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Phosphate transport across the outer membrane of Pseudomonas aeruginosa Poole, Raymond Keith 1986

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PHOSPHATE TRANSPORT ACROSS THE OUTER MEMBRANE OF PSEUDOMONAS AERUGINOSA by RAYMOND KEITH POOLE B.Sc,  The U n i v e r s i t y of B r i t i s h Columbia, 1980  A THESIS SUBMITTED IN PARTIAL FULFULLMENT OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in  THE  FACULTY OF GRADUATE STUDIES  (Department of M i c r o b i o l o g y )  We accept to  THE  this  t h e s i s as conforming  the r e q u i r e d s t a n d a r d  UNIVERSITY OF BRITISH COLUMBIA ^  (c)  April  1986  Raymond K e i t h Poole, 1986  In  presenting  degree  at  this  the  freely available copying  of  department publication  of  in  partial  fulfilment  University  of  British  Columbia,  for  this or  thesis  reference  thesis by  this  for  his thesis  and study. scholarly  or for  her  Department The University of British 1956 Main Mall Vancouver, Canada V6T 1Y3  DE-6(3/81)  Columbia  I further  purposes  gain  the  requirements  I agree  that  agree  may  representatives.  financial  permission.  of  be  It  shall not  that  the  Library  permission  granted  is  by  understood be  for  allowed  an  advanced  shall for  the that  without  make  it  extensive  head  of  my  copying  or  my  written  ABSTRACT  When wild-type  c e l l s of Pseudomonas aeruginosa  grown i n a p h o s p h a t e - l i m i t i n g they were derepressed  medium  (0.2 mM  f o r the production  membrane p r o t e i n , designated  p r o t e i n P.  were  orthophosphate)  of an outer T h i s p r o t e i n was  p u r i f i e d to homogeneity and demonstrated to form channels i n planar data, (due  lipid  b i l a y e r membranes.  In agreement with  previous  the channels formed by p r o t e i n P were a n i o n - s p e c i f i c to the presence of a b i n d i n g s i t e f o r anions i n the  channel) and e x h i b i t e d a marked s e l e c t i v i t y f o r phosphate 2(HP0  4  ) over other anions (e.g c h l o r i d e ) .  were not a l t e r e d i n p r o t e i n P p r e p a r a t i o n s  These p r o p e r t i e s purified  free of  lipopolysaccharide. P r o t e i n P was c o i n d u c i b l e with the enzymes a l k a l i n e phosphatase and phospholipase C, and with a p e r i p l a s m i c p r o t e i n of 34,000 molecular aeruginosa,  weight.  Mutants of P.  c o n s t i t u t i v e or non-inducible  c o n s t i t u e n t s , were i s o l a t e d .  f o r these  T h i s suggested that the genes  encoding these products were part of a phosphate  regulon.  A l k a l i n e phosphatase and phospholipase C were demonstrated to be s e c r e t e d  i n t o the e x t e r n a l medium upon i n d u c t i o n ,  although t h i s e x t r a c e l l u l a r r e l e a s e was s p e c i f i c and d i d not i n v o l v e an increase  i n outer membrane p e r m e a b i l i t y .  The 34K  p e r i p l a s m i c p r o t e i n was p u r i f i e d and demonstrated to bind phosphate i n v i t r o  (Kd=0.34 uM).  Specificity  studies  revealed that inorganic phosphate polymers (up to P15) and ii  arsenate could binding  i n h i b i t the binding  p r o t e i n , although organic  6-phosphate) could not. binding  p r o t e i n and  The  of orthophosphate to phosphates (e.g.  ability  the  glucose-  of the phosphate-  p r o t e i n P to a s s o c i a t e was  demonstrated  i n v i t r o , with i m p l i c a t i o n s concerning the means by which phosphate crosses Two  the outer membrane.  major inorganic  i d e n t i f e d , of low  phosphate t r a n s p o r t  systems were  (Km=l9.3 uM phosphate) and h i g h - a f f i n i t y  (Km=0.39 uM phosphate), r e s p e c t i v e l y .  Mutants d e f i c i e n t in  the phosphate-binding p r o t e i n were i s o l a t e d and  shown to  lack the h i g h - a f f i n i t y phosphate uptake system, the  r o l e of the binding  transport  protein  in P. aeruginosa.  confirming  in h i g h - a f f i n i t y phosphate  In a d d i t i o n , a r o l e f o r p r o t e i n  P in h i g h - a f f i n i t y phosphate t r a n s p o r t  was  confirmed by  the  i s o l a t i o n of a Tn501 i n s e r t i o n mutant l a c k i n g p o r i n  protein  P.  for  T h i s mutant e x h i b i t e d a t e n - f o l d increase  h i g h - a f f i n i t y phosphate t r a n s p o r t . proteins  i n the  growth defect  r e s p e c t i v e mutants was  in a phosphate-deficient  P r o t e i n P, oligomer  l i k e most p o r i n s , was  (trimer)  in i t s n a t i v e  d i s s o c i a t i n g to non-functional temperatures. t r i m e r s was  l o s s of  these  c o r r e l a t e d with a medium. i s o l a t e d as  an  (functional) state, monomers at  high  A p o l y c l o n a l antiserum s p e c i f i c f o r p r o t e i n P  r a i s e d and  shown to c r o s s - r e a c t  phosphate-starvation-inducible the  The  i n Km  outer membrane p r o t e i n s  f a m i l i e s Pseudomonadaceae and  c r o s s - r e a c t i v i t y was  with other  Enterobactereaceae.  observed only with the  native,  of This  oligomeric  forms of these p r o t e i n s .  No c r o s s - r e a c t i v i t y was  seen with the c o n s t i t u t i v e p o r i n s produced by these s t r a i n s , i n d i c a t i n g that the c r o s s - r e a c t i v i t y of phosphatel i m i t a t i o n - i n d u c i b l e oligomeric  outer  membrane p r o t e i n s was  not due to any homologies r e l a t i n g to p o r i n s t r u c t u r e i n general.  Using a p o l y c l o n a l antiserum s p e c i f i c  for protein  P monomers, no r e a c t i v i t y was observed with e i t h e r the oligomeric  or monomeric forms of any of the phosphate-  l i m i t a t i o n - i n d u c i b l e outer membrane p r o t e i n s p r o t e i n P monomers).  (except f o r  These data suggested that the common  a n t i g e n i c determinants present  i n these p r o t e i n s were  conserved i n the n a t i v e f u n c t i o n a l p r o t e i n s  only.  Examination of some of the p h y s i c a l p r o p e r t i e s of the phosphate-starvation-inducible  outer membrane p r o t e i n s (e.g.  molecular weight, peptidoglycan s o l u b i l i t y ) revealed  that these p r o t e i n s could be grouped  i n t o two c l a s s e s , represented and  a s s o c i a t i o n , detergent  by p r o t e i n P of P. aeruginosa  p r o t e i n PhoE of E s c h e r i c h i a c o l i .  resembling p r o t e i n P were i d e n t i f i e d  Those p r o t e i n s i n members of the  f l u o r e s c e n t Pseudomonads, i n c l u d i n g P. p u t i d a , P. fluorescens,  P. aureofaciens  purified proteins channels i n planar  and P. c h l o r o r a p h i s .  formed s m a l l ,  The  anion/phosphate-selective  l i p i d b i l a y e r s which were q u i t e s i m i l a r  to p r o t e i n P channels.  iv  TABLE OF CONTENTS  PAGE Abstract  i i  Table of Contents  v  L i s t of F i g u r e s  .x  L i s t of Tables  xiii  L i s t of A b b r e v i a t i o n s  xv  Acknowledgements  xvi  Introduction 1.  2.  1  The Gram-negative c e l l envelope  • . .1  a.  The cytoplasmic membrane  1  b.  The p e p t i d o g l y c a n  2  c.  The outer membrane  3  The r o l e of the outer membrane i n t r a n s p o r t . . . 6 a.  The LamB p r o t e i n  8  b.  The PhoE p r o t e i n  11  c.  I r o n - r e g u l a t e d outer membrane p r o t e i n s . . .13  d.  Others  14  3.  B a c t e r i a l phosphate t r a n s p o r t - with reference to E. c o l i  4.  The pho regulon of E. c o l i  Methods  specific  15 18 22  1.  Media and growth c o n d i t i o n s  22  2.  Bacterial strains  23  v  3.  C e l l f r a c t i o n a t i o n and sodium dodecyl polyacrylamide g e l e l e c t r o p h o r e s i s  4.  P u r i f i c a t i o n of p r o t e i n P  28  5.  A c e t y l a t i o n of p r o t e i n P  29  6.  Immunological methods .  30  7.  Preparation vesicles  of p r o t e i n P-phosphatidyl  8.  Preparation  of p r o t e i n a f f i n i t y columns  9.  10.  sulphate-  choline  23  30 31  a.  P r o t e i n F-Sepharose  31  b.  Protein P-Affigel-10  31  c.  Phosphate-binding protein-Sepharose  Preparation  of p r o t e i n P - s p e c i f i c a n t i s e r a  ... . .  .32 .32  a.  Trimer-specific  32  b.  Monomer-specific  34  c.  Antiserum to p r o t e i n P in phosphatidyl vesicles  I s o l a t i o n of a p r o t e i n P - d e f i c i e n t mutant  choline 35 . . .  .35  a.  Tn501 i n s e r t i o n mutagenesis  35  b.  S e l e c t i o n of a p r o t e i n P - d e f i c i e n t mutant using a p r o t e i n P t r i m e r - s p e c i f i c antiserum  36  11. Phosphate t r a n s p o r t  assays  37  12. Enzyme assays  38  13. N i t r o c e f i n p e r m e a b i l i t y 14. Osmotic shock and binding p r o t e i n  assay  p u r i f i c a t i o n of the  39 phosphate-  40  15. F i l t e r assay of phosphate binding  41  16. E q u i l i b r i u m d i a l y s i s  42  17.  I s o l a t i o n of mutants l a c k i n g the phosphate-binding protein 42  vi  18. C o n s t r u c t i o n of a r a b b i t a n t i - p r o t e i n P immunoadsorbant column  44  19. E l e c t r o p h o r e t i c e l u t i o n of p r o t e i n P from SDSpolyacrylamide g e l s  44  20.  P u r i f i c a t i o n of phosphate-starvation-inducible outer membrane p r o t e i n s of the f l u o r e s c e n t Pseudomonads  .45  21. Black l i p i d b i l a y e r experiments  47  22. M o d i f i e d ELISA procedure f o r demonstrating an a s s o c i a t i o n between p r o t e i n P and the phosphatebinding p r o t e i n 48 a.  Preparation  of p r o t e i n P  48  b.  Modified  ELISA procedure  49  23. A f f i n i t y chromatography method f o r determining an a s s o c i a t i o n between p r o t e i n P and the phosphatebinding p r o t e i n 50  24.  a.  Phosphate-binding protein-Sepharose 4B a f f i n i t y column  b.  Protein P-Affigel-10  .50  a f f i n i t y column . . . .50  I s o l a t i o n of r e g u l a t o r y mutants of a l k a l i n e phosphatase and phospholipase C  51  25. Other assays C h a p t e r One  52  Outer membrane p r o t e i n P: involvement i n h i g h - a f f i n i t y phosphate t r a n s p o r t i n Pseudomonas aeruginosa  S3  1.  Induction  2.  Co-regulation with a l k a l i n e phosphatase, phospholipase C and a 34K p e r i p l a s m i c p r o t e i n  3.  Outer membrane p e r m e a b i l i t y  4.  LPS-free p r o t e i n P forms channels i n planar b i l a y e r membranes  5.  I s o l a t i o n of a p r o t e i n P - d e f i c i e n t mutant . . . .73 a.  of p r o t e i n P by phosphate l i m i t a t i o n  Preparation antiserum  .53 . .60 63  lipid  69  of a p r o t e i n P t r i m e r - s p e c i f i c 73 vi i  b.  Tn501 mutagenesis of P. aeruginosa  75  c.  I s o l a t i o n of a Tn501-induced P - d e f i c i e n t mutant  80  protein  6.  Phosphate t r a n s p o r t  82  7.  Growth i n low phosphate medium  83  8.  Summary  85  Chapter Two  Role of a per.iplasmic phosphate-binding p r o t e i n i n phosphate t r a n s p o r t i n Pseudomonas aeruginosa  1.  P u r i f i c a t i o n and p r o p e r t i e s phosphate-binding p r o t e i n  2.  I s o l a t i o n of mutants l a c k i n g the phosphate-binding protein 95  3.  Phosphate t r a n s p o r t  4.  K i n e t i c s of phosphate t r a n s p o r t  5.  Growth i n phosphate-deficient  6.  P h y s i c a l a s s o c i a t i o n between outer membrane p r o t e i n P and the p e r i p l a s m i c phosphate-binding protein 103  7.  Summary  Chapter Three  1.  2.  of the p e r i p l a s m i c  89 89  97 . . . . . . . .  medium  100  . . .- . . 103  109  Immunological c r o s s - r e a c t i v i t y of phosphate-starvation-inducible outer membrane p r o t e i n s of the f a m i l i e s E n t e r o b a c t e r i a c e a e and Pseudomonadaceae 111  Phosphate s t a r v a t i o n - i n d u c t i o n of membrane p r o t e i n s of the Pseudomonadaceae and the Enterobacter iaceae . . Immunological c r o s s - r e a c t i v i t y of phosphates t a r v a t i o n - i n d u c i b l e outer membrane p r o t e i n s  111 . 118  a.  C r o s s - r e a c t i v i t y of p r o t e i n oligomers i n phosphate-limited c e l l envelopes  118  b.  I d e n t i f i c a t i o n of the c r o s s - r e a c t i v e proteins  124  viii  c. 3.  C r o s s - r e a c t i v i t y of induced monomers  phosphate-starvation-  Summary  Chapter Four  129 130  C h a r a c t e r i z a t i o n of p r o t e i n P - l i k e p o r i n s the f l u o r e s c e n t Pseudomonadaceae  from 132  1.  P u r i f i c a t i o n of the p h o s p h a t e - s t a r v a t i o n - i n d u c i b l e outer membrane p r o t e i n s of the f l u o r e s c e n t Pseudomonads 132  2.  S i n g l e channel experiments  3.  Ion-selectivity  4.  Phosphate i n h i b i t i o n of macroscopic conductance 145  5.  Summary  135 .142  152  Discussion  154  .1. . A phosphate regulon  i n Pseudomonas aeruginosa  2.  P r o p e r t i e s of outer  membrane p r o t e i n P  3.  The outer membrane of Pseudomonas aeruginosa as a p e r m e a b i l i t y b a r r i e r to phosphate under l i m i t i n g conditions 162  4.  P r o t e i n P and PhoE as members of two d i s t i n c t c l a s s e s of phosphate-regulated p o r i n s  167  5.  Conserved a n t i g e n i c determinants i n phosphates t a r v a t i o n - i n d u c i b l e outer membrane (porin) proteins  173  Literature  Cited  . 154  . . . . 158  176  ix  LIST OF FIGURES  Growth of P. aeruginosa i n a p h o s p h a t e - d e f i c i e n t medium Growth y i e l d of P. aeruginosa as a f u n c t i o n of the c o n c e n t r a t i o n of phosphate i n a d e f i n e d minimal medium SDS-polyacrylamide g e l electrophoretogram of p u r i f i e d p r o t e i n P and of outer membanes and shock f l u i d s of p h o s p h a t e - d e f i c i e n t c e l l s of P. aeruginosa Induction by phosphate l i m i t a t i o n and l o c a l i z a t i o n of a l k a l i n e phosphatase and phospholipase C of P. aeruginosa Hi 03 SDS-polyacrylamide g e l electrophoretogram of whole c e l l p r o t e i n e x t r a c t s and c e l l envelope and s o l u b l e (non-membrane) f r a c t i o n s of a l k a l i n e phosphatase r e g u l a t o r y mutants Outer membrane p e r m e a b i l i t y d u r i n g growth on p h o s p h a t e - d e f i c i e n t medium SDS-polyacrylamide g e l electrophoretogram of LPS a s s o c i a t e d with p r o t e i n P Immunoblots of e l e c t r o p h o r e t i c a l l y separated P. aeruginosa H103 c e l l envelopes and p u r i f i e d p r o t e i n P, and whole c e l l s SDS-polyacrylamide g e l electrophoretogram of outer membranes prepared from a p r o t e i n Pd e f i c i e n t mutant of P. aeruginosa and i t s w i l d type parent Induction of the 34K p e r i p l a s m i c p r o t e i n by phosphate l i m i t a t i o n  11  SDS-polyacrylamide g e l electrophoretogram of p u r i f i e d phosphate-binding p r o t e i n and whole c e l l p r o t e i n e x t r a c t s of a l k a l i n e phosphatase c o n s t i t u t i v e mutants of P. aeruginosa H242  91  12  Scatchard p l o t of phosphate-binding  93  13  Phosphate uptake  14  K i n e t i c s of phosphate uptake  15  Growth of a phosphate-binding p r o t e i n - d e f i c i e n t mutant i n a phosphate-limited medium  105  16  SDS-polyacrylamide g e l electrophoretogram of c e l l envelopes prepared from p h o s p h a t e - d e f i c i e n t and p h o s p h a t e - s u f f i c i e n t grown s t r a i n s of the f a m i l i e s Pseudomonadaceae and Enterobactereaceae  116  I n t e r a c t i o n of p r o t e i n P t r i m e r - s p e c i f i c or monomer-specific antiserum with Western b l o t s of p u r i f i e d p r o t e i n P and P. aeruginosa PA01 s t r a i n H103 c e l l envelopes  120  I n t e r a c t i o n of p r o t e i n P t r i m e r - s p e c i f i c antiserum with Western b l o t s of c e l l envelope p r e p a r a t i o n s of d i f f e r e n t b a c t e r i a grown under p h o s p h a t e - d e f i c i e n t or s u f f i c i e n t c o n d i t i o n s  122  Two-dimensional (unheated x heated) SDSpolyacrylamide g e l electrophoretogram of p u r i f i e d p r o t e i n P and c e l l envelopes prepared from phosphate-limited s t r a i n s of the Pseudomonadaceae and the E n t e r o b a c t e r i a c e a e  126  SDS-polyacrylamide g e l electrophoretogram of p u r i f i e d p h o s p h a t e - s t a r v a t i o n - i n d u c i b l e outer membrane p r o t e i n s of the f l u o r e s c e n t Pseudomonadaceae  134  17  18  19  20  activity  in P, aeruginosa  xi  i n P. aeruginosa  99 102  21  S t r i p chart recordings of stepwise increases i n the conductance of an o x i d i z e d c h o l e s t e r o l membrane caused by the p h o s p h a t e - s t a r v a t i o n i n d u c i b l e outer membrane p r o t e i n of P. putida  138  22  Histogram of the conductance f l u c t u a t i o n s observed with membranes of o x i d i z e d c h o l e s t e r o l i n the presence of the phosphate-starvation-inducible outer membrane p r o t e i n of P. putida 140  23  Average s i n g l e channel conductance of the phospates t a r v a t i o n - i n d u c i b l e p o r i n p r o t e i n of P. a u r e o f a c i e n s as a f u n c t i o n of the KC1 c o n c e n t r a t i o n i n the aqueous s o l u t i o n bathing an o x i d i z e d c h o l e s t e r o l membrane 147  24  Phosphate i n h i b i t i o n of c h l o r i d e f l u x through p r o t e i n P channels  xii  151  LIST OF  TABLES PAGE  Bacterial strains  24  II  Measurements of LPS a s s o c i a t e d with c o n v e n t i o n a l l y p u r i f i e d and e l e c t r o e l u t e d p r o t e i n P  71  III  F u n c t i o n a l p r o p e r t i e s of c o n v e n t i o n a l l y p u r i f i e d and e l e c t r o e l u t e d p r o t e i n P in planar l i p i d b i l a y e r membranes  72  Plasmids t e s t e d f o r u t i l i t y in transposon i n s e r t i o n mutagenesis of P. aeruginosa  77  K i n e t i c s of h i g h - a f f i n i t y phosphate t r a n s p o r t i n a p r o t e i n P - d e f i c i e n t mutant s t r a i n and i t s w i l d type parent  84  Growth of a p r o t e i n P - d e f i c i e n t mutant and s t r a i n s wild-type for p r o t e i n P in a phosphate-limited medium  86  Substrate s p e c i f i c i t y of the protein  94  IV  V  VI  VII  phosphate-binding  32 VIII  P-orthophosphate binding by p e r i p l a s m i c e x t r a c t s of wild-type and mutant s t r a i n s of P. aeruginosa  96  IX  In v i t r o a s s o c i a t i o n of the phosphate-binding p r o t e i n and outer membrane p r o t e i n P  107  X  P r o p e r t i e s of the phosphate-starvation-inducible membrane p r o t e i n s of the Enterobacteriaceae and the Pseudomonadaceae  113  Channel-forming p r o p e r t i e s of a f f i n i t y - p u r i f i e d and e l e c t r o e l u t e d phosphate-starvation-inducible outer membrane oligomers of the f l u o r e s c e n t Pseudomonads  141  XI  xi i i  XII  S i n g l e channel conductance of phosphate-starvationi n d u c i b l e porin p r o t e i n s of the f l u o r e s c e n t Pseudomonads i n s a l t s of v a r y i n g anion and c a t i o n size 144  XIII  Binding a f f i n i t i e s of phosphate-starvationi n d u c i b l e porin p r o t e i n s of the f l u o r e s c e n t Pseudomonads f o r c h l o r i d e and orthophosphate  xiv  148  LIST OF ABBREVIATIONS  A  405 600 /A  Absorbance at 405/600 nm  Cb  Carbenicillin  EDTA  Ethylenediaminetetraacetate  FCS  Fetal calf  Hepes  N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate  Kd  d i s s o c i a t i o n constant  KDO  2-keto-3-deoxy  Km  M i c h a e l i s constant  Kn  Kanamycin  LPS  Lipopolysaccharide  NPPC  para-Nitrophenyl p h o s p h o r y l c h o l i n e  P1 5  phosphate polymer comprising 15 phosphate.units  PBS  Phosphate b u f f e r e d s a l i n e  pNPP  para-Nitrophenyl phosphate  SDS  Sodium dodecyl sulphate  Tc  Tetracycline  Tp  Trimethoprim  Tris  Tris(hydroxymethyl)aminomethane  XP  5-bromo-4-chloro-3-indolyl phosphate-p-toluidine  serum  octulosonic  XV  acid  ACKNOWLEDGEMENTS  Firstly,  I thank Bob Hancock, who should a l r e a d y know  that he has my warmest a p p r e c i a t i o n f o r the guidance, encouragement and f r i e n d s h i p he has shown me. labmates,  As w e l l , my  past and present, have my enduring thanks f o r  t h e i r camaraderie  and f e l l o w s h i p , without which t h i s  experience would have been s o r e l y  lacking.  To J e r r i , my kindred s p i r i t , whom I f i r s t met a t the outset of t h i s -long journey, I am g r a t e f u l , f o r h e l p i n g me maintain a semblance of s a n i t y i n the insane world that i s graduate  school.  Without her understanding,  p a t i e n c e and  u n f a i l i n g confidence i n me I could not have come so f a r . L a s t l y , I thank my f a m i l y , e s p e c i a l l y my mom and dad, who have always encouraged but never pushed me, and who have always shown an i n t e r e s t i n my work, even when i t was a l l 'Greek' t o them.  xvi  INTRODUCTION N u t r i e n t a c q u i s i t i o n by p r o k a r y o t i c organisms n e c e s s a r i l y i n v o l v e s transmembrane t r a n s l o c a t i o n of s o l u t e molecules.  In the case of gram-negative  organisms  there are  two membranes which must be t r a v e r s e d during the u n i d i r e c t i o n a l movement of n u t r i e n t molecules environment  to the c e l l  interior.  from the  The mechanisms by which  t h i s u n i d i r e c t i o n a l t r a n s p o r t occur are s p e c i f i c t o the membranes being t r a v e r s e d . 1.  The gram-negative  microscopic  cell  envelope.  Electron  s t u d i e s have confirmed that the c e l l  gram-negative  envelope of  b a c t e r i a , i n c l u d i n g Psedomonas aeruginosa,  c o n s i s t s of three l a y e r s , the cytoplasmic or " i n n e r " membrane, the p e p t i d o g l y c a n or murein l a y e r and the outer membrane (Lugtenberg and van Alphen, i n s t a n c e s , and i n s p e c i f i c additional  1978; Troy, a.  bounding  In some  s t r a i n s , a capsule and/or  ( A ) - l a y e r i s a l s o present e x t e r n a l to the  t r i p a r t i t e c e l l envelope Schleytr,  1983).  (Glauert and Thornley, 1969;  1979).  The cytoplasmic membrane.  the cytoplasm  i s comprised  The membrane  of approximately  equimolar amounts of p h o s p h o l i p i d and p r o t e i n i n a t y p i c a l l i p i d bilayer  (Lugtenberg and van Alphen,  1983).  The  h y d r o p h o b i c i t y of t h i s membrane (Machtiger and Fox, 1973) makes i t a b a r r i e r to h y d r o p h i l i c molecules hydrophobic  molecules d i f f u s e r e l a t i v e l y 1  although  f r e e l y across i t  (Teuber et a l • , 1977). As such, the inner membrane f u n c t i o n s as a h i g h l y s p e c f i c p e r m e a b i l i t y b a r r i e r to h y d r o p h i l i c molecules,  with s o l u t e t r a n s l o c a t i o n dependent upon the  presence of s p e c i f i c e n e r g y - r e q u i r i n g the membrane  (Wilson,  water-soluble  1978).  t r a n s p o r t systems i n  In c e r t a i n t r a n s p o r t  binding p r o t e i n s present  i n the space between  the inner and outer membrane (the periplasm 1961)) f u n c t i o n i n concert with  systems,  (Mitchell,  inner membrane t r a n s p o r t  p r o t e i n s i n the uptake of s o l u t e molecules (Oxender, 1972; Wilson  and Smith, 1978; Hoshino and N i s h i o , 1982; Eisenberg  and Phibbs,  1982).  The energy f o r n u t r i e n t uptake i s  d e r i v e d from the e l e c t r o c h e m i c a l gradient of protons the inner membrane (the proton  across  motive f o r c e ) generated from  the primary a c t i v e t r a n s p o r t of H+ ions during  respiration  or ATP h y d r o l y s i s and/or from phosphate-bond energy i n the form of ATP or r e l a t e d metabolites Harold,  (Berger and Heppel, 1974;  1977; Hong et a l . , 1979). b.  The p e p t i d o q l y c a n .  The peptidoglycan  exists  as a network of l i n e a r amino sugars (N-acetyl glucosamine and N - a c e t y l muramic a c i d ) c o v a l e n t l y l i n k e d v i a c r o s s bridges between t e t r a p e p t i d e s attached acid  ( S c h l e i f e r and Kandler,  a r i g i d monolayer present al.,  1972).  to N-acetyl muramic  O r i g i n a l l y d e s c r i b e d as  i n the p e r i p l a s m i c space (Braun et  1973) the peptidoglycan  of e n t e r i c organisms has  r e c e n t l y been suggested to e x i s t as a hydrated probably  occupies  cytoplasmic  ' g e l ' which  the e n t i r e space between the outer and  membranes (Hobot et a l . , 1984).  2  In a d d i t i o n ,  the peptidoglycan  i s c o v a l e n t l y c r o s s - l i n k e d to the outer  membrane v i a a p r o t e i n analogous to the major l i p o p r o t e i n of Escherichia c o l i Although  (Braun,  1975; Lugtenberg et a l . , 1977).  the peptidoglycan  chemically d i f f e r e n t 1975), i t i s probably  of P. aeruginosa  from that of e n t e r i c organisms (Meadow, not c o v a l e n t l y l i n k e d to the outer  membrane (Hancock et a l . , 1981a). suggest  i s not  There i s no evidence to  that the peptidoglycan p r o v i d e s a b a r r i e r to s o l u t e  molecules  during t r a n s p o r t , although,  together  with  membrane-derived o l i g o s a c c h a r i d e s (MDOs) (van Golde et a l . , 1973)  i t p r o v i d e s f i x e d l o c a l i z e d charges w i t h i n the  periplasm which gives r i s e to a Donnan p o t e n t i a l across the outer membrane ( i n s i d e n e g a t i v e ) ( S t o c k et a l . , 1977).  This  p o t e n t i a l may play a r o l e i n the movement of a p p r o p r i a t e l y charged s u b s t r a t e molecules (Costerton, c.  across the outer membrane  1970). The outer membrane.  The outer membrane i s  comprised of p h o s p h o l i p i d s , p r o t e i n and l i p o p o l y s a c c h a r i d e (LPS) a l i p i d i c (Lugtenberg  molecule unique to gram-negative b a c t e r i a  and van Alphen, 1983).  LPS i s an amphipathic  molecule possessing a hydrophobic p o r t i o n , l i p i d A, embedded in the membrane, and a h y d r o p h i l i c p o l y s a c c h a r i d e p o r t i o n extending The  out from the c e l l  s u r f a c e ( L u d e r i t z et a l . , 1982).  d i s t a l p o r t i o n of the p o l y s a c c h a r i d e , the 0 antigen,  u s u a l l y c o n s i s t s of repeating o l i g o s a c c h a r i d e u n i t s which e x h i b i t wide v a r i a b i l i t y even w i t h i n a s i n g l e s p e c i e s (Luderitz e t a l . ,  1982).  L i k e the inner membrane, the outer  3  membrane appears as a b i l a y e r (Glauert and Thornley, 1969).  i n the e l e c t r o n  microscope  However, the outer membrane  i s unusual i n that the p h o s p h o l i p i d i s present e x c l u s i v e l y (except, perhaps,  i n c e r t a i n mutants) i n the inner l e a f l e t  of the b i l a y e r , while e s s e n t i a l l y a l l of the LPS occurs i n the outer l e a f l e t Nikaido,  1980).  (Muhlradt and G o l e c k i ,  1975; Funahara and  The p r o t e i n s of the outer membrane occur i n  both l e a f l e t s and i n some cases a c t u a l l y span the e n t i r e membrane (Lugtenberg and van Alphen,  1983).  U n l i k e the  inner membrane, the outer membrane l a c k s a hydrophobic uptake pathway (Nikaido, 1976), probably due to the presence of n e g a t i v e l y charged LPS molecules on i t s outer s u r f a c e , and f u c t i o n s as a n o n - s p e c i f i c p e r m e a b i l i t y b a r r i e r to h y d r o p h i l i c molecules  (Nikaido, 1979).  In the case of  e n t e r i c b a c t e r i a t h i s serves to p r o t e c t the organisms  from  the d e t e r g e n t - l i k e a c t i o n s of b i l e s a l t s , f a t t y a c i d s and g l y c e r i d e s , as well as from p r o t e o l y t i c and l i p o l y t i c enzymes and g l y c o s i d a s e s present i n the gut (Lugtenberg and van Alphen,  1983).  The P. aeruginosa outer membrane, which  has been i m p l i c a t e d i n the high i n t r i n s i c r e s i s t a n c e of t h i s organism to a n t i b i o t i c s  (Angus et a l . ,  1982; Yoshimura and  Nikaido, 1982; Nicas and Hancock, 1983), may well serve a s i m i l a r r o l e i n nature s i n c e P. aeruginosa i s commonly found i n the s o i l where a n t i b i o t i c producing organisms are also  found.  4  H y d r o p h i l i c s o l u t e molecules molecular weight c u t o f f permeating process  below a d e f i n e d  (the e x c l u s i o n l i m i t ) are capable of  the outer membrane v i a a passive d i f f u s i o n  (Nikaido, 1979)  molecular weights  mediated by a c l a s s of p r o t e i n s of  35,000-45,000, c a l l e d p o r i n s .  p r o t e i n s form w a t e r - f i l l e d channels through the core of the outer membrane 1979). P o r i n s e x i s t  These hydrophobic  (Hancock et a l . , 1979;  Nikaido,  i n the outer membranes as t r i m e r s  (Tokunaga et a l . , 1979;  Angus et a l . , 1983;  Maezawa et a l . ,  1983), are non-covalently attached to the p e p t i d o g l y c a n (Lugtenberg usually 1979;  et a l . , 1977;  isolated  Hancock et a l . , 1981a) and  i n a s s o c i a t i o n with LPS  S c h i n d l e r and Rosenbusch, 1978).  (Furukawa et a l . , LPS a s s o c i a t i o n i s  not, however, e s s e n t i a l f o r p o r i n f u n c t i o n i n v i t r o al.,  i n press) although i t has been proposed  in modulating  in vivo porin a c t i v i t y  are  (Parr et.  to be i n v o l v e d  ( K r o p i n s k i et a l . ,  1982). The e x c l u s i o n l i m i t of the P. aeruginosa membrane i s s i g n i f i c a n t l y  outer  l a r g e r than that of E. c o l i  or S.  typhimurium outer membranes (Mr 3,000-9,000 compared with 600-700) ( Nakae and N i k a i d o , 1975; Nikaido,  Nakae, 1976;  Hancock and  1978), an o b s e r v a t i o n c o n s i s t e n t with the formation  in model systems of l a r g e r channels by the major p o r i n p r o t e i n F of P. aeruginosa OmpF (1.1 nm)  (2.2 nm d i a . ) than by p r o t e i n s  and OmpC (1.0 nm),  (Benz et a l . , 1985).  the major E. c o l i  porins  I n t e r e s t i n g l y , however, the outer  membrane of P. aeruginosa  i s l e s s permeable than that of  5  E. c o l i  (Angus et a l . , 1982; Yoshimura and Nikaido, 1982;  Nicas and Hancock, 1983), which has l e a d to the suggestion that only a small percentage of p r o t e i n F molecules form f u c t i o n a l pores i n v i v o .  2.  The r o l e of the outer membrane i n t r a n s p o r t .  The  p e r m e a b i l i t y of the outer membrane, mediated by p o r i n s , provides a pathway f o r the entry of n u t r i e n t molecules (e.g. sugars, amino a c i d s , n u c l e o s i d e s ,  ions).  Because p o r i n s i n  general a r e n o n - s p e c i f i c , e x h i b i t i n g only weak i o n selectivity et  i n r e c o n s t i t u t e d planar b i l a y e r membranes (Benz  a l . , 1985), n u t r i e n t molecules c r o s s the outer membrane  down t h e i r r e s p e c t i v e c o n c e n t r a t i o n g r a d i e n t s . diffusion  The rate of  (V) of s o l u t e molecules a c r o s s the outer membrane  can be d e s c r i b e d by the equation V = P x A x [C (Fick's f i r s t  law of d i f f u s i o n )  - C^3  where P i s the p e r m e a b i l i t y  c o - e f f i c i e n t , A i s the area of the membrane and C  and C o  represent the c o n c e n t r a t i o n of s u b s t r a t e outside e x t e r n a l medium) and i n s i d e ( i n the periplasm) respectively  (Yoshimura and Nikaido,  1982).  1  ( i n the  the c e l l ,  The presence of  h i g h - a f f i n i t y p e r i p l a s m i c binding p r o t e i n s , and of cytoplasmic  membrane t r a n s p o r t systems of low Km,  to  low p e r i p l a s m i c c o n c e n t r a t i o n s  maintain  so that, i n the presence of s u f f i c i e n t concentrations  functions  of these  solutes  extracellular  of these n u t r i e n t s , t h i s gradient  i s usually  s u f f i c i e n t to t r a n s p o r t l e v e l s of n u t r i e n t s which are not l i m i t i n g f o r t r a n s p o r t or growth. 6  However, s u f f i c i e n t l y low  e x t e r n a l c o n c e n t r a t i o n s of n u t r i e n t molecules  or a decrease  in outer membrane p e r m e a b i l i t y 'resulting from, f o r example, the p o r i n - d e f i c i e n c y of mutant s t r a i n s  (Von Meyenburg, 1971;  Lutkenhaus, 1977; Nicas and Hancock, 1983), have been demonstrated to decrease  the rate of s o l u t e movement across  the outer membrane such that the o v e r a l l r a t e s of t r a n s p o r t (Lutkenhaus,  1977) and growth (Von Meyenburg, 1971) are  l i m i t e d by d i f f u s i o n across the outer membrane.  In the case  of p e r m e a b i l i t y mutants, r e d u c t i o n of outer membrane p e r m e a b i l i t y increases the Km of the o v e r a l l t r a n s p o r t process d e s p i t e the f a c t that upon d i f f u s i o n across the outer membrane a s u b s t r a t e i s t r a n s p o r t e d across the cytoplasmic membrane v i a a s p e c i f i c system with a very low Km.  Nonetheless,  the e f f e c t of p o r i n - d e f i c i e n c y on  t r a n s p o r t and growth i s seen only at lower c o n c e n t r a t i o n s of substrate since i t i s t h e o r e t i c a l l y and p r a c t i c a l l y  ("from P i c k ' s f i r s t law)  (Von Meyenburg, 1971; Lutkenhaus, 1977)  p o s s i b l e to r e s t o r e a n o n - l i m i t i n g r a t e of d i f f u s i o n  across  the outer membrane of p o r i n - d e f i c i e n t mutants simply by i n c r e a s i n g the e x t e r n a l s u b s t r a t e c o n c e n t r a t i o n . Pseudorevertants  of p o r i n - d e f i c i e n t mutants which  express novel p o r i n p r o t e i n s (Henning et a l . , 1977; Von Meyenburg and Nikaido,  1977; Van Alphen et a l . , 1978;  Pugsley and Schnaitman, 1978; Chai and Foulds,  1979) r e s t o r e  the c e l l ' s a b i l i t y to t r a n s p o r t n o n - l i m i t i n g c o n c e n t r a t i o n s of s o l u t e across the outer membrane, i n the presence of s u f f i c i e n t e x t e r n a l c o n c e n t r a t i o n s of n u t r i e n t molecule.  7  S i m i l a r l y , the r a t e - l i m i t i n g d i f f u s i o n of n u t r i e n t molecules across  the outer membrane r e s u l t i n g from low e x t r a c e l l u l a r  nutrient concentrations by the s y n t h e s i s function  i s compensated f o r i n some b a c t e r i a  of i n d u c i b l e outer membrane p r o t e i n s  i n the f a c i l i t a t e d  uptake of the l i m i t i n g  nutrient.  Examples i n c l u d e the p h o s p h a t e - s t a r v a t i o n - i n d u c i b l e p r o t e i n s of the Enterobacteriaceae  which  PhoE  (Overbeeke and  Lugtenberg, 1980; Sterkenburg e t a l . , 1984; Bauer et a l . , 1985)  and the i r o n regulated  i n many b a c t e r i a l species  outer membrane p r o t e i n s  found  (Ernst et a l . , 1978; Braun and  Hantke, 1982; S c i o r t i n o and F i n k l e s t e i n , 1983; W i l l i a m s et al.,  1984; Brown et a l . , 1984). In a d d i t i o n , novel membrane  p r o t e i n s are sometimes produced i n cases where the n u t r i e n t molecule permeates the outer membrane poorly porin proteins.  Such p r o t e i n s  v i a the major  include the  maltose/maltodextrin LamB (Ferenci and Boos, 1980) and the Tsx  nucleoside a.  coli  transport  (Hantke, 1976) p r o t e i n s of E. c o l i .  The LamB p r o t e i n .  Although i n d u c i b l e i n E.  s t r a i n s grown in n o n - l i m i t i n g c o n c e n t r a t i o n s  of maltose  (Braun and Krieger-Bauer, 1977), the LamB p r o t e i n , which f u n c t i o n s as the phage lambda receptor, e s s e n t i a l component of maltose t r a n s p o r t but  not at high  (> 1 mM) c o n c e n t r a t i o n s  appears to be an at low (< 10 uM) of maltose  (Szmelcman and Hofnung, 1975; Szmelcman et a l . , 1976). Studies  i n v o l v i n g the r e c o n s t i t u t i o n of the p u r i f i e d p r o t e i n  i n t o liposomes have demonstrated that the LamB p r o t e i n indeed e x h i b i t s a marked preference for maltose over other  8  •  disaccharides,  facilitating  the d i f f u s i o n of maltose i n t o  lipososmes 40 times f a s t e r than, f o r example, sucrose (Luckey and Nikaido, as an e f f i c i e n t  1980a).  The LamB p r o t e i n a l s o serves  channel f o r the uptake of m a l t o d e x t r i n s (up  to maltoheptaose) which exceed the e x c l u s i o n major E. c o l i p o r i n s Ishii,  1980).  1 mM)  defective  1980a; Nakae and  Mutants d e f i c i e n t i n LamB p r o t e i n e x h i b i t a  1000-fold increase uM t o  (Luckey and Nikaido,  l i m i t s of the  i n the Km f o r maltose t r a n s p o r t  (from 1.0  (Szmelcman et a l , 1976) and are s e v e r e l y  in transporting maltotriose  (Szmelcman et a l ,  1976), while maltotetraose and higher molecular weight m a l t o d e x t r i n s are not accumulated at a l l ( F e r e n c i , The  affinity  increases for  1980).  of the channel f o r maltose and m a l t o d e x t r i n s  with i n c r e a s i n g chain  length  of the d e x t r i n (Km  maltose=14 mM; Km for maltodecanose=75 uM) (Ferenci e_t  al.,  1980) and has been a t t r i b u t e d t o binding  sites  in/near  the channel (Ferenci et a l . , 1980; Luckey and Nikaido, 1980b). The  LamB p r o t e i n  i s a component of a maltose operon i n  E. c o l i which i n c l u d e s , transport  p r o t e i n s and cytoplasmic c a t a b o l i c enzymes, a  periplasmic et  i n a d d i t i o n to inner membrane  h i g h - a f f i n i t y maltose binding  a l . , 1978).  protein  (Dietzel  The binding p r o t e i n was demonstrated to bind  maltose and m a l t o d e x t r i n s i n the micromolar range (Wandersmann et a l . , 1979) i n agreement with the observed k i n e t i c s of t r a n s p o r t 1976).  (Ks = 1.0 uM) (Szmelcman et a l . ,  Furthermore, a p h y s i c a l a s s o c i a t i o n between the  9  b i n d i n g p r o t e i n and the LamB p o r i n was ( B a v o i l and Nikaido,  1981)  observed  in v i t r o  confirming e l e c t r o n microscopic  data which showed that the maltose-binding p r o t e i n a s s o c i a t e d with the p e r i p l a s m i c face of LamB-containing outer membranes (Boos and S t a e h l i n , 1981). a s s o c i a t i o n was  suggested  to be necessary  t r a n s p o r t of maltose and m a l t o d e x t r i n s  Such an f o r the  across the outer  membrane in v i v o (Wandersman et a l . , 1979; Nikaido,  Luckey  and  1983).  In a d d i t i o n to i t s r o l e in maltose and uptake, the LamB channel channel as w e l l .  maltodextrin  f u n c t i o n s as a general  diffusion  In v i t r o s t u d i e s have confirmed  a b i l i t y of a number of amino a c i d s and u n r e l a t e d (Nakae, 1979;  Luckey and N i k a i d o ,  (Boehler-Kohler et a l . , 1979) may  efficient  the sugars  1980a) as w e l l as ions  to permeate the channel.  LamB  a l s o be capable of r e p l a c i n g the major p o r i n s in v i v o i n  r e v e r t a n t s of p o r i n - d e f i c i e n t mutants (Von Meyenburg and Nikaido,  1977).  An analogous p r o t e i n , designated p r o t e i n D1, identified  i n the outer membrane of P. aeruginosa  growing i n g l u c o s e - c o n t a i n i n g media (Hancock and 1979).  has been cells  Carey,  Co-regulated with a b i n d i n g protein-dependent  a f f i n i t y glucose t r a n s p o r t system (Midley and Dawes,  high1973;  S t i n s o n et a l . , 1977), t h i s 46,000 molecular weight p r o t e i n forms channels to glucose  i n liposomes  which are s e l e c t i v e l y permeable  (Hancock and Carey, 1980).  10  b. conditions  The PhoE p r o t e i n .  Inducible  of p h o s p h a t e - l i m i t a t i o n  1980), p o r i n p r o t e i n PhoE was f i r s t  i n E. c o l i  (Overbeeke and Lugtenberg, identified  i n revertants  of p o r i n - d e f i c i e n t mutants (Henning et a l . , 1977; et  a l . , 1978;  1979) . large  under  Pugsley and Schnaitman, 1978;  Van Alphen  Chai and Foulds,  The p u r i f i e d p r o t e i n has been demonstrated to form (1 nm dia)  (Benz et a l . , 1985), weakly  (Benz et a l . , 1984) membranes.  channels i n r e c o n s t i t u t e d b i l a y e r  Examination of the t r a n s p o r t  mutants expressing p o r i n revealed  that a wide range of n u t r i e n t s  with the  (sugars,  amino  could permeate the channel i n v i v o  (Lugtenberg et a l . , 1978;  channel.  p r o p e r t i e s of  PhoE ( p r e v i o u s l y p r o t e i n e) as the sole  acids, nucleosides,ions)  consistent  anion-selective  Van Alphen et a l . , 1978),  formation of a general  diffusion  The p r o t e i n i s immunologically c r o s s - r e a c t i v e  with  the major p o r i n p r o t e i n s OmpF and OmpC (Overbeeke et a l . , 1980) , e x h i b i t i n g 70 % amino a c i d homology with OmpF (Tommassen et a l . , 1982)  and 61 % amino a c i d homology with  OmpC (Mizuno et a l . , 1983). and  In a d d i t i o n , the cloned  ompF genes have been demonstrated to h y b r i d i z e  along t h e i r e n t i r e lengths  phoE  i n regions  (Tommassen et a l . , 1982).  Despite these s i m i l a r i t i e s with the major p o r i n s , the PhoE channel e x h i b i t s p r o p e r t i e s c o n s i s t e n t r o l e i n phosphate a c q u i s i t i o n . (pho)  regulon i n E. c o l i  with a presumed  A component of the phosphate  (Tommassen and Lugtenberg, 1982)  (see s e c t i o n 4) the p r o t e i n forms a channel which i s more efficient  i n the uptake of a n i o n i c and phosphorylated  1 1  compounds than e i t h e r OmpF or OmpC (Overbeeke and Lugtenberg, 1982).  Overbeeke and Lugtenberg  (1982) a l s o  demonstrated that a mutant d e f i c i e n t i n PhoE grew more slowly  than w i l d type i n the presence of polyphosphate (P15)  as the sole phosphate source.  Furthermore, Korteland  (1982) have demonstrated that a PhoE-deficient e x h i b i t e d a 10-fold increase  mutant  i n the Km f o r phosphate  t r a n s p o r t compared with s t r a i n s expressing Unfortunately,  et a l  t h i s r e s u l t was obtained  a PhoE channel.  ina porin-deficient  background, rather than a background w i l d type f o r the major porins.  Therefore,  i t was not p o s s i b l e to conclude whether the  increase  i n Km f o r phosphate i n the P h o E - d e f i c i e n t  strain  r e f l e c t e d a s p e c i f i c r o l e f o r p r o t e i n PhoE i n phosphate t r a n s p o r t , or whether any p o r i n would have reversed  the poor  phosphate t r a n s p o r t of the p o r i n - d e f i c i e n t s t r a i n . PhoE p r o t e i n s have been i d e n t i f i e d Enterobacteriaceae,  i n c l u d i n g Salmonella typhimurium  et a l . , 1985) and Enterobacter 1984).  i n other  These p r o t e i n s  cloacae  (Bauer  (Verhoef e t a l . ,  form a n i o n - s e l e c t i v e channels  c o n s i s t e n t with t h e i r presumed r o l e s i n phosphate acquisition.  A 36 kDa outer membrane p r o t e i n i n d u c i b l e by  phosphate-limitation  has a l s o been i d e n t i f i e d i n K l e b s i e l l a  aerogenes (Sterkenburg et a l . , 1984) although i t was not assayed f o r p o r i n c.  function.  Iron-regulated  outer membrane p r o t e i n s .  Although i r o n i s an abundant metal i n nature i t occurs under aerobic c o n d i t i o n s  (at pH 7) as f e r r i c hydroxide with low  12  water s o l u b i l i t y  ( e q u i l i b r i u m c o n c e n t r a t i o n of 10  (Braun and Hantke,  1982).  C e l l s of E. c o l i ,  uM)  f o r example,  r e q u i r e an i r o n c o n c e n t r a t i o n of approximately 0.1 uM f o r growth and to gain s u f f i c i e n t chelators  i r o n they must produce  iron  (eg. ferrichrome; e n t e r o c h e l i n ) concomittant with  t r a n s p o r t systems f o r these c h e l a t e s 1975; Wayne and Neilands,  1975).  (Hantke and Braun,  A number of high molecular  weight outer membrane p r o t e i n s have.been  i d e n t i f i e d which  are c o - r e g u l a t e d with these c h e l a t o r s under conditions  (Braun et a l . ,  Mcintosh and E a r h a r t ,  iron-limiting  1976; Hancock and Braun, 1976;  1977).  Two of these, the products of  the fhuA (tonA) (Hantke and Braun, 1975) and fepA (feuB, c b r ) (Pugsley and Reeves, 1977; Wookey and Rosenberg,  1976; Mcintosh and Earhart,  1978) genes, f u n c t i o n i n the  uptake of f e r r i c - f e r r i c h r o m e and f e r r i c - ' e n t e r o c h e l i n , respectively.  This was supported by data which demonstrated  d i r e c t l y the a b i l i t y of the r e s p e c t i v e i r o n c h e l a t e s to bind to t h e i r receptor p r o t e i n s i n the outer membrane (Braun and Hantke,  1977; Ichihara and Mizushima,  1978) and by the  i n a c t i v a t i o n of s p e c i f i c t r a n s p o r t systems i n mutants deficient  i n the corresponding outer membrane p r o t e i n s  (Pugsley and Reeves,  1976; Wookey and Rosenberg,  S i m i l a r p r o t e i n s have been i d e n t i f i e d bacteria, including  i n other gram-negative  Salmonella typhimurium (Ernst et a l . ,  1978), N e i s s e r i a gonorrheae (Norqvist et a l . , cholerae  1978).  ( S c i o r t i n o and F i n k e l s t e i n ,  aerogenes (Williams et a l . ,  1978), V i b r i o  1983), K l e b s i e l l a  1984) and Pseudomonas aeruginosa  13  (Brown et a l . , 1984). d.  Others.  The t r a n s p o r t of vitamin B12  (cyanocobalamin) by E. c o l i  i s a b i p h a s i c process  involving  an energy-independent r a p i d b i n d i n g phase followed by a energy-dependent phase (DiGirolamo and Bradbeer, 1971).  The  i n i t i a l vitamin B12 b i n d i n g s i t e s are f i r m l y embedded i n the outer membrane (DiGirolamo et a l . , 1971; White et a l . ,  1973)  and have been i d e n t i f i e d as the p r o t e i n products of the btuB (bfe) gene (White et a l . , 1973; DiMasi and L i g g i n s , 1973).  et a l . , 1973; Kadner  A minor outer membrane p r o t e i n , the  btuB gene product has not been shown to e x h i b i t any p o r i n f u n c t i o n , although  , together with LPS and the OmpF p r o t e i n , i t has been  i d e n t i f i e d as a c o n s t i t u e n t of the c o l i c i n A receptor (Chai et a l . , 1982).  The p r o x i m i t y of the vitamin B12 receptor and major  p o r i n p r o t e i n OmpP i n v i v o may be s i g n i f i c a n t  i n terms of •  the mechanism by which vitamin B12 a c t u a l l y c r o s s e s the outer membrane. The  t r a n s p o r t of n u c l e o s i d e s by E. c o l i c e l l s r e p o r t e d l y  i n v o l v e s an outer membrane p r o t e i n , the tsx gene product, which forms an e s p e c i a l l y e f f i c i e n t channel (Hantke, 1976).  Although  f o r nucleosides  n u c l e o s i d e s are capable of  permeating the outer membrane v i a the OmpF and PhoE channels (van Alphen et a l , 1978), t h e i r d i f f u s i o n through channels  i s expected  nucleosides.  these  t o be slow due to the l a r g e s i z e of  Furthermore, the e x c e p t i o n a l l y high V  m a x  of  the n u c l e o s i d e a c t i v e t r a n s p o r t system (Koch, 1971) probably n e c e s s i t a t e s a s p e c i f i c channel  14  i n the outer membrane.  3.  B a c t e r i a l phosphate t r a n s p o r t - with s p e c i f i c  reference to E. c o l i . been c h a r a c t e r i z e d  i n o r g a n i c phosphate t r a n s p o r t has  i n a number of b a c t e r i a l  i n c l u d i n g Staphylococcus aureus ( M i t c h e l l ,  systems, 1954), B a c i l l u s  cereus (Rosenberg et a l , 1969), Micrococcus l y s o d e i k t i c u s (Friedberg,  1977), Streptococcus f a e c a l i s  Baarda, 1966), E. c o l i  (Harold and  (Medveczky and Rosenberg,  P. aeruginosa (LaCoste et a l . ,  1981).  1971) and  In each case, the  t r a n s p o r t i s c o n c e n t r a t i v e , energy-dependent and i n h i b i t a b l e to v a r y i n g degrees by arsenate, a phosphate analogue.  In  a d d i t i o n , the rate of and c a p a c i t y f o r phosphate uptake appears, i n many cases, to increase at low c o n c e n t r a t i o n s of phosphate suggesting that the t r a n s p o r t systems involved are inducible. The t r a n s p o r t of inorganic phosphate by E. c o l i has been c h a r a c t e r i z e d i n d e t a i l and two major systems of' uptake, the PST and PIT systems, have been ( W i l l s k y et a l . ,  1973).  identified  The PST system, which operates at  approximately 20 % of c a p a c i t y i n the presence of high l e v e l s of phosphate  (Rosenberg et a l . ,  derepressed under p h o s p h a t e - l i m i t i n g (Rosenberg et a l . , to  1977).  1977), i s completely  (< 1.0 mM)  conditions  A h i g h - a f f i n i t y system (Km = 0.16  0.43 uM) (Rosenber et a l . ,  1977; W i l l s k y and Malamy,  1980) the PST phosphate t r a n s p o r t system i s r e s p o n s i b l e f o r the  bulk of phosphate transport under l i m i t i n g  conditions.  The PIT system, which operates c o n s t i t u t i v e l y ,  i s of low  affinity  1977; W i l l s k y  (Km = 25 to 38 uM) (Rosenberg et a l . ,  15  and Malamy, 1980), and i s the major t r a n s p o r t system f o r phosphate under p h o s p h a t e - s u f f i c i e n t  conditions.  The PST system i s comprised of the products'of at l e a s t 5 genes  ( p s t , phoU, phoV, phoS, phoT) ( L e v i t z et a l , 1984),  one of which, the product of the phoS gene, f u n c t i o n s as a p e r i p l a s m i c phosphate-binding p r o t e i n 1974).  (Gerdes and Rosenberg,  The remaining gene products have not been  identified,  although they are probably l o c a l i z e d i n the cytoplasmic membrane (Tommassen and Lugtenberg, 1982).  The phosphate-  binding p r o t e i n i s i n d u c i b l e under c o n d i t i o n s of phosphatelimitation  ( Y a g i l et a l . ,  high-affinity  (Kd=0.8 uM)  1976) and binds phosphate with (Medveczky and Rosenberg,  1970),  accounting f o r the i n d u c i b i l i t y and low Km of the PST t r a n s p o r t system.  Osmotic shock and spheroplast  both of which r e s u l t  formation,  i n the r e l e a s e of the binding p r o t e i n  from whole c e l l s , have been demonstrated to i n a c t i v a t e the PST system (Gerdes et a l . ,  1977).  T y p i c a l of b i n d i n g  protein-dependent t r a n s p o r t systems in general, phosphate uptake v i a the PST system u t i l i z e s phosphate bond energy, i n the form of ATP or a r e l a t e d m e t a b o l i t e , as the energy source (Rosenberg et a l . ,  1979).  The recent demonstration  that the o r n i t h i n e - a r g i n i n e binding p r o t e i n of E. c o l i i s phosphorylated - dephosphorylated during substrate t r a n s p o r t (Celis,  1984) may  be a c l u e as to the r o l e ATP  (or a r e l a t e d  m e t a b o l i t e ) p l a y s in b i n d i n g protein-dependent t r a n s p o r t . Although arsenate was capable of i n h i b i t i n g phosphate uptake v i a the PST system (Ki = 0.39  uM)  16  ( W i l l s k y and Malamy,  1980)  i t was not t r a n s p o r t e d by t h i s system and c e l l s  expressing  only the PST phosphate t r a n s p o r t system were capable of growth  i n the presence of arsenate. The l o w - a f f i n i t y PIT phosphate transport  system  i n v o l v e s the product of a s i n g l e known gene, p i t (Bennet and Malamy, 1970; W i l l s k y et a l . , 1973; Sprague et a l . , 1975), which i s probably  an inner membrane p r o t e i n .  Inorganic  phosphate uptake v i a t h i s system i s not s e n s i t i v e to spheroplast  formation  (Rosenberg et a l . , 1977), c o n s i s t e n t  with the absence of a binding p r o t e i n a s s o c i a t e d with i t . C h a r a c t e r i s t i c of s h o c k - r e s i s t a n t t r a n s p o r t  systems,  phosphate t r a n s p o r t v i a the PIT system i s coupled proton  motive force (Rosenberg et a l . , 1979).  phosphate t r a n s p o r t to be t r a n s p o r t e d Malamy, 1980).  i s i n h i b i t e d by arsenate  to the  PIT-mediated which appears  e q u a l l y w e l l by t h i s system ( W i l l s k y and  C e l l s expressing  grow .in an a r s e n a t e - c o n t a i n i n g  only the PIT system cannot  medium i n which they s u f f e r  an almost t o t a l d e p l e t i o n of i n t r a c e l l u l a r ATP l e v e l s ( W i l l s k y and Malamy,  1980).  In a d d i t i o n to the two major inorganic phosphate uptake systems d e s c r i b e d above, three organophosphate  transport  systems have a l s o been i d e n t i f i e d i n E. c o l i with hexose phosphate  roles in  (the uhp operon) (Romberg and Smith, 1969)  and glcerol-3-phosphate  (the qlpT  ( L i n , 1976) and ugp  (Schweizer et a l . , 1982) operons) t r a n s p o r t .  Two of these,  i n v o l v i n g the c o n s t i t u e n t s of the glucose-6-phosphatei n d u c i b l e uhp operon and the  glycerol-3-phosphate-inducible  17  glpT operon appear to be pathways f o r the uptake of i n o r g a n i c phosphate as w e l l  ( W i l l s k y and Malamy, 1974).  The  other i n v o l v e s the products of the ugpA and uqpB genes (Schweizer et a l . ,  1982), which encode an inner membrane  permease and a p e r i p l a s m i c protein, respectively  glycerol-3-phosphate-binding  (Tommassen and Lugtenberg, 1982).  T h i s system i s derepressed upon p h o s p h a t e - l i m i t a t i o n  (Argast  and Boos,  regulon  1980) and forms part of a phosphate or pho  in E. c o l i  (Schweizer et a l . ,  Lugtenberg, 1982)  1982; Tommassen and  (see below).  I n t e r e s t i n g l y , P. aeruginosa a l s o appears to possess two major transport systems f o r inorganic phosphate, of low and h i g h - a f f i n i t y , r e s p e c t i v e l y  (LaCoste et a l . ,  1981), as  w e l l as an uptake system f o r glycerol-3-phosphate ( S i e g e l and Phibbs, 1979).  The t r a n s p o r t of inorganic phosphate by  P. aeruginosa i s somewhat s e n s i t i v e to osmotic shock, c o n s i s t e n t with the involvement of a p e r i p l a s m i c protein.  binding  In a d d i t i o n , the two uptake systems appear to  e x h i b i t d i f f e r e n t energy requirements s i m i l a r to the situation  4.  i n E. c o l i .  The pho regulon of E. c o l i .  Under c o n d i t i o n s of  p h o s p h a t e - l i m i t a t i o n , w i l d type c e l l s of E. c o l i  are  derepressed f o r the s y n t h e s i s of numerous p r o t e i n s (Tommassen and Lugtenberg, 1982), and at l e a s t  18 phosphate-  s t a r v a t i o n - i n d u c i b l e genes have been d e s c r i b e d  (Wanner et  al.,  1981).  The r o l e s of these gene products i n the  18  scavenging of phosphate and phosphate-containing molecules from a d i l u t e environment has, in many cases, been addressed (Tommassen and Lugtenberg, 1982).  The p h o s p h a t e - s t a r v a t i o n -  i n d u c i b l e p r o t e i n s which have been i d e n t i f i e d p e r i p l a s m i c b i n d i n g p r o t e i n s f o r phosphate product) (Gerdes and Rosenberg, phosphate  include  ( the phoS gene  1974) and g l y c e r o l - 3 -  (the uqpB gene product) ( Argast and- Boos,  1980),  a p e r i p l a s m i c a l k a l i n e phosphatase (the phoA gene product) (Torriani,  1960; Brickman and Beckwith, 1975), cytoplasmic  membrane permeases  f o r phosphate  (Rosenberg et a l . ,  1977) and glycerol-3-phosphate (the ugpA  gene product) (Argast and Boos, pore-forming p r o t e i n  ( the pst gene product)  1980), an outer membrane  (the phoE gene product) (Overbeeke and  Lugtenberg, 1980) and a cytoplasmic polyphosphatase ( Y a g i l , 1975).  S e v e r a l presumably cytoplasmic r e g u l a t o r y molecules,  i n c l u d i n g the products of the phoB (Makino et a l . ,  1982;  Tommassen and Lugtenberg, 1982), phoM (Ludtke et a l . , and phoR (Tommassen et a l . , identified.  Most  1984)  1982) genes have a l s o been  i f not a l l of these p r o t e i n s are part of a  s i n g l e regulon, designated the pho regulon, which e x h i b i t s some s i m i l a r i t y to the maltose regulon of E. c o l i which i s a l s o i n d u c i b l e f o r an outer membrane p r o t e i n , a p e r i p l a s m i c binding p r o t e i n , cytoplasmic membrane c a r r i e r p r o t e i n s and cytoplasmic c a t a b o l i c enzymes (Hengge and Boos,  1983).  P h o s p h a t e - s t a r v a t i o n - i n d u c i b l e p r o t e i n s i d e n t i f i e d i n P. aeruginosa i n c l u d e an e x t r a c e l l u l a r phospholipase C (Stinson and Hayden, 1979) and an a l k a l i n e phosphatase which occurs  19  in both the periplasm and the e x t r a c e l l u l a r medium (Hou e_t al.,  1966; Cheng et a l . ,  1979).  A number of a d d i t i o n a l  p h o s p h a t e - r e p r e s s i b l e p r o t e i n s have been i d e n t i f i e d i n phospholipase C r e g u l a t o r y mutants of Pseudomonas aeruginosa (Gray et a l . ,  1982) although t h e i r  functional  activities  have not been e l u c i d a t e d . The components  of the E. c o l i  pho regulon are under the  c o n t r o l of a complex network of r e g u l a t o r y p r o t e i n s which i n c l u d e s the products of three known genes - phoB, phoM and phoR (Tommassen and Lugtenberg, 1982).  As a model  f o r the  r e g u l a t i o n of pho regulon constutuents, production of the phoA gene product ( a l k a l i n e phosphatase) has been s t u d i e d i n detail  (Echols and Garen, 1961; Brickman and Beckwith, 1975;  Bracha and Y a g i l , Mutant s t u d i e s  1973; Wanner and L a t t e r e l l ,  1980 ).  and s t u d i e s i n v o l v i n g the cloned genes have  i n d i c a t e d that the phoB gene product f u n c t i o n s ' a s a t r a n s c r i p t i o n a l a c t i v a t o r of phoA (Bracha and Y a g i l ,  1973;  Brickman and Beckwith, 1975), while the phoR gene product a c t s as a repressor and a c t i v a t o r  (high phosphate) (Echols et a l . ,  (low phosphate) (Wanner and L a t t e r e l l ,  the  latter  the  phoM gene product (Wanner and L a t t e r e l l ,  1961) 1980),  f u n c t i o n being at l e a s t p a r t i a l l y r e p l a c e a b l e by  on r e s u l t s of mutant phoB gene product was  1980).  Based  s t u d i e s i t was a l s o concluded that the i t s e l f c o - r e g u l a t e d with a l k a l i n e  phosphatase and that phoB t r a n s c r i p t i o n was probably under the al.,  c o n t r o l of the phoR and phoM gene products (Tommassen et 1982).  I t remains to be seen whether phosphate a c t s  20  directly  (as c o - r e p r e s s o r ) or i n d i r e c t l y  process.  21  in regulating  this  METHODS  1.  Media and growth c o n d i t i o n s .  The minimal medium used i n  t h i s study contained 0.1 M N-2-hydroxy-ethyl 2-ethanesulfonate  (Hepes) (pH 7.0), 0.5 mMMgS0 , 7 mM 4  ( N H ) S 0 , 20 mM potassium 4  2  piperazine-N'-  4  s u c c i n a t e or 0.4 % (wt/vol)  glucose as the carbon source, 0.1 % (wt/vol) t r a c e ion solution  (as d e s c r i b e d by Hancock et a l . ,  0.2 mM potassium phosphate b u f f e r d e f i c i e n t medium  (pH 7.0) f o r phosphate-  (with exceptions, see below) or 0.6-1.0 mM  potassium phosphate b u f f e r medium.  1981b) and e i t h e r  (pH 7.0) f o r phosphate s u f f i c i e n t  Amino a c i d s were i n c l u d e d , as r e q u i r e d , at a f i n a l  c o n c e n t r a t i o n of 1 mM.  When the c u l t u r e organism was  Pseudomonas c e p a c i a , Pseudomonas pseudomallei or Pseudomonas acidovorans the p h o s p h a t e - d e f i c i e n t medium contained 0.1 mM phosphate.  When- the c u l t u r e organism  was K l e b s i e l l a  pneumoniae, Enterobacter aerogenes or S e r r a t i a marce'sens the p h o s p h a t e - d e f i c i e n t medium contained 0.15 mM Xanthamonas m a l t o p h i l i a  phosphate.  ( p r e v i o u s l y Pseudomonas m a l t o p h i l i a )  c u l t u r e s were supplemented with 1 mM methionine.  L-broth [1  % (wt/vol) t r y p t o n e / 0.5 % (wt/vol) yeast e x t r a c t / 0.05 % (wt/vol) NaCl] and proteose peptone No. 2 [1 % (wt/vol)] were used as the r i c h media  throughout.  L i q u i d c u l t u r e s were grown with vigorous a e r a t i o n at 37°C unless otherwise i n d i c a t e d . maintained on L-broth agar  S t r a i n s were r o u t i n e l y  (L-agar) or p h o s p h a t e - s u f f i c i e n t  medium agar p l a t e s prepared by i n c l u d i n g 2 % (wt/vol)  22  Bactoagar  (Difco) i n L-broth and p h o s p h a t e - s u f f i c i e n t  minimal medium r e s p e c t i v e l y . A n t i b i o t i c s were used i n s e l e c t i v e media at the following concentrations:  tetracycline  ( T c ) , 200  kanamycin (Kn), 300 ug/ml; c a r b e n i c i l l i n mercuric c h l o r i d e  (HgC^),  ug/ml;  (Cb), 1 mg/ml;  15 ug/ml and trimethoprim (Tp),  1 mg/ml.  2.  Bacterial strains.  The b a c t e r i a l s t r a i n s and  used i n t h i s study are l i s t e d was  in Table I.  introduced into s t r a i n H103  Equal volumes of mid-log  (grown i n L-broth) were  mixed and p e l l e t e d by c e n t r i f u g a t i o n . decanted and the p e l l e t broth.  resuspended  The supernatant  g e n t l y i n 0.05  was  ml of L-  The c e l l s were spread over approximately o n e - t h i r d  of the s u r f a c e of an L-agar p l a t e and 30°C.  Plasmid pMTlOOO  by c o n j u g a t i o n with  PA01594(pMT1000) on L-agar p l a t e s . phase donor and r e c i p i e n t c e l l s  plasmids  The mating mixture was  incubated f o r 2 h at .  then resuspended  in 1 ml of  p h o s p h a t e - s u f f i c i e n t medium, c e n t r i f u g e d and washed s e v e r a l times i n the same medium.  Transconjugants were s e l e c t e d at  30°C on p h o s p h a t e - s u f f i c i e n t minimal medium c o n t a i n i n g 100 ug/ml Tc.  3.  Cell  f r a c t i o n a t i o n and sodium dodecyl  polyacrylamide g e l e l e c t r o p h o r e s i s .  sulfate-  Whole c e l l  protein  e x t r a c t s were obtained as d e s c r i b e d by Nicas and Hancock (1980).  B r i e f l y , overnight c u l t u r e s were c e n t r i f u g e d and  23  Table I.  Bacterial  strains Description  Strain  Source/ Reference  P. aeruginosa PAO PA01  Hl03(pMT1000)  c o n t a i n s plasmid pMTlOO  T h i s study  H242  PA01  S. Benson  H287  ATCC #19305  H553  Tn501 i n s e r t i o n mutant of H103 n o n - d e r e p r e s s i b l e for a l k a l i n e phosphatase, phospholipase C, phosphatebinding p r o t e i n and p r o t e i n  T h i s study  H556  Tn501 i n s e r t i o n mutant of H103 r e q u i r i n g a r g i n i n e  T h i s study  H576  Tn501 i n s e r t i o n mutant of H103 n o n - d e r e p r e s s i b l e f o r protein P  T h i s study  H585  H586  H587  PA01594  wild  Hancock & Carey, 1979  HI 03  type  thr-102  Phosphate-binding p r o t e i n d e f i c i e n t mutant of H242 s e l e c t e d as c o n s t i t u t i v e for a l k a l i n e phosphatase A l k a l i n e phosphatase c o n s t i t u t i v e mutant of H242 producing a d e f e c t i v e phosphate-binding p r o t e i n Mutant s t r a i n of H242 constitutive for a l k a l i n e phosphatase, phospholipase C, phosphatebinding p r o t e i n and p r o t e i n P. met-28 ilv-202 str-1  rmo-53  24  T h i s study  T h i s study  T h i s study  M. Tsuda, Tokyo  Table I. - continued Strain  Description  Source/ Reference  P. aeruginosa PAO PAO1594(pMT1000)  c o n t a i n s plasmid pMTlOOO  Tsuda et a l , 1984  Pseudomonadaceae P. putida  ATCC # 12633  p. f l u o r e s c e n s  ATCC # 949  p. c h l o r o r a p h i s  ATCC # 9446  p. a u r e o f a c i e n s  ATCC # 13985  T  p. cepac i a  ATCC # 25609  T  p. pseudomallei  ATCC # 23343  T  p. ac idovorans  s t r a i n 29  p. s t u t z e r i  ATCC # 17588  T  p. syringae  ATCC # 19310  T  p. solanacearum  ATCC # 1 1696  p. m a l t o p h i l i a  ATCC # 13637  T  T  Warren, 1 979  T  T  Enterobacteriaceae Escherichia c o l i  K12  s t r a i n HMS174 OmpF  +  Escherichia c o l i  K12  R.A.J. Warren, U.B.C.  OmpC  +  s t r a i n JF700  Foulds and Chai, 1978  OmpF" OmpC  +  Escherichia c o l i  K12  s t r a i n JF694 OmpF" OmpC" PhoE  25  Foulds and Chai, 1979 c  Table I . - continued  Strain  Description  Salmonella  typhimurium  strain OmpC  Source/ Reference  SL1906 OmpD  OmpF  K l e b s i e l l a pneumoniae  ATCC # 13883  Enterobacter aeroqenes  ATCC # 13048  Serratia  ATCC # 13880  a  marcesens  Stocker et a l , 1979  T  T  T  ATCC, American Type C u l t u r e C o l l e c t i o n ; only the r e l e v a n t phenotypes are i n d i c a t e d ; T, type s t r a i n ; PhoE , c o n s t i t u t i v e f o r PhoE c  26  the c e l l s resuspended  i n 2 % (wt/vol) sodium dodecyl s u l f a t e  (SDS)/ 20 mM  (pH 8.0).  for  10 min,  Tris-HCl  A f t e r treatment at 100°C  r e s i d u a l c e l l s were removed by c e n t r i f u g a t i o n at  27,000 x g f o r 10 min and the r e s u l t i n g sonicated  (1 min,  supernatant  s e t t i n g 5, Biosonik s o n i c a t o r ,  S c i e n t i f i c , Rochester,NY) to shear DNA The p r e p a r a t i o n of c e l l  and reduce  envelopes was  method of Nicas and Hancock (1980).  Bronwill viscosity.  based on the  C e l l s from overnight or  l o g a r i t h m i c phase c u l t u r e s were c e n t r i f u g e d , resuspended i n 15 mM  Tris-HCl  (pH 8.0)  c o n t a i n i n g 10 ug/ml p a n c r e a t i c  deoxyribonuclease I (Sigma Chemical Co., broken psi.  i n a French pressure c e l l  S t . L o u i s , MO)  (Aminco) at 11,500-13,000  Unbroken c e l l s were removed by c e n t r i f u g a t i o n  g f o r 10 min)  (1,000 x  and the r e s u l t i n g supernatant c e n t r i f u g e d at  160,000 x g f o r 1 h. resuspended  and  The c e l l  envelope p e l l e t  was  i n d e i o n i z e d water.  Outer membranes were prepared using the  two-step  gradient method d e s c r i b e d by Hancock and Carey envelopes prepared i n 15 mM  Tris-HCl  (1979).  Cell  (pH 8.0)/ 20 % (wt/vol)  >-  sucrose were layered onto a sucrose step g r a d i e n t of 60 % (wt/vol) (top l a y e r ) and 70 % (wt/vol) (bottom l a y e r ) and c e n t r i f u g e d overnight at 183,000 x g in a Beckman SW41 rotor  (Beckman Instruments  outer membrane band was sucrose  Inc., Palo A l t o , CA).  Ti  A single  obtained at the i n t e r f a c e of the  two  solutions.  T r i t o n X-100-Tris, T r i t o n X-100-Tris-EDTA and T r i t o n 100-Tris-lysozyme e x t r a c t i o n of outer membranes was  27  X-  exactly  as d e s c r i b e d by Hancock et a l . (1981a). SDS-polyacrylamide g e l e l e c t r o p h o r e s i s was performed as d e s c r i b e d by Hancock and Carey (1979) using a 12 % (wt/vol) acrylamide running g e l . Two-dimensional  (unheated x heated) SDS-polyacrylamide  g e l e l e c t r o p h o r e s i s was previously  based on a method d e s c r i b e d  (Russel, 1976).  Samples s o l u b i l i z e d at room  temperature were e l e c t r o p h o r e s e d on an SDS-polyacrylamide s l a b g e l and the lanes e x c i s e d  (1st dimension).  The g e l  s t r i p s were then placed i n screw capped tubes conta-ining 2 % (wt/vol) SDS/20 mM 10 min.  Tris-HCl  (pH 6.8)  and heated at 88°C f o r  The heated g e l s t r i p s were l a i d h o r i z o n t a l l y a c r o s s  the top of a second SDS-polyacrylamide s l a b g e l , sealed i n place with 0.8  % agarose (Biorad, Richmond, CA)  e l e c t r o p h o r e s e d again (2nd dimension). urea was  Where i n d i c a t e d ,  included in the second dimension s l a b g e l s at a  f i n a l c o n c e n t r a t i o n of  4.  and  6M.  P u r i f i c a t i o n of p r o t e i n P.  The s o l u b i l i z a t i o n  in T r i t o n  X-100-EDTA of outer membranes from p h o s p h a t e - d e f i c i e n t and chromatography  on a DEAE-Sephacel  column was e x a c t l y as  d e s c r i b e d p r e v i o u s l y for p r o t e i n D1 p u r i f i c a t i o n and Carey, 1980).  cells  (Hancock  Protein P-containing fractions  ( e x h i b i t i n g some contamination with p r o t e i n F) were pooled and concentrated 5 - f o l d by d i a l y s i s against 20 % (wt/vol) p o l y e t h y l e n e g l y c o l 20,000 (Sigma Chemical Co.). pooled concentrate a 4 - f o l d excess of SDS  28  To  this  over T r i t o n  X-100  (i.e.  2 % (wt/vol) SDS) was added and the s o l u t i o n was made  3 mM f o r sodium a z i d e .  T h i s s o l u t i o n was added to a  Sepharose 4B column (46 x 2 cm) p r e - e q u i l i b r a t e d with 0.1 % (wt/vol) SDS/ 5 mM T r i s - H C l (column  b u f f e r ) and e l u t e d with column b u f f e r .  milliliter for  (pH 8.0)/ 3 mM sodium  Three-  f r a c t i o n s were c o l l e c t e d a t 12 ml/h and t e s t e d  absorbance  at 280 nm and for p r o t e i n composition on SDS-  polyacrylamide g e l s .  P r o t e i n P, s l i g h t l y contaminated  p r o t e i n F, e l u t e d j u s t a f t e r t h e v o i d T r i t o n X-100 e l u t e d i n subsequent containing  azide  with  volume, whereas the  fractions.  The p r o t e i n P-  f r a c t i o n s were again pooled, concentrated and  r e a p p l i e d to the Sepharose 4B column d e s c r i b e d  above.  The  r e s u l t a n t p r o t e i n P peak was homogeneous as determined by SDS-polyacrylamide  5.  gel electrophoresis.  A c e t y l a t i o n of p r o t e i n P.  P r o t e i n P was a c e t y l a t e d  using a c e t i c anhydride as described (1981).  by Tokunaga et a l  P r o t e i n P (500 ug i n 1.2 ml of 0.1 % (wt/vol) SDS/  10 mM T r i s - H C l  (pH 8.0)/ 3 mM sodium a z i d e ) was d i l u t e d i n  50 mM sodium phosphate b u f f e r to a f i n a l volume of 2 ml.  (pH 6.8)/ 0.1 % (wt/vol) SDS  The r e a c t i o n was s t a r t e d by the  a d d i t i o n of 2 u l of a c e t i c anhydride, which was subsequently added at 10 min i n t e r v a l s over a p e r i o d of 1 h. the s o l u t i o n was monitored  with a microprobe  and maintained  at approximately 7 with a l i q u o t s of 5 M NaOH. was  The pH of  The s o l u t i o n  allowed to s i t f o r an a d d i t i o n a l hour before i t was  d i a l y s e d against  one l i t e r  of 35 mM sodium phosphate b u f f e r  29  (pH 6.8)/ 0.1 % (wt/vol) SDS f o r 4 h. was changed and d i a l y s i s continued  The d i a l y s i s b u f f e r  overnight  i n the same  buffer.  6.  Immunological methods.  Antigen  s p e c i f i c i t y and t i t r e of  the v a r i o u s a n t i s e r a was determined by the enzyme-linked immunosorbent assay  (ELISA) as d e s c r i b e d by Mutharia and  .Hancock (1983) using 20 ug/ml f i n a l c o n c e n t r a t i o n of antigen in the w e l l s .  The Western immunoblot procedure,  involving  the e l e c t r o p h o r e t i c t r a n s f e r of SDS-polyacrylamide g e l electrophoretograms  to n i t r o c e l l u l o s e and subsequent  immunostaining, has been d e s c r i b e d p r e v i o u s l y (Mutharia and Hancock, 1983).  In cases where a peroxidase  used as the second antibody,  conjugate  azide was omitted  b u f f e r s and the b l o t s developed  was  from a l l  using the peroxidase  s u b s t r a t e descibed below.  7.  P r e p a r a t i o n of p r o t e i n P-phosphatidyl  choline vesicles.  Phosphatidyl c h o l i n e . ( 0 . 5 umol i n CHCl^) was d r i e d under N and d e s s i c a t e d f o r 30 min at room temperature.  Protein P  (200 ug) was added to the d r i e d l i p i d and vortexed sec.  2  f o r 30  Deionized water was added to the p r o t e i n - l i p i d  s o l u t i o n to make a f i n a l volume of 1 ml. scraped  The l i p i d was  from the s i d e s of the tube with a s p a t u l a and the  s o l u t i o n vortexed a f u r t h e r 30 sec.  Following s o n i c a t i o n (4  pulses of 15 sec each at s e t t i n g 50, Biosonik s o n i c a t o r , B r o n w i l l ) the v e s i c l e s o l u t i o n was cooled on i c e and stored  30  at -20°C.  8.  P r e p a r a t i o n of p r o t e i n a f f i n i t y columns. a.  P r o t e i n F-sepharose.  P r o t e i n F was  p u r i f i e d according to the procedure  partially  of Yoshimura et a l .  (1983), o m i t t i n g the column chromatography step.  The  r e s u l t a n t p r e p a r a t i o n , which had only minor contamination with p r o t e i n H2,  was  SDS-polyacrylamide  0.5 M NaCl/ 0.1 an absorbance  90 % pure as judged  gel electrophoresis.  p u r i f i e d p r o t e i n was column (15x1.5 cm)  approximately  e q u i l i b r a t e d with 0.1  % (wt/vol) SDS.  of 280 nm)  0.1  (PBS)  1 ml.  b.  (Pharmacia,  Upsalla,  The  f i n a l column volume  was  washed  to remove excess detergent.  Protein P - a f f i - g e l  10.  Purified protein P  was  (Biorad) column e q u i l i b r a t e d  M a c e t i c acid/sodium a c e t a t e b u f f e r , pH 5.0/  (wt/vol) SDS. 280 nm,  g dry weight) as  (Mutharia and Hancock 1983) c o n t a i n i n g  passed across a B i o g e l P-10 with 0.1  (measured at  P r i o r to use, the column was  e x h a u s t i v e l y with PBS  8.3)/  s t o r e d at 4°C i n phosphate-buffered  % ( v o l / v o l ) T r i t o n X-100.  approximately  (pH  c r o s s - l i n k e d to CNBr-activated  recommended by the manufacturer  (pH 7.4)  3  were pooled and the p r o t e i n  was  The column was  (Biorad)  M NaHC0  Peak f r a c t i o n s  Sepharose 4B beads (approximately 0.5  saline  partially  passed across a B i o g e l P-10  (approximately 3.5 mg)  Sweden).  The  by  0.1  Peak f r a c t i o n s , measured at an absorbance  were pooled and the p r o t e i n  approximately 0.65  ml of A f f i - g e l  31  (1.5 mg)  % of  c r o s s - l i n k e d to  10 (Biorad) f o r 2 h at  room temperature column was  as recommended by the manufacturer.  s t o r e d at 4°C  T r i t o n X-100.  P r i o r to use, the column was  e x h a u s t i v e l y with PBS c.  i n PBS c o n t a i n i n g 0.1  (wt/vol)  washed  to remove excess d e t e r g e n t .  Phosphate-binding  phosphate-binding  %  The  protein-Sepharose.  p r o t e i n was  Purified  passed a c r o s s a B i o g e l  P-10  (Biorad) column (at 20 % of column volume) e q u i l i b r a t e d with 0.1  M NaHC0 / 0.5 M NaCl 3  (pH 8.0)  ( c o u p l i n g b u f f e r ) and  the  p r o t e i n - c o n t a i n i n g f r a c t i o n s were pooled ( f i n a l volume of 5 ml at an absorbance  at 280 nm of 0.38)  a c t i v a t e d Sepharose 4B p r o t e i n F.  The  The column was  9.  (Pharmacia)  e x a c t l y as d e s c r i b e d f o r  f i n a l column volume was s t o r e d i n 20 mM  Tris-HCl  P r e p a r a t i o n of p r o t e i n P - s p e c i f i c a.  and coupled to CNBr-  approximately (pH 8.0)  1 ml.  at 4°C.  antisera.  T r i m e r - s p e c i f i c . • A n t i b o d i e s to p r o t e i n P t r i m e r s  were r a i s e d i n New immunization  Zealand White r a b b i t s using the  schedule.  P r o t e i n P (50 ug) was  following  injected  subcutaneously at weekly i n t e r v a l s over a three week p e r i o d . F o l l o w i n g t h i s , the r a b b i t s were r e s t e d , without for  three weeks.  immunization  injection,  T h i s c y c l e of three weeks of weekly  followed by three weeks without  injection  was  repeated twice more, before a f i n a l subcutaneous i n j e c t i o n of  p r o t e i n P (50 ug) was  given.  For the f i r s t  i n j e c t i o n s , p r o t e i n P ( d i l u t e d i n PBS) Freund's  Incomplete  otherwise i t was  Adjuvant  was  two  mixed 1:1  ( D i f c o , D e t r o i t , MI,  i n j e c t e d i n PBS  32  alone.  Two  with  USA),  weeks a f t e r  the  final  i n j e c t i o n , blood was  c o l l e c t e d and the serum obtained  a f t e r c e n t r i f u g a t i o n of c l o t t e d blood. The  r e s u l t a n t antiserum contained a n t i b o d i e s to  lipopolysaccharide p r o t e i n P.  (LPS) and p o r i n p r o t e i n F as w e l l as to  Thus i n order to generate a p r o t e i n P t r i m e r -  s p e c i f i c antiserum i t was  necessary to remove these  contaminating a c t i v i t i e s .  The antiserum was  against whole c e l l s of P. aeruginosa PA01 follows.  first  adsorbed  s t r a i n H103  as  C e l l s from a 10 ml overnight c u l t u r e i n L-broth  were harvested by c e n t r i f u g a t i o n  i n a t a b l e top c e n t r i f u g e  and washed twice with Hank's Balanced S a l i n e S o l u t i o n (Gibco, B u r l i n g t o n , Ont, resuspended  directly  Can).  The c e l l p e l l e t  i n t o 1.0 ml of the antiserum, p l a c e d i n  a 1.5 ml polypropylene c e n t r i f u g e tube S c i e n t i f i c , Los Angeles, CA, USA) at room temperature  was  and  (Evergreen incubated.for 45 min  i n an end-over-end shaker.  The  cells  were then p e l l e t e d and the antiserum-containing supernatant adsorbed a second time a g a i n s t a f r e s h batch of washed cells.  Whole c e l l a d s o r b t i o n e f f e c t i v e l y removed a l l  a n t i b o d i e s d i r e c t e d a g a i n s t smooth LPS as measured by ELISA and confirmed by Western immunoblots. no decrease  however,  i n antibody t i t r e to p r o t e i n F (or p r o t e i n P).  The'adsorbed  antiserum  45 min at room temperature column  There was,  (2.5x0.7 cm).  p e r i o d , the column was  (600 u l ) was  then incubated for  on a p r o t e i n F-Sepharose a f f i n i t y  At the completion of the i n c u b a t i o n washed with 4 ml of PBS and  unbound a n t i b o d i e s c o l l e c t e d  i n 400 u l f r a c t i o n s .  33  the Fractions  c o n t a i n i n g a n t i b o d i e s to p r o t e i n P, as determined were pooled to y i e l d a p r o t e i n F-adsorbed Adsorption of the antiserum  by ELISA,  antiserum.  on the p r o t e i n F-Sepharose  column f a c i l i t a t e d removal of 99 % of the antibody to p r o t e i n F, with no decrease P.  i n antibody  The p r o t e i n F-adsorbed antiserum,  t i t r e to p r o t e i n  however, c o u l d only  poorly d i s t i n g u i s h between phosphate-limited c e l l s p r o t e i n P (eg. s t r a i n H103) and phosphate-limited d e f e c t i v e i n p r o t e i n P production  producing cells  (eg. s t r a i n H553).  Therefore, the p r o t e i n F-adsorbed antiserum was adsorbed  activity  subsequently  twice a g a i n s t phosphate-limited P. aeruginosa  s t r a i n H553 c e l l s as d e s c r i b e d above f o r s t r a i n H103.  The  r e s u l t a n t antiserum was p r o t e i n P t r i m e r - s p e c i f i c (see Chapter One). b.  Monomer s p e c i f i c .  were r a i s e d as f o l l o w s .  A n t i b o d i e s to p r o t e i n P monomers  Purified protein P  was heated at  88°C f o r 15 min to promote the heat denaturation of t r i m e r s to form monomers (see Chapter protein  One). A f t e r c o o l i n g , the  (20 ug) , suspended i n 0.1 ml PBS (Mutharia and  Hancock, 1983), was i n j e c t e d i n t e r p e r i t o n e a l l y  i n t o Balb/c  mice (Department of M i c r o b i o l o g y breeding colony, U n i v e r s i t y of B r i t i s h Columbia, Vancouver, Canada). repeated.on  The i n j e c t i o n was  days 14, 28, 35, 42, 45 and 50.  c o l l e c t e d 7 days a f t e r the f i n a l  The blood was"  i n j e c t i o n and the serum  obtained a f t e r c e n t r i f u g a t i o n of c l o t t e d blood.  Antibodies  to LPS, as measured by ELISA using p u r i f i e d LPS as the a n t i g e n , were removed by adsorbtion against whole c e l l s of  34  P. aeruginosa PA01 s t r a i n HI 03 (see above). c. vesicles.  Antiserum to p r o t e i n P i n phosphatidyl c h o l i n e Antiserum to p r o t e i n P v e s i c l e s was r a i s e d i n  Balb/c mice as f o l l o w s .  Protein P vesicles  (20 ug p r o t e i n  P) were preincubated at 37°C i n the presence of monoclonal a n t i b o d i e s MA5-8 (LPS c o r e - s p e c i f i c ) (Hancock et a l . , and MA1-8  (LPS 0 a n t i g e n - s p e c i f i c ) (Hancock et a l . ,  f o r 30 min p r i o r t o subcutaneous  injection.  1983a)  1983a)  The  monoclonals were p r e v i o u s l y t i t r a t e d a g a i n s t p r o t e i n P v e s i c l e s i n the ELISA to determine the amounts of the 2 a n t i s e r a r e q u i r e d to block a l l LPS molecules present i n a given amount of p r o t e i n P-containing v e s i c l e s .  The  i n j e c t i o n s were repeated on days 14,21,28,35,42,56,70,84, and 98. One week a f t e r the f i n a l  i n j e c t i o n , the blood was  c o l l e c t e d and the antiserum obtained a f t e r c e n t r i f u g a t i o n of c l o t t e d blood.  10.  I s o l a t i o n of a p r o t e i n P - d e f i c i e n t mutant. a.  Tn50l i n s e r t i o n mutagenesis.  P. aeruginosa  Hl03(pMT1000) was c u l t u r e d overnight i n L-broth at 30°C i n the presence of H g C l .  D i l u t i o n s were p l a t e d onto L-agar  2  plates containing HgCl  2  and incubated at 42°C.  Colonies  growing up a f t e r 24 h ( i n s e r t i o n mutants) were p i c k e d onto g r i d s on f r e s h L-agar p l a t e s c o n t a i n i n g H g C l once again a t 42°C.  2  and incubated  A f t e r 24 h, these p l a t e s were then  r e p l i c a p l a t e d onto L-agar p l a t e s c o n t a i n i n g H g C l  2  and onto  p h o s p h a t e - d e f i c i e n t minimal medium p l a t e s , followed by  35  incubation  at 42°C.  The  r e p l i c a s on r i c h medium were  r e t a i n e d as a master set from which d e s i r e d mutants, once i d e n t i f i e d , could be rescued. phosphate-deficient  The  r e p l i c a s grown on  the  minimal medium p l a t e s were screened for  p r o t e i n P - d e f i c i e n t mutants. b.  S e l e c t i o n of a p r o t e i n P - d e f i c i e n t mutant using  p r o t e i n P t r i m e r - s p e c i f i c antiserum. r e s i s t a n t to H g C l  at 42°C and  2  phosphate-deficient by contact  Bacterial  clones  r e p l i c a p l a t e d onto  minimal medium p l a t e s were t r a n s f e r r e d  onto n i t r o c e l l u l o s e f i l t e r d i s c s  S c h u e l l Inc.,  a  Keene, NH,  USA,  (Schleicher  type BA85, 82 mm).  and  The  n i t r o c e l l u l o s e r e p l i c a s were subsequently screened, by a modification  of the procedure of Helfman et a l (1983), f o r  the absence of p r o t e i n P using  the above-described p r o t e i n P  t r i m e r - s p e c i f i c antiserum. B l o t t e d f i l t e r s were placed in  10 ml  of 50 mM  containing for  Tris-HCl  (pH 7.4)/  1 h at 37°C.  The  % NaCl/ 10 mM  NaCl/ 5 mM  Tris-HCl  7.4);  Towbin et a l  by three  5 min  washes i n 10 ml  albumin  (10 ml) and  2  gently  containing  PBS. of  to p r o t e i n P were was  3 % (wt/vol) bovine serum  then incubated on the  36  1979)  (with an ELISA t i t r e  at a 1/2000 d i l u t i o n of the antiserum) in PBS  shaken  (pH  2000, i n d i c a t i n g that a n t i b o d i e s  1:249  MgCl  i n 10 ml of T r i s - b u f f e r e d s a l i n e  P r o t e i n P t r i m e r - s p e c i f i c antiserum  diluted  dishes  f i l t e r s were then washed twice at room  with shaking, followed  detectable  t50 mM  3 % (wt/vol) bovine serum albumin and  temperature f o r 10 min (0.9  in i n d i v i d u a l P e t r i  filters  overnight  at  room temperature  with shaking.  The f i l t e r s  were  subsequently washed three times f o r 10 min at room temperature  i n 10 ml PBS.  A f f i n i t y p u r i f i e d goat  r a b b i t IgG (H+L)-peroxidase  conjugated antibody  L a b o r a t o r i e s , West Chester, PA, USA) d i l u t e d  anti-  (Cappel  1:999 i n 10 ml  PBS c o n t a i n i n g 3 % (wt/vol) bovine serum albumin  was then  incubated on the f i l t e r s at 37°C, again with shaking.  After  2 h of i n c u b a t i o n , the f i l t e r s were washed twice f o r 10 min each i n 10 ml of PBS at room temperature,  f o l l o w e d by three  washes of 10 min each i n 10 ml of T r i s - b u f f e r e d  saline.  Peroxidase s u b s t r a t e (30 mg chloro-4-naphthol (Sigma) i n 10 ml of methanol/ 50 ml of T r i s - b u f f e r e d s a l i n e / 0.02 ml H 0 2  2  (30 % ( v o l / v o l ) ) was then added (10 m l / f i l t e r ) and the filters  incubated at 37°C u n t i l c o l o u r developed.  colonies f a i l i n g  Those  to develop c o l o u r were i d e n t i f i e d and  picked from the master p l a t e s and screened f o r the absence of  p r o t e i n P i n SDS-polyacrylamide  cell  envelopes.  11.  Phosphate t r a n s p o r t assays.  g e l s of phosphate-limited  Overnight c u l t u r e s of  aeruginosa grown i n p h o s p h a t e - d e f i c i e n t medium harvested by f i l t r a t i o n  were  and washed with three volumes of  minimal Hepes-buffered medium without phosphate. c e l l s were resuspended  by v o r t e x i n g i n the same  l e s s medium at a f i n a l  absorbance  s t o r e d on i c e u n t i l needed. for  P.  Washed phosphate-  at 600 nm of 0.2-0.3 and  C e l l s c o u l d be s t o r e d on i c e  up to 3 h without any change i n c e l l d e n s i t y or any  37  signs of c e l l damage (measured as release of p e r i p l a s m i c a l k a l i n e phosphatase i n t o the medium).  Pr«ior to assaying  phosphate accumulation, c e l l s were shaken at 37°C f o r 5-25 min. To assay phosphate uptake, 1 ml samples of prewarmed c e l l s were added to 10 ml c u l t u r e tubes c o n t a i n i n g r a d i o a c t i v e l y l a b e l l e d orthophosphate.  The c e l l s were  vortexed to ensure adequate a e r a t i o n and 200 u l a l i q u o t s were removed at v a r i o u s times and f i l t e r e d on n i t r o c e l l u l o s e membrane f i l t e r cups (0.45 urn d i a , Amicon Corp) i n an Amicon vacuum m a n i f o l d .  F i l t e r e d c e l l s were washed twice with 1.5  ml of minimal Hepes-buffered medium c o n t a i n i n g u n l a b e l l e d phosphate.  1 mM  The f i l t e r s were then removed and  counted i n 3 ml of PCS s c i n t i l l a t i o n c o c k t a i l some cases i t was necessary to d i l u t e c e l l s  (Amersham). In  i n prewarmed  phosphate-less minimal Hepes-buffered medium a t the time of assay (1 ml f i n a l volume) due t o e x c e s s i v e l y r a p i d t r a n s p o r t r a t e s of u n d i l u t e d c e l l c u l t u r e s .  12.  Enzyme assays.  A l k a l i n e phosphatase was measured  p a r a - n i t r o p h e n y l phosphate  using  (pNPP) as the chromogenic  s u b s t r a t e a t a f i n a l c o n c e n t r a t i o n of 1 mg/ml i n 0.1 M T r i s HCl nm.  (pH 8.5).  The assay was read a t an absorbance of 410  Beta-lactamase was measured  using, as the s u b s t r a t e , the  chromogenic beta-lactam n i t r o c e f i n at a f i n a l c o n c e n t r a t i o n of  0.06 mg/ml i n 50 mM sodium phosphate b u f f e r  assay was read at an absorbance of 540 nm. activity  was measured  (pH 7 ) . The  Phospholipase C  using p a r a - n i t r o p h e n y l phosphorylcholine  38  (NPPC) as the chromogenic s u b s t r a t e at a f i n a l of 120 mg/ml in 60 % ( v o l / v o l ) g l y c e r o l / 0.25 7.4).  13.  The  assay was  Nitrocefin  p e r m e a b i l i t y was  p e r m e a b i l i t y assay.  millipore  Intact c e l l  of 0.22  One  taking  fraction  slowly squeezed through a  urn pore s i z e to obtain a c u l t u r e  while the other  f r a c t i o n was  left  unfiltered.  Equal volumes of each f r a c t i o n were t r a n s f e r r e d to cuvettes.  The  cuvette c o n t a i n i n g the c u l t u r e  was  placed  in the reference beam of a Perkin-Elmer  CT,  USA)  c u v e t t e , c o n t a i n i n g i n t a c t c e l l s and Nitrocefin,  a chromogenic  added to each cuvette  and  the d i f f e r e n t i a l rate of conversion  nitrocefoic  a c i d was  using a coupled  other  was  recorded  placed  beta-lactam,  ( f i n a l c o n c e n t r a t i o n of 0.06  Perkin-Elmer  mg/ml)  of n i t r o c e f i n  to  at an absorbance of 540  nm  model 561  recorded beta-lactamase a c t i v i t y was intact c e l l a c t i v i t y .  (Norwalk,  The  supernatant,  was  separate  supernatant  Lambda 3 dual beam spectrophotometer.  in the sample beam.  the  beta-lactamase  them i n two.  i n t o a syringe and  filter  supernatant  Outer membrane  measured on growing c u l t u r e s by f i r s t  taken up  (pH  nm.  determined using a m o d i f i c a t i o n of  a l i q u o t s of c e l l s and d i v i d i n g was  M Tris-HCl  read at an absorbance of 410  method of Angus et a l . (1982). a c t i v i t y was  concentration  chart r e c o r d e r .  The  a d i r e c t measure of  Because beta-lactamase has been shown  to be p e r i p l a s m i c , the a c t i v i t y of i n t a c t c e l l s at a given concentration lactam,  i s l i m i t e d by the d i f f u s i o n of the  beta-  in t h i s case n i t r o c e f i n , across the outer membrane  39  rather than by the amount of enzyme.  From  theory  (Zimmermann and R o s s e l e t ,  1977), the steady  h y d r o l y s i s of beta-lactam  in i n t a c t c e l l s  rate of beta-lactam  d i f f u s i o n across  s t a t e r a t e of  ( i v  n t  ) equals  the  the outer membrane  (Vp)  and hence provides a measure of outer membrane p e r m e a b i l i t y . P e r m e a b i l i t y parameters (C) were c a l c u l a t e d using formula  v  Rosselet  i  n  t  =  V  =  ^ out  c  S  D  ~ in^ S  a c C 0 E  "ding  to Zimmermann  (1977), where C = p e r m e a b i l i t y parameter; S  c o n c e n t r a t i o n of s u b s t r a t e  periplasm)  (which i s <<  and Q u t  =  ( n i t r o c e f i n ) o u t s i d e the c e l l  S. = c o n c e n t r a t i o n of s u b s t r a t e i n s i d e the c e l l in  14.  the  s t  ,  and  and  ( i . e . in the  thus n e g l i g i b l e ) .  Osmotic shock and p u r i f i c a t i o n of the phosphate-binding  protein.  P. aeruginosa  phosphate-deficient p e r i p l a s m i c 34K centrifugation  PA01  s t r a i n H103  medium to induce  protein.  was  grown in  the s y n t h e s i s of  the  Induced c e l l s were harvested  (10,000 x g for 10 min)  and  subjected to  by two  rounds of the T r i s - H C l / M g C l / c o l d shock procedure 2  d e s c r i b e d by Hoshino and Kageyama (1980).  The  shocked c e l l s  were removed by c e n t r i f u g a t i o n (10,000 x g f o r 10 min) the supernatant  concentrated  Amicon pressure  filtration  Corp, Danvers, MA,  USA).  approximately  using a PM10  50-fold via  microfilter  Remaining whole c e l l s and  were removed by c e n t r i f u g a t i o n in a c l i n i c a l centrifuge.  The  concentrated  e q u i l i b r a t e d with 20 mM  (Amicon debris  t a b l e top  shock f l u i d s were then  d e s a l t e d by passage over a B i o g e l P-10  (Biorad) column  T r i s - H C l (pH 7.4).  40  and  The  eluted  p r o t e i n peak was a p p l i e d to a DEAE-Sephacel column a l s o e q u i l i b r a t e d with 20 mM binding was  Tris-HCl  (Pharmacia) (pH 7.4).  The  p r o t e i n d i d not bind to DEAE-Sephacel at t h i s pH and  c o l l e c t e d i n the flow through f r a c t i o n .  containing Sepharose  f r a c t i o n s were pooled and a p p l i e d to a  CM-  (Pharmacia) column e q u i l i b r a t e d with 20 mM  acetate-acetic  acid buffer  (pH 5.0).  p r o t e i n bound to the column and was gradient  Binding p r o t e i n  of 0.1  between 0.1  to 0.4 M.  At t h i s pH the  sodium binding  e l u t e d with a NaCl  The binding  p r o t e i n e l u t e d at  and 0.2 M NaCl as a s i n g l e peak of homogeneous  p r o t e i n as determined by SDS-polyacrylamide g e l 32 electrophoresis.  P-orthophosphate-bindmg  activity  was  monitored at a l l stages of the p u r i f i c a t i o n . 15.  F i l t e r assay of phosphate  extracts  binding.  Periplasmic  (shock f l u i d s ) . a n d p u r i f i e d phosphate-binding  p r o t e i n were screened f o r t h e i r a b i l i t y to bind  phosphate  u t i l i z i n g a n i t r o c e l l u l o s e f i l t e r binding assay based on the methodology described binding  protein.  Eppendorf  by Lever (1972) for the h i s t i d i n e -  B r i e f l y , p r o t e i n e x t r a c t s were added to  tubes in a f i n a l volume of 250 u l c o n t a i n i n g  1 uM  32 P-orthophosphate phosphate,  ( s p e c i f i c a c t i v i t y = 1 mCi/umol  Amersham) and  10 mM  Tris-HCl  (pH 8.0).  After 5  min at 23°C 100 u l a l i q u o t s were removed and f i l t e r e d n i t r o c e l l u l o s e membrane f i l t e r s MA,  USA,  type HA,  0.45  with 600 u l of 10 mM  ( M i l l i p o r e Corp., Bedford,  urn pore s i z e ) .  LiCl,  on  A f t e r washing  once  the f i l t e r s were removed and 41  counted i n 5 ml of PCS aqueous s c i n t i l l a t i o n c o c k t a i l (Amersham). binding, the  To determine the s p e c i c i t y of the phosphate  various  reaction  16.  i n h i b i t o r s , as i n d i c a t e d , were included i n  mixture.  Equilibrium  dialysis.  orthophosphate binding equilibrium  To determine the Kd f o r  to the phosphate-binding p r o t e i n , the  d i a l y s i s technique was employed.  D i a l y s i s bags  (Spectrapor, 6.4 mm d i a , Spectrum Medical I n d u s t r i e s Inc., Los Angeles, CA, USA) were f i l l e d with 7 ug of p u r i f i e d phosphate-binding p r o t e i n binding  protein  i n a f i n a l volume of 300 u l .  s o l u t i o n s were d i a l y s e d against  The  40 ml of  r a d i o a c t i v e l y l a b e l l e d orthophosphate i n 50 ml c o n i c a l tubes.  The concentration  5.0 uM.  of phosphate ranged from 0.1 uM to  A f t e r d i a l y s i s f o r 24 h at 4°C, 25 u l a l i q u o t s ( i n  duplicate)  were removed from the d i a l y s i s bags and from the  s o l u t i o n s surrounding the bags and counted s e p a r a t e l y  in 3  ml of PCS aqueous s c i n t i l l a t i o n c o c k t a i l (Amersham).  17.  I s o l a t i o n of mutants l a c k i n g the phosphate-binding  protein.  D i e t h y l s u l f a t e mutagenesis of P. aeruginosa PA01  s t r a i n H242 was c a r r i e d out as d e s c r i b e d  by T. Nicas (Ph.D.  Thesis, U.B.C., Vancouver, Can, 1983) with An  overnight c u l t u r e  saturated buffer 1:49  modifications.  (0.1 ml) was resuspended i n 5 ml of a  s o l u t i o n of d i e t h y l s u l f a t e i n 50 mM sodium Hepes  (pH 7.0) f o r 30 min at 25°C.  C e l l s were then d i l u t e d  i n t o proteose peptone No. 2 broth and allowed to grow  42  overnight at 37°C.  A f t e r overnight growth,  c e l l s were harvested by c e n t r i f u g a t i o n  mutagenized  (10,000 x g f o r 10  min) and washed three times i n phosphate-less minimal Hepesb u f f e r e d medium.  The washed c e l l s were resuspended  same medium and d i l u t i o n s were p l a t e d onto s u f f i c i e n t medium agar p l a t e s  (1.0 mM  in the  phosphate-  phosphate) c o n t a i n i n g  20 ug/ml 5-bromo-4-chloro-3-indolyl-phosphate-p-toluidine (XP)  (Bachem).  I t was  necessary to d i s s o l v e the XP i n  d i m e t h y l s u l f o x i d e p r i o r to i t s a d d i t i o n to p l a t e s but the f i n a l c o n c e n t r a t i o n of d i m e t h y l s u l f o x i d e was (vol/vol).  T h i s medium i d e n t i f i e d a l k a l i n e  < 1 % phosphatase  c o n s t i t u t i v e mutants as blue-green c o l o n i e s , the c o l o u r r e s u l t i n g from the h y d r o l y s i s of XP by a l k a l i n e  phosphatase.  Wild-type c e l l s , which were repressed f o r a l k a l i n e phosphatase  production i n t h i s medium, were non-pigmented.  ( A l k a l i n e phosphatase  c o n s t i t u t i v i t y i s a phenotype of  phosphate-binding p r o t e i n - d e f i c i e n t mutants i n E. c o l i (Brickman and Beckwith,  1975)).  37°C blue-green pigmented  A f t e r overnight growth at  c o l o n i e s were p i c k e d and  cultured  overnight i n p h o s p h a t e - d e f i c i e n t medium (to induce the phosphate-binding p r o t e i n ) .  Shock f l u i d s and whole c e l l  e x t r a c t s were obtained and screened using g e l e l e c t r o p h o r e s i s f o r the absence p r o t e i n under inducing c o n d i t i o n s .  43  SDS-polyacrylamide  of the phosphate-binding  18.  Construction  column.  of a r a b b i t a n t i - p r o t e i n P immunoadsorbant  Protein P t r i m e r - s p e c i f i c antibodies  free of serum p r o t e i n s by  incubating  specific  r a b b i t antiserum  (200  affi-gel  a f f i n i t y column (1.8x0.7 cm).  room temperature for 45 min  and  n o n - s p e c i f i c a l l y bound m a t e r i a l ,  + 0.25  containing  0.1  respectively.  one  M glycine-HCl  l i t r e of 0.1  M borate b u f f e r  9.0)/0.1 M KC1) The  absorbance at 280  The  2.5)  enough  increase fractions  dialysed antibodies  nm  of 0.22)  9.0)  manufacturer.  as recommended by  stored at 4°C  19.  E l e c t r o p h o r e t i c e l u t i o n of p r o t e i n P from  one  an  in  0.5  M borate the 1.0  PBS.  SDS-  Phosphate-limited outer membranes of  s t r a i n H103  100-Tris-EDTA (pH 8.0)  M  approximately  The  P. aeruginosa PA01  with  (Pharmacia) in 0.1  ml.  polyacrylamide g e l s .  (0.1  (2.3 ml at  f i n a l column volume was  column was  9.0)  were then c r o s s - l i n k e d to  for 16 h at 4°C The  (pH  dialysed  f o r 24 h at 4°C,  CNBr-activated Sepharose 4B (pH  9.  to  Hancock, 1983), were pooled and  b u f f e r change.  buffer  (pH  ul) c o l l e c t e d in tubes c o n t a i n i n g  f r a c t i o n s to between 7 and  b o r i c a c i d (pH  gm  Bound  antibody to p r o t e i n P, as measured by ELISA  (Mutharia and against  at  washed with 3 ml  s o l i d T r i s base (Schwartz-Mann, Cambridge, MA) the pH of the  P-  M NaCl, to wash o f f unbound  were e l u t e d with 3 ml  f r a c t i o n s (300  trimer-  A f t e r incubation  the column was  followed  and  the p r o t e i n P  u l ) on the above p r o t e i n  PBS  antibodies  by 3 ml PBS  were p u r i f i e d  were s o l u b i l i z e d in T r i t o n  to r e l e a s e p r o t e i n P.  44  Protein  P-  X-  containing •  f r a c t i o n s , s o l u b i l i z e d at 23°C in a  s o l u b i l i z a t i o n mix  without 2-mercaptoethanol (Hancock and  Carey, 1979)", were loaded mm  thickness)  components.  and  onto SDS-polyacrylamide g e l s  electrophoresed  A p o r t i o n of the g e l was  blue to l o c a t e the appropriate and  the corresponding  unstained-unfixed gel  was  s t a i n e d with Coomassie  t r i m e r i c band of p r o t e i n P  region of the g e l was  p o r t i o n of the g e l .  then placed  (Mutharia  The  cut from the porin-containing  and  in d i a l y s i s bags i n the presence of  Hancock, 1983)  p r o t e i n s in the p r e p a r a t i o n s  for  protein  crushed with a g l a s s rod to increase i t s surface  area and  h at 4°C  to separate  (1.5  and  0.1  % (wt/vol) SDS.  e l e c t r o e l u t i o n was  The  were e l e c t r o e l u t e d at 50V  i n a Biorad t r a n s b l o t t i n g c e l l .  The  PBS  for 2  b u f f e r used  the same as that used f o r Western  immunoblots.  20.  P u r i f i c a t i o n of phosphate s t a r v a t i o n - i n d u c e d  outer  membrane p r o t e i n s of the f l u o r e s c e n t Pseudomonads.  Two  methods were used f o r the p u r i f i c a t i o n of phosphate starvation-induced  outer membrane p r o t e i n s .  an a f f i n i t y chromatography method u t i l i z i n g immunoadsorbant column.  T r i t o n X-100  The  first  was  an  insoluble c e l l  envelopes prepared from 100 ml s t a t i o n a r y phase c u l t u r e s of phosphate-limited  c e l l s were s o l u b i l i z e d  (wt/vol) T r i t o n X-100/20 mM containing  in 1 ml 2 %  T r i s - H C l , pH 8.0/10 mM  1 mg/ml lysozyme at 37 C f o r 30 min.  X-100-EDTA-lysozyme s o l u b l e f r a c t i o n s (300  45  EDTA  The T r i t o n  ul) were  subsequently incubated  on the a n t i - p r o t e i n P immunoadsorbant  column at room temperature. column was  A f t e r 45 min  incubation,  the  washed s u c c e s s i v e l y with 3 ml of 2 % TX-100/20  T r i s - H C l , pH 8.0  and  mM  3 ml of 2 % (wt/vol) T r i t o n X-100/20  T r i s - H C l , pH 8.0/0.5 M NaCl, to remove unbound and  non-  specifically  bound m a t e r i a l r e s p e c t i v e l y .  M a t e r i a l bound  specifically  to the column was  3 ml of  e l u t e d with  (wt/vol) T r i t o n X-100/0.1 M g l y c i n e - H C l  (pH 2.5),  mM  1 % and  f r a c t i o n s c o l l e c t e d in tubes c o n t a i n i n g s o l i d T r i s base as d e s c r i b e d above. polyacrylamide The  Peak f r a c t i o n s , as determined by  g e l e l e c t r o p h o r e s i s , were  solubilized  gels.  Phosphate-limited  in T r i t o n X-100/Tris-HCl  lysozyme treatment to r e l e a s e proteins.  c e l l envelopes were  (pH 8.0)/EDTA a f t e r  (300  without  u l ) were incubated  components.  electrophoresed  The  to separate  oligomers were then e x c i s e d from the gels and The  e l u t e d p r o t e i n s (5 ml  were concentrated  10-fold a g a i n s t  (20,000 molecular  weight, Sigma Chemical Co.,  USA)  before  a g a i n s t one Tris-HCl  mm  protein  phosphate-starvation-induced  as d e s c r i b e d above.  protein elecroeluted f i n a l volume)  s o l i d polyethylene  glycol  St. L o u i s ,  being d i a l y s e d at room temperature f o r 16 h l i t r e of 0.1  (pH 8.0)  % (wt/vol) T r i t o n X-100/20  with one  b u f f e r change.  46  at  2-mercaptoethanol,  onto i n d i v i d u a l SDS-polyacrylamide g e l s (1.5  t h i c k n e s s ) and  SDS-  phosphate-starvation-inducible  S o l u b i l i z e d proteins  23°C in a s o l u b i l i z a t i o n mix loaded  pooled.  second method u t i l i z e d e l e c t r o e l u t i o n from  polyacrylamide  SDS-  mM  MO,  21.  Black l i p i d b i l a y e r experiments.  The methods used f o r  black l i p i d b i l a y e r experiments have been d e s c r i b e d i n detail  (Benz et a l . ,  1978; Benz and Hancock, 1981).  The  apparatus c o n s i s t e d of a T e f l o n chamber with two compartments separated by a small hole (0.1 mm f o r s i n g l e channel experiments; 2 mm f o r macroscopic experiments).  conductance  A membrane was formed a c r o s s the hole by  p a i n t i n g on a s o l u t i o n of 1.5 % (wt/vol) o x i d i z e d c h o l e s t e r o l i n n-decane. the  B i l a y e r formation was i n d i c a t e d by  membrane t u r n i n g o p t i c a l l y black to i n c i d e n t l i g h t . In  s i n g l e channel conductance experiments, conductance the  through  pores was measured a f t e r a p p l i c a t i o n of a given v o l t a g e ,  using a p a i r of Ag/AgCl e l e c t r o d e s i n s e r t e d i n t o the aqueous s o l u t i o n s on both s i d e s of the membrane.  The c u r r e n t  through the pores was boosted by a p r e a m p l i f i e r ,  monitored  by a storage o s c i l l o s c o p e and recorded on a s t r i p c h a r t recorder. The procedure f o r macroscopic conductance experiments has been d e s c r i b e d submitted).  (Hancock  inhibition  and Benz,  B r i e f l y , the Ag/AgCl e l e c t r o d e s were r e p l a c e d  with Calomel e l e c t r o d e s and the c u r r e n t through the pores was monitored with a K e i t h l e y 610B e l e c t r o m e t e r .  After  a d d i t i o n of p u r i f i e d p r o t e i n P to the s o l u t i o n bathing the l i p i d b i l a y e r membrane the increase i n conductance  (measured  as c u r r e n t increase) was followed f o r 15-25 min or u n t i l the rate of increase had slowed c o n s i d e r a b l y .  At t h i s  time  membrane conductance had increased 2-4 orders of magnitude.  47  The  bathing  gently  s o l u t i o n s in both compartments were s t i r r e d  (approximately 60 r e v o l u t i o n s per minute) with a  magnetic s t i r bar and compartments. the new  a l i q u o t s of phosphate added to both  S u f f i c i e n t time (30-90 sec) was  current  allowed for  l e v e l to be e s t a b l i s h e d before a d d i t i o n of  subsequent a l i q u o t s .  22.  Modified  ELISA procedure for demonstrating  a s s o c i a t i o n between p r o t e i n P and  the  an  phosphate-binding  protein. a.  Preparation  of p r o t e i n P.  p r o t e i n P ( approximately 1 mg  A s o l u t i o n of p u r i f i e d  in 0.5  made 2 % ( v o l / v o l ) f o r T r i t o n X-100 and  passed across  cm)  e q u i l i b r a t e d with 0.1  Tris-HCl was at  (pH  a Sepharose 6B  7.4)/  3 mM  against  nm)  azide  (column b u f f e r ) . 600  were pooled and  second time across  The  The  mM  column  ul fractions collected  concentrated  5-fold  g l y c o l 20,000 (Sigma)  concentrated p r o t e i n was  passed a  the Sepharose 6B column described  protein-containing  ul)  f r a c t i o n s (as determined by  20 % (wt/vol) polyethylene  ( f i n a l volume 1 ml).  and  ( f i n a l volume of 500  % ( v o l / v o l ) T r i t o n X-100/ 10  Protein-containing  absorbance at 280  was  (Pharmacia) column (20x1.5  e l u t e d with column b u f f e r and 10 ml/h.  % (wt/vol) SDS)  f r a c t i o n s (600  above  ul) again pooled  and  concentrated to y i e l d a p u r i f i e d p r o t e i n P s o l u t i o n i n T r i t o n X-100. enriched  In some experiments, p r o t e i n P was  (but not pure) p r e p a r a t i o n  gradient-eluted,  p r o t e i n P-containing  48  used as  obtained from the NaCl f r a c t i o n s from  the  an  DEAE column described d i a l y s e d against  i n s e c t i o n 3.  10 mM  Tris-HCl  (pH  These p r e p a r a t i o n s were 7.4)  f o r 6-8  h p r i o r to  being used in the ELISA. b.  Modified  ELISA procedure.  p r o t e i n , p u r i f i e d as described carbonate/bicarbonate b u f f e r  Phosphate-binding  above and  (Ruitenberg et a l . , 1974),  used to coat the bottom of the w e l l s microtitre plates  (Falcon  3912  p r o t e i n P, was containing 7.4)  I I I , Becton  Hancock, 1983).  Tris-HCl  (pH  After  7.4)]  added to the phosphate-binding  wells d i l u t e d i n a s o l u t i o n of  containing  ( v o l / v o l ) FCS.  % ( v o l / v o l ) T r i t o n X-100  After  incubation  7.4)  (diluted  and  min,  protein-  10 mM  0.5-1  serum  f o r 45  Tris-HCl and  (pH  1 %  f o r 2 h at 37°C, the  were washed 4 times with 1 % T r i t o n X-100/ 10 mM (pH  for  unbound s i t e s i n the w e l l s with f e t a l c a l f  [1 % ( v o l / v o l ) in 10 mM  was  chloride  e x a c t l y as d e s c r i b e d  c o n v e n t i o n a l ELISA (Mutharia and  (FCS)  of p o l y v i n y l  Microtest  D i c k i n s o n Labware, Oxnard, CA)  blocking  d i l u t e d in  wells  Tris-HCl  the p r o t e i n P t r i m e r - s p e c i f i c antiserum  1:999  in T r i t o n - T r i s (pH  incubated on the wells  7.4)  + 1 % FCS)  f o r 2 h at 37°C.  The  again washed 4 times with T r i t o n - T r i s (pH  was  p l a t e s were  7.4)  and  subsequently incubated f o r 2 h at 37°C in the presence of a goat-anti-rabbit antibody 1 % FCS.  (Cappell)  (H+L)-alkaline phosphatase conjugated  d i l u t e d 1:999  in T r i t o n - T r i s (pH  A f t e r washing 4 times with T r i t o n - T r i s (pH  paranitrophenyl at  IgG  phosphate (pNPP) was  1 mg/ml f i n a l c o n c e n t r a t i o n  added to the  i n 10 %  49  (vol/vol)  7.4)/ 7.4),  wells  diethanolamine  b u f f e r (pH 9.8)  (Ruitenberg et a l . , 1974)  incubated at 3'7°C or 23°C u n t i l c o l o u r developed. c o l o u r r e a c t i o n was nm  assayed  The  by measuring absorbance at  using a T i t r e t e k M u l t i s c a n ELISA reader  (Flow  A l l incubations were at 37°C.  23.  chromatography method for determining  a s s o c i a t i o n between p r o t e i n P and phosphate-binding a. column.  Phosphate-binding  protein-Sepharose  4B  405  Labs,  McLean, VA).  Affinity  and  an protein.  affinity  T r i t o n - T r i s - E D T A s o l u b l e p h o s p h a t e - d e f i c i e n t outer  membrane (enriched for p r o t e i n P) the phosphate-binding 23°C or 37°C.  After  with 3 ml of 0.1 (pH8.0)/ 10 mM  (300 u l ) was  incubated  p r o t e i n Sepharose 4B column at 1 h i n c u b a t i o n , the column was  4°C, washed  % ( v o l / v o l ) T r i t o n X-100/ 20 mM T r i s - H C l  EDTA (pH 8.0),  followed by 3 ml of the same  s o l u t i o n c o n t a i n i n g '1 M NaCl. The e l u t e d f r a c t i o n s (400 were c o l l e c t e d , d i a l y s e d overnight in 20 mM 8.0)  and concentrated 5 - f o l d a g a i n s t 20 %  (wt/vol) SDS-  gels.  b. . P r o t e i n P - A f f i g e l - 1 0 a f f i n i t y column. binding p r o t e i n - c o n t a i n i n g crude T r i s - M g C l 2 ~ c o l d shock treatment  shockates,  column e q u i l i b r a t e d with 20 mM  Phosphate-  obtained  of phosphate-limited  were d e s a l t e d by passage across a B i o g e l P-10  incubated  ul)  T r i s - H C l (pH  p o l y e t h y l e n e g l y c o l 20,000 before being run on polyacrylamide  on  by cells,  (Biorad)  T r i s - H C l (pH 8.0)  and  (300 u l ) on a p r o t e i n P - A f f i g e l - 1 0 column  e q u i l i b r a t e d with 0.1  % ( v o l / v o l ) T r i t o n X-100  50  at 4°C,  23°C  or 37°C. A f t e r Tris-HCl  1 h, the column was washed with 3 ml 20 mM  (pH 8.0) + T r i t o n X-100, followed by 3 ml 20 mM  T r i s - H C l .(pH 8.0)/ 1 M NaCl + T r i t o n X-100.  Eluted  f r a c t i o n s (400 u l ) were d i a l y s e d overnight i n 20 mM T r i s - H C l (pH 8.0), concentrated 5 - f o l d a g a i n s t p o l e t h y l e n e g l y c o l as d e s c r i b e d above and run on SDS-polyacrylamide g e l s .  24.  I s o l a t i o n of r e g u l a t o r y mutants  and phospholipase C.  of a l k a l i n e  phosphatase  P. aeruginosa was mutagenized  d i e t h y l s u l p h a t e or Tn501 i n s e r t i o n  using  i n t o the chromosome (see  above) and mutagenized c e l l s were p l a t e d onto phosphates u f f i c i e n t minimal p l a t e s  ( f o r s e l e c t i o n of c o n s t i t u t i v e  mutants) or p h o s p h a t e - d e f i c i e n t minimal p l a t e s ( f o r s e l e c t i o n of n o n - i n d u c i b l e mutants) c o n t a i n i n g XP  (Bachem).  As d e s c r i b e d above, t h i s compound y i e l d s a blue-green c o l o u r when hydrolyzed by a l k a l i n e phosphatase.  Alkaline  phosphatase c o n s t i t u t i v e mutants were i d e n t i f i e d as bluegreen c o l o n i e s on p h o s p h a t e - s u f f i c i e n t p l a t e s and mutants non-inducible f o r a l k a l i n e phosphatase were i d e n t i f i e d as non-pigmented  c o l o n i e s on p h o s p h a t e - d e f i c i e n t p l a t e s .  d i s t i n g u i s h r e g u l a t o r y mutants  from mutants  To  i n the a l k a l i n e  phosphatase gene, mutant c o l o n i e s were t e s t e d f o r t h e i r phospholipase C phenotypes.  Thus, mutagenized c o l o n i e s were  t r a n s f e r r e d by contact onto Whatman 3M paper p r e v i o u s l y soaked i n NPPC (a chromogenic (see above)).  s u b s t r a t e f o r phospholipase C  C o l o n i e s demonstrating phospholipase C  a c t i v i t y turned r a p i d l y yellow on the f i l t e r  51  paper.  Regulatory mutants were i d e n t i f i e d as those c o l o n i e s constitutive  25.  or n o n - i n d u c i b l e f o r both enzyme a c t i v i t i e s .  Other assays.  P r o t e i n determinations were made by  e i t h e r the method of S c h a c t e r l e and P o l l a c k (1973), using bovine serum albumin as the standard, or the method of Warburg et a l (1941), using absorbance at 260 and 280 2-keto-3-deoxy-octulosonic method of Osborn (1963). protein  acid  (KDO) was measured using the  The s i l v e r  s t a i n i n g procedures f o r  (Wray et a l . , 1981) and LPS (Tsai and F r a s c h ,  have been d e s c r i b e d .  52  nm.  1982)  CHAPTER Outer membrane p r o t e i n P;  ONE  involvement  in h i g h - a f f i n i t y  phosphate t r a n s p o r t in Pseudomonas aeruginosa  1. of  Induction of p r o t e i n P by phosphate l i m i t a t i o n . P. aeruginosa  medium (0.2 mM  PA01  s t r a i n H103  phosphate) was  Growth  in a p h o s p h a t e - d e f i c i e n t  c h a r a c t e r i z e d by an  initial  l o g a r i t h m i c rate of growth i n d i s t i n g u i s h a b l e from that observed (60 min  f o r phosphate s u f f i c i e n t doubling time) ( F i g . 1).  limitation  (0.6 mM The  phosphate) c e l l s  onset  of phosphate-  in the p h o s p h a t e - d e f i c i e n t c e l l c u l t u r e  was  d e t e c t a b l e as a marked d e c l i n e in growth rate (> 2.5  h  doubling time) which c o n t r a s t e d with the phosphates u f f i c i e n t . c e l l c u l t u r e which continued doubling time of c a . 60 min of  phosphate below 0.2  mM  to grow with a  ( F i g . 1).  At  concentrations  the t o t a l growth y i e l d  was  dependent upon the c o n c e n t r a t i o n of phosphate in the medium (Fig.  2) i n d i c a t i n g that phosphate was  growth at these c o n c e n t r a t i o n s . membranes prepared  indeed  l i m i t i n g for  Examination of the  from P. aeruginosa  outer  c e l l s harvested  a f t e r the onset of phosphate l i m i t a t i o n  revealed  4 h  the  presence of a novel p r o t e i n ( F i g . 3, lane 2), h e r e a f t e r r e f e r r e d to as p r o t e i n P, not present  i n c e l l s grown i n a  p h o s p h a t e - s u f f i c i e n t medium ( F i g . 3, lane 1).  A major  p r o t e i n of c a . 22,000 molecular  i n the  weight present  outer  membranes of c e l l s grown in p h o s p h a t e - s u f f i c i e n t medium (Fig.  1, lane  1) was  c o n s i s t e n t l y absent  53  from the outer  •  Figure 1. Growth of P. aeruginosa i n a phosphate d e f i c i e n t medium. Overnight c u l t u r e s of P. aeruginosa PA01 s t r a i n H103 grown i n p h o s p h a t e - s u f f i c i e n t medium were harvested, washed twice with p h o s p h a t e - d e f i c i e n t medium and resuspended i n p h o s p h a t e - d e f i c i e n t ( X — X ) or p h o s p h a t e - s u f f i c i e n t ( 0 — 0 ) medium at an absorbance at 600 nm of 0.20 ( A 5 0 0 ) • Growth was followed by the time dependent increase i n A . f i n n  54  0  60  120  180 240 Time ( m i n )  55  300  360  F i g u r e 2. Growth y i e l d of Pseudomonas aeruginosa as a f u n c t i o n of the c o n c e n t r a t i o n of phosphate i n a d e f i n e d minimal medium. An overnight c u l t u r e of P. aeruginosa H103 grown i n p h o s p h a t e - s u f f i c i e n t medium was harvested, washed twice i n phosphate-free Hepesb u f f e r e d minimal medium and resuspended i n the o r i g i n a l volume of the same phosphate-free medium. A l i q u o t s (0.1 ml) were added to f l a s k s c o n t a i n i n g Hepes-buffered minimal medium and v a r y i n g c o n c e n t r a t i o n s of phosphate and allowed to grow overnight at 37 C. The growth y i e l d was determined from the c u l t u r e d e n s i t y (measured as A , ) obtained a f t e r overnight i n c u b a t i o n . 0 Q  56  0.8H  Phosphate Concentration ( J J M )  57  12  3 A 5 6  7  Figure 3. SDS-polyacrylamide g e l electrophoretogram of p u r i f i e d p r o t e i n P and of outer membranes and shock f l u i d s of p h o s p h a t e - s u f f i c i e n t and p h o s p h a t e - d e f i c i e n t c e l l s of P. aeruginosa H103. Lane 1, outer membrane of p h o s p h a t e - s u f f i c i e n t H103; lane 2, outer membrane of p h o s p h a t e - d e f i c i e n t H103; lane 3, p u r i f i e d p r o t e i n P s o l u b i l i z e d at 75°C; lane 4, p u r i f i e d p r o t e i n P s o l u b i l i z e d a t 55°C; lane 5, p u r i f i e d p r o t e i n P s o l u b i l i z e d a t 25°C; lane 6, unconcentrated shock f l u i d of p h o s p h a t e - s u f f i c i e n t H103; lane 7, unconcentrated shock f l u i d of p h o s p h a t e - d e f i c i e n t H103. Because of the d i l u t e nature of the samples i n lanes 6 and 7 only major p r o t e i n s were d e t e c t e d . Samples were s o l u b i l i z e d at 88°C p r i o r to e l e c t r o p h o r e s i s unless otherwise i n d i c a t e d . P, p r o t e i n P monomer; P* p r o t e i n P oligomer ( t r i m e r ; Angus et a l . , 1983); F, p r o t e i n F. f  membranes of a l l phosphate-limited P. aeruginosa  strains  examined, i n c l u d i n g a p r o t e i n P - d e f i c i e n t mutant  (H576)  ( F i g . 9).  A d d i t i o n a l minor a l t e r a t i o n s i n the outer  membrane p r o t e i n banding  p a t t e r n s of p h o s p h a t e - s u f f i c i e n t  and p h o s p h a t e - d e f i c i e n t grown c e l l s were c o n s i s t e n t l y observed,  and may  r e f l e c t d i f f e r e n c e s i n growth stage s i n c e  phosphate-limited c e l l s were r o u t i n e l y harvested s e v e r a l hours a f t e r the onset of l i m i t a t i o n when the growth rate s u b s t a n t i a l l y lower sufficient cells  than that observed  for phosphate-  ( F i g . 1).  By p r e v i o u s l y p u b l i s h e d c r i t e r i a p r o t e i n P was  was  (Hancock et a l . , 1981)  not a p e p t i d o g l y c a n - a s s o c i a t e d p r o t e i n  although  its inability  Tris-HCl  (pH 8.0)  to be s o l u b i l i z e d i n 2 % SDS/  suggested  that i t was  a s s o c i a t e d with the p e p t i d o g l y c a n .  20  mM  at l e a s t weakly  Using the  procedure  o u t l i n e d i n Methods, a h i g h l y p u r i f i e d p r e p a r a t i o n of t h i s p r o t e i n was  obtained  ( F i g . 3, lane 3)»  The  molecular weight of the p u r i f i e d p r o t e i n i n polyacrylamide g e l s a f t e r s o l u b i l i z a t i o n 48,000 (48K), corresponding  temperatures  SDS-  i n SDS  e x a c t l y to the  molecular weight i n outer membranes.  apparent  at 75°C  apparent  Upon s o l u b i l i z a t i o n at  < 60°C, however, the p r o t e i n ran at an  apparently higher molecular  weight ( F i g . 3, lanes 4 and  suggesting that the n a t i v e form of the p r o t e i n was oligomer.  The o l i g o m e r i c nature of p r o t e i n P was  confirmed  i n f a c t , a trimer (Angus et a l . 1983).  59  5)  an  by c r o s s - l i n k i n g data which i n d i c a t e d that the n a t i v e p r o t e i n was,  was  The  formation by p r o t e i n P of SDS-stable oligomers i n  polyacrylamide g e l s is» c o n s i s t e n t with p r o p e r t i e s of known e n t e r i c p o r i n p r o t e i n s (Lugtenberg and van Alphen,  1983), but  in c o n t r a s t to the p r e v i o u s l y d e s c r i b e d P. aeruginosa p o r i n p r o t e i n s F (Hancock and Carey, Carey,  1980)  and D1  which d i d not demonstrate  SDS-polyacrylamide  2.  1979)  (Hancock and  oligomer formation in  gels.  C o - r e g u l a t i o n with a l k a l i n e phosphatase,  and a 34K p e r i p l a s m i c p r o t e i n .  phospholipase C  When phosphate became  l i m i t i n g f o r growth as i n d i c a t e d by a d e c l i n e i n growth rate ( F i g . 4A) d e t e c t a b l e l e v e l s of the enzymes a l k a l i n e phosphatase  and phospholipase C were produced  by wild-type  P. aeruginosa c e l l s , and the l e v e l s increased with time ( F i g . 4, panels B and C). was  A p r o t e i n of molecular weight  34K  a l s o observed as the major p r o t e i n i n the p e r i p l a s m  ( r e l e a s a b l e by T r i s - M g C l ~ c o l d shock) of c e l l s grown i n 2  phosphate-deficient sufficient  ( F i g . 3, lane 7) but not  ( F i g . 3, lane 6) media.  supernatant a c t i v i t i e s  phosphate-  In a d d i t i o n to t h e i r  ( F i g . 4B), a l k a l i n e phosphatase  and  phospholipase C e x h i b i t e d c e l l - a s s o c i a t e d a c t i v i t y which  was  l o c a l i z e d to the periplasm ( F i g . 4C). The above data i n d i c a t e d that p r o t e i n P was regulated with the enzymes a l k a l i n e phosphatase phospholipase C and the 34K p e r i p l a s m i c p r o t e i n .  coand To  obtain support f o r t h i s g e n e t i c a l l y , mutants n o n - i n d u c i b l e (H553) or c o n s t i t u t i v e  (H587) f o r a l k a l i n e phosphatase  60  were  F i g u r e 4. Induction by p h o s p h a t e - l i m i t a t i o n and l o c a l i z a t i o n of a l k a l i n e phosphatase and phospholipase C of P. aeruginosa H103. A) Growth i n phosphated e f i c i e n t medium. B) Supernatant a c t i v i t i e s and C) P e r i p l a s m i c a c t i v i t i e s of a l k a l i n e phosphatase ( X — X ) and phospholipase C ( • — • ) . Logarithmic-phase c e l l s in p h o s p h a t e - s u f f i c i e n t medium (1 mM P i ) were washed and resuspended i n p h o s p h a t e - d e f i c i e n t medium (0.2 mM Pi) at time zero. C e l l s were harvested at v a r i o u s times d u r i n g growth. Supernatants were obtained a f t e r removal of c e l l s by c e n t r i f u g a t i o n and p e r i p l a s m i c e x t r a c t s were obtained using the T r i s - M g C l c o l d shock procedure of Hoshino and Kageyama (1980). Enzyme assays were c a r r i e d out as d e s c r i b e d i n Methods. The measurements i n panels B and C were r e p r e s e n t a t i v e data of 5 separate experiments. 2  61  62  isolated  (see legend t o F i g .  similarly of  non-inducible  for  t h e p r o t e i n complement  was a l s o n o n - i n d u c i b l e wild-type,  wild-type,  constitutive  levels type  5,  l a n e A; c f .  below t h a t  obtained  ( l a n e s D and F ) .  i n E. c o l i  characteristically  wild-type,-lane  in the f u l l y  5,  alkaline additionally  B), albeit  derepressed  a periplasmic  the a l k a l i n e  in  p h o s p h a t a s e and p h o s p h o l i p a s e C i n the periplasm (Fig.  of  4C).  However,  was  extracellular  r e l e a s e of  is  gram-negative  of p h o s p h a t e - l i m i t e d c e l l s  the c u l t u r e  supernatant  (see b e l o w ) .  t h e s e enzymes by  barrier,  the  The  phosphate-limited  c o u l d be e x p l a i n e d by a breakdown i n t h e  location.  1982).  of  released i n t o  of a  pho  p h o s p h a t a s e and t h e m a j o r i t y  phospholipase C a c t i v i t y  membrane p e r m e a b i l i t y  wild-  A l k a l i n e phosphatase marker  at  the existence  (Tommassen and L u g t e n b e r g ,  was a l w a y s d e t e c t a b l e  periplasmic  c f .  (Fig.  i n P. a e r u g i n o s a a n a l o g o u s t o t h e  p h o s p h a t e - l i m i t e d P. a e r u g i n o s a c e l l s  cells  the  s t r a i n H587 was  These d a t a s u p p o r t  b a c t e r i a and some a l k a l i n e  p o r t i o n of  protein  Likewise,  O u t e r membrane p e r m e a b i l i t y .  activity  l a n e C;  as w e l l as p r o t e i n P and t h e 34K p e r i p l a s m i c  phosphate regulon  3.  5,  it  f o r p h o s p h o l i p a s e C (measured by NPPC  (Fig.  regulon  mutant  examination  revealed that  protein P (Fig.  lane F ) .  phosphatase c o n s t i t u t i v e  protein  t h i s mutant  l a n e D) and t h e 34K p e r i p l a s m i c  l a n e E, c f .  hydrolysis)  s t r a i n H553 was  p h o s p h o l i p a s e C, and  of  for  5 ) . Mutant  outer  r e l e a s i n g them f r o m a  Conversely,  63  a mechanism o f  specific  a  A B  C D E F  F i g u r e 5. SDS-polyacrylamide g e l electrophoretogram of whole c e l l p r o t e i n e x t r a c t s and c e l l envelope and s o l u b l e (non-membrane) f r a c t i o n s of a l k a l i n e phosphatase r e g u l a t o r y mutants. Mutants of P. aeruginosa H103 c o n s t i t u t i v e (H587) and n o n - i n d u c i b l e (H585) f o r a l k a l i n e phosphatase were i s o l a t e d f o l l o w i n g d i e t h y l s u l p h a t e mutagenesis as d e s c r i b e d i n Methods. Lane A, whole c e l l p r o t e i n e x t r a c t of phosphates u f f i c i e n t H587; lane B, whole c e l l p r o t e i n e x t r a c t of p h o s p h a t e - s u f f i c i e n t H103; lane C, c e l l envelope of p h o s p h a t e - d e f i c i e n t H553; lane D, c e l l envelope of p h o s p h a t e - d e f i c i e n t H103; lane E, s o l u b l e f r a c t i o n of p h o s p h a t e - d e f i c i e n t H553; lane F, s o l u b l e f r a c t i o n of p h o s p h a t e - d e f i c i e n t H103. A l l samples were s o l u b i l i z e d at 88°C f o r 10 min p r i o r to e l e c t r o p h o r e s i s . P, p r o t e i n P; 34K, 34,000 molecular weight p e r i p l a s m i c p r o t e i n .  64  s e c r e t i o n across the outer membrane may be r e s p o n s i b l e f o r t h e i r r e l e a s e , i n the absence of any gross p e r m e a b i l i t y changes.  To t e s t the s p e c i f i c i t y of a l k a l i n e phosphatase  and phospholipase  C r e l e a s e , the d i s t r i b u t i o n of two other  p r o t e i n s normally  l o c a l i z e d w i t h i n the periplasm, the  c o n s t i t u t i v e RP1-encoded beta-lactamase  and the 34K p r o t e i n ,  were examined at the time of enzyme i n d u c t i o n and s e c r e t i o n . The  r e s u l t s i n F i g . 6 demonstrated that  beta-lactamase  remained almost wholly p e r i p l a s m i c (as T r i s - M g C ^ T e l e a s a b l e enzyme) during growth on p h o s p h a t e - d e f i c i e n t medium, with only 6 % of the t o t a l a c t i v i t y present  i n the supernatant  ( e x t r a c e l l u l a r medium) 2 h a f t e r the onset of phosphate limitation.  The 34K p r o t e i n , although present as the major  p r o t e i n i n the periplasm upon i n d u c t i o n ( F i g . 3 ) , was undetectable  i n the supernatant  as determined  by SDS-  polyacrylamide g e l e l e c t r o p h o r e s i s of 50-fold' concentrated supernatants.  In c o n t r a s t , up to 58 % of the t o t a l  alkaline  phosphatase a c t i v i t y and 87 % of the t o t a l phospholipase C a c t i v i t y were found  i n the supernatant  ( F i g . 4B). These r e s u l t s confirmed  2h p o s t - i n d u c t i o n  that the r e l e a s e of  a l k a l i n e phosphatase and phospholipase  C by whole phosphate-  l i m i t e d c e l l s was indeed s p e c i f i c and not e x p l a i n a b l e by a general increase i n outer membrane l e a k i n e s s . LPS  Furthermore,  (measured as KDO) or major outer membrane p r o t e i n s were  not detected i n 5 0 - f o l d concentrated  supernatants,  supporting the absence of membrane breakdown during r e l e a s e .  65  The al.,  periplasmic  l o c a t i o n of beta-lactamase (Hancock et  1981) and the demonstration by Angus et a l . (1982) that  nitrocefin provided  i s taken up by the h y d r o p h i l i c (porin) pathway,  a means by which outer membrane p e r m e a b i l i t y  could  be measured d i r e c t l y , as a f u n c t i o n of n i t r o c e f i n uptake and hydrolysis.  Furthermore, treatment of c e l l s with EDTA, an  agent known t o break down the outer membrane thus i n c r e a s i n g permeability  (Hague and Russel,  1974), i s a s s o c i a t e d  with  a 10-fold i n c r e a s e i n n i t r o c e f i n h y d r o l y s i s (Hancock et a l . , 1981).  From the r e s u l t s of n i t r o c e f i n p e r m e a b i l i t y  permeability c o e f f i c i e n t s  assays,  (C) were c a l c u l a t e d (see Methods)  as a f u n c t i o n of growth i n p h o s p h a t e - d e f i c i e n t  medium.  No  increase i n outer membrane p e r m e a b i l i t y was detected concomittant with, enzyme r e l e a s e only a l t e r a t i o n detected  ( F i g . 6B).  In f a c t , the  i n outer membrane p e r m e a b i l i t y which was  over the 2.5 h of the experiment was a general 2.8-  f o l d decrease i n p e r m e a b i l i t y  (Fig. 6).  Given the low p e r m e a b i l i t y  of the P. aeruginosa  membrane and the lack of an increase phosphate-limited hypothesize  in permeability  outer during  growth, i t seemed reasonable to  that p r o t e i n P may f u n c t i o n as a phosphate  p o r i n , mediating the uptake of phosphate from a d i l u t e environment.  66  F i g u r e 6. Outer membrane p e r m e a b i l i t y during growth on p h o s p h a t e - d e f i c i e n t medium. Panel A shows growth a f t e r t r a n s f e r to p h o s p h a t e - d e f i c i e n t medium at time zero as d e s c r i b e d i n the legend to Figure 4. Panel B shows beta-lactamase a c t i v i t y i n the supernatant (•—•) and periplasm (•—•) and the outer membrane p e r m e a b i l i t y c o e f f i c i e n t C ( X — X ) (expressed i n ml/min/mg whole c e l l p r o t e i n ) c a l c u l a t e d as d e s c r i b e d in Methods.  67  60  120 Time (min )  68  180  240  4.  LPS-free p r o t e i n P forms channels in planar  b i l a y e r membranes. P was  lipid  During the course of t h i s study, p r o t e i n  demonstrated to form small  (0.6  nm  diameter), water-  f i l l e d channels in l i p i d b i l a y e r membranes which were specific  f o r anions (Hancock et a l . , 1982).  t h i s s p e c i f i c i t y was the channel binding  shown to be  The  l y s i n e residues  b a s i s of in or near  (Hancock et a l . , 1983), which a l s o formed a  site  f o r phosphate (Hancock and  Benz, submitted).  Data i n the l i t e r a t u r e suggests that the a b i l i t y of  porins  to form channels i s dependent upon an a s s o c i a t i o n with ( S c h i n d l e r and  Rosenbusch, 1978).  Furthermore, i t has  proposed that LPS.may f u n c t i o n to modulate p o r i n (Kropinski  et a l . , 1982).  To determine i f any  LPS been  activity  of  the  p r o p e r t i e s h i t h e r t o a t t r i b u t e d to p r o t e i n P were r e l a t e d to i t s a s s o c i a t i o n with LPS p u r i f i e d preparations was  Methods.  i s i n v a r i a b l y detected  of p r o t e i n P (see below)) the  p u r i f i e d f r e e of LPS  trimer out  ( LPS  by e l e c t r o e l u t i n g the  LPS  (<2-3.8X10  as observed by ( F i g . 7).  in  mol/mol p r o t e i n )  as measured by  (Table  s i l v e r s t a i n i n g f o r LPS  (Table  II)  in polyacrylamide  In c o n t r a s t , the c o n v e n t i o n a l l y p u r i f i e d  p r o t e i n contained protein)  protein  of SDS-polyacrylamide g e l s as d e s c r i b e d  ELISA using L P S - s p e c i f i c monoclonal a n t i b o d i e s  gels  protein  P r o t e i n P i s o l a t e d by e l e c t r o e l u t i o n lacked  detectable  and  in  significant  I I ; F i g . 7).  l e v e l s of LPS  (1-1.7 mol/mol  LPS-free p r o t e i n P was  still  capable of forming channels i n l i p i d b i l a y e r membranes with a mean s i n g l e channel conductance i n 1 M KC1  69  (234  pS)  1  2  F i g u r e 7. SDS-polyacrylamide g e l electrophoretogram of LPS a s s o c i a t e d with p r o t e i n P. Conventionally p u r i f i e d (lane 1) and e l e c t r o e l u t e d (lane 2) p r o t e i n P were e l e c t r o p h o r e s e d on SDS-polyacryalimide g e l s a f t e r s o l u b i l i z a t i o n at 23°C f o r 10 min and s t a i n e d f o r LPS using the pocedure of T s a i and Frasch (1982). The area of densest s t a i n i n lane 1 occurs at the t r a c k i n g dye f r o n t .  70  Table  II.  Measurements of LPS a s s o c i a t e d with c o n v e n t i o n a l l y p u r i f i e d and e l e c t r o e l u t e d protein P  Associated Assay Method  SDS-PAGE ELISA  LPS (mol/mol p r o t e i n )  Conventionally Purified  Electroeluted  1.7  <2X10~  1.1  <3.8X10~  3  b  2  2  C o n v e n t i o n a l l y p u r i f i e d and e l e c t r o e l u t e d p r o t e i n P were e l e c t r o p h o r e s e d on SDS-polyacrylamide g e l s and s t a i n e d f o r LPS using the method of T s a i and Frasch (1982). Contaminating LPS was estimated by comparing s i l v e r s t a i n e d electrophoretograms of d i l u t i o n s of the p r o t e i n P p r e p a r a t i o n s with s i l v e r s t a i n e d electrophoretograms of d i l u t i o n s of pure LPS of known c o n c e n t r a t i o n . b  P r o t e i n P p r e p a r a t i o n s were s e r i a l l y d i l u t e d and used to coat the bottoms of m i c r o t i t r e w e l l s . Monoclonal a n t i b o d i e s s p e c i f i c f o r P. aeruginosa LPS were then used to d e t e c t the presence of LPS at each d i l u t i o n . Based on the d e t e c t i o n l i m i t s of the a n t i b o d i e s employed (approximately 50 ng LPS), d e r i v e d from ELISA a n a l y s i s of s e r i a l l y d i l u t e d pure LPS p r e p a r a t i o n s of known c o n c e n t r a t i o n , the LPS l e v e l s c o u l d be e s t i m t a t e d from the highest d i l u t i o n which s t i l l gave a p o s i t i v e LPS response.  71  Table  I I I . F u n c t i o n a l p r o p e r t i e s of c o n v e n t i o n a l l y p u r i f i e d and e l e c t r o e l u t e d p r o t e i n P i n planar l i p i d b i l a y e r membranes  Purification Procedure  Average s i n g l e channel conductance in 1 M KC1 (pS)  Number of events  Selectivity (Pc/Pa)  <0.0l  Conventional  235  317  Electroeluted  234  224  b  0.005  a P e r m e a b i l i t y r a t i o of K to C l d e r i v e d from the GoldmanHodgkin-Katz equation as d e s c r i b e d by Hancock et a l . (1982) +  b Taken from Benz et a l . (1985)  72  almost i n d i s t i n g u i s h a b l e from that obtained  f o r the  c o n v e n t i o n a l l y p u r i f i e d , LPS-contaminated p r o t e i n (Table I I I ) . anion-specific  (235 pS)  Furthermore, the LPS-free p r o t e i n remained (Pc/Pa = 0.005) and s i n g l e channel  conductance through LPS-free p r o t e i n P channels was observed to s a t u r a t e at high c o n c e n t r a t i o n s  of KC1, c o n s i s t e n t with a  b i n d i n g s i t e i n the channel f o r anions. were i n agreement with the published conventionally p u r i f i e d protein III).  These p r o p e r t i e s  p r o p e r t i e s of the  (Hancock et a l . , 1982; Table  LPS a s s o c i a t i o n was t h e r e f o r e not r e q u i r e d f o r  channel formation  by p r o t e i n P and was not r e s p o n s i b l e f o r  the f u n c t i o n a l p r o p e r t i e s of t h i s p r o t e i n i n v i t r o .  5.  I s o l a t i o n of a p r o t e i n P - d e f i c i e n t mutant.  a. antiserum.  Preparation  of a p r o t e i n P t r i m e r - s p e c i f i c  A p o l y c l o n a l r a b b i t antiserum r a i s e d a g a i n s t  p u r i f i e d protein P trimers  (see Methods) reacted  s p e c i f i c a l l y with the n a t i v e trimer form of the p r o t e i n ( F i g . 8A, lane 3), e x h i b i t i n g no r e a c t i o n with  heat-  d i s s o c i a t e d monomers ( F i g . 8A, lane 4 ) . The smearing p a t t e r n evident  i n the r e a c t i o n of the antibody  with  electrophoresed  p r o t e i n P t r i m e r s suggested some heterogeneity.  T h i s may be  due  t o an a s s o c i a t i o n of the trimer form of the p r o t e i n  LPS  or due to aggregation  of the t r i m e r s .  Nevertheless, a l l  of the m a t e r i a l i n the smear r e a c t i n g with the antibody p r o t e i n P as confirmed by the a b i l i t y  73  with  to convert  this  was  1  2 3 A  Figure 8. Immunoblots of e l e c t r o p h o r e t i c a l l y separated P.aeruginosa H103 c e l l envelopes and p u r i f i e d p r o t e i n P, and whole c e l l s . A) C e l l envelopes from phosphates u f f i c i e n t c e l l s (lane 1), phosphate-limited c e l l s (lane 2) and p u r i f i e d p r o t e i n P (lanes 3 and 4) were separated on SDS-polyacrylamide g e l s a f t e r s o l u b i l i z a t i o n at 23°C (lanes 1-3) or 88°C (lane 4) f o r 10 min. After e l e c t r o p h o r e t i c t r a n s f e r to n i t r o c e l l u l o s e , the b l o t s were i n t e r a c t e d with the p r o t e i n P - s p e c i f i c p o l y c l o n a l antiserum and subsequently immunostained using a peroxidase-conjugated g o a t - a n t i - r a b b i t IgG antibody and a h i s t o c h e m i c a l s t a i n f o r peroxidase (see Methods). B) A colony immunoblot showing the i n t e r a c t i o n of the p r o t e i n P - s p e c i f i c p o l y c l o n a l antiserum with phosphatel i m i t e d Tn501 i n s e r t i o n mutants of P. aeruginosa PA01 s t r a i n H103. The p r o t e i n P - d e f i c i e n t mutant, s t r a i n H576, i s i n d i c a t e d by the arrowhead. 74  m a t e r i a l to p r o t e i n P monomers by heating (see Chapter Three, F i g . 19A) . The s p e c i f i c i t y P was demonstrated  of the p o l y c l o n a l antiserum t o p r o t e i n  by the a b i l i t y of the antiserum to react  with a component present i n envelopes from phosphate-starved cells  ( F i g . 8A, lane 2) which was absent i n envelopes  phosphate-sufficient c e l l s profile  from  ( F i g . 8A, lane 1). The r e a c t i o n  was very s i m i l a r t o that seen with p u r i f i e d  protein  P t r i m e r s ( F i g . 8A, lane 3 ) .  b.  Tn50l mutagenesis  of P. aeruginosa.  In our  search f o r a s u i t a b l e v e h i c l e f o r use i n the transposon i n s e r t i o n mutagenesis  of P. aeruginosa PA01 (HI 03), a number  of v e c t o r s were t e s t e d  (see Table I V ) .  included plasmids pME9 and pME3l9, were  One c l a s s , which temperature  s e n s i t i v e f o r maintainance due to mutation, so that for  transposon-encoded  temperature chromosome.  selection  r e s i s t a n c e at the non-permissive  (42°C) r e s u l t e d i n i n s e r t i o n s i n t o the PAO Recovery  of the transposable element  non-permissive temperature  was u s u a l l y  with recovery of a l l plasmid a n t i b i o t i c  at the  (> 98 %) a s s o c i a t e d r e s i s t a n c e markers  as w e l l , i n d i c a t i n g that the e n t i r e plasmid had probably inserted. The second c l a s s of v e c t o r s t e s t e d included plasmids pUW942 (::Tn50l), pUW964 (::Tn5), pAS8Rep-1 (::Tn7) and pKPlOO (:;Tn5-132).  These plasmids were h y b r i d s comprising  the broad host range t r a n s f e r f u n c t i o n s of the Inc P-1  75  A b b r e v i a t i o n s : Cb , c a r b e n i c i l l i n r e s i s t a n t ; Kn , kanamycin r e s i s t a n t ; T p , trimethoprim r e s i s t a n t ; T c , t e t r a c y c l i n e r e s i s t a n t ; Hg , mercury r e s i s t a n t ; t s , temperature sensitive. Genotype symbols are a c c o r d i n g to Bachmann (1983). Only the r e l e v a n t phenotypes are i n d i c a t e d . r  r  r  r  r  b  A . d e r i v a t i v e of Tn5 c a r r y i n g a T c determinant the K n determinant (Berg and Berg, 1983). r  i n p l a c e of  r  c  d  The temperature s e n s i t i v e phenotype t r f A ( R e l l a et a l , 1985). A d e r i v a t i v e of Tn5 c a r r y i n g R751 ( R e l l a et a l , 1985).  i s due to a mutation i n  the Trf determinant of plasmid  76  Table IV. Plasmids t e s t e d f o r u t i l i t y i n transposon i n s e r t i o n mutagenesis of P. aeruginosa  Description'  Plasmid  pAS8Rep-1  pUW942  RP4-ColE1 hybrid/rep(RP4)::Tn7  Sato et a l ,  (Tra  1981  r  Kn  r  T c Tp ) s  r  +  Cb  r  Kn  r  Weiss and Falkow, 1983  T c T p Hg ) s  r  r  p u t a t i v e pAS8Rep-1:;Tn5-132 (Tra  pRK2013  Cb  +  pAS8Rep-1:;Tn501 (Tra  pKP100  Source/ Reference  +  Cb  r  Kn  r  b  T c Tp ) r  RK2-ColE1 h y b r i d  r  Tra  Kn  +  Figurski &  r  Helinski, 1979 pUW964  PME319  pRK20l3(Kan::Tn7)::Tn5  Weiss et a l ,  (Tra  1983  RP1  +  Cb  s  Kn  [Rep A  tS  r  T c Tp ) s  Rep B  t S  r  t S  ]  Haas et a l ,  ( c a r r i e s TnJ_) (Tra pME305  RP1  +  ts  Cb  r  1981  Kn T c ) r  r  with a 12 kb d e l e t i o n  in K n , primase and IS21 r  (Tra pME9  +  Cb  r  Rella et a l , 1985  Kn T c ) s  r  Kn  r  R e l l a et a l ,  T c Tp ) r  r  1985  a temperature-  Tsuda et a l ,  s e n s i t i v e mutant of  1984  R68 pMTlOOO  Cb  r  pME305::Tn5-751 (Tra  PMO190  +  c  (=RP4) Tsuda et a l ,  PMO190::Tn50l  1984  77  plasmids  (eg. RP1)  and the narrow host range r e p l i c a t i o n  f u n c t i o n s of the C o l E 1 - l i k e plasmids. Thus, these v e c t o r s could be t r a n s f e r r e d from E. c o l i unable 1979).  to r e p l i c a t e Selection  aeruginosa PAO  to P. aeruginosa but were  in t h i s r e c i p i e n t  (Bagdasarian et a l . ,  f o r transposon-encoded  r e s i s t a n c e s in P.  s t r a i n s mated with E. c o l i  harbouring these plasmids  strains  revealed c o l o n i e s with  in the chromosome. Almost without exception,  insertions  insertion  events a s s o c i a t e d with these v e c t o r s i n v o l v e d i n s e r t i o n of the whole plasmid, as i n d i c a t e d by the recovery of a l l plasmid markers.  The plasmid markers were s t a b l y  maintained  in P. aeruginosa and were not r e a d i l y t r a n s f e r r a b l e to a second host s t r a i n  i n d i c a t i n g that they were, indeed,  present as i n s e r t s i n the chromosome. Although c u r i n g of transposon-mediated inserts  whole plasmid  ( c o - i n t e g r a t e s ) , to leave a s i n g l e copy of the"  transposon  i n the mutated gene, has been documented  (Harayama et a l . , 1981;  Tsuda et a l . , 1984), the c o n s i s t e n t  i s o l a t i o n of whole plasmid i n s e r t s made the v e c t o r s o u t l i n e d above u n s u i t a b l e f o r our needs.  The  i s o l a t i o n of a p r o t e i n  P - d e f i c i e n t mutant, which i n v o l v e d a negative  selection,  r e q u i r e d the screening of thousands of p o t e n t i a l mutants. It was  thus d e s i r a b l e to have a system whereby mutants  generated would represent r e s o l v e d , s i n g l e i n s e r t s , without  the requirement  transposon  for additional  manipulations to obtain the d e s i r e d i n s e r t i o n a l  78  mutation.  Plasmid pMTlOOO, r e c e n t l y d e s c r i b e d by Tsuda et a l . (1984),  i s a t e m p e r a t u r e - s e n s i t i v e R68 plasmid c a r r y i n g a  Tn501 element.  I n s e r t i o n mutants i n P.aeruginosa  can be  r e a d i l y s e l e c t e d on H g C l ~ c o n t a i n i n g p l a t e s at 42°C.  After  2  r a i s i n g the incubation temperature of P. aeruginosa s t r a i n H103  (pMTlOOO) to the r e s t r i c t i v e  c o l o n i e s r e s i s t a n t to H g C l > 1x10~ /viable 3  2  PA01  temperature  (42°C),  were i s o l a t e d at a frequency of  c e l l . Of these, approximately  30 %  v i a b l e c e l l ) apparently represented whole plasmid  (3X10~ / 4  inserts in  that they were r e s i s t a n t to Cb, Tc and Kn, as w e l l as to HgCl2r  and t h i s p r o p o r t i o n decreased  to < 15 % a f t e r a  s i n g l e passage on H g C l ~ c o n t a i n i n g p l a t e s . 2  The remainder of  the mercury r e s i s t a n t c o l o n i e s were s e n s i t i v e to Cb, Tc and Kn.  T h i s , together with the high frequency  suggested  of i s o l a t i o n  that they were Tn501 i n s e r t i o n mutants.  Examination  of c o l o n i e s growing on H g C l ~ c o n t a i n i n g 2  p l a t e s a t 42°C revealed the e x i s t e n c e of two colony morphologies which c o u l d be c o r r e l a t e d to the type of i n s e r t i o n event which had occurred i n these c l o n e s i n the rescue of the Tn501 element. plasmid flat,  inserts  C o l o n i e s c o n t a i n i n g whole  (Hg , T c , K n , C b r  r  r  t r a n s l u c e n t and i r r e g u l a r l y  r  at 42°C) were t y p i c a l l y  shaped.  c o n t a i n i n g a r e s o l v e d Tn501 i n s e r t i o n  Colonies  (Hg , T c , K n , C b r  s  s  s  at  42°C) were opaque, dome-shaped and g e n e r a l l y c i r c u l a r , t y p i c a l of w i l d type. The Tn501  isolation,  i n the m a j o r i t y of cases, of r e s o l v e d  i n s e r t s i n P. aeruginosa  PA01 s t r a i n H103 meant that  79  the Tn501 i n s e r t i o n mutagenesis was than p u b l i s h e d procedures al.,  1984)  significantly  (Harayama et a l . ,  1981;  simpler Tsuda et  r e q u i r i n g no c u r i n g of plasmid sequences.  In  a d d i t i o n , the mutagenic c a p a b i l i t y of plasmid pMTlOOO et a l . , H103  1984)  was  confirmed i n P. aeruginosa PA01  by the i s o l a t i o n of auxotrophs  (Tsuda  strain  (frequency=2x10  /Hg —4  c o l o n y ) , mutants d e f i c i e n t  i n pigment production (6x10  c o l o n y ) , and a number of pho phosphate-binding  r  /Hg  including  p r o t e i n - d e f i c i e n t mutants (2x10  c o l o n y ) , a l k a l i n e phosphatase Hg  regulon mutants,  r  4  /Hg  r  c o n s t i t u t i v e mutants (1x10  /  colony) and a l k a l i n e p h o p h a t a s e - d e f i c i e n t mutants  (3.3x10~ /Hg 4  c.  r  colony).  I s o l a t i o n of a Tn501-induced p r o t e i n  d e f i c i e n t mutant.  In order to c o n f i r m a r o l e f o r p r o t e i n P  in phosphate t r a n s p o r t i n P. aeruginosa, i t was i s o l a t e a mutant d e f i c i e n t mediated HgCl  2  P-  i n p r o t e i n P.  necessary to  Plasmid pMTlOOO-  Tn501 i n s e r t i o n mutants, i s o l a t e d as r e s i s t a n t to  at 42°C, were t r a n s f e r r e d from p h o s p h a t e - d e f i c i e n t  minimal medium p l a t e s to n i t r o c e l l u l o s e by contact and screened f o r the absence of p r o t e i n P using a p r o t e i n s p e c i f i c antiserum. screened, only one  Of 3,200 mercury r e s i s t a n t c o l o n i e s f a i l e d to react s t r o n g l y with the p r o t e i n  P - s p e c i f i c antiserum i n the colony b l o t assay SDS-polyacrylamide t h i s mutant  P-  (see F i g . 8B).  g e l e l e c t r o p h o r e s i s of c e l l envelopes of  (designated s t r a i n H576), grown under  phosphate-  d e f i c i e n t c o n d i t i o n s , confirmed the absence of d e t e c t a b l e  80  12  3 4 5  Figure 9. SDS-polyacrylamide g e l electrophoretogram of outer membranes prepared from a p r o t e i n P - d e f i c i e n t mutant of P. aeruginosa and i t s w i l d type parent. Lane 1, p u r i f i e d p r o t e i n P. The outer membranes were prepared from: lane 2, p h o s p h a t e - s u f f i c i e n t H103 (wild type) c e l l s ; lane 3, p h o s p h a t e - d e f i c i e n t H103 c e l l s ; lane 4, p h o s p h a t e - s u f f i c i e n t H576 (mutant) c e l l s ; lane 5, p h o s p h a t e - d e f i c i e n t H576 c e l l s . A l l preparations were s o l u b i l i z e d at 88°C p r i o r to e l e c t r o p h o r e s i s such that p r o t e i n P (P) ran as the monomer.  81  p r o t e i n P (see F i g . 9, lane 5). s t r a i n H103  In c o n t r a s t , the parent  grown under the same c o n d i t i o n s produced  q u a n t i t i e s of p r o t e i n P ( F i g . 9, lane 3).  large  Western  immunoblots of e l e c t r o p h o r e t i c a l l y - s e p a r a t e d c e l l  envelope  and whole c e l l p r o t e i n s confirmed the absence of d e t e c t a b l e p r o t e i n P i n p h o s p h a t e - l i m i t e d mutant c e l l s u s i n g both a p r o t e i n P t r i m e r - s p e c i f i c and monomer-specific The mutant, l i k e  i t s parent, was  a l k a l i n e phosphatase  normally d e r e p r e s s i b l e f o r  and phospholipase C under c o n d i t i o n s of  phosphate d e f i c i e n c y . phosphate-binding  antiserum.  In a d d i t i o n ,  protein  the presence of the  i n shock f l u i d s and whole c e l l  e x t r a c t s of the mutant was  confirmed using  SDS-  polyacrylamide g e l e l e c t r o p h o r e s i s and Western immunoblotting antiserum  with a phosphate-binding  (data not shown).  protein  specific  These r e s u l t s supported the  s p e c i f i c l o s s of p r o t e i n P i n t h i s mutant, and  distinguished  t h i s s t r a i n from a c l a s s of mutants i s o l a t e d p r e v i o u s l y which were p l e i o t r o p i c a l l y d e f i c i e n t  i n the  phosphate-  r e g u l a t e d components of P. aeruginosa, i n c l u d i n g p r o t e i n P (see  6.  F i g . 5).  Phosphate t r a n s p o r t .  Phosphate t r a n s p o r t i n wild-type  P. aeruginosa i s c h a r a c t e r i z e d by the presence of two major systems of uptake, of low and h i g h - a f f i n i t y , (LaCoste et a l . ,  1981).  respectively  When s t a t i o n a r y phase,  phosphate-  s t a r v e d c e l l s of P. aeruginosa were pre-incubated, with a e r a t i o n , at 37°C f o r only 5 min p r i o r to t r a n s p o r t assays,  82  i t was  p o s s i b l e to examine h i g h - a f f i n i t y phosphate  alone,  since i t was  longer  periods  found necessary to incubate  (15-25 min)  transport  c e l l s for  at 37°C f o r the l o w - a f f i n i t y  uptake system to become o p e r a t i v e . p r e c i s e l y examine what r o l e ,  Thus i t was  i f any,  p o s s i b l e to  p r o t e i n P played  in  h i g h - a f f i n i t y phosphate t r a n s p o r t by comparing phosphate uptake in the p r o t e i n P - d e f i c i e n t mutant with that of i t s parent  H103.  Compared with the w i l d type parent s t r a i n , the P - d e f i c i e n t mutant was  s i g n i f i c a n t l y d e f e c t i v e in phosphate  t r a n s p o r t , e x h i b i t i n g a Km roughly No  protein  10 times greater  for h i g h - a f f i n i t y transport  than that of the parent  e f f e c t on the Vmax of the system was  (Table  seen, however, as a  r e s u l t of the l o s s of p r o t e i n P in the mutant (Table T h i s confirmed the phosphate t r a n s p o r t It was  V).  V).  involvement of p r o t e i n P in h i g h - a f f i n i t y i n P.  aeruginosa.  not p o s s i b l e to a c c u r a t e l y determine the k i n e t i c  parameters of l o w - a f f i n i t y phosphate t r a n s p o r t owing to the  simultaneous operation  uptake component (see Chapter Two,  in the mutant  of a h i g h - a f f i n i t y section 4 ) .  However, the  r a t e s of phosphate transport measured in the mutant (H576) and  wild-type  concentrations  (H103) were comparable at of phosphate (> 25 uM)  a f f i n i t y phosphate uptake was  higher  suggesting  that  low-  not a f f e c t e d by the p r o t e i n  d e f i c i e n c y of the mutant.  83  P-  T a b l e V.  Strain  K i n e t i c s of h i g h - a f f i n i t y phosphate t r a n s p o r t i n a p r o t e i n P - d e f i c i e n t mutant s t r a i n and i t s w i l d type parent  Km (uM)  Vmax (umol/min/mg c e l l  H1 03  0.39 + 0.07  5.34 + 0.59  H576  3.60 + 0.64  5.56 + 0.66  protein)  I n i t i a l r a t e s of phosphate t r a n s p o r t a t v a r i o u s c o n c e n t r a t i o n s o f p h o s p h a t e were p l o t t e d a s an E a d i e - H o f s t e e p l o t , f r o m w h i c h k i n e t i c p a r a m e t e r s were d e r i v e d by l e a s t s q u a r e s a n a l y s i s . The r e s u l t s a r e t h e mean v a l u e s + standard d e v i a t i o n s of four experiments.  84  7.  Growth i n low  phosphate medium.  determine i f the transport  I t was  of  interest  d i f f e r e n c e s a t t r i b u t a b l e to a  lack of p r o t e i n P were s i g n i f i c a n t  in terms of the growth  c a p a b i l i t i e s of the c e l l under p h o s p h a t e - l i m i t i n g (under which c o n d i t i o n s  conditions  p r o t e i n P i s normally d e r e p r e s s e d ) .  Thus wild-type s t r a i n H103  and  mutant s t r a i n H576 c e l l s were  grown in p h o s p h a t e - d e f i c i e n t  medium f o r 14-16  i n t e r n a l phosphate pools and  thus make growth dependent  transported  phosphate.  hrs to  50 uM  T y p i c a l l y , a l a g p e r i o d of 30-45 min  observed  logarithmic  growth f o r 2-4  this period  (Table  in a  phosphate. followed  Determination of the  revealed  d e f i c i e n t mutant grew more slowly s t r a i n H103  was  on  h at a very reduced r a t e , a f t e r  which the c e l l s stopped growing. of growth d u r i n g  deplete  These c e l l s were then placed  Hepes-buffered minimal medium c o n t a i n i n g  by  to  rate  that the p r o t e i n  P-  than i t s w i l d type parent  VI).  To e l i m i n a t e p o s s i b l e growth d i f f e r e n c e s a t t r i b u t a b l e to the presence of a Tn501 element in the chromosome of  the  p r o t e i n P - d e f i c i e n t mutant, an a r g i n i n e auxotroph,  strain  H556, obtained by Tn501 i n s e r t i o n mutagenesis, was  used as  the p r o t e i n P - d e r e p r e s s i b l e  control.  Again, the mutant  l a c k i n g p r o t e i n P e x h i b i t e d a slower rate of growth than s t r a i n producing w i l d type l e v e l s of p r o t e i n P (Table The  18-35  % increase  i n doubling  time c h a r a c t e r i z e d  p r o t e i n P mutant s t r a i n s t r e s s e s the  VI). the  importance of p r o t e i n  channels in the outer membrane of P. aeruginosa growing i n a phosphate-limited environment.  85  by  the  cells  P  Table V I .  Growth of a p r o t e i n P - d e f i c i e n t mutant and s t r a i n s w i l d type f o r p r o t e i n P i n a phosphatel i m i t e d medium  Experiment  3  Strain*  Doubling t i m e  5  c  H103  12.82 +1.18  H576  15.87 + 0.60  H103  9.98 + 0.90  H576  15.43 + 2.57  H556  13.55 + 0.33  H576  19.60 + 1.62  H556  11.27 + 0.53  H576  13.67 + 0.33  (h)  Overnight c u l t u r e s , grown i n p h o s p h a t e - d e f i c i e n t medium, were resuspended i n t r i p l i c a t e i n Hepes-buffered minimal medium c o n t a i n i n g 50 uM phosphate at A = 0.20 and growth measured by the increase i n A ^ Q Q . 6 Q 0  b  c  H103, w i l d type PA01; H556, a r g i n i n e r e q u i r i n g Tn50l i n s e r t i o n mutant; H576, Tn501 i n s e r t i o n mutant d e f i c i e n t i n p r o t e i n P. Doubling times represent the r e c i p r o c a l of growth r a t e , u, c a l c u l a t e d from a p l o t of l n A v s . time (min) using l e a s t squares a n a l y s i s . R e s u l t s are expressed as the mean doubling times + standard d e v i a t i o n s f o r three c u l t u r e s (see above). V a r i a t i o n s from experiment to experiment i n growth r a t e s obtained f o r a given s t r a i n r e f l e c t t e c h n i c a l d i f f i c u l t i e s i n o b t a i n i n g p r e c i s e l y the same degree of phosphate l i m i t a t i o n every time. For a given experiment, however, the degree of l i m i t a t i o n was the same f o r each strain. 6 0 Q  86  8. PA01  Summary.  When wild-type  c e l l s of Pseudomonas aeruginosa  were grown i n a medium c o n t a i n i n g 0.2 mM or l e s s  inorganic phosphate (phosphate-deficient medium) a new major outer membrane p r o t e i n , P, was induced.  The p r o t e i n was  p u r i f i e d and demonstrated to form SDS-resistant polyacrylamide  g e l s , a property  shared  oligomers i n  by most known p o r i n s .  enzymes a l k a l i n e phosphatase and phospholipase  C, as w e l l as  a major p e r i p l a s m i c p r o t e i n of 34K were co-induced i n a phosphate-deficient limitation  medium at the onset  of phosphate-  i d e n t i f i a b l e by a marked decrease i n growth r a t e .  Mutants c o n s t i t u t i v e or non-inducible phosphatase and phospholipase  for alkaline  C were i s o l a t e d and  demonstrated to be s i m i l a r l y c o n s t i t u t i v e and n o n - i n d u c i b l e , r e s p e c t i v e l y , f o r p r o t e i n P and the 34K p e r i p l a s m i c p r o t e i n c o n s i s t e n t with the existence of a phosphate i n P. aeruginosa.  regulon  The p h o s p h a t e - s t a r v a t i o n - i n d u c i b l e  enzymes a l k a l i n e phosphatase and phospholipase secreted i n t o the growth medium upon i n d u c t i o n  C were although  enzyme r e l e a s e was shown not to be a s s o c i a t e d with a breakdown i n the outer membrane or an increase i n outer membrane p e r m e a b i l i t y .  As such, an increase i n outer  membrane p e r m e a b i l i t y , which could c o n c e i v a b l y  increase the  r a t e of phosphate movement across the outer membrane, i s not the means by which P. aeruginosa l i m i t e d environment.  adapts to a phosphate-  P r o t e i n P has been demonstrated to  form s m a l l , a n i o n - s p e c i f i c channels and the p r o t e i n p u r i f i e d free of LPS e x h i b i t e d u n a l t e r e d channel-forming  87  properties  The  in planar  b i l a y e r membranes.  In order to demonstrate a r o l e  for p r o t e i n P channels in phosphate transport  in  P.  aeruginosa, a transposon i n s e r t i o n mutant d e f i c i e n t in p r o t e i n P was  sought.  A number of transposon d e l i v e r y  systems were t e s t e d which y i e l d e d , f o r the most p a r t , whole plasmid i n s e r t s .  Plasmid pMTlOOO (Tsuda et a l . , 1984), a  temperature-sensitive Tn501, was  R68  plasmid c a r r y i n g the  s u c c e s s f u l l y employed in the  transposon  i s o l a t i o n of a Tn501  i n s e r t i o n mutant l a c k i n g p r o t e i n P under normally conditions.  To  i d e n t i f y the mutant d e f i c i e n t i n p r o t e i n  a p r o t e i n P - s p e c i f i c p o l y c l o n a l antiserum was mutant, s t r a i n H576, was phosphate t r a n s p o r t ,  (0.39  used.  e x h i b i t i n g a Km  uM phosphate).  for uptake (3.60 than that of the  There was,  This  l o s s of protein. P in t h i s mutant.  uM wild-  however, no  change i n the Vmax f o r h i g h - a f f i n i t y t r a n s p o r t of the  P,  d e f i c i e n t in h i g h - a f f i n i t y  phosphate) almost ten times greater type s t r a i n  inducing  The  as a r e s u l t protein  P-  d e f i c i e n c y of the mutant c o r r e l a t e d with a growth d e f e c t a phosphate-limited medium, r e s u l t i n g in an in growth rate compared with the  88  wild-type.  18-35  in  % decrease  CHAPTER  TWO  Role of a p e r i p l a s m i c phosphate-binding phosphate t r a n s p o r t in Pseudomonas  1.  protein in  aeruginosa  P u r i f i c a t i o n and p r o p e r t i e s of the p e r i p l a s m i c  phosphate-binding aeruginosa  PA01  protein.  Phosphate l i m i t a t i o n of  s t r a i n HI 03 c e l l s r e s u l t e d in the i n d u c t i o n  of a major p r o t e i n of molecular Present  weight 34,000 ( F i g . 10).  as the major p r o t e i n i n the periplasm  c o l d osmotic  P.  shock) ( F i g . 11, lane A)  i t was  ( r e l e a s a b l e by readily  purified,  ( F i g . 11, lane B), using the procedure o u t l i n e i n  Methods.  A Scatchard  p l o t of the data obtained  from  e q u i l i b r i u m d i a l y s i s b i n d i n g s t u d i e s ( F i g . 12). r e v e a l e d that the p u r i f i e d p r o t e i n bound one molecule of phosphate per molecule of p r o t e i n (n = 0.91 a Kd of 0.34  + 0.05  uM  standard d e v i a t i o n ) . binding p r o t e i n was  from the Scatchard p l o t ) with  (mean of three Kd determinations The  +  s p e c i f i c i t y of the phosphate-  t e s t e d using a number of p o t e n t i a l  i n h i b i t o r s of phosphate binding  (Table V I I ) .  The  organic  phosphates glucose-6-phosphate, glycerol-3-phosphate  and  adenosine-5'-monophosphate d i d not compete with orthophosphate for b i n d i n g , even at 1000-fold orthophosphate (Table V I I ) . phosphate from P2 arsenate,  excess  over  In c o n t a s t , polymers of  (pyrophosphate) to P15,  as w e l l as  i n h i b i t e d the binding of orthophosphate to the  b i n d i n g p r o t e i n (Table V I I ) .  89  F i g u r e 10. Induction of the 34K p e r i p l a s m i c p r o t e i n by phosphate l i m i t a t i o n . C e l l s grown under phosphates u f f i c i e n t c o n d i t i o n s were harvested, washed i n phosphate-free minimal Hepes-buffered medium and resuspended i n p h o s p h a t e - d e f i c i e n t minimal Hepesbuf f ered medium at an absorbance at 600 nm of 0.20. C e l l s were shaken at 37 C, and a t 15 min i n t e r v a l s c e l l samples were removed and whole c e l l p r o t e i n e x t r a c t e d and run on SDS-polyacrylamide g e l s (lanes A-M). A l l samples were s o l u b i l i z e d at 88 C f o r 10 min p r i o r t o electrophoresis. P, p r o t e i n P.  90  ABC  D E F  G  F i g u r e 11. SDS-polyacrylamide g e l electrophoretogram of p u r i f i e d phosphate-binding p r o t e i n and whole c e l l p r o t e i n e x t r a c t s of a l k a l i n e phosphatase c o n s t i t u t i v e mutants of P. aeruginosa H242. Lane A, 5 0 - f o l d concentrated shock f l u i d of phosphate-limited s t r a i n H242 c e l l s ; lane B, p u r i f i e d phosphate-binding p r o t e i n ; lane C, whole c e l l p r o t e i n e x t r a c t of phosphate-limited s t r a i n H585; lane D, whole c e l l p r o t e i n e x t r a c t of p h o s p h a t e - l i m i t e d s t r a i n H586; lane E, whole c e l l p r o t e i n e x t r a c t of p h o s p h a t e - l i m i t e d s t r a i n H587; lanes F and G, whole c e l l p r o t e i n e x t r a c t s of phosphates u f f i c i e n t and p h o s p h a t e - d e f i c i e n t H242, r e s p e c t i v e l y . A l l samples were s o l u b i l i z e d at 88°C p r i o r to electrophoresis. P, p r o t e i n P; PBP, phosphate-binding potein.  91  F i g u r e 12. S c a t c h a r d p l o t o f phosphate-binding activity. E q u i l i b r i u m d i a l y s i s b i n d i n g a s s a y s were p e r f o r m e d w i t h 7 ug o f b i n d i n g p r o t e i n and v a r y i n g amounts o f p h o s p h a t e . V r e p r e s e n t s nmol p h o s p h a t e bound p e r nmol p h o s p h a t e - b i n d i n g protein. L r e p r e s e n t s the - c o n c e n t r a t i o n of phosphate.  92  93  Table V I I .  Substrate s p e c i f i c i t y of the protein  Inhibition  Inhibitor  0.1  mM  3  (%) 1 .0 mM  Arsenate  17  61  Pyrophosphate (P2)  40  75  Tripolyphosphate  50  80  65  93  Polyphosphate (P5)  62  N.D.  Polyphosphate  (P15)  41  N.D.  Orthophosphate (P1)  95  >99  Glucose-6-phosphate  0  0  Glycerol-3-phosphate  0  N.D.  Adenosine-5'-monophosphate  0  0  (P3)  Trimetaphosphate ( C y c l i c  a  phosphate-binding  P3)  Representative data from three determinations; N.D., determined  94  not  2.  I s o l a t i o n of mutants l a c k i n g the phosphate-binding  protein. E. c o l i  Mutants l a c k i n g the phosphate-binding p r o t e i n of (designated  phoS) are c o n s t i t u t i v e for a l k a l i n e  phosphatase ( W i l l s k y et a l . , 1973).  Therefore,  to obtain  mutants l a c k i n g the phosphate-binding p r o t e i n i n P. aeruginosa,  a l k a l i n e phosphatase c o n s t i t u t i v e mutants were  s e l e c t e d using the procedure of Brickman and (1975).  Of nine a l k a l i n e phosphatase c o n s t i t u t i v e mutants  obtained,  four lacked the phosphate-binding p r o t e i n on  polyacrylamide The  Beckwith  gels  (e.g. s t r a i n H585: F i g . 11,  lane  SDS-  C).  remainder were c o n s t i t u t i v e f o r a l l measured phosphate-  r e g u l a t e d c o n s t i t u e n t s in a d d i t i o n to a l k a l i n e phosphatase, i n c l u d i n g p r o t e i n P, phospholipase C and  the phosphate-  binding p r o t e i n , t y p i c a l of the r e g u l a t o r y mutants d e s c r i b e d i n Chapter One The  (e.g. H587 F i g . 11,  lane E ) .  absence of the phosphate-binding p r o t e i n i n  periplasmic  (and whole c e l l e x t r a c t s ) of  c e l l s of mutant s t r a i n H585 ( F i g . 11,  phosphate-limited  lane C) and  in  e x t r a c t s of the uninduced ( i . e . p h o s p h a t e - s u f f i c i e n t ) type parent  s t r a i n H242 ( F i g . 11,  lane F) was  wild-  correlated  32 with an  i n a b i l i t y of these e x t r a c t s to bind  orthophosphate (Table V I I I ) . e x t r a c t s of the induced (H242) ( F i g . 11,  In c o n t r a s t ,  (phosphate-limited)  lane G) and  Pperiplasmic parent  strain  the a l k a l i n e phosphatase  c o n s t i t u t i v e mutants r e t a i n i n g the phosphate-binding p r o t e i n (H587) ( F i g . 11,  lane E) demonstrated e x c e l l e n t binding of  32 P-orthophosphate (Table V I I I ) . 95  I n t e r e s t i n g l y , e x t r a c t s of  Table  VIII  32  a P - o r t h o p h o s p h a t e b i n d i n g by p e r i p l a s m i c e x t r a c t s of w i l d type and mutant s t r a i n s of aeruginosa  Strain  Phosphate-binding protein  32  P-orthophosphate "bound (cpm)  H585  2,370  H586  3,500  H587  18,100  H242  (Phosphatesufficient )  2,404  H242  (Phosphatedeficient )  17,100  P.  c  Ten ml c u l t u r e s were grown o v e r n i g h t under phosphatedeficient conditions (except as i n d i c a t e d ) and shock fluids o b t a i n e d as d e s c r i b e d i n Methods. Aliquots (25 u l ) were i n c u b a t e d i n t h e p r e s e n c e o f 0 . 5 uM o r t h o p h o s p h a t e (specific a c t i v i t y = 1 m C i / m l ) i n a f i n a l v o l u m e of 250 u l and p h o s p h a t e - b i n d i n g measured using the f i l t e r - b i n d i n g assay of L e v e r (1972) as d e s c r i b e d i n M e t h o d s . b The p r e s e n c e (+) o r absence ( - ) of the phosphate-binding p r o t e i n i n p e r i p l a s m i c e x t r a c t s was d e t e r m i n e d by SDSpolyacrylamide gel electrophoresis (see F i g . 5). c Representative  data  from  two  determinations  96  a l k a l i n e phosphatase-constitutive  s t r a i n H586  apparently  c o n t a i n the phosphate-binding p r o t e i n ( F i g . 11, lane D) yet 32 fail  to bind  P-orthophosphate (Table V I I I ) .  may w e l l represent a l t e r expression  T h i s mutant  a s t r u c t u r a l gene mutation which does not  of the binding p r o t e i n but does a f f e c t  activity. 3.  Phosphate t r a n s p o r t .  The involvement of the p e r i p l a s m i c  phosphate-binding p r o t e i n i n phosphate uptake i n v i v o was examined using a wild-type binding protein  s t r a i n c o n t a i n i n g the phosphate-  ( s t r a i n H242) and a mutant l a c k i n g the  b i n d i n g p r o t e i n ( s t r a i n H585).  The l o s s of the b i n d i n g  p r o t e i n i n H585 r e s u l t e d i n a marked d e f i c i e n c y i n phosphate t r a n s p o r t compared with the p a r e n t a l s t r a i n (Fig.  13). The r a p i d p l a t e a u i n g  (H242)  observed f o r the uptake  curve of the p a r e n t a l s t r a i n i n d i c a t e d that the a v a i l a b l e phosphate was being depleted, of an accurate  p r e c l u d i n g the determination  r a t e of t r a n s p o r t at the c o n c e n t r a t i o n of  phosphate used i n the experiment d e p i c t e d  i n F i g . 13.  Indeed, i t was necessary to d i l u t e the wild--type cells  1:4 compared with mutant c e l l s  i n order  parental  to o b t a i n a  comparable rate of phosphate uptake ( F i g . 13). At lower concentrations wild-type  cells  of phosphate i t was o f t e n necessary to d i l u t e 1:19 i n order  to obtain l i n e a r r a t e s of  transport.  97  F i g u r e 13. P h o s p h a t e u p t a k e i n P. a e r u g i n o s a . The procedure f o r transport assays i s described i n Methods. T h e c o n c e n t r a t i o n o f p h o s p h a t e was 2.5 uM. A l l c e l l s w e r e a s s a y e d a t a n a b s o r b a n c e a t 600 nm o f 0.30 e x c e p t a s i n d i c a t e d . S t r a i n H242 (• • ) ; strain H 5 8 5 (A A); s t r a i n H 2 4 2 d i l u t e d 1:4 ( f i n a l k^ = 0.06) ( 0 — 0 ) . nn  6  98  0  0  0.6  i 10  i  i  20 30 Time (seconds)  99  r AO  4.  K i n e t i c s of phosphate t r a n s p o r t .  In wild-type c e l l s of  P. aeruginosa, two major components of phosphate uptake were observable  ( F i g . 14B), c o n f i r m i n g p r e l i m i n a r y r e s u l t s  (LaCoste et a l . , 1981).  The h i g h - a f f i n i t y component of  uptake was c h a r a c t e r i z e d by an apparent  Km of 0.46 + 0.10 uM  phosphate and a Vmax of 5.4 + 0.2 nmol phosphate taken up/min/mg c e l l p r o t e i n while the l o w - a f f i n i t y component was c h a r a c t e r i z e d by an apparent  Km of 12.0 + 1.6 uM phosphate.  The e x t r a p o l a t e d Vmax value f o r the ' l o w - a f f i n i t y ' (16.0  + 1.5 nmol/min/mg c e l l p r o t e i n ) a c t u a l l y  curve  represented  the sum of both the high and l o w - a f f i n i t y parameters.  Given  that the e x t r a p o l a t e d Vmax of the h i g h - a f f i n i t y system i n the wild-type was 5.4 nmol/min/mg c e l l p r o t e i n , the a c t u a l Vmax of the l o w - a f f i n i t y system c o u l d be estimated as approximately  11 nmol/min/mg c e l l p r o t e i n .  T h i s was i n good  agreement with the value d e r i v e d from the phosphate-binding p r o t e i n - d e f i c i e n t mutant'strain H585 c o n t a i n i n g only the l o w - a f f i n i t y t r a n s p o r t system.  In the mutant H585, only a  s i n g l e phosphate uptake component with a Km of 19.3 + 1.4 uM phosphate and a Vmax of 12.1 +0.5 nmol/min/mg c e l l was  observable  protein  ( F i g . 14A). ( K i n e t i c constants represent the  mean of at l e a s t  three determinations + standard  deviation).  Thus, the l o s s of the phosphate-binding  by mutation  i n H585 c o r r e l a t e d with the l o s s of high-  affinity  phosphate  uptake.  100  protein  F i g u r e 14. K i n e t i c s of phosphate uptake i n P. aeruginosa. Data from uptake assays performed at v a r i o u s c o n c e n t r a t i o n s of phosphate was p l o t t e d as an Eadie-Hofstee p l o t with k i n e t i c constants d e r i v e d by l e a s t squares a n a l y s i s . A) phosphate-binding p r o t e i n d e f i c i e n t mutant s t r a i n H585 c o n t a i n i n g only a s i n g l e phosphate uptake system. B) wild-type s t r a i n H242 c o n t a i n i n g two phosphate uptake systems.  101  102  5.  Growth i n p h o s p h a t e - d e f i c i e n t medium.  In order to t e s t  whether the d e f e c t i n h i g h - a f f i n i t y phosphate t r a n s p o r t r e s u l t i n g from the phosphate b i n d i n g p r o t e i n - d e f i c i e n c y i n H585 c o u l d be c o r r e l a t e d with a growth d e f e c t , the growth of was  wild-type and mutant c e l l s i n p h o s p h a t e - d e f i c i e n t medium followed by measuring the time dependent increase i n  absorbance at 600 nm. sufficient cells  Upon resuspension of phosphate-  i n p h o s p h a t e - d e f i c i e n t medium (under  c o n d i t i o n s the phosphate-binding  which  p r o t e i n would be induced i n  the w i l d - t y p e ) , both s t r a i n s were seen to grow logarithmically  ( F i g . 15) a f t e r a short l a g (not shown).  However, the phosphate-binding  p r o t e i n - d e f i c i e n t mutant H585  grew at a markedly slower r a t e  (doubling time of 124  min)  compared with the wild-type H242 (doubling time of 67  min)  confiming the importance  of the b i n d i n g p r o t e i n to P.  aeruginosa c e l l s growing i n a l i m i t i n g  6.  environment.  P h y s i c a l a s s o c i a t i o n between outer membrane p r o t e i n P  and the p e r i p l a s m i c phosphate-binding  protein.  An  a s s o c i a t i o n between maltose-binding p r o t e i n and LamB p o r i n p r o t e i n of E. c o l i , demonstrated i n v i t r o Nikaido, for  1981), has been suggested  the e f f i c i e n t  a c r o s s the E. c o l i 1979).  to be necessary  t r a n s p o r t of maltose  and in vivo  and m a l t o d e x t r i n s  outer membrane (Wandersmann et a l • ,  To determine  phosphate-binding  (Bavoil  i f t h i s was  the case f o r p r o t e i n  P-  protein-mediated phosphate uptake i n P.  aeruginosa the phosphate-binding  103  p r o t e i n and p r o t e i n P were  F i g u r e 15. Growth of a phosphate-binding p r o t e i n . d e f i c i e n t mutant and i t s wild-type parent i n phosphate-limited medium. Overnight c u l t u r e s of H242 (wild type parent s t r a i n ) ( 0 — 0 ) and H585 (phosphate b i n d i n g p r o t e i n - d e f i c i e n t mutant) ( X — X ) grown i n p h o s p h a t e - s u f f i c i e n t medium were harvested, washed twice i n p h o s p h a t e - d e f i c i e n t medium and resuspended i n p h o s p h a t e - d e f i c i e n t medium at an absorbance at 600 nm of 0.20. Growth was followed by the time-dependent i n c r e a s e i n A,-.,..  104  examined f o r t h e i r a b i l i t i e s to a s s o c i a t e in v i t r o .  Using  a  modi fried ELISA procedure (see Methods) the phosphate-binding p r o t e i n was  immobilized  m i c t o t i t r e p l a t e s and specifically  on the bottom of the w e l l s of  examined f o r i t s a b i l i t y  r e t a i n p r o t e i n P f o l l o w i n g incubation  p r o t e i n P-containing  e x t r a c t s . The  demonstrated that p r o t e i n P and were apparently  capable of a s s o c i a t i n g in v i t r o .  (approximately  r e q u i r e d to At  this  0.14  uM)  when as l i t t l e as 20 ug/ml  of p r o t e i n P was  binding p r o t e i n - c o n t a i n i n g w e l l s of 20 ug/ml (0.14  16 % above background and background at 100  <10  uM)  added, to the  (Table IX). uM),  At a  p r o t e i n P binding  was  t h i s increased t o 33 % above  ug/ml (0.69  ug/ml (1.74  b i n d i n g was  At l e a s t 3  of b i n d i n g p r o t e i n , p r o t e i n P r e t e n t i o n i n  the w e l l s c o u l d be detected  concentration  IX  the phosphate-binding p r o t e i n  demonstrate p r o t e i n P r e t e n t i o n in the w e l l s . concentration  with  r e s u l t s in Table  ug/well of phosphate-binding p r o t e i n was  at 250  to  uM)  (Table I X ) .  and  42 % above background  In c o n t r a s t , p r o t e i n P  % above background i n the absence of  the phosphate-binding p r o t e i n (Table IX).  Increasing  the  amount of phosphate-binding p r o t e i n in the w e l l s d i d not increase the amount of p r o t e i n P which bound (at a given concentration)  suggesting  that l e v e l s of b i n d i n g p r o t e i n > 3  ug were s a t u r a t i n g the w e l l s .  The  observation  that  phosphate-binding protein-dependent p r o t e i n P binding d e t e c t a b l e at uM c o n c e n t r a t i o n s implied that the Kd  of p r o t e i n P (Table  f o r p r o t e i n P-binding  106  protein  was  IX)  Table IX.  In v i t r o a s s o c i a t i o n of the phosphate-binding p r o t e i n and outer membrane p r o t e i n P  [ P r o t e i n P]  1 A  Experiment ug/ml  1  2  d  e  Absorbance assays b  uM  + PBP  b  C  405 - PBP  C  0  0.00  0.27+0.02  0.21+0.01  20  0.14  0.32+0.01  0.22+0.01  100  0.69  0.36+0.01  0.23+0.01  250  1.74  0.12+0.03  0.07+0.02  at 405 nm + standard d e v i a t i o n of d u p l i c a t e  Assuming a molecular weight of 144,000 [deduced from the p r o t e i n P monomer molecular weight of 48,000 and the demonstrated trimer form of the n a t i v e p r o t e i n (Angus et a l , 1983)] P r o t e i n P b i n d i n g to m i c r o t i t r e w e l l s pre-coated with 3 ug of phosphate-binding p r o t e i n (+PBP) or no phosphate-binding p r o t e i n (-PBP) was d e t e c t e d using a p r o t e i n P t r i m e r s p e c i f i c antiserum. Antibody b i n d i n g , which was expected to be p r o p o r t i o n a l to the amount of p r o t e i n P present i n the w e l l s , was measured at A 4 0 5 f o l l o w i n g incubation with an a l k a l i n e phosphatase-conjugated second antibody and a chromogenic s u b s t r a t e , p a r a - n i t r o p h e n y l phosphate (pNPP).  d  e  A f t e r a d d i t i o n of the pNPP, c o l o u r developement was allowed to proceed f o r 18 h at room temperature ( i n the dark) before measuring absorbance at 405 nm A f t e r a d d i t i o n of pNPg, c o l o u r development was allowed to proceed f o r 2 h at 37 C before measuring absorbance at 405 nm 107  a s s o c i a t i o n was i n the uM range. data obtained and  f o r the a s s o c i a t i o n of maltose-binding  with protein  p r o t e i n LamB (Rd = 0.15 uM) (Neuhaus et a l . , 1983). One  was  T h i s was c o n s i s t e n t  of the problems with the ELISA method used above  the very low l e v e l s of p r o t e i n P which were i n f a c t  being  r e t a i n e d i n the w e l l s  ( i n one experiment  overnight  incubation was necessary to detect p r o t e i n P r e t e n t i o n ) . Since p r o t e i n P r e t e n t i o n was apparently  dependent upon the  presence of phosphate-binding p r o t e i n i n the w e l l s  (Table  IX) t h i s c o u l d be a t t r i b u t e d to s a t u r a t i o n of the m i c r o t i t r e w e l l s by phosphate-binding p r o t e i n at low l e v e l s  (> 3 u g ) .  T h i s was compounded by an observed decrease i n the a f f i n i t y of the antiserum used to detect p r o t e i n P b i n d i n g presence of detergent was  (e.g. T r i t o n X-100).  i n the  As a r e s u l t , i t  d i f f i c u l t to a c c u r a t e l y and c o n s i s t e n t l y measure b i n d i n g  between p r o t e i n P and the b i n d i n g p r o t e i n s i n c e background l e v e l s o f t e n e q u a l l e d or exceeded l e v e l s due t o s p e c i f i c binding.  Thus, i n order  to o b t a i n higher  l e v e l s of b i n d i n g  i t was deemed necessary to devise a method whereby l a r g e r amounts of the binding p r o t e i n c o u l d be  immobilized.  B a v o i l et a l . (1981) demonstrated an a s s o c i a t i o n between the maltose-binding immobilizing  p r o t e i n and p r o t e i n LamB by  l a r g e amounts. (10 mg) of the b i n d i n g p r o t e i n on  Sepharose beads and using a f f i n i t y chromatography t o demonstrate s p e c i f i c binding of the LamB p r o t e i n .  Using  t h i s methodology, a phosphate-binding p r o t e i n Sepharose 4B column was c o n s t r u c t e d  (see Methods)  108  and examined f o r the  a b i l i t y to s p e c i f i c a l l y bind p r o t e i n P. experiment, p r o t e i n P was  immobilized  i t s a b i l i t y to s p e c i f i c a l l y bind the p r o t e i n was  a l s o examined.  columns f a i l e d to bind any  sufficiently binding  7.  on Sepharose beads  these a f f i n i t y  p r o t e i n s , i n c l u d i n g the The  and  phosphate-binding  Unfortunately,  phosphate t r a n s p o r t p r o t e i n s . p r o t e i n s to Sepharose may  In a r e c i p r o c a l  relevant  c r o s s - l i n k i n g of these  well d i s t o r t  the  proteins  to prevent the adoption of the  necessary  conformations.  Summary.  purified  A b i n d i n g p r o t e i n f o r inorganic phosphate  to apparent homogeneity from the shock f l u i d s  phosphate-limited  Pseudomonas aeruginosa.  The  was of  purified  p r o t e i n bound one molecule of phosphate per molecule of binding p r o t e i n with a Kd of 0.34 pyrophosphate and long could  + 0.05  uM.  i n o r g a n i c polyphosphates up to 15 u n i t s  i n h i b i t the binding of phosphate to the  p r o t e i n , although organic  phosphates such as  phosphate, glycerol-3-phosphate and monophosphate could not.  distinct  observed i n wild-type uM  glucose-6-  adenosine-5'-  shown to be d e f i c i e n t i n  phosphate t r a n s p o r t compared with wild-type kinetically  binding  Mutants l a c k i n g the phosphate-  binding p r o t e i n were i s o l a t e d and  + 0.10  Arsenate,  cells.  Two  systems f o r phosphate uptake could c e l l s , with apparent Km  (high a f f i n i t y ) and  12.0  + 1.6  uM  values  of  be 0.46  (low a f f i n i t y ) .  In c o n t r a s t , only a s i n g l e l o w - a f f i n i t y t r a n s p o r t  system  was  observable in mutants l a c k i n g the phosphate-binding p r o t e i n  109  (Km  apparent = 19.3  + 1.4  uM phosphate), suggesting  the  involvment of the binding p r o t e i n i n the h i g h - a f f i n i t y phosphate uptake system of P. aeruginosa.  Mutants d e f i c i e n t  in the binding p r o t e i n were a l s o d e f e c t i v e in t h e i r to grow i n a p h o s p h a t e - l i m i t i n g specific  i n d u c t i o n and  medium c o n s i s t e n t with  requirement for the  protein-dependent h i g h - a f f i n i t y t r a n s p o r t limiting conditions.  ability the  phosphate-binding system under  An apparent a s s o c i a t i o n between the  phosphate-binding p r o t e i n and  the  phosphate-limitation-  i n d u c i b l e outer membrane p r o t e i n P was vitro.  110  demonstrated i n  CHAPTER THREE Immunological c r o s s - r e a c t i v i t y of phosphate-starvationinduced outer membrane p r o t e i n s of the f a m i l i e s E n t e r o b a c t e r i a c e a e and Pseudomonadaceae  1.  Phosphate-starvation-induction  of membrane p r o t e i n s of  the Pseudomonaceae and the E n t e r o b a c t e r i a c e a e .  Under  c o n d i t i o n s of p h o s p h a t e - l i m i t a t i o n , P. aeruginosa i s derepressed selective  f o r the s y n t h e s i s of p r o t e i n P, a phosphate-  (Hancock and Benz, manuscript  forming outer membrane p r o t e i n .  submitted),  channel-  Growth of other  Pseudomonads as w e l l as members of the E n t e r o b a c t e r i a c e a e (Table X) i n a p h o s p h a t e - d e f i c i e n t medium r e s u l t e d , i n many of  these s t r a i n s ,  (Fig.  i n the i n d u c t i o n of novel membrane p r o t e i n s  16), many of which e x i s t e d as the major c e l l  protein.  envelope  The observation that these p r o t e i n s were e n r i c h e d  in cation-aggregated  membrane p r e p a r a t i o n s demonstrated  that they were probably outer membrane p r o t e i n s .  The  phosphate s t a r v a t i o n - i n d u c t i o n of the PhoE outer membrane p r o t e i n s of E. c o l i S. typhimurium previously.  (Bauer et a l . , 1985) has been demonstrated  In experiments reported here, however, the PhoE  p r o t e i n of E. c o l i strain  (Overbeeke and Lugtenberg, 1980) and  co-migrated  with the OmpF p r o t e i n of t h i s  ( F i g . 16, lanes 17 and 18) making i t necessay to use  an ompF mutant s t r a i n induction  (JF700) (Table 1) to demonstrate PhoE  ( F i g . 16, lanes 15 and 16). New membrane p r o t e i n s  111  Enriched, s o l u b l e p r e p a r a t i o n s of the phosphate s t a r v a t i o n - i n d u c e d p r o t e i n s were s o l u b i l i z e d at 23°C or 88°C p r i o r to e l e c t r o p h o r e s i s on SDS-polyacrylamide s l a b g e l s . Low molecular weight standards (Sigma Chemical Co, St. L o u i s , Mo.) were co-electrophoresed and a p l o t of log molecular weight vs R f (measured as d i s t a n c e migrated in cm) f o r the standards was d e r i v e d , from which molecular weights of the phosphate s t a r v a t i o n - i n d u c e d p r o t e i n s were determined from t h e i r r e s p e c t i v e Rf v a l u e s . Because the p r o t e i n s s o l u b i l i z e d at 23°C occurred as smeared bands (see text) the d i s t a n c e migrated ( R f ) was determined f o r the midpoint of the area of densest s t a i n . 48K e.g. s i g n i f i e s a molecular weight of 48,000 b  A, e x t r a c t a b l e from c e l l envelopes i n 2 % (wt/vol) T r i t o n X- 100/20 mM T r i s - H C l pH 8.0/0.5 M EDTA; B, e x t r a c t a b l e from c e l l envelopes in 2 % (wt/vol) T r i t o n X-100/20 mM T r i s - H C l pH 8.0 a f t e r 30 min incubation at 37°C i n the presence of 1 mg/ml of lysozyme; C, e x t r a c t a b l e from c e l l envelopes i n 2 % (wt/vol) SDS/0.5 M NaCl Probable cross'-reactive p r o t e i n ( i n the oligomer form); see text for d i s c u s s i o n  d not e  f  determined  The 37K p r o t e i n of P. c h l o r o r a p h i s was e x t r a c t a b l e from c e l l envelopes i n 2 % (wt/vol) T r i t o n X-100 alone and i s probably an inner membrane p r o t e i n The 37K and 24K p r o t e i n s c o - p u r i f i e d , using a l l methods t e s t e d , such that the r e s u l t a n t oligomers at 23° C c o u l d not be d i s t i n g u i s h e d , but appeared as a high molecular smear of approximately 115K  11 2  Table X.  P r o p e r t i e s of phosphate s t a r v a t i o n - i n d u c e d membrane p r o t e i n s of the E n t e r o b a c t e r i a c e a e and the Pseudomonadaceae  Strain  Apparent Molecular Weight ( i n thousands) a f t e r s o l u b i l i z a t i o n at 88°C 23°C 3  Solubility p r o p e r t i e s of n a t i v e oligomers  P. aeruginosa  48  97  p. f l u o r e s c e n s  50  102°  A  22  22  N.D.  p. putida  45.5  97  p. c h l o r o r a p h i s  49.5  1 10  37  N.D.  _e  p. a u r e o f a c i e n s  48  107  A  p. cepacia  37  1!5  f  C  24  1l5  f  C  20.5  20.5  N.D.  p. pseudomallei  39  104  C  E. c o l i  37  83  C  B,C  S. typhimurium  36  82  c  B,C  K. pneumoniae  36.5  83  C  B,C  E. aerogenes  36  85  C  B,C  S. marcesens  37  87  C  C  11 3  A  c  d  A  c  C  C  C  A  were not detected  i n c e l l envelopes of P. m a l t o p h i l i a (now  Xanthomonas m a l t o p h i l i a ) , P. acidovorans  and P. solanacearum  s t r a i n s grown i n a p h o s p h a t e - d e f i c i e n t medium, although these s t r a i n s were derepressed  f o r the s y n t h e s i s of a l k a l i n e  phosphatase i n t h i s medium (data not shown). which apparently  P. s t u t z e r i ,  f a i l e d to produce a new membrane p r o t e i n  when grown i n p h o s p h a t e - d e f i c i e n t medium, grew  extremely  p o o r l y i n t h i s medium, and P. syrinqae d i d not grow at a l l , although both of these s t r a i n s grew q u i t e w e l l i n phosphates u f f i c i e n t medium. While most s t r a i n s expressed  a s i n g l e phosphate  s t a r v a t i o n - i n d u c e d membrane p r o t e i n band ( F i g . 16), P. f l u o r e s c e n s and P. c h l o r o r a p h i s each apparently  expressed  two ( F i g . 16, lanes 4 and 8, r e s p e c t i v e l y ) , and P. c e p a c i a apparently expressed  three ( F i g . 16, lane 12). With the  exception of the 20.5K p r o t e i n of P. cepacia  ( F i g . 16, lane  12) and the 22K p r o t e i n of P. f l u o r e s c e n s ( F i g . 16, lane 2 ) , a l l of the phosphate s t a r v a t i o n - i n d u c e d membrane p r o t e i n s were heat-modifiable electrophoretograms  (Table X ) .  SDS-polyacrylamide g e l  of s o l u b l e p r e p a r a t i o n s of these  p r o t e i n s revealed that the n a t i v e or unheated forms of the p r o t e i n s ran as higher molecular  weight oligomers  which  could be d i s s o c i a t e d to monomers upon heating at temperatures greater than  60°C , a property shared by the  m a j o r i t y of p o r i n s d e s c r i b e d to date Alphen, 1983).  (Lugtenberg  and van  In a d d i t i o n , the monomer molecular  weights  ranged from 36R to 39K or from 46K to 50K (Table X ) ,  1 14  F i g u r e 16. SDS-polyacrylamide g e l electrophoretogram of c e l l envelopes prepared from p h o s p h a t e - d e f i c i e n t and p h o s p h a t e - s u f f i c i e n t grown s t r a i n s of the f a m i l i e s Pseudomonadaceae and E n t e r o b a c t e r i a c e a e . Cell envelopes (lanes 1 to 14) and T r i t o n X-100 i n s o l u b l e c e l l envelopes (lanes 15-27) were prepared from: lane 1, p h o s p h a t e - s u f f i c i e n t and lane 2,phosphate-deficient P. aeruginosa; lane 3, p h o s p h a t e - s u f f i c i e n t and lane 4, p h o s p h a t e - d e f i c i e n t P. f l u o r e s c e n s ; lane 5, p h o s p h a t e - s u f f i c i e n t and lane 6, p h o s p h a t e - d e f i c i e n t P. p u t i d a ; lane 7, p h o s p h a t e - s u f f i c i e n t and lane 8, p h o s p h a t e - d e f i c i e n t P. c h l o r o r a p h i s ; lane 9, p h o s p h a t e - s u f f i c i e n t and lane 10, p h o s p h a t e - d e f i c i e n t P. a u r e o f a c i e n s ; lane 11, p h o s p h a t e - s u f f i c i e n t and lane 12, p h o s p h a t e - d e f i c i e n t P. c e p a c i a ; lane 13, p h o s p h a t e - s u f f i c i e n t and lane 14, p h o s p h a t e - d e f i c i e n t P. pseudomallei; lane 15, p h o s p h a t e - s u f f i c i e n t and lane 16, p h o s p h a t e - d e f i c i e n t E. c o l i K-12 s t r a i n JF700; lane 17, L-broth grown E. c o l i K-12 s t r a i n HMS174; lane 18, L-broth grown E. c o l i K-12 s t r a i n JF694; lane 19, p h o s p h a t e - s u f f i c i e n t and lane 20, p h o s p h a t e - d e f i c i e n t S. typhimurium LT2; lane 21, p h o s p h a t e - s u f f i c i e n t and lane 22, p h o s p h a t e - d e f i c i e n t 5. marcesens; lane 23, p h o s p h a t e - s u f f i c i e n t and lane 24, p h o s p h a t e - d e f i c i e n t K. pneumoniae; lane 25, p h o s p h a t e - s u f f i c i e n t E. aerogenes; lanes 26 and 27, p h o s p h a t e - d e f i c i e n t E. aerogenes. The arrows i n d i c a t e the phosphate-regulated p r o t e i n s . A l l samples were s o l u b i l i z e d at 88 °C p r i o r to e l e c t r o p h o r e s i s . The g e l in lanes 15-27 contained urea at a f i n a l c o n c e n t r a t i o n of 6M.  1 15  1  1  6  c h a r a c t e r i s t i c of the major E n t e r o b a c t e r i a l p o r i n p r o t e i n s (Lugtenberg and van Alphen, 1983) and p r o t e i n P of P.aeruginosa  (see Chapter One), r e s p e c t i v e l y .  T y p i c a l l y , p o r i n p r o t e i n P can be e x t r a c t e d n a t i v e s t a t e from p e p t i d o g l y c a n - a s s o c i a t e d  in i t s  c e l l envelopes or  outer membranes with T r i t o n X-100 i n the presence of EDTA (Chapter One), i n c o n t r a s t to the major p o r i n p r o t e i n F of P. aeruginosa  and the major p o r i n s of E. c o l i  typhimurium, which are s o l u b i l i z e d  and S.  ( i n t h e i r n a t i v e forms)  in T r i t o n X-100 only a f t e r lysozyme d i g e s t i o n of the peptidoglycan  (Hancock et a l . , 1981) or i n the presence of  SDS and high s a l t 1976;  ( > 0.4 M NaCl) (Nakamura and Mizushima,  Tokunaga et a l . , 1979; Yoshimura et a l . , 1983).  A  number of the phosphate s t a r v a t i o n - i n d u c i b l e p r o t e i n s examined were s o l u b l e i n T r i t o n X-100-EDTA, i n c l u d i n g those of P. p u t i d a , P. f l u o r e s c e n s , P. a u r e o f a c i e n s 49.5K p r o t e i n of P. c h l o r o r a p h i s phosphate-starvation-induced  (Table X ) .  and the The 37K  p r o t e i n of P. c h l o r o r a p h i s was  e x t r a c t a b l e with T r i t o n X-100 i n the absence of EDTA.  In  c o n t r a s t _ t o the other p r o t e i n s d e s c r i b e d here, t h i s p r o t e i n may w e l l be from the cytoplasmic  membrane.  The remainder of  the phosphate-starvation-induced  membrane p r o t e i n s were  i n s o l u b l e i n T r i t o n X-100-Tris-EDTA but c o u l d be s o l u b i l i z e d in t h e i r o l i g o m e r i c  forms i n SDS/0.5 M NaCl (Table X ) .  I n t e r e s t i n g l y , lysozyme d i g e s t i o n of the peptidoglycan d i d not  facilitate  T r i t o n X-100 s o l u b i l i z a t i o n of the phosphate  s t a r v a t i o n - i n d u c e d membrane p r o t e i n s of P. c e p a c i a ,  117  P. pseudo-mallei or S. marcesens, although such treatment d i d y i e l d T r i t o n - s o l u b l e phosphate-starvation-induced p r o t e i n s . in the cases of E. c o l i ,  S. typhimurium,  K. pneumoniae and  E. aerogenes (Table X ) .  2.  Immunological  c r o s s - r e a c t i v i t y of phosphate  starvation-  induced outer membrane p r o t e i n s . a.  C r o s s - r e a c t i v i t y of p r o t e i n oligomers i n phosphate-  limited c e l l  envelopes.  To t e s t  f o r immunological  cross-  r e a c t i v i t y of the phosphate s t a r v a t i o n - i n d u c e d membrane p r o t e i n s of the v a r i o u s s t r a i n s examined , SDSpolyacrylamide g e l electrophoretograms of p h o s p h a t e - l i m i t e d c e l l envelopes were e l e c t r o p h o r e t i c a l l y t r a n s f e r r e d t o n i t r o c e l l u l o s e and probed with a p r o t e i n P t r i m e r - s p e c i f i c p o l y c l o n a l antiserum Fig.,  ( f o r s p e c i f i c i t y of the antiserum see  17 lane 1; c f . lane 2 ) .  demonstrated  T h i s antiserum was  to s p e c i f i c a l l y d e t e c t p r o t e i n P t r i m e r s i n  c e l l envelopes of p h o s p h a t e - l i m i t e d P. aeruginosa c e l l s (Fig.  17, lane 4 ) . The p r o t e i n P - s p e c i f i c antiserum was  capable of r e a c t i n g with a component present i n the p h o s p h a t e - l i m i t e d c e l l envelopes of a l l s t r a i n s which had produced  a phosphate-starvation-induced membrane p r o t e i n and  with a component present i n c e l l  envelopes of E. c o l i K-12  s t r a i n JF694 which l a c k s the major p o r i n p r o t e i n s OmpF and OmpC but i s c o n s t i t u t i v e f o r PhoE ( F i g . 1 8 ) . In each case smeared bands of high molecular weight antiserum,  r e a c t e d with the  suggesting that the n a t i v e (unheated) oligomers  118  F i g u r e 17. I n t e r a c t i o n of p r o t e i n P t r i m e r - s p e c i f i c or monomer-specific antiserum with Western b l o t s of p u r i f i e d p r o t e i n P and Pseudomonas aeruginosa PA01 s t r a i n H103 c e l l envelopes. C e l l envelopes or p u r i f i e d p r o t e i n s were e l e c t r o p h o r e t i c a l l y t r a n s f e r r e d from SDS-polyacrylamide g e l electrophoretograms to n i t r o c e l l u l o s e and incubated with a p r o t e i n P t r i m e r s p e c i f i c (lanes 1 to 5) or monomer-specific (lanes 6 to 10) antiserum. Antibody b i n d i n g was detected using an a l k a l i n e phosphatase-conjugated g o a t - a n t i - r a b b i t IgG antibody ( f o r the t r i m e r - s p e c i f i c antiserum) or an a l k a l i n e phosphatase-conjugated goat-anti-mouse IgG antibody ( f o r the monomer-specific antiserum) and a h i s t o c h e m i c a l a l k a l i n e phosphatase s u b s t r a t e . Lanes 1 and 7, p u r i f i e d p r o t e i n P s o l u b i l i z e d at 23°C (trimer form); lanes 2 and 6, p u r i f i e d p r o t e i n P s o l u b i l i z e d at 880c (monomer form); lane 3, c e l l envelope p r e p a r a t i o n of p h o s p h a t e - s u f f i c i e n t P. aeruginosa s o l u b i l i z e d a t 23°C; lanes 4 and 10, c e l l envelope p r e p a r a t i o n of p h o s p h a t e - d e f i c i e n t P. aeruginosa s o l u b i l i z e d at 23°C; lanes 5 and 9, c e l l envelope p r e p a r a t i o n of p h o s p h a t e - d e f i c i e n t P. aeruginosa s o l u b i l i z e d a t 88°C; lane 8, c e l l envelope p r e p a r a t i o n of p h o s p h a t e - s u f f i c i e n t P. aeruginosa s o l u b i l i z e d at 88°C. A small amount of monomer p r o t e i n P can be seen in the t r i m e r p r e p a r a t i o n i n lane 7. The p r o t e i n P oligomer ( t r i m e r ) band observable i n Coomassie-staihed g e l s (e.g. F i g . 3, lanes 4 and 5) migrates at a p o s i t i o n corresponding to the bottom of the smeared band i n lane 1.  119  120  Figure 18. I n t e r a c t i o n of p r o t e i n P t r i m e r - s p e c i f i c antiserum with Western b l o t s of c e l l envelope p r e p a r a t i o n s of d i f f e r e n t b a c t e r i a grown under p h o s p h a t e - d e f i c i e n t or s u f f i c i e n t c o n d i t i o n s . Lane 1, p h o s p h a t e - d e f i c i e n t and lane 2, p h o s p h a t e - s u f f i c i e n t P. aeruginosa; lane 3, p h o s p h a t e - d e f i c i e n t and lane 4, p h o s p h a t e - s u f f i c i e n t P. f l u o r e s c e n s ; lane 5, p h o s p h a t e - d e f i c i e n t and lane 6, p h o s p h a t e - s u f f i c i e n t , P. p u t i d a ; lane 7, p h o s p h a t e - d e f i c i e n t and lane 8, p h o s p h a t e - s u f f i c i e n t P. c h l o r o r a p h i s ; lane 9, p h o s p h a t e - d e f i c i e n t and lane 10, p h o s p h a t e - s u f f i c i e n t P. a u r e o f a c i e n s ; lane 11, p h o s p h a t e - d e f i c i e n t and lane 12, p h o s p h a t e - s u f f i c i e n t P. c e p a c i a ; lane 13, p h o s p h a t e - d e f i c i e n t P. c e p a c i a ; lane 14, phosphated e f i c i e n t P. cepacia c e l l envelopes s o l u b i l i z e d i n 2% SDS/0.5 M NaCl to i n a c t i v a t e contaminating a l k a l i n e phosphatase; lane 15, p h o s p h a t e - d e f i c i e n t and lane 16, p h o s p h a t e - s u f f i c i e n t P. pseudomoallei; lane 17, p h o s p h a t e - d e f i c i e n t and lane 18, p h o s p h a t e - s u f f i c i e n t K. pneumoniae; lane 19, p h o s p h a t e - d e f i c i e n t and lane 20, p h o s p h a t e - s u f f i c i e n t E. aeroqenes; lane 21, p h o s p h a t e - d e f i c i e n t and lane 22, p h o s p h a t e - s u f f i c i e n t S. marcesens; lane 23, p h o s p h a t e - d e f i c i e n t and lane 24, p h o s p h a t e - s u f f i c i e n t S. typhimurium; lane 25, Lbroth grown E. c o l i K-12 s t r a i n JF694; lane 26, Lbroth grown E. c o l i K-12 s t r a i n HMS174; lane 27, p h o s p h a t e - d e f i c i e n t and lane 28, p h o s p h a t e - s u f f i c i e n t E. c o l i K-12 s t r a i n JF700. The c e l l envelopes were s o l u b i l i z e d at 23 °C p r i o r to e l e c t r o p h o r e s i s . The b l o t s , with the exception of lane 11, were developed with the p r o t e i n P - t r i m e r - s p e c i f i c antiserum as d e s c r i b e d i n the legend to F i g . 17. The b l o t i n lane 13 was incubated d i r e c t l y with an a l k a l i n e phosphatase h i s t o c h e m i c a l s u b s t r a t e to detect contaminating c e l l envelope bound a l k a l i n e phosphatase. S i m i l a r c o n t r o l s were negative f o r a l l other s t r a i n s shown here.  121  i i  1  L  12 3 4 5 6 7 8 910 11 12 13 14  1516 17 18 19 20 21 22 23 24 25 26 2728  1 22  of the  phosphate s t a r v a t i o n - i n d u c e d  In support  p r o t e i n s were r e a c t i n g .  of t h i s , the non-heat-modified o l i g o m e r i c  of the phosphate s t a r v a t i o n - i n d u c e d i d e n t i f i a b l e as high-molecular  forms  membrane p r o t e i n s were  weight smeared bands i n  Coomassie s t a i n e d SDS-polyacrylamide g e l electrophoretograms of enriched,  soluble preparations  S o l u b i l i z a t i o n of c e l l  of these p r o t e i n s .  envelopes at 8-8°C f o r 10 min, which  converted  o l i g o m e r i c p r o t e i n s to monomers (Table X ) ,  destroyed  this reactivity.  inability  T h i s was c o n s i s t e n t with the  of the antiserum to react with p r o t e i n P monomers  in heat t r e a t e d , phosphate-limited lane 5) or p u r i f i e d  i n detergent  cell  envelopes  (Fig.17,  ( F i g . 17, lane 2 ) . These  data excluded the non-heat-modifiable 20.5K and 22K p r o t e i n s of P. cepacia and P. f l u o r e s c e n s , r e s p e c t i v e l y , as the c r o s s - r e a c t i v e species i n these s t r a i n s . In the case of P. c e p a c i a , a strong c r o s s - r e a c t i v i t y o r i g i n a l l y seen ( F i g . 18, lane  11) was demonstrated to be due, i n p a r t , to the  presence of a l k a l i n e phosphatase a s s o c i a t e d with the c e l l envelope ( F i g . 18, lane soluble preparation  13). Using  a 2 % SDS/0.5 M NaCl  of a phosphate-limited  envelope, which contained  the phosphate  P. cepacia  cell  starvation-induced  p r o t e i n s but lacked a l k a l i n e phosphatase, a weak r e a c t i v i t y with the p r o t e i n P t r i m e r - s p e c i f i c antiserum was detected (Fig.  18, lane  14). No r e a c t i v i t y was observed with any c e l l  envelopes d e r i v e d from p h o s p h a t e - s u f f i c i e n t c e l l s  123  ( F i g . 18).  b.  I d e n t i f i c a t i o n of the c r o s s - r e a c t i v e p r o t e i n s .  c o n f i r m that the c r o s s - r e a c t i v i t y limited c e l l  envelopes was  seen in n a t i v e  To  phosphate-  indeed due to the o l i g o m e r i c  forms of the phosphate-starvation-induced p r o t e i n i n each case, we attempted to convert the m a t e r i a l present in the c r o s s - r e a c t i v e smeared bands to the a p p r o p r i a t e monomeric p r o t e i n s by h e a t i n g .  Thus, SDS-polyacrylamide g e l  electrophoretograms of n a t i v e cell  (unheated) p h o s p h a t e - l i m i t e d  envelopes ( f i r s t dimension) were heated at 88°C and  e l e c t r o p h o r e s e d on f r e s h SDS-polyacrylamide s l a b g e l s (second dimension).  A typical result  i s shown i n F i g . 19.  P r o t e i n s which were not-heat m o d i f i a b l e t y p i c a l l y  (Russel,  1976) appeared on the d i a g o n a l of a 2-dimensional (unheated vs heated) SDS-polyacrylamide g e l , since t h e i r molecular weights would remain unchanged i n the second dimension a f t e r heating.  P r o t e i n s which form n a t i v e oligomers which  d i s s o c i a t e i n response to h e a t i n g would  t y p i c a l l y appear at  a p o s i t i o n to the l e f t of the d i a g o n a l at t h e i r monomer molecular weights. to  the l e f t  As expected, p r o t e i n P occured  of the d i a g o n a l ( F i g . 19B) since i t ran as a  trimer i n the f i r s t dimension the  appropriate  second dimension  (unheated) and a monomer i n  (heated).  Furthermore, the p r o t e i n P  monomers ran as a broad band in the second dimension, c o n s i s t e n t with the apparent h e t e r o g e n e i t y of p r o t e i n P trimers in phosphate-limited c e l l purified  i n detergent ( F i g . 19A).  124  envelopes ( F i g . 19B) or  Figure 19. Two-dimensional (unheated x heated) SDSpolyacrylamide gel electrophoretogram of p u r i f i e d p r o t e i n P and c e l l envelopes prepared from phosphatel i m i t e d s t r a i n s of the f l u o r e s c e n t Pseudomonads. A) p u r i f i e d p r o t e i n P; B) P. aeruginosa; C) P. c h l o r o r a p h i s ; D) P. pseudomallei; E) §7 marcesens; and F) P. c e p a c i a . SDS-polyacrylamide g e l electrophoretograms of c e l l envelopes s o l u b i l i z e d at 23°C f o r 10 min p r i o r to e l e c t r o p h o r e s i s were e x c i s e d (1st dimension) were heated at 88°C f o r 10 min, l a i d across the top of a second SDS-polyacrylamide s l a b g e l with (4E) or without (4A,B,C,D,F) urea, and e l e c t r o p h o r e s e d i n the second dimension as d e s c r i b e d in M a t e r i a l s and Methods. Western immunoblots of f i r s t dimension gels (e.g. F i g 3) are included above the 2-D g e l s to i n d i c a t e the p o s i t i o n of the c r o s s r e a c t i n g m a t e r i a l i n n a t i v e c e l l envelopes p r i o r to heating i n the second dimension. The phosphates t a r v a t i o n - i n d u c e d p r o t e i n monomers are i n d i c a t e d by arrows.  1 25  126  For  a l l s t r a i n s expressing a h e a t - m o d i f i a b l e phosphate  s t a r v a t i o n - i n d u c e d p r o t e i n , i t was  p o s s i b l e to  demonstrate  the presence of phosphate-starvation-induced monomers to the left  of the diagonal i n the second dimension of a  dimensional unheated In  two-  vs heated SDS-polyacrylamide  slab g e l .  a l l cases these monomeric p r o t e i n s ran as broad bands,  the p o s i t i o n s of which corresponded with the p o s i t i o n of the c r o s s - r e a c t i v e smeared oligomer bands present i n n a t i v e phosphate-limited c e l l (Fig. of  19).  envelopes i n the f i r s t  Since the phosphate  dimension  starvation-induced proteins  P. p u t i d a , P. f l u o r e s c e n s and P. a u r e o f a c i e n s represented  the only h e a t - m o d i f i a b l e membrane p r o t e i n s i n these s t r a i n s , demonstrating broad bands which appeared  to the l e f t  of the  d i a g o n a l i n the second dimension and which corresponded with the c r o s s - r e a c t i v i t y seen in . f i r s t dimension g e l s  (e.g. F i g .  19B), they were r e a d i l y confirmed as the c r o s s - r e a c t i v e s p e c i e s i n these s t r a i n s . produced  S i m i l a r l y , the c o n s t i t u t i v e l y  PhoE p r o t e i n of the p o r i n - d e f i c i e n t E. c o l i  strain  JF694 e x i s t e d as the lone h e a t - m o d i f i a b l e , o l i g o m e r i c protein  i n c e l l envelopes of t h i s s t r a i n  o c c u r r i n g to the l e f t for  ( F i g . 16; Table X),  of the d i a g o n a l and thus accounting  the c r o s s - r e a c t i v i t y observed with c e l l  this strain.  envelopes of  Of the two h e a t - m o d i f i a b l e phosphate  s t a r v a t i o n - i n d u c e d membrane p r o t e i n s produced  by  P.  c h l o r o r a p h i s , only the 49.5K monomer p r o t e i n ran as a broad band i n the second dimension, whose p o s i t i o n a l s o corresponded with the c r o s s - r e a c t i v i t y seen in f i r s t  127  dimension  g e l s ( F i g . 1 9 C ) , c o n s i s t e n t with i t s being the  c r o s s - r e a c t i v e membrane p r o t e i n  in t h i s s t r a i n .  The remaining  s t r a i n s , which i n c l u d e d P. c e p a c i a , P. pseudomallei, E. c o l i K-12,  S. typhimurium,  K. pneumoniae, E. aerogenes and S.  marcesens, a l l produced  a number of c o n s t i t u t i v e membrane  p r o t e i n s which, l i k e the v a r i o u s p h o s p h a t e - s t a r v a t i o n induced membrane p r o t e i n s , were h e a t - m o d i f i a b l e , appearing to the l e f t of the d i a g o n a l i n the second dimension two-dimensional (e.g. F i g s .  unheated  vs heated SDS-polyacrylamide g e l  19D and 19E). Furthermore,  broad bands of monomer molecular weight a l s o corresponded f i r s t dimension envelopes  of a  they occurred as i n a p o s i t i o n which  with the c r o s s - r e a c t i v e smears observed i n  g e l s of n a t i v e p h o s p h a t e - l i m i t e d c e l l  (e.g. F i g s . 19D and 19E).  However, the'  expression of these c o n s t i t u t i v e p r o t e i n s i n the c e l l envelopes of phosphate s u f f i c i e n t c e l l s , which had p r e v i o u s l y f a i l e d to react with the p r o t e i n P-trimer s p e c i f i c antiserum  ( F i g . 17), excluded these p r o t e i n s as the  c r o s s - r e a c t i v e components i n these s t r a i n s .  Thus the s i n g l e  h e a t - m o d i f i a b l e phosphate s t a r v a t i o n - i n d u c i b l e present i n each s t r a i n  protein  (except P. cepacia) must be  r e s p o n s i b l e f o r the c r o s s - r e a c t i v i t y observed with the p r o t e i n P - s p e c i f i c antiserum.  Nonetheless,  demonstrated  (heterogeneity) on SDS-  that the smearing  this  polyacrylamide g e l s of n a t i v e oligomers was not r e s t r i c t e d to phosphate-starvation-induced membrane p r o t e i n s .  Rather,  i t may w e l l be a property of o l i g o m e r i c membrane p r o t e i n s ,  128  specifically The  porins.  expression  by P. cepacia  phosphate s t a r v a t i o n - i n d u c e d  of two  membrane p r o t e i n s , both of  which migrated as broad bands to the the  second dimension, and  heat-modifiable  l e f t of the diagonal  in a p o s i t i o n which corresponded  with the c r o s s - r e a c t i v e smeared band seen i n the dimension ( F i g . 19F),  made i t d i f f i c u l t  first  to unambiguously  i d e n t i f y the c r o s s - r e a c t i v e s p e c i e s . Furthermore, proteins  the  were i n v a r i a b l y c o - p u r i f i e d by a l l t e s t e d methods,  making i t impossible  to i n d i v i d u a l l y examine t h e i r  r e a c t i v i t i e s with the p r o t e i n P - s p e c i f i c antiserum . on molecular weight, however, the c r o s s - r e a c t i v e species was molecular weight was that of the 24K  c. monomers. porin  the  37K  l i k e l y candidate for  the  p r o t e i n , whose monomer  more t y p i c a l of porins  i n general  than  protein.  C r o s s - r e a c t i v i t y of phosphate-starvation-induced Porin monomers, obtained by heat denaturation  (Nakamura and Mizushima, 1976)  the b e t a - s t r u c t u r e  of the n a t i v e p r o t e i n  Mizushima, 1976).  Consistent  be  Based  of  t r i m e r s , have been shown to e x h i b i t an a l p h a - h e l i c a l  structure  trimer  in  very d i s t i n c t (Nakamura  from  and  with t h i s , the monomer  and  forms of i n d i v i d u a l p o r i n s have been demonstrated to  immunologically n o n - c r o s s - r e a c t i v e  1981). The  (Hofstra and  Dankert,  demonstration, then, that the p o r i n monomers of  d i f f e r e n t species  of the  family Enterobacteriaceae  immunologically c r o s s - r e a c t  (Hofstra and  1 29  Dankert,  could 1980;  Overbeeke and Lugtenberg, 1980), although l i n e a r epitopes present  implying that  i n p o r i n monomers had been  conserved  during p o r i n e v o l u t i o n , d i d not demonstrate the e x i s t e n c e of conserved  epitopes  i n the n a t i v e oligomers.  In order to  determine whether the c r o s s - r e a c t i v i t y of phosphate s t a r v a t i o n - i n d u c e d membrane p r o t e i n s could be a t t r i b u t e d to conserved  l i n e a r e p i t o p e s , the monomer and oligomer  forms of  the v a r i o u s p h o s p h a t e - s t a r v a t i o n - i n d u c i b l e membrane p r o t e i n s were t e s t e d f o r t h e i r a b i l i t y to r e a c t with an antiserum r a i s e d a g a i n s t h e a t - d i s s o c i a t e d p r o t e i n P monomers. antiserum  was demonstrated to r e a c t s p e c i f i c a l l y  p r o t e i n P monomers i n heat denatured phosphate-limited  P. aeruginosa  cell  The  with  envelopes of  ( F i g . 17, lane 9) or with  p u r i f i e d p r o t e i n P monomers ( F i g . 17, lane 6), e x h i b i t i n g no r e a c t i v i t y with the trimer form of the p r o t e i n ( F i g . 17, lanes 7 and 10) or with uninduced ( p h o s p h a t e - s u f f i c i e n t ) cell  envelopes ( F i g . 17 lane 8 ) .  T h i s antiserum  f a i l e d to  react with the p h o s p h a t e - s t a r v a t i o n - i n d u c i b l e membrane p r o t e i n s , i n monomer or oligomer  form (data not shown),  i n d i c a t i n g that the p h o s p h a t e - s t a r v a t i o n - i n d u c i b l e membrane p r o t e i n s do not c r o s s - r e a c t immunologically  with p r o t e i n P  monomers.  3.  Summary.  B a c t e r i a from the f a m i l i e s  Enterobacteriaceae  and Pseudomonadaceae were grown under p h o s p h a t e - d e f i c i e n t (0.1 for  - 0.2 mM inorganic phosphate) c o n d i t i o n s and examined the production of novel membrane p r o t e i n s .  130  Twelve of  the seventeen s t r a i n s examined expressed a phosphatestarvation-induced modifiable,  outer membrane p r o t e i n which was  i n that a f t e r s o l u b i l i z a t i o n  i n SDS  heat-  at  low  temperature the p r o t e i n s ran on g e l s as d i f f u s e bands of higher  apparent molecular weight, presumably oligomer forms,  which s h i f t e d to t h e i r apparent monomer forms a f t e r s o l u b i l i z a t i o n at high temperature. i n t o two and  These p r o t e i n s  fell  c l a s s e s based on t h e i r monomer molecular weights  the detergent c o n d i t i o n s r e q u i r e d to r e l e a s e  p r o t e i n s from the p e p t i d o g l y c a n . by species of the P. Pseudomonadaceae, was  fluorescens  The  f i r s t c l a s s , expressed  branch of the  s i m i l a r to the  the  family  phosphate-starvation-  i n d u c i b l e channel-forming p r o t e i n P of P. aeruginosa.  The  second c l a s s resembled the major E n t e r o b a c t e r i a l p o r i n p r o t e i n s and coli.  the phosphate-regulated PhoE p r o t e i n of  E.  Using a p r o t e i n P t r i m e r - s p e c i f i c p o l y c l o n a l  antiserum i t was the o l i g o m e r i c Western b l o t s .  p o s s i b l e to demonstrate c r o s s - r e a c t i v i t y of  forms of both c l a s s e s of these p r o t e i n s However, t h i s antiserum d i d not  the monomeric forms of any p r o t e i n P monomers. antiserum, no  of these p r o t e i n s ,  on  react with  including  Using a p r o t e i n P monomer-specific  r e a c t i v i t y was  seen with any  s t a r v a t i o n - i n d u c i b l e membrane p r o t e i n s or monomeric form) with the exception  of the phosphate-  (in either  oligomeric  of p r o t e i n P monomers.  These r e s u l t s suggest the presence of conserved a n t i g e n i c determinants only i n the n a t i v e , f u n c t i o n a l p r o t e i n s .  131  CHAPTER FOUR C h a r a c t e r i z a t i o n o§ p r o t e i n P - l i k e p o r i n s from the fluorescent  1.  Pseudomonadacea  P u r i f i c a t i o n of the p h o s p h a t e - s t a r v a t i o n - i n d u c i b l e  membrane p r o t e i n s of the f l u o r e s c e n t Pseudomonads. previous chapter  outer  In the  a number of b a c t e r i a l s t r a i n s were  demonstrated to synthesize outer membrane p r o t e i n s .  phosphate-starvation-inducible Members of the f l u o r e s c e n t  Pseudomonadaceae, i n c l u d i n g P. p u t i d a , P. f l u o r e s c e n s , P. a u r e o f a c i e n s and P. c h l o r o r a p h i s synthesized an o l i g o m e r i c , heat-modifiable  outer membrane p r o t e i n which e x h i b i t e d a  number of p r o p e r t i e s i n common with p r o t e i n P of P. aeruginosa.  Using  the observed immunological c r o s s -  r e a c t i v i t y of these p r o t e i n s with p r o t e i n P, an attempt was made t o p u r i f y these phosphate-regulated specific  p r o t e i n s by  r e t e n t i o n on an immunoadsorbent column c o n s t r u c t e d  using the p r o t e i n P t r i m e r - s p e c i f i c antiserum Chapter one.  described in  P r o t e i n P, the o r i g i n a l antigen, was r e a d i l y  p u r i f i e d and i n reasonable ( F i g . 20, lanes  q u a n t i t i e s by t h i s method  1 and 2 ) . I t was a l s o p o s s i b l e to i s o l a t e  the other p h o s p h a t e - s t a r v a t i o n - i n d u c i b l e p r o t e i n s using t h i s column, although  outer membrane  the y i e l d s were  s u b s t a n t i a l l y lower, r e q u i r i n g a s e n s i t i v e s i l v e r s t a i n i n g procedure to detect the p r o t e i n s i n SDS-polyacrylamide g e l s (eg. F i g . 20, lanes 3 and 4 ) . Apparently,  the c r o s s -  r e a c t i v e a n t i b o d i e s i n the p r o t e i n P t r i m e r - s p e c i f i c  132  Figure 20. SDS-polyacrylamide g e l electrophoretogram of p u r i f i e d p h o s p h a t e - s t a r v a t i o n - i n d u c i b l e outer membrane p r o t e i n s of the f l u o r e s c e n t Pseudomonads. Phosphates t a r v a t i o n - i n d u c i b l e p r o t e i n s were p u r i f i e d from the outer membranes of lanes 1,2,11 and 12, P. aeruginosa ( i . e . p r o t e i n P); lanes 3 and 4, P. f l u o r e s c e n s ; lanes 5 and 6, P. p u t i d a ; lanes 7 and 8, P. aureofacTens; lanes 9 and 10, P. c h l o r o r a p h i s . The p r o t e i n s i n lanes 3,4,11 and 12 were p u r i f i e d v i a a f f i n i t y chromatography using a r a b b i t a n t i - p r o t e i n P immunoadsorbant column. The p r o t e i n s i n lanes 1,2 and 5-10 were p u r i f i e d by e l e c t r o e l u t i o n from polyacrylamide g e l s . Samples were s o l u b i l i z e d at 88°C (lanes 1,3,5,7,9,11) or 23°C (lanes 2,4,6,8,10,12) p r i o r to e l e c t r o p h o r e s i s . Lanes 3 and 4 were s t a i n e d for p r o t e i n using a s e n s i t i v e s i l v e r s t a i n i n g procedure (Wray et a l 1981). A r t i f a c t bands v i s i b l e in these lanes are a product of the s i l v e r s t a i n i n g procedure. A l l other lanes were s t a i n e d by Coomassie b r i l l i a n t blue. The f a i n t continuous band seen i n the middle of the g e l i s an a r t i f a c t and was observable i n lanes were no p r o t e i n was loaded.  133  -  1 2  f  3  i  4  5  6  134  7 8  9  10  11 12  antiserum represent  only a minor or l o w - a f f i n i t y component  of t h i s antiserum.  The p u r i f i e d p r o t e i n s occurred  molecular  as higher  weight oligomers i n SDS-polyacrylamide gels when  s o l u b i l i z e d at room temperature p r i o r to e l e c t r o p h o r e s i s (e.g. F i g . 20, lane 4), d i s s o c i a t i n g to lower  molecular  weight monomers when s o l u b i l i z e d at 88°C (e.g. F i g . 20, lane 3).  T h i s was c o n s i s t e n t with the observed p r o p e r t i e s of  these p r o t e i n s i n phosphate-limited preparations  cell  envelope  (see Chapter Three, F i g . 16) and with the  p r o p e r t i e s of p u r i f i e d p r o t e i n P ( F i g . 20,- lanes  1 and 2 ) .  To improve y i e l d s , these p r o t e i n s were a l s o p u r i f i e d using a procedure f o r the e l e c t r o e l u t i o n of p r o t e i n s out of SDS-polyacrylamide g e l s (Parr et a l . , 1986). starvation-induced  Phosphate-  p r o t e i n - c o n t a i n i n g e x t r a c t s prepared from  each of the above s t r a i n s were run on SDS-polyacrylamide g e l s , the r e l e v a n t p r o t e i n oligomer bands e x c i s e d and the p r o t e i n e l e c t r o e l u t e d from the g e l . substantially  increased y i e l d s of a l l p r o t e i n s  lanes 5-12) which were e a s i l y v i s i b l e gels.  T h i s method produced ( F i g . 20,  i n Coomassie s t a i n e d  Again, the p u r i f i e d p r o t e i n s r e t a i n e d t h e i r  oligomeric  s t r u c t u r e , as a t t e s t e d by t h e i r r e s i s t a n c e to SDS  denaturation  ( F i g . 20, lanes 6,8,10,12) unless heated at  high temperature ( F i g . 20, lanes 5,7,9,11).  2.  S i n g l e channel experiments.  phosphate-starvation-inducible  When the p u r i f i e d outer membrane p r o t e i n s were  added i n small q u a n t i t i e s (5-10 ng/ml) to the aqueous  1 35  solutions  bathing a black l i p i d b i l a y e r membrane, membrane  conductance was seen to increase (e.g.  i s a stepwise  fashion  F i g . 21), presumably due to the i n c o r p o r a t i o n of  individual protein for  other p o r i n s  and  Hancock, 1981).  oligomers i n t o the membrane as suggested  (Benz et a l . , 1978; Benz et a l . , 1979; Benz The observed s i n g l e channel conductance  increments were d i s t r i b u t e d about a mean (e.g. F i g . 22), although l a r g e r and  increments were a l s o seen at 2 ( F i g . 22), 3  4 (not shown) times-the average s i n g l e channel  conductance.  These probably represented m u l t i p l e  of the p r o t e i n  oligomers i n t o the b i l a y e r membrane as has  been observed f o r other p o r i n s , et a l . , 1982).  insertions  including protein  P (Hancock  The average s i n g l e channel conductances i n  1 M KC1 measured f o r a given p r o t e i n  p u r i f i e d by e i t h e r  a f f i n i t y chromatography or e l e c t r o e l u t i o n were not s i g n i f i c a n t l y d i f f e r e n t (Table X I ) , c o n f i r m i n g both the u t i l i t y of the a n t i - p r o t e i n  P immunoadsorbent column i n  purifying functional cross-reactive  molecules and the  general a p p l i c a b i l i t y of the e l e c t r o e l u t i o n procedure i n purifying functionally active porin proteins. the d e r i v e d  In a d d i t i o n ,  average s i n g l e channel conductance values  obtained f o r each of the p h o s p h a t e - s t a r v a t i o n - i n d u c i b l e proteins 233  were not s i g n i f i c a n t l y d i f f e r e n t , f a l l i n g  and 252 pS (Table X I ) .  between  These values were s u b s t a n t i a l l y  l e s s than those obtained f o r the E. c o l i  porins,  including  the p h o s p h a t e - s t a r v a t i o n - i n d u c i b l e PhoE porin of t h i s s t r a i n (approximately 2 nS) (Benz et a l . , 1985), and f o r the major  136  F i g u r e 21. S t r i p chart recordings of stepwise i n c r e a s e s in the conductance of a small (0.1 mm ) o x i d i z e d c h o l e s t e r o l membrane (1.5 % i n n-decane) caused by the a d d i t i o n of 10 ng/ml of the p h o s p h a t e - s t a r v a t i o n induced outer membrane p r o t e i n from P. p u t i d a t o the aqueous phase (1 M KC1, pH 6.0). The a p p l i e d v o l t a g e was 50 mV and the temperature was 25°C. 2  1 37  I—  1  ho tn  cn O  >  -° CO  -o  1 38  °  F i g u r e 22. Histogram of the conductance f l u c t u a t i o n s observed with membranes of o x i d i z e d c h o l e s t e r o l (1.5 % in n-decane) i n the presence of the phosphates t a r v a t i o n - i n d u c e d outer membrane p r o t e i n of P. p u t i d a and 1 M KC1 (pH 6.0) i n the aqueous phase. The a p p l i e d v o l t a g e was 50 mV and the temperature was 25°C. P(A) i s the p r o b a b i l i t y of a given conductance increment A ~ taken from recorder t r a c i n g s such as that shown i n Figure 21.  1 39  A  1 =  247 pS  n = 152  A = 4 9 0 pS n=7 2  0  120  240  360  A/pS  140  480  600  Table XI.  Channel-forming p r o p e r t i e s of a f f i n i t y - p u r i f i e d and e l e c t r o e l u t e d p h o s p h a t e - s t a r v a t i o n - i n d u c i b l e outer membrane oligomers of the f l u o r e s c e n t Pseudomonads  Affinity Purified  Electroeluted  Strain Single channel conductance (pS)  n  3  b  S i n g l e channel conductance (pS)  n  P. aeruginosa  239  317  234  224  p. putida  233  74  247  307  p. f l u o r e s c e n s  241  1 17  -  p. aureofaciens  237  54  252  198  p. c h l o r o r a p h i s  243  45  237  201  a Average value  from n events  b Number of s i n g l e channel events measured  141  -  p o r i n p r o t e i n F of P. aeruginosa (5 nS) (Benz and 1981).  They were, however, i n e x c e l l e n t agreement  Hancock, with the  observed s i n g l e channel conductance of p r o t e i n P i n 1 M KC1 (Table XI) suggesting that the phosphate-regulated p o r i n p r o t e i n s of the f l u o r e s c e n t Pseudomonads a l l form small channels t y p i c a l of p r o t e i n P and i n c o n t r a s t to the m a j o r i t y of p o r i n s descibed to date (Benz et a l . ,  1981),  i n c l u d i n g other p h o s p h a t e - s t a r v a t i o n - i n d u c i b l e p o r i n s proteins al.  3.  f  (Benz et a l . ,  1981; Verhoef et a l . ,  1984; Bauer et  1985).  Ion-selectivity.  To examine the i o n - s e l e c t i v i t y of  these channels, s i n g l e channel conductance was. measured i n s a l t s of v a r y i n g c a t i o n or anion s i z e .  The a n i o n - s p e c i f i c  p r o t e i n P channel has p r e v i o u s l y been demonstrated to y i e l d average s i n g l e channel conductances, the magnitudes of which were dependent e x c l u s i v e l y upon the s i z e of the anion (Benz et a l . ,  1983).  Thus conductance through p r o t e i n P  channels was demonstrated to be i n v e r s e l y r e l a t e d to the s i z e of the anion (Benz et a l . ,  1983), while remaining  b a s i c a l l y u n a f f e c t e d by changes  i n c a t i o n s i z e of the s a l t  bathing a p r o t e i n P-containing l i p i d b i l a y e r membrane (Hancock et a l . ,  1982).  By comparing the average s i n g l e  channel conductance values obtained i n K C1 +  example, T r i s C l +  with, f o r  and K Hepes , i n which cases the c a t i o n +  and anion s i z e s , r e s p e c t i v e l y , are increased, i t should be p o s s i b l e to gain some idea of the ion s e l e c t i v i t y of each of  142  the phosphate-starvation-induced p o r i n p r o t e i n s .  The  r e s u l t s i n Table XII suggested that i n a l l cases the observed s i n g l e channel conductance  was  dependent upon anion  s i z e o n l y , such that i n c r e a s i n g the anion s i z e i n the case of K Hepes +  (anion dimensions of 1.4x0.6x0.5 nm compared  with a r a d i u s of 0.181  nm  f o r K+Cl ) r e s u l t e d i n no  d e t e c t a b l e conductance  increments, while i n c r e a s i n g the  c a t i o n s i z e in the case of T r i s C l  ( c a t i o n r a d i u s of  +  nm compared with 0.133 channel conductance  nm  f o r K C1 +  which was  that observed i n K C1 +  .  0.67  ) yielded a single  not d i s c e r n a b l y d i f f e r e n t  from  These data were c o n s i s t e n t with the  formation of a n i o n - s e l e c t i v e , i f not s p e c i f i c , channels by these p r o t e i n s . The a n i o n - s p e c i f i c i t y of p r o t e i n P has been shown to be due to the presence of an anion-binding s i t e w i t h i n the channel  (Benz et a l . ,  1983).  Thus conductance  through  p r o t e i n P channels s a t u r a t e s at high s a l t c o n c e n t r a t i o n s (Benz et a l . ,  1983),  i n c o n t r a s t to p o r i n p r o t e i n s which  lack b i n d i n g s i t e s and t y p i c a l l y r e v e a l a l i n e a r dependence of s i n g l e channel conductance and Hancock, 1981;  on s a l t c o n c e n t r a t i o n (Benz  Benz et a l . ,  1984).  To determine  i f the  a n i o n - s e l e c t i v i t y of the phosphate-regulated p o r i n p r o t e i n s could be a t t r i b u t e d to b i n d i n g s i t e s w i t h i n t h e i r channels, s i n g l e channel conductance f u n c t i o n of s a l t  was measured as a  (KC1) c o n c e n t r a t i o n .  channel conductance  was  respective  In every case, s i n g l e  seen to s a t u r a t e at high s a l t  c o n c e n t r a t i o n s (e.g. F i g . 23) c o n s i s t e n t with the presence  143  Table X I I .  S i n g l e channel conductance of phosphates t a r v a t i o n - i n d u c i b l e p o r i n p r o t e i n s of the f l u o r e s c e n t Pseudomonads i n s a l t s of v a r y i n g anion and c a t i o n s i z e  Average s i n g l e channel conductance (pS) i n  Strain K Cl" +  Tris Cl"  b  +  b  K Hepes" +  P. aeruginosa  158  141  <25  p. putida  144  1 43  <25  p. f l u o r e s c e n s  156  149  <25  p. a u r e o f a c i e n s  164  169  <25  p. c h l o r o r a p h i s  166  1 67  <25  Average b  b  of at l e a s t 60 s i n g l e channel events  S a l t s were employed at a c o n c e n t r a t i o n of 0.5 M. Ion r a d i i ( i n nm) are as f o l l o w s : K , 0.133; C l " , 0.181; T r i s , 0.670. Hepes , an e l l i p s o i d molecule, has dimensions 1.4x0.6x0.5 nm. +  -  144  of  a b i n d i n g s i t e w i t h i n these channels.  data as an Eadie-Hofstee p l o t values f o r C l  By p l o t t i n g the  (e.»g. F i g . 23  i n s e t ) , Kd  b i n d i n g were r e a d i l y d e r i v e d (Table X I I I ) .  While some v a r i a b i l i t y  i n the a f f i n i t y of C l  f o r each of  the channels was observed, there was only a 2 - f o l d range i n Kd values f o r C l  b i n d i n g f o r a l l channels,  including  p r o t e i n P, demonstrating that the r e l a t i v e a f f i n i t i e s of each of the channels f o r C l  4.  were s i m i l a r to p r o t e i n P.  Phosphate i n h i b i t i o n of macroscopic  Because the s i n g l e channel conductance in phosphate  conductance. of p r o t e i n P channels  i s low (6-9 pS i n 1 M H P 0 2  4  ) (Hancock et a l . ,  1982), approaching the r e s o l u t i o n l i m i t s of the black  lipid  b i l a y e r apparatus, the presence of a phosphate-binding  site  w i t h i n p r o t e i n P channels was supported by the a b i l i t y of orthophosphate channels.  to i n h i b i t C l  conductance  The d e r i v e d I^Q value f o r phosphate  the c o n c e n t r a t i o n of phosphate of  c h l o r i d e conductance)  which y i e l d e d 50 % i n h i b i t i o n had a  f o r p r o t e i n P channels than d i d  (Hancock and Benz, submitted) the anion p r e v i o u s l y  e x h i b i t i n g the h i g h e s t a f f i n i t y et  (defined as  i n d i c a t e d that orthophosphate  60-100 f o l d higher a f f i n i t y Cl  through p r o t e i n P  al.,  submitted).  phosphate-binding  f o r p r o t e i n P channels  In order to i d e n t i f y  (Benz  potential  s i t e s i n the phosphate-starvation-induced  p o r i n p r o t e i n s of P. p u t i d a , P. f l u o r e s c e n s , P. a u r e f a c i e n s and P. c h l o r o r a p h i s , t h i s s t r a t e g y was a l s o a p p l i e d . a f t e r formation of a l i p i d b i l a y e r  145  ( i n d i c a t e d by the  Thus,  Figure 23. Average s i n g l e channel conductance of the phosphate-starvation-induced p o r i n p r o t e i n of P. a u r e o f a c i e n s as a f u n c t i o n of the KC1 c o n c e n t r a t i o n i n the aqueous s o l u t i o n bathing an o x i d i z e d c h o l e s t e r o l (1.5 % i n n-decane) membrane. The a p p l i e d v o l t a g e was 50 mV and the temperature was 25°C. The aqueous phase contained approximately 10 ng/ml of p o r i n p r o t e i n at KC1 c o n c e n t r a t i o n s of 300 mM and h i g h e r . For KC1 c o n c e n t r a t i o n s below 300 mM, 100 ng/ml of p r o t e i n had to be added to obtain a s u f f i c i e n t number of s i n g l e channels. I n s e t . An Eadie-Hofstee p l o t of the data obtained from measurements of s i n g l e channel conductance (V) as a f u n c t i o n of the KC1 c o n c e n t r a t i o n (S). Binding constants (Table XIII) were obtained from such a p l o t using l e a s t squares a n a l y s i s .  146  KCl  Concentration  147  (mM)  Table X I I I .  Binding a f f i n i t i e s of p h o s p h a t e - s t a r v a t i o n i n d u c i b l e p o r i n p r o t e i n s of the f l u o r e s c e n t Pseudomonads f o r c h l o r i d e and orthophosphate  IgQ f o r phosphate (mM) Strain  Kd f o r C l (mM)  aeruginosa  at  a  40 mM P.  b  Cl"  1 M Cl"  153  0.59  12.7  P. p u t i d a  192  1 .08  -  P. f l u o r e s c e n s  220  -  9.7  P. a u r e o f a c i e n s  297  -  27.0  P. c h l o r o r a p h i s  204  2.40  -  The average s i n g l e channel conductance from at l e a s t 75 recorded events was determined at each of 5 c o n c e n t r a t i o n s of KC1 between 50 and 1000 mM. The data was p l o t t e d as an Eadie-Hofstee p l o t (Figure 23 i n s e t ) from which Kd values were obtained by l e a s t squares a n a l y s i s . b  I n h i b i t i o n of macroscopic c h l o r i d e conductance by phosphate was c a r r i e d out as d e s c r i b e d i n Methods. The % i n h i b i t i o n of i n i t i a l conductance was measured f o r d i f f e r e n t c o n c e n t r a t i o n s of phosphate and the data p l o t t e d as an Eadie-Hofstee p l o t (see F i g . 24 i n s e t ) from which I values were obtained by l e a s t squares a n a l y s i s 5 Q  148  membrane's t u r n i n g o p t i c a l l y b l a c k ) a small amount of p r o t e i n was added to the aqueous s o l u t i o n bathing the l i p i d membrane and conductance followed u n t i l the rate of increase had slowed c o n s i d e r a b l y  (usually  15-25 min).  At t h i s time  membrane conductance had u s u a l l y increased 2-4 orders of magnitude and > 1000 channels were present i n the membrane. A l i q u o t s of phosphate were added s e q u e n t i a l l y and the new conductance l e v e l measured a f t e r each a d d i t i o n . For each p r o t e i n s t u d i e d , phosphate a d d i t i o n was seen to decrease the l e v e l of conductance o r i g i n a l l y observed i n the presence of KC1 alone, and the magnitude of t h i s decrease was d i r e c t l y r e l a t e d to the c o n c e n t r a t i o n  added  (e.g. F i g . 24). By p l o t t i n g the data as % i n h i b i t i o n of c h l o r i d e conductance as a f u n c t i o n of % i n h i b i t i o n of c h l o r i d e conductance/phosphate c o n c e n t r a t i o n  ( i . e an Eadie-  Hofstee p l o t ) ( F i g . 24 i n s e t ) i t was p o s s i b l e to d e r i v e an apparent I ^ Q value f o r phosphate  i n h i b i t i o n of c h l o r i d e  conductance f o r each of the phosphate-regulated p o r i n s (Table  X I I I ) .  These data were c o n s i s t e n t with the presence  of a phosphate-binding s i t e w i t h i n each of these channels. The apparent I ^ Q values v a r i e d with the c o n c e n t r a t i o n of KC1,  ranging from 9.7 to 27 mM phosphate  0.59 t o 2.5 mM phosphate X I I I ) .  i n 1 M KC1 and from  i n 40 mM KC1 (at pH 7) (Table  At a given c o n c e n t r a t i o n of KC1, however, the  variation  i n I ^ Q values obtained f o r a l l of the channels d i d  not exceed 4 - f o l d , i n d i c a t i n g that the r e l a t i v e a f f i n i t i e s of these channels f o r phosphate were q u i t e  149  similar.  F i g u r e 24. Phosphate i n h i b i t i o n of c h l o r i d e ( C l ) f l u x through p r o t e i n P channels. P r o t e i n P (100 ng/ml) was added to the aqueous s o l u t i o n (40 mM KC1/ 1 mM T r i s HCl, pH 7.0) bathing an o x i d i z e d c h o l e s t e r o l (1.5 % i n n-decane) membrane and the membrane conductance allowed to increase u n t i l i t had s t a b i l i z e d ( u s u a l l y at a l e v e l 2-4 orders of magnitude higher than the i n i t i a l l e v e l ) . At t h i s time a l i q u o t s of potassium phosphate b u f f e r pH 7.0 were added t o the aqueous phase on both sides of the membrane and the new conductance l e v e l recorded. The % decrease i n conductance was c a l c u l a t e d and p l o t t e d as a f u n c t i o n of the aqueous phase phosphate c o n c e n t r a t i o n [ P i ] . The a p p l i e d voltage was 20 mV and the temperature was 25 C. I n s e t . An Eadie-Hofstee p l o t of the data d e r i v e d from measurements of the % i n h i b i t i o n of c h l o r i d e conductance as a f u n c t i o n of phosphate c o n c e n t r a t i o n . I 5 0 values f o r phosphate i n h i b i t i o n of c h l o r i d e conductance (Table XIII) were c a l c u l a t e d using l e a s t squares a n a l y s i s .  150  100  10OH 0  1  |  1  1  r  1  2  3  k  5  Phosphate  Concentration  151  (mM)  5.  Summary.  Phosphate-starvation induced o l i g o m e r i c  p r o t e i n s from the outer membranes of P. f l u o r e s c e n s , P. p u t i d a , P. a u r e o f a c i e n s and P. c h l o r o r a p h i s were p u r i f i e d to homogeneity.  The i n c o r p o r a t i o n of p u r i f i e d p r o t e i n s  into  planar l i p i d b i l a y e r membranes r e s u l t e d i n stepwise increases i n membrane conductance. conductance  S i n g l e channel  experiments demonstrated  that these p r o t e i n were  a l l capable of forming s m a l l , p r o t e i n P - l i k e channels with an average s i n g l e channel conductance 233 and 252 pS.  The conductance  when the p r o t e i n s were p u r i f i e d  i n 1 M KC1 of between  p r o p e r t i e s were not a l t e r e d f r e e of LPS p r i o r t o  r e c o n s t i t u t i o n i n l i p i d b i l a y e r membranes. conductance  S i n g l e channel  measurements made i n s a l t s of v a r y i n g c a t i o n or  anion s i z e i n d i c a t e d that the channels were u n i f o r m l y anionselective.  The measurement of s i n g l e channel conductance  as  a f u n c t i o n of KC1 c o n c e n t r a t i o n r e v e a l e d that a l l channels saturated at high s a l t c o n c e n t r a t i o n s , c o n s i s t e n t with the presence of a b i n d i n g s i t e i n the channel. values f o r C l fold  Apparent  were c a l c u l a t e d and shown to vary only two-  (180 - 297 pS) amongst a l l channels, i n c l u d i n g  P channels. conductance  Kd  Phosphate was capable of i n h i b i t i n g through a l l channels, with apparent  between 0.59 and 2.5 mM phosphate 9.7 and 27.0 mM phosphate  protein  chloride I ^ Q values of  at 40 mM C l , and between  at 1 M C l .  These data were  c o n s i s t e n t with the presence of a phosphate-binding  site in  the channels of these phosphate-regulated p r o t e i n s . Furthermore,  they i n d i c a t e d that these channels had at l e a s t 152  .a 20 to 8 0 - f o l d  higher a f f i n i t y  f o r phosphate over  153  chloride.  DISCUSSION  The  t r a n s p o r t of i n o r g a n i c phosphate i n the gram-  negative bacterium Pseudomonas aeruginosa i n v o l v e s t r a n s l o c a t i o n across two membranes.  In t h i s study only  those c o n s t i t u e n t s e x t e r n a l to the cytoplasmic membrane were examined i n d e t a i l ,  i n an attempt  to address the  mechanism(s) by which phosphate overcomes the p e r m e a b i l i t y b a r r i e r of the outer membrane under p h o s p h a t e - l i m i t i n g conditions.  1.  A phosphate regulon i n Pseudomonas aeruginosa.  response  In  to p h o s p h a t e - d e f i c i e n c y , w i l d type c e l l s of P.  aeruginosa were shown to be derepressed f o r the s y n t h e s i s of the enzymes a l k a l i n e phosphatase and phospholipase C ( F i g . 4), i n a d d i t i o n to a p e r i p l a s m i c phosphate-binding and an outer membrane channel-forming and  10).  protein  protein, P (Figs. 3  The o b s e r v a t i o n that c o l l e c t i v e l y these species  were s i m i l a r l y c o n s t i t u t i v e l y produced mutants of P. aeruginosa  or n o n - i n d u c i b l e i n  ( F i g . 5) suggested  that these  c o n s t i t u e n t s were indeed c o - r e g u l a t e d , forming a phosphate regulon analogous  to the pho regulon of E s c h e r i c h i a c o l i  (Tommassen and Lugtenberg,  1982).  A d d i t i o n a l , as yet  u n c h a r a c t e r i z e d , orthophosphate-regulated i d e n t i f i e d i n phospholipase  p r o t e i n s have been  C r e g u l a t o r y mutants of P.  aeruginosa PAO (Gray et a l . , 1981, 1982) suggesting that a phosphate regulon i n P. aeruginosa PAO may be s i g n i f i c a n t l y  1 54  more extensive than d e s c r i b e d here.  The coordinate  r e g u l a t i o n of the c o n s t i t u e n t genes of operons or regulons u s u a l l y r e f l e c t s the r o l e s of t h e i r gene products common process.  The maltose regulon  in a  i n E. c o l i , f o r  example, i n v o l v e s c o n s t i t u e n t s of the t r a n s p o r t and c a t a b o l i s m of maltose and maltodextrins  (Bedouelle,  while many of the components of the pho regulon function  1984),  i n E. c o l i  i n the a c q u i s i t i o n of i n o r g a n i c phosphate  (Tommassen and Lugtenberg, 1982). L i k e E. c o l i , P. aeruginosa  demonstrates two major  uptake systems f o r i n o r g a n i c phosphate, of low and h i g h affinity, The  r e s p e c t i v e l y (LaCoste  et a l . , 1981;  observation that the l o w - a f f i n i t y system  constitutively  F i g . 14B). . operates  i s c o n s i s t e n t with a r o l e i n the t r a n s p o r t of  phosphate i n a phosphate-rich  medium.  I t s high c a p a c i t y  (Vmax = 12.1 nmol/min/mg c e l l p r o t e i n ) undoubtedly  reflects  the ready a v a i l a b i l i t y of phosphate i n a r i c h medium, as w e l l as the growth p o t e n t i a l of the organism under conditions.  these  In a d i l u t e environment, however, phosphate  uptake v i a the l o w - a f f i n i t y system w i l l be l i m i t i n g f o r growth (compare the r a t e of growth of wild-type P. aeruginosa  with that of a mutant expressing only the low-  affinity  system ( F i g . 13)). The derepression of a h i g h -  affinity  system permits  efficient  from a d i l u t e environment. limitation  s t r a i n s capable  t r a n s p o r t of phosphate  Thus, during phosphate of expressing a h i g h - a f f i n i t y  uptake system t r a n s p o r t phosphate at s i g n i f i c a n t l y  155  greater  r a t e s than mutants d e f i c i e n t lower c a p a c i t y of t h i s system  i n t h i s system (Vmax =5.4  ( F i g . 12).  The  nmol/min/mg c e l l  p r o t e i n ) compared with the l o w - a f f i n i t y system  undoubtedly  r e f l e c t s the decreased a v a i l a b i l i t y of phosphate and a consequently reduced growth p o t e n t i a l i n a p h o s p h a t e - l i m i t e d environment  ( F i g . 2).  A l k a l i n e phosphatase  (a phosphate monoesterase)  f u n c t i o n to provide the i n o r g a n i c orthophosphate  may  (Pi)  s u b s t r a t e f o r these t r a n s p o r t systems by i t s a c t i o n on phosphate-containing molecules.  I t s concerted a c t i o n with  phospholipase C, which r e l e a s e s the phosphoryl c h o l i n e moiety  from s p e c i f i c p h o s p h o l i p i d molecules,  probably  f u n c t i o n s to make p h o s p h o l i p i d s a v a i l a b l e as a phosphate source as w e l l .  The d e r e p r e s s i o n of these enzymes i n  p h o s p h a t e - l i m i t i n g media suggests that i n an r i c h environment,  orthophosphate-  l a r g e r phosphate-containing molecules  not normally be used as phosphate sources. d e p l e t i o n of the a v a i l a b l e orthophosphate  may  However, upon supply, other  sources are made a v a i l a b l e by the a c t i o n of these h y d r o l y t i c enzymes.  S i m i l a r l y , the i n d u c t i o n of p h o s p h a t e - s e l e c t i v e  outer membrane p o r i n p r o t e i n P i n a p h o s p h a t e - l i m i t e d environment  i s a response  to the consequently low rate of  d i f f u s i o n of phosphate across the outer membrane which would otherwise be l i m i t i n g f o r t r a n s p o r t and growth (see H i g h - a f f i n i t y t r a n s p o r t i n P. aeruginosa demonstrated  below).  was  to o b l i g a t e l y r e q u i r e a p e r i p l a s m i c phosphate-  b i n d i n g p r o t e i n , such t r a n s p o r t being absent  156  in a  phosphate-  binding p r o t e i n - d e f i c i e n t mutant affinity  ( F i g . 14A).  f o r phosphate (Kd=0.34 uM)  was  The  binding  in good agreement  with the observed k i n e t i c s of h i g h - a f f i n i t y phosphate transport  (Km=0.46 uM).  T h i s i s c h a r a c t e r i s t i c of  protein-dependent transport  binding  systems where the k i n e t i c s  appear to be d i c t a t e d by the b i n d i n g p r o t e i n s which f u n c t i o n as the rate determining step. a b i l i t y to be  The  binding a f f i n i t y  r e l e a s e d by cold-osmotic  shock are t y p i c a l of  binding proteins- i s o l a t e d from P. aeruginosa al.,  1977;  1982)  and  1976).  Hoshino and  Kageyama, 1 980;  of b i n d i n g p r o t e i n s i n general  Binding  (Berger and  (Stinson et  Eisenberg  by phosphate-bond energy (e.g.  Heppel, 1974).  Thus, an  (LaCoste et a l . , 1981)  that i t was  (Ki=0.24 mM;  might be  in P.  due  to i t s nature of  LaCoste et a l . , 1981)  suggests  a c t i n g at some component of the h i g h - a f f i n i t y  t r a n s p o r t system. The  observation  that arsenate was  of i n h i b i t i n g the binding of phosphate to the p r o t e i n at c o n c e n t r a t i o n s  p r o t e i n stage of  capable  binding  comparable to the. Ki (Table  implied that the e f f e c t of arsenate was  It  ATP)  i n h i b i t o r y e f f e c t of  a f f e c t on e n e r g i z a t i o n , although the competitive the i n h i b i t i o n  Phibbs,  systems are  arsenate on h i g h - a f f i n i t y phosphate t r a n s p o r t aeruginosa  and  (Oxender and Quay,  protein-dependent t r a n s p o r t  t y p i c a l l y energized  and  at the  VII)  binding  transport.  i s noteworthy t h a t , upon i n d u c t i o n  in phosphate-  d e f i c i e n t medium, the enzymes a l k a l i n e phosphatase phospholipase C were secreted  from the c e l l  157  and  ( F i g . 4B).  Theories  of e x t r a c e l l u l a r p r o t e i n r e l e a s e g e n e r a l l y held  that outer membrane breakdown was  a means of r e l e a s i n g  p r o t e i n s from, for example, a p e r i p l a s m i c l o c a t i o n . Obviously,  i f such a mechanism were r e s p o n s i b l e  for the  observed enzyme r e l e a s e , the concomittant increase  in outer  membrane p e r m e a b i l i t y c o u l d have functioned to increase  the  rate of d i f f u s i o n of phosphate across the outer membrane without the need for a s p e c i f i c channel. enzyme s e c r e t i o n was  Nevertheless,  s p e c i f i c , as demonstrated by  f a i l u r e to observe any concomittant r e l e a s e of  the  the  p e r i p l a s m i c beta-lactamase and phosphate-binding p r o t e i n , and  the b a r r i e r p r o p e r t i e s of the outer membrane were  maintained  ( F i g . 6).  phosphate-selective  T h i s implied that the s y n t h e s i s of a outer membrane p o r i n p r o t e i n was  superfluous.  I n t e r e s t i n g l y , i t has  et a l . , 1973;  B h a t t i and  been suggested  Ingram, 1981)  phosphatase r e l e a s e by P. aeruginosa change in outer membrane p e r m e a b i l i t y release.  Unfortunately,  not (Ingram  that a l k a l i n e i s a s s o c i a t e d with a in a d d i t i o n to  LPS  t h e i r use of a T r i s - b u f f e r e d medium  i n v a l i d a t e d t h e i r r e s u l t s , given the observed a b i l i t y of T r i s to permeabilize  and cause s i g n i f i c a n t s t r u c t u r a l  r e o r g a n i z a t i o n of the outer membrane ( I r v i n et__al.,  2.  P r o p e r t i e s of outer membrane p r o t e i n P..  p r o t e i n P of P. aeruginosa  was  Outer membrane  demonstrated to form  s t a b l e oligomers ( t r i m e r s ; Angus and Hancock, 1983) polyacrylamide  g e l s , a property  1981).  SDSin  shared by the majority  158  of  porin proteins described  to date.  The monomer molecular  weight of t h i s p r o t e i n , 48,000, was s i g n i f i c a n t l y  greater  than that of most E n t e r o b a c t e r i a l p o r i n s and p o r i n p r o t e i n F of P. aeruginosa (36-39,000) (Lugtenberg and van Alphen, 1983;  Hancock and Carey, 1979).  molecular weight of the n a t i v e  Attempts at determining the (trimer) form of the p r o t e i n  on SDS-polyacrylamide g e l s [from Ferguson p l o t s of the e l e c t r o p h o r e t i c m o b i l i t y of the t r i m e r s as a f u n c t i o n of the acrylamide c o n c e n t r a t i o n  (Tokunaga e t a l . , 1979)] have  f a i l e d because the n a t i v e p r o t e i n migrates anomalously i n t h i s g e l system.  The anomalous migration  of p o r i n  in SDS-polyacrylamide g e l s has been described  oligomers  previously  (Tokunaga et a l . , 1979) and has been a t t r i b u t e d to the high degree of b e t a r s t r u c t u r e present  i n the n a t i v e  porins  (Nikaido and Vaara, 1985), which r e s u l t s i n the b i n d i n g of l e s s SDS (wt/wt) by these p r o t e i n s With few exceptions  (Rosenbusch, 1974).  (e.g. p r o t e i n s F and D1 of P.  aeruginosa; Hancock and Carey, 1979; Hancock and Carey, 1980), p o r i n s e x i s t as undenatured t r i m e r s when e x t r a c t e d with SDS.  The observation  that p r o t e i n P formed SDS-stable  t r i m e r s , although t y p i c a l of p o r i n s  i n general,  c o n t r a s t to the other p o r i n p r o t e i n s described aeruginosa. majority  i n P.  However, p r o t e i n P was d i s t i n g u i s h a b l e from the  of p o r i n s ,  readily solubilized 100)  i s thus i n  i n c l u d i n g p r o t e i n F, i n that i t was i n a non-denaturing detergent  i n the presence of EDTA.  ( T r i t o n X-  Most p o r i n p r o t e i n s are  s o l u b l e i n T r i t o n only a f t e r d i g e s t i o n of the peptidoglycan  159  with lysozyme,  or i n SDS and high s a l t  (Lugtenberg and van Alphen,  1983).  (> 0.4 M NaCl)  T h i s has been  i n t e r p r e t e d as i n d i c a t i n g a strong non-covalent of  these p o r i n s to the p e p t i d o g l y c a n .  attachment  In a d d i t i o n , the  channels formed by p r o t e i n P i n l i p i d b i l a y e r membranes (0.6 nm d i a . ) were s i g n i f i c a n t l y smaller than those formed by p r o t e i n F (2.2 nm) and the major E n t e r o b a c t e r i a l p o r i n s (11.4 nm) (Benz et a l . ,  1985).  F i n a l l y , while the m a j o r i t y of  p o r i n s s t u d i e d to date, i n c l u d i n g the p h o s p h a t e - s t a r v a t i o n i n d u c i b l e PhoE p o r i n p r o t e i n of E. c o l i , ion  selectivity  al., et  e x h i b i t only weak  i n planar l i p i d b i l a y e r membranes (Benz et  1985), p r o t e i n P channels are a n i o n - s p e c i f i c  al.,  1982;  (Hancock  Table I I I ) .  As confirmed i n t h i s study, (Table X I I I ) , the anions p e c i f i c i t y of p r o t e i n P channels can be a t t r i b u t e d to the presence of a binding s i t e f o r anions i n the channel (Benz et a l . ,  1983).  A c e t y l a t i o n of a v a i l a b l e amino groups  on p r o t e i n P (see Methods), of  which d i d not a f f e c t the a b i l i t y  p r o t e i n P to form SDS-stable oligomers ( t r i m e r s )  in  polyacrylamide g e l s (not shown), r e s u l t e d i n the l o s s of the a n i o n - b i n d i n g s i t e and a concomittant l o s s of the anions p e c i f i c i t y of the channel  (Hancock et a l . ,  1983b).  From  these s t u d i e s i t was concluded that the a n i o n - s p e c i f i c i t y of p r o t e i n P channels was a f u n c t i o n of epsilon-amino groups of l y s i n e r e s i d u e s present on the p r o t e i n 1983b). coli  (Hancock et a l . ,  S i m i l a r l y , the phosphate-regulated PhoE p o r i n of E.  has been demonstrated  to possess a c e t y l a t a b l e  160  lysine  residues which were r e s p o n s i b l e  for the observed, a l b e i t  weaker, a n i o n - s e l e c t i v i t y of t h i s channel i n black  lipid  b i l a y e r membranes (Darveau et a l . , 1984). The LPS  contamination of p u r i f i e d p o r i n p r e p a r a t i o n s  i s w e l l documented (Nikaido and Vaara, 1985)  with  and  suggests a strong tendency for these molecules to a s s o c i a t e . Indeed, an  i n v i v o a s s o c i a t i o n of LPS  by the marked reduction  with p o r i n s ,  in p o r i n l e v e l s i n the  outer  membranes of L P S - d e f i c i e n t mutants (e.g. Koplow Goldfine,  1974), has  al.,  ( S c h i n d l e r and  preparations  Although c o n v e n t i o n a l l y i n v a r i a b l y contained  in the formation  by t h i s p r o t e i n Lugtenberg heptose-less  (Table I I I ) .  significant  l e v e l s of  LPS  demonstrated  to  of a n i o n - s p e c i f i c channels  Recently,  (1984) have reported LPS  (Kropinski e_t  p u r i f i e d protein P  ( F i g . 7; Table I I ) , such an a s s o c i a t i o n was be dispensable  not  Rosenbusch, 1978),  a l s o for the modulation of p o r i n a c t i v i t y 1982).  and  been suggested to be necessary,  only f o r p o r i n f u n c t i o n but  suggested  Korteland  and  that E. c o l i mutants with  produce PhoE p o r i n s which f a c i l i t a t e a  6-  to 8 - f o l d more e f f i c i e n t permeation of a n i o n i c s o l u t e s . contrast  to r e s u l t s presented here with p r o t e i n P,  data suggest that LPS  may  indeed i n f l u e n c e p o r i n  In a d d i t i o n to being c o - r e g u l a t e d  In  these  activity.  with components of a  h i g h - a f f i n i t y phosphate uptake system in P. aeruginosas  (see  above), p r o t e i n P channels have r e c e n t l y been demonstrated to possess a binding and  s i t e f o r inorganic phosphate (Hancock  Benz, submitted; Table X I I I ) .  161  The  observation  that  p r o t e i n P channels e x h i b i t e d a 60 to 8 0 - f o l d higher for phosphate over c h l o r i d e (Hancock and Benz,  affinity  submitted;  Table XIII) was c o n s i s t e n t with the demonstrated r o l e f o r p r o t e i n P i n phosphate uptake (Table V) (see below).  3.  The outer membrane of P. aeruginosa  as a p e r m e a b i l i t y  b a r r i e r to phosphate under p h o s p h a t e - l i m i t i n g c o n d i t i o n s . The  low i n t r i n s i c p e r m e a b i l i t y of the P. aeruginosa  membrane i s w e l l documented, supported  outer  by observations of  the i n c r e a s e d c r y p t i c i t y of p e r i p l a s m i c enzymes i n P. aeruginosa  compared with the analogous enzymes i n , f o r  example, E. c o l i  (Yoshimura and Nikaido,  1982) as well as  d i r e c t measurements of outer membrane p e r m e a b i l i t y al.,  1982; Yoshimura and Nikaido,  1983).  (Angus et  1982; Nicas and Hancock,  Thus, the apparent t r a n s p o r t and growth Km values  for given n u t r i e n t molecules are o f t e n higher aeruginosa  than i n E. c o l i .  i n P.  The r e s u l t s of t h i s  study  demonstrated that the outer membrane of P. aeruginosa  indeed  f u n c t i o n s as a p e r m e a b i l i t y b a r r i e r to phosphate molecules under p h o s p h a t e - l i m i t i n g c o n d i t i o n s .  This was supported by  the observed increase i n the apparent Km f o r h i g h - a f f i n i t y phosphate t r a n s p o r t i n the p r o t e i n P - d e f i c i e n t mutant  (Table  V), e x p l a i n a b l e by a decrease (at a given e x t e r n a l phosphate concentration)  i n the r a t e of d i f f u s i o n of phosphate  molecules across the p r o t e i n P - d e f i c i e n t outer membrane compared with the w i l d type outer membrane.  The f a c t that  the Vmax of h i g h - a f f i n i t y uptake remained u n a l t e r e d i n the  162  mutant (Table V) f u r t h e r i n d i c a t e d that the d i f f u s i o n of phosphate across the p r o t e i n P - d e f i c i e n t outer membrane was only r a t e - l i m i t i n g phosphate.  f o r t r a n s p o r t at low c o n c e n t r a t i o n s of  At higher c o n c e n t r a t i o n s of phosphate (e.g. > 25  uM) the r a t e of phosphate t r a n s p o r t was not d e t e c t a b l y different  i n the mutant compared with the wild-type.  Furthermore, a defect i n the growth c a p a b i l i t i e s of the p r o t e i n P - d e f i c i e n t mutant s t r a i n was only seen at a very low c o n c e n t r a t i o n of phosphate (50 uM) (Table V I ) , i n d i c a t i n g that phosphate d i f f u s i o n a c r o s s the outer membrane was dependent upon p r o t e i n P at low e x t e r n a l phosphate c o n c e n t r a t i o n s only.  At higher c o n c e n t r a t i o n s of  phosphate, d i f f u s i o n across the outer membrane through the major p o r i n p r o t e i n F of P. aeruginosa of s a t i s f y i n g the c e l l u l a r requirements  i s apparently  capable  for this nutrient.  In a d d i t i o n , the small s i z e and the s e l e c t i v i t y of the p r o t e i n P channels low  would undoubtedly serve to maintain the  i n t r i n s i c p e r m e a b i l i t y of t h i s membrane to other  constituents. Whether the p r o t e i n P channels  are s u f f i c i e n t to  f a c i l i t a t e the e f f i c i e n t movement of phosphate from a d i l u t e environment across the outer membrane remains i n q u e s t i o n . The apparent  a b i l i t y of the phosphate-binding  p r o t e i n to  a s s o c i a t e i n v i t r o with p r o t e i n P may have p h y s i o l o g i c a l relevance i n v i v o concerning the mechanism by which phosphate d i f f u s i o n across the outer membrane i s f a c i l i t a t e d in a d i l u t e environment.  C e r t a i n l y the a b i l i t y of the  163  p e r i p l a s m i c maltose-binding  p r o t e i n to a s s o c i a t e with the  c o r e g u l a t e d LamB outer membrane p o r i n p r o t e i n has been documented ( B a v o i l and Nikaido,  1981).  Such an a s s o c i a t i o n ,  demonstrated i n v i t r o , has been suggested  t o be necessary i n  v i v o f o r the t r a n s l o c a t i o n , across the outer membrane, of maltose,  when present at low c o n c e n t r a t i o n s , and  maltodextrins 1983).  (Wandersman et a l . , 1979; Luckey and Nikaido,  From these s t u d i e s i t was suggested  that a p h y s i c a l  a s s o c i a t i o n between a h i g h - a f f i n i t y b i n d i n g p r o t e i n and an outer membrane p o r i n c o u l d f u n c t i o n t o b r i n g a h i g h - a f f i n i t y b i n d i n g s i t e near the outer s u r f a c e of the outer membrane. Such an a s s o c i a t i o n might a l s o ensure r a p i d binding of s u b s t r a t e once i t entered the periplasm, m a i n t a i n i n g a c o n c e n t r a t i o n gradient across the outer membrane, even under d i l u t e c o n d i t i o n s , by reducing the l e v e l s of free s u b s t r a t e in the periplasm.  In the case of the phosphate-binding  p r o t e i n and p r o t e i n P of P. aeruginosa,  such an a s s o c i a t i o n  would c e r t a i n l y not be o b l i g a t o r y given the a b i l i t y of the phosphate-binding  protein-dependent  h i g h - a f f i n i t y uptake  system t o operate  in., the absence of p r o t e i n P.  The i n c r e a s e  in the Km f o r h i g h - a f f i n i t y phosphate t r a n s p o r t observed f o r the p r o t e i n P - d e f i c i e n t mutant s t r a i n may r e f l e c t  higher  f r e e p e r i p l a s m i c c o n c e n t r a t i o n s of phosphate and a subsequently  smaller c o n c e n t r a t i o n g r a d i e n t to d r i v e  d i f f u s i o n across the outer membrane owing t o the i n a b i l i t y of the phosphate-binding  p r o t e i n and p r o t e i n P to a s s o c i a t e  at the outer membrane.  164  A f u r t h e r analogy with the maltose t r a n s p o r t system of E. c o l i  i s seen i n the a b i l i t y of long phosphate  bind to the phosphate-binding p r o t e i n .  polymers to  I f polyphosphates  are capable of being t r a n s p o r t e d i n t a c t by the h i g h - a f f i n i t y phosphate  t r a n s p o r t system  by a l k a l i n e phosphatase permeation  i n v i v o , without p r i o r  or other phosphatases,  hydrolysis  their  of p r o t e i n P channels w i l l undoubtedly  depend  upon the proper l i n e a r o r i e n t a t i o n of the molecules at the channel mouth, s i n c e these molecules exceeed l i m i t of p r o t e i n P channels.  the e x c l u s i o n  Such o r i e n t a t i o n c o u l d be  c a r r i e d out by the phosphate-binding s i t e of p r o t e i n P and/or  the h i g h - a f f i n i t y phosphate-binding s i t e present on  the phosphate-binding p r o t e i n , i n a s s o c i a t i o n with the outer membrane channel-forming p r o t e i n .  S i m i l a r l y , the a b i l i t y of  the LamB p r o t e i n to allow the d i f f u s i o n of m a l t o d e x t r i n s which exceed the apparent  e x c l u s i o n l i m i t of the LamB pore,  has been p o s t u l a t e d to depend upon b i n d i n g s i t e s both i n the channel and on the maltose-binding p r o t e i n and N i k a i d o , 1977; F e r e n c i and Boos,  (Von Meyenburg  1980).  Growth of P. aeruginosa on polyphosphates as the s o l e phosphate 1966).  source has been demonstrated  Confirmed  ( V a l e t t e et a l . ,  i n t h i s study, i t was f u r t h e r  that growth i n media c o n t a i n i n g phosphate  demonstrated  polymers  of up to  5 u n i t s i n l e n g t h occurred without the d e r e p r e s s i o n of a l k a l i n e phosphatase  (data not shown).  T h i s suggested that  polyphosphates c o u l d be t r a n s p o r t e d i n t a c t , without h y d r o l y s i s to c o n s t i t u e n t orthophosphate  165  molecules.  prior As  such, phosphatase(s) must be present i n the cytoplasm to convert these polymers phosphate,  orthophosphate.  gram-negative demonstrated 1975).  to »the usual currency of i n o r g a n i c  organisms,  In t h i s regard, a number of  i n c l u d i n g E. c o l i , have been  to posses cytoplasmic polyphosphatases  (Yagil,  I n t e r e s t i n g l y , growth of P. aeruginosa i n a medium  c o n t a i n i n g a phosphate phosphate  polymer  of 15 u n i t s as the sole  source was, i n f a c t , accompanied  of d e t e c t a b l e a l k a l i n e phosphatase  by the i n d u c t i o n  i n both the p e r i p l a s m and  i n the e x t e r n a l medium (data not shown).  Whether t h i s  r e f l e c t s an i n a b i l i t y of polyphosphate P15 to be t r a n s p o r t e d i n t a c t or a rate of uptake so slow as t o mimic deficiency  i s uncertain.  s i n c e a l k a l i n e phosphatase  phosphate-  I t may be a moot p o i n t , however, h y d r o l y s i s of such large  polyphosphates was apparently necessary f o r growth. In c o n t r a s t to P. aeruginosa, the observed higher intrinsic  p e r m e a b i l i t y of the E. c o l i outer membrane leads  one to conclude that the major p o r i n s themselves may be capable of s a t i s f y i n g the phosphate cell,  without the need f o r a f a c i l i t a t e d d i f f u s i o n channel  for phosphate. the  requirements of the  According to F i c k ' s f i r s t  rate of phosphate  law of d i f f u s i o n ,  t r a n s p o r t across the E. c o l i outer  membrane v i a the c o n s t i t u t i v e p o r i n pathway w i l l be higher, at a given c o n c e n t r a t i o n of phosphate, T h i s makes the need of a phosphate obvious.  than i n P. aeruginosa  channel i n E. c o l i  less  Despite the f a c t that E. c o l i c e l l s deprived  of phosphate  are derepressed f o r a channel-forming outer  166  membrane p r o t e i n , PhoE (Overbeeke and Lugtenberg, 1980), a s p e c i f i c r o l e i n phosphate uptake has not been demonstrated and u n l i k e p r o t e i n P, PhoE does not bind phosphate al.,  1984).  (Benz et  Thus, although the PhoE p o r i n f u n c t i o n s as a  more e f f i c i e n t channel f o r phosphate than do the OmpF or OmpC p o r i n s (Rorteland e t al.,1982), i t does not provide any advantage to c e l l s growing i n medium c o n t a i n i n g orthophosphate as the s o l e phosphate source (Overbeeke and Lugtenberg, 1982).  Furthermore, a 10-fold i n c r e a s e i n the  Km f o r phosphate t r a n s p o r t r e p o r t e d f o r a P h o E - d e f i c i e n t mutant compared  with a PhoE  +  strain  (Korteland et a l . , 1982)  was obtained i n a background d e f i c i e n t  i n the major p o r i n s .  Thus i t was p o s s i b l e that the increase i n Km of the PhoE mutant  simply r e f l e c t e d the o v e r a l l p o r i n - d e f i c i e n c y of the  PhoE mutant  s t r a i n such that e.g. an OmpF PhoE +  s t r a i n might  have t r a n s p o r t e d phosphate as w e l l as the above OmpF PhoE strain.  The E. c o l i  +  PhoE channel does, however, provide a  growth advantage t o c e l l s growing i n a medium c o n t a i n i n g polyphosphate as the s o l e phosphate source (Overbeeke and Lugtenberg, 1982), and i t i s a l s o an e f f i c i e n t channel f o r the  uptake of organic phosphates.  Possibly i t s  p h y s i o l o g i c a l r o l e i s i n the t r a n s p o r t of l a r g e r phosphorylated molecules.  4.  P r o t e i n P and p r o t e i n PhoE as members of two d i s t i n c t  c l a s s e s of phosphate-regulated p o r i n s . coli  In a d d i t i o n to E.  and P. aeruginosa, a number of other gram-negative  167  bacteria,  i n c l u d i n g S, typhimurium  E. cloacae  (Verhoef  et a l . , 1984),  (Bauer et a l . , 1985) and have been demonstrated  to s y n t h e s i z e novel outer membrane p o r i n p r o t e i n s i n response to p h o s p h a t e - l i m i t a t i o n .  These p r o t e i n s form  a n i o n - s e l e c t i v e channels i n r e c o n s t i t u t e d l i p i d  bilayer  - membranes (Bauer et a l . , 1985; Verhoef et a l . , 1984; Benz et al.,  1984) c o n s i s t e n t with t h e i r presumed r o l e s i n phosphate  acquisition.  The r e s u l t s of t h i s study  extend the l i s t of  bacteria synthesizing phosphate-starvation-inducible membrane p r o t e i n s .  E x i s t i n g as oligomers  outer  (probably  trimers)  in SDS, these p r o t e i n s can be d i s s o c i a t e d to monomers when subjected  to temperatures above 60°C (Table X ) , a property  c h a r a c t e r i s t i c of p o r i n s  (Lugtenberg and van Alphen,  Furthermore, based on the monomer molecular peptidoglycan  1983).  we-ight,  a s s o c i a t i o n and abundance of these p r o t e i n s ,  they are l i k e l y to be p o r i n s . Two c l a s s e s of these p r o t e i n s were d i s t i n g u i s h a b l e , based on monomer molecular and  detergent  peptidoglycan  weight (36 t o 39K or 45.5 to 50K)  requirements to remove the p r o t e i n s from the (SDS-high s a l t or T r i t o n - T r i s - E D T A ) .  The  former c l a s s , t y p i f i e d by p r o t e i n PhoE, i n c l u d e d the phosphate-starvation-inducible pseudomallei  p r o t e i n s of P. c e p a c i a , P.  and the E n t e r o b a c t e r i a c e a e .  The l a t t e r c l a s s  i n c l u d e d the p h o s p h a t e - s t a r v a t i o n - i n d u c i b l e  p r o t e i n s of the  P. f l u o r e s c e n s branch of the family Pseudomonadaceae and was e x e m p l i f i e d by p r o t e i n P.  These two p r o t e i n s were a l s o  d i s t i n g u i s h a b l e from a f u n c t i o n a l point of view.  168  Thus,  while p r o t e i n PhoE forms l a r g e selective  channels  (1 nm), weakly anion-  (Benz et a l . , 1985) which lack binding  s i t e s f o r anions and f o r phosphate (Benz et a l . , 1984), p r o t e i n P channels were small (0.6 nm) (Hancock et a l . , 1982), possessing b i n d i n g s i t e s f o r anions and phosphate (Benz et a l . , 1983; Hancock and Benz, submitted; Table XIII) c o n s i s t e n t with the observed a n i o n - s p e c i f i c i t y al.,  1982) and p h o s p h a t e - s e l e c t i v i t y  (Hancock e_t  (Hancock and Benz,  submitted; Table XIII) of t h i s channel.  Furthermore, the  observed channel-forming p r o p e r t i e s of the phosphates t a r v a t i o n - i n d u c i b l e outer membrane p r o t e i n s of the f l u o r e s c e n t Pseudomonads were indeed c o n s i s t e n t with the formation of p r o t e i n P type p o r i n s by these p r o t e i n s . One can only s p e c u l a t e on the purpose  of s y n t h e s i z i n g  one or the other of these phosphate-regulated proteins.  porin  I t seems probable that the production of a small  s p e c i f i c channel l i k e p r o t e i n P, i n P. aeruginosa and the f l u o r e s c e n t Pseudomonads, r e f l e c t s the need to maintain low outer membrane p e r m e a b i l i t y .  Because these  fluorescent  Pseudomonads occur n a t u r a l l y i n the s o i l , where many a n t i b i o t i c - p r o d u c i n g microorganisms necessary f o r t h e i r antibiotics.  Indeed,  has been i m p l i c a t e d organism  survival  i t may be  that they maintain a b a r r i e r to  the outer membrane of P. aeruginosa  i n the high i n t r i n s i c  to a n t i b i o t i c s  and Hancock, 1983).  are found,  (Yoshimura  r e s i s t a n c e of t h i s  and N i k a i d o , 1982; Nicas  In c o n t r a s t , E. c o l i  demonstrates  s i g n i f i c a n t l y higher outer membrane p e r m e a b i l i t y , implying  169  that an outer membrane of extremely low p e r m e a b i l i t y i s not e s s e n t i a l f o r E. c o l i c e l l s .  The production of a  phosphate-  s t a r v a t i o n - i n d u c i b l e p o r i n p r o t e i n , PhoE, whose channel s i z e i s not s i g n i f i c a n t l y d i f f e r e n t p o r i n s of E. c o l i  from the major c o n s t i t u t i v e  (Benz et a l • ,  1985)  would o b v i o u s l y  f u n c t i o n to maintain the same degree of p e r m e a b i l i t y , e s p e c i a l l y s i n c e t o t a l p o r i n l e v e l s i n the outer membrane are  regulated in a stringent fashion  Alphen, 1983).  (Lugtenberg and van  U n l i k e the p r o t e i n P channel of P.  aeruginosa, which  i s too small to act as a channel f o r  l a r g e r phosphate-containing molecules [except perhaps polymers of i n o r g a n i c phosphate  linear  (see above)], the E. c o l i  PhoE channel has been shown to allow the passage of l a r g e organic phosphate 1982). the  molecules (Overbeeke  and  Given the p r e d i c t e d high i n t r i n s i c  E. c o l i  Lugtenberg, p e r m e a b i l i t y of  outer membrane to i n o r g a n i c phosphate,  t r a n s p o r t of l a r g e r phosphorylated molecules may,  the in fact,  be the most important f u n c t i o n of the PhoE channel. E. c o l i  Indeed,  i s o f t e n i s o l a t e d from sewage e f f l u e n t where  polyphosphates  (and organic phosphates) w i l l  abundance, as a r e s u l t of the a c t i o n of  be i n  anaerobic organisms  which t y p i c a l l y make and s t o r e l a r g e q u a n t i t i e s of polyphosphate  (Kulaev, 1975).  has been i d e n t i f i e d  i n E. c o l i  A cytoplasmic polyphosphatase (Yagil,  1975), suggesting  that polyphosphates are a p o t e n t i a l source of phosphate in t h i s organism. act  Because p r o t e i n P channels are too small to  as channels f o r large phosphate-containing molecules,  170  the a b i l i t y  of P. aeruginosa to u t i l i z e large phosphorylated  molecules under d i l u t e c o n d i t i o n s , where uptake across the outer membrane v i a the major p o r i n ( s ) w i l l be l i m i t i n g f o r t r a n s p o r t and growth, may  be dependent upon p r i o r  h y d r o l y s i s of these molecules by a l k a l i n e phosphatase, r e l e a s e orthophosphate. has been demonstrated cells  Interestingly, alkaline  phosphatase  to be s e c r e t e d from P. aeruginosa  i n t o the e x t e r n a l medium (Cheng et a l . ,  1970;  F i g . 4B)  where i t c o u l d , by cleavage of organic phosphates, source of i n o r g a n i c phosphate f o r t r a n s p o r t .  provide a  E. c o l i ,  the other hand, s y n t h e s i z e s a p o r i n p r o t e i n i n  l a r g e r organic phosphates,  of  t h i s bacterium  on  phosphate-  l i m i t i n g media (PhoE) which i s capable of t r a n s p o r t i n g  a l k a l i n e phosphatase  to  and t h i s might e x p l a i n  these  why  i s e x c l u s i v e l y l o c a t e d i n the p e r i p l a s m  (Malamy and Horecker,  1961).  The production of phosphate-starvation-induced p o r i n s by the f l u o r e s c e n t Pseudomonads, a l l of which form p r o t e i n P - l i k e channels, suggests an evolutionary/taxonomic relationship.  T h i s i s borne out by s e v e r a l p i e c e s of data.  A l l of these s t r a i n s produce  a major, c o n s t i t u t i v e p r o t e i n  (porin) which does not form SDS-stable oligomers.  This i s  in c o n t r a s t to the E n t e r o b a c t e r i a c e a e and other Pseudomonads, i n c l u d i n g P. c e p a c i a and P. pseudomallei, which produce  major p o r i n p r o t e i n s which are s t a b l e to  d e n a t u r a t i o n at temperatures Alphen,  1983).  SDS  below 60°C (Lugtenberg and  T h i s "heat-unmodifiable" phenotype i s  t y p i c a l of p o r i n p r o t e i n F of P. aeruginosa  171  (Hancock and  van  Carey, 1979).  In a d d i t i o n , these f l u o r e s c e n t  a l s o produce a l i p o p r o t e i n which  Pseudomonads  cross-reacts  immunologically with outer membrane p r o t e i n H2  of  aeruginosa PA01  Furthermore,  (Mutharia and  based on the r e s u l t s of DNA  Hancock, 1985).  and  rRNA h y b r i d i z a t i o n  these s t r a i n s were s i m i l a r l y c l a s s i f i e d group and  were c l e a r l y d i s t i n c t  f a m i l y Pseudomonadaceae (De Vos  evolutionary  studies,  in the same homolgy  from other species and  De Ley,  be  of  1983).  data suggest that outer membrane p r o t e i n p r o f i l e s immunological r e l a t i o n s h i p s may  P.  These and  important i n d i c a t o r s of  links.  Because a number of d i s t i n c t  b a c t e r i a l species  have  been demonstrated to produce a p r o t e i n P - l i k e p o r i n , opportunity and  the  e x i s t s to. study the biogenesis  to i d e n t i f y f u n c t i o n a l domains.  of the  the  protein  Given the  similarities  of the f u n c t i o n a l p r o p e r t i e s of these p r o t e i n s  in different  s t r a i n s , which were q u i t e d i s t i n c t regulated  porins,  i t should be p o s s i b l e to i d e n t i f y regions  of the p r o t e i n i n v o l v e d s e l e c t i v i t y as regions  i n , f o r example, anion/phosphateof c l o s e homology i n the genes  encoding these p r o t e i n s . contribute aeruginosa.  from other phosphate-  Such data w i l l  to e l u c i d a t i n g the topology of t h i s p r o t e i n Furthermore, by examining the expression  these f o r e i g n genes in P. aeruginosa PA01 regions  undoubtedly  of the p r o t e i n  we  172  of  can i d e n t i f y  important i n s y n t h e s i s ,  assembly.  in P.  secretion  and  5.  Conserved  a n t i g e n i c determinants  i n phosphate-  s t a r v a t i o n - i n d u c i b l e outer membrane (porin) p r o t e i n s .  The  demonstration here that phosphate-starvation-induced membrane oligomers c r o s s - r e a c t e d  immunologically  i n d i c a t e d that these p r o t e i n s express conserved sites  (regions of s t r u c t u r a l homology).  The  ( F i g . 18), antigenic  observed  d e s t r u c t i o n of t h i s c r o s s - r e a c t i v i t y by the heatd i s s o c i a t i o n of oligomers to monomers, which a l s o d e s t r o y s p o r i n f u n c t i o n , and the f a i l u r e of phosphate  starvation-  induced monomers to c r o s s - r e a c t with a p r o t e i n P monomers p e c i f i c antiserum, suggested that the conserved regions of homology only.  were maintained i n the n a t i v e , f u n c t i o n a l p r o t e i n s  Furthermore,  the i n a b i l i t y  of the p r o t e i n P t r i m e r -  s p e c i f i c antiserum to react with the major p o r i n p r o t e i n s i n these s t r a i n s ( F i g . 18) i n d i c a t e d that the c r o s s - r e a c t i v i t y was  distinct  general.  from any homologies  Such homologies  the observed  immunological  and PhoE p o r i n s of E. c o l i  do,  r e l a t i n g to p o r i n s t r u c t u r e i n  i n f a c t , e x i s t as i n d i c a t e d by  c r o s s - r e a c t i v i t y of OmpF, OmpC (Overbeeke  et a l . , 1980)  and of  p o r i n p r o t e i n s F and P of P. aeruginosa (K. Poole, unpublished  result).  The c r o s s - r e a c t i v i t y of phosphate oligomeric  p r o t e i n s may  starvation-induced  w e l l r e l a t e to s p e c i f i c  functional  p r o p e r t i e s of these phosphate-regulated p r o t e i n s .  In t h i s  v e i n , a number of the phosphate  starvation-induced  membrane  p r o t e i n s have been demonstrated  to form a n i o n - s e l e c t i v e  channels  Benz et a l . , 1984;  (Hancock et a l . , 1982;  1 73  Verhoef  et a l . , 1984; Bauer et a l . , 1985; Table X I I I ) , i n c o n t r a s t with the major E n t e r o b a c t e r i a l p o r i n p r o t e i n s and the major p o r i n p r o t e i n F of P. aeruginosa, which are weakly c a t i o n selective  (Benz et a l . , 1985).  The a n i o n - s e l e c t i v i t y of  some of these p r o t e i n s has been a t t r i b u t e d to f i x e d charges, p o s s i b l y epsilon-amino  groups of l y s i n e r e s i d u e s ,  in or near the mouth of the r e s p e c t i v e channels al., least  positive  1983b; Darveau et a l . , 1984).  (Hancock et  The demonstration  of at  14 exposed/accessible l y s i n e r e s i d u e s i n the p r o t e i n P  and PhoE channels  (R.E.W. Hancock, unpublished r e s u l t ) i s  c o n s i s t e n t with t h i s i d e a . By comparison,  p o r i n p r o t e i n OmpF  c o n t a i n s 5 a c c e s s i b l e l y s i n e r e s i d u e s (R.E.W. Hancock, unpublished r e s u l t ) .  I t i s tempting  to hypothesize that  exposed l y s i n e r e s i d u e s present i n these, and perhaps other, phosphate-regulated  p o r i n p r o t e i n s may be i n v o l v e d i n the  observed c r o s s - r e a c t i v i t y  of phosphate-starvation-induced  membrane p r o t e i n s , perhaps forming p a r t of the conserved antigenic s i t e ( s ) .  In support of t h i s , p r o t e i n PhoE  channels are f u n c t i o n a l l y  i n d i s t i n g u i s h a b l e from OmpF and  OmpC channels upon a c e t y l a t i o n of a v a i l a b l e l y s i n e r e s i d u e s . The observed proteins  immunological  c r o s s - r e a c t i v i t y of these  (Overbeeke et a l . , 1980), the 60-70 % amino a c i d  homology (Tommassen et a l . , 1982; Mizuno et a l . , 1983), and the a b i l i t y of the ompF and phoE genes to h y b r i d i z e i n regions along t h e i r e n t i r e lengths (Tommassen et a l . , 1982) implies that the property of a n i o n - s e l e c t i v i t y as a f u n c t i o n of l y s i n e residues i n the channel may w e l l be the major  174  d i s c r i m i n a t i n g f e a t u r e of PhoE compared with the other major p o r i n s in E. c o l i . 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