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Partial characterization of the molecular basis for carotovoricin-379 sensitivity in Erwinia carotovora Smith, Donna Susan 1986

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PARTIAL CHARACTERIZATION OF THE MOLECULAR BASIS FOR CAROTOVORICIN-379 IN ERWINIA CAROTOVORA SENSITIVITY By Donna Susan Smith B . S c , The U n i v e r s i t y of B r i t i s h Columbia, 1981 A THESIS SUBMITTED IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of P l a n t Science) We accept t h i s theses as conforming to the req u i r e d 1 standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1986 © Donna Susan Smith ® 3 In p r e s e n t i n g this thesis in part ial fu l f i lment o f the requ i rements for an a d v a n c e d d e g r e e at the Univers i ty o f Br i t ish C o l u m b i a , I agree that the Library shall m a k e it f reely avai lable for re fe rence a n d s tudy . I further agree that p e r m i s s i o n for e x t e n s i v e c o p y i n g o f this thesis for scho la r l y p u r p o s e s may b e g r a n t e d by the h e a d o f m y d e p a r t m e n t o r by his o r her representat ives . It is u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n of this thesis for f inanc ia l ga in shal l no t b e a l l o w e d w i t h o u t m y w r i t t e n p e r m i s s i o n . D e p a r t m e n t T h e Un ivers i ty o f Brit ish C o l u m b i a 1956 M a i n M a l l V a n c o u v e r , C a n a d a V 6 T 1Y3 DE-6(3/81) i i ABSTRACT In t h e e v a l u a t i o n o f s e v e r a l methods o f q u a n t i f y i n g t h e a c t i v i t y o f c a r o t o v o r i c i n - 3 7 9 , t h e t e t r a z o l i u m a s s a y and t h e s p o t - p l a t e a s s a y were r e j e c t e d b e c a u s e o f i n s e n s i t i v i t y and i r r e p r o d u c i b i l i t y , r e s p e c t i v e l y . An a d a p t i o n o f t h e c r i t i c a l d i l u t i o n method, c a l l e d t h e m i c r o t i t r e p l a t e a s s a y , overcame t h e weaknesses o f t h e o t h e r methods. Found t o be a c c e p t a b l y r a p i d , s e n s i t i v e , and r e p r o d u c i b l e , t h i s new method f a c i l i t a t e d f u r t h e r i n v e s t i g a t i o n s o f c a r o t o v o r i c i n - 3 7 9 s e n s i t i v i t y i n Erwlnia carotovoza s u b s p e c i e s a t r o s e p t i c a (Eca). R e p r e s e n t a t i v e s t r a i n s o f t h e s e r o g r o u p s o f Eca d i s p l a y e d d i f f e r e n t l e v e l s o f s e n s i t i v i t y t o c a r o t o v o r i c i n - 3 7 9 . B e c a u s e a l l s e n s i t i v e t e s t s t r a i n s a d s o r b e d c a r o t o v o r i c i n - 3 7 9 , t h e s e d i f f e r e n c e s were a t t r i b u t e d t o t o l e r a n c e r a t h e r t h a n r e s i s t a n c e . T h i s i m p l i e d t h a t a l t h o u g h l e s s s e n s i t i v e s t r a i n s were c a p a b l e of a d s o r b i n g c a r o t o v o r i c i n - 3 7 9 , t h e e v e n t s l e a d i n g t o t h e d e a t h o f t h e c e l l were n o t t r i g g e r e d . A l i n e a r r e l a t i o n s h i p between t h e c a r o t o v o r i c i n - 3 7 9 dosage and t h e l o g o f t h e p r o p o r t i o n o f s u r v i v i n g c e l l s i n t e s t c u l t u r e s i n d i c a t e d t h a t c a r o t o v o r i c i n - 3 7 9 f o l l o w e d s i n g l e - h i t k i l l i n g k i n e t i c s . T h i s r e s u l t showed t h a t o n l y one a d s o r b e d p a r t i c l e w h i c h i s a b l e t o r e a c h i i i i t s t a r g e t of a c t i o n i s r e q u i r e d to k i l l a c e l l . In l e s s s e n s i t i v e s t r a i n s , t h e r e f o r e , a high p r o p o r t i o n of adsorbed p a r t i c l e s are unable to reach the s i t e of a c t i o n . Increased r e l a t i v e f l u o r e s c e n c e of 8-ani1ino-1-naphthalene s u l f o n i c a c i d (ANS) was observed upon treatment of s e n s i t i v e c e l l s with c a r o t o v o r i c i n - 3 7 9 . These i n c r e a s e s were not observed when n o n - s e n s i t i v e c e l l s were t r e a t e d i n an i d e n t i c a l manner. Because ANS f l u o r e s c e n c e i s a q u a l i t a t i v e i n d i c a t o r of membrane p o l a r i z a t i o n , these r e s u l t s i n d i c a t e d t h a t c a r o t o v o r i c i n -379 a c t s by e f f e c t i n g a c o l l a p s e of the proton motive f o r c e . The cy t o p l a s m i c membrane, t h e r e f o r e , was t e n t a t i v e l y i d e n t i f i e d as the t a r g e t of a c t i o n of c a r o t o v o r i c i n - 3 7 9 . Sodium d o d e c y l s u l f a t e polyacryamide g e l e l e c t r o p h e s i s of membrane p r o t e i n s e x t r a c t e d from s e n s i t i v e c e l l s r e v e a l e d t h a t these p r o t e i n s were d i s t r i b u t e d i n t o e i t h e r the T r i t o n - s o l u b l e or i n s o l u b l e f r a c t i o n s . C a r o t o v o r i c i n - 3 7 9 n e u t r a l i z a t i o n a c t i v i t y , however, was d i s t r i b u t e d e v e n ly between them, c o r r e l a t i n g with the d i s t r i b u t i o n of l i p o p o l y s a c c h a r i d e (LPS). Membrane p r o t e i n f a i l e d to n e u t r a l i z e c a r o t o v o r i c i n - 3 7 9 a c t i v i t y . LPS from s e n s i t i v e c e l l s , however, e x h i b i t e d i v n e u t r a l i z a t i o n a c t i v i t y . N e u t r a l i z a t i o n a c t i v i t y was s e n s i t i v e to p e r i o d a t e , s u g g e s t i n g t h a t the r e c e p t o r s i t e i s carbohydrate. D i r e c t b i n d i n g of c a r o t o v o r i c i n - 3 7 9 to the LPS was demonstrated with an i n d i r e c t enzyme-linked immunosorbent assay using p o l y c l o n a l antiserum r a i s e d a g a i n s t c a r o t o v o r i c i n - 3 7 9 . Strong p o s i t i v e r e a c t i o n s were obtained with LPS from s e n s i t i v e s t r a i n s ; however the r e a c t i o n obtained with n o n - s e n s i t i v e s t r a i n s were e i t h e r extemely weak or n e g a t i v e . From these r e s u l t s i t was concluded t h a t the c a r o t o v o r i c i n - 3 7 9 r e c e p t o r s i t e i n Erwinia carotovora i s l o c a t e d on the LPS. V TABLE OF CONTENTS T i t l e Page i A b s t r a c t i i Table of Contents v L i s t of Tables v i i L i s t of F i g u r e s v i i i Acknowledgement x i INTRODUCTION 1 LITERATURE REVIEW 2 A. B a c t e r i o c i n P r o d u c t i o n 2 B. Q u a n t i f i c a t i o n of B a c t e r i o c i n A c t i v i t y 6 C. B a c t e r i o c i n S e n s i t i v i t y 9 1. B a c t e r i o c i n A d s o r p t i o n 9 2. B a c t e r i o c i n Receptors 11 3. B a c t e r i o c i n Mode of A c t i o n 16 D. B a c t e r i o c i n s of Erwinia cazotovora 18 CHAPTER 1. THE QUANTIFICATION OF CAROTOVORICIN-379 ACTIVITY 22 I n t r o d u c t i o n 2 2 M a t e r i a l s and Methods 25 B a c t e r i a l S t r a i n s and Growth C o n d i t i o n s 25 P r e p a r a t i o n of Carotovor i c i n - 3 7 9 26 T r i p h e n y l T e t r a z o l i u m C h l o r i d e Assay 27 Sp o t - p l a t e Assay 27 M i c r o t i t r e P l a t e Assay 28 R e s u l t s 29 D i s c u s s i o n 35 v i CHAPTER 2. CHARACTERISTITCS OF CAROTOVORICIN-379 SENSITIVITY IN ERWINIA CAROTOVORA 41 I n t r o d u c t i o n 41 M a t e r i a l s and Methods 43 B a c t e r i a l S t r a i n s and Growth C o n d i t i o n s 43 P r e p a r a t i o n of C a r o t o v o r i c i n - 3 7 9 43 C a r o t o v o r i c i n - 3 7 9 A c t i v i t y Assay 43 Measurement of C a r o t o v o r i c i n - 3 7 9 Adsorption...43 E f f e c t of C a r o t o v o r i c i n - 3 7 9 on C e l l S u r v i v a l . . 4 5 Determination of C a r o t o v o r i c i n - 3 7 9 Adsorption.46 F l u o r i m e t r y 47 R e s u l t s 47 D i s c u s s i o n 58 CHAPTER 3. LOCALIZATION OF THE CAROTOVORICIN-379 RECEPTOR SITE IN ERWINIA CAROTOVORA 64 I n t r o d u c t i o n 64 M a t e r i a l s and Methods 66 B a c t e r i a l S t r a i n s and Growth C o n d i t i o n s 66 P r e p a r a t i o n of C a r o t o v o r i c i n - 3 7 9 68 Q u a n t i f i c a t i o n of C a r o t o v o r i c i n - 3 7 9 A c t i v i t y . . 6 8 C e l l Wall F r a c t i o n a t i o n 68 Li p o p o l y s a c c h a r ide E x t r a c t i o n 70 Determination of T o t a l P r o t e i n 72 Determination of Ketodeoxyoctanate 73 Determination of C a r o t o v o r i c i n - 3 7 9 N e u t r a l i z a t i o n A c t i v i t y 74 Pe r i o d a t e I n a c t i v i a t i o n of Receptor A c t i v i t y . . 7 5 Enzyme-linked Immunosorbent Assay 75 Sodium D o d e c y l s u l f a t e P o l y a c r y l a m i d e Gel E l e c t r o p h o r e s i s 77 R e s u l t s 79 D i s c u s s i o n 91 GENERAL DISCUSSION 9 8 SUMMARY 103 LITERATURE CITED 105 v i i LIST OF TABLES Chapter 1: The Q u a n t i f i c a t i o n of C a r o t o v o r i c i n - 3 7 9 A c t i v i t y Table 1. Absorbance at 500 nm of i n d i c a t o r suspension c o n t a i n i n g d i f f e r e n t r e l a t i v e ^ c o n c e n t r a t i o n s (RC) of c a r o t o v o r i c i n - 3 7 9 (C-379) with (+) and without (-) t r i p h e n y l t e t r a z o l i u m c h l o r i d e (TTC) 30 Table 2. Means and standard d e v i a t i o n s (SD) of c a r o t o v o r i c i n - 3 7 9 a c t i v i t y i n a r b i t r a r y u n i t s (au) over e i g h t r e p l i c a t e s i n three d i f f e r e n t samples as determined by the s p o t - p l a t e and m i c r o t i t r e p l a t e assay method , 36 Chapter 2. C h a r a c t e r i s t i c s of C a r o t o v o r i c i n - 3 7 9 S e n s i t i v i t y i n E r w i n i a carotovora Table 1. S t r a i n s of E r w i n i a cazotovora subspecies c a r o t o v o r a (Ecc) and Erwinia c a r o t o v o r a subspecies a t r o s e p t i c a (Eca) i s o l a t e d i n B r i t i s h Columbia (BC) and Wisconsin (Wn) used i n t h i s chapter , 44 Table 2. C a r o t o v o r i c i n - 3 7 9 (C-379) a d s o r p t i o n c a p a c i t i e s of v a r i o u s s t r a i n s of E r w i n i a carotovora , 55 Chapter 3. L o c a l i z a t i o n of the C a r o t o v o r i c i n - 3 7 9 Receptor S i t e i n Erwinia carotovora Table 1. S t r a i n s of E r w i n i a carotovora subspecies c a r o t v o r a i s o l a t e d i n B r i t i s h Columbia and Wisconsin and E s c h e r i c h i a c o l i (E. c o l i ) used i n t h i s chapter 67 Table 2. Y i e l d s and s p e c i f i c c a r o t o v o r i c i n -379 n e u t r a l i z a t i o n a c t i v i t i e s of l i p o p o l y s a c c h a r i d e s e x t r a c t e d from s t r a i n s of E r w i n i a carotovora 86 v i i i LIST OF FIGURES Chapter 1. The Q u a n t i f i c a t i o n of C a r o t o v o r i c i n - 3 7 9 A c t i v i t y F i g u r e 1. S p o t - p l a t e d e t e r m i n a t i o n of c a r o t o v o r i c i n - 3 7 9 a c t i v i t y i n ( l e f t to r i g h t ) u n d i l u t e d , 1/3, and 1/10 r e l a t i v e c o n c e n t r a t i o n s 31 F i g u r e 2. R e p l i c a t e s p o t - p l a t e assays of a s i n g l e t w o - f o l d d i l u t i o n s e r i e s of c a r o t o v o r i c i n - 3 7 9 (shown l e f t to r i g h t i n descending order beginning at 1/2) demonstrating the v a r i a b i l i t y i nherent i n response of the E r w i n i a c a r o t o v o r a subspecies a t r o s e p t i c a i n d i c a t o r s t r a i n 530 33 F i g u r e 3. A t y p i c a l m i c r o t i t r e p l a t e assay showing the extent of i n h i b i t i o n of i n d i c a t o r (Erwinia carotovora subspecies a t r o s e p t i c a s t r a i n 530) growth by two-fold d i l u t i o n s e r i e s ( l e f t to r i g h t s t a r t i n g at 1/2) of a c a r o t o v o r i c i n - 3 7 9 suspension. The d i l u t i o n endpoint i s 1/256 i n a l l e i g h t r e p l i c a t e s 34 Chapter 2. C h a r a c t e r i s t i c s of C a r o t o v o r i c i n - 3 7 9 S e n s i t i v i t y i n Erwinia carotovora F i g u r e 1. S p o t - p l a t e s of c a r o t o v o r i c i n - 3 7 9 demonstrating plaque morphology on v a r i o u s t e s t s t r a i n s of Erwinia carotovora 48 F i g u r e 2. R e s i d u a l c a r o t o v o r i c i n - 3 7 9 (C-379) a c t i v i t y i n the f i l t e r s t e r i l i z e d supernatant f o l l o w i n g the i n t r o d u c t i o n of c e l l s of e i t h e r Erwinia carotovora subsp. atroseptica s t r a i n 530 or Erwinia carotovora subsp. carotovora s t r a i n 379 50 i x F i g u r e 3. D e c l i n e i n the number of col o n y forming u n i t s per ml (CFU/ml) of Erwinia carotovora subsp. atroseptica s t r a i n 530 over time f o l l o w i n g exposure to 1280 a r b i t r a r y u n i t s (au)/ml of c a r o t o v o r i c i n -379 (C-379) . F i g u r e 4 Fi g u r e 5 F i g u r e 6 51 E f f e c t of c a r o t o v o r i c i n - 3 7 9 (C-379) i n a r b i t r a r y u n i t s (au)/ml on the s u r v i v a l of E r w i n i a carotovora subsp. a t r o s e p t i c a s t r a i n 530 20 min a f t e r exposure. E r r o r bars r e p r e s e n t the range of values obtained over three experiments 52 Demonstration of the v a r i o u s l e v e l s of s e n s i t i v i t y to c a r o t o v o r i c i n - 3 7 9 e x h i b i t e d among s t r a i n s of Erwinia caxotovoza as i n d i c a t e d by the number of c o l o n y forming u n i t s (CFU)/ml remaining a f t e r the a d d i t i o n of 128 a r b i t r a r y u n i t s (au)/ml ( f i n a l c o n c e n t r a t i o n ) of c a r o t o v o r i c i n - 3 7 9 . E r r o r bars r e p r e s e n t the range of values obtained over three experiments 54 Fluorescence emission s p e c t r a of E r w i n i a cazotovoza subspecies atroseptica s t r a i n 530 (530) and 8-a n i l i n o - l - n a p h t h a l e n e s u l f o n i c a c i d (ANS) with and without the a d d i t i o n of 320 a r b i t r a r y u n i t s / m l ( f i n a l c o n c e n t r a t i o n ) c a r o t o v o r i c i n - 3 7 9 (C-379) F i g u r e 7. E f f e c t of c a r o t o v o r i c i n - 3 7 9 (C-379) dosage on the r e l a t i v e f l u o r e s c e n c e of 8 - a n i l i n o - l - n a p h t h a l e n e s u l f o n i c a c i d (ANS) at an emission wavelength of 500 nm alone ( C o n t r o l ) , and i n a s s o c i a t i o n with c a r o t o v o r i c i n - 3 7 9 - s e n s i t i v e ( E r w i n i a c a r o t o v o r a subspecies a t r o s e p t i c a s t r a i n 530) and - i n s e n s i t i v e (Erwinia cazotovoza subspecies carotovora s t r a i n 379) c e l l s 56 57 X Chapter 3 F i g u r e 1. F i g u r e 2. F i g u r e 3 F i g u r e 4 F i g u r e 5, L o c a l i z a t i o n of the C a r o t o v o r i c i n - 3 7 9 Receptor S i t e i n E r w i n i a carotovora Sodium dodecyl s u l f a t e p o l y a c r y l a m i d e g e l electophoretogram of T r i t o n -i n s o l u b l e p r o t e i n e x t r a c t e d from two s t r a i n s of Erwinia carotovora subspecies atroseptica with and without lysozyme pretreatment , 80 Sodium dodecyl sulphate p o l y a c r y l a m i d e e l e c t r o p h o r e t o g r a m of T r i t o n - s o l u b l e and - i n s o l u b l e f r a c t i o n s of E r w i n i a c a r o t o v o r a subspecies a t r o s e p t i c a s t r a i n s 530 and 496 81 R e l a t i v e d i s t r i b u t i o n s of p r o t e i n , ketodeoxyoctanate (KDO), and c a r o t o v o r i c i n - 3 7 9 n e u t r a l i z a t i o n a c t i v i t y (C-379 NA) i n f r a c t i o n s obtained through T r i t o n X-100 s e l e c t i v e s o l u b i l i z a t i o n of crude c e l l w a l l s of Erwinia carotovora subspecies atroseptica 83 S p o t - p l a t e a c t i v i t y d e t e r m i n a t i o n of c a r o t o v o r i c i n - 3 7 9 untreated (a) and pre-mixed 10:1 with l i p o p o l y s a c c h a r i d e e x t r a c t e d from Erwinia carotovora s e n s i t i v e s t r a i n s 530 (b) and 496 (c) and E r w i n i a carotovora n o n - s e n s i t i v e s t r a i n 379 (d) 85 S p o t - p l a t e demonstrating the e f f e c t of p e r i o d a t e on the c a r o t o v o r i c i n - 3 7 9 n e u t r a l i z a t i o n a c t i v i t y of l i p o p o l y s a c c h a r i d e e x t r a c t e d from Erwinia carotovora subspecies a t r o s e p t i c a s t r a i n 530 88 x i F i g u r e 6. Sodium d o d e c y l s u l f a t e p o l y a c r y l a m i d e g e l e lectrophoretogram of l i p o p o l y s a c c h a r i d e e x t r a c t e d from E r w i n i a carotovora (Be) and Escherichia coli (B. coli) 89 F i g u r e 7. Enzyme-linked immunosorbent assay of c a r o t o v o r i c i n - 3 7 9 bound to 1 i p o p o l y s a c c h a r i d e e x t r a c t e d from v a r i o u s s t r a i n s of Erwinia carotovora subspecies carotovora (Ecc), Erwinia c a r o t o v o r a s u b s p e c i e s a t r o s e p t i c a , (Eca) and E s c h e r i c h i a c o l i 92 x i i ACKNOWLEGDEMENT I wish to thank my s u p e r v i s o r Dr. R.J. Copeman f o r h i s support and encouragement throughout the course of t h i s work. I would a l s o l i k e to thank the other members of my committee, Dr. S.H. De Boer, Dr. R.A.J. Warren, Dr. R.E.W. Hancock, and Dr. V.C. Runeckles f o r t h e i r c r i t i c i s m and s u g g e s t i o n s . Thanks are a l s o extended to Mr. L. J . Ward f o r the generous g i f t of the p o l y c l o n a l antiserum; to Mr. Leroy Scrubb f o r t e c h n i c a l i n s t r u c t i o n ; and to Mr. B r i a n MacMillan f o r help with photography. F i n a l l y , I would l i k e to thank Dr. M. Shaw f o r the use of h i s u l t r a c e n t r i f u g e . 1 INTRODUCTION E r w i n i a carotovora, a s p e c i e s of gram negative bacterium, causes s o f t - r o t d i s e a s e s of many vegetable crops which can r e s u l t i n s i g n i f i c a n t economic l o s s e s both i n the f i e l d and i n po s t - h a r v e s t s t o r a g e . E. carotovora subspecies a t r o s e p t i c a (van H a l l ) Dye {Eca) i s s p e c i f i c a l l y the c a u s a l agent of b l a c k l e g of potato. T h i s d i s e a s e i s system i c , and the i n f e c t i o n u s u a l l y spreads r a p i d l y through the v a s c u l a r t i s s u e and i n t o the t u b e r s . E r w i n i a carotovora subspecies carotovora (Jones) Dye (E c c ) , having a wider host range, causes s o f t - r o t of a v a r i e t y of v e g e t a b l e s . F o l l o w i n g i n v a s i o n of the p l a n t t i s s u e , these organisms m u l t i p l y w i t h i n the i n t e r c e l l u l a r spaces of the p l a n t and produce a wide v a r i e t y of macerating enzymes. These enzymes, mainly p e c t o l y t i c and c e l l u l o l y t i c , degrade the components of the p l a n t c e l l w a l l s , e v e n t u a l l y c a u s i n g the c o l l a p s e and death of the t i s s u e ( A g r i o s 1978). The organisms may be a s s o c i a t e d with the p l a n t t i s s u e or rh i z o s p h e r e i n a l a t e n t s t a t e . Although many of the environmental f a c t o r s which a f f e c t d i s e a s e development have been i d e n t i f i e d , the mechanism by which the d i s e a s e process i s t r i g g e r e d i s unknown (Perombelon and Kelman 1980). 2 L i k e many other phytopathogenic b a c t e r i a , E r w i n i a spp. are capable of producing b a c t e r i o c i n s . Because b a c t e r i o c i n s posses many c h a r a c t e r i s t i c s d e s i r a b l e i n a b i o l o g i c a l c o n t r o l agent, they have been the s u b j e c t of study f o r s e v e r a l years. With regard to E r w i n i a carotovora, however, l i t t l e i s known about the b a s i s of t h e i r a c t i o n or t h e i r r o l e i n the ecology of the s p e c i e s . The o b j e c t i v e of t h i s t h e s i s was to c h a r a c t e r i z e the molecular b a s i s of s e n s i t i v i t y of Eca to a b a c t e r i o c i n produced by Ecc s t r a i n 379. LITERATURE REVIEW A. B a c t e r i o c i n P r o d u c t i o n . B a c t e r i o c i n s are d e f i n e d as n o n - r e p l i c a t i n g b a c t e r i c i d a l agents, composed of p r o t e i n , which are produced by c e r t a i n s t r a i n s of b a c t e r i a and are a c t i v e a g a i n s t others of the same or c l o s e l y r e l a t e d s p e c i e s (Nomura 1967). ' C o l i c i n s , b a c t e r i o c i n s which are produced by and a c t i v e a g a i n s t s t r a i n s of Escherichia coli, were d i s c o v e r e d by G r a t i a i n 1925. Subsequent d i s c o v e r i e s soon 3 demonstrated t h a t b a c t e r i o c i n s are g e o g r a p h i c a l l y u b i q u i t o u s and are produced by a broad range of both Gram p o s i t i v e and Gram negative b a c t e r i a . B a c t e r i o c i n s are h i g h l y potent, l e t h a l agents, s u g g e s t i n g t h a t they confer a s e l e c t i v e advantage to the producing s t r a i n and may p l a y an important r o l e i n b a c t e r i a l e v o l u t i o n and ecology (Reeves 1972). In a s i x month study of i n t e s t i n a l E. c o l i of f i v e people, l o n g -term r e s i d e n t s t r a i n s were u s u a l l y c o l i c i n o g e n i c while t h i s was l e s s common i n t r a n s i e n t s t r a i n s (Branche et a l . 1963). V i r i d i c i n s , produced by Aerococcus viridans have been i m p l i c a t e d i n redu c i n g p o p u l a t i o n s of pathogenic s t r e p t o c o c c i i n t h r o a t t i s s u e (Dajani and wannamaker 1969). When b a c t e r i o c i n - p r o d u c i n g s t r a i n s are co-i n o c u l a t e d with s e n s i t i v e s t r a i n s i n planta or i n vitro, the s e n s i t i v e s t r a i n p o p u l a t i o n i s u s u a l l y e l i m i n a t e d while the producing s t r a i n s u r v i v e s . T h i s has been demostrated with phytopathogenic s p e c i e s of both Pseudomonas and Corynebacterium (Vidaver 1976). Although by d e f i n i t i o n a l l b a c t e r i o c i n s are pro t e i n a c e o u s , a wide range of s t r u c t u r a l d i v e r s i t y e x i s t s among them. Bradley (1967) c l a s s i f i e d b a c t e r i o c i n s i n t o two major groups designated the high and low molecular weight b a c t e r i o c i n s . Low molecular weight b a c t e r i o c i n s , o f t e n r e f e r r e d to as " t r u e " b a c t e r i o c i n s , are g l o b u l a r 4 p r o t e i n s g e n e r a l l y c o n s i s t i n g of one or two p o l y p e p t i d e c h a i n s . Molecular s i z e w i t h i n t h i s group ranges from 8000 d a l t o n s (d) ( s t r e p t o c o c c i n - A F F 2 2 produced by group A s t r e p t o c o c c i ) to 97,000 d (pyocin-AP41 produced by Pseudomonas aeruginosa) and 150,000 d (staphylococcin-1580 produced by Staphylococcus e p i d e r m i d i s ) (Tagg et a l . 1976). Most c o l i c i n s ( E l , E2, E3, K, Ia, and L) f a l l w i t h i n the 45,000 to 67,000 d s i z e range (Nomura 1967). In c o n t r a s t to the low molecular weight b a c t e r i o c i n s , high molecular weight b a c t e r i o c i n s are r e a d i l y sedimented i n the u l t r a c e n t r i f u g e . When viewed by e l e c t r o n microscopy, these p a r t i c u l a t e b a c t e r i o c i n s d i s p l a y a h i g h l y complex s t r u c t u r e resembling bacteriophage or phage components. Because s e r o l o g i c a l r e l a t i o n s h i p s between p a r t i c u l a t e b a c t e r i o c i n s and bacteriophage are not uncommon, these b a c t e r i o c i n s are thought to be d e f e c t i v e phage (Ackerman and Brochu 1978). They are , however, d i s t i n g u i s h a b l e from phage i n t h a t they do not reproduce i n i n f e c t e d c e l l s . Genes encoding the p r o d u c t i o n of both s m a l l and l a r g e molecular weight b a c t e r i o c i n s r e s i d e w i t h i n the producing c e l l , and u n l i k e phage genes, are not t r a n s f e r r e d to s e n s i t i v e c e l l s v i a the i n f e c t i o u s p a r t i c l e . Of a l l the b a c t e r i o c i n s s t u d i e d , the g e n e t i c s of the c o l i c i n s i s p a r t i c u l a r l y w e l l understood. 5 The s t r u c t u r a l genes of c o l i c i n s are c a r r i e d on plasmids c a l l e d Col f a c t o r s . These plasmids a l s o code f o r the r e g u l a t i o n of c o l i c i n b i o s y n t h e s i s and r e l e a s e . Furthermore, Col f a c t o r s u s u a l l y c a r r y the genes r e q u i r e d f o r t r a n s f e r ( s e l f - t r a n s m i s s i b i l i t y ) a l l o w i n g the spread of Col f a c t o r s throughout e n t i r e p o p u l a t i o n s by means of co n j u g a t i v e t r a n s f e r . Col f a c t o r s a l s o confer c o l i c i n immunity to the host or producing s t r a i n . Genes f o r the pr o d u c t i o n of other b a c t e r i o c i n s such as those produced by Rhizobium ( H i r s c h 1979) and Bacillus (Tagg et a l . 1976) are a l s o c a r r i e d on plasmids, while the R-type pyocins are known to be chromosomally encoded (Kageyama 1974 ) . B i o s y n t h e s i s of b a c t e r i o c i n s can be s t r o n g l y i n f l u e n c e d by c u l t u r a l c o n d i t i o n s such as media composition, pH, and a e r a t i o n (Reeves 1972, Tagg et a l . 1976). Jacob et a l . (1952) d i s c o v e r e d t h a t c o l i c i n s y n t h e s i s could be induced by u l t r a v i o l e t l i g h t . Although c o l i c i n i n d u c t i o n i s the most tho r o u g h l y c h a r a c t e r i z e d , b a c t e r i o c i n p r o d u c t i o n i s i n d u c i b l e i n a number of s p e c i e s . S e v e r a l DNA-damaging agents such as mitomycin-C and n i t r o s o g u a n i d i n e are capable of ind u c i n g or enhancing b a c t e r i o c i n p r o d u c t i o n , probably i n a manner s i m i l a r to th a t of prophage i n d u c t i o n . 6 B. Q u a n t i f i c a t i o n o f B a c t e r i o c i n A c t i v i t y . B a c t e r i o c i n a c t i v i t y i s t h e p o t e n c y o f t h e l e t h a l e f f e c t or k i l l i n g a c t i o n d i s p l a y e d a g a i n s t c e l l s o f s e n s i t i v e s t r a i n s . I n o r d e r t o s t u d y i n t e r a c t i o n s between b a c t e r i o c i n s and t h e s e n s i t i v e c e l l s t h e y a c t upon, i t i s n e c e s s a r y t o employ a t e c h n i q u e w h i c h w i l l y i e l d q u a n t i t a t i v e v a l u e s o f b a c t e r i o c i n a c t i v i t y . T h i s i s u s u a l l y a c h i e v e d u s i n g a s e n s i t i v e s t r a i n as a b i o l o g i c a l i n d i c a t o r . T h r e e b a s i c a s s a y t e c h n i q u e s have been d e v e l o p e d i n a p p l y i n g t h i s a p p r o a c h . The f i r s t i s t h e c r i t i c a l d i l u t i o n a s s a y method (CDA). O r i g i n a l l y u s e d t o measure t h e a c t i v i t y o f c o l i c i n s ( J a c o b e t al 1 9 5 2 ) , i t has become t h e most g e n e r a l l y a p p l i e d a s s a y method f o r a wide v a r i e t y o f b a c t e r i o c i n s ( M a y r - H a r t i n g e t a l 1 9 7 2 ) . T h i s method i n v o l v e s t h e p r e p a r a t i o n o f s e r i a l d i l u t i o n s , u s u a l l y t w o - f o l d , o f t h e b a c t e r i o c i n s u s p e n s i o n . A c o n s t a n t volume o f e a c h d i l u t i o n i s d e p o s i t e d on t h e s u r f a c e of an a g a r p l a t e u n i f o r m l y s e e d e d w i t h c e l l s o f t h e s e n s i t i v e i n d i c a t o r s t r a i n . The d e g r e e t o w h i c h t h e o r i g i n a l s u s p e n s i o n c a n be d i l u t e d w h i l e s t i l l i n h i b i t i n g t h e g r o w t h o f t h e i n d i c a t o r lawn i s a measure o f t h e s t r e n g t h o r t i t r e o f t h e o r i g i n a l p r e p a r a t i o n . T h i s measure i s u s u a l l y e x p r e s s e d i n t e r m s o f a r b i t r a r y u n i t s (au) w h i c h 7 are d e f i n e d as the r e c i p r o c a l of the l a s t d i l u t i o n to give complete i n h i b i t i o n of i n d i c a t o r growth f o l l o w i n g a s t a n d a r d i z e d p e r i o d of i n c u b a t i o n . A second method of q u a n t i f y i n g b a c t e r i o c i n a c t i v i t y i s the d i f f u s i o n zone assay (He a t l y and F l o r e y 1946). A l a y e r of agar u n i f o r m l y seeded with i n d i c a t o r b a c t e r i a i s allowed to s o l i d i f y i n a l a r g e f l a t d i s h . Wells are then punched i n the medium with a s t e r i l e c o r k - b o r e r . Constant amounts of b a c t e r i o c i n suspension are added to the w e l l s and the p l a t e i s incubated a l l o w i n g the b a c t e r i o c i n to d i f f u s e i n t o the agar. The diameters of the i n h i b i t i o n zones produced by the l e t h a l a c t i o n of the b a c t e r i o c i n are taken as d i r e c t measures of b a c t e r i o c i n a c t i v i t y . The t h i r d method of assay i s c a l l e d the s u r v i v o r count method, an approach developed from s t u d i e s i n v o l v i n g the k i n e t i c s of a d s o r p t i o n (Mayr-Harting et a l . 1972). Because the a d s o r p t i o n of b a c t e r i o c i n to the s u r f a c e s of s e n s i t i v e b a c t e r i a f o l l o w s a P o i s s o n i a n d i s t r i b u t i o n , the number of l e t h a l u n i t s can be c a l c u l a t e d u s i n g the f o l l o w i n g e q uation: Lo = InR x No where Lo i s the number of l e t h a l u n i t s , R i s the r a t i o of s u r v i v i n g c e l l s , and No i s the o r i g i n a l number of c e l l s 8 i n the p o p u l a t i o n . A f o u r t h method i n v o l v e s a m o d i f i c a t i o n of both the s u r v i v o r count and c r i t i c a l d i l u t i o n methods. With t h i s approach, the p r o p o r t i o n of s u r v i v i n g c e l l s i s measured as a f u n c t i o n of b a c t e r i o c i n c o n c e n t r a t i o n . The number of s u r v i v i n g c e l l s i s measured by e s t i m a t i n g t h e i r a b i l i t y to reduce c o l o u r l e s s t r i p h e n y l t e t r a z o l i u m c h l o r i d e (TTC) to red formazan by a c o l o u r i m e t r i c assay. Absorbance i s p l o t t e d a g a i n s t b a c t e r i o c i n d i l u t i o n to y i e l d a t i t r a t i o n curve. The mean common sl o p e of the l i n e a r r e g i o n s of t e s t and standard samples i s then used to c a l c u l a t e the po t e n c i e s of the t e s t samples r e l a t i v e to t h a t of the standard (Shannon and Hedges 1970). A technique chosen f o r measuring the a c t i v i t y of any b a c t e r i o c i n must ensure high l e v e l s of both s e n s i t i v i t y and p r e c i s i o n . For r o u t i n e use, r e s u l t s must be o b t a i n a b l e w i t h i n a reasonable amount of time. The p h y s i c a l and b i o l o g i c a l p r o p e r t i e s of both the b a c t e r i o c i n and s e n s i t i v e s t r a i n must t h e r e f o r e be c a r e f u l l y c o nsidered i n choosing the technique best s u i t e d to th a t p a r t i c u l a r system. 9 C. B a c t e r i o c i n S e n s i t i v i t y . The events r e s u l t i n g from the i n t e r a c t i o n between b a c t e r i o c i n s and s e n s i t i v e c e l l s can g e n e r a l l y be d i f f e r e n t i a t e d i n t o two stages - the a d s o r p t i o n phase and the l e t h a l phase. The a d s o r p t i o n phase occurs upon i n i t i a l c o n t a c t between the b a c t e r i o c i n and the s e n s i t i v e c e l l . T h i s phase i s d e f i n e d by a r a p i d a d s o r p t i o n or b i n d i n g of the b a c t e r i o c i n to s p e c i f i c r e c e p t o r s on the c e l l s u r f a c e . The l e t h a l phase, which may be induced by a d s o r p t i o n , i s c h a r a c t e r i z e d by the onset of p h y s i o l o g i c a l or bi o c h e m i c a l changes i n the s e n s i t i v e c e l l , i r r e v e r s i b l y l e a d i n g to the death of the c e l l (Mayr-Harting et a l . 1972). C . l . B a c t e r i o c i n A d s o r p t i o n . The phenomenon of a d s o r p t i o n can be demonstrated by the o b s e r v a t i o n of a decrease i n measured b a c t e r i o c i n a c t i v i t y upon the a d d i t i o n of s e n s i t i v e c e l l s . When b a c t e r i o c i n i s i n excess, the r a t e of d e c l i n e i n r e s i d u a l b a c t e r i o c i n a c t i v i t y depends on the number of a v a i l a b l e r e c e p t o r s i t e s . I f a d s o r p t i o n i s i r r e v e r s i b l e , the l e v e l of r e s i d u a l a c t i v i t y over time w i l l f o l l o w f i r s t order r e a c t i o n k i n e t i c s . The r a t e of a d s o r p t i o n depends on the 10 number of a v a i l a b l e s i t e s . As these s i t e s become occupied, the p r o b a b i l i t y of a d s o r p t i o n and t h e r f o r e the r a t e of a d s o r p t i o n decrease. The s p e c i f i c i t y of the a d s o r p t i o n s i t e i s u s u a l l y i n d i c a t e d by demonstrating a lack of a d s o r p t i o n i n a r e l a t e d , but i n s e n s i t i v e s t r a i n . The k i n e t i c s of the a d s o r p t i o n phase and i t s e f f e c t s on c e l l v i a b i l i t y can be f u r t h e r a n a l y s e d . The a d s o r p t i o n of a b a c t e r i o c i n p a r t i c l e to s e n s i t i v e c e l l s i s a d i s c r e t e event, the p r o b a b i l i t y of which f o l l o w a Poisson d i s t r i b u t i o n . The P o i s s o n i a n p r o b a b i l i t y e q u a t i o n : (1) P(Y=k)=e- u x u*/k! i n d i c a t e s t h a t the p r o b a b i l i t y (P) that the random v a r i a b l e Y takes the value k i s equal to the e~" where u i s the mean of the d i s t r i b u t i o n ; i . e . the d i s t r i b u t i o n of b a c t e r i o c i n molecules over a p o p u l a t i o n of s e n s i t i v e c e l l s . In t h i s case, Y r e f e r s to the a d s o r p t i o n of a l e t h a l dose of b a c t e r i o c i n . I f a c e l l f a i l s to adsorb a l e t h a l dose of b a c t e r i o c i n (k=0), th a t c e l l w i l l s u r v i v e . The average p r o b a b i l i t y of t h i s o c c u r i n g i s equal to the p r o p o r t i o n of s u r v i v i n g c e l l s (Nt) to the o r i g i n a l number of c e l l s (No) i n the t e s t p o p u l a t i o n . The mean of the 11 d i s t r i b u t i o n of b a c t e r i o c i n over the p o p u l a t i o n of s e n s i t i v e c e l l s i s the average number of b a c t e r i o c i n molecules per c e l l (Lo/No). Equation (1) t h e r e f o r e can be r e w r i t t e n : Nt /No = e < L o / ' N o > I f f o r a p a r t i c u l a r b a c t e r i o c i n system, l o g Nt/No i s p r o p o r t i o n a l to the b a c t e r i o c i n c o n c e n t r a t i o n , then the i n t e r a c t i o n between the b a c t e r o i c i n and the c e l l s i n t h a t system i s s a i d to f o l l o w s i n g l e - h i t k i n e t i c s . T h i s i m p l i e s t h a t a s i n g l e p a r t i c l e of b a c t e r i o c i n i s capable, with a c e r t a i n p r o b a b i l i t y , of k i l l i n g a s e n s i t i v e c e l l upon a d s o r p t i o n . A s i n g l e - h i t mechanism can be confirmed by the f a c t t h a t at low m u l t i p l i c i t i e s , the number of c e l l s k i l l e d should be r o u g h l y equal to the number of l e t h a l u n i t s present i n the sample. A m u l t i -h i t mechanism i s one i n which b a c t e r i o c i n molecules must a c t c o - o p e r a t i v e l y or c u m u l a t i v e l y i n order to k i l l s e n s i t i v e c e l l s . T h i s i s i n d i c a t e d by a p l a t e a u near the o r i g i n of the l o g Nt/No vs Lo c o n c e n t r a t i o n p l o t , as low m u l t i p l i c i t i e s are not able to e f f e c t c e l l death as e f f i c i e n t l y . C.2. B a c t e r i o c i n Receptors. As p r e v i o u s l y i n d i c a t e d , the b a c t e r i o c i n must f i r s t n 12 adsorb to r e c e p t o r s on the s u r f a c e of the b a c t e r i a l envelope i n order to e f f e c t the l e t h a l phase. The c e l l envelope i t s e l f i s a s t r u c t u r e of remarkable complexity. In Gram negative b a c t e r i a , the envelope c o n s i s t s of the cyt o p l a s m i c membrane, the p e r i p l a s m i c space and the outer membrane. The c y t o p l a s m i c , or innermost membrane c o n t a i n s the enzyme systems of the e l e c t r o n t r a n s p o r t c h a i n , f o r a c t i v e t r a n s p o r t of s o l u t e s , and f o r e x c r e t i o n of waste products. The b i o s y n t h e t i c machinery necessary f o r the pr o d u c t i o n and t r a n s l o c a t i o n of the s t r u c t u r a l components of the envelope a l s o r e s i d e s w i t h i n the c y t o p l a s m i c membrane (Hammond et al. 1984). Between the cy t o p l a s m i c and outer membranes i s the p e r i p l a s m i c space. Found w i t h i n t h i s space i s the p e p t i d o g l y c a n l a y e r , a s t r u c t u r e which c o n f e r s mechanical s t r e n g t h and s t a b i l i t y to the c e l l envelope. A l s o l o c a t e d here are scavenging enzymes and b i n d i n g p r o t e i n s which are i n v o l v e d i n both n u t r i e n t uptake and chemotaxis. The outermost l a y e r of the c e l l envelope i s the outer membrane. I t c o n s i s t s of p r o t e i n , p h o s p h o l i p i d , and l i p o p o l y s a c c h a r i d e . U n l i k e the c y t o p l a s m i c membrane, the outer membrane pl a y s no r o l e i n energy t r a n s d u c t i o n and possesses l i t t l e enzyme a c t i v i t y . 13 L i p o p o l y s a c c h a r i d e (LPS) i s a molecular s p e c i e s unique to the outer membranes of Gram negative b a c t e r i a . I t c o n s i s t s of a l i p i d moiety (the l i p i d A), the core, and the O-antigen s i d e c h a i n . The l i p i d A, c o n s i s t i n g of hydroxy or s t r a i g h t c h a i n f a t t y a c i d s , remains f i r m l y anchored i n the matrix of the membrane. The core i s a complex o l i g o s a c c h a r i d e c o n s i s t i n g of a branched c h a i n of heptoses and hexoses. The l i p i d A and core are i n d i s p e n s i b l e to the c e l l as Gram negative b a c t e r i a l a c k i n g these s t r u c t u r e s have never been i s o l a t e d . The O-antigen i s attached to the core and i s the outermost p o r t i o n of the molecule. I t c o n s i s t s of r e p e a t i n g o l i g o s a c c h a r i d e u n i t s and i s the s e r o l o g i c a l l y domininant pa r t of the molecule (Hammond et al. 1984). Both the composition of the i n d i v i d u a l s a c c h a r i d e u n i t s and t h e i r c o n f i g u r a t i o n w i t h i n the molecule determine i t s a n t i g e n i c s p e c i f i c i t y . In the E n t e r o b a c t e r i a c e a e , LPS makes up the e n t i r e outer l e a f l e t of the outer membrane l i p i d b i l a y e r , while p h o s p h o l i p i d comprises the inner l e a f l e t (Hancock 1984) . The s t r u c t u r a l asymmetry of the outer membrane i s a r e f l e c t i o n of i t s f u n c t i o n s . I t serves as a p e r m e a b i l i t y b a r r i e r , p r o v i d i n g p r o t e c t i o n from h y d r o l y t i c enzymes, a n t i b i o t i c s and e m u l s i f y i n g agents, while outer membrane p r o t e i n s a l l o w the s e l e c t i v e u n i d i r e c t i o n a l d i f f u s i o n of 14 n u t r i e n t s . The outer membrane a l s o serves as an i n t e r f a c e between the c e l l and i t s environment, o f t e n f a c i l i t a t i n g s u r f a c e adhesion and mediating communication beween the c e l l and i t s surroundings. B a c t e r i o c i n r e c e p t o r s i t e s must face the outer s u r f a c e of the c e l l ; t h e r e f o r e , they are always found i n the outer membrane of Gram negative b a c t e r i a and i n the capsule or w a l l of Gram p o s i t i v e b a c t e r i a . Although l i t t l e i s known about the chemical nature of the bond between b a c t e r i o c i n s and t h e i r cognate r e c e p t o r s , the r e c e p t o r s themselves have been c h a r a c t e r i z e d to some exte n t . In E s c h e r i c h i a c o l i , c o l i c i n r e c e p t o r s have been e s p e c i a l l y w e l l d e s c r i b e d . The r e c e p t o r f o r the E-group c o l i c i n s was the f i r s t to be p u r i f i e d and was shown to be a 6 0 , 0 0 0 d outer membrane p r o t e i n (Sabet and Schnaitman 1971). Receptors fo r c o l i c i n s M (Braun et al. 1973), K (Manning and Reeves 1978), I (Konisky and L i u 1974) and D (Pugsely and Reeves 1976) were a l s o found to be outer membrane p r o t e i n s . The r e c e p t o r f o r c o l i c i n A, although a p r o t e i n , i s o n l y a c t i v e when a s s o c i a t e d with LPS (Chai et al. 1982). Although r e c e p t o r s i t e s f o r many bacteriophages have been i d e n t i f i e d , l i t t l e work has been done on the b i n d i n g s i t e s of p a r t i c u l a t e b a c t e r i o c i n s . A notable e x c e p t i o n i s the R-pyocin group, produced by Pseudomonas 15 aeruginosa. Receptors of the R-type pyocins have been l o c a l i z e d on the LPS (Ikeda and Egami 1969). Various sugar groups w i t h i n the core component have been t e n t a t i v e l y i d e n t i f i e d as b i n d i n g s i t e s f o r the i n d i v i d u a l members of the R-group (Meadow and Wells 1978) . B a c t e r i o c i n r e s i s t a n t s t r a i n s a r i s e when s e n s i t i v e c e l l s l o s e the f u n c t i o n a l r e c e p t o r s i t e , thereby p r e c l u d i n g a d s o r p t i o n . Because r e s i s t a n t mutants can u s u a l l y be i s o l a t e d i n the l a b o r a t o r y , i t i s c l e a r t h a t b a c t e r i o c i n r e c e p t o r f u n c t i o n i s seldom an e s s e n t i a l c h a r a c t e r . The f a c t t h a t these p o t e n t i a l l y d e t r i m e n t a l s u r f a c e s t r u c t u r e s are maintained i n wil d - t y p e c e l l s ; however, suggests that they provide a f u n c t i o n b e n e f i c i a l to the c e l l . In E. c o l i , the outer membrane p r o t e i n which binds the E group c o l i c i n s a l s o p r o v i d e s a d i f f u s i o n pathway f o r v i t a m i n B i 2 / while the c o l i c i n K rec e p t o r s p e c i f i c a l l y f a c i l i t a t e s the uptake of n u c l e o s i d e s (Konisky 1982). C o l i c i n s B, D I, and M bind to outer membrane p r o t e i n s which a l s o serve as r e c e p t o r s f o r i r o n s i d e r o p h o r e s (Hantke and Braun 1981). C o l i c i n -r e s i s t a n t c e l l s which have undergone osmotic shock (and thus rendered leaky) become s e n s i t i v e . C o l i c i n s a l s o r e a d i l y penetrate c y t o p l a s m i c membrane-derived v e s i c l e s . T h i s suggests t h a t c o l i c i n s (and probably other 16 b a c t e r i o c i n s ) u t i l i z e s u r f a c e s t r u c t u r e s as r e c e p t o r s i n order to overcome the p e r m e a b i l i t y b a r r i e r of the outer membrane. C.3. B a c t e r i o c i n Mode of A c t i o n . C e l l s d e r i v e d from s e n s i t i v e s t r a i n s which adsorb b a c t e r i o c i n but are k i l l e d at e f f i c i e n c i e s between 0-5 % of t h a t of the wild-type are s a i d to be b a c t e r i o c i n -t o l e r a n t (Nomura 1967). C o l i c i n - t o l e r a n t s t r a i n s have been i s o l a t e d and c h a r a c t e r i z e d with the aim of understanding the mechanism by which the l e t h a l phase i s t r i g g e r e d by the a d s o r p t i o n phase. Although t h i s approach has provided some c l u e s , d y s f u n c t i o n s which l e a d to t o l e r a n c e are so d i v e r s e and can have many v a r i e d p l e i o t r o p i c e f f e c t s , t h a t these aims have yet to be r e a l i z e d (Hantke and Braun 1981). The mechanism by which the l e t h a l phase i s excecuted, or the mode of a c t i o n , of c o l i c i n s and many other b a c t e r i o c i n s i s w e l l documented. The a c t i o n of some b a c t e r i o c i n s i s d i r e c t e d to c y t o p l a s m i c macromolecules. C o l i c i n - E 3 a f f e c t s the 16s ribosomal RNA, r e s u l t i n g i n a c e s s a t i o n of p r o t e i n s y n t h e s i s (Nomura 1964). C o l i c i n E2 s p e c i f i c a l l y i n h i b i t s DNA s y n t h e s i s and induces d e g r a d a t i o n (Nomura 1964). F o l l o w i n g uptake, these c o l i c i n s d i r e c t l y a f f e c t t h e i r 17 t a r g e t s , as E2 and E3 r e s p e c t i v e l y e x h i b i t endo-DNase and ribosomal RNase a c t i v i t i e s i n v i t r o . C l o a c i n DF13, produced by Enterobactez cloacae and pyocin AP41 are a l s o known to have nuclease a c t i v i t y (Konisky 1982). C o l i c i n s A, E, I, and K form ion-permeable channels a c r o s s the c e l l envelope. Because t r e a t e d c e l l s leak potassium and become permeable to other i o n s , the e l e c t r o c h e m i c a l g r a d i e n t a c r o s s the membrane c o l l a p s e s . The e l e c t r o c h e m i c a l g r a d i e n t or proton motive f o r c e (pmf) i s maintained i n l i v i n g c e l l s by the energy generated from carbohydrate c a t a b o l i s m . The pmf d r i v e s the s y n t h e s i s of adenosine t r i p h o s p h a t e (ATP) which i n t u r n d e l i v e r s the energy r e q u i r e d f o r b i o s y n t h e s i s and growth. F o l l o w i n g treatment with these c o l i c i n s , ATP pools become d e p l e t e d and macromolecular s y n t h e s i s i s s e v e r e l y i n h i b i t e d . S t a p h y l o c o c c i n 1580 and the p a r t i c u l a t e R-type pyocins a l s o e x e r t t h e i r e f f e c t s toward the cy t o p l a s m i c membrane. Although these b a c t e r i o c i n s cause the c o l l a p s e of the pmf, i t i s u n c e r t a i n whether they do so by the formation of ion-permeable channels (Konisky 1982). Treatment of c e l l s with c o l i c i n M or p e s t i c i n A1122 r e s u l t s i n l y s i s (Konisky 1982). Under i s o t o n i c c o n d i t i o n s , however, these b a c t e r i o c i n s induce the formation of s p h e r o p l a s t s , s u g g e s t i n g t h a t the t a r g e t of a c t i o n i s the p e p t i d o g l y c a n . P e s t i c i n A1122, but not 18 c o l i c i n M, d i s p l a y s murein hydrolase a c t i v i t y i n v i t r o and d i r e c t l y degrades the p e p t i d o g l y c a n . C o l i c i n M i s probably an a c t i v a t o r of murein hydrolase or an i n h i b i t o r of p e p t i d o g l y c a n s y n t h e s i s , or both (Konisky 1982). D. B a c t e r i o c i n s of Erwinia carotovora. In 1961, Hamon and Peron d i s c o v e r e d b a c t e r i o c i n - l i k e a c t i v i t y i n E r w i n i a carotovora. In Japan, a b a c t e r i o c i n from E. carotovora, c a l l e d c a r o t o v o r i c i n - E r , was p a r t i a l l y p u r i f i e d , and e l e c t r o n microscopy of the a c t i v e f r a c t i o n r e v e a l e d s t r u c t u r e s resembling phage t a i l s (Endo et al. 1975). P u r i f i c a t i o n and f u r t h e r c h a r a c t e r i z a t i o n confirmed t h a t c a r o t o v o r i c i n - E r was ph a g e - l i k e , c o n s i s t i n g of a core, base p l a t e , c o n t r a c t i l e sheath and t a i l f i b e r s . That c a r o t o v o r i c i n - E r c l o s e l y resembled R-type pyocins was a l s o noted (Itoh et al. 1978). Treatment of s e n s i t i v e c e l l s with c a r o t o v o r i c i n -Er r e s u l t e d i n ex t e n s i v e c e l l l y s i s (Itoh et al. 1980a) accompanied by p h o s p h o l i p i d d e g r a d a t i o n c a t a l y s e d by membrane-bound phosphlipase A (Itoh et a l . 1980b). l n mutants d e f i c i e n t i n phospholipase A a c t i v i t y , the extent of p h o s p h o l i p i d d e g r a d a t i o n as w e l l as c e l l l y s i s were markedly decreased, s u p p o r t i n g the c o n c l u s i o n t h a t c a r t o v o r i c i n - E r a c t i v a t e s phopholipase A (Itoh et al. 19 1981). These mutants, however, r e t a i n e d s e n s i t i v i t y to the k i l l i n g a c t i o n of c a r o t o v o r i c i n - E r , i n d i c a t i n g t h a t phospholipase a c t i v a t i o n i s not the primary mode of a c t i o n (Itoh et al. 1981). D e p l e t i o n of i n t r a c e l l u l a r ATP f o l l o w e d by the a r r e s t of a l l macromolecular s y n t h e s i s was observed upon treatment of s e n s i t i v e c e l l s with c a r t o v o r i c i n - E r . Uptake of t r i p h e n y l methyl ammonium ion was a l s o i n h i b i t e d , s u g g e s t i n g t h a t c a r o t o v o r i c i n - E r a c t s i n a manner s i m i l a r to pyocin-R, by d i s t u r b i n g the ener g i z e d s t a t e of the c y t o p l a s m i c membrane and uncoupling ATP s y n t h e s i s (Itoh et a l . 1982). Concurrent r e s e a r c h i n North America e s t a b l i s h e d t h a t b a c t e r i o c i n p r o d u c t i o n i s a common c h a r a c t e r i s i t i c of Erwinia carotovora. Two types of c a r o t o v o r i c i n s were d i s t i n g u i s h e d on the b a s i s of heat, p r o t e o l y t i c enzyme a c t i v i t y and d i f f u s i b i l i t y i n agar (Campbell and Echandi 1979). A b a c t e r i o c i n which produced a n o n - d i f f u s e plaque was p a r t i a l l y p u r i f i e d by u l t r a c e n t r i f u g a t i o n . E l e c t r o n microscopy r e v e a l e d t h a t i t was p a r t i c u l a t e , resembling a p h a g e - t a i l . Crowley and De Boer (1980) a l s o found t h a t many s t r a i n s of E. ca r o t o v o r a produce p a r t i c u l a t e b a c t e r i o c i n s , while a few s t r a i n s produce both l a r g e and sma l l types. J a i s (1982) and Ward (1986) found at l e a s t three f r a c t i o n s i n Ecc s t r a i n 379 c u l t u r e supernatants with b a c t e r i o c i n a c t i v i t y . The l a r g e molecular weight 20 f r a c t i o n was comprised of p a r t i c l e s a l s o resembling phage t a i l s . T h i s b a c t e r i o c i n was c a l l e d c a r o t o v o r i c i n - 3 7 9 (Ward 1986 ) . S t r a i n s of E. carotovora have been c l a s s i f i e d on the b a s i s of s e r o l o g i c a l s p e c i f i c i t y (De Boer et al. 1979). To date, 36 serogroups of Ecc and 4 serogroups of Eca have been i d e n t i f i e d (De Boer, p e r s o n a l communication). Serogroup XI i s one of the more f r e q u e n t l y d e t e c t e d i n B r i t i s h Columbia. The f a c t t h a t many a g g r e s s i v e l y c a r o t o v o r i c i n o g e n i c s t r a i n s are c l a s s i f i e d w i t h i n t h i s serogroup suggests t h a t c a r o t o v o r i c i n may confer such a s e l e c t i v e advantage to the producing s t r a i n . T h i s hypothesis i s supported by experiments i n which potato s l i c e s (Campbell and Echandi 1979) or growth media ( J a i s 1982) were c o - i n o c u l a t e d with c a r o t o v o r i c i n - p r o d u c i n g and s e n s i t i v e s t r a i n s . The producing s t r a i n grew s u c c e s s f u l l y , while growth of the s e n s i t i v e s t r a i n was prevented, even when the s e n s i t i v e s t r a i n inoculum was ten times t h a t of the producer ( J a i s 1982) . Some serogroups of Eca are a l s o found more commonly than o t h e r s . Crowley and De Boer (1980) noted t h a t there was a p a r t i a l c o r r e l a t i o n between s e n s i t i v i t y to some c a r o t o v o r i c i n s and serogroup d e s i g n a t i o n among s t r a i n s of Eca. An attempt was made to e l u c i d a t e i f s e n s i t i v i t y to 21 serogroup XI c a r o t o v o r i c i n s was a f a c t o r i n determining d i s t r i b u t i o n of Eca i n B r i t i s h Columbia. S u r p r i s i n g l y , J a i s (1982) found t h a t the serogroups of Eca most commonly de t e c t e d a l s o d i s p l a y e d the hi g h e s t l e v e l of s e n s i t i v i t y to c a r o t o v o r i c i n - 3 7 9 . Furthermore, i n a broad survey of Eca s t r a i n s i s o l a t e d from b l a c k - l e g i n f e c t e d potato p l a n t s , s t r a i n s r e s i s t a n t to c a r o t o v o r i c i n - 3 7 9 were never found (Copeman, pers o n a l communication). T h i s suggested t h a t c a r o t o v o r i c i n - 3 7 9 s e n s i t i v i t y may be an important f a c t o r i n the ecology and/or p a t h o g e n i c i t y of Eca. In l i g h t of these f i n d i n g s , t h i s p r o j e c t was undertaken t o determine the molecular b a s i s of s e n s i t i v i t y of Eca to c a r o t o v o r i c i n - 3 7 9 . The experimental approach was d i v i d e d i n t o three s p e c i f i c o b j e c t i v e s : 1) To develop a procedure f o r the q u a n t i f i c a t i o n of c a r o t o v o r i c i n - 3 7 9 a c t i v i t y . 2) To examine p o s s i b l e mechanisms which might e x p l a i n the d i f f e r e n t l e v e l s of s e n s i t i v i t y to c a r t o v o r i c i n - 3 7 9 . 3) To i s o l a t e and c h a r a c t e r i s e the c a r o t o v o r i c i n - 3 7 9 r e c e p t o r i n Eca. 22 CHAPTER 1. QUANTIFICATION OF CAROTOVORICIN-379 ACTIVITY I n t r o d u c t i o n Erwinia carotovora subspecies carotovora s t r a i n 379 (Ecc 379) produces a l a r g e molecular weight b a c t e r i o c i n under c o n d i t i o n s of mitomycin i n d u c t i o n ( J a i s 1982). These b a c t e r i o c i n p a r t i c l e s , which resemble phage t a i l s , e x e r t a l e t h a l e f f e c t on most other s t r a i n s of Ecc and Erwinia carotovora subspecies a t r o s e p t i c a (Eca). In order to more c l o s e l y examine the e f f e c t s of t h i s b a c t e r i o c i n , c a l l e d c a r o t o v o r i c i n - 3 7 9 (Ward 1986), on s e n s i t i v e c e l l p o p u l a t i o n s , i t was necessary to develop an assay system which co u l d be used r o u t i n e l y , r a p i d l y , and e c o n o m i c a l l y while p r o v i d i n g a high degree of both s e n s i t i v i t y and p r e c i s i o n . Although b i o a s s a y s are n o t o r i o u s l y imprecise and v a r i a b l e , they remain the best method of measuring the potency of b i o l o g i c a l l y a c t i v e substances such as b a c t e r i o c i n s . B a c t e r i o c i n a c t i v i t y , t h e r e f o r e , i s c o n v e n t i o n a l l y measured us i n g a s e n s i t i v e s t r a i n as a b i o l o g i c a l i n d i c a t o r . Three b a s i c b i o a s s a y techniques have been developed i n a p p l y i n g t h i s approach. These are 23 the c r i t i c a l d i l u t i o n assay, the s u r v i v o r count assay, and the d i f f u s i o n zone assay. The c r i t i c a l d i l u t i o n assay (CDA) method, o r i g i n a l l y used to measure the a c t i v i t y of c o l i c i n s (Jacob et a l . 1952), has become the most g e n e r a l l y a p p l i e d assay method fo r a wide v a r i e t y of b a c t e r i o c i n s . I t i n v o l v e s d i l u t i n g the b a c t e r i o c i n suspension and s p o t t i n g the d i l u t i o n s onto a s o f t agar p l a t e seeded with s e n s i t i v e i n d i c a t o r b a c t e r i a . The r e c i p r o c a l of the l a s t d i l u t i o n to completely i n h i b i t the growth of the i n d i c a t o r lawn i s c a l l e d the b a c t e r i o c i n t i t r e , and i s expressed i n a r b i t r a r y u n i t s (au). T h i s form of the c r i t i c a l d i l u t i o n assay, the s p o t -p l a t e assay, i s a t t r a c t i v e because i t i s simple, and r e s u l t s can be obtained w i t h i n 24 hours. The main disadvantage, however i s the inherent lack of p r e c i s i o n . For example, a b a c t e r i o c i n suspension which can be d i l u t e d 1/64 before l o s i n g i t s potency i s s a i d to have a t i t r e of 64 au. T h i s r e a l l y means, however, t h a t the minimal value i s 64 au, and t h a t the a c t u a l value l i e s somewhere between 64 and the r e c i p r o c a l of the next d i l u t i o n , 128 au. In the s u r v i v o r count method, the number of s e n s i t i v e c e l l s to s u r v i v e exposure to a suspension of b a c t e r i o c i n i s a l s o used as a measure of the potency of 24 t h a t p r e p a r a t i o n . S u r v i v i n g c e l l s are counted by standard p l a t e enumeration techniques. T h i s method has been found to be h i g h l y p r e c i s e , given enough r e p l i c a t i o n s , and i s extremely u s e f u l i n s t u d y i n g k i n e t i c parameters of b a c t e r i o c i n - s e n s i t i v e c e l l i n t e r a c t i o n s . I t i s , however, cumbersome, i m p r a c t i c a l and uneconomical, as i t i s t e d i o u s and time-consuming to perform. Furthermore, l a r g e volumes of c u l t u r e media and incubator space are r e q u i r e d when a s s a y i n g s e v e r a l r e p l i c a t e d samples. F i n a l l y , Eca r e q u i r e s at l e a s t 48 hours to produce v i s i b l e c o l o n i e s i n standard p l a t e enumeration, making t h i s assay method unacceptably slow f o r r o u t i n e use. The t e t r a z o l i u m r e d u c t i o n assay (TRA) i s e s s e n t i a l l y a s u r v i v o r count assay i n which the p r o p o r t i o n of s u r v i v i n g c e l l s i n a p o p u l a t i o n f o l l o w i n g treatment with b a c t e r i o c i n i s determined by measuring the extent of t r i p h e n y l t e t r a z o l i u m c h l o r i d e r e d u c t i o n r a t h e r than by p l a t e enumeration. T e t r a z o l i u m r e d u c t i o n i s i n d i c a t e d by the development of a red c o l o u r i n the r e a c t i o n tube. The t e t r a z o l i u m r e d u c t i o n assay o f f e r s a more p r e c i s e i n d i c a t i o n of t r u e d i l u t i o n end-point than the s p o t - p l a t e assay. Furthermore t h i s assay can be performed r a p i d l y , as r e s u l t s with E s c h e r i c h i a c o l i systems are o b t a i n a b l e w i t h i n a few hours. (Shannon and Hedges 1970). 25 In the d i f f u s i o n zone method, the zone on an i n d i c a t o r p l a t e i s taken as a d i r e c t measure of b a c t e r i o c i n a c t i v i t y . In s p i t e of r e q u i r i n g a leng t h y i n c u b a t i o n p e r i o d , t h i s assay i s an e f f e c t i v e l y p r e c i s e method of measuring the a c t i v i t y of low molecular weight b a c t e r i o c i n s . Large molecular weight b a c t e r i o c i n s such as c a r o t o v o r i c i n - 3 7 9 , however, do not d i f f u s e w e l l , even through s o f t agar. Because i t i s g e n e r a l l y accepted t h a t t h i s method i s f a r too i n s e n s i t i v e when a p p l i e d to l a r g e molecular weight b a c t e r i o c i n s (Mayr-Harting et a l . 1972) i t s use i n the de t e r m i n a t i o n of c a r o t o v o r i c i n - 3 7 9 a c t i v i t y would be of l i t t l e v a l u e . In t h i s chapter, experiments e v a l u a t i n g both the TRA and s p o t - p l a t e assay methods f o r the de t e r m i n a t i o n of c a r o t o v o r i c i n - 3 7 9 a c t i v i t y , as w e l l as the development of a mo d i f i e d c r i t i c a l d i l u t i o n assay c a r r i e d out i n a m i c r o t i t r e p l a t e are d e s c r i b e d . M a t e r i a l s and Methods B a c t e r i a l S t r a i n s , Media and Growth C o n d i t i o n s : The c a r o t o v o r i c i n producer s t r a i n was Erwinia carotovora subspecies carotovora s t r a i n 379. The i n d i c a t o r s t r a i n was E r w i n i a carotovora subspecies 26 atroseptica s t r a i n 530. The producer s t r a i n was grown i n L u r i a broth c o n s i s t i n g of 1% (w/v) Bacto-Tryptone ( D i f c o ) , 0.5% (w/v) Yeast E x t r a c t ( D i f c o ) , and 0.65% (w/v) NaCl (Sigma). The i n d i c a t o r s t r a i n was grown i n N u t r i e n t Broth ( D i f c o ) . S t a r t e r c u l t u r e s of both s t r a i n s were incubated on a r o t a r y shaker (150 rpm) a t 20 C. P r e p a r a t i o n of C a r o t o v o r i c i n - 3 7 9 : Overnight s t a r t e r c u l t u r e s of Erwinia carotovora subspecies carotovora s t r a i n 379 were i n o c u l a t e d i n t o f r e s h L u r i a broth to a l e v e l of 1% ( v / v ) . Mitomycin C (Sigma) was added to a f i n a l c o n c e n t r a t i o n of 0.2 ug/ml. The c u l t u r e was incubated on a r o t a r y shaker (150 rpm) a t 20 C f o r 48 h. C e l l s were removed from the c u l t u r e by c e n t r i f u g a t i o n at 10,000 x g f o r 20 min. A c h i l l e d , s t e r i l e s o l u t i o n of p o l y e t h y l e n e g l y c o l 8000 (Sigma) was added to the supernatant to a f i n a l c o n c e n t r a t i o n of 12% (w/v). F o l l o w i n g overnight i n c u b a t i o n at 0 C, the p r e c i p i t a t e d p r o t e i n was conce n t r a t e d by c e n t r i f u g a t i o n a t 10,000 x g r a v i t y f o r 20 minutes. The p e l l e t was g e n t l y resuspended i n 1/50 to 1/100 times the o r i g i n a l volume i n i c e - c o l d 10 mM potassium phosphate b u f f e r pH 7.2. P a r t i c u l a t e m a t e r i a l was removed from the concen t r a t e by c e n t r i f u g a t i o n a t 10,000 x g f o r 5 min. The supernatant (2.5 ml) was loaded on a 2.5 x 35 cm 27 column packed with Sephacryl S-300 i n lOmM potassium phosphate b u f f e r pH 7.2. The flow r a t e was maintained at about 1.0 ml/min. Column e l u a t e was c o n t i n u o u s l y monitored f o r absorbance at 280 nm. The peak c o n t a i n i n g c a r o t o v o r i c i n - 3 7 9 a c t i v i t y was c o l l e c t e d and f i l t e r s t e r i l i z e d . T r i p h e n y l T e t r a z o l i u m C h l o r i d e Assay: D i l u t i o n s of c a r o t o v o r i c i n - 3 7 9 were prepared i n 3 ml volumes of 10 mM potassium phosphate b u f f e r pH 7.2. An equal volume of s e n s i t i v e i n d i c a t o r c e l l suspension was added to each d i l u t i o n to a f i n a l ODsso of 0.2. I n d i c a t o r suspension was a l s o added to s t e r i l e d i l u e n t c o n t a i n i n g no c a r o t o v o r i c i n - 3 7 9 . These samples were dispensed i n t o s i x 1 ml samples. T r i p h e n y l t e t r a z o l i u m c h l o r i d e was added to h a l f the samples at a f i n a l c o n c e n t r a t i o n of 0.02 %. F o l l o w i n g a 24 or 48 h i n c u b a t i o n , 0.5 ml of 0.25 % f o r m a l i n was added to each sample. Absorbance was measured a t 500 nm with a Bausch and Lomb S p e c t r o n i c 20. Spot - P l a t e Assay: I n d i c a t o r p l a t e s were prepared by adding s e n s i t i v e i n d i c a t o r b a c t e r i a at a f i n a l c o n c e n t r a t i o n of about 10 7 c e l l s / m l i n t o molten i n d i c a t o r agar which c o n s i s t e d of 1% 28 (w/v) Bacto-Peptone, 0.5% Bacto-Agar ( D i f c o ) and 0.6% NaCl. T h i s i n d i c a t o r agar was poured i n t o s t e r i l e p e t r i p l a t e s i n 5 ml volumes and the agar was l e f t to s o l i d i f y . Two-fold s e r i a l d i l u t i o n s of c a r o t o v o r i c i n - 3 7 9 were prepared i n 10 mM potassium phosphate b u f f e r pH 7.2. Ten m i c r o l i t e r s of c a r o t o v o r i c i n from each d i l u t i o n was spo t t e d onto e i g h t r e p l i c a t e i n d i c a t o r p l a t e s . The p l a t e s were incubated o v e r n i g h t at 24 C. The t i t r e of c a r o t o v o r i c i n - 3 7 9 a c t i v i t y was taken as the r e c i p r o c a l of the l a s t d i l u t i o n to cause complete i n h i b i t i o n of growth on the i n d i c a t o r lawn. This a c t i v i t y was expressed i n terms of a r b i t r a r y u n i t s (au). Means and sample d e v i a t i o n s of a r b i t r a r y u n i t s were c a l c u l a t e d w i t h i n each d i l u t i o n s e r i e s . M i c r o t i t r e P l a t e Assay: Two-fold s e r i a l d i l u t i o n s of c a r o t o v o r i c i n - 3 7 9 were made i n 100 u l volumes d i r e c t l y i n the rows of a s t e r i l e m i c r o t i t r e p l a t e u s i n g s t e r i l e 10 mM potassium phosphate b u f f e r pH 7.2 as a d i l u e n t . M i c r o t i t r e p l a t e s were s t e r i l i z e d by a 5 min exposure to short wave u l t r a - v i o l e t l i g h t from a GE-15 g e r m i c i d a l tube at a d i s t a n c e of 20 cm. A suspension of i n d i c a t o r b a c t e r i a was prepared i n 1% (w/v) Bacto-Peptone 0.6% NaCl to a f i n a l c o n c e n t r a t i o n 29 of about 10 7 c e l l s / m l . One hundred u l of c e l l suspension was dispensed i n t o each one of the w e l l s . P l a t e s were incubated 24 h a t 24 C. The t i t r e of the c a r o t o v o r i c i n -379 p r e p a r a t i o n was taken as the r e c i p r o c a l of the l a s t d i l u t i o n to produce a c l e a r (non-turbid) w e l l . A c t i v i t y was expressed as a r b i t r a r y u n i t s (au). Means and standard d e v i a t i o n s were c a l c u l a t e d f o r au between rows. R e s u l t s The TRA method proved to be slower and l e s s s e n s i t i v e than the s p o t - p l a t e assay. No measurable red c o l o u r developed i n the TRA tubes at 24 h i n c u b a t i o n (Table 1). There was no d i f f e r e n c e i n the readings between t e s t samples, and c o n t r o l s with and without TTC, i n d i c a t i n g t h a t the absorbances were due mainly to c u l t u r e t u r b i d i t y . At 48 hours, s i g n i f i c a n t c o l o u r had developed i n c o n t r o l s with TTC but l a c k i n g c a r o t o v o r i c i n -379. Absorbance readings i n samples with TTC and c a r o t o v o r i c i n at 1 and 1/3 r e l a t i v e standard c o n c e n t r a t i o n s d i d not change, i n d i c a t i n g t h a t the c a r o t o v o r i c i n - 3 7 9 had prevented growth i n these samples. Although c a r o t o v o r i c i n - 3 7 9 a c t i v i t y was d e t e c t e d i n the 1/3 standard samples, i t was not d e t e c t e d i n the 1/10 30 Table 1. Absorbance a t 500 nm of i n d i c a t o r suspension c o n t a i n i n g d i f f e r e n t r e l a t i v e c o n c e n t r a t i o n s (RC) of c a r o t o v o r i c i n - 3 7 9 (C-379) with (+) and without (-) t r i p h e n y l t e t r a z o l i u m c h l o r i d e (TTC). A s o o (+/-.02 1) a f t e r C-379 24 hours 48 hours RC + TTC - TTC + TTC - TTC 1 .31 .29 .30 .34 1/3 .30 .28 .36 .32 1/10 .32 .30 .50 .35 0 .32 .29 .49 .35 ^-indicates the range of values obtained over three r e p l i c a t e s . 31 F i g u r e 1. S p o t - p l a t e d e t e r m i n a t i o n of c a r o t o v o r i c i n -379 a c t i v i t y i n ( l e f t to r i g h t ) u n d i l u t e d , 1/3, and 1/10 r e l a t i v e c o n c e n t r a t i o n s . 32 standard samples, even though a c t i v i t y i n these samples was d e t e c t e d on an i n d i c a t o r p l a t e ( F i g . 1). The s p o t - p l a t e assay, although r e l a t i v e l y more r a p i d and s e n s i t i v e than the TRA, was found to be h i g h l y i r r e p r o d u c i b l e because of the v a r i a b i l i t y i n h e r e n t i n the i n d i c a t o r response ( F i g . 2). F i g u r e 2a shows the i d e a l r e s u l t obtained from t h i s assay. The c r i t i c a l d i l u t i o n endpoint i s c l e a r l y 2~s or 32 au/10 u l . In c o n t r a s t , an i d e n t i c a l l y t r e a t e d p l a t e shown i n F i g . 2b i n d i c a t e d t h a t the t i t r e of t h i s sample was 2~ 4 or 16 au/10 u l . More commonly, however, r e s u l t s were obtained i n which the d i l u t i o n endpoint was not so c l e a r l y d e f i n e d . For example, i n F i g . 2c, the l a s t d i l u t i o n to cause complete i n h i b i t i o n of growth was 2~"*, t h e r e f o r e the t i t r e of t h i s sample was recorded as 16 au/10 u l even though s u b s t a n t i a l i n h i b i t i o n occurred to d i l u t i o n s of 2 - s and 2~ 7 ( F i g . 2 c ) . H i g h l y ambiguous r e s u l t s ( F i g . 2d) r e q u i r i n g a degree of s u b j e c t i v e i n t e r p r e t a t i o n were a l s o commonly obtained. The m i c r o t i t r e method, a modified c r i t i c a l d i l u t i o n assay, was found to be an e f f e c t i v e means of q u a n i t i f y i n g c a r o t o v o r i c i n - 3 7 9 a c t i v i t y . F i g u r e 3 shows a t y p i c a l r e s u l t when b a c t e r i o c i n i s d i l u t e d i n the w e l l s a long the rows of the p l a t e . In the dark w e l l s , 33 F i g u r e 2. R e p l i c a t e s p o t - p l a t e assays of a s i n g l e two-fold d i l u t i o n s e r i e s of c a r o t o v o r i c i n - 3 7 9 (shown l e f t to r i g h t i n descending order beginning at 1/2) demonstrating the v a r i a b i l i t y inherent i n the response of the E r w i n i a carotovora subspecies a t r o s e p t i c a i n d i c a t o r s t r a i n 530. a. D i l u t i o n endpoint i s 1/32. b. D i l u t i o n endpoint i s 1/16. c. D i l u t i o n endpoint i s not c l e a r l y d e f i n e d , but recorded as 1/16. d. D i l u t i o n endpoint cannot be i n t e r p r e t e d o b j e c t i v e l y . 34 F i g u r e 3. A t y p i c a l m i c r o t i t r e p l a t e assay showing the extent of i n h i b i t i o n of i n d i c a t o r (Erwinia carotovora subspecies a t r o s e p t i c a s t r a i n 530) growth by two-fold d i l u t i o n s e r i e s ( l e f t to r i g h t ) s t a r t i n g at 1/2) of a c a r o t o v o r i c i n - 3 7 9 suspension. The d i l u t i o n endpoint i s 1/256 i n a l l e i g h t r e p l i c a t e s . 35 c a r o t o v o r i c i n - 3 7 9 has completely i n h i b i t e d the growth of the i n d i c a t o r organism. The l i g h t c o l o r e d w e l l s however are t u r b i d , i n d i c a t i n g t h a t the c o n c e n t r a t i o n of b a c t e r i o c i n i s no longer s u f f i c i e n t to i n h i b i t growth at a d i l u t i o n of 2 _ e . The t i t r e of t h i s p r e p a r a t i o n i s t h e r e f o r e 256 au/100 u l or 2560 au/ml. T h i s t i t r e was found to be c o n s i s t e n t over e i g h t d i f f e r e n t d i l u t i o n s e r i e s ( F i g . 3) i n three d i f f e r e n t samples. When the s p o t - p l a t e and m i c r o t i t r e assays were compared (Table 2), the standard d e v i a t i o n s i n the m i c r o t i t r e assays were much l e s s than those found i n the s p o t - p l a t e assays. The means of the t i t r e s over e i g h t r e p l i c a t e s appeared to be higher when the spot assay was used; however, these values o n l y represented the lower l i m i t of a range of p o s s i b l e a c t i v i t y v a l u e s , t h e r e f o r e the s e n s i t i v i t i e s of the two methods were comparable. Discuss ion The number of methods which could be a p p l i e d i n q u a n t i f y i n g c a r o t o v o r i c i n - 3 7 9 a c t i v i t y was l i m i t e d 36 Table 2. Means and standard d e v i a t i o n s (SD) of c a r o t o v o r i c i n - 3 7 9 a c t i v i t y i n a r b i t r a r y u n i t s (au) over e i g h t r e p l i c a t e s i n each of three d i f f e r e n t samples as determined by the spot p l a t e and m i c r o t i t r e assay methods Ca r o t o v o r i c i n - 3 7 9 A c t i v i t y Sample Spot P l a t e Mean SD au/ml M i c r o t i t r e Mean SD au/ml 1 2 3 3600 2800 2400 17.4 15.5 8.0 2560 2560 1440 0 0 5.0 37 because of the b a c t e r i o c i n ' s l a r g e s i z e and the r e l a t i v e l y slow growth r a t e of the i n d i c a t o r s t r a i n . These two f a c t o r s r u l e d out the p o s s i b i l t y of u s i n g of the d i f f u s i o n zone and s u r v i v o r count methods f o r r o u t i n e a c t i v i t y assays. As a consequence, on l y the TRA and SPA were s e l e c t e d f o r e v a l u a t i o n . The t e t r a z o l i u m r e d u c t i o n assay was the more a t t r a c t i v e of the two because i t was p o t e n t i a l l y capable of p r o v i d i n g a more p r e c i s e value of a c t i v i t y when compared to a standard than a d i l u t i o n end-point. In p r a c t i s e , however, the t e t r a z o l i u m r e d u c t i o n assay proved to be slow and i n s e n s i t i v e , r e q u i r i n g 48 h f o r r e l i a b l e c o l o u r development. Because of t h i s , f u r t h e r a n a l y s i s of the data obtained was not undertaken. The spot p l a t e assay had the advantage of being s i m p l e r , f a s t e r and more s e n s i t i v e than the t e t r a z o l i u m r e d u c t i o n assay. I t was, however, a l s o h i g h l y v a r i a b l e , even w i t h i n the same d i l u t i o n s e r i e s ( F i g . 2). Because of the frequent lack of a d i s c r e t e d i l u t i o n end-point, t h i s method of assay was found to be too v a r i a b l e f o r a p p l i c a t i o n s r e q u i r i n g reasonable p r e c i s i o n . V a r i a b i l i t y was found to be extremely high when the end-point d i l u t i o n of c a r o t o v o r i c i n - 3 7 9 was s t r i c t l y taken as the l a s t d i l u t i o n to cause complete i n h i b i t i o n of growth. I f 3 8 t h i s d e f i n i t i o n was not p r a c t i s e d i n the s t r i c t e s t sense, then the i n t e r p r e t a t i o n of the r e s u l t became h i g h l y s u b j e c t i v e . In the spot p l a t e assay, the number of c e l l s the b a c t e r i o c i n i s exposed to d i r e c t l y a f f e c t s the r e s u l t of the assay. Obviously the fewer i n d i c a t o r c e l l s , the higher the t i t r e . The v a r i a b i l i t y observed when the assay i s used d i r e c t l y r e s u l t s from an i n h e r e n t i n a b i l i t y to s t a n d a r d i z e the number of i n d i c a t o r c e l l s exposed to the b a c t e r i o c i n . I d e a l l y , the agar p l a t e i s u n i f o r m l y seeded with a s t a r d a r d i z e d number of s e n s i t i v e c e l l s . Although a constant volume of b a c t e r i o c i n suspension i s s p o t t e d onto the suface of the p l a t e , there i s no way the area over which the drop spreads can be c o n t r o l l e d or s t a n d a r d i z e d . T h i s can a f f e c t the outcome of the assay, e s p e c i a l l y i f the p l a t e s are d i s t u r b e d before the drops are dry. A more common problem and a g r e a t e r source of v a r i a b i l i t y , however, i s the s m a l l d i f f e r e n c e s i n the t h i c k n e s s of the agar i n d i f f e r e n t areas of the p l a t e . V a r i a t i o n i n t h i c k n e s s i s v i r t u a l l y impossible to a v o i d , unless there i s a p e r f e c t l y l e v e l s u r f a c e a v a i l a b l e on which the p l a t e s can dry. These sma l l v a r i a t i o n s i n t h i c k n e s s r e s u l t i n a non-uniform d i s t r i b u t i o n of c e l l s over the s u r f a c e of the p l a t e . T h i s a f f e c t s the outcome 39 of the assay because a drop c o n t a i n i n g b a c t e r i o c i n s p o t t e d on a t h i c k area of the p l a t e must d i f f u s e f u r t h e r i n t o the agar and k i l l more c e l l s than t h a t s p o t ted onto a t h i n n e r a r e a . Thus, c r i t i c a l d i l u t i o n end-points can d i f f e r s u b s t a n t i a l l y between p l a t e s of the same d i l u t i o n s e r i e s , a c c o u n t i n g f o r the high v a r i a n c e s observed. In the m i c r o t i t r e assay, the r e a c t i o n between the b a c t e r i o c i n and s e n s i t i v e c e l l s takes p l a c e w i t h i n a constant volume and area ( i . e . the w e l l s of a m i c r o t i t r e p l a t e ) . Thus f a c t o r s which can a f f e c t the outcome of the s p o t - p l a t e assay are avoided i n the m i c r o t i t r e assay. Furthemore, because the i n d i c a t o r b a c t e r i a are not added to molten agar, the e f f e c t of high temperature on the v i a b i l i t y of the i n d i c a t o r s t r a i n i s not a concern. As a consequence, the m i c r o t i t r e p l a t e assay was found to be both s e n s i t i v e and r e p r o d u c i b l e . In a d d i t i o n , the m i c r o t i t r e assay was easy to perform, r e q u i r i n g l i t t l e p r e p a r a t i o n time, fewer m a t e r i a l s and l e s s c u l t u r e media than the s p o t - p l a t e assay. The s e n s i t i v i t y and p r e c i s i o n of the m i c r o t i t r e p l a t e assay c o u l d probably be improved. S e n s i t i v i t y c o u l d be i n c r e a s e d by u s i n g a lower number of i n d i c a t o r c e l l s per w e l l , f a c i l i t a t i n g the d e t e c t i o n of v e r y low l e v e l s of b a c t e r i o c i n . T h i s cannot be done with the s p o t - p l a t e assay, because i f fewer i n d i c a t o r c e l l s are 40 used, a c o n f l u e n t lawn of b a c t e r i a w i l l not grow. P r e c i s i o n c o u l d be improved by u s i n g d i f f e r e n t s t a r t i n g c o n c e n t r a t i o n s of c a r o t o v o r i c i n - 3 7 9 i n geometric d i l u t i o n s e r i e s . T i t r e s could be estimated to +/- 10% of the a c t u a l v a l u e , p r o v i d i n g a degree of p r e c i s i o n comparable to t h a t obatined by the t e t r a z o l i u m r e d u c t i o n assay (Shannon and Hedges 1970). The m i c r o t i t r e p l a t e assay i s t h e r e f o r e , the method of choice f o r q u a n t i f y i n g c a r o t o v o r i c i n - 3 7 9 a c t i v i t y . 41 CHAPTER 2. CHARACTERISTICS OF CAROTOVORICIN-379 SENSITIVITY IN ERWINIA CAROTOVORA I n t r o d u c t i o n B a c t e r i o c i n s are proteinaceous agents produced by a wide v a r i e t y of b a c t e r i a l s p e c i e s which e x e r t a l e t h a l e f f e c t on s t r a i n s c l o s e l y r e l a t e d to the producer. B a c t e r i o c i n s e n s i t i v i t y i s determined by the presence of re c e p t o r s i t e s and by the a b i l i t y of the b a c t e r i o c i n to reach i t s t a r g e t of a c t i o n . C o n s e r v a t i o n of b a c t e r i o c i n s e n s i t i v i t y g e n e r a l l y suggests t h a t the s e n s i t i v e phenotype pro v i d e s some f u n c t i o n b e n e f i c i a l to the c a r r y i n g s t r a i n (Reeves 1972). C o l i c i n r e c e p t o r s , f o r example, are a l s o r e q u i r e d f o r the uptake of i r o n , v i t a m i n B i 2 , n u c l e o s i d e s and other s o l u t e s (Hantke and Braun 1981). The f a c t o r s i n f l u e n c i n g the c o l i c i n uptake, however, are not w e l l c h a r a c t e r i z e d . Many s t r a i n s of the phytopathogen Erwinia carotovora produce b a c t e r i o c i n s of v a r y i n g s i z e s (Campbell and Echandi 1979, Endo et a l . 1975) and a c t i v i t y s p e c t r a (Crowley and De Boer 1980, J a i s 1982). C a r o t o v o r i c i n - E r was the f i r s t of these to be p u r i f i e d and c h a r a c t e r i z e d 42 as a l a r g e molecular weight b a c t e r i o c i n resembling a bacteriophage t a i l of an R-type pyocin (Itoh et a l . 1978). E. carotovora subspecies c a r o t o v o r a s t r a i n 379 produces a s i m i l a r p a r t i c u l a t e c a r o t o v o r i c i n ( J a i s 1982) c a l l e d c a r o t o v o r i c i n - 3 7 9 (Ward 1986). C a r o t o v o r i c i n s e n s i t i v i t y appears to be h i g h l y conserved among E. carotovora subspecies atroseptica (Eca), as no r e s i s t a n t s t r a i n s have ever been i s o l a t e d (R.J. Copeman, pers o n a l communication). The four serogroups of Eca do d i s p l a y , however, d i f f e r e n t l e v e l s of s e n s i t i v i t y to c a r o t o v o r i c i n - 3 7 9 ( J a i s 1982) as w e l l as to other p a r t i c u l a t e c a r o t o v o r i c i n s (Crowley and De Boer 1980). When the d i s t r i b u t i o n of each serogroup of Eca was compared with the l e v e l of C-379 s e n s i t i v i t y , J a i s (1982) found t h a t the serogroup most f r e q u e n t l y found i n b l a c k l e g i n f e c t e d potato t i s s u e a l s o d i s p l a y e d the h i g h e s t l e v e l of s e n s i t i v i t y to c a r o t o v o r i c i n - 3 7 9 . In t h i s chapter, i n t e r a c t i o n s between c a r o t o v o r i c i n -379 and the s e n s i t i v e c e l l s they a c t upon were examined i n an attempt to e l u c i d a t e those f a c t o r s which determine the extent of s e n s i t i v i t y to c a r o t o v o r i c i n - 3 7 9 among the four serogroups of Eca 43 M a t e r i a l s and Methods B a c t e r i a l S t r a i n s and Growth C o n d i t i o n s : The b a c t e r i a l s t r a i n s used i n t h i s chapter are l i s t e d i n Table 1. Test s t r a i n s were grown i n n u t r i e n t b r o t h o v e r n i g h t at 20 C and were used i n mid or l a t e - l o g phase. For the p r o d u c t i o n of c a r o t o v o r i c i n - 3 7 9 , s t a r t e r c u l t u r e s of Ecc 379 were grown i n L u r i a broth o v e r n i g h t at 20 C. P r e p a r a t i o n of C a r o t o v o r i c i n - 3 7 9 : C a r o t o v o r i c i n - 3 7 9 was prepared as d e s c r i b e d i n Chapter 1. C a r o t o v o r i c i n - 3 7 9 A c t i v i t y Assay: C a r o t o v o r i c i n a c t i v i t y was determined by the m i c r o t i t r e p l a t e assay as d e s c r i b e d i n Chapter 1. I n d i c a t o r spot p l a t e s used to demonstrate the presence or absence of a c t i v i t y were prepared as d e s c r i b e d i n Chapter 1. Measurement of C a r o t o v o r i c i n - 3 7 9 A d s o r p t i o n : Log-phase t e s t c u l t u r e s (Eca s t r a i n 530 and Ecc s t r a i n 379) grown i n n u t r i e n t broth were d i l u t e d 1/50 44 Table 1. S t r a i n s of E r w i n i a carotovora subspecies carotovora (Ecc) and Erwinia carotovora subspecies a t r o s e p t i c a (Eca) i s o l a t e d i n B r i t i s h Columbia (BC) and Wisconsin (Wn) used i n t h i s chapter Species S t r a i n Serogroup Source Ecc 379 XI Copeman, BC Ecc 504 XVIII Copeman, BC Eca SR8 I Kelman, Wn Eca 530 XX Copeman, BC Eca 496 XXII Copeman, BC 45 i n t o 5 ml of s t e r i l e potassium phosphate b u f f e r pH 7.2. An equal volume of c a r o t o v o r i c i n - 3 7 9 was added and q u i c k l y mixed. A l i q u o t s (1 ml) of the Eca s t r a i n 530 sample were drawn every 15 seconds (sec) over a two minute p e r i o d and immediately expressed through a 0.2 um c e l l u l o s e a c e t a t e f i l t e r . A l i q u o t s (1 ml) of the Ecc s t r a i n 379 sample were drawn at 1 and 2 minutes and a l s o f i l t e r - s t e r i l i z e d . Each a l i q u o t was then assayed f o r c a r o t o v o r i c i n - 3 7 9 a c t i v i t y u s i n g the m i c r o t i t r e p l a t e method. E f f e c t of C a r o t o v o r i c i n - 3 7 9 on S e n s i t i v e C e l l S u r v i v a l : Log-phase c u l t u r e s of Eca 530 were grown over n i g h t i n n u t r i e n t broth at 20 C. In order to determine the e f f e c t of c a r o t o v o r i c i n - 3 7 9 dosage on Eca s u r v i v a l , a two-fold d i l u t i o n s e r i e s of c a r o t o v o r i c i n - 3 7 9 was prepared i n s t e r i l e t e s t - t u b e s u s i n g s t e r i l e 10 mM potassium phosphate b u f f e r pH 7.2 as a d i l u e n t . An equal volume of c e l l suspension was added to each type of the c a r o t o v o r i c i n - 3 7 9 d i l u t i o n s e r i e s as w e l l as to s t e r i l e d i l u e n t b u f f e r as a c o n t r o l . The mixtures were incubated a 24 C f o r 20 min with g e n t l e a g i t a t i o n . A l i q u o t s from each sample were d i l u t e d i n s e r i e s of 100-fold d i l u t i o n s and dispensed onto n u t r i e n t agar p l a t e s i n order to measure the number of v i a b l e c e l l s . C o l o n i e s were 46 c o u n t e d f r o m t r i p l i c a t e p l a t e s f o l l o w i n g a 48 h i n c u b a t i o n a t 24 C. T h i s e x p e r i m e n t was r e p e a t e d t h r e e t i m e s u s i n g d i f f e r e n t c a r o t o v o r i c i n - 3 7 9 and s e n s i t i v e c e l l p r e p a r a t i o n s . The s l o p e of t h e l i n e In r a t i o of s u r v i v i n g c e l l s v s a r b i t r a r y u n i t s (au) c a r o t o v o r i c i n - 3 7 9 was u s e d t o c a l c u l a t e t h e a v e r a g e number o f l e t h a l u n i t s p e r a r b i t r a r y u n i t u s i n g t h e P o l s s o n p r o b a b i l i t y e q u a t i o n . I n o r d e r t o d e t e r m i n e t h e p r o g r e s s i o n o f c e l l d e a t h o v e r t i m e , t e s t c u l t u r e s were mixed i n e q u a l volumes w i t h c a r o t o v o r i c i n - 3 7 9 s u s p e n s i o n s o f known p o t e n c y . A t v a r i o u s t i m e s , a l i q u o t s were t a k e n and d i l u t e d 1/100. V i a b l e c e l l s were enum e r a t e d as d e s c r i b e d a b o v e . D e t e r m i n a t i o n o f C a r o t o v o r i c i n - 3 7 9 A d s o r p t i o n C a p a c i t y : C u l t u r e s o f t h e t e s t s t r a i n s were d i l u t e d 1/10 i n t o a p r e p a r a t i o n o f c a r o t o v o r i c i n - 3 7 9 and i n c u b a t e d f o r 20 min. F o l l o w i n g t h i s i n c u b a t i o n p e r i o d , t h e m i x t u r e was e x p r e s s e d t h r o u g h a 0.2 um c e l l u l o s e a c e t a t e f i l t e r i n t o s t e r i l e t u b e s . R e s i d u a l c a r o t o v o r i c i n - 3 7 9 a c t i v i t y was d e t e r m i n e d by t h e m i c r o t i t r e p l a t e method. The d i f f e r e n c e i n t h e o r i g i n a l t i t r e and t h e t i t r e a f t e r e x p o s u r e t o t h e t e s t s t r a i n s was c o n s i d e r e d t o be t h e amount a d s o r b e d i n a r b i t r a r y u n i t s . T h i s number was c o n v e r t e d t o l e t h a l u n i t s . C e l l numbers were d e t e r m i n e d 47 by d i l u t i o n p l a t i n g of v i a b l e c e l l s . F l u o r i m e t r y : A l l f l u o r e s c e n c e data were obtained u s i n g a Turner Model 430 s p e c t r o f l u o r o m e t e r . The f l u o r e s c e n t probe, 8-a n i l i n o - l - n a p h t h a l e n e s u l f o n i c a c i d hemimagnesium s a l t (ANS) (Sigma), was used a t a c o n c e n t r a t i o n of 50 uM. For a l l experiments, the e x c i t a t i o n wavelength was 350 nm. Test c u l t u r e s were washed and the c e l l s resuspended i n 10 mM HEPES b u f f e r pH 7.4 and used a t a f i n a l d e n s i t y of 0.125 O . D . s s o . Experiments were c a r r i e d out i n 5 ml f i n a l volumes. C a r o t o v o r i c i n - 3 7 9 was added i n 200 u l volumes and r a p i d l y mixed. A l i q u o t s of 1 ml were used f o r f l u o r e s c e n c e measurements, a l l of which were repeated a t l e a s t once. C u l t u r e t u r b i d i t y was measured before and a f t e r a l l experiments u s i n g a Bausch and Lomb Spec 20 spectrophotometer at 660 nm. R e s u l t s C a r o t o v o r i c i n - 3 7 9 formed n o n - d i f f u s e , c l e a r plaques on p l a t e s seeded with d i f f e r e n t s t r a i n s of s e n s i t i v e i n d i c a t o r b a c t e r i a ( F i g . l a - c ) . The producer s t r a i n , Ecc s t r a i n 379 was n o n - s e n s i t i v e ( F i g . I d ) . 48 V o F i g u r e 1. Spot p l a t e s o f c a r o t o v o r i c i n - 3 7 9 d e m o n s t r a t i n g p l a q u e m o r p h o l o g y on v a r i o u s t e s t s t r a i n s of Erwinia carotovora. a. Erwinia carotovora s u b s p . atroseptica s t r a i n 530. b. Erwinia carotovora s u b s p . atroseptica s t r a i n 496. c. E r w i n i a carotovora s u b s p . carotovora s t r a i n 504. d. Erwinia carotovora s u b s p . c a r o t o v o r a s t r a i n 379. 49 There was no n e u t r a l i z a t i o n of c a r o t o v o r i c i n - 3 7 9 a c t i v i t y upon the a d d i t i o n of the n o n - s e n s i t i v e s t r a i n Ecc 379 ( F i g . 2). A d d i t i o n of a suspension of s e n s i t i v e c e l l s (Eca s t r a i n 530), however, r e s u l t e d i n a r a p i d d e c l i n e i n a c t i v i t y with time ( F i g . 3). Whereas the i n i t i a l r a t e of a c t i v i t y l o s s or n e u t r a l i z a t i o n was high, the r a t e dropped with time i n a l o g a r i t h m i c f a s h i o n to zero by one minute. The h a l f - l i f e of b a c t e r i o c i n a c t i v i t y i n f r e e suspension under these c o n d i t i o n s was found to be about 15 sees. The i n i t i a l r a t e of n e u t r a l i z a t i o n appeared to depend on the number of s e n s i t i v e c e l l s present, as i t was too r a p i d to measure when c e l l numbers (of Eca 530) exceeded 1 x 10 9 (data not shown). The number of v i a b l e c e l l s over time f o l l o w i n g a d d i t i o n of c a r o t o v o r i c i n - 3 7 9 a l s o dropped i n a l o g a r i t h m i c f a s h i o n ( F i g . 3). When the p r o p o r t i o n of s u r v i v i n g c e l l s was measured f o l l o w i n g a 20 min exposure to v a r y i n g l e v e l s of b a c t e r i o c i n , the r e l a t i o n s h i p between b a c t e r i o c i n c o n c e n t r a t i o n and l o g percent s u r v i v a l was found to be c l e a r l y l i n e a r ( F i g . 4). V i a b l e c e l l numbers i n c o n t r o l s l a c k i n g b a c t e r i o c i n i n d i c a t e d t h a t there was no d e t e c t a b l e growth i n the samples over the d u r a t i o n of the experiment. The average number of l e t h a l u n i t s per a r b i t r a r y u n i t was found to be 2.1 x 10 G (from F i g . 2). 50 F i g u r e 2. R e s i d u a l c a r o t o v o r i c i n - 3 7 9 (C-379) a c t i v i t y i n the f i l t e r s t e r i l i z e d supernatant f o l l o w i n g the i n t r o d u c t i o n of c e l l s of e i t h e r Erwinia carotovora subsp. a t r o s e p t i c a s t r a i n 530 or E r w i n i a carotovora subsp. c a r o t o v o r a s t r a i n 379. 51 Time minutes F i g u r e 3. D e c l i n e i n the number of colo n y forming u n i t s per ml (CFU/ml) of E r w i n i a carotovora subsp. a t r o s e p t i c a s t r a i n 530 over time f o l l o w i n g exposure to 1280 a r b i t r a r y u n i t s (au)/ml of c a r o t o v o r i c i n - 3 7 9 (C-379) 52 1400 C—379 Concentration au/ml F i g u r e 4. E f f e c t of c a r o t o v o r i c i n - 3 7 9 (C-379) dosage i n a r b i t r a r y u n i t s (au)/ml on the s u r v i v a l of E r w i n i a carotovora subsp. atroseptica s t r a i n 530 20 min a f t e r exposure. E r r o r bars r e p r e s e n t the range of values obtained over three experiments. 53 S u r v i v a l over time of s t r a i n s r e p r e s e n t i n g the four serogroups of Eca f o l l o w i n g exposure to the same l e v e l of c a r o t o v o r i c i n - 3 7 9 ( F i g . 5) i n d i c a t e d t h a t each s t r a i n d i s p l a y e d a d i f f e r e n t l e v e l of s e n s i t i v i t y to the b a c t e r i o c i n . Ecc s t r a i n 504 d i s p l a y e d the l e a s t s e n s i t i v i t y , followed by Eca s t r a i n s 496, 530 and SR8. The r a t e of decrease of v i a b l e c e l l s dropped to zero w i t h i n one hour i n the case of s t r a i n s 504 and 496. When the a d s o r p t i o n c a p a c i t i e s of the s t r a i n s were compared, s t r a i n 504 had the h i g h e s t a d s o r p t i o n c a p a c i t y while 496 was s l i g h t l y lower than 530 and SR8 (Table 2). These data i n d i c a t e d that although s t r a i n 504 and 496 adsorbed more c a r o t o v o r i c i n - 3 7 9 than the other s t r a i n s , they were k i l l e d at a much lower e f f i c i e n c y than more s e n s i t i v e s t r a i n s . Again, Ecc s t r a i n 379 d i d not adsorb d e t e c t a b l e amounts of c a r o t o v o r i c i n - 3 7 9 . Treatment of s e n s i t i v e c e l l s with c a r o t o v o r i c i n - 3 7 9 r e s u l t e d i n an i n c r e a s e i n the r e l a t i v e f l u o r e s c e n c e of ANS accompanied by a 15 nm blue s h i f t i n the emission spectrum ( F i g . 6) The magnitude of t h i s i n c r e a s e was dependent on the amount of c a r o t o v o r i c i n - 3 7 9 used i n the treatment ( F i g . 7). There was no e f f e c t of c a r o t o v o r i c i n - 3 7 9 on c u l t u r e t u r b i d i t y as the O . D . s s o remained at 0.125 before and a f t e r a l l experiments. Treatment of s t r a i n 379 with c a r o t o v o r i c i n - 3 7 9 r e s u l t e d 54 F i g u r e 5. Demonstration of the v a r i o u s l e v e l s of s e n s i t i v i t y to c a r o t o v o r i c i n - 3 7 9 e x h i b i t e d among s t r a i n s of E r w i n i a cazotovoza as i n d i c a t e d by the number of col o n y forming u n i t s (CFU)/ml remaining a f t e r the a d d i t i o n of 128 a r b i t r a r y u n i t s (au)/ml ( f i n a l c o n c e n t r a t i o n ) of c a r o t o v o r i c i n - 3 7 9 . E r r o r bars r e p r e s e n t the range of value s obtained over three experiments. 55 Table 2. C a r o t o v o r i c i n - 3 7 9 (C-379) a d s o r p t i o n c a p a c i t i e s of v a r i o u s s t r a i n s of Erwinia carotovora S t r a i n SR8 504 530 496 379 C-379 A d s o r p t i o n Mean l u V c e l l 203 298 162 140 ND 2 xi l e t h a l u n i t s 2 ; none detected 56 400 450 500 550 600 650 Emission Wavelength nm F i g u r e 6. F l u o r e s c e n c e emission s p e c t r a of Erwinia carotovora subspecies a t r o s e p t i c a s t r a i n 530 (530) and 8-a n i l i n o - l - n a p h t h a l e n e s u l f o n i c a c i d (ANS) with and without the a d d i t i o n of 320 a r b i t r a r y u n i t s / m l ( f i n a l c o n c e n t r a t i o n ) c a r o t o v o r i c i n - 3 7 9 (C-379). 1 0 0 A 5 3 0 X 3 7 9 • C o n t r o l 3 5 0 Carotovoricin-379 au/ml F i g u r e 7. E f f e c t of c a r o t o v o r i c i n - 3 7 9 (C-379) dosage on the r e l a t i v e f l u o r e s c e n c e of 8-ani1ino-l-naphthalene s u l f o n i c a c i d (ANS) a t an emission wavelength of 500 nm alone ( C o n t r o l ) , and i n a s s o c i a t i o n with c a r o t o v o r i c i n -3 7 9 - s e n s i t i v e ( E r w i n i a c a r o t o v o r a subsp. atroseptica s t r a i n 530) and - r e s i s t a n t (Erwinia carotovora subsp. carotovora s t r a i n 379) c e l l s . 58 i n a sma l l i n c r e a s e i n f l u o r e s c e n c e , however a s i m i l a r i n c r e a s e was induced by c a r o t o v o r i c i n - 3 7 9 i n the absence of c e l l s ( F i g . 7). D i s c u s s i o n E. carotovora s t r a i n s 504 (serogroup XVIII) and 496 (serogroup XXII) are more t o l e r a n t to the a c t i o n of c a r o t o v o r i c i n - 3 7 9 than s t r a i n s 530 (serogroup XX) and SR8 (serogroup I ) . Tolerance i s d e f i n e d as a p r o p e r t y of c e l l s which, although endowed with f u n c t i o n a l r e c e p t o r s i t e s , d i s p l a y an i n c r e a s e d or ab s o l u t e r e s i s t a n c e to the a c t i o n of b a c t e r i o c i n (Nomura 1967). C a r o t o v o r i c i n - 3 7 9 appears to adsorb t o s e n s i t i v e c e l l s . The observed d e c l i n e i n c a r o t o v o r i c i n - 3 7 9 a c t i v i t y f o l l o w i n g the a d d i t i o n of s e n s i t i v e c e l l s i s most probably due to a d s o r p t i o n . The f a c t t h i s d e c l i n e ( F i g . 2) resembles a f i r s t order r e a c t i o n i n d i c a t e s t h a t the p r o b a b i l i t y of a d s o r p t i o n decreases as a v a i l a b l e s i t e s on the c e l l s u r f a c e become occupied and suggests t h a t the c a r o t o v o r i c i n - 3 7 9 binds to s p e c i f i c c e l l s u r f a c e r e c e p t o r s . L i k e the r a t e of a d s o r p t i o n , the r a t e of c e l l death decreases e x p o n e n t i a l l y with time ( F i g . 3), sug g e s t i n g t h a t c e l l death i s a t l e a s t p a r t i a l l y 59 dependent on the a b i l i t y to adsorb c a r o t o v o r i c i n . C e l l s of the n o n - s e n s i t i v e s t r a i n Ecc 379 do not e f f e c t d e t e c t a b l e n e u t r a l i z a t i o n of c a r o t o v o r i c i n - 3 7 9 a c t i v i t y , s u g g e s t i n g t h a t t h i s s t r a i n possesses few, i f any, r e c e p t o r s i t e s . C a r o t o v o r i c i n - 3 7 9 , l i k e c a r o t o v o r i c i n - E r (Itoh et al. 1980a), e x h i b i t e d s i n g l e - h i t k i l l i n g k i n e t i c s . I t i s now w e l l e s t a b l i s h e d t h a t t h i s behavior i n d i c a t e s t h a t the i n i t i a t i o n of the l e t h a l phase depends upon a d s o r p t i o n , and t h a t the a d s o r p t i o n of o n l y a s i n g l e p a r t i c l e of c a r o t o v o r i c i n i s r e q u i r e d to k i l l a c e l l (Nomura 1967, Mayr-Harting et a l . 1972). T h i s i s d i s t i n c t from a m u l t i - h i t mechanism i n which a d s o r p t i o n events must a c t c o - o p e r a t i v e l y i n order to e f f e c t the l e t h a l phase (Nomura 1967). The reduced s e n s i t i v i t y d i s p l a y e d by Ecc s t r a i n 504 ( F i g . 5) may have been due to a l a r g e p r o p o r t i o n of r e s i s t a n t i n d i v i d u a l s i n the p o p u l a t i o n . T h i s i s u n l i k e l y , however, as i n d i v i d u a l c o l o n i e s were never observed growing w i t h i n i n h i b i t i o n zones on i n d i c a t o r p l a t e s ( F i g . I c ) . The c l o u d i n e s s i n one of the plaques was not commonly observed, and i s probably due to the t h i c k n e s s of the agar, or poor mixing of the i n d i c a t o r inoculum. Because the apparent potency depends p a r t l y on the a b i l i t y of the s e n s i t i v e s t r a i n to adsorb the 60 b a c t e r i o c i n , i t i s a l s o p o s s i b l e t h a t the reduced s e n s i t i v i t y was due to a r e l a t i v e lack of r e c e p t o r s i t e s . T h i s i s a l s o u n l i k e l y , because enumeration of s u r v i v i n g c e l l s f o l l o w i n g exposure to c a r o t o v o r i c i n - 3 7 9 suggested t h a t a l l the b a c t e r i o c i n had been adsorbed. Furthermore, s t r a i n 504 had the h i g h e s t a d s o r p t i o n c a p a c i t y per c e l l of a l l the t e s t s t r a i n s . The v a r i o u s l e v e l s of s e n s i t i v i t y d i s p l a y e d by the serogroups of Eca are best e x p l a i n e d by v a r y i n g degrees of t o l e r a n c e to c a r o t o v o r i c i n - 3 7 9 . T h i s i m p l i e s t h a t i n the l e s s s e n s i t i v e s t r a i n s such,as 504 and 496, fewer a d s o r p t i o n events are capable of t r i g g e r i n g the events r e q u i r e d to k i l l the c e l l . C a r o t o v o r i c i n - E r (Itoh et al. 1982) and the R-type pyocins (Kageyama 1978) k i l l s e n s i t i v e c e l l s by e f f e c t i n g a c o l l a p s e of the proton-motive f o r c e (pmf). Fluorescence data suggested t h a t c a r o t o v o r i c i n - 3 7 9 e x e r t s i t s l e t h a l e f f e c t s i n a s i m i l a r manner. Treatment of s e n s i t i v e c e l l s with b a c t e r i o c i n r e s u l t e d i n a dose-dependent i n c r e a s e i n the f l u o r e s c e n c e of ANS, while the r e s i s t a n t s t r a i n appeared to be u n a f f e c t e d ( F i g . 7). T h i s i n c r e a s e i n r e l a t i v e f l u o r e s c e n c e was not due to c e l l l y s i s , because c u l t u r e t u r b i d i t y remained unchanged throughout the experiments. The use of ANS f l u o r e s c e n c e as a q u a l i t a t i v e 61 i n d i c a t o r of membrane d e p o l a r i z a t i o n i s now w e l l e s t a b l i s h e d (Conti 1975). In E. coli, channel-forming c o l i c i n s which d i s r u p t membrane p o l a r i z a t i o n induce i n c r e a s e d ANS f l u o r e s c e n c e . Increased f l u o r e s c e n c e i s not observed when r e s i s t a n t c e l l s are t r e a t e d . Furthermore, no changes i n f l u o r e s c e n c e occur when s e n s i t i v e c e l l s are t r e a t e d with enzymatic c o l i c i n s which do not a f f e c t energy c o u p l i n g ( P h i l l i p s and Cramer 1973). A blue s h i f t i n the a d s o r p t i o n maximum, occured upon a d d i t i o n of c a r o t o v o r i c i n - 3 7 9 ( F i g . 6), an o b s e r v a t i o n a l s o noted with s t u d i e s of channel-forming c o l i c i n s ( P h i l l i p s and Cramer 1973) and pyocins ( U r a t a n i and Kageyama 1977), and i s thought to r e f l e c t s t r u c t u r a l changes i n the c e l l envelope r e s u l t i n g from de-e n e r g i z a t i o n (Konisky 1982). Because i t i s hollow, p e n e t r a t i o n of the c e n t r a l core of c a r o t o v o r i c i n - 3 7 9 through the outer and cy t o p l a s m i c membranes would d i s r u p t the i n t e g r i t y of the c e l l envelope and render i t permeable to protons and io n s . As a consequence, the c e l l would no longer be able to m a i n t ain the proton-motive f o r c e r e q u i r e d to support metabolic and r e p r o d u c t i v e a c t i v i t y . Thin s e c t i o n e l e c t r o n micrographs of i n f e c t i n g bacteriophage show that the c e n t r a l core of the phage p a r t i c l e p e n e trates the c e l l w a l l of the bacterium i n order to f a c i l i t a t e genome 62 i n j e c t i o n (Birge 1981). Furthermore, headless phage p a r t i c l e s d i s p l a y b a c t e r i o c i n - l i k e a c t i v i t y a g a i n s t s e n s i t i v e c e l l s and induce an in c r e a s e i n ANS f l u o r e s c e n c e (Hantke and Braun 1981) i n a manner s i m i l a r to c a r o t o v o r i c i n - 3 7 9 . T h i s suggests that c a r o t o v o r i c i n -379, c a r o t o v o r i c i n - E r , p y o c i n s , and headless bacteriphages may be s i m i l a r i n t h e i r a c t i o n s a g a i n s t s e n s i t i v e c e l l s . C e l l death may r e s u l t from a c o l l a p s e of the pmf induced by the p e n e t r a t i o n of the c e n t r a l core through the cy t o p l a s m i c membrane. B a c t e r i o c i n t o l e r a n c e does not a f f e c t r e c e p t o r s i t e s or a d s o r p t i o n , but i s a r e s u l t of mutations which block the l e t h a l phase. Although c o l i c i n - t o l e r a n t mutants are w e l l c h a r a c t e r i z e d , the mechanism by which the l e t h a l phase i s i n i t i a t e d remains to be e l u c i d a t e d . The degree of t o l e r a n c e to c a r o t o v o r i c i n - 3 7 9 observed of some s t r a i n s of E. ca r o t o v o r a may a l s o r e s u l t from the i n a b i l i t y of a l a r g e p r o p o r t i o n of a d s o r p t i o n events to t r i g g e r the sequence of events r e q u i r e d to k i l l the c e l l . These s t r a i n s , f o r example, may be more capable of a v o i d i n g core p e n e t r a t i o n than l e s s t o l e r a n t s t r a i n s . In E. carotovora, the degree of s e n s i t i v i t y to c a r o t o v o r i c i n - 3 7 9 i s c o r r e l a t e d with serogroup d e s i g n a t i o n ( J a i s 1982). Because the composition and s t r u c t u r e of the O-antigens of the l i p o p o l y s a c c h a r i d e are 63 s e r o l o g i c a l l y dominant (De Boer et a l . 1985), these outer membrane components may p l a y a r o l e i n determining t o l e r a n c e to c a r o t o v o r i c i n - 3 7 9 . 6 4 CHAPTER 3. LOCALIZATION OF THE CAROTOVORICIN-379 RECEPTOR SITE IN ERWINIA CAROTOVORA Introduction Bacteriocins d i f f e r from conventional a n t i b i o t i c s in that their l e t h a l action is directed againt strains of only one or a few species. In order to reach t h e i r target of action, bacteriocins must f i r s t overcome the permeability barrier of the outer membrane. As they are generally too large to pass f r e e l y through d i f f u s i o n pores, bacteriocin action is mediated by s p e c i f i c receptor s i t e s on the c e l l surface. The presence of functional receptor s i t e s i s a major factor in determining the s p e c i f i c i t y of any bacteriocin. Receptors in v i t r o generally r e t a i n the a b i l i t y to t i g h t l y bind bacteriocin. This binding results in the n e u t r a l i s a t i o n of bacteriocin k i l l i n g a c t i v i t y . The conventional approach in l o c a l i s i n g receptor a c t i v i t y has been to fractionate the membrane and evaluate the a b i l i t y of the various fractions to neutralize bacteriocin a c t i v i t y . Because the outer membrane of Gram negative bacteria is s t a b i l i z e d by divalent cations, the inner 65 membrane can be s e l e c t i v e l y s o l u b i l i z e d with a n o n - i o n i c detergent such as octylphenoxy polyethoxy ethanol ( T r i t o n X-100) i n the presence of magnesium. The outer membrane can then be s o l u b i l i z e d by T r i t o n X-100 i n the presence of EDTA (Schnaitman 1971). T h i s technique was f i r s t used to l o c a l i z e and p u r i f y the c o l i c i n E r e c e p t o r (Sabet and Schnaitman 1971), a 60,000 d outer membrane p r o t e i n . I t has been a p p l i e d s u c c e s s f u l l y i n the i d e n t i f i c a t i o n of many b a c t e r i o c i n r e c e p t o r s , most of which are outer membrane p r o t e i n s (Hantke and Braun 1981). Although not as e x t e n s i v e l y s t u d i e d , r e c e p t o r s i t e s f o r p a r t i c u l a t e b a c t e r i o c i n s are more commonly found on the l i p o p o l y s a c c h a r i d e (LPS). B a c t e r i o c i n s from Proteus v u l g a r i s (Smit et al.1969), Rhizobium l u p i n i ( P f i s t e r and Loders t a e d t 1977) and Pseudomonas aeruginosa (Ikeda and Egami 1969) are n e u t r a l i z e d by LPS from s e n s i t i v e s t r a i n s . S t r a i n s of E r w i n i a carotovora subspecies a t r o s e p t i c a (Eca) and Erwinia carotovora subspecies carotovora (Ecc). are s e n s t i v e to a l a r g e molecular weight b a c t e r i o c i n , c a l l e d c a r o t o v o r i c i n - 3 7 9 , produced by Ecc s t r a i n 379 ( J a i s 1982). In Chapter 2, c a r o t o v o r i c i n - s e n s i t i v e s t r a i n s were shown to r a p i d l y adsorb c a r o t o v o r i c i n - 3 7 9 . Furthermore, k i l l i n g a c t i o n followed s i n g l e - h i t k i n e t i c s . T h i s evidence suggested the presence of a s p e c i f i c 66 c a r o t o v o r i c i n - 3 7 9 r e c e p t o r on the s u r f a c e of s e n s i t i v e c e l l s . In t h i s chapter, T r i t o n X-100 membrane s o l u b i l i z a t i o n and LPS e x t r a c t i o n s were c a r r i e d out i n order to l o c a l i z e and i d e n t i f y the c a r o t o v o r i c i n - 3 7 9 r e c e p t o r i n s e n s i t i v e c e l l s . M a t e r i a l s and Methods B a c t e r i a l S t r a i n s and Growth C o n d i t i o n s : B a c t e r i a l s t r a i n s used i n t h i s chapter are shown i n Table 1. The c a r o t o v o r i c i n producing s t r a i n Ecc s t r a i n 379 was grown i n L u r i a broth overnight at 20 C before i n o c u l a t i o n i n t o f r e s h b r o t h . L u r i a broth c o n s i s t e d of 1.0 % (w/v) Bacto-Tryptone ( D i f c o ) , 0.5 % (w/v) Yeast E x t r a c t ( D i f c o ) and 0.65 % NaCl (w/v) (Sigma). S t a r t e r c u l t u r e s , grown i n N u t r i e n t Broth ( D i f c o ) o v e r n i g h t at 20 C, were used to i n o c u l a t e f r e s h broth f o r i n d i c a t o r p l a t e s , membrane f r a c t i o n a t i o n or LPS p r e p a r a t i o n . I n d i c a t o r p l a t e s c o n s i s t e d of 0.5 % (w/v) Bacto-Agar ( D i f c o ) , 1.0 % (w/v) Bacto-Peptone ( D i f c o ) and 0.65 % (w/v) NaCl (Sigma), seeded with about 10"' c e l l s per ml. A f t e r the samples were s p o t t e d , p l a t e s were incubated at 67 T a b l e 1: S t r a i n s o f Erwinia carotovora s u b s p e c i e s carotovora (Ecc) and E r w i n i a carotovora s u b s p e c i e s a t r o s e p t i c a (Eca) i s o l a t e d i n B r i t i s h C o l u m b i a (BC) and W i s c o n s i n (Wn) and Escherichia coli (E. coli) u s e d i n t h i s c h a p t e r . S p e c i e s S t r a i n S e r o g r o u p S o u r c e Ecc 379 XI Copeman, BC Ecc 504 X V I I I Copeman, BC Eca SR8 I Kelman, Wn Eca 530 XX Copeman, BC Eca 496 XXII Copeman, BC E. coli HB101 S u t t o n 68 23 C o v e r n i g h t . P r e p a r a t i o n o f C a r o t o v o r i c i n - 3 7 9 : C a r o t o v o r i c i n - 3 7 9 was p r e p a r e d as d e s c r i b e d i n C h a p t e r 1. Q u a n t i f i c a t i o n o f C a r o t o v o r i c i n - 3 7 9 A c t i v i t y : C a r o t o v o r i c i n - 3 7 9 a c t i v i t y was measured u s i n g t h e m i c r o t i t r e p l a t e t e c h n i q u e , d e s c r i b e d i n C h a p t e r 1. C e l l W a l l F r a c t i o n a t i o n : T e s t s t r a i n s were i n o c u l a t e d i n t o 1 t o 1.5 1 b a t c h e s o f f r e s h n u t r i e n t b r o t h a t a l e v e l o f 1 % ( v / v ) , and i n c u b a t e d 24 h on a r o t a r y s h a k e r (150 rpm) a t 20 C. C e l l s were h a r v e s t e d by c e n t r i f u g a t i o n a t 4 C, 10,000 x g f o r 10 min and washed t w i c e w i t h c o l d , s t e r i l e 10 mM p h o s p h a t e b u f f e r pH 7.2 f o l l o w e d by one wash i n c o l d , s t e r i l e 10 mM N - 2 - h y d r o x y e t h y l p i p e r i z i n e - N ' - 2 - e t h a n e s u l f o n i c a c i d (HEPES) b u f f e r pH 7.2. C e l l s were r e s u s p e n d e d i n t h e same HEPES b u f f e r t o a f i n a l volume o f 25 ml. D e o x y r i b o n u c l e a s e (DNase) and r i b o n u c l e a s e (RNase) were added t o f i n a l c o n c e n t r a t i o n s o f 100 ug/ml and 50 ug/ml, r e s p e c t i v e l y . The c e l l s u s p e n s i o n s were p a s s e d t w i c e t h r o u g h a 69 F r e n c h P r e s s (Aminco) a t a p r e s s u r e of 20,000 pounds p e r s q u a r e i n c h . Magnesium i n t h e f o r m o f M g C l 2 was added t o a f i n a l c o n c e n t r a t i o n o f 2 mM. These s u s p e n s i o n s were t h e n c e n t r i f u g e d a t low s p e e d (5000 x g) f o r 5 m i n u t e s t o remove u n b r o k e n c e l l s . The p e l l e t was d i s c a r d e d and t h e s u p e r n a t a n t was c e n t r i f u g e d a t 100,000 x g. The r e s u l t i n g p e l l e t was g e n t l y r e s u s p e n d e d i n 4 ml c o l d , s t e r i l e 10 mM HEPES b u f f e r pH 7.2 p l u s 2mM M g C l z . T h i s s u s p e n s i o n was r e f e r r e d t o as t h e c r u d e c e l l w a l l f r a c t i o n . To t h e c r u d e c e l l w a l l f r a c t i o n , 1 ml o f 10 % T r i t o n - X 100 i n lOmM HEPES b u f f e r p l u s 2mM M g C l 2 was added t o a f i n a l c o n c e n t r a t i o n o f 2 % T r i t o n X-100 and i n c u b a t e d a t 20 C f o r 15 min w i t h g e n t l e a g i t a t i o n . The i n c u b a t i o n was f o l l o w e d by c e n t r i f u g a t i o n a t 100,000 x g f o r two h o u r s . The s u p e r n a t a n t was s a v e d and t h e p e l l e t was r e - e x t r a c t e d two more t i m e s . The p o o l e d s u p e r n a t a n t s were r e f e r r e d t o as t h e T r i t o n - s o l u b l e f r a c t i o n w h i l e t h e p e l l e t f r a c t i o n was c a l l e d t h e T r i t o n - i n s o l u b l e f r a c t i o n . The T r i t o n - i n s o l u b l e f r a c t i o n was t h e n g e n t l y r e s u s p e n d e d i n 4 ml o f 10 mM HEPES b u f f e r pH 7.2. One ml of lOmM HEPES b u f f e r pH 7.2 c o n t a i n i n g 10 % ( v / v ) T r i t o n X-100, 25 mM e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (EDTA) ( S i g m a ) , and 250 ug/ml l y s o z y m e (Sigma) were added so t h a t t h e f i n a l c o n c e n t r a t i o n s o f EDTA, l y s o z y m e and T r i t o n - X 100 were t h e n 5 mM, 50 ug/ml, and 2 %, 70 r e s p e c t i v e l y . The T r i t o n - i n s o l u b l e f r a c t i o n was t h e n i n c u b a t e d f o r 15 min a t 20 C w i t h g e n t l e a g i t a t i o n and c e n t r i f u g e d a t 97,000 x g f o r 2 h. The s u p e r n a t a n t was s a v e d and t h e p e l l e t s u b j e c t e d t o t h i s e x t r a c t i o n p r o c e d u r e two a d d i t i o n a l t i m e s . P r o t e i n f r o m t h e T r i t o n - s o l u b l e and - i n s o l u b l e f r a c t i o n s was e x t r a c t e d by e t h a n o l p r e c i p i t a t i o n . The s u p e r n a t a n t f r a c t i o n s were c o o l e d t o 0 C i n an i c e - w a t e r b a t h . Two volumes o f i c e c o l d e t h a n o l were added d r o p w i s e w h i l e g e n t l y a g i t a t i n g t h e s a m p l e . The p r o t e i n was s t o r e d i n p r e c i p i t a t e d f o r m a t -20 C. L i p o p o l y s a c c h a r i d e E x t r a c t i o n : T e s t s t r a i n s were i n o c u l a t e d i n t o f r e s h n u t r i e n t b r o t h a t a l e v e l o f 1% and i n c u b a t e d on a r o t a r y s h a k e r 24 h a t 20 C. C e l l s were h a r v e s t e d and washed t h r e e t i m e s i n c o l d d i s t i l l e d w a t e r . C e l l s were r e s u s p e n d e d i n 5 ml d i s t i l l e d w a t e r . L i p o p o l y s a c c h a r i d e (LPS) was e x t r a c t e d i n p u r e f o r m by one or b o t h o f t h e methods w h i c h f o l l o w . A f t e r e x t r a c t i o n , a l l LPS s a m p l e s were f i l t e r s t e r i l i z e d by p a s s a g e t h r o u g h 0.2 m i c r o n c e l l u l o s e a c e t a t e f i l t e r s ( N a l g e n e ) and s t o r e d a t 4 C. 1) Hot p h e n o l e x t r a c t i o n ( W e s t p h a l and J a n n 1965) A 90 % aqueous s o l u t i o n o f d i s t i l l e d p h e n o l was 71 p r e p a r e d and warmed t o 60 C. The 5 ml c e l l s u s p e n s i o n was a l s o warmed t o 60 C. An e q u a l volume o f t h e p h e n o l s o l u t i o n was added t o t h e c e l l s u s p e n s i o n and t h e m i x t u r e was h e a t e d and h e l d a t 70 +/- 2 C f o r 15 min w i t h o c c a s i o n a l m i x i n g . The s a m p l e s were t h e n c o o l e d r a p i d l y i n an i c e - w a t e r b a t h t o 10 C and c e n t r i f u g e d a t 30,000 x g f o r 60 min a t 10 C. The c e n t r i f u g a t i o n r e s u l t e d i n a s e p a r a t i o n o f t h e s a m p l e s i n t o a p h e n o l phase and a l i g h t e r aqueous p h a s e . The aqueous phase was drawn o f f and s a v e d w h i l e t h e p h e n o l phase was r e - e x t r a c t e d w i t h an a d d i t o n a l volume of warm w a t e r . The aqueous p h a s e s were p o o l e d and d i a l y s e d a g a i n s t 400 volumes o f d i s t i l l e d w a t e r p e r day f o r 3 d a y s . To t h e d i a l y s a t e , 1 ml o f 100 mM T r i s - H C l pH 7.4 c o n t a i n i n g 100 ug DNase and RNase was a d d e d . The s a m p l e s were i n c u b a t e d a t 37 C f o r s e v e r a l h o u r s and t h e n c e n t r i f u g e d a t 100,000 x g f o r 4 h. The s u p e r n a t a n t was d i s c a r d e d and l i q u i d d r a i n e d f r o m t h e p e l l e t . 2) P h e n o l / c h l o r o f o r m / e t h e r e x t r a c t i o n ( G a l a n o s e t a l . 1969) An e x t r a c t i o n m i x t u r e of 90 % aqueous p h e n o l , c h l o r o f o r m and p e t r o l e u m e t h e r i n 2:5:8 p r o p o r t i o n s was f r e s h l y p r e p a r e d . The 5 ml c e l l s u s p e n s i o n was washed s e q u e n t i a l l y i n e t h a n o l , a c e t o n e , and d i e t h y l e t h e r and 72 t h e s u p e r n a t a n t s d i s c a r d e d . A f t e r t h e f i n a l wash, t h e p e l l e t was r e s u s p e n d e d i n e x t r a c t i o n m i x t u r e and c e n t r i f u g e d a t 10,000 x g f o r 10 m i n u t e s . The p e l l e t was r e s u s p e n d e d i n t h e e x t r a c t i o n m i x t u r e and washed an a d d i t i o n a l two t i m e s . The s u p e r n a t a n t s were p o o l e d and p o u r e d t h r o u g h a s h e e t o f Whatman # 4 f i l t e r p a p e r . The e t h e r and c h l o r o f o r m were removed f r o m t h e f i l t e r e d s u p e r n a t a n t i n a r o t a r y e v a p o r a t o r . Water was t h e n added d r o p w i s e t o t h e s u p e r n a t a n t i n o r d e r t o e f f e c t p r e c i p i t a t i o n o f t h e LPS, w h i c h was c o l l e c t e d by c e n t r i f u g a t i o n a t 10,000 x g f o r 10 min. The p r e c i p i t a t e d LPS was t h e n washed t w i c e w i t h d i e t h y l e t h e r t o remove t h e p h e n o l . LPS was d i s s o l v e d i n 10 mM HEPES b u f f e r pH 7.2 warmed t o 40 C. D i s s o l u t i o n o f t h e LPS c o u l d be i m p r o v e d by a p p l y i n g a vacuum t o t h e s u s p e n s i o n . D e t e r m i n a t i o n o f T o t a l P r o t e i n : T o t a l p r o t e i n was d e t e r m i n e d by t h e method o f S p e c t o r ( 1 9 7 8 ) . The p r o t e i n - d e t e c t i o n r e a g e n t was p r e p a r e d by d i s s o l v i n g 100 mg C o o m a s s i e G-250 (Sigma) i n 50 ml o f 95 % e t h a n o l . To t h i s , 100 ml 85 % p h o s p h o r i c a c i d were a d d e d . The m i x t u r e was d i l u t e d t o 1 l i t e r w i t h d i s t i l l e d w a t e r . Samples were d i l u t e d i n 0.1 N NaOH t o f i n a l c o n c e n t r a t i o n s o f 2-10 ug/ml i n 1 ml. An e q u a l volume o f 73 p r o t e i n r e a g e n t was added and t h e a b s o r b a n c e r e a d a t 595 nm a f t e r 2-30 m i n u t e s i n c u b a t i o n . B o v i n e serum a l b u m i n a t 1-10 ug/ml was u s e d t o p r e p a r e a s t a n d a r d c u r v e . D e t e r m i n a t i o n o f K e t o d e o x y o c t o n a t e (KDO) C o n t e n t : KDO was d e t e r m i n e d by t h e method d e s c r i b e d by Hanson and P h i l l i p s ( 1 9 8 1 ) . Samples were d i l u t e d t o f a l l w i t h i n 2-8 ug/ml KDO i n a f i n a l volume of 50 u l and 50 u l o f 0.50 N HaSO-n were added t o e a c h s a m p l e . The s a m p l e s were t h e n p l a c e d i n a b o i l i n g w a ter b a t h f o r 15 min. A f t e r a l l o w i n g t h e s a m p l e s t o c o o l t o room t e m p e r a t u r e , 50 u l o f 2.28 % p e r i o d i c a c i d were added and t h e s a m p l e s were i n c u b a t e d 10 min a t room t e m p e r a t u r e . N e x t , 200 u l o f a r s e n i t e r e a g e n t (4 % s o d i u m a r s e n i t e i n 50 ml 0.5 N H C l ) were added f o l l o w e d by t h e a d d i t i o n o f 800 u l o f f r e s h l y p r e p a r e d 0.6 % t h i o b a r b i t u r i c a c i d (Kodak E a s t m a n ) . The s a m p l e s were t h e n p l a c e d i n a b o i l i n g w a t e r b a t h f o r 10 m i n u t e s and c o o l e d i n a c o l d water b a t h . F o l l o w i n g t h e a d d i t o n o f 1.5 ml o f b u t a n o l - H C l ( 5 % ( v / v ) c o n e . H C l i n b u t a n o l ) e a c h sample was mixed and c e n t r i f u g e d a t 5000 x g f o r 5 min. The t o p phase ( c o l o u r e d ) was drawn o f f and a b s o r b a n c e measured a t 549 nm i n a Beckman d u a l beam s p e c t r o p h o t o m e t e r . P u r i f i e d KDO (Sigma) a t 1-10 ug/ml was u s e d t o p r e p a r e a s t a n d a r d c u r v e . 74 D e t e r m i n a t i o n o f C a r o t o v o r i c i n - 3 7 9 N e u t r a l i z a t i o n : A c t i v i t y : N e u t r a l i z a t i o n o f c a r o t o v o r i c i n - 3 7 9 was q u a n t i t a t e d by d i l u t i n g w a l l m a t e r i a l i n 50 u l 10 mM HEPES b u f f e r pH 7.2 a c r o s s t h e w e l l s o f a m i c r o t i t r e p l a t e . 50 u l o f c a r o t o v o r i c i n - 3 7 9 o f known p o t e n c y was added t o e a c h w e l l and t h e m i x t u r e was i n c u b a t e d f o r 15 m i n u t e s a t room t e m p e r a t u r e . 100 u l i n d i c a t o r c e l l s u s p e n s i o n c o n s i s t i n g o f 10"7 c e l l s / m l Eca s t r a i n 530 s u s p e n d e d i n 1.0 % B a c t o -P e p t o n e ( D i f c o ) and 0.65 % N a C l (Sigma) was added t o e a c h w e l l . The p l a t e s were i n c u b a t e d a t 23 C f o r 24 h. The s p e c i f i c n e u t r a l i z a t i o n a c t i v i t y was t a k e n a s t h e t i t r e o f c a r o t o v o r i c i n - 3 7 9 a c t i v i t y n e u t r a l i z e d p e r ug o f w a l l m a t e r i a l . Membrane p r o t e i n e x t r a c t e d f r o m t h e f r a c t i o n s was r e - d i s s o l v e d i n 2% T r i t o n X-100 i n 10 mM HEPES b u f f e r pH 7.2 a t a f i n a l c o n c e n t r a t i o n o f 5-10 mg/ml p r o t e i n . B e c a u s e c a r o t o v o r i c i n - 3 7 9 was i n a c t i v a t e d by l e v e l s o f T r i t o n X-100 g r e a t e r t h a n 0.5%, s a m p l e s had t o be d i l u t e d i n HEPES b u f f e r t o 1-2 mg/ml p r o t e i n b e f o r e t e s t i n g n e u t r a l i z a t i o n a c t i v i t y . C a r o t o v o r i c i n - 3 9 7 a c t i v i t y was d e t e r m i n e d u s i n g e q u a l volumes o f 0.4% T r i t o n X-100 i n HEPES b u f f e r pH 7.2 and lOmM p o t a s s i u m p h o s p h a t e b u f f e r pH 7.2 as a d i l u e n t . 75 P e r i o d a t e I n a c t i v a t i o n o f R e c e p t o r A c t i v i t y : P e r i o d i c a c i d ( F i s h e r ) was d i s s o l v e d i n 50 mM p o t a s s i u m p h o s p h a t e b u f f e r pH 7.2 t o a f i n a l c o n c e n t r a t i o n o f 0.2 M. T h i s was mixed i n e q u a l 100 u l v o l u m e s w i t h LPS ( f r o m Eca 530) s u s p e n s i o n e q u i v a l e n t t o 80 ug o f KDO and i n c u b a t e d 10 m i n u t e s . T h i s m i x t u r e was d i l u t e d 1/10 i n 1 ml o f c a r o t o v o r i c i n - 3 7 9 s u s p e n s i o n c o n s i s t i n g o f 320 a u . T h i s m i x t u r e was t h e n s p o t t e d o n t o an i n d i c a t o r p l a t e . As c o n t r o l s , c a r o t o v o r i c i n - 3 7 9 was mixed w i t h LPS p r e - t r e a t e d w i t h s t e r i l e 50 mM p h o s p h a t e b u f f e r pH 7.2 and p e r i o d a t e (0.01 M f i n a l c o n c e n t r a t i o n ) . These m i x t u r e s were a l s o s p o t t e d o n t o i n d i c a t o r p l a t e s . E n z y m e - l i n k e d Immunosorbent A s s a y ( E L I S A ) : The E L I S A was p e r f o r m e d u s i n g a s h e e t o f n i t r o c e l l u l o s e ( B i o - R a d ) as a s o l i d p h a s e . LPS (5 ug) was s p o t t e d o n t o a pre-dampened s h e e t o f n i t r o c e l l u l o s e . B i n d i n g o f t h e LPS t o t h e n i t r o c e l l u l o s e s h e e t was f a c i l i t a t e d by d r y i n g t h e s h e e t under a s t r e a m o f f i l t e r e d a i r i n a l a m i n a r f l o w hood. A l l s u b s e q u e n t s t e p s were p e r f o r m e d i n a p e t r i p l a t e w i t h g e n t l e a g i t a t i o n a t room t e m p e r a t u r e (23 C ) . The n i t r o c e l l u l o s e was washed i n 3% (w/v) g e l a t i n 76 d i s s o l v e d i n 10 mM p o t a s s i u m p h o s p h a t e b u f f e r pH 7.2 f o r 2 h i n o r d e r t o b l o c k a l l unbound s i t e s . T h i s s t e p was f o l l o w e d by a s i n g l e 10 min wash i n t h e p h o s p h a t e b u f f e r . The n i t r o c e l l u l o s e was t h e n i n c u b a t e d i n 20 ml o f c a r o t o v o r i c i n - 3 7 9 (640 au/ml) f o r 30 min. A f t e r d i s c a r d i n g t h e c a r o t o v o r i c i n s u s p e n s i o n , t h e n i t r o c e l l u l o s e was s u b j e c t e d t o t h r e e 10 min washes i n 10 mM p o t a s s i u m p h o s p h a t e b u f f e r pH 7.2. T h i s s t e p was f o l l o w e d by t h e a d d i t i o n o f a 1/100 d i l u t i o n o f r a b b i t p o l y c l o n a l a n t i s e r u m s p e c i f i c a l l y d i r e c t e d a g a i n s t c a r o t o v o r i c i n - 3 7 9 ( p r o v i d e d by L . J . Ward, D e p t . P l a n t S c i e n c e , UBC). The n i t r o c e l l u l o s e was i n c u b a t e d i n t h e p r i m a r y a n t i s e r u m f o r 1 h f o l l o w e d by t h r e e more 10 min washes i n 10 mM p o t a s s i u m p h o s p h a t e b u f f e r pH 7.2. Goat a n t i - r a b b i t h o r s e r a d i s h p e r o x i d a s e c o n j u g a t e (Sigma) d i l u t e d 1/1000 i n 10 mM p o t a s s i u m p h o s p h a t e b u f f e r pH 7.2 was added and i n c u b a t e d f o r 1 h, f o l l o w e d by t h r e e a d d i t i o n a l 10 min washes i n 10 mM p o t a s s i u m p h o s p h a t e b u f f e r pH 7.2. The n i t r o c e l l u l o s e was t h e n washed once (10 min) i n s u b s t r a t e b u f f e r , c o n s i s t i n g o f 25.7 % ( v / v ) 0.2 M s o d i u m p h o s p h a t e and 24.3 % ( v / v ) 0.IM c i t r i c a c i d , f i n a l pH 5.0. o - P h e n l y e n e - d i a m i n e (Sigma) was d i s s o l v e d i n t h e s u b s t r a t e b u f f e r a t 0.04 % (w/v) i n t h e d a r k and h y d r o g e n p e r o x i d e (BDH) was added t o a f i n a l c o n c e n t r a t i o n of 0.012 %. T h i s m i x t u r e was i m m e d i a t e l y 77 a dded t o t h e n i t r o c e l l u l o s e and i n c u b a t e d i n d i m l i g h t u n t i l t h e a p p e a r a n c e o f r e d s p o t s and a d a r k e n i n g o f t h e b a c k g r o u n d . The n i t r o c e l l u l o s e s h e e t was r i n s e d i n d i s t i l l e d w a t e r and d r i e d and s t o r e d between p a p e r t o w e l s . Sodium D o d e c y l P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s (SDS-PAGE): SDS-PAGE was p e r f o r m e d u s i n g t h e d i s c o n t i n u o u s s y s t e m o f Laemmli ( 1 9 7 0 ) . A l l a c r y l a m i d e g e l s were p r e p a r e d f r o m a s t o c k s o l u t i o n o f 30:0.8 % a c r y l a m i d e : b i s - a c r y l a m i d e . S e p a r a t i n g g e l s were b u f f e r e d w i t h 0.375 M T r i s - H C l pH 8.8. S t a c k i n g g e l s c o n s i s t e d o f 4% a c r y l a m i d e s t o c k and 0.1% SDS b u f f e r e d by 0.125 M T r i s - H C l pH 6.8. Sample b u f f e r c o n s i s t e d o f 1% g l y c e r o l , 5% b e t a - m e r c a p t o e t h a n o l , 0.01% b romophenol b l u e , and 2% SDS b u f f e r e d by 0.125 M T r i s - H C l pH 6.8. 1) SDS-PAGE o f P r o t e i n : E t h a n o l p r e c i p i t a t e d membrane p r o t e i n was d i s s o l v e d i n sample b u f f e r and p l a c e d i n a b o i l i n g w a ter b a t h f o r 5 min. A l i q u o t s were l o a d e d i n t o t h e sample w e l l s and e l e c t r o p h o r e s e d t h r o u g h an 11% a c r y l a m i d e g e l i n a m i n i -g e l s y s t e m ( B i o - R a d model 360) a t 100 v o l t s and 50 mA. P r o t e i n bands were s t a i n e d w i t h 1 % C o o m a s s i e b l u e R-250 78 and d e s t a i n e d i n 50 % m e t h a n o l and 5 % a c e t i c a c i d . 2) SDS-PAGE o f LPS: LPS s a m p l e s were mixed w i t h sample b u f f e r a t an a p p r o p r i a t e d i l u t i o n and p l a c e d i n a b o i l i n g w a ter b a t h f o r 5 m i n u t e s . A l i q u o t s were l o a d e d i n t o sample w e l l s . The r u n n i n g g e l us e d was 14% a c r y l a m i d e s t o c k i n t h e s t a n d a r d b u f f e r e d SDS p l u s 4 M u r e a . The s a m p l e s were r u n i n a B i o - R a d model 360 o r P h a r m a c i a model GE-214 LS e l e c t r o p h o r e s i s u n i t a t 100 v o l t s and 50 mA. F o l l o w i n g e l e c t r o p h o r e s i s , t h e LPS bands were v i s u a l i z e d by t h e s i l v e r s t a i n i n g method o f T s a i and F r a s c h ( 1 9 8 2 ) . The g e l s were f i r s t washed i n d e i o n i z e d w a t e r and f i x e d i n 50% e t h a n o l and 5% a c e t i c a c i d f o r f r o m 1 h t o o v e r n i g h t . The g e l was t h e n p l a c e d f o r i n 0.7% p e r i o d i c a c i d d i s s o l v e d i n 50% e t h a n o l f o r 5 min. T h i s s t e p was f o l l o w e d by t h r e e 15 min washes i n d e i o n i z e d w a t e r . The g e l was t h e n p l a c e d i n s i l v e r s t a i n f o r 15 min. The s i l v e r s t a i n c o n s i s t e d o f 1% (w/v) s i l v e r n i t r a t e p l u s 2% ( v / v ) o f ammonium h y d r o x i d e and 20% ( v / v ) IN NaOH. A f t e r a r i n s e w i t h d e i o n i z e d w a t e r , t h e d e v e l o p i n g s o l u t i o n w h i c h c o n s i s t e d o f .005 % ( v / v ) c i t r i c a c i d and 0.018 % ( v / v ) f o r m a l d e h y d e was p o u r e d o v e r t h e g e l . The d e v e l o p e r was d i s c a r d e d when t h e bands had s u f f i c i e n t l y d a r k e n e d and t h e g e l was q u i c k l y washed 79 s e q u e n t i a l l y w i t h 1% ( v / v ) g l a c i a l a c e t i c a c i d and d e i o n i z e d w a t e r . R e s u l t s When c r u d e w a l l s o f Eca s t r a i n s 496 and 530 were f r a c t i o n a t e d by t h e T r i t o n X-100 s e l e c t i v e s o l u b i l i z a t i o n p r o c e d u r e , T r i t o n - i n s o l u b l e p r o t e i n s were n o t r e s o l v e d i n SDS-PAGE. M o d i f i c a t i o n o f t h e p r o c e d u r e by t h e a d d i t i o n o f l y s o z y m e t o t h i s f r a c t i o n , however, f a c i l i t a t e d t h e r e s o l u t i o n o f t h e p r o t e i n s ( F i g . 1 ) . B e c a u s e most o f t h e p r o t e i n s i n t h e T r i t o n - i n s o l u b l e f r a c t i o n were p r o b a b l y o u t e r membrane p r o t e i n , t h i s s u g g e s t e d t h a t t h e o u t e r membrane i n E c a may be t i g h t l y a s s o c i a t e d w i t h t h e p e p t i d o g l y c a n . In b o t h s t r a i n s 530 and 496, t h e p r o t e i n c o m p o s i t i o n between t h e s o l u b l e and i n s o l u b l e f r a c t i o n s a p p e a r e d t o be d i f f e r e n t ( F i g . 2 ) . Bands a p p e a r i n g i n one f r a c t i o n were n o t p r e s e n t i n t h e o t h e r f r a c t i o n , or were p r e s e n t i n much s m a l l e r amounts, s u g g e s t i n g t h a t i n d i v i d u a l p r o t e i n s were s e p a r a t e d i n t o one f r a c t i o n or t h e o t h e r d u r i n g t h e s o l u b i l i z i n g p r o c e d u r e . P r o t e i n and k e t o d e o x y o c t o n a t e (KDO) y i e l d s d e t e r m i n e d t h a t w a l l p r o t e i n made up an a v e r a g e of 10.4% 80 F i g u r e 1. Sodium d o d e c y l s u l f a t e p o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f T r i t o n - i n s o l u b l e p r o t e i n e x t r a c t e d f r o m two s t r a i n s of E r w i n i a c a r o t o v o r a s u b s p e c i e s atroseptica w i t h and w i t h o u t l y s o z y m e p r e t r e a t m e n t . L e f t t o r i g h t , l a n e s : 1) S t r a i n 496 (1 ug) w i t h l y s o z y m e p r e t r e a t m e n t . 2) S t r a i n 530 (1 ug) w i t h l y s o z y m e p r e t r e a t m e n t . 3) S t r a i n 496 (2.5 ug) w i t h l y s o z y m e p r e t r e a t m e n t . 4) S t r a i n 530 (2.5 ug) w i t h l y s o z y m e p r e t r e a t m e n t . 5) S t r a i n 496 (5 ug) w i t h l y s o z y m e p r e t r e a t m e n t . 6) S t r a i n 530 (5 ug) w i t h l y s o z y m e p r e t r e a t m e n t . 7) S t r a i n 496 (5 ug) w i t h o u t l y s o z y m e p r e t r e a t m e n t . 8) S t r a i n 530 (5 ug) w i t h o u t l y s o z y m e p r e t r e a t m e n t . F i g u r e 2. Sodium d o d e c y l s u l f a t e p o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m of T r i t o n - s o l u b l e and - i n s o l u b l e f r a c t i o n s o f Erwinia carotovora s u b s p e c i e s a t r o s e p t i c a s t r a i n s 530 and 496. L e f t t o r i g h t , l a n e s : 1 and 5) T r i t o n - i n s o l u b l e p r o t e i n f r o m s t r a i n 496. 2 and 6) T r i t o n - i n s o l u b l e p r o t e i n f r o m s t r a i n 530. 3 and 7) T r i t o n - s o l u b l e p r o t e i n f r o m s t r a i n 496. 4 and 8) T r i t o n - s o l u b l e p r o t e i n f r o m s t r a i n 530. 9) Low m o l e c u l a r w e i g h t s t a n d a r d s ( B i o - R a d ) r a n g i n g f r o m 14,400 t o 92,500 d a l t o n s . 82 and 11.8% t o t a l d r y w e i g h t and KDO c o m p r i s e d 0.25% and 0.27% d r y w e i g h t i n s t r a i n s 496 and 530 r e s p e c t i v e l y . R e l a t i v e d i s t r i b u t i o n s o f p r o t e i n , KDO and n e u t r a l i z a t i o n a c t i v i t y f o r t h e w a l l f r a c t i o n s ( F i g s . 3a and 3b f o r s t r a i n s 530 and 496 r e s p e c t i v e l y ) r e p r e s e n t t h e mean v a l u e s d e r i v e d f r o m t h e f r a c t i o n a t i o n o f t h r e e d i f f e r e n t c u l t u r e b a t c h e s . The T r i t o n - i n s o l u b l e f r a c t i o n c o n t a i n e d 19% and 17% mean t o t a l p r o t e i n i n s t r a i n s 530 and 496 r e s p e c t i v e l y , w h i l e mean KDO y i e l d s i n b o t h s t r a i n s were 49%. S o l u b i l i z a t i o n o f h a l f t h e LPS w i t h t h i s p r o c e d u r e i n E. coli has a l s o been o b s e r v e d ( S c h n a i t m a n 1 9 7 1 ) . T w i c e as much T r i t o n - i n s o l u b l e m a t e r i a l as c r u d e w a l l m a t e r i a l was r e q u i r e d t o n e u t r a l i z e t h e same amount o f c a r o t o v o r i c i n - 3 7 9 . N e u t r a l i z a t i o n a c t i v i t y o f t h e T r i t o n - i n s o l u b l e f r a c t i o n was t h e r e f o r e o n l y 50% t h a t of t h e c r u d e w a l l . A t c o n c e n t r a t i o n s o f 1-2 mg/ml, p r o t e i n e x t r a c t e d f r o m e i t h e r T r i t o n - s o l u b l e or i n s o l u b l e f r a c t i o n s d i s p l a y e d no n e u t r a l i z a t i o n a c t i v i t y . LPS e x t r a c t e d f r o m a l l f o u r s e n s i t i v e s t r a i n s n e u t r a l i z e d c a r o t o v o r i c i n - 3 7 9 a c t i v i t y , w h i l e LPS f r o m r e s i s t a n t s t r a i n Ecc 379 d i d n o t ( F i g . 4 ) . LPS f r o m Ecc s t r a i n s 530 and 496 p r e v e n t e d t h e f o r m a t i o n o f an i n h i b i t o n zone p r o d u c e d by c a r o t o v o r i c i n - 3 7 9 , w h i l e LPS e x t r a c t e d f r o m r e s i s t a n t Ecc s t r a i n 379 f a i l e d t o do s o . LPS y i e l d s ( T a b l e 2) f r o m Eca s t r a i n s 530 and 496 were 83 F i g u r e 3. R e l a t i v e d i s t r i b u t i o n s o f p r o t e i n , k e t o d e o x y o c t a n a t e (KDO), and c a r o t o v o r i c i n - 3 7 9 n e u t r a l i z a t i o n a c t i v i t y (C-379 NA) i n f r a c t i o n o b t a i n e d t h r o u g h T r i t o n X-100 s e l e c t i v e s o l u b i l i z a t i o n o f c r u d e c e l l s w a l l s o f E r w i n i a carotovora s u b s p e c i e s a t r o s e p t i c a . CCW ( c r u d e c e l l w a l l ) , TIW ( T r i t o n - i n s o l u b l e w a l l ) , TSP ( T r i t o n - s o l u b l e p r o t e i n ) , T IP ( T r i t o n - i n s o l u b l e p r o t e i n ) . a) Erwinia carotovora s u b s p e c i e s a t r o s e p t i c a 530. b) Erwinia carotovora s u b s p e c i e s atroseptica 496. 84 Wall Fraction b Wall Fraction 85 F i g u r e 4. S p o t - p l a t e a c t i v i t y d e t e r m i n a t i o n o f c a r o t o v o r i c i n - 3 7 9 u n t r e a t e d (a) and p r e m i x e d 10:1 w i t h l i p o p o l y s a c c h a r i d e e x t r a c t e d f r o m Erwinia carotovora s e n s i t i v e s t r a i n s 530 ( b ) , 496 (c ) and Erwinia carotovora n o n - s e n s i t i v e s t r a i n 379 ( d ) . 86 T a b l e 2: Y i e l d s and s p e c i f i c c a r o t o v o r i c i n - 3 7 9 n e u t r a l i z a t i o n a c t i v i t i e s (C-379 NA) of 1 i p o p o l y s a c c h a r i d e e x t r a c t e d f r o m s t r a i n s o f Erwinia carotovora by t h e methods o f W e s t p h a l and J a n n (1965) (A) and G a l a n o s e t al. (1969) ( B ) . S t r a i n LPS Y i e l d by S p e c i f i c C-379 NA A B ug KDOVmg dw 2 A o b t a i n e d by B l u 3 / u g KDO 530 SR8 504 496 379 1.4 0.8 0.9 1.6 0.0 ND 4 ND 1.4 0.1 0.2 1000-1200 1000-1200 ND 3500-4000 0 ND ND >1600 ND 0 1KDO; k e t o d e o x y o c t a n a t e 2 dw; d r y w e i g h t 3 l u ; l e t h a l u n i t s 4 ND; n o t d e t e r m i n e d 87 a b o u t 50%, b a s e d on KDO d e t e r m i n a t i o n s f r o m c r u d e w a l l s . F o r s e n s i t i v e s t r a i n s 530, 496, and SR8, t h e h o t p h e n o l method was t h e most e f f e c t i v e . LPS f r o m s t r a i n 379 c o u l d be e x t r a c t e d by t h e method o f G a l a n o s e t a l . ( 1 9 6 9 ) , a l t h o u g h y i e l d s were o f t e n v e r y low. Q u a n t i t a t i o n o f c a r o t o v o r i c i n - 3 7 9 n e u t r a l i z a t i o n a c t i v i t y r e v e a l e d t h a t among t h e Eca s t r a i n s , 496 had t h e h i g h e s t n e u t r a l i z a t i o n a c t i v i t y w h i l e SR8 had t h e l o w e s t ( T a b l e 2). Ecc s t r a i n 379 a g a i n d i s p l a y e d no m e a s u r a b l e n e u t r a l i z a t i o n a c t i v i t y . LPS p r e - t r e a t e d w i t h s t e r i l e p o t a s s i u m p h o s p h a t e b u f f e r n e u t r a l i z e d c a r o t o v o r i c i n - 3 7 9 a c t i v i t y w h i l e t h e LPS p r e - t r e a t e d w i t h p e r i o d a t e had l o s t a l l a b i l i t y t o n e u t r a l i z e ( F i g . 5 ) . C o n t r o l s i n d i c a t e d t h a t 0.01 M p e r i o d a t e d i d n o t a f f e c t t h e a c t i v i t y o f t h e c a r o t o v o r i c i n nor t h e g r o w t h o f t h e i n d i c a t o r s t r a i n ( F i g . 5 ) . When LPS e x t r a c t s were s u b j e c t e d t o e l e c t r o p h o r e t i c a n a l y s i s i n 4 M u r e a 14% a c r y l a m i d e g e l s , LPS f r o m t h e s e n s i t i v e s t r a i n s were f o u n d t o d i s p l a y a g r e a t d e a l o f s t r u c t u r a l h e t e r o g e n e i t y ( F i g . 6a and 6 b ) . R e p e a t i n g band p a t t e r n s o f i n c r e a s i n g m o l e c u l a r w e i g h t r e p r e s e n t e d i n d i v i d u a l LPS m o l e c u l e s w i t h components of i n c r e a s i n g l e n g t h s o f O - a n t i g e n , l i k e l y p r e s e n t w i t h i n t h e same c e l l . The l o w e r m o s t bands on t h e g e l r e p r e s e n t e d t h e 88 F i g u r e 5. S p o t - p l a t e d e m o n s t r a t i n g t h e e f f e c t o f p e r i o d a t e on t h e c a r o t o v o r i c i n - 3 7 9 n e u t r a l i z a t i o n a c t i v i t y o f LPS e x t r a c t e d f r o m E r w i n i a c a r o t o v o r a s u b s p e c i e s a t r o s e p t i c a s t r a i n 530. (a) c a r o t o v o r i c i n -379 t r e a t e d w i t h 0.01 M p e r i o d i c a c i d d i s s o l v e d i n 50 mM p o t a s s i u m p h o s p h a t e b u f f e r pH 7.2; (b) LPS p r e t r e a t e d w i t h s t e r i l e p h o s p h a t e b u f f e r and d i l u t e d 1/10 i n t o c a r o t o v o r i c i n - 3 7 9 s u s p e n s i o n ; (c) p e r i o d i c a c i d (0.1 M) d i s s o l v e d i n s t e r i l e p h o s p h a t e b u f f e r ; (d) LPS p r e -t r e a t e d w i t h 0.1 M p e r i o d a t e d i l u t e d 1/10 i n c a r o t o v o r i c i n - 3 7 9 s u p e n s i o n . 89 F i g u r e 6. Sodium d o d e c y l s u l f a t e p o l y a c r y l a m i d e g e l e l e c t r o p h o r e t o g r a m o f l i p o p o l y s a c c h a r i d e e x t r a c t e d f r o m E r w i n i a c a r o t o v o r a ( E c ) and Escherichia coli ( E . c o l i s t r a i n s . a. Run on P h a r m a c i a E l e c t r o p h o r e s i s U n i t , Model GE 214 L S. L e f t t o r i g h t , l a n e s : 1) E c s u b s p e c i e s carotovora 504. 2) Ec s u b s p e c i e s a t r o s e p t i c a 530. 3) Ec s u b s p e c i e s a t r o s e p t i c a SR8. 4) E c s u b s p e c i e s a t r o s e p t i c a 496. b. Run on B i o - R a d E l e c t r o p h o r e s i s U n i t Model 360. L e f t t o r i g h t , l a n e s : 1) E . c o l i HB101. 2) Ec s u b s p e c i e s c a r o t o v o r a 3 7 9 1 . 3) E c s u b s p e c i e s atroseptica 4 9 6 2 . 4) E c s u b s p e c i e s carotovora 5 0 4 1 . 5) E c s u b s p e c i e s a t r o s e p t i c a 5 3 0 2 . 6) E c s u b s p e c i e s carotovora 5 0 4 2 . E x t r a c t e d by t h e method of G a l a n o s e t a l . 1969. 2 E x t r a c t e d by t h e method o f w e s t p h a l and Ja n n 1965. 91 s m a l l e s t components, p r o b a b l y i n d i v i d u a l c o r e components l a c k i n g O - a n t i g e n . In s t r i k i n g c o n t r a s t , LPS e x t r a c t e d f r o m E . coli HB101 and t h e r e s i s t a n t Ecc s t r a i n 379 was f a r more homogeneous ( F i g . 6 b ) . T h e r e i s o n l y one major band, a p p a r e n t l y o f low m o l e c u l a r w e i g h t . F u r t h e r m o r e , Ecc s t r a i n 379 LPS c o u l d o n l y be e x t r a c t e d when t h e method o f G a l a n o s e t a l , w h i c h p r e f e r e n t i a l l y e x t r a c t s r o u g h LPS, was employe d . T h i s s u g g e s t e d t h a t l i k e E. coli HB101, Ecc 379 was r o u g h , c o m p l e t e l y l a c k i n g O - a n t i g e n s i d e c h a i n s . I n an E L I S A w h i c h was p e r f o r m e d on n i t r o c e l l u l o s e , p o s i t i v e r e a c t i o n s were o b t a i n e d w i t h LPS f r o m s e n s i t i v e s t r a i n s ( F i g . 7 ) . Weakly p o s i t i v e r e s u l t s were o b t a i n e d w i t h Ecc s t r a i n 379. N e g a t i v e r e s u l t s were o b t a i n e d w i t h LPS e x t r a c t e d E . coli HB101. B e c a u s e t h e c a r o t o v o r i c i n -379 was p r e v e n t e d f r o m b i n d i n g t o t h e n i t r o c e l l u l o s e , t h e o b s e r v e d p o s i t i v e r e a c t i o n s p r o b a b l y r e s u l t e d f r o m t h e d i r e c t and i r r e v e r s i b l e b i n d i n g o f c a r o t o v o r i c i n - 3 7 9 t o t h e LPS. D i s c u s s i o n U t i l i z a t i o n o f LPS as a r e c e p t o r s i t e i s e x t r e m e l y r a r e among s m a l l m o l e c u l a r w e i g h t b a c t e r i o c i n s , a l t h o u g h 92 F i g u r e 7. E n z y m e - l i n k e d immunosorbent a s s a y o f c a r o t o v o r i c i n - 3 7 9 bound t o 1 i p o p o y s a c c h a r i d e e x t r a c t e d f r o m v a r i o u s s t r a i n s of E r w i n i a carotovora s u b s p e c i e s c a r o t o v o r a (Ecc), Erwinia carotovora s u b s p e c i e s a t r o s e p t i c a , ( E c a ) and Escherichia c o l i . Top row, l e f t t o r i g h t : Ecc s t r a i n 379, E c c s t r a i n 504, E c a s t r a i n 496. B o t t o m row, l e f t t o r i g h t : Eca s t r a i n SR8, Eca s t r a i n 530, E. coli s t r a i n HB101. 93 LPS i s known t o enhance t h e a c t i v i t y o f t h e p r o t e i n r e c e p t o r o f c o l i c i n A ( C h a i e t a l . 1 9 8 2 ) . P y o c i n s , ( I k e d a and Egami 1969) as w e l l a s p a r t i c u l a t e b a c t e r i o c i n s p r o d u c e d by P r o t e u s v u l g a r i s ( S m i t e t al. 1969) and Rhizobibium lupini ( P f i s t e r and L o d e r s t a e d t 1 9 7 7 ) , however, a l l a d s o r b t o s i t e s on t h e LPS o f s e n s i t i v e c e l l s . The r e c e p t o r s i t e f o r c a r o t o v o r i c i n -379 i n s e n s i t i v e c e l l s a l s o a p p e a r s t o be l o c a t e d on some component o f t h e LPS. T h r e e main l i n e s o f e v i d e n c e s u p p o r t t h i s c o n c l u s i o n . P r o t e i n e x t r a c t e d f r o m T r i t o n - s o l u b l e and - i n s o l u b l e f r a c t i o n s f a i l e d t o n e u t r a l i z e c a r o t o v o r i c i n - 3 7 9 . F u r t h e r m o r e , w h i l e SDS-PAGE r e v e a l e d t h a t i n d i v i d u a l membrane p r o t e i n s were s e p a r a t e d i n t o one f r a c t i o n or t h e o t h e r , b o t h LPS and n e u t r a l i z a t i o n a c t i v i t y were d i s t r i b u t e d e v e n l y between t h e t h e f r a c t i o n s . The r e c e p t o r s i t e , t h e r e f o r e , d i d n o t a p p e a r t o be a membrane p r o t e i n . LPS e x t r a c t e d f r o m s e n s i t i v e s t r a i n s s p e c i f i c a l l y n e u t r a l i z e d c a r o t o v o r i c i n a c t i v i t y . Q u a n t i t a t i o n o f LPS s p e c i f i c n e u t r a l i z a t i o n a c t i v i t y r e v e a l e d t h a t LPS f r o m E c a s t r a i n 496 n e u t r a l i z e d more c a r o t o v o r i c i n - 3 7 9 p e r ug KDO t h a n t h a t f r o m s t r a i n s 530 or SR8. Some o f t h e n e u t r a l i z a t i o n c a p a c i t y o f t h e LPS, however, was p r o b a b l y i n a c t i v a t e d a s a r e s u l t o f t h e e x t r a c t i o n p r o c e d u r e . 94 C o n v e n t i o n a l l y , n e u t r a l i z a t i o n of b a c t e r i o c i n a c t i v i t y i s c o n s i d e r e d t o be an i n d i c a t o r of r e c e p t o r a c t i v i t y . A l t h o u g h LPS f r o m t h e r e s i s t a n t s t r a i n Ecc 379 f a i l e d t o n e u t r a l i z e c a r o t o v o r i c i n - 3 7 9 , t h e r e was no d i r e c t e v i d e n c e t o i n d i c a t e t h a t t h e o b s e r v e d n e u t r a l i z a t i o n i n s e n s i t i v e s t r a i n s was due t o d i r e c t b i n d i n g t o t h e LPS and n o t t o some l e s s s p e c i f i c c h e m i c a l i n a c t i v a t i o n . U s i n g a n t i s e r u m d i r e c t e d a g a i n s t c a r o t o v o r i c i n - 3 7 9 , d i r e c t b i n d i n g o f t h e b a c t e r i o c i n t o LPS was d e m o n s t r a t e d . C a r o t o v o r i c i n - 3 7 9 bound o n l y t o LPS e x t r a c t e d f r o m s e n s i t i v e c e l l s and n o t t o t h a t o f r e s i s t a n t s t r a i n E. coli HB101. The r e a c t i o n o b s e r v e d w i t h Ecc s t r a i n 379 was e x t r e m e l y weak, and may have been due t o a p a r t i a l c r o s s - r e a c t i o n w i t h E c c 379 LPS. In a d d i t i o n , t h e r e may be a weak s p e c i f i c or n o n - s p e c i f i c a s s o c i a t i o n between Erwinia LPS and c a r o t o v o r i c i n - 3 7 9 . The i n a c t i v a t i o n o f r e c e p t o r a c t i v i t y by p e r i o d a t e o x i d a t i o n s u g g e s t e d t h a t t h e r e c e p t o r s i t e i s composed o f c a r b o h y d r a t e . The c a r o t o v o r i c i n - 3 7 9 r e c e p t o r s i t e , t h e r e f o r e , i s p r o b a b l y l o c a t e d on t h e O - a n t i g e n or c o r e p o r t i o n of t h e LPS m o l e c u l e . B e c a u s e of t h e c o r r e l a t i o n between s e r o g r o u p d e s i g n a t i o n and t h e d e g r e e o f s e n s i t i v i t y t o l a r g e m o l e c u l a r w e i g h t b a c t e r i o c i n s , C r o w l e y and De Boer (1980) s u g g e s t e d t h a t t h e r e c e p t o r s i t e was l o c a t e d on t h e O - a n t i g e n . A l t h o u g h many 95 b a c t e r i o p h a g e s s u c h as P22 a d s o r b t o s i t e s on t h e 0-a n t i g e n , t h i s i s u n l i k e l y i n t h e c a s e o f c a r o t o v o r i c i n -379. I n t h e E n t e r o b a c t e r i a c e a e , O - a n t i g e n s commonly d i s p l a y a h i g h d e g r e e o f v a r i a b i l i t y between s t r a i n s o f t h e same s p e c i e s , w h i l e t h e v a r i a b i l i t y o f t h e c o r e s t r u c t u r e i s f a r l e s s e xtreme (Hantke and B r a u n 1 9 8 1 ) . O - a n t i g e n s t r u c t u r e and c o m p o s i t i o n d e t e r m i n e s e r o g r o u p d e s i g n a t i o n i n E. c a r o t o v o r a (De Boer e t a l . 1 9 8 5 ) . P o l y c l o n a l a n t i s e r a d i r e c t e d t o w a r d s one s e r o g r o u p o f Eca ( I ) d i d n o t c r o s s - r e a c t w i t h t h e LPS o f a n o t h e r ( X V I I I ) i n E L I S A (De Boer e t a l . 1 9 8 5 ) , s u g g e s t i n g t h a t t h e 0-a n t i g e n s o f t h e two s e r o g r o u p s do n o t s h a r e common s i t e s . B e c a u s e t h e c o r e p o r t i o n o f t h e LPS i s l o c a t e d b e n e a t h t h e O - a n t i g e n p o l y s a c c h a r i d e , i t i n i t i a l l y seems u n l i k e l y t h a t t h i s component c o u l d s e r v e a s a r e c e p t o r s i t e f o r c a r o t o v o r i c i n - 3 7 9 . F o r example, c o r e - s p e c i f i c b a c t e r i o p h a g e s s u c h as T4 i n f e c t r o u g h E . c o l i s t r a i n s B and K12 (Hantke and B r a u n 1 9 8 1 ) . Smooth s t r a i n s of P. a e r u g i n o s a and Salmonella s p . , however, a r e r e s p e c t i v e l y s e n s i t i v e t o c o r e - s p e c i f i c p y o c i n s (Meadow and W e l l s 1978) and b a c t e r i o p h a g e s (Hantke and B r a u n e t a l . 1 9 7 1 ) . E l e c t r o p h o r e t i c a n a l y s i s o f t h e LPS f r o m s e n s i t i v e Eca s t r a i n s r e v e a l e d t h a t i n d i v i d u a l LPS m o l e c u l e s v a r y g r e a t l y i n s i z e . The f a c t t h a t t h e l o w - m o l e c u l a r w e i g h t c o r e components a p p e a r w i t h o u t a t t a c h e d O - a n t i g e n 96 s u g g e s t s t h a t b o t h c o r e and O - a n t i g e n s i d e c h a i n s a r e ex p o s e d t o t h e o u t e r s u r f a c e o f t h e c e l l . I t i s t h e r e f o r e n o t u n r e a s o n a b l e t o s u g g e s t t h a t t h e c a r o t o v o r i c i n - 3 7 9 r e c e p t o r s i t e i s l o c a t e d on t h e c o r e o l i g o s a c c h a r i d e . To d a t e , t h e r e have been no s t r a i n s o f Eca f o u n d w h i c h a r e r e s i s t a n t t o c a r o t o v o r i c i n - 3 7 9 . The c o n s e r v a t i o n o f t h e c a r o t o v o r i c i n - 3 7 9 r e c e p t o r s i t e s u g g e s t s t h a t t h i s s t r u c t u r e p r o v i d e s some f u n c t i o n b e n e f i c i a l t o t h e c e l l . I n n a t u r e , b a c t e r i a l s u r f a c e p o l y s a c c h a r i d e s c o n f e r t o t h e c e l l m e c h a n i c a l s t a b i l i t y , r e s i s t a n c e t o t o x i c compounds, and f a c i l i t a t e s u r f a c e a d h e s i o n . S i n c e a l l t h e s t r a i n s s t u d i e d were o r i g i n a l l y i s o l a t e d f r o m b l a c k l e g i n f e c t e d p o t a t o s t ems, t h e c a r o t o v o r i c i n - 3 7 9 r e c e p t o r s i t e may c o n t r i b u t e t o t h e v i r u l e n c e o f t h e c a r r y i n g s t r a i n . T h i s i s an i n t r i g u i n g p o s s i b i l i t y i n l i g h t o f r e c e n t f i n d i n g s w h i c h s u g g e s t t h a t i n E r w i n i a carotovora, c e l l s u r f a c e components i n f l u e n c e p o s s i b l e v i r u l e n c e f a c t o r s s u c h a s r e s i s t a n c e t o p h y t o a l e x i n s . ( L y o n e t a l . 1985). D e t a i l e d g e n e t i c and b i o c h e m i c a l a n a l y s i s o f Eca LPS w i l l be r e q u i r e d t o d e t e r m i n e t h e p r e c i s e l o c a t i o n and s t r u c t u r e o f t h e c a r o t o v o r i c i n - 3 7 9 r e c e p t o r s i t e . T h i s work w i l l c o n t r i b u t e t o t h e e l u c i d a t i o n of t h e r o l e o f c a r o t o v o r i c i n - 3 7 9 s e n s i t i v i t y i n t h e e c o l o g y o f E. 97 carotovora. The a p p l i c a t i o n o f h y b r i d o m a t e c h n o l o g y t o t h i s p r o b l e m c o u l d a l s o y i e l d r e s u l t s o f p r a c t i c a l v a l u e , as a m o n o c l o n a l a n t i b o d y d i r e c t e d a g a i n s t t h e c a r o t o v o r i c i n - 3 7 9 r e c e p t o r s i t e may p r o v i d e a d i a g n o s t i c t o o l f o r t h e d e t e c t i o n of E. carotovora i n p l a n t t i s s u e . 98 GENERAL DISCUSSION B a c t e r i o c i n s e n s i t i v i t y i s i n f l u e n c e d by t h e p r e s e n c e o f r e c e p t o r s i t e s and t h e a b i l i t y o f t h e b a c t e r i o c i n t o r e a c h t h e t a r g e t o f a c t i o n . The p r e s e n c e o f r e c e p t o r s was d e m o n s t r a t e d i n a l l t h e s e n s i t i v e s t r a i n s ( C h a p t e r s 2 and 3 ) , t h e r e f o r e t h e d i f f e r e n t l e v e l s o f c a r o t o v o r i c i n s e n s i t i v i t y o b s e r v e d among s e r o g r o u p s o f Eca a p p e a r t o be due t o v a r y i n g d e g r e e s o f t o l e r a n c e . T h i s i m p l i e s t h a t i n l e s s s e n s i t i v e s t r a i n s , much o f t h e b a c t e r i o c i n a d s o r b e d t o c e l l s u r f a c e r e c e p t o r s i s u n a b l e t o r e a c h t h e a p p r o p r i a t e t a r g e t o f a c t i o n . I n t h e c a s e o f c a r o t o v o r i c i n - 3 7 9 , t h e r e c e p t o r s i t e i s l o c a t e d on t h e 1 i p o p o l y s a c c h a r i d e (LPS) and t h e t a r g e t o f a c t i o n a p p e a r s t o be t h e c y t o p l a s m i c membrane. The d i f f e r e n t l e v e l s o f c a r o t o v o r i c i n - 3 7 9 s e n s i t i v i t y o b s e r v e d by J a i s (1982) i n s p o t - p l a t e a s s a y s were c o n f i r m e d i n C h a p t e r 2 by d e t e r m i n i n g t h e number o f v i a b l e c e l l s o v e r t i m e f o l l o w i n g t h e a d d i t i o n o f c a r o t o v o r i c i n - 3 7 9 . I n C h a p t e r 1, t h e c r i t i c a l d i l u t i o n a s s a y method was m o d i f i e d and a d a p t e d so t h a t i t c o u l d be p e r f o r m e d i n a m i c r o t i t r e p l a t e . T h i s method, b e c a u s e o f i t s s e n s i t i v i t y and r e p r o d u c i b i l i t y , f a c i l i t a t e d t h e f u r t h e r i n v e s t i g a t i o n o f c a r o t o v o r i c i n - 3 7 9 s e n s i t i v i t y i n 99 Eca. The p r e s e n c e o f r e c e p t o r s was c o n f i r m e d by m e a s u r i n g n e u t r a l i z a t i o n o f c a r o t o v o r i c i n - 3 7 9 by whole c e l l s ( C h a p t e r 2) and c r u d e w a l l f r a c t i o n s ( C h a p t e r 3 ) . R e s i d u a l c a r o t o v o r i c i n - 3 7 9 a c t i v i t y r a p i d l y d e c l i n e d upon t h e a d d i t i o n o f s e n s i t i v e c e l l s , b u t n o t upon t h a t o f r e s i s t a n t c e l l s ( C h a p t e r 2 ) . L i p o p o l y s a c c h a r i d e ( L P S ) , b u t n o t membrane p r o t e i n , n e u t r a l i z e d c a r o t o v o r i c i n - 3 7 9 a c t i v i t y i n v i t r o ( C h a p t e r 3 ) , s u g g e s t i n g t h a t t h e r e c e p t o r s i t e i s l o c a t e d on LPS. N e u t r a l i z a t i o n a c t i v i t y was i n a c t i v a t e d by p e r i o d a t e o x i d a t i o n , s u g g e s t i n g t h a t t h e r e c e p t o r s i t e i s composed o f c a r b o h y d r a t e ( C h a p t e r 3) . N e u t r a l i z a t i o n o f c a r o t o v o r i c i n - 3 7 9 by whole c e l l s f o l l o w e d f i r s t o r d e r r e a c t i o n k i n e t i c s , s u g g e s t i n g t h a t t h e o b s e r v e d n e u t r a l i z a t i o n was i r r e v e r s i b l e ( C h a p t e r 2 ) . C a r o t o v o r i c i n - 3 7 9 , d e t e c t e d w i t h p o l y c l o n a l a n t i s e r u m , bound t o LPS e x t r a c t e d f r o m s e n s i t i v e s t r a i n s , b u t n o t w i t h t h a t f r o m r e s i s t a n t s t r a i n s ( C h a p t e r 3 ) . T h i s s u g g e s t s t h a t c a r o t o v o r i c i n - 3 7 9 n e u t r a l i z a t i o n by b o t h whole c e l l s and LPS was due t o d i r e c t b i n d i n g . The f a c t t h a t t h e c a r o t o v o r i c i n - 3 7 9 d i d n o t wash o f f d u r i n g t h e E L I S A p r o c e d u r e ( C h a p t e r 3) f u r t h e r s u g g e s t s t h a t t h e b i n d i n g between c a r o t o v o r i c i n - 3 7 9 and t h e LPS r e c e p t o r i s i r r e v e r s i b l e . 100 The mode o f a c t i o n o f c a r o t o v o r i c i n - E r i n v o l v e s d i s r u p t i o n o f c e l l u l a r e n e r g i z a t i o n ( I t o h e t al. 1 9 8 2 ) . C a r o t o v o r i c i n - 3 7 9 a p p e a r s t o a c t i n a s i m i l a r manner ( C h a p t e r 2 ) . When s e n s i t i v e c e l l s a r e t r e a t e d w i t h c a r o t o v o r i c i n - 3 7 9 i n t h e p r e s e n c e o f t h e f l u o r e s c e n t p r o b e 8 - a n i l i n o - l - n a p h t h a l e n e s u l f o n i c a c i d t h e r e l a t i v e f l u o r e s c e n c e o f t h e p r o b e i n c r e a s e s i n a d o s e - d e p e n d e n t f a s h i o n ( C h a p t e r 2 ) . T h i s d o e s n o t o c c u r when r e s i s t a n t c e l l s ( E c c 379) a r e t r e a t e d w i t h c a r o t o v o r i c i n - 3 7 9 . T hese r e s u l t s i n d i c a t e d t h a t c a r o t o v o r i c i n - 3 7 9 e f f e c t s a c o l l a p s e o f t h e p r o t o n m o t i v e f o r c e , s u g g e s t i n g t h a t t h e t a r g e t o f a c t i o n i s t h e c y t o p l a s m i c membrane. I t was p o s t u l a t e d t h a t c a r o t o v o r i c i n - 3 7 9 e f f e c t s i t s l e t h a l a c t i o n by f o r c i n g i t s h o l l o w c o r e t h r o u g h o u t e r w a l l and c y t o p l a s m i c membrane, c a u s i n g l e a k a g e o f c e l l u l a r c o n t e n t s and c o l l a p s e o f t h e p r o t o n m o t i v e f o r c e (pmf) ( C h a p t e r 2 ) . T h i s model i s c o n s i s t e n t w i t h t h e s i n g l e -h i t mechanism o b s e r v e d i n C h a p t e r 2, as s i n g l e c h a n n e l s t h r o u g h t h e c y t o p l a s m i c membrane o f E. c o l i were s u f f i c i e n t t o p r e v e n t c e l l d i v i s i o n ( S c h e i n e t a l . 1 9 7 8 ) . C a r o t o v o r i c i n - 3 7 9 must overcome t h e b a r r i e r o f t h e o u t e r membrane b e f o r e r e a c h i n g t h e c y t o p l a s m i c membrane. A d s o r p t i o n t o t h e LPS a p p e a r s t o be t h e f i r s t s t e p i n t h i s p r o c e s s . A d s o r p t i o n o f t h e b a c t e r i o p h a g e T4 p a r t i c l e t o LPS t r i g g e r s s t r u c t u r a l c h a n g e s i n t h e 101 b a s e p l a t e w h i c h c a u s e t h e s h e a t h t o c o n t r a c t . T h i s c o n t r a c t i o n a p p e a r s t o be n e c e s s a r y f o r i n f e c t i o n (Hantke and B r a u n 1 9 8 1 ) . S h e a t h c o n t r a c t i o n i s a l s o n e c e s s a r y f o r t h e a c t i o n o f p y o c i n s (Govan 1974) and p r o b a b l y a l s o c a r o t o v o r i c i n - E r ( I t o h e t a l . 1 9 7 8 ) . In l i g h t o f t h e s e o b s e r v a t i o n s , i t does n o t seem u n r e a s o n a b l e t o s u g g e s t t h a t a d s o r p t i o n o f c a r o t o v o r i c i n - 3 7 9 t o i t s c o g n a t e LPS r e c e p t o r a l s o c a u s e s s h e a t h c o n t r a c t i o n , and t h a t t h i s c o n t r a c t i o n f o r c e s t h e h o l l o w c o r e t h r o u g h t h e o u t e r and c y t o p l a s m i c membranes. The mechanism by w h i c h some s e r o g r o u p s o f E c a e x h i b i t r e l a t i v e l y h i g h l e v e l s o f t o l e r a n c e t o c a r o t o v o r i c i n - 3 7 9 ( C h a p t e r 2) r e m a i n s t o be e l u c i d a t e d . P a r t i a l c o r r e l a t i o n s o b s e r v e d between s e r o g r o u p d e s i g n a t i o n and t h e d e g r e e o f s e n s i t i v i t y t o p a r t i c u l a t e c a r o t o v o r i c i n s ( C r o w l e y and De Boer 1980, J a i s 1982) s u g g e s t t h a t t h e O - a n t i g e n p o l y s a c c h a r i d e i n f l u e n c e s c a r o t o v o r i c i n t o l e r a n c e . O - a n t i g e n c h a i n l e n g t h p r o f o u n d l y a f f e c t s LPS-LPS i n t e r a c t i o n s w i t h r e g a r d t o f l u i d i t y i n v i t r o and i t i s s u g g e s t e d t h a t 0-a n t i g e n c h a i n i n t e r a c t i o n s may a l s o m o d u l a t e t h e s t r u c t u r e and f u n c t i o n s o f t h e o u t e r membrane. ( P e t e r s o n e t a l . 1 9 8 6 ) . A l t h o u g h i t i s u n l i k e l y t h a t t h e c a r o t o v o r i c i n r e c e p t o r s i t e i s l o c a t e d on t h e O - a n t i g e n , t h e c a r o t o v o r i c i n - 3 7 9 r e c e p t o r s i t e i s n e v e r t h e l e s s 102 l o c a t e d on t h e LPS ( C h a p t e r 3 ) . I t does n o t seem "\ u n r e a s o n a b l e , t h e r e f o r e , t o s u g g e s t t h a t O - a n t i g e n s t r u c t u r e and c o m p o s i t i o n may a f f e c t t h e i n t e r a c t i o n between t h e c a r o t o v o r i c i n - 3 7 9 p a r t i c l e and i t s r e c e p t o r s i t e . C a r o t o v o r i c i n s e n s i t i v i t y a p p e a r s t o be a h i g h l y c o n s e r v e d p h e n o t y p e among s t r a i n s o f E. carotovora i s o l a t e d f r o m i n f e c t e d p l a n t m a t e r i a l . T h i s s u g g e s t s t h a t s e n s i t i v i t y may c o n f e r one or more b e n e f i c i a l a t t r i b u t e s t o t h e c a r r y i n g s t r a i n . M o r e o v e r , t h e e v o l u t i o n o f c a r o t o v o r i c i n t o l e r a n c e r a t h e r t h a n r e s i s t a n c e i s i n d i c a t i v e o f what may be a s i g n i f i c a n t r o l e f o r t h e c a r o t o v o r i c i n - 3 7 9 r e c e p t o r s i t e i n t h e e c o l o g y o f E. carotovora. T h i s s u g g e s t i o n i s s u p p o r t e d by r e c e n t i n v e s t i g a t i o n s i m p l i c a t i n g a r o l e f o r c e l l s u r f a c e components a s v i r u l e n c e f a c t o r s i n E. carotovora ( L y o n e t al. 198 5 ) . 103 SUMMARY 1) The m i c r o t i t r e p l a t e a s s a y method p r o v i d e d an a c c e p t a b l y h i g h s t a n d a r d o f s e n s i t i v i t y and r e p r o d u c i b i l i t y f o r q u a n t i f y i n g c a r o t o v o r i c i n - 3 7 9 a c t i v i t y . 2) S t r a i n s r e p r e s e n t i n g t h e f o u r s e r o g r o u p s o f Erwinia carotovora d i s p l a y e d v a r y i n g l e v e l s o f s e n s i t i v i t y t o c a r o t o v o r i c i n - 3 7 9 . 3) A l l s e n s i t i v e t e s t s t r a i n s a d s o r b e d c a r o t o v o r i c i n -379 r e g a r d l e s s o f t h e e x t e n t o f s e n s i t i v i t y , t h e r e f o r e t h e d i f f e r e n c e s i n s e n s i t i v i t y were a t t r i b u t e d t o v a r y i n g d e g r e e s o f t o l e r a n c e . 4) C a r o t o v o r i c i n - 3 7 9 f o l l o w e d s i n g l e - h i t k i l l i n g k i n e t i c s , i n d i c a t i n g t h a t o n l y one p a r t i c l e i s r e q u i r e d t o e f f e c t c e l l d e a t h . 5) The f l u o r e s c e n t p r o b e r e s p o n s e t o c e l l s t r e a t e d w i t h c a r o t o v o r i c i n - 3 7 9 i n d i c a t e d t h a t t h e mode o f a c t i o n i n v o l v e s a d i s r u p t i o n o f e n e r g y c o u p l i n g and t h a t t h e t a r g e t o f a c t i o n i s l i k e l y t h e c y t o p l a s m i c membrane. 0 104 6 ) L i p o p o l y s a c c h a r i d e e x t r a c t e d f r o m s e n s i t i v e c e l l s n e u t r a l i z e d c a r o t o v o r i c i n - 3 7 9 a c t i v i t y . 7 ) C a r o t o v o r i c i n - 3 7 9 b i n d s d i r e c t l y and i r r e v e r s i b l y t o l i p o p o l y s a c c h a r i d e f r o m s e n s i t i v e c e l l s . 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