeability  permeability  of broken  of d i f f e r i n g  This  hydrolysis  hand,  chapter).  activity  crypticity  the  s t r a i n s WT3, on  partial  this  calculation  that  should  G  determined  a  membrane  more p e r m e a b l e  excellent  ( s  q  other  appeared  although  ch  S U  type  phenotype demonstrated  (part I of  periplasmic  the  distinguished also  S  to  C ranged  Mutant M1,  approximately  the  equilibrium, S  at  )  periplasmic  concentration.  for wild  WT1-1.  Q  that  to o u t e r  parameter  / min-mg c e l l s  parent  high  C = v / S  rate  a specified  calculated  e  compared  and  is directly  h  Assuming  experimentally),  at  t  of  may  hydrolysis  S  at 0  ,  leak rate.  or  low at from  Table  Strain  VI.  Chromosomal b e t a - l a c t a m a s e a c t i v i t y and n i t r o c e f i n p e r m e a t i o n t h r o u g h the o u t e r membrane o f w i l d t y p e and antibiotic h y p e r s u s c e p t i b l e P_;_ a e r u g i n o s a strains.  Beta-lactamase Broken  cells  activity  Crypticity  Whole c e l l s  "C" (ml/min-mg c e l l s X 10~3)  WT3  11.1  1 .3  9.3  1 .5  WT1  9.6  1 .4  6.9  1  WT1-1  9.4  0.92  10.2  1 .1  P1 -4  7.0  1 .5  4.7  1 .8  P1-6  7.8  2 .1  3.7  2.5  M1  6.2  4.7  1.3  5.6  .6  The beta-lactamase activity i s the mean o f three to five separate trials and i s expressed as nmol of nitrocefin hydrolysed/mg of c e l l d r y weight per minute. The crypticity represents the r a t i o of beta-lactamase activity of broken cells/ a c t i v i t y o f whole c e l l s . The p e r m e a b i l i t y parameter "C" was c a l c u l a t e d from F i c k ' s Law o f d i f f u s i o n and i s equal to the b e t a - l a c t a m a s e a c t i v i t y i n whole c e l l s / nitrocefin concentration. The b e t a - l a c t a m a s e a c t i v i t y o f whole c e l l s o f strains M1 , P1-4 and P1-6 were shown to be statistically d i f f e r e n t f r o m t h a t o f s t r a i n WT1 by S t u d e n t t - t e s t (p < 0.02, < 0.01 and < 0.001, respectively). Standard d e v i a t i o n s f o r the same s t r a i n s were 40$, 20$ and 7$ o f the average whole c e l l beta-lactamase a c t i v i t y , and f o r s t r a i n WT1 was 18$. The 1 - o c t a n o l / w a t e r p a r t i t i o n c o e f f i c i e n t o f n i t r o c e f i n (ratio of n i t r o c e f i n p a r t i t i o n i n g i n t o o c t a n o l to that partitioning i n t o w a t e r ) was e q u a l t o 1.0.  70  The  mutant  activity  /  whole  comparison In  with  fact,  showed  the  that  nitrocefin. in  this  the as  ratio  than  M1  T h i s agreed  strains  well with  calculated  mutant  and t h e f u l l  also  consistent  (Part  I,  attempt  this  their  chapter).  higher  than  closer  d a t a were  two  strains  cryptic  toward  value  those  f o r "C"  for of  WT1-1,  to wild  M1  both but  although  type  values,  c o n s i s t e n t with  full  i n WT1  antibiotic  The f u l l  and p a r t i a l  the  and p a r t i a l restoration  (Sykes  t o i n c r e a s e enzyme a c t i v i t y  outside  T h i s assay  strains,  P2-6  WT2, were  also values  profiles  r e v e r t a n t s had o f the  whole  barrier  and P2-8,  hydrolysis,  f o r C were  Cells  of  by  the wild  another added t o  transconjugant type  parent,  study.  approximately  71  RP1-  1976) i n an  of n i t r o c e f i n  as w e l l as t h e i r  included i n this  using  and, i n t u r n , the a c c u r a c y  involved  cells.  measured  and Matthew,  located beta-lactamase,  of  and was  membrane.  TEM-2 b e t a - l a c t a m a s e  assay.  and WT1-1, resistance  membrane p e r m e a b i l i t y was a l s o  periplasmically  The  (Table V I ) .  ( b a r r i e r ) values  r e v e r t a n t s than  of the outer  the  less  revertant strain  permeation  with  correspondingly  encoded,  the  1.0) i n  t h a t t h e o u t e r membrane was more p e r m e a b l e i n t h e  and p a r t i a l  Outer  >  cell  f o r "C".  nitrocefin  function  between  the c a l c u l a t e d  The c r y p t i c i t y t e s t e d were  o f broken  WT3 and WT1  was f i v e - f o l d  p e r m e a b i l i t y o f P1-4 was a g a i n  hypothesis  the  type  (ratio  was o n l y s l i g h t l y  of c r y p t i c i t y  strain  WT1  cryptic  activity  the wild  revertants  The  of  cell  mutant.  partial lower  M1 was n o t v e r y  five-fold  higher  in  Table  Strain  VII.  Outer membrane p e r m e a b i l i t y o f whole cells n i t r o c e f i n u s i n g TEM-2 b e t a - l a c t a m a s e .  Beta-lactamase Broken  cells  activity  Crypticity Ratio  Whole c e l l s  "C" (ml/mg c e l l s - m i n x 10"3)  WT1  371  1.60  233  9.44  P1-4  257  1.43  180  8.56  M1  210  4.13  WT2  387  1.93  201  P2-6  408  1.60  256  9.44  P2-8  447  0.44  1015  2.60  50.9  to  24.4 11.4  The beta-lactamase activity i s expressed as nmol of nitrocefin hydrolysed/mg c e l l dry weight per minute. The crypticity ratio i s d e f i n e d as t h e r a t i o of hydrolysis of nitrocefin i n b r o k e n c e l l s o v e r t h a t i n whole cells. The permeability parameter "C was c a l c u l a t e d as described in Table VI. Assays were p e r f o r m e d on cells containing the plasmid RP1, which e x p r e s s e s a h i g h l e v e l o f b e t a - l a c t a m a s e . R e s u l t s r e p r e s e n t t h e means o f f o u r t o f i v e t r i a l s . Standard deviations f o r whole c e l l b e t a - l a c t a m a s e a c t i v i t y o f strains WT1 and M1 were 65$ and 3%, respectively, of the total. Whole cell a c t i v i t y r e s u l t s f r o m s t r a i n M1 were shown t o be s t a t i s t i c a l l y d i f f e r e n t from t h o s e o f s t r a i n WT1 by S t u d e n t t t e s t (p < 0.02 ) .  72  this  assay  possible  than  reasons  hydrolysis lactamase the  i n the  for this  enhanced  of cells  v a l u e s , the  permeability  caused  significantly < 0.02  by  displayed to  be  reason  P1-6  strains  values  was  that  by  the  RP1  enzyme  which  In s p i t e  of  When  calculated M1  was  from  found  3 - f o l d ) more p e r m e a b l e Strains  P1-H,  than  wild  pursued.  than  WT2  Strain  P2-8  P2-6  appeared but  of  could  not  be  exhibited  leakage  adequately  controlled  hydrolysis,  the  derivatives  Table  and  be WT1  and  type,  RP1  the  to  in  VII  the  TEM-2  date  of supernatant  t h e r e f o r e these  of than  betafor  by  strains  included.  The  crypticity  very  low  crypticity  results  also  p e r m e a b i l i t y t o the  values  strains  in  Table  ( c f . Table  VI)  be  M1  five-fold  approximately  that wild  substrate  due  detected  73  showed  V I I were much  o f enzyme w h i c h c o u l d had  ii)  P2-6,8 were c o n s t r u c t e d a t a l a t e r  subtraction  carrying  beta-  and  which  had  mutant  not  higher  or  were m a i n t a i n e d .  to n i t r o c e f i n  result  of  Two  r e s p e c t to p e r m e a b i l i t y of  f o r C as WT1.  lactamase,  were n o t  some l e a k a g e  t test).  permeable  for this  strains the  similar  less  data,  VI).  TEM-2  produced  ( T a b l e V I I ) was  (approximately Student  the  upon c e n t r i f u g a t i o n .  C  beta-lactamase-derived  by  o f the measurements,  to n i t r o c e f i n  parameter  (Table  i ) t h a t the  generated  same t r e n d s w i t h  strains  are  beta-lactamase  cell-associated  different  (p  discrepancy  accuracy  amount  plasmid-carrying  higher  set of data  r a t e s w h i c h c o u l d be  large  remained  previous  t o the  i n broken  higher  type  cells  nitrocefin. higher  for  much h i g h e r cells.  The RP1-  amounts  The  p e r m e a b i l i t y than  mutant wild  type  WT1  with  as measured  results  Table for  using  to  strains  be s i g n i f i c a n t l y  0.01  by S t u d e n t  partial  calculated  which  using  access is  only f i v e - f o l d higher  method  that of wild  The  type  type  chromosomal  is  inducible  and  Matthew,  type  1976).  concert with  (p  <  the  (p > 0 . 0 5 ) .  equation  i n mutant M1  P_;_  Also,  seen  t h a t i n WT1 ,  from may  data  in  represent  allowing far f o r E_;_  coli,  aeruginosa  to  most  p e r m e a b i l i t y measured  by  this  only  twelve-fold greater  aeruginosa.  beta-lactamase  from  strains  Id b e t a - l a c t a m a s e of beta-lactam  This property  the b a r r i e r  cells  c o u l d n o t be shown t o be  o f chromosomal  by a v a r i e t y  found  periplasmic concentration  Pseudomonas  and mutant  was  and  beta-lactamase,  g r e a t e r than  1983) was  properties  type  f o r the B-lactamase,  membrane  ( N i c a s and Hancock,  wild  wild  to the t a r g e t p r o t e i n s .  outer  Induction  the mutant M1  the M i c h a e l i s - M e n t e n  challenge  calculated  membrane p e r m e a b i l i t y  f a r more s u s c e p t i b l e t h a n  antibiotics,  in  WT1  value  - see  for a l lrevertant  t h e RP1  V I I - n o t shown) o f B - l a c t a m  greater  2.  was  five-fold  insurmountable  than  Using  t h e change i n o u t e r  t h e mutant M1  a potentially  an  t test).  well  (five-fold  the  more p e r m e a b l e t h a n  more p e r m e a b l e t h a n  Although  Table  analysed,  r e v e r t a n t and t r a n s c o n j u g a n t s  significantly  in  than  When t h e d a t a were  which agreed  beta-lactamase  somewhat h i g h e r  (three-fold).  transconjugant  calculation,  chromosomal  V I ) b u t was "C"  by t h i s  effect  74  of  P_;_  aeruginosa  antibiotics  (Sykes  of the b e t a - l a c t a m a s e o f t h e o u t e r membrane,  gene, may  Table  VIII. Induction o f chromosomal b e t a - l a c t a m a s e i n P. a e r u g i n o s a w i l d t y p e and a n t i b i o t i c h y p e r s u s c e p t i b l e strains.  Strain  Concentration of (ug/ml)  Inducer  Induction  WT3  200  21  WT1  200  13  WT1-1  200  18  P1-6  2.0  2.4  M1  0.2  0.7  Ratio  C e l l s were grown f o r two h o u r s i n t h e p r e s e n c e o f t h e g i v e n concentration o f b e n z y l p e n i c i l l i n as i n d u c e r . Nitrocefin assays were p e r f o r m e d as p r e v i o u s l y d e s c r i b e d on F r e n c h p r e s s e d cells. The above i n d u c t i o n r a t i o s ( b e t a - l a c t a m a s e a c t i v i t y in induced b r o k e n c e l l s o v e r t h a t i n u n i n d u c e d b r o k e n c e l l s ) were o b t a i n e d .  75  play  an  important  It type  was  strain  18-fold of  shown t h a t WT1  by  role in resistance  and  the  full  growth of  b e n z y l p e n i c i l l i n (200  Similar  results  although  in  l a c t a m a s e was WT1. and  strains  partial  revertant  the  to  a  2.4-fold  and  phenylalanine,  tyrosine  experiments  workers  used  background. beta-lactamase  P_;_  using  method was a  The  An  could  by  wild be  was a  reported  mutant w i l l  76  be  these  10  ug/ml,  addition  of  the  MIC,  in  but  was  t h e n made  to  tryptophan  as  as  Attempts  to  strain  used.  of  discussed.  reproduce failed induction  since  undetermined lack  well  WT1  Possibly  of  i n the  since  of  type  the  mutant  induction  clinical  of  strain  non-beta-lactam  phenomenon,  significance  beta-  for  P1-6  a strain-specific strain  for  and  for  using  histidine.  not  1.0  attempt  (1984) had  the  P1-6  WT3 ,  of  used  The  induction  aeruginosa and  be  to  VIII).  strain  that  o n e - f i f t h of  o f mutant M1  Cullmann  in  amino a c i d s  I).  i n d u c e mutant M1.  Dalhoff  this  part  a concentration  caused to  II,  beta-lactamase  therefore  not  13-  presence  level  than  MICs f o r b e n z y l p e n i c i l l i n o f (Table  wild  (Table  type  induced  a representative  beta-lactamase  by  wild  could  insufficient  to  f o r the  fully  the  i n the  inducer  inducer  case,  these  the  an  hours  l e v e l s of  had  inducer.  f o r two  ug/ml) as  l e v e l s of  were i n c r e a s e d  greater  benzylpenicillin  induce  WT1-1  100$  respectively  each  case  between 50  Similar  M1  cells  were o b t a i n e d  this  beta-lactams.  beta-lactamase  revertant  the  to  these genetic  induction  of  3.  Outer  membrane p e r m e a b i l i t y  Permeability (hydrophobic  through  outer  i n wild  since  the a n t i b i o t i c  quite  chapter, The  type c e l l s  fluorescent  probe,  through  but  in  envelope.  the c e l l  cells  in buffer,  time  point  strongly  was  was  and  weakly  added  antibiotic  tool  for assessing  tendency  to f l u o r e s c e  a membrane,  promoted  uptake i n NPN  (described  and  which  can  be  had  a link  to i d e n t i f y The  seen  type c e l l s ,  at a  was  by  aqueous such  prevented  al.. ,  to  1984).  hydrophobic patterns  was  used due  of  by  of  as  to  a  its such  i n the  observed  addition  as  specified  environment  alterations  latter  outer  been f o u n d  (MIC) NPN  of  azide-treated  permeability,  any  f l u o r e s c e n c e caused  the  between  resistance  this  role.  an  (Loh e_t  only i n a hydrophobic  pathway.  in section  in  measured  to  indicator  pre-treatment  o f NPN,  hydrophobic  I of  environment,  hypersusceptible strains. both  shown  of  to whole,  Azide  the a n t i b i o t i c  as an  portion  f l u o r e s c e n c e was  addition.  was  i n part  used  i n a hydrophobic  made t o e s t a b l i s h  the  wild  NPN,  a  However,  a more p r o m i n e n t  fluoresces  NPN  then  was  permeability  the  play  w i t h f l u o r e s c e n c e measurements  attempt  As  it  after  agents,  the h y d r o p h o b i c  energised secretion  interfere  bilayer  smooth LPS.  h y p e r s u s c e p t i b l e mutant M1  pathway may  uptake  environment  changes  possess  this  because  as  which  to hydrophobic  membrane,  An  LPS-phospholipid  susceptible  permeability  possible  the  compounds.  membrane p e r m e a b i l i t y ) i s n o t n o r m a l l y  factor  be  to hydrophobic  selfnoting  gentamicin  5). i n T a b l e IX,  NPN  reacted  but d e m o n s t r a t e d  77  o n l y weakly  enhanced  with  fluorescence  Table  IX.  Strain  Fluorescence cells.  Total  Increase  due  fluorescence increase (arbitrary units)  to uptake  Ratio  o f NPN  i n whole  of mutant/wild  WT1  1.43  1.0  P1-4  3.45  2.4  P1-6  4.88  3.4  M1  8.47  5.9  WT2  1.03  1.0  P2-6  4.43  4.3  P2-8  0.87  0.84  P2-6.8  2.80  2.7  type  T o t a l f l u o r e s c e n c e i n c r e a s e was measured two m i n u t e s a f t e r a d d i t i o n o f NPN. R a t i o s of mutant/wild type f o r s t r a i n s P1-4, P1-6 and M1 were i n c o m p a r i s o n w i t h s t r a i n WT1 and f o r s t r a i n s P2-6, P2-8 and P2-6,8 were i n c o m p a r i s o n w i t h strain WT2. NPN, 1-N-phenylnaphthylamine. R e s u l t s r e p r e s e n t t h e means o f t h r e e to f o u r t r i a l s .  78  Fig. 2. Gentamicin enhancement of NPN uptake  Figure  2. G e n t a m i c i n enhancement  o f NPN  u p t a k e by whole  cells.  Cells were s u s p e n d e d t o a f i n a l o p t i c a l d e n s i t y a t 600 nm o f 0.5. NPN was added t o g i v e a f i n a l c o n c e n t r a t i o n o f 10 >iM then f l u o r e s c e n c e e m i s s i o n was measured a t 420 nm. Gentamicin was added to g i v e a f i n a l c o n c e n t r a t i o n o f 4 jug/ml. , WT1; , P1-4; M1 . 79  emission Fig.  intensity  2).  phenotype for  A  correlation  P1-6  than  elicited  strain  increase  due  to  P2-6  type  parent,  less  fluorescence  with  MIC  experiments  where  Due  respective  by  to  wild  the  In  to present  in  environment,  as j u d g e d  (even  of  increased  NPN  t test)  P1-4,  chapter.  80  agreed  well  were  more  from  comparitive day),  was  shown  from  that  strains  to  only be  of t h e i r  P1-4 and P2-6,8  type.  P1-6, P2-6 and P2-6,8  to hydrophobic  by u p t a k e  will  marginally  results  on a g i v e n  and M1  wild  antibiotic,  though  of the  hypersusceptibility  hypothesis  of t h i s  The  elicited  than w i l d  a lower b a r r i e r  and t h e c o r r e l a t e d  sections  NPN  s t r a i n s M1 ,  appeared  P1-4.  hydrophobic  (by S t u d e n t  t o t a k e up more  This  strain  of t h e i r respective  s t r a i n s , although  conclusion,  substances.  fluorescence  P2-6 and P2-6,8  P2-6  P1-4  "partial"  variability  s t r a i n s P1-6,  observed  revertants  These r e s u l t s a l s o  on d i f f e r e n t d a y s  type  also  P2-6,8 a l s o c a u s e d  strains  resistance  fluorescence  into  relatively  different  appeared  other  NPN  between s t r a i n s were c o n s i s t e n t  significantly  probe  the  also  NPN  whereas s t r a i n P2-8  the  done  uptake  the  of  t h a n WT2.  to  was  the p a r t i a l  to 4.3-fold that  results,  trimethoprim.  2 shows  and  WT2),  susceptible  also  uptake  (2.7-  fluorescence  NPN  Fig.  (see  antibiotic  fluorescence  i n that  cells  to 3.4-fold higher  2.4-  WT1.  transconjugants  results  o f M1,  t o mutant M1 between  and i n t e n s i t y o f NPN  the r e v e r t a n t s  and  when added  be d e a l t  to with  substances hydrophobic hydrophobic further  in  4.  Surface From  charge p r o p e r t i e s the  hydrophobic mutant  M1  previous  probe,  differently For t h i s  reason,  to  into  or the o t h e r  phase  polymer  s y s t e m was  phase  in rich  medium, c o n c e n t r a t e d  applied  to  examined.  the polymer  separate.  Each  was  presence  of c e l l s .  dextran  system s e p a r a t e s m a t e r i a l  and  hydrophobicity  phase  provided  whereas  a relatively  be  hydrophobicity, particular uncharged  molecule. phase  might  surface.  useful mutant  for  identifying  changes  glycol  The  to the  (PEG)  -  P E G - r i c h top environment  had  were  characteristics  because  respect  to  separation charge  or  more i n f l u e n c e  on a  separation of l i k e  of charged m o i e t i e s  this  to  m o l e c u l e s which These  instance,  Nevertheless,  left  due  hydrophilic  force  a lack  and  ^^QQ  be c a u s e d by r e p u l s i o n o r by  centrifugation,  mixed  1977).  difficult with  For  by  the bases of both charge  charged.  either  environment  were grown t o m i d - l o g  attracted  depending which  t h e d e x t r a n phase  cell  phase  of  two-  for  uncharged,  of r e s u l t s  achieved  on  surface  o f an a q u e o u s ,  polyethylene  e_t al,  h y d r o p h o b i c and/or  interpretation  would  in  (Magnusson  the d e x t r a n - r i c h  relatively made  two-phase,  a  cells  ten-fold  measured  of  the tendency o f  system, c a r e f u l l y  phase The  the c e l l  phase Cells  cells  uptake  w i t h t h e aqueous  o f WT1. one  mutant  involving  apparent that  than d i d t h a t partition  t y p e and  experiments,  i t was  interacted  of wild  method was  i n the c e l l  into  charges on  shown  surface  an  to of  the be the  strain.  Mutant  strain  M1  (Table  X) 81  separated  preferentially  into  Table  X.  Strain  Phase p a r t i t i o n i n g P . aeruginosa.  Ratio  o f A 600  of wild  i  n  type  and mutant  c e l l s of  t o p phase (PEG) btm phase ( D e x t r a n )  WT1  0.175  M1  2.80  Phase p a r t i t i o n o f whole c e l l s was p e r f o r m e d i n an aqueous polyethyleneglycol/dextran system as d e s c r i b e d i n t h e text. Ratios were p r e s e n t e d as t h e means o f t h r e e s e p a r a t e trials. Partition o f mutant s t r a i n M1 c e l l s i n t o t h e u p p e r phase was f o u n d t o be s i g n i f i c a n t l y d i f f e r e n t f r o m w i l d t y p e by Student t t e s t (p < 0 . 0 5 ) .  82  the  uncharged,  strain  WT1  top  phase,  p a r t i t i o n e d i n t o the  dextran-rich mutant  PEG-rich  bottom  wild  somewhat n e g a t i v e l y  was  altered in i t s presentation  environment, although  the  exact  5.  be  determined  Self-promoted  by  outer  Aminoglycoside to a h y d r o p h o b i c Having was  altered,  for  increased a  pathway  determine  (postulated  to  was  final  e_t a_l.  cells.  The  could  permeability  of  final  LPS  the  be  involved to  1980,  then g e n t a m i c i n  that gentamicin intensity  83  of  uptake  altered in  different  the the  methods  enhancement  compounds.  was  cuvette  2).  then  to e x p l a i n  (previously  4 ug/ml i n the  (self-  self-promoted  hydrophobic  (Fig.  (nitrocefin  mechanism  three  NPN  responsible  agents  aminoglycoside  compound  strain  m o l e c u l e s ) was  Hancock,  mutants,  measured  (1984),  and  may  uptake  surface  o f mutant  o f a m i n o g l y c o s i d e s ) was  to c e l l s ,  was  surface  different  whether  Nicas  first  concentration  fluorescence  the  alteration  membrane  alteration  two  cell  fluorescent  added  cell  a third  membrane p e r m e a b i l i t y  p.77)  Loh  this  hypersusceptible  hydrophobic  on  by  effect  The  the  probe),  To  used.  outer  that  through  antibiotic were  and  that  permeability  permeabilising  to  compound  uptake  examined.  the  the  method.  enhancement o f o u t e r  hydrophobic  promoted  of  that  membrane p e r m e a b i l i t y j_  established  M1  and  this  nature  type  charged  These r e s u l t s i n d i c a t e d  surface  not  cell  phase.  whereas the  I t had  stimulated  The  described,  added and  to g i v e  the  NPN  a  effect  been f o u n d ,  fluorescence  of  uptake  by by  emission  Table  XI.  Strain  Rate o f f l u o r e s c e n c e i n c r e a s e due t o u p t a k e o f NPN by whole c e l l s a f t e r a d d i t i o n o f 4 ug/ml g e n t a m i c i n .  Rate o f f l u o r e s c e n c e ( a r b . u n i t s / m i n . - 10^  increase cells)  Ratio:mutant/wild  WT1  0 .65  1 .0  P1-4  0.36  0 .55  P1-6  1 .35  2.1  M1  a  a  WT2  0.58  1 .0  P2-6  1 .97  3.4  P2-8  1.10  1 .9  2.23  3.8  P2-6  ,8  type  R e s u l t s r e p r e s e n t the means o f t h r e e s e p a r a t e trials, a: the effect o f g e n t a m i c i n p e r m e a b i l i s a t i o n on strain M1 could n o t be examined b e c a u s e NPN a l o n e caused near-maximal f l u o r e s c e n c e i n t h i s s t r a i n (see F i g . 2 ) .  84  stimulated  in this  gentamicin, which al.  most r e l i a b l y  1984),  Table  was  however i t was  was  ,  manner  therefore  similar  the r a t e of f l u o r e s c e n c e  c o r r e l a t e d with  strains  WT1  fluorescence arbitrary exert  (4  WT2  the  (3.4-fold  a  modest on  greatest greater  wild  type  effect  on  the met+-transconjugant  strain than  P1-6  P1-4,  (Loh  e_t  Fig. before  and  gentamicin  on  with  wild  o f NPN  a d d i t i o n of  type  was  P2-6 P2-  could  These r e s u l t s i n d i c a t e d t h a t u p t a k e pathway,  gentamicin  The  in  for  t  be test.  effect because  this  to hydrophobic  there  to  measurements  n o t be j u d g e d intensity  the  revertant,  by S t u d e n t  gentamicin.  to  influence  shown  fluorescence  had no f u r t h e r e f f e c t  85  appeared  Another  to this  transductant,  XI).  P2-6  to i t s s u b s t a n t i a l p e r m e a b i l i t y  self-promoted  at  ( 1 . 9 - f o l d ) and on one o f  maximal f l u o r e s c e n c e  thus gentamicin  thus appeared  intermediate  of (0.65  cells  p e r m e a b i l i s a t i o n by  from  t h e mutant M1  by  rate  pro+-transconjugant,  strain  example c u r v e s after  caused  Only  different  2 shows  less  low  Gentamicin  ( 2 . 1 - f o l d , see T a b l e  WT1.  significantly  P2-8  o f the w i l d  effect,  and t h e d o u b l e  t h a n WT2),  displayed  strain  Gentamicin  the  a  uptake  cells.  on  t h a n WT2)  greater  NPN  only  permeabilising  (3.8-fold  revertants,  to  cells).  6,8  the  values  ug/ml) t o c e l l s  produced  due  units/min-108  only  have  and  increase  concentration,  and  increase  XI.  type  due  MIC  of  r e s u l t s f o r r a t e s have been r e p o r t e d i n  The a d d i t i o n o f g e n t a m i c i n  alone  at a l l concentrations  of NPN  strain,  compounds,  ( F i g .2). might the  be a d e f e c t i n AbsA  phenotype  strains, more  which  obvious  permeation  6.  was  either  defect  (section  related  found  in  aminoglycoside  1ysozyme-mediated The  effect  "permeabilisers" 1984),  examined. access the  cell of  cation  e_t aJL. ,  and  Hancock and  particularly such  as  lysozyme  amount o f l y s i s  permeabiliser saturation.  Both  Wong,  polymyxin  lysozyme  was  Therefore,  lysozyme-mediated  aid  this  i n self-promoted  not  have  due  to  that  or  many  divalent  method  was  uptake  also  in  the  strains. compounds t o whole c e l l s ,  caused  (Fig. lysis  86  cell  varied  lower  EDTA  could  also  Hancock e_t  (1984) showed compounds  on  by  observed a  by  B,  a c r o s s t h e o u t e r membrane b a r r i e r  of lysozyme,  from  and  n o r m a l l y would  Wong,  of p e r m e a b i l i s i n g  The  to  known  of  hypersusceptible  presence  Hancock  Experiments  EDTA,  assess a l t e r a t i o n s  the  of  permeation  i t s integrity.  Addition  other  (EDTA) and  polycationic  disrupting  antibiotic  enhancement  p e p t i d o g l y c a n i n the p e r i p l a s m ,  or  to  B  two  1957;  s u c h as l y s o z y m e  nitrocefin  caused  and  o f t h e o u t e r membrane.  chelators  hydrophobic  lysis.  o u t e r membrane  to i t s t a r g e t ,  compounds,  used  polymyxin  gentamicin  the  A protein  (1981b)  to  the  permeability:  and  (Warren  of  presence  al. ,  resistance  ethylenediaminetetraacetate  permeability  or s e p a r a t e from,  4).  S e l f - p r o m o t e d o u t e r membrane EDTA,  to,  3).  (see F i g u r e  w i t h the c o n c e n t r a t i o n  threshold 3A)  lysis  in  and  up  to  polymyxin  o f between 40  and  of  a  point  of  B  (Fig.  3C)  85?  of  all  A. EDTA  B. Gentamicin  C. Polymyxin B  fPX]  Figure  (>jg/ml)  3. P e r m e a b i l i s a t i o n o f whole c e l l s g e n t a m i c i n o r p o l y m y x i n B.  A l i q u o t s o f p e r m e a b i l i s e r were added cell suspensions. Lysozyme-mediated d e c r e a s e i n o p t i c a l d e n s i t y a t 600 nm.  87  to lysozyme  by EDTA,  t o lysozyme p r e - t r e a t e d l y s i s was followed as o, WT1; •, M1.  Table  Strain  XII.  Permeabilisation o f whole c e l l s EDTA, g e n t a m i c i n o r p o l y m y x i n B.  Maximal L y s i s EDTA GM  (%) PX  EDTA  to  I  lysozyme  ( M) GM  by  U  PX  WT1  70.0  53.0  63.0  32.0  4.75  1 .45  M1  43.0  15.0  61 .0  77.0  8.86  1 .70  WT2  84.0  42 .0  83.0  15.9  14.3  1 .64  P2-6  74 .5  44.0  63 .5  10.9  11.0  1 .45  P2-8  85 .0  20.0  57 .0  15.0  35 .4  1 .82  P 2 - 6 ,8  86 .0  28.0  55.0  9.9  22 .5  2.40  The effect o f v a r i o u s p e r m e a b i l i s e r s on l y s i s of whole cells by l y s o z y m e was f o l l o w e d s p e c t r o p h o t o m e t r i c a l l y as i n Fig. 3R e s u l t s r e p r e s e n t t h e means o f t h r e e t r i a l s . EDTA, ethylenediaminetetraacetate; GM, gentamicin sulphate; PX, polymyxin B sulphate; I concentration of permeabiliser c a u s i n g 50% maximal l y s i s .  88  cells  as  (Fig. 50?  measured  3B)  was  less  of c e l l s  examined order  uptake,  was  and  maximum  wild  amount  of  cells  lysis  at which  data  particular  from  the  polymyxin  B promoted  this  trend  (Table X I I ) . well  with  difference The EDTA in  permeabiliser higher there  (Fig. was  displayed wild  type  necessary 3,  Table  a l s o a marked percent strain  WT2,  first-  theory  behind  permeabiliser  WT1  observed  (I^Q).  Although  3C  showed  than  in  and  and  5  was M1  only  strain  89  values P2-8,  trials  inhibitory  by  mutant the  grown a  on  four-fold  (Table I I ) . gentamicin strain  and  M1  than  concentrations  maximal l y s i s  Q  that strain  separate  minimal  With r e s p e c t  I  the  permeation c o r r e l a t e d  promoted  i n the  mutant  the  three  and  and  f o r 50?  the  aeruginosa  lysis  WT1,  of  were  P2-6,  c l o s e to those w h i c h had  of  two-fold  to g e n t a m i c i n  d i f f e r e n c e between s t r a i n  lysis  be  in  in Fig.  where t h e r e  XII).  not  either  in  from  lower  strain  B,  occurred  t r u e over  between s t r a i n s  type  or  for  medium,  and  follow  of the  f o r polymyxin  amount o f l y s o z y m e  wild  the  i n mutant M1  obtained  p e r m e a b i l i s a t i o n was the  lysis  hold  experiments  i n MIC  with  f o r polymyxin  experiment  results  polymyxin-containing  d i d not  15  cooperative.  more l y s i s  results  concentration  lysis  attained  50?  d i d not  The  o f between  d i f f e r e n c e between any  type  gentamicin  parameters could  where i n t e r a c t i o n  little  whereas  lysis  i s compatible  concentrations  WT1 ,  Kinetic  s i n c e the  This  nm,  causing  lipopolysaccharide i s  There strains  effective,  assay  kinetics.  the  a b s o r b a n c e a t 600  (Table X I I ) .  in this  self-promoted with  by  only, which  of  the  intermediate  susceptibility assay,  in  strain  P2-6  to  p e r m e a b i l i s a t i o n by  c o n t r a s t to gentamicin showed w i l d  promoted  pathway.  promoted  uptake  type  less  NPN  Despite  this  anomaly,  between  antibiotic  strains  gentamicin Given  the  lysis  lipopolysaccharide  were  explored  stable  in  part  uptake,  the  assay  P2-6  of  due  strain  selfself-  to  observed  differences  and  the  wild  cell  indeed  surface  one  might  did of  the  have  in fact  the reflect  compounds,  o u t e r membrane. three of  exist.  greater  apparently a  number o f b i n d i n g s i t e s  these  of  antibiotic  expected  However,  the  (Table IX).  interaction  o b t a i n e d may  for  this  some d e f e c t i n the  a f f i n i t y or a reduced  less  this  the  permeabiliser.  results  consequently  by  susceptibility  strains,  less  contradictory binding  that  greater  with  uptake  EDTA a t the  hypersusceptible  NPN  h y p e r s u s c e p t i b l e mutant M1  indicated  and  the  accurate for s t r a i n  high background apparent  that  In  o f NPN  p e r m e a b i l i s a t i o n by  already  type  enhancement  It i s possible was  gentamicin.  reduced on  producing  the a  These p o s s i b i l i t i e s  chapter  one  and  in  the  Discussion.  7.  SUMMARY The  to  be  i)  o u t e r membrane o f t h e mutant  more p e r m e a b l e Beta-lactamase  indicator compounds. five  times  of  outer The  than  the w i l d  activity membrane  type  strain by  than 90  was  o f whole c e l l s was permeability  that  o f the  observed  three c r i t e r i a :  o u t e r membrane o f mutant M1  more p e r m e a b l e  M1  used  to was  as  an  hydrophilic approximately  parent  strain  WT1  using  permeability  measurement cells  that  type  strain  WT1  significantly  d i f f e r e n t from w i l d  outer  using  a hydrophobic  cells  caused  did  the  portions  of  type  fluorescent  the c e l l  fluorescence)  partial  revertants  transconjugant, which  outer  between t h a t  of  strain  M1.  s t r a i n s was  permeability  probe,  NPN.  (within  was  not  measured  Mutant  more NPN  displayed  their  broken  type.  strain  fluorescence the  w h i c h NPN  and t h e t r i m e t h o p r i m  with  and  mutant  were more a c c e s s i b l e  also  Also,  s t r a i n s had  i n d i c a t i n g that  surface  P2-6,  correlated  the  six-fold  strain,  enhanced  i n whole  i n transconjugant  membrane  approximately  wild  criterion.  revertant  and  to n i t r o c e f i n  Hydrophobic  a  w h i c h was i n t e r m e d i a t e  Permeability  ii)  as  activity  the p a r t i a l  permeability  wild  nitrocefin  beta-lactamase  indicated  membrane the  of  to  M1  than  hydrophobic demonstrated  i n t h e m u t a n t . The susceptible  enhanced  susceptibility  uptake to  (AbsA) o f NPN,  hydrophobic  antibiotics. iii) be  Mutant M1 less  effects  greater  susceptible of  lysozyme.  to the  gentamicin Strain  and  P2-6  permeabilisation  permeabilisation these  and t h e t r a n s d u c t a n t outer EDTA, (AbsA) to  P2-8  NPN  (AbsB) were f o u n d t o  membrane based  displayed by  two r e s u l t s a r e n o t n e c e s s a r i l y  permeation  or  but  EDTA,  a t odds w i t h  promoted u p t a k e c h a r a c t e r i s t i c s o f t h e AbsA  to  proportionately  gentamicin  t o l y s o z y m e by g e n t a m i c i n  91  on  permeabilising  the  phenotype.  less however self-  PART  III.  CHARACTERISATION  OF  ALTERATIONS IN  COMPONENTS INVOLVED IN  Having phenotype  of  increases outer  established mutant M1  i n outer  membrane  altered  components  structures.  were t e s t e d .  other  and  responsible  1.  to  major  outer  M1  phase  cells As  protein  patterns  protein  I staining  transconjugant  strain  WT1.  non-reducing strains  WT1  properties  with  c a r r i e d out  WT1  consistent  to  identify  proteins  were  any first  functional properties  were compared  strains  to  both  identify  of  to  of  each  mutations  patterns.  Outer  examined  be  seen  difference  of  WT1  and  i n the  strains.  (PA01  membranes  and  can  significant  WT3  and  susceptible  characterisation  membrane c o n s t i t u e n t s  proteins.  strain  correlated  s t r u c t u r a l and  Strains  SUSCEPTIBILITY  antibiotic  membrane  transconjugant  electrophoresis. no  was  MEMBRANE  f o r each s t r u c u r a l a l t e r a t i o n .  first  logarithmic  be  Outer  O u t e r membrane p r o t e i n  The  be  could  the  membrane p e r m e a b i l i t y ,  examined, then v a r i o u s LPS  that  ANTIBIOTIC  OUTER  The  by  the  except  demonstrated  (experiments  for  shown) a l s o  conditions  i n these  92  outer the  by  from  of  no  absence WT2  and  pattern  to  of all of  that  of  solubilisation  and  difference  A.  gel  membrane  resembled  a d d i t i o n a l outer  performed  were  appeared  membrane p r o t e i n  i n temperature  M1  there  s t r a i n s WT1,  Alterations  and  examined  SDS-polyacrylamide  between  type  be  prepared  4,  outer  - d a t a not  were  in Fig.  M1  wild  to  membrane  Carey,  between protein  1980;  not  A B C D E F  Figure  4.  G H I  SDS-PAGE o f o u t e r membranes and c e l l e n v e l o p e s wild type and antibiotic hypersusceptible s t r a i n s o f P_j_ a e r u g i n o s a .  from mutant  Outer membranes or cell envelopes were isolated as described i n Methods, s o l u b i l i s e d by h e a t i n g a t 88°C, and r u n on a 14$ p o l y a c r y l a m i d e g e l . Lane A , WT1; l a n e B, M1; l a n e C, WT2; l a n e D, P 2 - 6 ; l a n e E , P 2 - 8 ; l a n e F , P 2 - 6 . 8 ; l a n e G, WT11: lane H, P1-4; lane I, P1-6. L a n e s A - F , o u t e r membrane samples; lanes G - I, c e l l envelope samples. The banding p o s i t i o n s o f p r o t e i n F and l i p o p r o t e i n I a r e shown.  93  shown).  Most  importantly,  amount o r b a n d i n g porin  protein  examined The  was no d i f f e r e n c e i n t h e  c h a r a c t e r i s t i c s o f t h e major o u t e r  F  between mutant  and w i l d  type  apparent to  caused  coincident  strains  presence  or absence o f  was  be due t o a d i f f e r e n c e i n t h e s t a i n i n g o f  the  position  in  the  gel.  (LPS)  migrating  Preliminary  n o t shown),  indicated that  not  cell,  envelope  stain  in  preparations  whole  of wild  unless  type  20  staining  proposed  mix.  concentrations,  that  phenomenon The  rough  major outer  (Fig.4).  As  noticeable  were noted  core  2  w  a  revertant  protein Idid  outer  membrane  Mg  causes aggregation LPS was i n v o l v e d  the  in  (which,  + +  o f LPS)  in  A vs.  in  Because the a l t e r a t i o n  membrane p r o t e i n s  the  band in  i t was  staining  a l s o compared  of the r e v e r t a n t s  and  i n SDS-polyacrylamide  gels  f o r s t r a i n s WT1  M1,  proteins  WT1-1  d i d not s t a i n  and P1-6 had C o o m a s s i e B l u e - s t a i n a b l e conclusion  94  no  Coomassie Blue  on  o f p r o t e i n I of the w i l d  P1-4  The  were  The l i p o p r o t e i n I o f  with  strain  M1.  In c o n t r a s t ,  there  i n any o f t h e s t r a i n s  of the l i p o p r o t e i n I .  s i m i l a r to the behaviour WT1.  and  type  mutant  experiments  present  s  be e f f e c t e d by  d i f f e r e n c e s between  the exception  SDS-PA g e l s ,  to  or  a  (cf. section 2).  transconjugants  full  MgCl  of p r o t e i n I could  appropriate  the  cell  to  s t r a i n s ( s e e a l s o F i g . 4, l a n e  mM  solubilisation/reduction  with  when  I  by l i p o p o l y s a c c h a r i d e  lipoprotein  (R.E.W. Hancock, d a t a  B)  membrane  i n SDS-PAGE.  considered protein  there  the p a r t i a l  from  revertant  strains  protein I,  similar  these  data  was  that  reversion be  o f the cause  concomitant  resistance  that  WT2  staining  with  the  reversion  phenotype.  For  the  differences strain  of t h i s  could  be  neither  t h e met*- l i n k e d  p_ro -linked  mutation  changes  protein  +  in  full  the w i l d  strains.  mutation  of s t r a i n banding  to  strains,  type  of s t r a i n  no  recipient  I t thus  appeared  P2-8 n o r t h e  P2-6 was r e s p o n s i b l e  or i n banding  to  antibiotic  transconjugant  s e e n between  and any o f t h e p r o g e n y  phenomenon a p p e a r e d  for  position  of  any  rough  c o r e LPS.  2.  Densitometry samples The  stains:  stained  protein  5.  the  Brilliant  rough  c o r e LPS would absence  procedure)  5B).  phenomenon was p r o b e d  carbohydrate-selective  appeared than that  Coomassie  t h e bottom be e x p e c t e d  o f Mg++,  a broad  of p a r t i a l l y  protein  staining,  staining  for strain  material  co-electrophoresing  (Fig.  5D).  Coomassie staining.  This  Blue  WT1,revealed  Possibly  the  a large  as shown i n  scanned. by  5D)  the  band  acid  band  performed treatment  In  Schiff  with  lipoprotein  k,  (where  lower  I (Fig.  to  carbohydrate  of  purple-blue  with the LPS-carbohydrate  was  95  (Fig.  subsequent  purple-blue-staining staining  (stained  purified  two  stain,  of the g e l s  t o band) were  band  using  to scans of F i g u r e  portions  t h e LPS o f WT1 in  membrane  Schiff  or both,  P a n e l s A and D were e q u i v a l e n t Only  outer  or c a r b o h y d r a t e  Blue f o r p r o t e i n ,  A and B.  mobility  electrophoresed  for protein  lanes  the  of  I staining  either  Coomassie Fig.  scans  appeared after  only  band when  carbohydrate  involved  in  the  Figure  5. D e n s i t o m e t r y s c a n s o f SDS-PA g e l s o f P_;_ a e r u g i n o s a o u t e r membranes s t a i n e d f o r c a r b o h y d r a t e a n d / o r p r o t e i n .  Outer membrane s a m p l e s ( c o n t a i n i n g 20 ug o f p r o t e i n and a p p r o x i m a t e l y 0.23 umol o f l i p o p o l y s a c c h a r i d e were s o l u b i l i s e d and r u n on a 14$ a c r y l a m i d e g e l as d e s c r i b e d i n M e t h o d s . EDTA (to i m p r o v e LPS r e s o l u t i o n ) o r M g C l a g g r e g a t e LPS) were added to the d e s i g n a t e d samples. Gels were stained for carbohydrate using the Schiff-periodate method and subsequently f o r p r o t e i n w i t h Coomassie blue. Densitometry s c a n s were p e r f o r m e d on the r e g i o n between p r o t e i n H2 (20,500 d a l t o n s ) and p r o t e i n I . The b a n d i n g p o s i t i o n s o f l i p o p r o t e i n s H2 and I are i n d i c a t e d . D e n s i t o m e t r y o f the carbohydrates t a i n e d g e l s was p e r f o r m e d a t a g a i n o f 9-5, whereas p r o t e i n s t a i n e d g e l s were s c a n n e d a t a g a i n o f 5.5 and t h u s t h e s e are n o t i n t e n d e d t o be q u a n t i t a t i v e l y c o m p a r a b l e . .... = p r o t e i n s t a i n a l o n e ; = carbohydrate s t a i n alone = c a r b o h y d r a t e s t a i n f o l l o w e d by p r o t e i n s t a i n . 2  Where p r o t e i n s t a i n the same c u r v e as t h e  (  t  o  a l o n e (....) i s n o t shown, i t f o l l o w e d carbohydrate plus protein s t a i n ( ).  Figure  5A.  Mutant s t r a i n 2mM EDTA.  M1  Figure  5B.  Partially purified lipoprotein I of s t r a i n (containing some protein H2 but no LPS) solubilised i n t h e p r e s e n c e o f 2 mM EDTA.  WT3  Figure  5C.  L P S - d e f i c i e n t (rough) t h e p r e s e n c e o f 40 mM  strain EDTA.  in  Figure  5D.  Wild type s t r a i n o f 2 mM EDTA.  WT1  solubilised  i n the  presence  Figure  5E.  Wild type s t r a i n o f 20 mM M g C l .  WT1  solubilised  i n the  presence  2  96  solubilised  i n the presence  AK43  solubilised  of  carbohydrate (Kropinski  stain  allowed  e_t a_l. , 1 9 8 2 ) .  I c o u l d be e x p l a i n e d i f , LPS  In  direct  solubilisation lipoprotein protein  M1  prevented  demonstrated  The  rough  mask I,  and  I  There apparent mutant,  treatment  these  + +  this  treatment  I stained  with  the  the l i p o p r o t e i n  treatment  during  the  g e l and  partially  purified  by  Coomassie with  this  Blue mutant  material ran  to  of l i p o p r o t e i n  partially  purified  pretreatment.  i n the presence  Similar (Fig.  - d a t a n o t shown)  i n which,  5A).  a p p a r e n t l y unable  mobility  a  with  (Fig.  rough  5A)  o f LPS.  carbohydrate-staining  material  LPS-deficient  as w i t h mutant M1,  Coomassie b l u e w i t h o u t  patterns of  more e x t e n s i v e e x a m i n a t i o n F,  WT1,  Mg  + +  the pre-  t o mutant M1.  digestion  protein  like  experiments  with  region of gels of 5C),  of lipoprotein  of the l i p o p r o t e i n  f o r M1 b o t h  Mg  of carbohydrate  less carbohydrate-staining  without  measurable  similarly  Protease  porin,  that  stained  AK 43 ( F i g .  lipoprotein  A  and  no  in  with  the e l e c t r o p h o r e t i c  (after  was  + +  entering  t h e g e l s and t h a t  were o b t a i n e d  absence  Mg  from  co-electrophoresed  lipoprotein results  LPS  masked  LPS o f mutant M1 was t h u s  or i n f l u e n c e which  this,  significantly  g r e a t e r than  core  strains  and t h e r e b y  Repeating  that  had e n t e r e d  mobility  absence  5E) and c o u l d be s t a i n e d  pretreatment.  material  type  co-electrophoresed  I (Fig.  without  3.  i n wild  agreement w i t h  I  staining  The a p p a r e n t  co-electrophoresed with  I.  a  Coomassie  was p e r f o r m e d 98  outer  membrane  o f t h e major by  outer  examination  of  membrane peptide  A B C D E Figure  6.  F G H I J  K L  SDS-PAGE o f p r o t e a s e d i g e s t i o n and i m m u n o r e a c t i v i t y of o u t e r membrane samples from s t r a i n s WT1 and M1.  Lanes A - J : o u t e r membrane samples a t a c o n c e n t r a t i o n o f 0.5 mg/ml were d i g e s t e d f o r 1 h a t 37°C with S_;_ g r i s e u s protease Type XIV a t a r a t i o o f 0.4 mg p r o t e a s e / mg outer membrane protein. D i g e s t e d samples were s o l u b i l i s e d and r u n on a 14$ a c r y l a m i d e g e l and s t a i n e d f o r p r o t e i n . Lanes A - E were solubilised i n the p r e s e n c e o f 2-ME and lanes F - J were solubilised i n t h e a b s e n c e o f 2-ME to help identify f r a g m e n t s o f p r o t e i n F. Lanes A and F WT1 c o n t r o l ; l a n e s B and G - WT1 + S_^ g r i s e u s p r o t e a s e XIV ( P r o n a s e ) ; l a n e s C and H - P r o n a s e c o n t r o l ; l a n e s D and I - M1 + P r o n a s e ; l a n e s E and J - M1 c o n t r o l . The banding p o s i t i o n s of unhydrolysed protein F i n the p r e s e n c e ( F ^ ) and a b s e n c e ( F ) o f 2 - m e r c a p t o e t h a n o l as w e l l as of p r o t e o l y s i s fragments are noted with s m a l l arrows. Lanes K - L: o u t e r membrane s a m p l e s were r u n on an 11$ SDS-polyaerylamide gel, blotted onto nitrocellulose then reacted with monoclonal a n t i b o d y MA5-8 a g a i n s t protein F. Only s t r a i n s WT1 and M1 a r e shown, however a l l s t r a i n s r e a c t e d equally well with t h i s antibody. Lane K, WT1; l a n e L, M1.  99  fragments  produced  by  protease  digestion  produced  with  There  to  complete  digestion.  the  digestion  degradation daltons Without was  2-ME  protein bonds this  fragment  dalton  was  Whole c e l l s  EDTA  WT3  a  digestion and  for  mutant  approximately no  difference  F.  Pronase  one  cleaved  samples.  20,000 d a l t o n  or b o t h  possesses,  9,000  34,000, 12,000 and  Possibly this  was  of the  and  to produce  (strains  o f whole c e l l s  aeruginosa  proteolysis  addition  (data not  a  fragment  two  of  disulphide  when 2-ME the  product  was  added 9,000  12,000 and  was  WT1  - L.  particular  attempted. to  proteases  when t h e  LPS  was  (200mM) c o n c e n t r a t i o n o f Mg++  samples were a l s o specific  Mutharia,  a l l strains  and  also  resistant  only observed  of a high  monoclonal antibody  F from  were v e r y  was  shown).  membrane  (MA5-8  Protein  the  o f P_^  by  Outer with  the  porin protein  only a s i n g l e ,  digestion  g e n e r a l , and  disturbed or  griseus.  i n 2-mercaptoethanol-treated  protein then  membranes  S_;_  type  the  fragments.  Protease  in  from  between  required  of approximately  which c o n t a i n e d this  outer  i n the w i l d  p a t t e r n s f o r the  i n the g e l .  which  M1  Most i m p o r t a n t l y , t h e r e was  treatment,  F  difference  Pronase  observed  visible  and  F i g u r e 6 shows  (Pronase)  l e n g t h of time  products  were  Type XIV no  by  or i n t h e  in  be  produced  strains  digestion.  p a t t e r n s o f WT1  Protease  appeared  patterns  protease  M1  for  for protein Thesis,  reactivity F of  strain  U.B.C.,  1985).  reacted s t r o n g l y with  o n l y are  epitope  Ph.D.  tested  this  antibody  shown i n F i g . 6 ) , i n d i c a t i n g  was  1 00  conserved  in  that  hypersusceptible  strains.  Since  conformational considered  the  epitope  to be  o f p r o t e i n F was  4.  epitope (L.  good  Mutharia,  evidence  unaltered  Self-promoted  outer  Interaction  recognised  by Ph.D.  t h a t the  i n mutant  this  antibody  was  Thesis), this  native  a was  configuration  M1.  membrane p e r m e a b i l i t y ;  of polymyxin  B_ w i t h  wild  type  and  mutant  cell  surfaces. Since LPS  the  above d a t a  alteration,  involves 1986),  a  and  LPS  was  promoted  uptake  polymyxin  B as  sites  on  Teuber,  1975;  Nicas  enhanced  own  to  LPS  the  both  on  binding  extended  i n the  outer  Hancock,  to c e l l s ,  (Moore  using  B had  membrane  polymyxin  a  which  e_t  a_l. ,  the  self-  dansylated  been p o s t u l a t e d with  1980;  had  pathway,  c h a r a c t e r i s a t i o n of  was  dansyl  mutant M1  specific  to Mg  (Schindler  + +  and  Moore e_t a l . , 19 86 ).  enabled  measurement  of  s i n c e b i n d i n g was  accompanied  by  fluorescence. of d a n s y l  probe  strain  concentrations were  polymyxin  differences  WT1  fluorescent  binding  membrane  Polymyxin  and  mechanism between w i l d of  outer  uptake v i a i n t e r a c t i o n  group  Binding cells  t h a t the  self-promoted  3),  a probe.  binding  polymyxin  the  pathway  its  dansyl  on  (Fig.  promote  The  s i n c e the  site  altered  suggested  type  and  M1  the  same.  in  and  first  the  (data  polymyxin  in  not  whole uptake  (Table X I I I ) .  to bind  similar shown),  w h i c h gave  This correlated  101  examined  self-promoted  mutant M1  appeared  compound  of d a n s y l  was  with  Cells  amounts and  of the  50$  saturated  the  MICs  for  polymyxin,  w h i c h were o n l y  four-fold  different  between  t h e two  strains. After  near  s a t u r a t i n g amounts o f DPX were bound  aliquots  of M g  the  surface  cell  A higher by  removed.  The  maximal for  concentrations  fluorescence  effect  seen  indicated  than  with  that  f o r wild  5.  I n t e r a c t i o n o f polymyxin  major  site  o f Dansyl  (Nicas  )  w  e  r  site  polymyxin caused  50%  moderately  e  whole  of  higher  w h i c h was s i m i l a r in  was  to the  cells.  This  lower a f f i n i t y f o r  s i n c e both  interact  (Moore e_t al_. , 1 986 )  B with l i p o p o l y s a c c h a r i d e :  polymyxin  lipopolysaccharide  interaction  uptake  lysis  competitor  also for gentamicin,  t h e same b i n d i n g  Since  type,  5 Q  increasing  t h e mutant M1 had a somewhat  at  Binding  (I  be r e v e a l e d  which  + +  polymyxin.  would  as d a n s y l  of Mg  inhibition  lysozyme  and p r e s u m a b l y  + +  molecule  intensity  a f f i n i t y of  and d a n s y l  + +  (as a f u n c t i o n o f  fluorescence  t h e mutant  the r e l a t i v e  for Mg  f o r the competing  a r a p i d decrease in  to assess  o f each s t r a i n  affinity  molecule)  Mg  were added  + +  to c e l l s ,  has been p r o p o s e d  of aminoglycosides  and Hancock,  during  1980), b i n d i n g  to  be  the  self-promoted  of dansyl  polymyxin  B t o l i p o p o l y s a c c h a r i d e and t o one o f i t s components, L i p i d was  next  examined.  ethanol  precipitation  Aliquots  of dansyl  LPS  LPS was i s o l a t e d method  polymyxin  using  o f Darveau  B were added  up t o t h e p o i n t o f s a t u r a t i o n .  102  the  cold  and Hancock to a f i x e d  Mg  A, + +  -  (1983). amount o f  Table  XIII.  Strain  Binding o f dansyl polymyxin and L i p i d A, and c o m p e t i t i o n  Binding (S  Whole  o f DPX  B t o whole c e l l s , LPS o f b i n d i n g by Mg++.  Competition Maximal I n h i b ' n  0.5)  by  Mg  (?) I  5  0  + +  (mM)  cells  WT1  1 -39  75.0  1 .25  M1  1 -39  83-3  1 .67  WT1  0 .491  67.0  2.06  M1  0.674  61.0  2.26  WT 1  0.674  68.0  0.83  M1  0.614  63.0  1 .40  LPS  Lipid A  Dansyl polymyxin was bound t o n e a r t h e s a t u r a t i o n point (as measured by f l u o r e s c e n c e i n c r e a s e ) , t h e n M g was t i t r a t e d in to assess e f f e c t i v e c o m p e t i t i o n as d e s c r i b e d fully in Methods. Results were c a l c u l a t e d f r o m H i l l p l o t s ( f o r DPX binding) o r L i n e w e a v e r - B u r k p l o t s ( f o r Mg++ c o m p e t i t i o n ) of fluorescence measurement d a t a c o l l e c t e d f r o m three separate t r i a l s . DPX, d a n s y l p o l y m y x i n B; S c o n c e n t r a t i o n o f DPX a t 50? maximal f l u o r e s c e n c e ; I concentration of permeabiliser c a u s i n g 50? maximal i n h i b i t i o n o f f l u o r e s c e n c e . + +  Q  5 Q >  103  5  >  Measurement polymyxin showed  B  no  of  (DPX)  to w i l d  difference  maximal f l u o r e s c e n c e type  and  DPX  mutant  was  that  25$  the  LPS  The  caused  and  between the  (DPX  seemed  than had  loss  S  umol o f  Q  ^  f  o  r  affinity  f r a c t i o n of  the  total  LPS,  however.  An  was  therefore  binding  k i n e t i c s i n the  the  6.  next  sites  being  s  t  r  mutant  a specific  of  respect  LPS.  similar  only  attempt  tested,  by  innermost  dansyl  with  f o r the  binding  of  strains  ain  WT1  wild of  (Table indicating  for  probably  dansyl affected on  the  made t o r e d u c e  the  examining portion  Both  M1 ,  number o f  to  amounts  a somewhat l o w e r a f f i n i t y of  number  binding  lipopolysaccharide  to b i n d  The  that  by  mutant  b i n d i n g ) per  molecules.  lower  mutant  polymyxin.  type  therefore  to whole LPS  XIII)  fluorescence  sites  dansyl  of  LPS,  polymyxin Lipid  A,  in  section.  Interaction Binding  of of  polymyxin DPX  to  B with  the  lipopolysaccharide:  isolated  Lipid  A  fraction  of  LPS. The  Lipid  moieties  which  A o f gram n e g a t i v e may  be  capable  These p h o s p h a t e g r o u p s a r e may  be  the  critical  outer  points  membrane.  substituted  with  retain  phosphate  the  other  of  located  contains  phosphate  binding  divalent  cations.  i n the  for divalent The  backbone o f  cation  phosphate  charge  groups  c h a r a c t e r i s t i c s and  o u t e r membrane s t a b i l i t y .  to  A was  therfore  examined  104  LPS  are or may  can  wild  t y p e and  of  often not  therefore  Dansyl polymyxin  i n the  and  stabilisation  f u n c t i o n a l g r o u p s , w h i c h may  influence Lipid  bacteria  binding mutant  strains. Lipid samples Lipid of  A  was p r e p a r e d  suspended A from  t h e mutant  affinity  type of  Lipid  these  equal  Lipid  tested  from  further using  Competition  by  of Mg  stabilise  polymyxin sites  Mg±±  competition regarding  the by  strain.  sites  was  + +  which  could  of Mg  The  relative  ( S . ._) u.b  A.  DPX  w  a  s  to  o f DPX b i n d i n g t o It  appeared  from  a particular  This hypothesis sites  by M g  binding  to  + +  was  .  isolated  of  on LPS has been P_^_  + +  (Table X I I I ) ,  + +  provide i n outer  effect  LPS w i t h  Mg  by near  also  Dansyl  i n part to  binds.  meaningful  Therefore information  membrane s t a b i l i t y  of competition adding  proposed  aeruginosa.  (Moore e_t a.1. , 1986) t o b i n d  to + +  from  the  be m i s s i n g  f o r these  of  membrane  examined  unfractionated Mg  Mg  the r o l e  mutant  For  LPS  A  and L i p i d A  the outer  was r e p o r t e d  on  on L i p i d  to several s i t e s  + +  curve  i n Methods.  competition  less  The amount o f DPX bound  a standard  sites  25%  did Lipid  although  LPS  i n Methods.  i n each s t r a i n  t h a t t h e mutant M1 might  lipopolysaccharide  to  sites  from  approximately  7),  (Table X I I I ) .  o f DPX b i n d i n g  Binding  bound  (Figure  A as d e s c r i b e d  results  portion  7.  strain  A was d e t e r m i n e d  excess  strain  DPX f o r b i n d i n g  approximately  hydrolysis  p o r t i o n o f DPX t h a n  + +  wild  acid  t o a known m o l a r i t y as d e s c r i b e d  the Mg -competible  the  by m i l d  f o r LPS  aliquots  of  o f the binding  Mg  + +  to  s a t u r a t i n g amounts o f DPX bound.  t h e maximum  105  percent  inhibition  of  Figure  7.  Binding of dansyl polymyxin to Lipid A and c o m p e t i t i o n o f b i n d i n g by Mg++. Binding o f DPX t o L i p i d A was measured as i n c r e a s e I n fluorescence emission a t 485 nm. Mg++ was added t o DPXsaturated Lipid A a t the i n d i c a t e d concentrations, and competition f o r DPX b i n d i n g s i t e s was measured by l o s s of fluorescence emission i n t e n s i t y . o, WT1; • , M1.  106  fluorescence that Mg  f o r t h e mutant M1. was n e c e s s a r y  + +  the  seen f o r the w i l d  mutant  Mg++  to those  Methods.  For Lipid strain  consistently Thus,  the  specific, Lipid  these  potential  moieties for  0  M1  strain  A,  is  the a f f i n i t y ,  addressed inability the  in  the molar  ratios  r  Mg  appeared DPX  binding  may  be  outer  n  be  were  measurements. deficient  site  in  doubled i n  These e f f e c t s  located  in on  a  the  binding  of Mg  A was l o w e r  Mg  + +  14.  to a n t i b i o t i c s  .  i n the  However,  substituted This  could  available  An a l t e r n a t i v e  than  either  site  case,  This  with  account  number  binding  + +  problem i s  the  relative  may i n f l u e n c e s t a b i l i t y o f p o s s i b l y accounting f o r  which use the s e l f - p r o m o t e d  membrane o f P_^ a e r u g i n o s a .  1 07  of  explanation  t h e number, o f M g  i n the mutant. In  at this  + +  sites for  groups.  partially  h y d r o p h o b i c u p t a k e mechanisms as t h e i r the  stated  was a l m o s t  + +  to  l i p o p o l y s a c c h a r i d e i n whole c e l l s ,  susceptibility  ±  performed  t o be two p o t e n t i a l b i n d i n g  rather  section  to bind  A were  independent  f o r t h e mutant M1.  on t h e L i p i d  forLipid  XIII).  three  25% r e d u c t i o n  sites  sites  of  T h i s was s i m i l a r t o  a t t h e two p h o s p h a t e  sites  apparent  concentration  of the l i p o p o l y s a c c h a r i d e .  binding that  in  which p r e c l u d e  an  o  (see Table  a r e proposed  on t h e L i p i d  + +  using  A, t h e I,_ f  + +  There Mg  o f LPS,  Mg -competable  A component  to  inhibition  type.  experiments  obtained mutant  higher  similar  cells.  competition  mutant  A slightly  to the wild  i n whole  similarly  the  WT1 was q u i t e  t o produce half-maximal  as compared  what was s e e n  type  means o f p a s s a g e  and  across  o-  0  Figure  10  20  [GM]  8.  30  AO  (ug/ml)  50  60  Inhibition by gentamicin of dansyl polymyxin b i n d i n g t o L i p i d A. Gentamicin was added to DPX-saturated L i p i d A at the indicated concentrations, and c o m p e t i t i o n f o r DPX binding sites was measured by decrease of fluorescence emission i n t e n s i t y a t 485 nm. o, WT1; •, M1.  108  Gentamicin using  the  competed  same methods as poorly  inhibition to  c o m p e t i t i o n was for  Mg  a t 50  ug/ml g e n t a m i c i n ) as  type  Lipid  A  (35$  Table  Fig.  3 i n which  XII  and  ability  8. Whole c e l l  Whole  fatty  acid  in  response  M  contains five acid,  their  for fatty  methanolic  fatty  acids  acid These  20%  compared  the  w i t h the  same  data  in  demonstrated to  acid  a  lysozyme.  HC1.  calculated  LPS  acid,  esters from  A  and  for  2-  and  quantitated using  3acid  i n T a b l e XIV  each,  P.  fraction:  hexadecanoic  were i d e n t i f i e d times  methyl  i n the L i p i d  and  retention  factors  (only  at  gentamicin  3-hydroxydecanoic  1975).  A  composition  2  hydroxydodecanoic  of  i s consistent  were a s s a y e d  hydrolysis  dodecanoic  inhibition  t o p e r m e a b i l i s e t h e mutant c e l l s  cells  aeruginosa  Lipid  A,  Gentamicin  of f l u o r e s c e n c e  result  basis  8).  t o mutant  This  (Meadow,  (Fig.  + +  for Lipid  bound  concentration).  after  examined  w i t h DPX  wild  reduced  also  on  by  the using  commercial  standards. These  a n a l y s e s demonstrated  seven  major  test)  reductions  hexadecanoic  fatty  major  the w i l d fatty  significantly  in  type, acid  amounts  and  acid  a  different  of  The in (P  levels  the > 0.5)  109  i n four  (P<0.1 by  acid  increase  in  o f mutant M1 of each  full from  of  o f the  Student  dodecanoic  slight  i n whole c e l l s  WT1. peaks  differences  but s i g n i f i c a n t  the  acid,  hydroxydodecanoic with  acids  no  t and 2-  compared the  revertant t h e amounts  seven  were  not  in  WT1  Table  XIV.  Fatty acid and M1.  Strain  composition  o f whole c e l l s  o f P. a e r u g i n o s a  F a t t y acid composition (% t o t a l f a t t y a c i d s )  strains  WT1 , P1-1, P1-4  T o t a l f a t t y acid content (nmol/mg c e l l d r y wt.)  LPS/PL ratio  30H10:0  12 :0  20H12:0  30H12:0  16 : 1  16 :0  18 : 1  WT1  5.5  4 .3  5.9  6.9  15 . 1  33 . 1  29 .7  315.8  0.290  WT1-1  6.5  4 .0  5.6  7.3  13 .6  33 . 1  29 .8  300.6  0 .306  P1-4  7.7  3 .3  6.7  7.7  13 .7  30 .4  29 .5  290.4  0.345  M1  5.9  2 .2  6.3  7.1  14 .7  29 .9  32 .5  285.7  0 .279  For fatty acid nomenclature, t h e number t o t h e l e f t o f t h e c o l o n represents the number o f c a r b o n atoms, and the number t o the r i g h t r e p r e s e n t s t h e number o f d o u b l e b o n d s . OH refers to hydroxy f a t t y a c i d s . The r e s u l t s r e p r e s e n t t h e mean of three separate determinations. LPS/PL, r a t i o o f L P S - s p e c i f i c f a t t y a c i d s (30H10:0, 12:0, 20H12:0 and 30H12:0) t o p h o s p h o l i p i d - l o c a t e d f a t t y a c i d s (16:1, 16:0 and 1 8 : 1 ) .  (Table XIV),  whereas t h e  intermediate the  total  differences  reflected  in  (Kropinski level  for  whole c e l l of  LPS  et^ al.. , 1 9 8 2 ) . In f a c t ,  whole  cell  Lipid  in  LPS  A.  causing a l t e r a t i o n s a d a p t a t i o n to another  L i p o p o l y s a c c h a r i d e rough  Isolated  fatty  first  examined  LPS  sugar  structure  by  carbohydrate  or f u n c t i o n .  major  acids  a 40  t o 50$  decrease  i n the  and  oligosaccharide carbohydrates portion sulphuric reflect  (Meadow, acid the  of  portions are  the  observed  relative  for Lipid  surface  of  primary  strains  for  or  inner  in  LPS  glucose/rhamnose and  and  was  alterations  differences  and  outer  core  amino-substituted  scans  amounts o f i n n e r and  a  composition  mutant  sugars  constant  susceptibility  to look  absorbance  The  distinguished  were  sugar  and  LPS,  for neutral  A.  alteration.  reflect  the  be  content  assay  i n c r e a s e i n the  approximately  main c o n s t i t u e n t s  1975),  assays  a 20$  Since heptoses  components  also  fatty  type  which might  were  A  core n e u t r a l  o f the w i l d  content  acids  in antibiotic  LPS  that  Lipid  acid  cell  showed  an  identical.  I t c o u l d not  fatty  showed  and  i n the mutant,  and  changes  were an  are  in  P1-4,  ratios  approximately  of 3-hydroxydodecanoate remained  mutation  in  was  o f 2 - h y d r o x y d o d e c a n o a t e was  whether  9.  revertant,  LPS:phospholipid  analyses  of dodecanoate  level level  phenotype.  amount o f LPS  The  partial  of  the  0-antigen  of  the  cysteine-  were outer  considered core  to  sugars,  respectively. The  wild  type  strains  WT3 111  and  WT1  gave a l m o s t  identical  I  1  1  I  550  500  450  400  r —  350  Absorbance (nm)  Figure  9.  Spectrophotometeric scans of c a r b o h y d r a t e assay on purified lipopolysaccharide from aeruginosa s t r a i n s WT1, WT3 and M1. A Wright and Rebers c y s t e i n e - s u l p h u r i c acid assay was performed as s t a t e d i n Methods on p u r i f i e d l i p o p o l y s a c c h a r i d e from w i l d t y p e and mutant s t r a i n s . Scans were p e r f o r m e d w i t h non-carbohydrate-containing assay fluid as a reference. Heptose sugars produced coloured products of maximum absorbance at 505nm. Hexoses and methylpentoses produced a b s o r b a n c e maxima a t 410 and 396 nm, r e s p e c t i v e l y .  112  products  as  revealed  densities  at  suggested  that  sugar  550  that  the  that  strains  from  Fig.  400 nm  nm  type  range  and  w h i l e M1  LPS  appeared  pink.  neutral of  the  antibiotic  difference by  The  was  was HgSOjj  by  of products  products  505  nm  (yellow),  enough  eye.  Wild  i n both  absorbed  Standards  L-rhamnose had  than  difference  obvious  distinguished  to absorbance  415  f o r the  produced  assay  and  (pink),  similar  strains.  in this  strongly  considerably less  mannoheptose, D - g l u c o s e nm  This  observed  was  optical  profiles  altered  colour  c o l o u r s c o u l d be  range,  9).  the  Another  the w i l d  between  extremely  o f mutant M1  due  scans  contrast,  t y p e s o f LPS  were o r a n g e ,  500  had  background  o f t h e LPS  t h e two  and  In  of  the produced  type  (see  mutant M1.  amount  produced  between  nm  s c a n were s u b s t a n t i a l l y  hydrolysis  the  350  these  hypersusceptible  500  and  compositions.  wavelength  that  by w a v e l e n g t h  the  mainly  in  L-glycero-D-  peak a b s o r p t i o n s o f  and  396  nm  (yellow),  respectively. All  strains  were  lipopolysaccharide presented  in  methylpentose three-fold M1  and  derived  from  methylpentose. strains. et  al.  (neutral)  tested  sugars,  The  (rhamnose)  sugars/heptose  than w i l d  partial strain  ratio  and  T a b l e XV.  lower  t h e two  subsequently  type  levels  Levels  These r e s u l t s  had of  wild  heptose  (1982) u s i n g gas  liquid  113  sugars  was  P1-6.  levels  were t h e  were c o n s i s t e n t  core  (glucose)  f o r the mutant and  type  rough  these r e s u l t s  of hexose  r e v e r t a n t s P1-4 WT2  for  are plus  two  to  strain  A l l strains  of  hexose  same  for  and all  with data of K r o p i n s k i  chromatography  to  quantitate  T a b l e XV.  Strain  Analysis  of  LPS  neutral  sugars.  Measured absorbance at a b s o r b a n c e maxima Heptose  U  5  0  5  )  Hexose  Ratio  of  hexose heptose  (A1J05)  WT1  0 .24  0 .76  3-2  P1-4  0 .22  0.31  1 .4  P1-6  0.21  0 .22  1 .0  M1  0 .20  0.24  1 .2  WT2  0 .24  0.65  2.7  P2-6  0.26  0.73  2.8  P2-8  0.32  0.96  3-0  A m o d i f i e d c y s t e i n e - s u l p h u r i c a c i d a s s a y was p e r f o r m e d as stated in Methods on p u r i f i e d l i p o p o l y s a c c h a r i d e which had been standardised for ketodeoxyoctonate content. V a l u e s were derived from data analogous to t h a t i n F i g . 9» using peak h e i g h t s from e a c h r e s p e c t i v e b a s e l i n e , and numbers r e p o r t e d i n this table for hexose actually represent hexose plus (A methylpentose, where the absorbance maxima and 410 A 3 9 6 > r e s p e c t i v e l y ) c o u l d n o t be separated.  114  LPS  carbohydrates. These  and  the  region  r e s u l t s seemed two  of  amounts  partial  their  of  indicating  revertants  and  that  the  in  wild  The  t y p e and  structure  strains  was  gel  (Palva  and  Makela,  found,  by  this  polysaccharide same  separate  the 0-  method,  roughly  (Peterson  lipopolysaccharide  length,  no  differences aeruginosa First, corresponding  (rough  al.,  wild  length  core  type  the  band LPS,  115  SDS-urea-  many  bacteria  1986)  of  has  been  respect  LPS  in this  all  from  plus  the  (WT1)  gel  this with was  slowest,  fastest.  method  observed  and Two  mutant  with  in a  of  rough major  (M1)  (Figure  highest  the  system  l a r g e s t number  and  to  molecules  n  migrating  using  to r o u g h c o r e  and  a " l a d d e r - l i k e pattern,  were o b s e r v e d major  in  preparations  population  chain  core,  (AbsA  (LPS)  in  sugar u n i t s ) m i g r a t i n g  substitutions  between LPS  to  1 986 ) f o r m i n g  repeating  with  even w i t h i n  LPS  using  LPS et  normal.  changes.  heterogeneous with  heterogeneous  l a r g e s t molecules  core  to be  according  e_t a_l. ,  antigen  characterised  Peterson  were  the  lipopolysaccharide  of  1980;  The  LPS  of rough core  electrophoresis.  chain  strain.  f o r core  outer  since  mutations  +  strain  the  type  strains.  the  in  (KDO)  mutant  further  polyacrylamide  me_t -linked  mutant  cores,  to have w i l d  responsible  pattern  the  rough  ketodeoxyoctonate  pro+- and  SDS-PAGE b a n d i n g  that  were d e f i c i e n t  s t r a i n s appeared  B p h e n o t y p e s ) were n o t  10.  Indicate  lipopolysaccharide  heptose  Transconjugant  to  P.  10).  mobility, different  A  Figure  B  C  D  E  F  G  H  1  10.  J  SDS-PAGE o f LPS from w i l d t y p e and antibiotic hypersusceptible strains. LPS was i s o l a t e d as d e s c r i b e d i n Methods, then samples were s t a n d a r d i s e d f o r KDO c o n t e n t and s o l u b i l i s e d i n 4$ SDS, 10$ 2 - m e r c a p t o e t h a n o l , 40 mM EDTA f o r 5 min at 1 0 0 C . Samples were r u n on a S D S - u r e a - 1 5 $ p o l y a c r y l a m i d e g e l and s t a i n e d for c a r b o h y d r a t e u s i n g p e r i o d a t e and s i l v e r n i t r a t e . L a n e s A and E , WT1 ; l a n e s B and F , M1; l a n e C, P 1 - 4 ; l a n e D, P 1 - 6 ; l a n e G, WT2; l a n e H, P 2 - 6 ; l a n e I , P 2 - 8 ; l a n e J , P 2 - 6 , 8 . Lanes A - D were overloaded to show the 0 - a n t i g e n b a n d i n g p a t t e r n s more c l e a r l y i n s t r a i n s M1, P1-4 and P 1 - 6 . Banding p o s i t i o n s of wild t y p e (WR) and mutant (MR) r o u g h c o r e LPS as w e l l as w i l d type (WS) and mutant (MS) smooth LPS a r e indicated on the margin. P  116  position  on  mobility  seen  from  f o r mutant  the carbohydrate  that  some s u g a r  missing, two  showed  were  also  visible  staining The  was t h u s  rough  core  core  higher  core  core  strain  on  (hexoses)  were  LPS s t r u c t u r e . The  M1,  P1-4 and  carbohydrate  banding  data  suggested  assay  gels.  P1-6,  results,  These  the c o n c l u s i o n that p r o t e i n  membrane  data I  was  p a t t e r n s o f above s t r a i n s ( F i g .  above t h e lower  core  no i n t e r f e r e n c e from  core  to  that  There  migration wild  type  core  pr_o -  linked  phenotypic  LPS  of f u l l  of the w i l d  was  between  rough  also  LPS band LPS i n  revertant  type  ( F i g . 5)  the  protein  strains  WT2  LPS a l t e r a t i o n mutation,  (data  not  rough  core  LPS  and t r a n s d u c t a n t s  and  in  This confirmed  was n o t due t o e i t h e r  and  migrated  WT1  no d i f f e r e n c e  o r WT1.  WT1-1  strain  a l l transconjugants  that  the  was t h e r e f o r e g i v e n  the  me_t+- o r  the  "AbsC"  designation.  SDS-PAGE  banding  antigen-containing  to  The  procedure.  shown).  +  rough  i t banded  similarly  the  the outer  r e v e r t a n t s o f mutant  altered  M1.  c o r e was c o n s i s t e n t w i t h  cause a shortened  i n the outer  there  strain  o f r o u g h c o r e , w h i c h had  c o n s i s t e n t with  because  and  assay  had d i s p l a y e d s i m i l a r  also  4)  rough  components from  w h i c h would  partial  which  11.  t h e g e l f o r t h e mutant  The  next  be  a  corresponding  p a t t e r n o f major  lipopolysaccharide  species.  major o b s e r v a t i o n was t h a t t h e r e  region to  0-  of  altered  LPS  appeared  in  SDS-PAGE  banding  O-antigen-substituted  117  also  rough  core  (ie.  "smooth") (Figure  LPS. 10)  major  seen  chain  spacing the  It  and  various  one  LPS  the  isolation LPS  mutant M1), the  not  pro*,  this  with  studies  linked  respect.  mutations to  the  P2-6 the  linked  and  phenotypes.  118  to  there  structural  trials,  the  than  altered (as  the  "AbsA"  rough  core  confirming 1983). (AbsA)  rough core strains  appeared  to  alteration  antibiotic  well  responsible  alteration  transconjugant  AbsC  higher  more  chains, Hancock,  the  strains.  P2-6,8  fewer  0-antigen  Thus,  a  type  M1 ,  linked  P2-6,8,  had  mutation  a l s o had  the  mutant  over  and  WT2)  p_ro+-  Since  of a shortened  causing  AbsA  and  in multiple  shown).  was  the  since a l l of  LPS,  gel  and  ( D a r v e a u and  that  the  LPS  particular  (WT1  in wild  0-antigen  AbsC p h e n o t y p e ) ,  separate  in  o f LPS  in strains  to p o s s e s s i o n  two  band  ( l a n e F)  concluded  latter  type  i n the  ( d a t a not  related  i n the  wild  a  me_t+- t r a n s c o n j u g a n t  property  substituted  was  bands had  and  indicated that  M1  represent  heterogeneous  r e p r o d u c i b l y observed  occurred  from e a r l i e r  It  P1-6,  mobility  to  (P2-8) however, i n t h e  and  attempt  Mutant  molecules results  with  corresponding  structural  phenotype.  the  these  loading concentrations  length  normal  of  that  bands o f l o w e r  were presumed  upper p o s i t i o n  phenomenon was  for  noted  P1-4  the  than  at  was  P2-6  occupying  chain  species  mutation  revertants  mobility  as  was  met*- l i n k e d  transcon jugant  This  i n each s t r a i n  between them i n s t r a i n s  partial  band  heavily staining  length  population.  or  Two  (the were exist  of  the  resistance  12.  Immunoreactivity monoclonal  10  moieties the  another from  (Tsai  purported  t o be  specific  1982),  i t was n e c e s s a r y  and F r a s c h ,  of the a l t e r e d  To do t h i s ,  10) was made.  antibodies  raised  of the monoclonals Mutharia,  nature  WT2-derived  their  reaction  WT2,  represent  strains  from  as t h e s e c o n d a r y  the  o f LPS gel in  monoclonal  a PA01  strain  conjugated  antibody.  One  serotype s p e c i f i c (L. f o r the  P2-6, P2-8 and P2-6,8, and n o t f o r P1 -1 ,  P1-4, P1-6 o r M1,  t o which i t  in Fig.  (Figure  monoclonal  stained  t o be  11,  LPS,  bands due  to  l a n e s E - H) w i t h  a n t i b o d y MA1-8.  These  bands  l o n g c h a i n p o p u l a t i o n o f LPS m o l e c u l e s ( E .  personal  a similar  heavily  was d e t e r m i n e d  on W e s t e r n b l o t s  an e x t r a  McGroarty,  0-antigen  to  as LPS by  blot  phosphatase  o f t h e low m o b i l i t y ,  O-antigen-specific  0-antigen  with a l k a l i n e  strains,  from  produced  to confirm  low r e a c t i v i t y .  The  the  carbohydrate  reacted with  (MA1-8) was 0 - a n t i g e n  strains,  the WT1-derived very  gel (similar  gel in  Ph. D. T h e s i s , 1985) and c o u l d o n l y be used  WT2-derived  had  for  i n t h e mutant  a g a i n s t LPS d e t e r m i n a n t s  Fab' fragment  f o r the  an e l e c t r o p h o r e t i c  T h i s was t h e n  and s u b s e q u e n t l y  Goat-anti-mouse  method used  components  with  lipopolysaccharide.  stain  SDS-urea-polyacrylamide  Figure  against  components  silver  criterion.  a  (WT3)  the was  identity  o u t e r membrane  antibodies directed  Although Figure  of altered  communication).  p a t t e r n to that  seen  10, c o n f i r m i n g t h a t  containing  LPS  molecules  119  The  antibody  by s i l v e r  reaction  staining  of  a p o p u l a t i o n o f smooth, was a l t e r e d  in  strains  ws  WR MR  B  Figure  C  D  11.  E  F  G  H  Immunoreactivity of e l e c t r o p h o r e t i c a l l y blotted LPS f r o m w i l d t y p e and mutant s t r a i n s . LPS samples were t r e a t e d as i n F i g . 10, except that the gel was not s t a i n e d , but was b l o t t e d o n t o n i t r o c e l l u l o s e and r e a c t e d w i t h a m o n o c l o n a l a n t i b o d y s p e c i f i c f o r P^. aeruginosa rough c o r e LPS (MA3-5 - l a n e s A - D), or w i t h a monoclonal a n t i b o d y s p e c i f i c f o r P_j_ a e r u g i n o s a s e r o t y p e 5 0 - a n t i g e n (MA18 - l a n e s E - H). Lane A, WT1; l a n e B, M1 ; l a n e C, P1 - 4; l a n e D P1-6; l a n e E, WT2; l a n e F, P2-6; l a n e G, P2-8; l a n e H, P26,8. The banding positions of r o u g h and smooth LPS are provided for reference from Fig. 10, which was run concurrently.  120  demonstrating The  AbsA  LPS  by  and  but  WT1-derived  with  strains  are  core  did  antigenic  phosphate  important determined  was  t h e mutant this  present and  was  type  XVI).  For  contained  Lipid  phosphate  phosphates  and  partial  r e g i o n of  the  present  i n the  moieties.  WT1  and  M1,  phosphate  i n both assayed  80?  two as  did  WT1.  The  did  was  restricted  Isolated  as much p h o s p h a t e  an  not  involve  121  LPS  Lipid  rough  as  (Table  even g r e a t e r had  disparity  t o the  the  (1960).  r e d u c t i o n i n amounts o f a s s a y a b l e M1  was  ascorbic  mutant M1  This  and  be  the  there existed  strains.  to  content  whole LPS  using  Dubin  approximately  presumed  A fraction,  mutant  the  the  the  i f the  for  that  s a m p l e s were s t a n d a r d i s e d f o r KDO  as  the  and  when b o t h  between t h e  explained  M1  outer  were  the  P h o s p h a t e was  disparity much  P2-8  P2-6,  Perhaps  strain  core  moieties  / m o l y b d a t e method o f Ames and  wild  D)  (only r e s u l t s  t h e r e f o r e not  binding s i t e s ,  + +  for strains  LPS  WT2,  m o n o c l o n a l was  LPS  -  Monoclonal  11).  i n the  shortened  phosphate  A fraction.  mutant M1  in  or M1  be  content  LPS  as M g  from  with  P1-6  to  lanes A  (MA3-5).  shown i n F i g u r e  core,  strains  Because  acid  confirmed  ( F i g u r e 11,  antibody  P1-4,  react with  determinant  susceptible  Lipid  LPS  not  lipopolysaccharide  LPS  blots  monoclonal  not  t h a t the  revertants  13.  on  were a l s o  MA3-5 r e a c t e d w i t h LPS c o r e from WT1,  P2-6,8  reason  s t a i n e d areas  reaction  a core-specific  antibody  phenotype.  high m o b i l i t y  rough core with  the  only  62%  could  be  phosphate  A-associated  core-associated  T a b l e XVI.  Strain  Phosphate and mutant  LPS phosphate  c o n t e n t o f LPS strain M1.  Ratio  mutant w i l d type  (mg/ml)  and  Lipid  A from w i l d  Lipid A phosphate  Ratio  type  mutant w i l d type  (mg/ml)  WT1  1.39  N/A  1.38  N/A  M1  1.10  0.79  0.85  0.616  Values for the amounts of phosphate were spectrophotometrically determined by a b s o r b a n c e a t 820 nm. Samples had been p r e v i o u s l y s t a n d a r d i s e d f o r k e t o d e o x y o c t o n a t e content to ensure e q u i m o l a r i t y .  122  phosphate. the  phosphate  result  of  lower  + +  artifacts  dansyl  (sections  particular  these r a t i o s  from  the i s o l a t i o n  c o n t e n t o f mutant polymyxin  6 and 7)  binding  that  were r e p r e s e n t a t i v e o f  c o n t e n t o f LPS i n whole c e l l s ,  phosphate  altered  Mg  Assuming  and M g  Lipid + +  rather  procedure, A could  binding  rather  site.  123  than  reduced  the  then  the  explain i t s characteristics  as h a v i n g been due t o a r e d u c e d  sites,  than  number  affinity  of  of a  14.  SUMMARY  Examination strain  WT1  o u t e r membrane  and mutant M1  alterations protein  of  in  except  porin  proteins  F o r any o t h e r  f o r l i p o p r o t e i n I,  Coomassie Blue i n t h e w i l d  type  strains.  staining for  from  protein  type s t r a i n  treatment  strains  protein  WT1  and  F  showed no d i f f e r e n c e  to  proteolysis  and  r o u g h c o r e LPS the banding  of  F,  monoclonal antibody  chemical  carbohydrate  content  between  type  wild hexose  indicating  that  therefore  the p r o t e i n  itself  was  rough core  LPS.  Reaction  indicated  demonstrated  and m u t a n t .  M1  of by  also  as  i n the a c c e s s i b i l i t y produced  The two p a r t i a l  structural  of that  to  identity, form .  lipopolysaccharide  there  The mutant  compared  was a l t e r e d  of protein F  F i n i t s native  analysis  content  and  fragments  between t h e two s t r a i n s , o f p r o t e i n Crude  type  or i n the  a l i k e i n t h e two s t r a i n s .  were  strain  differences had a  heptose  much  content,  i n the outer  region  revertants,  P1-4 and  of i t s P1-6,  this alteration.  There for  revertant  o f o u t e r membranes f r o m w i l d  structurally  retained  that  by  i n the  lipopolysaccharide  i n t e r f e r e d with  of protein  lower  partial  showed  proteolysis  with  stained  I i n the g e l .  Protease mutant  M1  i n SDS-polyacrylamide gels  the w i l d  membrane  s t r a i n s b u t was s t a i n e d  mutant  type  showed no  outer  w h i c h was n o t  antibiotic-susceptible  protein  wild  on S D S - p o l y a e r y l a m i d e g e l s  protein  Differential  from  appeared  separate  t o be a t l e a s t two m u t a t i o n s  a l t e r a t i o n s i n LPS s t r u c t u r e 124  i n the  responsible antibiotic  hypersusceptible caused LPS be  higher  ( t h e p_ro +  directly  mutant  mobility linked  i n the The  AbsC), partly across of  and  other  responsible  which  susceptibility  loss  AbsA),  appeared  to  antibiotic  permeability,  as  (causing the  phenotype  designated  i n a t r u n c a t e d rough core hydrophobic  LPS, t o be  permeability  and was n o t r e p r e s e n t e d strains.  in  The s t r u c t u r a l  mutation  i n the other  caused  beta-lactam  nature of  transcon jugant and  either  strain  aminoglycoside  ( d e s i g n a t e d A b s B ) , was n o t a p p a r e n t .  M1  was a l s o  of the L i p i d  of a f f i n i t y  separate  which  O-antigen-containing  wide s p e c t r u m  f o r increased  t h e o u t e r membrane,  affinity  mutations,  i n c r e a s e d hydrophobic  to r e s u l t  +  the  of  phenotype, d e s i g n a t e d  mutation  appeared  Mutant  the  t r a n s c o n j u g a n t P2-6.  me_t - l i n k e d  P2-8,  a  of a species  t h e two t r a n s c o n j u g a n t  the  One o f  responsible f o rpartial,  susceptibility, seen  M1.  for Mg  mutation  + +  found  t o be a l t e r e d  A f o r Mg  + +  .  o f t h e mutant  125  respect  i t i s n o t known Lipid  t o t h a t which caused  structure.  with  to  whether  A was c a u s e d  by  t h e c h a n g e ( s ) i n LPS  DISCUSSION  To  study  the  intrinsic  aeruginosa,  I have used  intrinsic  (wild  outer may  membrane be  there  will  good  indication  that  this  because  and t h e l a r g e  was s u s c e p t i b l e . a  I chose of  that  was  nitrosoguanidine,  i t was l i k e l y had  taken  that  make t h e i r  membrane  and  responsible  for  undertaken outer  gives  to  cause  M1 , was  to which  outer  more t h a n  membrane  the  candidate membrane  126  this  single  easier.  questions:  permeability  are r e s p o n s i b l e  i t  with  one m u t a t i o n t o  For  identification  t o answer  a  antibiotic  i t an i d e a l  to i s o l a t e  h y p e r s u s c e p t i b i l i t y and, components  nevertheless  to  N-methyl-N'-nitro-N-  place.  and  antibiotic  there  responsible,  in  and  s t r a i n s were used  in  Although  t h e mutant M1 had been m u t a g e n i s e d  transconjugant  increase  which  i t s extreme  altered  methanesulphonate  was  determine  variety of a n t i b i o t i c s  both  study  antibiotic  to c h a r a c t e r i s e ,  both  Because  therefore  to  P.  i t has l o s t  o f changes a r e a b l e  permeability.  susceptibility  of study  These two f a c t o r s made  mutant  ethyl  an  the s t r u c t u r e s  type  The mutant  interesting  be  (.ie.  components  o f what k i n d s  susceptibility.  to  that  of  n e c e s s a r i l y be t h e same ones w h i c h mutate  susceptibility,  sensitivity  such  an a t t e m p t  membrane  guarantee  cause  most  in  resistance  s t r u c t u r e s m i g h t be i n v o l v e d .  no  resistance  mutated  resistance  strain)  s e v e r a l outer is  a strain  type)  hypersusceptible  antibiotic  i f so,  reason, mutations A plan  of  i)  an  responsible i i ) which  f o r such a  is  for outer  permeability  alteration? the  In t h i s  antibiotic  Alterations  pursuit,  an e x t e n s i v e c h a r a c t e r i s a t i o n  h y p e r s u s c e p t i b l e mutant has  i n both hydrophobic  and  been  of  presented.  s e l f - p r o m o t e d uptake  were  observed. Another the  property  functional (Table  VI),  the  Although  that  the l e v e l above b a s a l  considerable  loss  level  inducibility  the uninduced  keep  with permeation  may  membrane. well  be  permeable using  artifact  and  necessary  which w i l l  inhibit  (1974), type  concentration  of periplasmic  the apparent caused  required  by  As  ug  lack  of permeation  80  min  reflects  through  the  of  period.  a  more  uninducible of the  betaamount  Nordstrom P_^  at  and  aeruginosa an  p e r ml. level  inducer The  time  exposure  A combination  of b e n z y l p e n i c i l l i n  127  not  distinguish,  by  low  mean  could  i s g r e a t e r than  min  induced  inducibility  to  in  data  induction  where t h e amount  of b e n z y l p e n i c i l l i n  this  of  between an  of beta-lactamase  a  antibiotics,  enzyme  demonstrated  presumably  not  could  possession  a p p a r e n t l y t a k e s 80  for induction  to b e t a - l a c t a m over rate  induction  induction  o f 200  case,  It i s d i f f i c u l t  the c e l l .  strains  of  to b e t a - l a c t a m  the s i t u a t i o n for  n o t be  Lack  were t h e  these experimental c o n d i t i o n s ,  lactam  wild  However,  an  could  of beta-lactam molecules  o u t e r membrane.  beta-lactamase  Sykes  level  could  permeability  (Table V I I I ) .  i f this  of  shown t o p o s s e s s  nitrocefin  of r e s i s t a n c e  because  outer  from  o f enzyme a c t i v i t y  beta-lactamase,  up  t h e mutant was  beta-lactamase,  increase  of  low  was  Type Id Pseudomonas chromosomal b e t a - l a c t a m a s e  be d e m o n s t r a t e d .  to  o f mutant M1  a c r o s s the  of  the  outer  membrane, surface  the n o n s p e c i f i c and  periplasmic  benzylpenicillin uninduced  of  result  o f mutant M1  i n binding  result  cells.  binding  of  - Table  I I ) could  R.E.W.  Hancock,  from  permeable  mutant  cellular  periplasmic  inactivation beta-lactam  beta-lactamase type  from time,  hydrolysis  decrease strain  induction  strain,  result  influx  its  target  could  of a n t i b i o t i c  occur.  by t h e u n i n d u c e d  i n induction  of this  either  VI) allowed  128  the  F i r s t , the M1  (1.0 level  Soga  and  although  this  i n a highly might  result  hydrolysis  of  beta-lactamase  In c o n t r a s t ,  could,  beta-lactamase. membranes as  f o r treatment  in  or the  of beta-lactam  beta-lactamase  of outer  of  mutant  t h e e s c a p e o f a few m o l e c u l e s  (Table  quite  view.  Thus,  by t h e u n i n d u c e d  i n the p e r m e a b i l i t y P1-6  to  the  would  for induction,  before  this  by t h e u n i n d u c e d  results).  high  to saturate  sites,  to k i l l  to  i n t h e amount  1 08 c e l l s i n 2 t o 4 min ( C .  be s u f f i c i e n t  the  prevent  and c o n s e q u e n t k i l l i n g  be h y d r o l y s e d  the r a p i d  of  the r e l a t i v e l y  of t h i s  of in  available  an i n c r e a s e  required  unpublished  might n o r m a l l y  fraction  being  binding  argue i n favour  benzylpenicillin  beta-lactamase  wild  a small  of b e n z y l p e n i c i l l i n  proteins  cell  beta-lactamase  sufficient  nonspecific  Two t h i n g s  amount  since  by  hydrolysis  membranes m i g h t  i n the periplasm,  rapidly  penicillin  outer  exposure,  and  in  i n only  In c o n t r a s t ,  penicillin  level  of  the beta-lactamase.  beta-lactamase  of  level  concentration  o f low l e v e l  ug/ml  low  beta-lactam  and t h e  benzylpenicillin  permeability period  of t h i s  components,  the  c e l l s would  external induce  by  binding  with  over  Second, a seen a  for level  (i.e. and  2 ug/ml) o f i n d u c e r h i g h e r t h a n consequent  VIII). than  Even the  induce  so, t h i s  minimum e x t e r n a l  has  loss  that of  antibiotics  aeruginosa  (1976) by  the  t o t a k e up wild  implied of II  that  I I I ) showing  hydrophobic  that  antibiotics  (Nikaido,  very  low  1976),  The by  i n mutant  uptake  antibiotic  hypersusceptible  permeation  of  studies  M1  were  probe  than  This  w i t h MIC  of  more  evidence uptake  data (Tables  susceptible WT1 .  to  Since  t h e o u t e r membrane i s with  smooth  be  mutant  allowed  129  al.  strain  must t h e r e f o r e which  t h e s e t y p e s o f compounds.  e_t  of  M1.  2).  i n gram n e g a t i v e c e l l s  some d e f e c t  loss  loss  type s t r a i n  through  where  in  i n hydrophobic  correlated  the w i l d  in  hydrophobic  Fig.  in  non-beta-  Ohmori  o f the mutant  t h e mutant was  than  of  total  (1973) d i d n o t r e s u l t  which  the  inducible,  inducibility  some a l t e r a t i o n  t h e amount o f h y d r o p h o b i c normally  in studies  cells  to  of  situation,  (Table I I ) .  ( T a b l e IX and  t h e r e was  lower  p l a y s a major r o l e  t o a wide r a n g e  more o f a f l u o r e s c e n t  type s t r a i n  longer  this  of beta-lactamase  t h e o u t e r membrane, and  in  i s involved  experiment,  10-fold  strains.  i t i s no  to the e x t e n t o c c u r r i n g  In a n o t h e r  type  an a l t e r a t i o n  Zimmermann  still  M1  (Table  i f the beta-lactamase  that  beta-lactamase  R o s s e l e t and  shown  even  o f mutant M1  or the l o s s  resistance  i n wild  resistance  hypersusceptibility  mutant  inducer concentration required  such  such  for  of the b e t a - l a c t a m a s e  o f i n d u c e r was  that  been a l t e r e d  i s doubtful  causing  P•  fully  s h o u l d be n o t e d  mutant  lactam  induction level  beta-lactamase  It  it  slight  t h e MIC  The  LPS  p r e s e n t i n the enhanced  mechanism  of  enhanced  hydrophobic  presumably assumed  through  to  structure  be  for  increased  LPS-phospholipid  to at l e a s t  deep  the  outer  of the  rough  f o r outer  model, the  hydrophobic  leaflet  of  shown banding  mobility  strains  P2-6  more h y d r o p h o b i c  probe  LPS  ratios  therefore by  Smit  Salmonellae  o f the  outer e_t  al.  was  not  M1.  outer  In  membrane  proposed  to  exist.  and  to  (NPN  and  - Table  This  seem  either  i)  i n g e n e r a l or i i ) e x p o s i n g  on  outer  from  up  2.5-  were  than  which  1 30  might  be  M1,  the  the to  other of  AbsA 6-fold  strains.  the  strains  a normally  could  1 0 - f o l d more  destabilising  permitted  Such a s i t e  dansyl polymyxin  AbsA  of  agents.  (  and  that possession  membrane  compounds.  took  I X ) , and  c h a i n l e n g t h s p e c i e s of  membrane  species  P1-6,  (Table I I I ) ,  to suggest  by  hydrophobic  0-antigen  P2-6,8) e a c h  to trimethoprim  uptake  be  the  p a t t e r n o f t h e mutant  P1-4  susceptible  LPS  to  susceptibility  transconjugants  Evidence  LPS/phospholipid  i n p a r t i c u l a r ) was  were  higher  revertant  hydrophobic  in  Firstly,  the  would  was  agents  with  the  alterations  suggested  strains  and  permeation  correlated with  type  was  p o s s i b l e e x p l a n a t i o n s f o r enhanced  be  hydrophobic  The  M1  mutants.  0-antigen  mobility  bilayer  of the  phospholipid content  characteristic  partial  one  mutant  membrane p e r m e a b i l i t y i n s t r a i n  i n deep r o u g h  Several  Strains  in  were u n a l t e r e d i n t h e mutant M1,  responsible  (and  due  perturbation  (1975)  their  the  permeability  w h i c h were r e p o r t e d .  ( T a b l e XIV) membrane  type  higher allowed  the  outer  unexposed  site  partitioning  of  lipid  binding favoured  in  nature.  the  latter  hypothesis To might  and  will  understand  influence  not d i s p l a y  length  smaller between  in  two  suggested  by  and  could  study,  two  wild  several  Leive,  on g e l s sugar  amino g r o u p  charges  on  reaction  raised  likely, M Groarty, G  such  smooth LPS  0-antigen. composition were  difference during  in  species:  between  synthesis.  the  monoclonal  column  a^l. ,  1975;  no  out.  e_t a_l. , two  groups, and  P2-  antibody  MA1-  seem  less  two  long  chain"  the  sugar  in  of s t r a i n s  1982).  (E.  demonstrated  "extra  s p e c i e s o f LPS  In any must  process, long 0-antigen  131  These capping  P2-6  makes t h i s  differences fractions  for  chromatography  " l o n g c h a i n " and  In t h i s  size,  acetyl  strains  ( F i g . 11)  by  o f t h e smooth LPS (Kropinski  t o be  reasons  m o i e t i e s by AbsA  with  difference  e_t  other  p r e p a r a t i o n ) has  Furthermore,  observed  from  LPS  fractionation  manuscript  The  c o n t e n t s or d i f f e r e n t i a l  0-antigen  type  along  have n o t been r u l e d  serotype-specific  against wild and  however  of 0-antigen  with 0-antigen  M1,  (Jann  as i s  populations  the s u r f a c e ,  researchers  19 8 0 ) ,  mobility  mutant  on  chain  Instead,  populations.  s p e c i e s appears,  to  Although  of a l l 0-antigen 1983).  and  gels  S a l m o n e l l a LPS i t  t y p e s o f smooth LPS  type  on  on g e l s .  unlike  Hancock,  altered  6,8 8,  and  banding  i t i s necessary  o f LPS  even d i s t r i b u t i o n  include  however  banding  o f o t h e r LPS  these  differential  of  an  both  amounts  Goldman  permeability,  i s heterogeneous,  in this  predominate  further.  a change i n 0 - a n t i g e n  s p e c i e s (Darveau  confirmed  as  how  the a l t e r e d  a e r u g i n o s a LPS  does  discussed  hydrophobic  know what c a u s e d P.  be  WT1  and  case, be  M1 the  created  chains  are  first  assembled,  acceptor  molecules  distributions aeruginosa which onto  of  transfer  the rough  be  species  O-antigens  mean t h a t  t h e AbsA  of  these  shorter such one  chain chain may  quite  portion in  molecular to  LPS  10  substantial,  o f M1  core  on  since  correspond  0-antigen  units with There  0-antigen  species  caused  by  that  i t  to  which  a mutation  in  recognised  core  a  composition  0-antigen  units  from  or m o d i f i e d . SDS  gels  of  i s indeed  i t was  extra  due  in M  observed p  a  u  c  h  r o u g h l y to  100  single  sugar  moieties  units).  In t h e o r y , t h e r e f o r e ,  antigen  observed  for  The  molecules),  s e g r e g a t e d . T h i s would  0-antigen  for  P.  to  a  s  shorter  a  strains  i n the t  h  a  t  long  upper  observed  difference  in  ( f o r p r o t e i n s ) o f r o u g h l y 20,000, e q u i v a l e n t  approximately  responsible  that  i n l e n g t h between t h e two  A difference  would  weight  be  defective  molecules  o f the g e l .  Fig.  rough  seen  major  i n S_j_ t y p h i m u r i u m .  to accept completed was  core  a r e more s t r i n g e n t  enzymes s u c h  l e n g t h , the d i f f e r e n c e  be  completed  those  two  higher mobility  smooth  two  i t appears  s p e c i e s o r i n the r o u g h  the a b i l i t y  the  Since  are p r e f e r e n t i a l l y  transfer  rough  which r e c o g n i s e d i f f e r e n t  or  of the t r a n s f e r a s e s If  than  p h e n o t y p e might  0-antigen  that  type  to  ( o r membrane c a r r i e r  acceptor molecule,  different  one  1979).  in transferring  enzyme v a r i a n t s  length  intact  types predominate,  enzymes  to 0-antigen two  chain  LPS  transferred  (Osborn,  LPS  are i n v o l v e d  respect may  then  enhanced  o t h e r LPS  mutant  strain  ( o r 20  t o 30  the a l t e r a t i o n M1  could  LPS in  easily  0be  hydrophobic p e r m e a b i l i t y .  structural  132  change,  that  of the  truncated  rough but  core  not  ( AbsC),  i n e i t h e r the  suggested  that  c o r e ) were n o t toward  the  partial  slightly  related.  because  seemed  of  The  have  or  LPS  mutations  The  c o n t r i b u t i o n of  i t was  could  the  t o NPN  than  the  (ie.  permeability  result  of  the  appear  mutation and  should  hydrophobic  1983;  d i d not be  the was  0-antigen  permeability)  obtained  by  selected  f o r mutants of  lengths  and  could  not  out,  appear  only  to be  aeruginosa  with  0-antigen. Studies  LPS  these  this  by  membrane  contrast  of  LPS  chain  core  Kropinski e_t  to  workers  permeability  whereas B r y a n for  results  These  portions  alterations in strains isolated  133  AbsB  hydrophobic  various  enhanced  along  found  strain  hydrophobic  outer  (1985).  were removed  similar results,  M1  core  The  to  in direct  when s i g n i f i c a n t  showed  that  bearing  affecting  compounds  (1978)  F).  that  hydrophobic  with  AbsC  appreciably.  Wells  observe  the  rough  to enhanced  however,  length  Meadow and P_^  lane  susceptible NPN  only  in this  transconjugant  t a k e up  pointed  results  10,  were  s e e n i n mutant  0-antigen  Fig.  be  beyond  l o w e r number o f  to c o n t r i b u t e  since  shorter  strains,  hydrophobic  mutation  mutations  revertants  AbsA  rough  not  other  acute  permeability,  It  with  additional effect  phenotype d i d not  antibiotics  core  have l i t t l e  Hancock,  unidentified  the  This  and  therefore  partial  m o l e c u l e s w h i c h were s u b s t i t u t e d w i t h and  (0-antigen  i n combination  Since  revertants,  strains.  to  been t h e  (Darveau  in partial  AbsB r e c o m b i n a n t  permeability  revertants.  mutation  may  two  more p e r m e a b l e  AbsA.  also present  AbsA  hydrophobic  ascertained in  was  a_l  to LPS  e_t a l . (1984)  resistance  to  aminoglycosides.  P o s s i b l y the r e s u l t s  other  mutations  underlying  Peterson coli  e_t a_l.  may  studies with  strain.  (1 986 ) i n d i c a t e d t h a t  actually affect  were done u s i n g LPS  i n this  of varying  f o r M1 may r e f l e c t t h e  0-antigen  cation binding  a cationic chain  spin  lengths  Work  length  to L i p i d  label  done in  A.  from  E.  These  probe,  isolated  by  CAT-12,  wild  type  cells. LPS  probably  fluidity, as  and 0 - a n t i g e n  well.  Membrane  susceptibility serum  WT1  to  with found  (L.  mutant  short  chain  t o be c o n s i d e r a b l y  Mutharia, could  The  therefore  (Taylor,  1983)  outer  be a s s e s s e d . was o b s e r v e d  partial permeable had  phenotype  revertants  results).  themselves or could by a n o t h e r  membrane  only P1-4  type  therefore  permeability  via  to  influence  and  so-called tend  to  be  Mutant  M1  LPS c h a n g e s i n t h e outer  membrane  adaptations  hydrophobic  M1  to  Since  d i d not appear  was  permeability  i n t h e mutant  (and t o a  and P1-6)  to  lesser  degree  significantly  nitrocefin,  to a f f e c t as  can  to n i t r o c e f i n ,  the transconjugant  p o r i n pathway,  134  be  for permeability  permeability  the  property  mutation.  permeability  mutant  to the beta-lactam. wild  this  cells  e i t h e r cause p o s s i b l e  From d a t a  that  membrane  more serum s e n s i t i v e compared t o  c o n t r i b u t i o n w h i c h enhanced  to g e n e r a l  outer  has been r e p o r t e d  unpublished  changes caused  P2-6  may a f f e c t  length  in  LPS o r r o u g h LPS m u t a t i o n s .  fluidity  it  chain  complement  alterations  also  role  fluidity  fluidity  made  an i m p o r t a n t  (complement) - s e n s i t i v e gram n e g a t i v e  those was  has  outer  measured  more strain  the  AbsA  membrane by  this  method.  Therefore,  permeability antibiotic  fact  affected  n o t be s o l e l y  by  that  outer  enhanced  nitrocefin  did  not  consistent  with  studies  postulated  that  beta-lactam  porin in  aeruginosa  enhanced  nitrocefin  therefore  reflect  a  this  strain.  protease  was  through  identical  d i g e s t i o n fragment porin  interaction  with  peptidoglycan  out,  activity  or  interactions ruled  in  with  porin  membrane  with  peptidoglycan  mutant, based  on  1983).  The  M1  could  conceivably  b i l a y e r , due integrity F  the  membrane  no a p p a r e n t  appeared basis  (Fig.  was a l s o  4).  135  by LPS,  alteration  proteins  and WT1  could  in  not  pattern  be of  were o b s e r v e d on  The i n t e r a c t i o n o f p r o t e i n  superficially  isolation  of  antibody  example  changes i n the  i n s t r a i n s M1  in  be r e g u l a t e d  An  outer  may  or i i )  a n a l y s i s and m o n o c l o n a l  proteins.  on s e p a r a t e  periplasm for  protein  both s t r a i n s ,  who  confirmed  membrane  other  SDS-polyacrylamide gels  the  the LPS-phospholipid  for  proteins  (1983),  activity,  molecules,  although  was  mutant  other  other  observation  and Hancock,  t h e major p o r i n  argued the  enter  the  not  by  e_t al.. ,  of  was  alone  further  by N i c a s  overall  periplasm  This  antibiotics  permeability  Although  reactivity,  F  i n t o the  more e x t r e m e d i s r u p t i o n o f o u t e r  structurally  outer  permeability  e i t h e r i ) higher  uptake of n i t r o c e f i n  f o r the  permeability  type c e l l s .  This  hydrophobic  M1.  done by N i k a i d o  proteins.  nitrocefin  membrane  enter  pathway i n w i l d  to  responsible  hydrophobic  hydrophobic  through  enhanced  h y p e r s u s c e p t i b i l i t y o f mutant  The  that  could  AbsA-mediated  unchanged  procedures  in  performed  the  by A.  Carey  and  R.  depended  on  complexes. with  Darveau  association  protein  (R.  variable.  Two  pore  been  (Woodruff  ejt a_l. ,  specific  regulation.  regulatory another  from  be  the major  in  mutant  to  in  and  of  In  R.E.W.  summary,  for  Hancock,  higher  strain  moderately  hydrophobic  (legend,  type b a r r i e r  Vesicle  studies  strains  was  but r e s u l t s  were have  experiments  may  subject  and  be  unknown  tested  to  porin  pore  size,  although,  dansyl  activity  polymyxin  i s not b e l i e v e d  o u t e r membrane  black l i p i d  F  bilayer  n o t be  bilayer  to  permeability studies  (R.P.  unpublished observations) f a i l e d  circumstantial  permeation  M1. by  of  Nitrocefin  of channel s i z e s  evidence  points  as t h e major  nitrocefin  of  was  Table VI),  therefore uptake  the hydrophobic  could  interior  o f Yamaguchi e_t a_l.  136  factor  determined  a disruption  to  into  1-octanol-water p a r t i t i o n i n g  to hydrophobic  into  mutant  interaction  M1.  then,  in  nitrocefin  could  i n the d i s t r i b u t i o n  and  periplasm  o f LPS  functional  of the L P S / p h o s p h o l i p i d b i l a y e r  responsible  study  regulate  in porin  Furthermore,  peptidoglycan  i n an as y e t  mechanism o f enhanced  Fs f r o m WT1  disruption  which  which  hydrophobic  show an a l t e r a t i o n  protein  and  could  isolation  aeruginosa protein  i n black l i p i d  Mutation  an i n c r e a s e  M1.  t y p e and  f o r P_j_  press),  which  data  permeability,  Darveau  sizes  possibility  the  gel analysis  unpublished r e s u l t s )  identified  system,  pelletted  F from w i l d  Darveau,  recently  is  with  A two-dimensional  isolated  attempted  (unpublished r e s u l t s ) wherein  the  to  be  in  this  o f the  wild  allow p a r t i t i o n i n g o f the c e l l  (1982) showed  of  envelope. that  beta-  lactam  antibiotics  through  the  effective  w h i c h were a b l e  walls  of p h o s p h o l i p i d v e s i c l e s  against  porin-deficient  1977).  These r e s u l t s  primary  path  is  through  on  the  cell  appear  f o r beta-lactam porins,  LPS/phospholipid physical  these  bilayer  with  can  also  characteristics  of both  the  more  e_t  al. ,  although  the  outer  pass  v a r y i n g degrees  easily  also  (Sawai  that,  uptake a c r o s s  agents  were  mutants  to i n d i c a t e  more  the  membrane  through  of success  antibiotic  the based  and  the  study  of  surface. An  interesting  antibiotic activity central  problem which  i s raised  h y p e r s u s c e p t i b l e mutants which  role  antibiotic  i s a s c r i b e d to which  this  a l l antibiotic  far  described  have  been  employed  mutations  in  1984)  (Hancock,  porin-defective.  the  given  aeruginosa  porins  the  poor  and  the  apparently  plays  in  1986),  i t is  odd  Hancock,  are  thought  P o s s i b l y the  for  results LPS  the  h y p e r s e n s i t i v e m u t a n t s o f P_^ a e r u g i n o s a  than  duplicate  by  i s that,  poor a c t i v i t y  r e s i s t a n c e ( N i k a i d o and  that  cell  t o permeate  P_^  occur  for  more e a s i l y  LPS-  wrong s e l e c t i o n  aeruginosa,  obtained  t o be  in  or a r e  rather methods  attempting  coli, less  so  or  to  perhaps  damaging  to  i n mutant M1  was  viability. The  related  third  alteration  to c a t i o n  discussed crosslink  crossbridging especially  stabilisation  previously, LPS  molecules of  w h i c h was  o f the  outer  divalent  cations  in  negative  adjacent  important  observed  i n P_^  gram LPS  molecules  aeruginosa  137  are  membrane.  As  proposed  to  bacteria. appears  (Nicas  and  Mg++ to  be  Hancock,  1980),  where  induction  exposure  Polymyxin  function  to bind,  sites  No and of  dansyl  binding. binding  sites  and mutant  LPS  has  at least  change  i n dansyl  by c o m p l e t e  between w i l d  respect  to the  inhibition  the hydrophobic  each  four dansyl  type  extent  of  this  1 986 ) .  core  both  A,  binding at  o f these  would  aeruginosa  M1 ,  site The  in  outer  s i n c e i t i s t h e backbone  membrane.  o f t h e medium Each L i p i d A at  each  of binding  (Moore e_t a l . ,  be a p o t e n t i a l  138  one  sites.  role  a t the i n t e r f a c e  of the outer  P_^  binding  I n t h e mutant  o f which a r e capable  i n w i l d type  P.  t o be u n a l t e r e d , t h e r e f o r e a  polymyxin  was L i p i d  because  polymyxin  head g r o u p h a s a p h o s p h a t e m o i e t y  1979),  polymyxin  samples  binding a t the other  o f LPS and i s f o u n d  diglucosamine  i n these  l i k e l y t o have a c r i t i c a l  stabilisation  structure  probed  were presumed  o f LPS most  (Osborn,  LPS s i t e s  cells.  Mg++  (Moore e_t a_l. ,  sites  be masked  membrane  1 9 6 0 ; Moore e_t  t o examine t h e c a t i o n  be d e t e c t e d  or  i n the outer  probe o f these  LPSs w i t h  binding  were b e i n g  per molecule  portion  sites  However, i t was l i k e l y t h a t a l a r g e number o f c a t i o n  significant  Thus,  type  membrane.  i srelatively susceptible,  polymyxin  c e l l s or i s o l a t e d  most o f t h e s e  and  i n wild  l e d to  to r e p l a c e the  the outer  1986) was t h e r e f o r e used  polymyxin  aeruginosa  could  in  b a c t e r i a (Newton,  major d i f f e r e n c e s c o u l d  mutant  sites  Mg++  i n part, to cation binding  A dansylated  (Moore e_t a_l. , binding  of  o f gram n e g a t i v e  1 986 ).  conditions  w h i c h was p r o p o s e d  B, t o w h i c h JP^ a e r u g i n o s a  membrane al. .  H1,  of a protein,  crossbridging  appears  to Mg++_deficient  Mg++  binding  end  dansyl 1 986 ) . site  depending  on  synthesis  with  portions  of  whether  or  not  i t was  a charge-neutralising residue.  adjacent  LPS m o l e c u l e s  v i a these  phosphate r e s i d u e s  (Nicas  Hancock,  19 80) .  isolated  Lipid  A  from w i l d  Dansyl type  The  during Lipid  have been p r o p o s e d  crossbridged and  substituted  by d i v a l e n t  polymyxin  and  mutant  A  to  be  cations  binding  was  to  therefore  examined. It  appeared  approximately type  cells  rise  to  25% l e s s  (Fig. the  strain  M1  sites,  because  rapidly level  7).  Lipid  for  and one m i g h t  polymyxin  binding  Logic  was q u i t e  capacity  from  and,  sites  expect  gave  s t o i c h i o m e t r i c a l l y modified  139  WT1  fell  the  same of  as o c c u r r i n g due A binding  i n fact,  exist  sites,  f o r dansyl the S  Lipid  per  Lipid  Q  ^  As. A  a 50% l o s s o f d a n s y l in reality  1 9 7 9 ) , much  A particular  (eg_. a t t h e 4' p o s i t i o n o f one g l u c o s a m i n e )  necessarily  binding  one t h i n k  f o r both  (Osborn,  1980).  + +  mutant  Mg++  l o s s o f one s i t e ,  structure  whole LPS (Goldman and L e i v e ,  Mg  from  of a f f i n i t y  similar  therefore  wild  t o assume  polymyxin  bound  from  forstrain  demands t h a t  f o rdansyl  A i s a heterogeneous  residue  region  two p o t e n t i a l b i n d i n g  molecule,  Lipid  curve  a s i m u l t a n e o u s 25% l o w e r i n g  only  missing  change i n one o f t h e L i p i d  polymyxin  that  affinity  a t both phosphate r e s i d u e s ,  dansyl  Although  the s i t e s  affinity  f o r M1.  than  cells  experiments with  competition  + +  mutant  polymyxin  that  the high  some m u t a t i o n a l  polymyxin  from  A were t h e h i g h e s t  the M g  through  than  dansyl  A  Competition  a loss of a f f i n i t y  rather  Lipid  conclusion  as t h e c u r v e  such to  that  with  other  like  phosphate i s not  moieties i n  each L i p i d may  be  50$  A molecule.  the  populations necessary binding  Lipid  of  Lipid  sites. from  provided.  Dr.  A  Lipid  data  with  strain  less  evidence during  in  to support isolation  crossbridging collection more  of  phosphate  strain.  of smaller  the  indicated  Mg  + +  site  which  aggregation 1983).  sites.  apparently  M1, w h i c h a l s o a g r e e d  sensitivity with  this  1 40  was  a  of  observed  employs  and LPS  Mg  + +  that  longer from  the  t o be  due  caused  by l o s s  to  lysis,  (Table X I I ) ,  to removal of M g proposal.  piece  f o r lysozyme  c h e l a t o r EDTA as p e r m e a b i l i s e r  a lessened  had  I t was n o t i c e d  to p e l l e t  Data  LPS and L i p i d  for centrifugal  conditions  LPS a g g r e g a t e s ,  indeed  i n whole LPS and  phenomenon was presumed  a p o r t i o n o f Mg++ b i n d i n g  using  a method  I  carbohydrate  anecdotal  binding  were n e c e s s a r y  This  which  t h e mutant  (20$ l e s s  + +  temperature  times  that  An a d d i t i o n a l  ( D a r v e a u and Hancock,  stringent  formation  X V I ) showed  + +  diglucosamine  unidentified from  Mg  31p_NMR  WT1 and M1  o f phosphate  to cause  A  t h a t mutant M1 does  yet  the  unpublished  a t the 1 p o s i t i o n of  loss of a Mg  LPS  Lipid  i s c o n s i s t e n t with  A from s t r a i n s  o f LPS u s i n g  of  centrifugation mutant  A).  block  Univ.,  on  distinct  effectively  (McQuarie  an a s s a y  (Table  Lipid  would  phosphates two  a t 25$ o f t h e t o t a l  an as  l o w e r amount o f a s s a y a b l e 40$  creating  suggested  residue  Furthermore, each  This  Bately  using  substituted  of  A.  explanation  M  These  molecules,  charge  This  have t h e p h o s p h a t e  moiety.  A  negative  observations)  25$  o f mutant M1, t h e L i p i d A  s u b s t i t u t e d a t one o f t h e two a t t a c h e d  of  data  In the case  + +  in  mutant  The  total  number o f a l t e r e d Mg++  could  result  would  be 1 X 1()6 / c e l l  molecules et  al.,  LPS  from  a b o l i t i o n o f 25$ o f t o t a l (based  ( o r 4 X 10& L i p i d 1982).  o f the crossbridging  by  a  on  the  molecules  in  t h e membrane.  Vaara  reason  their  Lipid  Mg++  on L i p i d  assays  polymyxin  was  binding  between cause  by V a a r a  species.  The  i n whole  cell  presumably  sites  A,  on  because  LPS  retained  ability.  M1 c a u s e d  causing  the other  a t l e a s t ) served  i n doing  i t i s therefore  hypersusceptibility  a l t e r a t i o n s i n the phosphate  Possibly  phenotype,  destroyed  bridging  bacterial  a l l o f t h e above e v i d e n c e ,  A.  i o n and  between 25 and  sites  e f f e c t s were n o t n o t i c e d  t h e major m u t a t i o n s  mutant  of  LPS  6  - Angus  Such an a l t e r a t i o n would  binding  dansyl  From  and,  these  binding  that  organisation  cell  i sa divalent  binding  sites  of 2 X 10  membrane d e s t a b i l i s a t i o n , as s u g g e s t e d  polymyxin  additional  + +  which  A  p o t e n t i a l l y be  (1983a and b) f o r o t h e r  that  dansyl  Mg  could  polymyxin  depending  and  since  sites  25$ l o s s o f d a n s y l  outer  Lipid  i snecessarily cooperative,  50$  profound  on an e s t i m a t e  sites  A phosphate s i t e s ) /  In a d d i t i o n ,  crossbridging  binding  so,  also  likely i n the  substituents of  LPS s t r u c t u r a l a l t e r a t i o n s to s t a b i l i s e  contributed  the primary  t o enhanced  outer  t h e mutant  M1  (AbsA  mutation membrane  permeability. Certain  other  transconjugants vivo  were  (whole c e l l )  Firstly,  properties  although  of  discovered  self-promoted mutant  from e x p e r i m e n t s outer  membrane  probing  in  permeability.  M1 was more s u s c e p t i b l e  141  and i t s  than  were  wild  type  cells  gentamicin less  (Table I I ) ,  sensitive  antibiotic XII).  to k i l l i n g  t o o u t e r membrane  Although  these  of  readily  explained  binding  site.  likely  caused  which  would  gentamicin,  two r e s u l t s  within  profound ease  of  of  susceptibility  Mg  + +  binding  Gentamicin  is  stabilising  proposed site,  barrier. of  LPS  gentamicin  attempts of this  gentamicin  used  molecules  can  to  fill  Mg  loss  of this  site  outer  including  However, l o s s mean  loss  disruption ( L o h e_t  i s proportional a_l. ,  1984).  on mutant  Lipid  process  o c c u r r i n g so t h a t ,  membrane  is  more p e r m e a b l e  to gentamicin  affected  by  t h e more s e v e r e  142  physical  Mg  + +  when  .  The of  of  the  A may p r e v e n t  this  mutant  itself,  permeabilising effects  lysozyme).  at  outer  to the l e v e l  the  ( i . e . p e r m e a b i l i s a t i o n t o t h e 14,000  + +  of the  as  an  sites.  with M g  Loss  whereas  of a  problems  niche  in  of  interaction  steric  + +  membrane,  resulting  disruption  t h e same  be  missing  molecules,  due t o  the  the  the  may a l s o  Table  conflict,  t o compete s u c c e s s f u l l y  binding site  agent  in  most  causing severe  and  permeabilisation  gentamicin  aforementioned from  two-fold  T h i s m i g h t be c o n c e p t u a l i s e d as  separation  magnitude  to  phenotype.  sites  number o f s p e c i f i c  membrane  3  a c r o s s t h e o u t e r membrane b a r r i e r ,  equivalent  the  appear  previously,  instability  passage  least  (see F i g .  the context of  mentioned  antibiotic,  p e r m e a b i l i s a t i o n by t h e same  t h e mutant M1 t o  As  heightened  portion  i t was a p p a r e n t l y a t  (and by EDTA) t o l y s o z y m e  insensitivity  the  by t h e a m i n o g l y c o s i d e  dalton  outer  i t i s not of  this  protein,  Another that by  apparent  anomaly w h i c h needed  the transconjugant gentamicin  gentamicin  but  to  P2-6 had h i g h  displayed  lysozyme.  showed  intermediate  (Table  X I ) and l y s o z y m e  these  two a s s a y s ,  In  t h e same p a r a m e t e r  for  the  behaviour  wild  type p e r m e a b i l i s a t i o n  high  resulted  in  added.  however Lipid  uptake  even  this  relatively of  A Mg  binding  + +  of dansyl  theory. in  the  Lipid  mutation  (data The  AbsB s t r a i n pattern,  alteration. case  change was o b s e r v e d A Mg  + +  sites.  143  (Table  XI)  gentamicin  was  by g e n t a m i c i n  seen  P2-8,  by  an  in strain  M1,  binding  to  failed  to  gentamicin  just  like i t s  be p o i n t e d the  to  possessed  n o t be a s c r i b e d  to a  out that  mutant  i n v o l v i n g only  Therefore  c a u s e s a more s u b t l e  outer pre-  f o r low  I t should  afforded  caused  the  not included)  could  fact  explanation  o f NPN,  polymyxin  reason  self-  o f the  strain  site  that  in  gentamicin  compound  this  seems  One  NPN u p t a k e when  studies  extreme  modest  the t o t a l  AbsB  high  t o b o t h NPN  not  disruption)  of the hydrophobic  susceptibility  the  P2-6.  but i n the case  P2-8  t o measure  were  P2-6 i s t h a t  seem t o i n d i c a t e t h a t  phenotypic  in  transconjugant  P2-8's low p e r m e a b i l i s a t i o n  permeabilisation  specific  by  strains,  i n strain  the transconjugant  substantiate  permeabilisation  I t therefore  (positive  strain,  of the L i p i d  A from  type  of s t r a i n  preliminary  antibiotic  wild  aberrantly  l y s o z y m e would alteration  in  this  Strain  NPN  by g e n t a m i c i n  XII).  was  to  w h i c h were b o t h d e s i g n e d  measuring  existing  contrast,  (Table  uptake  in  permeabilisation  type  permeabilisation  promoted  membrane  wild  explanation  M1,  a  a portion  i t may be t h a t t h e  alteration  i n these  sites  (affecting case  eg.  1C-5 s i t e s  o f M1) and t h a t  inadequate The that  an a p p a r e n t l y  binding of  cell  crosslinking  small  nature  example,  the  periplasmic  plays  an i m p o r t a n t  role  slow  beta-lactam  permeation  will  greatly increase  the  antibiotic  proteins  Lipid  membrane an  permeability  Mg  f u n c t i o n s have  susceptibility.  to a n t i b i o t i c s  which  Maintenance the  of a very  outer  membrane  with  and C u r t i s , M1,  (loss  i n self-promoted  in  combined  outer  to  membrane  of  1974).  both  the  major  o f Mg++ b i n d i n g  site  i n hydrophobic  outer  uptake  (due  cause  the  observed  permeability.  i s postulated  t o have  turn,  in antibiotic  susceptibility  by  three i)  increase  interdependent destabilisation  as  to the  enhancement o f p e r m e a b i l i t y t h e extreme  of  biosynthetic target  (due t o t h e AbsA p h e n o t y p e ) as w e l l  presumably  + +  also  f o r complete h y d r o l y s i s  mutant  mutation  + +  (eg_. b e t a - l a c t a m a s e )  rate across  - Richmond  in  alteration  changes  cooperative  antibiotic  A) and t h e a d d i t i o n a l i n c r e a s e  mutation)  moderate  the  i t can i n t e r a c t  susceptibility  undefined  AbsB  before  Mg  t o cause major d i s r u p t i o n s  enzymes  the chances  combination,  antibiotic  in  means  as a 25% l o s s o f  i n resistance.  (see I n t r o d u c t i o n  The  on  such  rate of permeability  destructive  proved  interactions  interdependent  face  i n the  change.  Beside  t o have a r o l e  sites  6  a s s a y s used  o f LPS - LPS  envelope. other  of 10  polymyxin  minor change,  o f LPS,  ascribed  For  this  instead  c a p a c i t y , c a n be e n v i s i o n e d  the  been  the dansyl  f o r probing cooperative  per c e l l  This  caused,  in  o f M1  mechanisms: o f the outer  144  membrane v i a a l a c k  of  interaction ii)  interaction  (truncated iii) and  between  enhanced  uptake  A b a c k b o n e , and  l o s s o f s y n e r g y between  membrane  beta-lactamase,  across  the outer  the outer  barrier  due t o t h e e f f e c t o f enhanced  membrane as w e l l  as on  inducibility  beta-lactamase.  These a l t e r a t i o n s a r e summarised From  study  o f an a n t i b i o t i c  can  draw c e r t a i n c o n c l u s i o n s  for  antibiotic  been  general LPS  resistance  demonstrated  undoubtedly  a  o f P_^  general. capacity  i n wild  resistance  appears  and  addition,  Mg  o f LPS m o l e c u l e s w i t h  length  i s important  wild  outer + +  seems t o be c r i t i c a l ,  therefore  hypersusceptible  type  P_^  low  porin  that  this  to play  membrane  I f there  o f LPS  a  that  role  permeability  long  in in  to  0-antigen  to hydrophobic  even m i n o r  full  chain  agents.  s t r u c t u r a l changes i n the  as an e f f e c t i v e b a r r i e r t o  145  is  and m a i n t e n a n c e o f a t l e a s t one  especially  agents.  is  i tis  a powerful  t y p e LPS c a n p r o d u c e major r e p e r c u s s i o n s the outer  As had  activity  study,  membrane  one  responsible  aeruginosa.  crossbridging  for resistance  apparent  mutant  the s t r u c t u r e s  one c a n draw f r o m  class  is  i n t h e model i n F i g . 12.  factor i n resistance.  aeruginosa also  In  about  previously,  major  conclusion  antibiotic  of  hydrophobic  0 - a n t i g e n ) and d e s t a b i l i s e d L i p i d  periplasmic  diffusion of  between LPS m o l e c u l e s ,  It in  function  destructive  Figure  12. Model suggesting how LPS a l t e r a t i o n s i n an a n t i b i o t i c h y p e r s u s c e p t i b l e mutant o f P_^ a e r u g i n o s a (M1) a f f e c t p e r m e a b i l i t y .  The o u t e r l e a f l e t o f t h e o u t e r membrane i s diagrammed above, showing t h e two p r e d o m i n a n t 0 - a n t i g e n c h a i n l e n g t h s p e c i e s of LPS m o l e c u l e s ( L ) ( w i t h r e p r e s e n t a t i v e Mg++ c r o s s b r i d g i n g ) , as w e l l as a p o r i n m o l e c u l e ( P ) . The p u t a t i v e s i t e s o f e n t r y o r i n t e r a c t i o n o f v a r i o u s t y p e s o f a g e n t s w i t h the o u t e r membrane are a l s o shown f o r b o t h w i l d t y p e and mutant s t r a i n M1. A, wild type; B, mutant M1; AG, aminoglycosides; HPHI, h y d r o p h i l i c a g e n t s ; HPHO, h y d r o p h o b i c a g e n t s .  146  CHAPTER CHEMICAL  CROSSLINKING  ESCHERICHIA NATIVE  COLI  OF  OUTER  TWO PSEUDOMONAS  MEMBRANE PORIN  AERUGINOSA  PROTEINS  TO  AND REVEAL  OLIGOMERS  Introduction Early indicated  that  oligomers. of  studies these  Certain  porin  coli  porin,  (Ishii  data 1977;  studies  (Dorset  Palva  were  solubilisation  weights under  temperatures  < 60°C).  porin  F formed  protein  electrophoresis  porin,  D1,  even  (Hancock was  also  after  major  and  directly,  a_l. ,  1 979 ) of  porin  this of  al.,  optical  by  gel  and  diffraction  porins virtue  S.  1979).  (Reithmeier  E^_ c o l i  (OmpC  and  of  their  SDS-polyacrylamide gels  after  contrast,  conditions  Pseudomonas  1 9 7 9 ) . One  s e n s i t i v e to SDS,  (ie.  aeruginosa  SDS-polyacrylamide  solubilisation  147  moderate  more et_  as  mobility  association  oligomeric  Carey,  low  Tokunaga e t  monomer bands on  and  (Yu  non-denaturing In  form  at  and,  the  1978) Other  on  the  coli  polyacrylamide  crosslinking  Randall,  shown to be  as  1980;  e_t a_l. , 1 9 8 3 ) .  also  1979 )  well  by  as  by  trimeric  Nakae,  Escherichia  incubation  experiments  as  and  molecular  temperature  e_t a_l. ,  were c o n f i r m e d  Bragg,  such  analysis  a native  and  of  exist in their native  after  equilibrium to  E_^  apparent  (Nakae  porins  evidence  p r i o r to  pointed  typhimurium  LamB)  physical  i n SDS  sedimentation  These  proteins  OmpF  electrophoresis  major  the  protein  temperatures  strongly  on  in  other  SDS P_;_  at  gel room  aeruginosa  whereas a n o t h e r ,  protein  P,  showed  oligomers  a f t e r low  Sedimentation  equilibrium  P.  porin  aeruginosa  analysis  protein  F due  lipopolysaccharide  seen  (Hancock  1 979 ; P a r r  and  Carey,  hampered  therefore  alternative  method  Pseudomonas p o r i n Chemical systems  1979;  available g r o u p s on  the  to  different amino or have  allow  involves  1979;  Conditions unproductive  of  the  a  of  protein have  results.  most  viable  associations  been w i d e l y  the  used  associations  in  of  the  groups,  to  1979).  shifts  addition  species  monofunctional 148  be  bonds  crosslinked  reagent. of  on  SDS-  A  more  crosslinked  two-dimensional  gels  where t h e  relative  can  give  oligomeric adjusted  reaction  of  d)  linking  azido  in mobility  the  1976),  favoured must  Reagents  alignment  Randall,  reaction  of  components on  crosslinked  hydrophobicity  c l e a v a b i l i t y and  Analysis  of  become  functional  photoactivatible  involves  most  c)  crosslinker. and  have  crosslinkable  environment,  variety  proteins ( J i ,  agents  d i f f e r e n t degrees of  due  of  of  in a  of  b)  and  each  of  be  choice  t h e i r cleaved Palva  indication  the  w h i c h would  native  variety  observations  gels  of  of  1 986 ) ,  to  for  tight association  a)  technique  products with  intensity  A  employed • ( J i ,  sophisticated  (Ji,  1979).  lengths  polyacrylamide  the  attempted  proteins.  sulphhydryl  been  not  solubilisation.  sedimentation  appeared  protein's  span  products  of  for determining  protein,  the  to  SDS  isolation  to demonstrate n a t i v e  which  was  e_t a_l. ,  c r o s s l i n k i n g has  Freedman,  suited  during  interpretation  Crosslinking  of  temperature  of  also  an  association. to  minimise  crosslinker,  spontaneous due  to  aggregation  chance  contact  potential  hazards  corrected  simply  relative for  of isolated proteins, between p r o t e i n s ,  of the technique. by  adjusting  amounts o f r e a g e n t  reaction.  crosslinking oligomers,  With  however  solubilising  and p r o t e i n ,  technique  i t cannot  a l l of  which  Most o f t h e s e  are  c a n be  conditions,  and t h e t i m e  stringently  i s a valuable  and c r o s s l i n k i n g  applied  allowed  conditions,  f o r i n d i c a t i o n of native  provide  sole  proof  of  such  associations. For  this  reactive,  1.2  (succinimidyl Reithmeier integral reaction in  close  could  study,  long,  propionate)  and B r a g g , outer  membrane  proximity,  cleavable  proteins. to allow  Fairbanks,  The b r i d g e  reaction  only  any  of proteins.  i n a two-dimensional  1976;  length with  and  subunits  reaction  which  A cleavage  bond was a l s o p r e s e n t  d i s s o c i a t i o n of crosslinked  thus a n a l y s i s  dithio-bis-  t o be most s u i t a b l e f o r  to t r y to eliminate  of a disulphide  amino g r o u p -  reagent,  (DSP - Lomant and  be due t o random c o l l i s i o n  o f DSP a l l o w i n g  hydrophobic,  1977) was deemed  t i m e were c h o s e n  comprised  and  nm  a moderately  site  i n the middle  product  with  2-ME  SDS-polyacrylamide g e l  system. Chemical proteins  of  typhimurium associations for  E_^_ c o l i  (Palva,  proteins  studies  (Reithmeier  1980) were used  f o r proteins  t h e 45K p r o t e i n  porin  crosslinking  and B r a g g ,  OmpF and OmpC  Pseudomonas of  membrane  1977) and  (E_^ c o l i )  characteristics  1 49  outer  to demonstrate  o f JLi. t y p h i m u r i u m .  possess  on  S.  oligomeric as w e l l  as  aeruginosa  similarity  and  dissimilarity porins. to  with respect  F o r example,  be  of s i m i l a r  ( 3 8 K ) ,  i t has  (O.36  nS)  P,  induced  and  SDS-stable  to  E^_  coli  smaller et  stable  and  al. ,  1982)  e_t a_l. ,  e_t a_l. ,  more a n i o n - s p e c i f i c i s of a l a r g e r  the n a t i v e  in  this  (as  bilayer mentioned  crosslinking association  (48K  vs.  study.  150  analogous  Lugtenberg, been shown  pores  apparent  than  PhoE  associations  19 8 0;  to  form  (Hancock  m o l e c u l a r weight  3 8 K ) . I t was  technique. o f each  has  form.  conditions  i s therefore and  OmpF  smaller  artificial  starvation  1 982),  1 9 8 0 ) but  interest  oligomeric  and  phosphate  gels  the  (5nS)  under  SDS-polyacrylamide  using  appears  d e n a t u r a t i o n t o the monomer  Lugtenberg,  t o compare  an  F  protein  i n p r e s s ) and  (Overbeeke  and  coli  larger  in  PhoE  (0.2nS),  Salmonella  t o SDS  (Hancock protein  as E_^  both  channels  (Woodruff  and  aeruginosa protein  m o l e c u l a r weight  hydrophilic  above) i s l e s s  Tommassen  although  been shown t o f o r m  membrane s y s t e m  Protein  t o the above E_^ c o l i  therefore  of these  Evidence  was  of  proteins  supporting  of these p r o t e i n s  on  an  obtained  RESULTS  1 . Crosslinking  and  o f E. c o l i  As  a control  to  demonstrate  undertook  outer  f o rstudies  with  the v a l i d i t y  crosslinking studies  PhoE o f E_^ c o l i  has r e c e n t l y  protein  which  cations  (Korteland  i s three-  et^ a_l. ,  similar electrophoretic  been  protein  would  dimension  SDS  second  the  like  purified  t o break  gels  to  other  coli  t h e s t r u c t u r a l gene and  primary  OmpF  porin  shown  amino of  to acid  E_;_  coli  i t may be e x p e c t e d  that  T h i s was  analysis  PhoE p r e p a r a t i o n s were  i n both  evidence (Figure  silver  s t a i n i n g were c o n f i r m e d outer  2-  and t h e n r u n i n  The major s p o t s  t o be p r o t e i n a c e o u s  membranes  151  with  o f m u l t i m e r s was o b s e r v e d  13).  gels  r u n on f i r s t  treated  the c r o s s l i n k bridges  stained  blue-stained  In the  E_j_  t h e OmpF p o r i n .  gels  silver  methionine-labelled  over  (Figure 1 3 ) .  polyacrylamide  dimension,  Protein  pore-forming  and  Therefore,  be a t r i m e r  crosslinked  mercaptoethanol  Coomassie  behaviour  acid  with  1982).  and two d i m e n s i o n s  first  i t forms an o l i g o m e r  by c r o s s l i n k i n g and e l e c t r o p h o r e t i c  When  the  gels,  In a d d i t i o n ,  nucleic  ( 6 4 $ ) homology  confirmed one  1984).  strong  (Tommassen e_t a_l, , this  PhoE.  i s o l a t e d by Tommassen and c o l l e a g u e s  demonstrate sequence  protein  I  1 9 8 2 ; Benz et; a^l. , 1 9 8 4 ) .  on SDS p o l y a c r y l a m i d e  (Benz e_t a^l. ,  techniques,  porins,  to f o u r - f o l d s e l e c t i v e f o r anions  with  has  o f my  PhoE  aeruginosa  been shown t o be a  form  porins  t h e P_^_  of porin  unheated a  membrane p r o t e i n  ( f o r protein  (for a l l proteins).  seen  in by  using 35sF)  and  Artifact  Figure  13-  One- and t w o - d i m e n s i o n a l c r o s s - l i n k i n g p u r i f i e d E. c o l i p r o t e i n Pho E.  analysis of  Purified protein Pho E was t r e a t e d w i t h 500 ug DSP/mg protein and r u n on an 11$ p o l y a c r y l a m i d e g e l . The first d i m e n s i o n g e l was c u t o u t , s o a k e d i n r e d u c t i o n mix c o n t a i n i n g 2-mercaptoethanol t o b r e a k DSP c r o s s l i n k s and t h e n t h e l a n e laid on t o p o f a n o t h e r 11$ a c r y l a m i d e g e l and r u n i n t h e s e c o n d d i m e n s i o n as d e s c r i b e d i n M e t h o d s . The f i r s t d i m e n s i o n gel showed crosslinked products: Lane A - Pho E u n t r e a t e d control ; l a n e B - Pho E + DSP. Products observed i n the first and/or second dimension gels were an internally c r o s s l i n k e d monomer ( E i 1 ) , dimers (E2), t r i m e r s (E3)» and a small amount o f h i g h e r m o l e c u l a r w e i g h t p r o d u c t ( s ) o f unknown derivation. The r u n n i n g p o s i t i o n s o f t h e monomer o f t h e Pho E protein i n t h e f i r s t d i m e n s i o n and t h e monomer and spots d e r i v e d from c r o s s l i n k e d o l i g o m e r s i n the second d i m e n s i o n a r e l a b e l l e d on t h e l e f t hand s i d e .  152  PhoE PhoE 3  2  PhoE  67 K 45 K  30 K  streaks 60,000  seen  across  and 67,000  g e l s a t apparent  d a l t o n s were enhanced  molecular  weights  of  by s i l v e r  staining  and  were p o s s i b l y due t o i m p u r i t i e s i n t h e r e d u c i n g  agent  and  two-dimensional  Dessev,  1983).  polyacrylamide other  authors  These  blemishes  of  (Tasheva  g e l e l e c t r o p h o r e s i s have a l s o been r e p o r t e d ( G u e v a r a e_t ajL. ,  1982;  M a r s h a l l and  by  Williams,  1984). Multimers uncrosslinked mobility and  aligned  the  seen  high  The p u r i f i e d  and  (14) f r e e  proportion  100,000  cleavage spots  which  a 34,000 bridge since  from  38,000  dimensional  analysis.  (called  using  purified  due  to  band  could  daltons  large  C) were s e e n  154  in  weight  dimensional  w h i c h was result  gels  to  or  higher possess  trimer  of  in a  an  of  two-  similar  experiments  by t h e a u t h o r s as  A  a band o f  seen  in crosslinking  gels  probably  i n press).  and a  amounts  freely  in  In a d d i t i o n ,  and e x p l a i n e d  mobility  originally  molecular  140,000 d a l t o n s was  OmpF p r o t e i n ,  aberrant  were  (Hancock e_t a_l. ,  Since  first  horizontally  demonstrated  o f d i m e r o f 73,000  weight  were  of  higher  d a l t o n monomer was  since this  amino g r o u p s  molecular  one  their  between t h e  they  d a l t o n band,  PhoE was  to  line  t h e PhoE p r o t e i n and were  d a l t o n s were a l s o o b s e r v e d .  estimated  product  due  t h a t the h i g h e r  A) were d e r i v e d  due t o an i n t r a c h a i n  several  13,  i n the Coomassie-stained  to form  mobility  from  confirmed  13, l a n e  crosslinked  below t h e d i a g o n a l  The c l e a v e d  protein  artifacts.  gel  i n Figure  2-mercaptoethanol  This  products  as s p o t s  dimensions.  to  derived.  not  proteins  after  second  (Fig.  appeared  as  aberrant  conformation this  o f an o l i g o m e r  product  Therefore,  2. C r o s s l i n k i n g Protein starvation  on  were sound  polyacrylamide  gels  that  fold  (Hancock  protein  The  daltons  (Figs.  P  dalton  PhoE,  over  by p h o s p h a t e P_^ a e r u g i n o s a oligomers at  It differs  t h e PhoE  from  monomer  i n that  low  molecular  13 and 1 4 ) , a s m a l l e r f o r anions  channel  i tis  cations i n black l i p i d  products  (of estimated  and  145,000  44,000  lower  f o r anions  crosslinked  crosslink a  that  100-  bilayers  e_t a_l. , 1982; Benz e_t aJL. , 1 9 8 3 ) .  Four  90,000  basic  solubilisation  i t has a h i g h e r a p p a r e n t  and a h i g h e r s e l e c t i v i t y selective  our  i t forms s t a b l e  after  protein  i s induced  Unlike other  t h e PhoE p r o t e i n ) ,  (Hancock e_t aJL. , 1 9 8 2 ) .  size  and s u g g e s t e d  t h e PhoE p r o t e i n ,  (Hancock e_t . a l . , 1 9 8 2 ) .  o f 48,000  that  tetramer.  p r o t e i n P_  temperatures  weight  a  1978),  p o r i n s , was a t r i m e r .  P, l i k e  in  represent  demonstrated  o f P. a e r u g i n o s a  (but l i k e  SDS  necessarily  studies  techniques  o t h e r E_j_ c o l i  porins  not  these  crosslinking like  may  i n SDS ( P a l v a and R a n d a l l ,  molecular  weights  d a l t o n s p o t was p r e s u m a b l y  apparent band  was  band  was  protein  P monomer.  tighter  molecular  folding  weight.  possibly  crosslink.  dalton  d a l t o n monomer o f 44,000,  d a l t o n s c o u l d be v i s u a l i s e d  which caused  intrachain  o f t h e 48,000  a  Based  a dimer  spot  155  seen  (Figure an  Similarly, of  of  the  on b o t h  and t h u s  the  subunits weight,  14).  intrachain  of the p r o t e i n  on m o l e c u l a r  most l i k e l y Another  dimer  due t o  80,000,  80,000  with  an  t h e 90,000  48,000  dalton  t h e one and two-  Figure  14.  One- and t w o - d i m e n s i o n a l c r o s s - l i n k i n g a n a l y s i s o f p u r i f i e d P. a e r u g i n o s a p r o t e i n P.  P u r i f i e d p r o t e i n P was t r e a t e d as i n F i g . 13 e x c e p t t h a t a 9% p o l y a c r y l a m i d e g e l was u s e d . Crosslinked products were o b s e r v e d i n b o t h t h e f i r s t amd s e c o n d d i m e n s i o n g e l s : Lane A, uncrosslinked control ; lane B, first dimension DSP crosslinked protein P . Products included a monomer o f protein P^ with an i n t e r n a l c r o s s l i n k r e s u l t i n g in lower a p p a r e n t m o l e c u l a r w e i g h t ( P ) , a d i m e r o f p r o t e i n P., ( P ) , a d i m e r w i t h a t l e a s t one i n t e r n a l c r o s s l i n k ( ) trimer of p r o t e i n P. ( p , ) . The r u n n i n g p o s i t i o n s o f t h e monomer o f protein P i n t h e f i r s t d i m e n s i o n and t h e monomer and spots derived from c r o s s l i n k e d p r o d u c t s i n t h e t w o - d i m e n s i o n a l g e l a r e i n d i c a t e d on t h e l e f t hand s i d e . ± 1  2  p  a  i 2  156  n  d  a  /57  dimensional P,  with  This  gels probably  an  estimated  product  trimer  of  was  the  insufficient intrachain 80,000  crosslinks  after  P  were  was  observed  F,  see  complexes  not  the  over case)  protein can  Randall, saturation  o f DSP  and  one  no  the  tendency  data  as  PhoE  t o the  i n the  crosslinker  Freedman,  gel  o f amino g r o u p s on  158  in  its  used  an  In o r d e r  to  more  higher protein  monomers  was  probably  stacking  gel).  have shown t h a t molar  not  and  did This  higher excess  P i n the  crosslinking 1979)  or  SDS-insoluble  c o m p l e x e s s i n c e we  protein.  0-  at  (and  of  for protein  the  c)  co-purified  as  used  Ji,  or  still P  to 5 0 0 - f o l d  1979;  the was  two-fold  At  (up  in non-specific  form  amounts o f t r i m e r  formation  of l a r g e c r o s s l i n k e d  c o n c e n t r a t i o n was result  reason,  gels.  o f o t h e r w o r k e r s who  of  which  P o f 60.  (the p r o t e i n  to  protein)  only l i m i t e d  the  a)  product  amino g r o u p s  the  the  l a r g e amount o f  most o f p r o t e i n  staining  daltons.  either  C r o s s l i n k e r was  dimensional  due  to  corresponding  longer entered  protein  1978;  the  : protein  still  crosslinker  form  concentrations  by  1975).  of c r o s s l i n k e r  with  observe  due  higher  free  crosslinker  presumably  i n the  confirms  ratio  added  below)  oligomers,  simply  Meadow,  on  concentrations  a  of  w h i c h has  insufficient  crosslinker  possibly  b)  145,000  protein  amounts i n t h e g e l t h a n  f o r w h i c h no  and  ( K o v a l and  out  of  l i p o p o l y s a c c h a r i d e (LPS)  approximate molar rule  at lower  crosslinking  to  to a t r i m e r of  weight  (as e v i d e n c e d  band  protein  antigen  present  crosslinker,  crosslinking with  molecular  PhoE p r o t e i n ,  dalton  observed  corresponded  (Palva  above and  presumably  3. C r o s s l i n k i n g The  of p u r i f i e d  purification  P. a e r u g i n o s a  procedure  solubilisation  in Triton  retained  the  p r o t e i n ' s pore-forming  bilayers  and p r o t e i n  its  n a t i v e or a c t i v e  protein (Figure  F clearly 15).  molecular were  F was  was  corresponding  to cleavage  of c r o s s l i n k s ) .  were  varied  a 5 0 0 - f o l d range  (see  Figure  16  demonstrated), varied  fron  were s h a r p  DSP  the first  oligomer  the  time  dimension spots  with  demonstrated  mg  with  protein membranes range  is  crosslinker  although  the p r o t e i n  on t h e t r e a t m e n t  molecular  products  weights  as d i s c u s s e d a b o v e . performed  159  F monomer  c o n c e n t r a t i o n s (0.2 - 1 times).  At  were r u n i n t h e  c l e a v e d and r u n i n t h e second  i n F i g u r e 15 was  (ie.  At no c r o s s l i n k e r c o n c e n t r a t i o n  depending  d a l t o n s appeared  products  concentrations  concentration  crosslinker  average  broad  gels  (4 ug t o 2 mg/  when t h e c r o s s l i n k e d then  oligomers  one d i m e n s i o n a l  of treatment  average  although  Crosslinker  in  daltons  an  These  analyses,  150-fold  at higher  with  ( n o t shown) and o u t e r  bands s e e n ,  protein  same t i m e ,  100,000  a  F  30 s e c t o 30 min.  p e r mg  broad  where and  band d i s a p p e a r e d mg  stained  protein  lipid  purified  t o d i m e r and t r i m e r  prior  purified  of  o f 70,000  observed.  observed  both  silver  1979)  i n black  a smear o f p r o t e i n  were  for  activity  Crosslinking  i n two d i m e n s i o n a l  in  (involving  t h e r e f o r e presumed t o be s t i l l  o f 120,000  of s t a i n i n g  F  Hancock e_t a_l. ,  r e v e a l e d a dimer product  weight  over  protein  - EDTA;  conformation.  In a d d i t i o n ,  o n l y seen  areas  X-100  for  p r o t e i n F_  of The  dimension, 70,000  and  experiment  at a concentration  at  Figure  15.  Two-dimensional c r o s s l i n k i n g P. a e r u g i n o s a p r o t e i n F .  analysis  of  purified  Purified protein F was treated as in Fig. 13* Crosslinked p r o d u c t s i n c l u d e d d i n e r s (F2) and t r i m e r s (F3) o f protein F as w e l l as h i g h e r molecular weight crosslinked p r o d u c t s o f p r o t e i n F (F ) . n  160  which  60$  of  position. a  higher  higher  PhoE  weight  to  including  the oligomers  In  these  experiments, slightly  the above  and  40$ a p p e a r e d a t  ( F ) and 20$  appeared  2  apparent  monomer  ( F 3 ) .  trimers  causing  apparent  molecular  E_^  coli  porins  a l l other  diagonal  which  bridges,  of high molecular  weight  The  may seen  be for  ( P a l v a and R a n d a l l ,  protein  F  crosslinking  38,000 d a l t o n monomer o f p r o t e i n F a p p e a r e d the  mercaptoethanol,  second  the  were n o t f u r t h e r c h a r a c t e r i s e d but  ( F i g u r e 13) and o t h e r  1978) .  from  that approximately  e q u i v a l e n t t o dimer  oligomers  oligomers  related  protein F disappeared  I t was e s t i m a t e d  molecular  as  the  broke  due  to  native  the p r o t e i n to u n f o l d  dimension,  weight  the  effect  intrachain  of  disulphide  f u r t h e r and r a i s i n g i t s  f r o m 35,000 t o 38,000 d a l t o n s  as d e s c r i b e d  2-  i n the  p r e v i o u s l y (Hancock and C a r e y ,  1979) .  4.  Crosslinking membranes Because  with  the  more  difficult  PhoE  and  P,  of  proteins  various  data  obtained  was  P.  concentrations  to i n t e r p r e t there  in  those  protein  obtained  possibility  that  the  apparent  association  o f p r o t e i n F monomers  The  use  allowed Figure F,  of lower  outer  membranes  molar r a t i o s  15 and F i g u r e  for  crosslinking  of crosslinker  17) t h a n  those  p r e s u m a b l y due t o t h e h i g h e r  161  used  artifactual  purification. experiments  t o be used with  were  proteins  a s s o c i a t i o n o f p r o t e i n F was due t o an during  F  for  oligomeric  induced  outer  of c r o s s l i n k e r  for purified  than  a  aeruginosa  purified  (compare protein  concentration of p r o t e i n F i n  Figure  16.  One-dimensional SDS-polyacrylamide gel electrophoresis of o u t e r membrane samples treated with v a r i o u s c o n c e n t r a t i o n s o f DSP f o r 2 min.  Crosslinked outer membranes were run on an 11$ polyacrylamide gel as described in Methods. Concentrations o f DSP i n d i c a t e d b e n e a t h t h e individual lanes a r e e x p r e s s e d as jug/mg p r o t e i n . C r o s s l i n k i n g was visualised in the f i r s t d i m e n s i o n as a d e c r e a s e i n s t a i n i n g i n t e n s i t y o f protein bands and a c o n c o m i t a n t i n c r e a s e i n amount of high m o l e c u l a r weight m a t e r i a l unable to e n t e r the s e p a r a t i n g g e l .  162  outer  membrane c r o s s l i n k i n g At  concentrations  causing (see  16),  dimensional 17).  of c r o s s l i n k e r  minimal a l t e r a t i o n  Figure  the only  analyses  Using  another  20% o f  (40 ug DSP/mg  protein)  o f o u t e r membrane p r o t e i n  patterns  crosslinked  cleavable  (dimethyl  t h e monomer  analysis  (data  was l e s s t h a n form  and  a  (Figure  18),  associations order  LPS,  of higher  with  other  to confirm  for  method  of  staining  Tsai  seen  gels  to other  of  F could  the c r o s s l i n k e d  the  here  70,000  weight  products  crosslinked  outer  weights  be  to  crosslinked for  crosslinked  products of gels  stained  block  were  the p r o t e i n  appeared  By  stain  spots not  protein  than  I t thus  to  membranes was  which appeared  Several  also  be  or to p r o t e i n s .  163  weight  periodate/silver  15 and 17.  LPS m o l e c u l e s  t i m e used  (LPS).  molecular  LPS c o u l d  concentration  or l i p o p o l y s a c c h a r i d e  silver-stained  seen i n F i g u r e s  species  aligning  on  i n the  due t o  (1982),  proteins.  a t lower apparent  F oligomers certain  19) by  dimer  and some a p p a r e n t l y  protein  and F r a s c h  o f a l l major  previously observed  LPS ( F i g u r e  high  molecular  proteins  that  products  molecular  some due t o m u l t i m e r s  to  F was c r o s s l i n k e d t o  a two-dimensional g e l of c r o s s l i n k e d  stained  either  17), p r o t e i n  3,3'-  up t o an  converted  the treatment  of estimated  streak  be  crosslinked  N.B.  i n Figure  dimers  could  i n two (Figure  concentrations,  At a r e l a t i v e l y  protein;  that  visible  daltons  In  n o t shown).  (200 ug/mg  crosslinker  at higher  the appearance o f other  DSP  observed daltons  without  of  product  was a d i m e r o f 70,000  dithiobis-propionimidate) estimated  experiments.  by  that DSP,  vertically f o r protein  Figure  17.  Two-dimensional c r o s s l i n k i n g o f u s i n g 40 jjg DSP/mg p r o t e i n .  outer  membranes  Outer membranes were treated with 40 jug DSP/mg p r o t e i n f o r 30 s e c and r u n on an 11$ p o l y a c r y l a m i d e g e l . the lane was c u t o u t , soaked i n r e d u c t i o n mix c o n t a i n i n g 2m e r c a p t o e t h a n o l t o b r e a k c r o s s l i n k s and t h e n t h e l a n e l a i d on top o f a n o t h e r 11$ a c r y l a m i d e g e l . The l o w e r e d c r o s s l i n k e r concentration p r o d u c e d o n l y one p r o d u c t , a dimer o f F ( F 2 ) . An unknown, high molecular weight p r o t e i n (X) was 2-MEmodified but not c r o s s l i n k e d (see a l s o F i g . 2 0 ) .  164  F2FL  Figure  18.  F|  Two-dimensional c r o s s l i n k i n g of u s i n g 200 DSP/mg p r o t e i n .  outer  membranes  Outer membranes were crosslinked with 200 ,ug DSP/mg p r o t e i n f o r 30 s e c o n d s , run on a 12.5$ polyaerylamide g e l , and the l a n e c u t out , soaked i n r e d u c t i o n mix containing 2-mercaptoethanol to b r e a k DSP c r o s s l i n k s and t h e n the lane l a i d on top o f a n o t h e r 12.5$ acrylamide running g e l , and run i n the second d i m e n s i o n as d e s c r i b e d i n M e t h o d s . Crosslinked products r e v e a l e d and l a b e l l e d on the F i g u r e i n c l u d e d a d i m e r of p r o t e i n F ( F 2 ) , a p r o p o s e d c r o s s l i n k between p r o t e i n F and a p a r t i c u l a r s p e c i e s o f LPS (FL -see a l s o F i g . 5D) as w e l l as higher molecular weight m a t e r i a l unable to e n t e r the first dimension g e l . The monomer o f F (F1) was 2-mercaptoethanolmodifiable, accounting for i t s s l i g h t l y off-diagonal p o s i t i o n .  165  and  LPS we were a b l e  spots  seen  in  crosslinking. interaction the for the  Figure  At  18  least  (labelled  were  one  spot  spots  noted  by p r o t e i n F:LPS c r o s s l i n k s . 0-antigen  Two  protein  I t should  determined,  Although  i t  P_j_  is  F  LPS:protein  way,  b u t none be  F  F of  accounted  be n o t e d  that  aeruginosa  both  contain  1975; K r o p i n s k i e_t a^l. , 1 9 8 2 ) .  t o r e v e a l any  Only  X) was o b s e r v e d  below t h e d i a g o n a l .  to  a n a l y s i s of u n c r o s s l i n k e d outer  (Figure 20).  (labelled  LPS:protein  above c o u l d  was done as a c o n t r o l  multimers  to  due  LPS o f  and Meadow,  dimensional  preparations linked  and r o u g h c o r e  (Koval  due  F L ) was s e e n i n t h i s  p r o t e i n F multimer  amino g r o u p s  not  t o examine w h e t h e r any o f t h e p r o t e i n  one h i g h  likely  disulphide-  molecular  which c o n s i s t e n t l y  the i d e n t i t y  of this  t h a t i t forms  membrane  weight  appeared  p r o t e i n was  an  interchain  disulphide-linked  d i m e r w h i c h was c l e a v e d  by 2 - m e r c a p t o e t h a n o l  between t h e f i r s t  and s e c o n d  No o t h e r d i s u l p h i d e -  linked were  or S D S - r e s i s t a n t  dimensions.  multimers  of outer  membrane  proteins  detected. Whole  confirm  P^  that  aeruginosa  cells  were c r o s s l i n k e d  the c r o s s l i n k e d products  e x p e r i m e n t s were a c t u a l l y rearrangement  occurring during  The  results  more e x t e n s i v e  molecules 70,000 another,  in  dalton  whole  i n outer  further membrane  n a t i v e s t r u c t u r e s and n o t t h e r e s u l t  of  showed  seen  to  cells  than  outer  membrane  crosslinking i n outer  isolation.  of c e l l  membranes  surface but  d i m e r o f p r o t e i n F and 95,000 d a l t o n d i m e r  glucose-induced  protein,  XVII).  166  D1, were i d e n t i f i e d  the of  (Table  F  Figure  19.  I 2  I  FL  T~  L  Two-dimensional membrane samples  I  L  SDS-PAGE o f crosslinked stained for carbohydrate.  outer  O u t e r membranes were t r e a t e d as i n F i g . 17 e x c e p t t h a t the gel was stained for LPS (see Methods). Crosslinked products w h i c h a p p e a r e d t o be due t o L P S r L P S i n t e r a c t i o n (L) as well as one p r o p o s e d L P S : p r o t e i n F i n t e r a c t i o n ( F L ) were observed. The s p o t l a b e l l e d FL was i d e n t i f i e d by i t s v e r t i c a l alignment w i t h a p r o t e i n F s p o t which d i d not s t a i n by this p r o c e d u r e but i s c l e a r l y seen i n F i g . 18, s t a i n e d f o r p r o t e i n . The position of the dimer o f p r o t e i n F ( F 2 ) , w h i c h was not s t a i n e d by s i l v e r / p e r i o d a t e , i s m a r k e d .  167  Figure  20.  T w o - d i m e n s i o n a l SDS-PAGE o f u n c r o s s l i n k e d o u t e r membrane s a m p l e s .  control  Uncrosslinked c o n t r o l o u t e r membranes were r u n on an 11$ p o l y a c r y l a m i d e g e l as i n F i g . 17. No v i s i b l e o f f - d i a g o n a l s p o t s o t h e r than p r o t e i n X ( p o s s i b l y a d i s u l p h i d e - b o n d e d dimer c l e a v e d by 2 - m e r c a p t o e t h a n o l t r e a t m e n t ) were o b s e r v e d .  168  To  confirm  that  result  of chemical  native  conformation  the observed  bridging  a control  prior  crosslinking.  protein  F  Carey, in  It  after  was  outer  SDS-heat  control  same  protein all with  crosslinking  were o b s e r v e d  outer  membrane  outer  (data  proteins  membrane  (Figure  monomers  n o t shown). (Nicas  t o be a p o r i n ,  conditions.  (data  between  No  (Hancock  and  be c r o s s l i n k e d ,  17) and whole  experiments.  169  cell  SDS that SDSand  P.  Hancock,  was s u b j e c t e d  multimers  free  observed  Another  n o t shown), d e m o n s t r a t i n g could  with  on  p r o d u c t s were  H1  i n their  observed  treatment  protein  membrane p r o t e i n ,  1980), w h i c h was n o t s u s p e c t e d the  previously  exclusively  No c r o s s l i n k e d  the SDS-pretreated  aeruginosa  subunits  F sample was p r e t r e a t e d  almost  gels  1979).  between p r o x i m a l  protein  formed  polyacrylamide  F m u l t i m e r s were t h e  and n o t due t o random c o n t a c t  monomers, to  protein  of  to this  that not  i n agreement crosslinking  DISCUSSION  The When  results  analysing  of t h i s the  crosslinked  experiment,  i t was  or  e t c . based  trimers,  second  dimension  frequency  of  instance,  one  crosslinking requiring appear  cannot about  appearance would  (intensity  expect  consistent  of  and  of  the  absence  of  2-mercaptoethanol  of p r o t e i n  crosslinked  F would  be  mercaptoethanol-unmodified) PhoE,  however,  difficult on  by  dimer  for with  protein  one  could  F,  be  multimers would  i f the  native  of  intrachain  170  involved  were  in  the  dalton  35,000 d a l t o n proteins  P  spots  formed  I t was  concluded,  dimer  weight  70,000  products,  was  that  (90,000  crosslink  in  molecular  measured  For  of c r o s s l i n k e d likely  species  information  S i n c e the  o f two  one  products  the  and For  the m o l e c u l a r  treatment,  subunits.  i n the  dimers  specific  forms.  composed  P were most  at l e a s t  which  crosslinks.  the m o l e c u l a r w e i g h t  seen  of  p r o d u c t s were  interpretation  intrachain  even  of c r o s s l i n k e d  trimer  weights  dimer  dimers  only  than  therefore  For p r o t e i n  appearance  w i t h dimer  requiring  exact f r e q u e n c y o f each  i n the a b s e n c e  each  staining).  than t r i m e r s ,  the number o f amino g r o u p s  frequency  in  of i n t e r e s t  more f r e q u e n t l y  molecules,  The  reactions.  of  multimers  to appear  crosslinking  predicted  crosslinking and  w i t h monomers o f the p r o t e i n  were a t r i m e r .  be  produced  XVII.  on m o l e c u l a r w e i g h t s , a l i g n m e n t  much more f r e q u e n t l y  structure  products  i n Table  n e c e s s a r y t o d e s i g n a t e o l i g o m e r s as  molecule  two  s t u d y a r e summarised  (2and made  based spots  dalton),  (80,000)  and  Table  XVII.  Summary proteins  Protein  o f the c r o s s l i n k e d multimers observed i n t h i s study.  Assignment  and M"  r  — Monomer  Dimer  Trimer  70,000  120,000  of  porin  N a t i v e SDSresistant Other oligomer  P.aeruginosa protein F  38,000  35,000 (minus  P.aeruginosa p r o t e i n D1  47,000  95,000  145,000  29,000 (unheated)  No  P.aeruginosa protein P  48,000  90,000  145,000  44,000 (internal crosslink)  Yes  No 2-ME)  80 ,000 E-  coli protein  38,000  73,000  PhoE  100,000  34,000 (internal crosslink)  Yes  ( r e l a t i v e m o l e c u l a r w e i g h t ) v a l u e s were determined comparison with the m o l e c u l a r weight standards stated Methods. M  r  171  by in  trimer  (145,000)  dalton  spot  was  spot  a trimer.  with  intrachain  73,000  assumed  in  with  an  two  crosslinks,  three  molecule, lysine  dimensional  and  different  of  respectively) consequently explains  the  these  relative  to  polypeptides  or  the  protein  P,  (1.1nm  and  due  positioning In  t o the or  below a p r o t e i n  or LPS  spot  products  commonly  Proteins easily  i n the  crosslinked analysed  P  visualised  by  not  the seen  of a  dimer of  protein  due  products  by  suggested  0.6nm  to  than  those  of p r o t e i n  eliminated  and  by  from  a porin  directly  i t had  i n the  spot above  co-migrated Although  gels  the  were  not  crosslinked  interactions.  multimers F.  chains  of p o r i n p r o t e i n s  involved several protein-LPS PhoE c r o s s l i n k e d  and  heterologous  complexity,  and  diameter,  side  cases,  which  their  XVII),  dimension.  their  172  in  (Table  were  seen  two  as  dimension  first  the  different  all  with  of  P o s s i b l y the  were i d e n t i f i e d  second  stringently  dimers  each  of l y s i n e  LPS  i n the  heterologous  on  crosslinking  to  Such p r o d u c t s  to c r o s s l i n k i n g )  was  i n each  weights  diagonal  (due  to  presence  groups  1986).  molecular  products  below  dalton  necessitate possession  amino  observations.  consideration.  The  would  e_t a^,  crosslinked other  100,000  a spot  g r o u p s were found  sizes  and  the  have been o b s c u r e d  analyses.  PhoE and  channel  73,000  14 t r i n i t r o b e n z e n e s u l p h o n a t e - a c c e s s i b l e  above p r o t e i n s (Hancock conformations  the  PhoE,  corresponding  however s u c h  accessible  epsilon-amino  a spot i t may  crosslink  at l e a s t  protein  a d i m e r and  I f t h e r e was  intrachain  least  For  t o be  d a l t o n dimer s p o t ,  even  at  products.  Since  were the  more  ease  of  crosslinking  reflects  the  availability  reactive  amino g r o u p s w i t h i n  may  that  be  proteins these may  P  and  reflect  al • ,  1982).  filters  in  selectivity  of  of  f o r the  the  pore  E.  coli,  the  P  present  different protein  channels  P  toward  The  did  the  easily  porin  P of  lipid  Given P_^  the the  appear  portion  interior might  the  of  also  anionic but,  apparent  be  and  PhoE  explained  larger  the  at size  of  the  if  protein  channel.  explain  PhoE p r o t e i n  be  developmentally  the  dimeric  to  above-mentioned  are  of  the  channels.  the  aeruginosa  proteins  e_t  channels)  bilayers  change  of  uptake  that  anion-selective  not  yielded  while  both  Korteland 1984)  it  weight,  phosphate  i n pore s i z e c o u l d  structure  crosslinks to  in  mouths o f  i n black  10,000 d a l t o n  more  the  as  two  difference  on  i n molecular  (Benz et; a_l. ,  e_t a_l. , 1984).  these  channel  internal readily  the  extra  accessible  partly neutralised  between p r o t e i n s  some o f was  function  PhoE p r o t e i n ,  The  each s u b u n i t ) ,  (Hancock e_t al^. , 1982;  these groups  perhaps  related.  more  crosslinker-  PhoE have amino g r o u p s w h i c h  (Darveau  similarities  on  functions  been shown  their of  two  s i m i l a r c r o s s l i n k i n g patterns  (presumably  P and  Acetylation  least  (one  their differences  membrane  I t has  proteins  important  The  despite  outer  selectivity of  PhoE.  t h e i r analogous  the  nm  s u c h amino g r o u p s a r e  proteins,  through  1.2  of  This  fact  that  associations  with  crosslinked  more  trimers. crosslinked  visualise  than  primarily  due  those to  the  bands of  of  protein  proteins  fact that  173  P  protein  F  and  were  harder  to  PhoE.  This  was  result  in  F did  not  distinct  bands  dimension over  a  500-fold  observed Chromatium  vinosum  cleavage  demonstrated formed  about  spots  the  corresponded  to  dimension and  their  (as  products  relative analysis,  by  or LPS  seen  such  1980;  Palva  weights  using  m o b i l i t y of  of  174  gels  f o r the  also using stain  dimer  to  spots the  limiting  Using of  the  as  with were  second  oligomers  1978), we  spots  than  align  in  i t .  regions  rather  the  were seen  35$_  reveal  weight  weights  either  either  vertically  Randall,  weights  the  using  molecular  o f the  products  observed  Although  the m u l t i m e r s .  molecular  dimension  d i m e r was  that smaller spots centre  the  Hurlbert,  second  crosslinked porin and  of  These  d i d not  spots.  be  trimer.  and  i n these  can  crosslinking  regions  or s i l v e r  f o r other  outer  f o r the  of m o l e c u l a r  t a k i n g the  the  s i n c e the  s i n c e they  range  molecular  or  F  homologous c r o s s l i n k e d p r o d u c t s  Morse,  crosslinker, 17)  a  whole  trimer crosslinked  covered  blue  varied  patterns  of p r o t e i n  130,000 d a l t o n s  products  over  and  of c r o s s l i n k e r  polypeptide  spread  either  60,000 t o 80,000 d a l t o n s  Coomassie  heterologous  dimer  first  Similar d i f f i c u l t i e s  products  artifacts  Furthermore,  using  of c r o s s l i n k i n g  which  to a b o u t  concentrations  methionine,  Leith  the  spots  not  the  c o n c e n t r a t i o n was  e l e c t r o p h o r e s i s i n the  110,000 t o  were  other  p r o t e i n F.  of the  that  corresponding  ( F i g u r e 16)  in  42,000 d a l t o n p r o t e i n (Lane and  and  smeared  polypeptides  crosslinker  examination  Analysis  after  low  range  purified  from  1980).  crosslinked  even when t h e  membranes or  and  of  estimated  amounts (e.g. the  either  of  Figure apparent  method  multimers  of were  approximately fold  m u l t i p l e s of  Although protein the  the P,  dimer  that  lambda t o be  dimeric  ( P a l v a and since  on  (since  trimer 14)  no  spaced Thus,  at  amino  probability  of seeing  accessible,  properly  oriented  smearing  the  and  could  have  covalent variety  a  v a r i e t y of  interaction  of  o f bands i n t h i s  1982),  b)  intrinsic  molecules,  since  demonstrated  (Benz  will  and  amino  groups which are  of  coli  DSP  simply if  are a v a i l a b l e  p r o t e i n F with of  the  Hancock, 1 986 ),  gel  and  175  as  1981;  trimer  the  the  three the  number  first  of The  dimension  including:  a)  LPS  forms  which  (Kropinski of  Nicas  has and  c) m o n o f u n c t i o n a l but  e_t  are  nona  al.. ,  protein  heterogeneity  d)  a single  increases.  heterogeneity  a c c e s s i b l e t o DSP  crosslinking,  only  of  bands i n the  explanations  area  properly  Similarly,  amino g r o u p s  trimer  only  understood  c r o s s l i n k e d to a two  is  demonstrated  distance  up  for fact  results  occur  increase  functional  W o o d r u f f e_t al^. ,  for  nm  seen the  oligomer).  physical  1983 ;  oriented  i n the  a trimer  dimer  1.2  would use  accessible  of  only  n e v e r be  product groups  will  be  for  despite  species  can  (and  than  i f e a c h monomer has  p r o t e i n can  dimeric  porin)  This  crosslinking  the  that,  trimer  1979).  subunits.  less  c r o s s l i n k i n g with  with  trimer) weight.  for protein F  noted  (maltose  ( f o r the  molecular  considerably be  receptor  amino g r o u p s  a  species  was  i t should  Westermann,  such group,  three-  monomeric p r o t e i n F  aggregates,  adjacent  d i m e r ) and  a trimer,  chemical  oriented  the  Figure  species,  the  ( f o r the  amount o f  see  recognised in  two-  F  been  Hancock,  reaction  of  improperly  p r o t e i n F i s known t o  be  able  to  occupy  positions of  the  proteins  studied  demonstrated  dimers, i n  Leith likely  structure  proving  this,  i s  L.  some  temperature  and  size,  form  I feel  results,  with  and  products  thesis,  which  This  small  additional  of protein  F.  level  evidence  at other 1979;  t h e most  while  not  proposal. form  I f  may  far  be  despite  physicochemical U.B.C.,  treatment  immunoblots  176  this  selectivity  after  with  that  of have  Westermann,  so  even  I  oligomers,  the trimeric  D.  any  trimer,  examined  (Ph.  Western  provided  the  that  that  By a n a l o g y and  consistent  oligomeric  an o l i g o m e r i c  weight  concentrations  are  1980) ,  a l l porins  t o p r o t e i n F.  treatment  proteins  Palva,  p r o t e i n F,  using  a specific  (Palva  Mutharia  that  to  be c o n c l u d e d  states.  a trimer  i n channel  demonstrated  antibodies  native  1980;  of  properties.  visualised  these  i ti s interesting  characteristic  contained  that  forms  are at least  i s so,  differences  definitely  organisms  and Morse,  according  2-mercaptoethanol  here  i n their  other  gels  molecular  1979).  i tcannot  porins  this  and  Although  least  s i x different  o n SDS p o l y a c r y l a m i d e  and C a r e y ,  clearly  of  least  solubilisation  (Hancock  SDS  at  could with  with only  SDS, be  monoclonal  of oligomers f o r the  1985)  after  existence  LITERATURE  CITED  Abraham, E.P. 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