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Bacteriocin production in Erwinia carotovora, subspecies carotovora, strain 379 1986

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BACTERIOCIN PRODUCTION IN ERWINIA CAROTOVORA SUBSPECIES CAROTOVORA STRAIN 37 By LEONARD JOHN WARD 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 FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Pla n t Science) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA ' October, 1986 ©Leonard John Ward I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my 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 o f VWV Sex The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main Ma l l V a n c o u v e r , C a n a d a V6T 1Y3 IE-6 (3/81) . i i ABSTRACT Erwinia carotovora subsp. carotovora s t r a i n 379 (Ecc 379) produced a p a r t i c u l a t e 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 which resembled a bact e r i o p h a g e t a i l . C a r o t o v o r i c i n - 3 7 9 , producing both c l e a r and d i f f u s e zones of growth i n h i b i t i o n , was a c t i v e a g a i n s t s e v e r a l other Erwinia carotovora s t r a i n s . I t was dete c t e d i n the f i l t e r - s t e r i l i z e d supernatant of Ecc 379 under standa r d c u l t u r a l c o n d i t i o n s . I t s c o n c e n t r a t i o n s i n the supernatant f r a c t i o n of c u l t u r e d Ecc 379 were i n c r e a s e d by i n d u c t i o n with mitomycin C (0.2 ug/ml). I n d u c t i o n was f o l l o w e d by c e l l l y s i s , which was r e f l e c t e d by a sharp r e d u c t i o n i n c u l t u r e t u r b i d i t y . Growth o f Ecc 379 at 37 C with or without mitomycin C r e s u l t e d i n the l o s s of p a r t i c u l a t e c a r o t o v o r i c i n - 379 p r o d u c t i o n . Under these c o n d i t i o n s , a low molecular weight, h i g h l y d i f f u s i b l e b a c t e r i o c i n component was de t e c t e d which produced l a r g e d i f f u s e zones of i n h i b i t i o n w i t h t h r e e of the f o u r sta n d a r d Erwinia carotovora i n d i c a t o r s t r a i n s used. C e l l l y s i s f o l l o w i n g i n d u c t i o n and a w i l d - t y p e r e s i s t a n c e to erythromycin and chloramphenicol were i i i a l s o temperature s e n s i t i v e . C a r o t o v o r i c i n - 3 7 9 p r o d u c t i o n was i n v e s t i g a t e d by i s o l a t i n g i n t e r m e d i a t e s at s e v e r a l times a f t e r i n d u c t i o n . These i n t e r m e d i a t e s were analysed by e l e c t r o n microscopy (EM) and Sephacryl S-300 column chromatography. EM showed a s u b u n i t - l i k e arrangement of b a c t e r i o c i n components around a c e n t r a l core. C a r o t o v o r i c i n - 3 7 9 p a r t i c l e s were i n i t i a t e d as f i m b r a e - l i k e p r o j e c t i o n s which matured by the gradual e x t e r n a l a d d i t i o n o f b a c t e r i o c i n components. T h i s maturation process was accompanied by a gene r a l i n c r e a s e i n molecular weight, b i o a c t i v i t y and u l t r a s t r u c t u r a l appearance of p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 . By u s i n g a mo d i f i e d negative s t a i n i n g p r o t o c o l , c e l l p r o j e c t i o n s which resembled c a r o t o v o r i c i n - 3 7 9 i n t e r m e d i a t e s were seen p h y s i c a l l y a t t a c h e d to i n t a c t producing c e l l s . On the b a s i s of these r e s u l t s a h y p o t h e t i c a l model f o r c a r o t o v o r i c i n - 3 7 9 p r o d u c t i o n was o u t l i n e d . G e n e t i c a n a l y s i s of a temperature s e n s i t i v e , p a r t i c u l a t e b a c t e r i o c i n p r o d u c t i o n i n Erwinia carotovora subsp. carotovora s t r a i n 379 {Ecc 379) was performed w i t h and without m o b i l i z a t i o n v e c t o r iv R68.45. E. coli t r a n s c o n j u g a n t s which both produced p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 and degraded c r y s t a l v i o l e t p e c t a t e medium (CVP) were produced o n l y when R68.45 was used. In a d d i t i o n , the t r a n s f e r f r e q u e n c i e s obtained were i n d i c a t i v e of chromosomally d e r i v e d determinants. Erythromycin- and c h l o r a m p h e n i c o l - r e s i s t a n t t r a n s c o n j u g a n t s were o b t a i n e d r e g a r d l e s s of R68.45 mediation. DNA a n a l y s i s by agarose g e l e l e c t r o p h o r e s i s showed that Ecc c o n t a i n e d a r e s i d e n t megaplasmid which was s e l f - t r a n s m i s s i b l e . T h i s plasmid coded f o r erythromycin and chloramphenicol r e s i s t a n c e along with the pr o d u c t i o n of the low molecular weight c a r o t o v o r i c i n - 3 7 9 component s i m i l a r to that produced by heat t r e a t e d Ecc 379. A n a l y s i s of transc o n j u g a n t s by e l e c t r o n microscopy showed a protuberance of s u r f a c e " b l e b s " or v e s i c l e s . In a d d i t i o n , a l k a l i n e phosphatase (a p e r i p l a s m i c enzyme) was detected i n supernatants of tra n s c o n j u g a n t s . These f a c t s i m p l i e d that the megaplasmid may a l s o be i n v o l v e d i n the r e l e a s e of many exo- p r o t e i n s of Erwinia. P o l y c l o n a l r a b b i t antiserum was developed a g a i n s t p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 . This V antiserum was tested against culture supernatants from wild-type Erwinia and several Erwinia x E. coli transconjugants using r a d i a l immunodiffusion (RID), immunosensitive electron microscopy (ISEM) and Western b l o t t i n g . The polyclonal antiserum against p a r t i c u l a t e carotovoricin-379 reacted with supernatant fractions from a l l transconjugants. CVP+ transconjugants consistently showed a wider range of reactive bacteriocin components than CVP" transconjugants. A low molecular weight p a r t i a l l y active carotovoricin-379 component was detected in CVP- transconjugants by antiserum raised against p a r t i c u l a t e carotovoricin. This suggested a relatedness between the two major bacteriocin components of carotovoricin-379. vi TABLE OF CONTENTS T i t l e Page . i A u t h o r i z a t i o n i i Ab s t r a c t i i i Table of Contents v i i L i s t of Tables x L i s t of Fi g u r e s . . x i Acknowledgements x i v General I n t r o d u c t i o n 1 C h a p t e r 1 U l t r a s t r u c t u r a l Evidence f o r B a c t e r i o c i n S e c r e t i o n by Erwinia carotovora I n t r o d u c t i o n 19 M a t e r i a l s and Methods 22 A. C u l t u r e of Erwinia carotovora s t r a i n s and i n d u c t i o n of b a c t e r i o c i n product ion 22 B. B a c t e r i o c i n p l a t e assays 22 C. 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 . . . . 24 D. Column chromatography 25 E. E l e c t r o n microscopy 25 F. M o d i f i e d negative s t a i n i n g of whole e e l Is 25 G. F i x a t i o n and embedding 26 Res u l t s 27 A. Mitomycin C induced c u l t u r e s 27 B. Examination of the supernatant f r a c t ions 27 C. S e r i a l subunit s t r u c t u r e of p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 30 D. Developmental stages of c a r o t o v o r i c i n - 3 7 9 p r o d u c t i o n by mitomycin C induced c e l l s 33 v i i R e s u l t s (cont.) E. E x t r u s i o n of c a r o t o v o r i c i n through the membrane 38 F. Sephacryl S-300 column chromatography 39 D i s c u s s i o n . 44 C h a p t e r 2 Genetic Determinants of C a r o t o v o r i c i n P r o d u c t i o n i n Erwinia carotovora I n t r o d u c t i o n 53 M a t e r i a l s and Methods 55 A. Media and growth c o n d i t i o n s 55 B. Mating p r o t o c o l s 57 C. B a c t e r i o c i n p l a t e assays 58 D. T o t a l DNA e x t r a c t i o n and e l e c t r o p h o r e s i s 58 Re s u l t s 61 A. Sephacryl S-300 column chromatograpy and b i o a s s a y s 61 B. DNA content 67 C. E l e c t r o n microscopy 67 D i s c u s s i o n 70 C h a p t e r 3 S e r o l o g i c a l R e l a t i o n s h i p s Among the D i f f e r e n t Forms of Erwinia B a c t e r i o c i n Detected by P o l y c l o n a l Antiserum Against P a r t i c u l a t e C a r o t o v o r i c i n - 3 7 9 I n t r o d u c t i o n 76 M a t e r i a l s and Methods 79 A. B a c t e r i a l s t r a i n s 79 B. Development of antiserum 79 C. R a d i a l immunodiffusion 80 D. Immuno-sensitive e l e c t r o n microscopy (ISEM) 80 E. Sodium dodecyl sulphate 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 (SDS-PAGE) 81 F. Western b l o t t i n g 82 v i i i R e s u l t s 83 A. Immuno-sensitive e l e c t r o n microscopy (ISEM) 85 B. SDS-PAGE 87 C. Western b l o t t i n g 90 D i s c u s s i o n 92 General D i s c u s s i o n 96 Summary 105 References 108 ix L I S T OF TABLES Chapter 1 Table 1. C a r o t o v o r i c i n producing and s e n s i t i v e s t r a i n s of Erwinia carotovora 23 Chapter 2 Table 1. B a c t e r i a l s t r a i n s used 56 X L I S T OF FIGURES Chapter 1 Fig u r e 1. Fig u r e 2 Fig u r e 3 Fig u r e 4, Fig u r e 5. Fig u r e 6, E l e c t r o n micrographs of mitomycin C induced c u l t u r e s of Erwinia carotovora subsp. carotovora s t r a i n 379 48 h a f t e r i n d u c t i o n at 20 C 28 Negative s t a i n e d p r e p a r a t i o n s of p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 c o n c e n t r a t e d from the supernatant f r a c t i o n of mitomycin C induced Erwinia carotovora subsp. carotovora s t r a i n 379 , 31 Negative s t a i n e d immature c a r o t o v o r i c i n - 3 7 9 sheared from producing c e l l s of Erwinia carotovora subsp. carotovora s t r a i n 379 8 h a f t e r mitomycin C i n d u c t i o n and the plaque types they induce i n s e n s i t i v e i n d i c a t o r s t r a i n s Negative s t a i n e d c a r o t o v o r i c i n - 3 7 9 p a r t i c l e s and c e l l u l a r p r o j e c t i o n s produced by mitomycin C induced c u l t u r e s of Erwinia carotovora subsp. carotovora s t r a i n 379 34 36 Sephacryl S-300 column chromatograms of c a r o t o v o r i c i n - 3 7 9 at (a) 44, (b) 52, (c) 58 and (d) 62 h a f t e r mitomycin C i n d u c t i o n i n conc e n t r a t e d supernatants of Erwinia carotovora subsp. carotovora s t r a i n 379 40 Sephacryl S-300 column chromatograms of c a r o t o v o r i c i n - 3 7 9 produced by Erwinia carotovora subsp. carotovora s t r a i n 379 48 h a f t e r mitomycin C i n d u c t i o n and e l e c t r o n micrographs of the corresp o n d i n g n e g a t i v e s t a i n e d peak contents 42 x i Figure 7. Sephacryl S-300 chromatograms of carotovoric in-379 produced by Erwinia carotovora subsp. carotovora s t r a i n 379 in mitomycin C induced cul tures grown under d i f f erent condi t ions 45 Figure 8. Hypothet ical model for the production of c a r o t o v o r i c i n by Erwinia carotovora subsp. carotovora s t r a i n 379 49 C h a p t e r 2 Figure 1. Sephacryl S-300 column chromatograms of caro tovor i c in from Erwinia and E. coli transcon jugants 62 Figure 2. Carotovor ic in plate assays of colonies and Sephacryl S-300 fract ionated peaks from concentrated supernatants of E. coli transconjugants and E. carotovora subsp. carotovora (Ecc) s t r a i n 379. . .63 Figure 3. Total DNA analys i s of wi ld-type E. coli, wi ld-type E. carotovora subsp. carotovora s t r a i n 379 (Ecc 379) and Ecc 379 x E. coli transconjugants with and without R68.45 media t ion . . , 68 Figure 4. E lectron micrographs of c e l l s and supernatants of E. coli transconjugants 69 C h a p t e r 3 Figure 1. Radial immunodiffusion analys is of the r e l a t i o n s h i p between p a r t i c u l a t e carotovoric in-379 produced by Erwinia carotovora subsp. carotovora s t r a i n 379 (Ecc 379) and Ecc 379 x E. coli transconjugants 84 x i i F i g u r e 2. D e t e c t i o n of b a c t e r i o c i n s produced by mitomycin C induced Erwinia carotovora subsp. carotovora s t r a i n 379 by immunosensitive e l e c t r o n microscopy employing p o l y c l o n a l antiserum r a i s e d a g a i n s t the l a r g e molecular weight peak obtained by Sephacryl S-300 f r a c t i o n a t i o n of supernatants , 86 F i g u r e 3. SDS-polyacrylamide g e l e l e c t r o p h o r e s i s of con c e n t r a t e d supernatants of Erwinia carotovora subsp. carotovora s t r a i n 379 and Escherichia coli 88 F i g u r e 4. Western b l o t on n i t r o c e l l u l o s e of supernatant p r o t e i n s from Erwinia carotovora subsp. carotovora s t r a i n 379 and Escherichia coli transconjugants f o l l o w e d by immunodetection u s i n g p o l y c l o n a l antiserum a g a i n s t p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 91 x i i i ACKNOWLEDGEMENTS I wish to thank my s u p e r v i s o r Dr. R.J. Copeman f o r f i n a n c i a l support and c r i t i c a l readings and the other members of my committee namely, Dr. M. Shaw, Dr. R.E .W. Hancock and Dr. V.C. Runeckles f o r t h e i r help and suggestions. I would a l s o l i k e to thank Mrs. B. V a l e n t i n e and Mr. F. Skelton f o r help with e l e c t r o n microscopy. Thanks are a l s o extended to B r i a n MacMillan f o r gra p h i c s help, Donna Smith and Robyn DeYoung f o r t y p i n g , Leroy Scrubb f o r help with b i o c h e m i c a l and a n a l y t i c a l techniques and Dr. R. Stace-Smith f o r h e l p f u l s u g g e s t i o n s and comments. F i n a l l y , I would l i k e to thank A g r i c u l t u r e Canada Vancouver Research S t a t i o n f o r the use of t h e i r e l e c t r o n microscope. 1 GENERAL INTRODUCTION Erwinia carotovora s u b s p . carotovora ( J o n e s ) B e r g e y et al. i s r e s p o n s i b l e f o r s o f t - r o t o f a v a r i e t y o f e c o n o m i c a l l y i m p o r t a n t a g r i c u l t u r a l c r o p s . T hese b a c t e r i a a r e o p p o r t u n i s t i c p a t h o g e n s and s u r v i v e as s a p r o p h y t e s . They do n o t a t t a c k h e a l t h y p l a n t m a t e r i a l b u t a r e a b l e t o i n v a d e wounded, weakened o r o l d p l a n t t i s s u e s . W i t h i n i n f e c t e d m a t e r i a l , t h e b a c t e r i a m u l t i p l y i n t h e i n t e r c e l l u l a r s p a c e s and s e c r e t e a v a r i e t y o f enzymes w h i c h d e g r a d e t h e m i d d l e l a m e l l a and c e l l w a l l components. T h i s r e s u l t s i n a s o f t e n i n g and s e p a r a t i o n o f p l a n t c e l l s f o l l o w e d by w a t e r l o s s and e v e n t u a l p l a n t c e l l d e a t h . B e c a u s e t h e s e p o t e n t i a l p a t h o g e n s may be f o u n d i n s o i l as n a t u r a l m i c r o f l o r a , one p o s s i b l e means o f c o n t r o l i n v o l v e s t h e s e l e c t i v e i n h i b i t i o n o f t h e s e o r g a n i s m s by non- p a t h o g e n i c r h i z o s p h e r e i n h a b i t a n t s . The s i m p l e s t c o n c e i v a b l e means o f t h i s t y p e o f b i o - c o n t r o l w o u l d be t h e p r o d u c t i o n o f an i n h i b i t o r y s u b s t a n c e ( i e . a b a c t e r i o c i n ) by some n o n - p a t h o g e n i c o r g a n i s m w h i c h was s p e c i f i c a l l y a c t i v e a g a i n s t Erwinia carotovora. An a l t e r n a t i v e a p p r o a c h might i n v o l v e t h e 2 transfer of genetic information for bac ter ioc in production to a non-pathogenic rhizosphere inhabitant . This l a t t e r approach requires a genetic segregation of bac ter ioc in production from other c e l l funct ions . In some extensively invest igated animal-pathogen systems, th i s segregation has been shown to be a natural rather than a r t i f i c i a l l y constructed separat ion. In some such systems bac ter ioc in and/or v irulence factors have been shown to be segregated on independent genetic elements c a l l e d plasmids. In Erwinia the genetic basis for bac ter ioc in production has not been establ ished; however, cer ta in species of Erwinia have been checked for plasmid content. Erwinia stewartii was shown to contain between 11 to 13 plasmids ranging in s ize from 2.8 to 210 megadaltons (Coplin er al. 1980). Likewise in Erwinia carotovora both large and small molecular weight plasmids have been noted (Zink et al. 1984, Forbes 1981). In a l l cases noted, the plasmids of Erwinia have been c l a s s i f i e d as c r y p t i c with no a t t r i b u t a b l e phenotypes. However, the maintenance of these plasmids suggests that some yet un ident i f i ed t r a i t s may be plasmid coded in 3 Erwinia. Plasmids are d e f i n e d as independently r e p l i c a t i n g extrachromosomal g e n e t i c s t r u c t u r e s (Brock 1979). The e s s e n t i a l f e a t u r e of a plasmid i s that i t must c o n t a i n the g e n e t i c i n f o r m a t i o n f o r i t s own r e p l i c a t i o n and maintenance, u t i l i z i n g the metabolic machinery of the harbouring organism. Most plasmids can be d i l u t e d to the p o i n t of e l i m i n a t i o n u s i n g v a r i o u s s o - c a l l e d c u r i n g treatments. These approaches r e l y upon the f a c t that the e l i m i n a t i o n of most plasmids i s not l e t h a l to the host organism. Thus, plasmids code f o r f u n c t i o n s that are not e s s e n t i a l f o r the s u r v i v a l of the host. They have been i s o l a t e d from a wide v a r i e t y of organisms, s u g g e s t i n g that although plasmid-encoded f e a t u r e s are not e s s e n t i a l , they are probably b e n e f i c i a l . In e v o l u t i o n a r y terms, the s u r v i v a l of the plasmid must be due to an o v e r a l l i n c r e a s e i n the f i t n e s s of the harbouring organism. P h y s i c a l evidence f o r the presence of a plasmid hinges upon the f a c t that a l l plasmids are s u b s t a n t i a l l y s m a l l e r than the c i r c u l a r b a c t e r i a l chromosome and e x i s t as a c o v a l e n t l y c l o s e d 4 s u p e r c o i l e d or t w i s t e d c i r c l e (CCC). This i s the most compact form w i t h i n a c e l l ; however, DNA i s o l a t i o n procedures can i n t r o d u c e a nick or break i n one or both of the DNA s t r a n d s . A n i c k i n one s t r a n d r e s u l t s i n an open c i r c u l a r (OC) DNA which i s not s u p e r c o i l e d , while a n i c k i n both strands r e s u l t s i n a l i n e a r DNA molecule. DNA e x t r a c t i o n and subsequent s e p a r a t i o n of plasmid molecules i n v o l v e s c e l l l y s i s f o l l o w e d by orga n i c e x t r a c t i o n of p r o t e i n and carbohydrates. Even the most g e n t l e l y s i s p r o t o c o l s r e s u l t i n the sh e a r i n g of chromosomal DNA i n t o l i n e a r fragments. Plasmid DNA i s however, l a r g e l y l e f t i n t a c t i n a s u p e r c o i l e d conformation. Plasmid DNA can e a s i l y be separated from l i n e a r DNA u s i n g cesium c h l o r i d e - ethidium bromide (C s C l - E t B r ) d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . This technique i s based upon the f a c t that when a homogeneous s o l u t i o n of cesium c h l o r i d e i s s u b j e c t e d to e x c e s s i v e g r a v i t a t i o n a l f o r c e s such as u l t r a c e n t r i f u g a t i o n , a g r a d i e n t of the s a l t s o l u t i o n i s formed. This s a l t g r a d i e n t s e t s up a d e n s i t y g r a d i e n t and DNA d i s s o l v e d i n the s a l t s o l u t i o n i s separated upon the b a s i s of t h i s d e n s i t y g r a d i e n t . The i n c o r p o r a t i o n of ethidium 5 bromide into the sa l t gradient serves two purposes. F i r s t l y , ethidium bromide in terca la tes into DNA and RNA and provides a v i s u a l , r evers ib l e tag for l o c a l i z a t i o n of the nuc le ic ac ids . Secondly, ethidium bromide i n t e r c a l a t i o n d i f f e r e n t i a l l y lowers the density of the nuc le ic ac ids . Linear and open c i r c u l a r DNA bind more ethidium than highly compact supercoi led DNA. This resu l t s in a banding of plasmid supercoi led DNA at a higher density than l i n e a r or open c i r c u l a r DNA. A l l species of plasmid DNA's which are supercoi led w i l l band at approximately the same densi ty . S i m i l a r i l y , nicked plasmid molecules w i l l band in the same region as l inear chromosomal DNA. Thus, the successful separation of plasmid from chromosomal DNA r e l i e s upon an e f fec t ive l y s i s / e x t r a c t i o n protocol which minimizes the amount of plasmid n i ck ing . A plasmid molecule may be nicked by phys ica l shearing or enzymatic cleavage; The s u s c e p t i b i l i t y of a plasmid to n ick ing i s due p r i m a r i l y to i t s phys ica l s i z e . Larger plasmids provide more s i t e s for enzymatic cleavage and are more suscept ible to shearing. In add i t ion , larger plasmids are in 6 general under a more str ingent contro l by the harbouring c e l l and are found at a l eve l of one to two copies per c e l l (low copy number). In contrast , small plasmids are under a more relaxed contro l and are found in much higher copy number. Furthermore, larger plasmids require cer ta in host components and are thus not ampli f ied to higher leve ls by the addit ion of a prote in synthesis i n h i b i t o r such as chloramphenicol. In s t r i k i n g contrast , smaller plasmids may be ampli f ied to l eve ls of one thousand copies per c e l l . Although large plasmids present p r a c t i c a l problems with respect to t h e i r p u r i f i c a t i o n and subsequent inves t iga t ion , they are usual ly conjugative which f a c i l i t a t e s t h e i r rapid spread throughout a populat ion. In add i t ion , large plasmids may spend a cer ta in proportion of time integrated or covalent ly l inked to the b a c t e r i a l chromosome. Subsequent conjugation and transfer of these integrated plasmids f a c i l i t a t e s a transfer of both plasmid and l inked chromosomal determinants. Thus plasmids, as mobile and var iab le genetic elements, provide important tools for the understanding of prokaryot ic metabolism and 7 r e g u l a t i o n . Plasmids are a l s o important t o o l s f o r the i n v e s t i g a t i o n of s e v e r a l b i o l o g i c a l and e c o l o g i c a l phenomena. The best s t u d i e d and most widespread group of plasmids c o n t a i n r e s i s t a n c e t r a n s f e r f a c t o r s (R f a c t o r s ) which confer m u l t i p l e r e s i s t a n c e to a n t i b i o t i c s . These R plasmids came i n t o prominence at the end of the Second World War when a n t i b i o t i c s were used widely to c o n t r o l dysentery. U n f o r t u n a t e l y , t h i s c r e a t e d a s t r o n g s e l e c t i o n f o r b a c t e r i a harbouring plasmids coding f o r a n t i b i o t i c r e s i s t a n c e . These R f a c t o r s were able to t r a n s f e r v i a co n j u g a t i o n i n a s u r p r i s i n g l y short time to v a r i o u s human and animal pathogens. Plasmids are a l s o i n v o l v e d i n the p r o d u c t i o n of va r i o u s v i r u l e n c e f a c t o r s and t o x i n s r e l a t e d to dis e a s e m a n i f e s t a t i o n . In some e n t e r i c pathogens, the a b i l i t y to c o l o n i z e the small i n t e s t i n e i s due to the presence of a s u r f a c e p r o t e i n (K antigen) which i s coded f o r by a plasmid. Alpha-hemolysin which l y s e s red blood c e l l s , and e n t e r o t o x i n which causes e x c e s s i v e s e c r e t i o n of water and s a l t s are two plasmid-coded t o x i n s produced by enteropathogenic Escherichia coli. 8 The c o n t r o l of both p l a n t and animal pathogens has i n v o l v e d the use of substances which i n h i b i t t h e i r growth. A n t i b i o t i c s were f i r s t i s o l a t e d from n a t u r a l i n h a b i t a n t s of s o i l . These substances had i n general a wide spectrum of a c t i v i t y , i n h i b i t i n g many d i f f e r e n t organisms. The e f f e c t s of a n t i b i o t i c s were e i t h e r b a c t e r i o s t a t i c (stopped growth) or b a c t e r i o c i d a l ( k i l l e d a f f e c t e d organisms). The compounds themselves were simple organic molecules which t a r g e t e d p r i m a r i l y on the p r o t e i n - s y n t h e s i z i n g machinery. However as mentioned above, the widespread use of a n t i b i o t i c s to combat dise a s e s p r o v i d e d an extremely high s e l e c t i o n pressure f o r the development of an e f f i c i e n t r e s i s t a n c e f a c t o r . The r e s u l t was the e v o l u t i o n of R plasmids which u s u a l l y c o ntained m u l t i p l e drug r e s i s t a n c e and were s e l f - t r a n s m i s s i b l e (Broda 1979). The b a s i s of R f a c t o r r e s i s t a n c e was the pr o d u c t i o n of enzymes which m o d i f i e d the incoming a n t i b i o t i c s and rendered them u s e l e s s (Brock 1979). S i m i l a r i l y the s o l u t i o n to R f a c t o r r e s i s t a n c e , from a di s e a s e c o n t r o l s t a n d p o i n t , was the o r g a n i c s y n t h e s i s of a l t e r e d a n t i b i o t i c s which were not 9 recogni z e d by the a n t i b i o t i c - m o d i f y i n g enzymes. This p r a c t i c e set up an ongoing p o s i t i v e feedback c y c l e between chemist and pathogen. As a r e s u l t , b a c t e r i o c i n s were c o n s i d e r e d as a p o s s i b l e a l t e r n a t i v e to a n t i b i o t i c therapy. In 1925, G r a t i a found that a c e r t a i n s t r a i n of E. coli produced a h i g h l y s p e c i f i c a n t i b i o t i c which i n h i b i t e d another s t r a i n ( B i r g e 1981). Fur t h e r r e s e a r c h by many workers showed that most b a c t e r i a produced proteinaceous agents which k i l l e d or i n h i b i t e d c l o s e l y r e l a t e d s p e c i e s . These b a c t e r i o c i n s , as d e f i n e d by Nomura i n 1967, formed a d i v e r s e group of substances f r e q u e n t l y of high molecular weight (with r e s p e c t to a n t i b i o t i c s ) , but u n l i k e a n t i b i o t i c s , showed a very narrow a c t i v i t y spectrum. B a c t e r i o c i n nomenclature r e f l e c t s attempts to name these u b i q u i t o u s agents s y s t e m a t i c a l l y . I n d i v i d u a l b a c t e r i o c i n s are u s u a l l y named a f t e r the producing organisms. For example: i n f l u e n z a c i n s are produced by H. influenzae^ s u b t i l i n s by B. subtilis, pyocins by P. aeruginosa ( o r i g i n a l l y P. pyocyanea) , c o l i c i n s by E. coli and c a r o t o v o r i c i n s by Erwinia carotovora. Subclasses of b a c t e r i o c i n 10 are i d e n t i f i e d by l e t t e r s and/or numbers a f t e r the c l a s s d e s i g n a t i o n . In a d d i t i o n , as s l i g h t l y d i f f e r e n t forms of a p a r t i c u l a r b a c t e r i o c i n may be produced by d i f f e r e n t s t r a i n s , a complete d e s i g n a t i o n a l s o i n c l u d e s the s t r a i n of producer. Thus, c o l i c i n V-K357 i n d i c a t e s a c o l i c i n V produced by s t r a i n K357. The nomenclature i s an important f a c t o r i n the d e s c r i p t i o n of a b a c t e r i o c i n as c e l l s from a producing s t r a i n are immune to t h e i r own b a c t e r i o c i n , but may be s e n s i t i v e to b a c t e r i o c i n produced by another s p e c i e s ( B i r g e 1981). Most b a c t e r i o c i n o g e n i c s t r a i n s produce low l e v e l s under normal c u l t u r a l c o n d i t i o n s . However, b a c t e r i o c i n y i e l d s may be i n c r e a s e d from s e v e r a l to a thousand-fold by the same s o r t s of treatments which cause i n d u c t i o n of lambda prophage ( B i r g e 1981). Such i n d u c t i o n s come from u l t r a v i o l e t (UV) l i g h t or treatment with DNA-damaging chemicals such as mitomycin C. In some temperature s e n s i t i v e or auxotrophic mutants of E. coli, i n d u c t i o n can a l s o be accomplished by heat treatment or n u t r i e n t d e p r i v a t i o n r e s p e c t i v e l y (Mayr-Harting et al. 1972). P a r a l l e l s to phage i n d u c t i o n can a l s o be drawn from the f a c t that most producing c e l l s 11 undergo l y s i s or q u a s i - l y s i s i n r e l e a s i n g b a c t e r i o c i n . T h i s i s probably due to the f a c t that the l a r g e s i z e of these p r o t e i n s exceeds the f u n c t i o n a l p e r m e a b i l i t y of most membranes. Genetic a n a l y s i s of b a c t e r i o c i n p r o d u c t i o n has shown that these determinants are plasmid and/or chromosomally coded ( B i r g e 1981). D i f f u s i b l e lower molecular weight b a c t e r i o c i n s such as c o l i c i n s ( u s u a l l y l e s s than 100 KD) were found to be coded f o r by plasmids whereas, l a r g e p a r t i c u l a t e b a c t e r i o c i n s (>500 KD) were found to be p r i m a r i l y coded f o r by the b a c t e r i a l chromosome. As expected, c o l i c i n s or E. coli b a c t e r i o c i n s have been the most e x t e n s i v e l y s t u d i e d group. Genetic a n a l y s i s has shown that i n a l l cases i n v e s t i g a t e d so f a r , c o l i c i n s have been coded f o r by plasmids (Broda 1979). The plasmids f a l l i n t o two major c l a s s e s . Some of these plasmids are l a r g e ( g r e a t e r than 60 megadaltons), c o n j u g a t i v e , and present i n low copy number (one to two per c e l l ) while others are small ( l e s s than 6 megadaltons), non- c o n j u g a t i v e and present i n high copy numbers (10-30 per c e l l ) . C o l i c i n - p r o d u c i n g plasmids have always been denoted by Col f o r c o l i c i n f o l l o w e d by an 12 a p p r o p r i a t e l e t t e r . These plasmids have been made i n v a l u a b l e by g e n e t i c d e l e t i o n and i n s e r t i o n of markers forming v a r i o u s u s e f u l c l o n i n g v e h i c l e s with c o l i c i n plasmid r e p l i c a t i v e f u n c t i o n ( c o l r e p l i c o n s ) . C o l i c i n s were c l a s s i f i e d on the b a s i s of a c t i v i t y a g i n s t s e v e r a l E. coli s t r a i n s . T h i s was however complicated by the f a c t that most s t r a i n s produced s e v e r a l c o l i c i n s . As an a l t e r n a t i v e , c o l i c i n s have been c l a s s i f i e d by a c t i v i t y on o r i g i n a l l y s e n s i t i v e d e r i v a t i v e s of p a r t i c u l a r s t r a i n s ( c r o s s r e s i s t a n c e grouping) (Broda 1979). Thus, d e r i v a t i v e s of c e l l s which are t o l e r a n t to a l l c o l i c i n s i n the cr o s s r e s i s t a n c e group A are designated t o l A. C e l l s designated phage T l r e s i s t a n t (tonB) or e n t e r o c h e l i n e x c r e t i o n (exbB) are r e s i s t a n t to a l l c o l i c i n s i n the cross r e s i s t a n c e group B. S e v e r a l c o l i c i n r e c e p t o r s have been l o c a l i z e d on the outer membrane and r e s i s t a n c e to a p a r t i c u l a r c o l i c i n can i n most but not a l l cases be t r a c e d to an a l t e r a t i o n or l o s s of t h i s r e c e p t o r (Broda 1979). Bi n d i n g and subsequent a c t i o n of a c o l i c i n to a s e n s i t i v e c e l l u s u a l l y r e s u l t s i n a k i l l i n g of that 13 c e l l . There are three general ways i n which c o l i c i n s are known to k i l l c e l l s (Broda 1979). Some c o l i c i n s act at the c e l l membrane and act as energy uncouplers ( c o l E l and K), while others cause a degradation of DNA ( c o l E2) or RNA ( c o l E3) (Broda 1979). As mentioned above, producing c e l l s are immune to the b a c t e r i o c i n they produce. The molecular b a s i s f o r t h i s immunity has been i d e n t i f i e d f o r both c o l i c i n E2 and E3. C e l l s producing these p r o t e i n s a l s o produce a small molecular weight immunity p r o t e i n (about 10,000 D) which complexes with each c o l i c i n i n a r a t i o of 1:1. D i s s o c i a t i o n of t h i s p r o t e i n from the a p p r o p r i a t e c o l i c i n r e s u l t s i n an i n c r e a s e i n the i n v i t r o a c t i v i t y of the c o l i c i n ( B i r g e 1981). The marked s i m i l a r i t i e s i n i n d u c t i o n p r o t o c o l s and assay c o n d i t i o n s f o r both b a c t e r i o c i n s and bacteriophage, together with t h e i r p o s s i b l e co- e x i s t e n c e i n a p a r t i c u l a r c e l l n e c e s s i t a t e s a method of d i s t i n c t i o n . The o l d e s t and p o s s i b l y s i m p l e s t method takes advantage of the f a c t that bacteriophage, u n l i k e b a c t e r i o c i n s , m u l t i p l y i n t h e i r host c e l l s r e s u l t i n g i n l o c a l i z e d areas of i n c r e a s e d t i t r e . Thus, a s e r i e s of d i l u t i o n s of 14 the sample under t e s t , s p o t t e d on an i n d i c a t o r lawn of b a c t e r i a produces d i f f e r e n t i a l r e s u l t s f o r phage and b a c t e r i o c i n . D i l u t i o n of phage r e s u l t s i n a de c r e a s i n g number of d i s c r e t e phage plaques whereas b a c t e r i o c i n d i l u t i o n r e s u l t s i n a gradual t h i n n i n g of growth, which i s more marked at the highest d i l u t i o n s (Mayr-Harting et al. 1972). The d i s t i n c t i o n between b a c t e r i o c i n s and bacteriophage became p a r t i c u l a r l y d i f f i c u l t with P. aeruginosa. This organism produces a l a r g e p a r t i c u l a t e p y ocin whose estimated molecular weight was s e v e r a l m i l l i o n . E l e c t r o n microscopy of pyocin r e v e a l e d a phage t a i l - l i k e p a r t i c l e complete with base p l a t e , f i b r e s , core and sheath. Routine spot assays of producing s t r a i n s on lawns of i n d i c a t o r showed no zones of c l e a r i n g due to the poor d i f f u s i b i l i t y of pyoc i n . In a d d i t i o n , almost a l l s t r a i n s of P. aeruginosa contained one or more bacteriophage with v a r i o u s degrees of d e f e c t i v e n e s s (Mayr-Harting et al. 1972). These bacteriophage t a i l s had b a c t e r i o c i d a l a c t i v i t y , s i m i l a r to pyocin (Shinomiya et al, 1979). Both types of p a r t i c l e s were induced by standard i n d u c t i o n regimes u s i n g mitomycin C or UV l i g h t . 15 A comparative study of the s o - c a l l e d R-type pyocins of P. aeruginosa showed that a l l types c r o s s r e a c t e d immunologically and appeared almost i d e n t i c a l under EM. In a d d i t i o n a l l pyocins were shown to have a s i m i l a r mode of a c t i o n which r e s u l t e d i n a shut-down of p r o t e i n and macromolecular s y n t h e s i s (Ohsumi et al. 1980). P r o t e i n subunit composition of v a r i o u s pyocins was found to be almost i d e n t i c a l except f o r a p r o t e i n component of the t a i l f i b e r which d i f f e r e d i n molecular weight. It has been i m p l i e d that d i f f e r e n c e s i n t h i s t a i l f i b e r r e g i o n are r e s p o n s i b l e f o r the s p e c i f i c b i n d i n g of d i f f e r e n t pyocins to the l i p o p o l y s a c c h a r i d e r e c e p t o r s of s e n s i t i v e s t r a i n s (Ohsumi et al. 1980). Due to r e s i s t a n c e problems encountered i n medicine, the widespread use of a n t i b i o t i c s i n pl a n t pathology has not been a common p r a c t i c e . As an a l t e r n a t i v e , the prospect f o r c o n t r o l of phytopathogenic b a c t e r i a by bacteriophage and b a c t e r i o c i n s was i n v e s t i g a t e d (Vidaver 1976). B a c t e r i o c i n s were i d e a l narrow spectrum s p e c i f i c a n t i b a c t e r i a l compounds which were l e s s p e r s i s t e n t , and e n v i r o n m e n t a l l y a c c e p t a b l e . U n f o r t u n a t e l y few 16 b a c t e r i o c i n s of phytopathogenic b a c t e r i a were w e l l c h a r a c t e r i z e d (Vidaver 1976). The use of agocin-84 f o r the c o n t r o l of crown g a l l was the only w e l l c h a r a c t e r i z e d and f i e l d t e s t e d " b a c t e r i o c i n " . However agrocin-84 by d e f i n i t i o n d i d not f a l l i n t o the category of a b a c t e r i o c i n as i t was not proteinaceous (Vidaver 1976). In 1961 Hamon and Peron showed that Erwinia carotovora subspecies carotovora (van H a l l ) Dye produced a b a c t e r i o c i n which they c a l l e d c a r o t o v o r i c i n . An ap p a r e n t l y s i m i l a r b a c t e r i o c i n was p u r i f i e d to homogeneity and c h a r a c t e r i z e d i n 1978 ( I t o h et al. 1978). C a r o t o v o r i c i n - E R was shown to be a t h e r m o l a b i l e , p a r t i c u l a t e p r o t e i n , s e n s i t i v e to sodium dodecyl sulphate (SDS), unstable to high or low pH, but s t a b l e to h y d r o l y t i c d i g e s t i o n by v a r i o u s p r o t e o l y t i c enzymes i n n a t i v e conformation ( I t o h et al. 1978). Syn t h e s i s of c a r o t o v o r i c i n - E R was i n d u c i b l e by UV l i g h t and was accompanied by c e l l l y s i s 3-5 hours a f t e r i n d u c t i o n . C a r o t o v o r i c i n - E R v i s u a l i z e d by EM showed a s t r i k i n g resemblance to pyocin R ( I t o h et al. 1978, Kamimiya et al. 1977) B a c t e r i o c i n a d d i t i o n to s e n s i t i v e c e l l s caused 17 a r a p i d and e x t e n s i v e l y s i s . T h i s l y s i s was i n h i b i t e d by the a d d i t i o n of magnesium (I t o h et al. 1980). Further s t u d i e s with a phospholipase A" mutant s t r a i n of Erwinia showed that c a r o t o v o r i c i n - ER b i n d i n g r e s u l t e d i n the a c t i v a t i o n of a membrane bound phospholipase A l e a d i n g to c e l l l y s i s . T h i s l y s i s was v i r t u a l l y i n h i b i t e d i n the phospholipase A mutant. However the c e l l s although i n t a c t , were s t i l l k i l l e d by c a r o t o v o r i c i n - E R , s u g g e s t i n g that phospholipase A a c t i v a t i o n i s not the primary b a c t e r i c i d a l a c t i o n of t h i s c a r o t o v o r i c i n ( I t o h et al. 1981). C a r o t o v o r i c i n - E R was l a t e r shown to cause a r e d u c t i o n i n i n t e r n a l ATP l e v e l which was not due to an a c t i v a t i o n of ATPase but to an i n a c t i v a t i o n of the e n e r g i z e d s t a t e of the c y t o p l a s m i c membrane necessary f o r ATP s y n t h e s i s and t r a n s p o r t of amino a c i d s ( I t o h et al. 1982). E s s e n t i a l l y c a r o t o v o r i c i n - E R reduced the proton motive f o r c e (combination of chemical p o t e n t i a l and proton g r a d i e n t ) which d r i v e s ATP s y n t h e s i s most probably by the i n t r o d u c t i o n of n o n - s p e c i f i c membrane channels. In order to use a b a c t e r i o c i n as a p o s s i b l e c o n t r o l agent, a thorough understanding of the 18 a c t i v i t y spectrum, mode of a c t i o n and g e n e t i c and p h y s i c a l b a s i s f o r p r o d u c t i o n i s necessary. The a c t i v i t y spectrum of c a r o t o v o r i c i n - 3 7 9 produced by Erwinia carotovora subsp. carotovora s t r a i n 379 has been i n v e s t i g a t e d p r e v i o u s l y ( J a i s 1982). In r a d d i t i o n , the mode of a c t i o n of c a r o t o v o r i c i n - E R had been e x t e n s i v e l y i n v e s t i g a t e d ( I t o h et al. 1980a, 1980b, 1980c, 1981, 1982). The aim of t h i s t h e s i s was to study the pr 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 p e c i f i c o b j e c t i v e s were: 1) To i n v e s t i g a t e the s t r u c t u r a l b a s i s of c a r o t o v o r i c i n - 3 7 9 p r o d u c t i o n u s i n g e l e c t r o n microscopy. 2) To determine i f c a r o t o v o r i c i n - 3 7 9 p r o d u c t i o n i s chromosomally and/or plasmid coded. 3) To determine the s e r o l o g i c a l r e l a t i o n s h i p s of the v a r i o u s components of c a r o t o v o r i c i n - 3 7 9 . 19 CHAPTER 1 ULTRASTRUCTURAL EVIDENCE FOR BACTERIOCIN SECRETION BY ERWINIA CAROTOVORA INTRODUCTION B a c t e r i o c i n s are p roteinaceous a n t i m i c r o b i a l agents 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 which are d i r e c t e d a g a i n s t c l o s e l y r e l a t e d s t r a i n s (Nomura 1967). The b i o l o g i c a l r o l e of b a c t e r i o c i n s i n v o l v e s c o n f e r r i n g a s e l e c t i v e advantage to producing s t r a i n s , by k i l l i n g r e l a t e d s t r a i n s . Some b a c t e r i o c i n s , such as pyocins, have h i g h l y complex p r o t e i n s t r u c t u r e s which resemble bacteriophage t a i l s . In f a c t , most b a c t e r i o c i n s are induced by prophage-inducing agents such as mitomycin C or u l t r a v i o l e t i r r a d i a t i o n . L i k e lambda prophage, b a c t e r i o c i n s may be r e l e a s e d e i t h e r by a l y s i s , or q u a s i - l y s i s mechanism depending on the molecular weight of the p r o t e i n and inherent p e r m e a b i l i t y of the producing s t r a i n ( B i r g e 1981). These o b s e r v a t i o n s imply that b a c t e r i o c i n p r o d u c t i o n may have e v o l u t i o n a r y l i n k s 20 to d e f e c t i v e phage components. B a c t e r i o c i n p r o d u c t i o n by Erwinia carotovora, which causes potato b l a c k l e g and s o f t r o t of a v a r i e t y of economically important a g r i c u l t u r a l crops, was f i r s t shown by Hamon and Peron i n 1961. Subsequently they coined the term c a r o t o v o r i c i n f o r b a c t e r i o c i n s produced by Erwinia carotovora (Ecc). F u r t h e r r e s e a r c h has demonstrated the e x i s t e n c e of two types of b a c t e r i o c i n a c t i v i t y i n some s t r a i n s . One i s c h a r a c t e r i z e d by l a r g e d i f f u s e zones of i n h i b i t i o n and the other by small c l e a r zones of i n h i b i t i o n (Crowley and DeBoer 1980, J a i s 1982). P h y s i c a l c h a r a c t e r i z a t i o n of p u r i f i e d c a r o t o v o r i c i n p r e p a r a t i o n s having the l a t t e r a c t i v i t y e s t a b l i s h e d that t h e r m o l a b i l e , p a r t i c u l a t e , t r y p s i n - r e s i s t a n t p r o t e i n s were i n v o l v e d ( I t o h et al. 1978). Phage t a i l - l i k e p a r t i c l e s with a c o n t r a c t i l e sheath, core, b a s e - p l a t e and f i b r e s were observed by e l e c t r o n microscopy (EM) (It o h et al. 1978). I n i t i a l work with c a r o t o v o r i c i n s i m p l i e d that r e l e a s e o f these b a c t e r i o c i n s i s due to c e l l l y s i s f o l l o w i n g i n d u c t i o n . However, some s t r a i n s of Erwinia produce s u b s t a n t i a l amounts of c a r o t o v o r i c i n c o n s t i t u t i v e l y with no s i g n i f i c a n t 21 r e d u c t i o n i n t u r b i d i t y or c e l l v i a b i l i t y . Moreover, l y s i s was dete c t e d only a f t e r i n d u c t i o n by mitomycin C or u l t r a v i o l e t i r r a d i a t i o n and f o l l o w i n g an i n c r e a s e i n c a r o t o v o r i c i n t i t e r s ( I t o h et al. 1978). These o b s e r v a t i o n s suggested that p o s s i b l y some mechanism other than c e l l l y s i s was a l s o i n v o l v e d i n c a r o t o v o r i c i n l i b e r a t i o n . In t h i s study, c a r o t o v o r i c i n - 3 7 9 p r o d u c t i o n i n Erwinia carotovora subspecies carotovora s t r a i n 379 (Ecc 379) was i n v e s t i g a t e d by u l t r a s t r u c t u r a l examination of b a c t e r i o c i n - p r o d u c i n g c e l l s at va r i o u s times a f t e r i n d u c t i o n . The obs e r v a t i o n s made support the hypothesis that the p a r t i c u l a t e component of c a r o t o v o r i c i n - 3 7 9 i s s e c r e t e d on membrane enclosed, f i m b r a e - l i k e p r o j e c t i o n s , the t i p s of which s w e l l to form detachable v e s i c u l a r heads. 22 MATERIALS AND METHODS Culture of Erwinia carotovora S tra ins and Induction of Bacter ioc in Production: Erwinia carotovora subsp. carotovora s t r a i n 379 was chosen as a b a c t e r i o c i n producer. This s t r a i n c o n s t i t u t i v e l y produces p a r t i c u l a t e b a c t e r i o c i n but can be induced to produce i t at much higher t i t r e s . Three s t r a i n s of E. carotovora subspecies atroseptica and one s t r a i n of E. carotovora subsp. carotovora were used as i n d i c a t o r s (Table 1). B a c t e r i a were grown i n L u r i a Broth (Ma n i a t i s et al. 1984) pH 7.4 or minimal M9 medium pH 7.4 (Man i a t i s et al. 1984) at 20 C on a r o t a r y shaker (100 rpm). Enhanced b a c t e r i o c i n p r o d u c t i o n was induced by the a d d i t i o n of mitomycin C at 0.2 ug/ml 2 hours (h) a f t e r i n o c u l a t i o n . Bacter ioc in Plate Assays: C u l t u r e s were vortexed at hig h speed f o r 30 seconds (s) at 8, 12 and 24 h a f t e r i n d u c t i o n , and supernatants were viewed by e l e c t r o n microscopy (EM) as d e s c r i b e d below. B a c t e r i o c i n a c t i v i t y was T a b l e 1: 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 o f Erwinia carotovora P r o d u c e r : Erwinia carotovora s e r o g r o u p XI S e n s i t i v e s t r a i n s : Erwinia carotovora s e r o g r o u p I Erwinia carotovora s e r o g r o u p XXII Erwinia carotovora s e r o g r o u p XX Erwinia carotovora s e r o g r o u p X V I I I s u b s p . carotovora s t r a i n 379 s u b s p . atroseptica s t r a i n SR8 s u b s p . atroseptica s t r a i n 496 s u b s p . atroseptica s t r a i n 530 s u b s p . carotovora s t r a i n 504 24 assayed by p l a c i n g 5 u l of t e s t samples on a lawn of i n d i c a t o r s t r a i n s seeded i n peptone s o f t agar (0.85% NaCl, 1% Bacto-Peptone, 0.45% Bacto-Agar pH 7.4) . Concentration of Carotovoric in-379: A l i q u o t s of induced c u l t u r e s were p e l l e t e d at 10,500 x g f o r 20 min. The supernatant f r a c t i o n s were b r i e f l y vortexed (15 s at high speed) and f i l t e r - s t e r i l i z e d u s i n g a 0.22 um f i l t e r . S t e r i l i z e d 18% (w/v) p o l y e t h y l e n e g l y c o l (PEG) M.W. 8000 was d i s s o l v e d i n the s t e r i l e supernatants at 22 C and the mixtures were allowed to stand on i c e overnight to f a c i l i t a t e p r e c i p i t a t i o n . The p r e c i p i t a t e s were c o l l e c t e d by c e n t r i f u g a t i o n at 13,500 x g f o r 25 min and the p e l l e t s were d i s s o l v e d i n 1/100 the o r i g i n a l volume of 5OmM sodium phosphate b u f f e r pH 7.4. I n s o l u b l e m a t e r i a l was removed by low speed c e n t r i f u g a t i o n at 8000 x g f o r 10 min at 4 C. The r e s u l t i n g c a r o t o v o r i c i n - 3 7 9 p r e p a r a t i o n s were analysed by column chromatography or e l e c t r o n microscopy. 25 Column Chromatography: Concentrated c a r o t o v o r i c i n - 3 7 9 (0.5 ml) was a p p l i e d to the top of a 35 cm x 1.8 cm column packed with S e p a c r y l S-300. The running b u f f e r was 5OmM sodium phosphate pH 7.4 and the flow r a t e was maintained at about 0.5 ml/min. E l u a t e was monitored at 280 nm, and peaks were c o l l e c t e d and bioassayed as d e s c r i b e d above. Electron Microscopy: Concentrated c a r o t o v o r i c i n - 3 7 9 (10 u l ) was loaded onto copper g r i d s coated with c o l l o d i o n - carbon and incubated at room temperature f o r 3 to 5 min. G r i d s were then s t a i n e d with 10 to 12 drops of 2% phosphotungstic a c i d (PTA) pH 7.0, allowed to dry, and viewed i n a P h i l l i p s EM-300 e l e c t r o n microscope ( H i l l 1984). Modified Negative Sta in ing of Whole C e l l s : In order to i n c r e a s e the r e s o l u t i o n of n e g a t i v e l y s t a i n e d whole c e l l p r e p a r a t i o n s , two p r o t o c o l s f o r c e l l pretreatment were used. In the f i r s t procedure, c u l t u r e s were p r e f i x e d i n 0.2% osmium t e t r a o x i d e ( f i n a l c o n c e n t r a t i o n added 26 d i r e c t l y to growth media) f o r 1 h at 20 C, washed t h r i c e with s t e r i l e b r o t h and s t a i n e d with 2% PTA pH 7.0. In the second pretreatment procedure, reagent grade toluene was added at 1:2 (v/v) to an a l i q u o t of b a c t e r i a l c e l l s . The tubes were capped and g e n t l y i n v e r t e d 10 times a l l o w i n g complete s e p a r a t i o n of the two phases a f t e r each i n v e r s i o n . The c e l l suspension was then removed and s t a i n e d as be f o r e with PTA. Fixation and Embedding: C e l l s were f i x e d i n agar a c c o r d i n g to the R y t e r - K e l l e n b e r g e r procedure (1958) u s i n g \% osmium t e t r o x i d e i n v e r o n a l a c e t a t e b u f f e r supplemented with c a l c i u m c h l o r i d e ( K e l l e n b e r g e r b u f f e r ) , f o r 14 to 16 h at room temperature. Blocks of c e l l s were s u b j e c t e d to a s e r i a l a l c o h o l dehydration and embedded i n EPON. Thin s e c t i o n s were s t a i n e d f o r 15 to 20 min i n 5* u r a n y l a c e t a t e . 27 RESULTS Mitomycin C Induced Cultures: U l t r a s t r u c t u r a l examination of induced c u l t u r e s of E. carotovora subsp. carotovora a f t e r 48 h at 20 C showed b a c t e r i a l c e l l s with a p r o l i f e r a t i o n of f i m b r a e - l i k e p r o j e c t i o n s of v a r i o u s lengths ( F i g . 1). Low l e v e l s of p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 were seen i n c l o s e a s s o c i a t i o n with i n t a c t b a c t e r i a l c e l l s ( F i g . I d ) . Thin s e c t i o n s of induced c e l l s r e v e a l e d p r o j e c t i o n s with a subunit s t r u c t u r e at v a r i o u s stages of e l o n g a t i o n ( F i g . I c ) . In some p r e p a r a t i o n s , v e s i c u l a r p r o j e c t i o n s were a l s o present i n a d d i t i o n to f i m b r a l p r o j e c t i o n s ( F i g . l a - b ) . Examination of the Supernatant Fract ions: F i l t e r s t e r i l i z a t i o n (0.22 um f i l t e r ) of supernatants from induced c u l t u r e s markedly reduced b a c t e r i o c i n a c t i v i t y . However, a b r i e f (15 s) sh e a r i n g by vortex b e f o r e f i l t r a t i o n r e s u l t e d i n a r e t e n t i o n of a c t i v i t y . M i c r o s c o p i c examination of n o n - f i l t e r - s t e r i l i z e d p r e p a r a t i o n s showed that 28 F i g u r e 1. E l e c t r o n micrographs of mitomycin C induced c u l t u r e s of Erwinia carotovora subsp. carotovora s t r a i n 379 48 h a f t e r i n d u c t i o n at 20 C (Bars=100nm). a-b. Negative s t a i n e d c e l l s showing both v e s i c u l a r and f i m b r a e - l i k e p r o j e c t i o n s ; c. Thin s e c t i o n of a producing c e l l showing subunit s t r u c t u r e of f i m b r a e - l i k e p r o j e c t - ions at v a r i o u s stages of e l o n g a t i o n . I n s e r t : area c o n t a i n i n g e a r l y stages of p r o j e c t i o n ; d. Negative s t a i n e d c e l l showing f i m b r a e - l i k e p r o j e c t i o n s and c l o s e a s s o c i a t i o n of p a r t i c u l a t e c a r o t o v o r i c i n with i n t a c t c e l l . 29 30 c a r o t o v o r i c i n p a r t i c l e s were aggregated and p h y s i c a l l y attached by a c e n t r a l core to v e s i c l e s ( F i g . 2a) This o b s e r v a t i o n e x p l a i n e d the l o s s of a c t i v i t y upon s t e r i l i z a t i o n as v e s i c l e s and attached c a r o t o v o r i c i n - 3 7 9 would be r e t a i n e d on the f i l t e r . The b r i e f s h e a r i n g presumably detached these p a r t i c l e s . V e s i c l e s o f t e n had long t h r e a d - l i k e p r o j e c t i o n s attached to them which were of e x a c t l y the same diameter as c a r o t o v o r i c i n cores ( F i g . 2b). S e r i a l Subunit Structure of P a r t i c u l a t e Carotovoric in-379: C a r o t o v o r i c i n p a r t i c l e s c o n s i s t e d of subunits arranged around a c e n t r a l core which extended through the sheath ( F i g . 2c-g). In r a r e i n s t a n c e s the core was attached t e r m i n a l l y to a v e s i c u l a r head ( F i g . 2g). Although attached heads were uncommon, numerous detached heads were found i n concentrated p r e p a r a t i o n s s u g g e s t i n g a r a t h e r weak a s s o c i a t i o n with the core ( F i g . 2g). 31 F i g u r e 2. Negative s t a i n e d p r e p a r a t i o n s of p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 concentrated from the supernatant f r a c t i o n of mitomycin C induced Erwinia carotovora subsp. carotovora s t r a i n 379 (Bars=100nm). a. C a r o t o v o r i c i n - 3 7 9 attachment to v e s i c l e s by c e n t r a l core; b. V e s i c l e with long c o r e - l i k e p r o j e c t i o n s attached; c. C o n t r a c t e d form of c a r o t o v o r i c i n - 3 7 9 showing core surrounded by sheath. Note d i a g o n a l l y d i s p l a c e d , d i s r u p t e d p a r t i c l e i l l u s t r a t i n g a c e n t r a l core with a modular arrangement of surrounding sheath; d. P a r t i a l l y d i s r u p t e d c a r o t o v o r i c i n - 3 7 9 sheath exposing a connecting core. Note the ova l upper terminus of the core; 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 with an i n t a c t sheath surrounding the lower h a l f and a p a r t i a l l y d i s r u p t e d sheath surrounding the upper h a l f . This d i s r u p t e d sheath p a r t i a l l y obscures a c e n t r a l core which terminates i n a b l o c k - l i k e s t r u c t u r e ; f. Two separated sheath components each c o n t a i n i n g a p r o t r u d i n g core; g. C o n t r a c t e d form of c a r o t o v o r i c i n - 3 7 9 showing atta c h e d head. Attachment i s f a c i l i t a t e d by ste m - l i k e p r o j e c t i o n s . Note numerous f r e e f l o a t i n g v e s i c u l a r heads. 3 2 33 Developmental Stages of C a r o t o v o r i c i n - 3 7 9 Production by Mitomycin C Induced C e l l s : P a r t i c l e s found i n the concentrated supernatants 8 h a f t e r i n d u c t i o n appeared to be immature c a r o t o v o r i c i n on the b a s i s of appearance and a c t i v i t y on known i n d i c a t o r s s t r a i n s ( F i g . 3a- f ) . In some cases these immature p a r t i c l e s were f u l l y e ncapsidated while others had a d e f i n i t e s w e l l i n g ( F i g . 3b). Other p a r t i c l e s were open at both ends sugge s t i n g i n t e r m e d i a t e p o s i t i o n s on a b a c t e r i o c i n p a r t i c l e ( F i g . 3c and d). These p a r t i c l e s were observed to aggregate end to end at a low frequency, s u g g e s t i n g a s e r i a l a s s o c i a t i o n ( F i g . 3d). P a r t i c l e s found i n 12 h p r e p a r a t i o n s resembled mature c a r o t o v o r i c i n - 3 7 9 . They were l a r g e r i n diameter and more elongate than 8 h p a r t i c l e s with a d e f i n i t e beaded s t r u c t u r e ( F i g . 4a). At t h i s stage, cores i n the process of t h i c k e n i n g were observed ( F i g . 4b). Whole c e l l p r e p a r a t i o n s were a l s o observed with r e l e a s e d p a r t i c l e s , or p a r t i a l l y formed p a r t i c l e s with subunit a d d i t i o n to surround and enclose the numerous p r o j e c t i n g core f i l a m e n t s ( F i g . 4 c ) . F u l l y mature p a r t i c l e s were r e l e a s e d 34 Fi g u r e 3. Negative s t a i n e d immature c a r o t o v o r i c i n - 3 7 9 sheared from producing c e l l s of Erwinia carotovora subsp. carotovora s t r a i n 379 8 h a f t e r mitomycin C i n d u c t i o n and the plaque types they induce i n s e n s i t i v e i n d i c a t o r s t r a i n s . a. Negative s t a i n e d p a r t i c l e s found i n supernatant (Bar=100nm); b. Immature p a r t i c l e s showing t e r m i n a l v e s i c u l a r s w e l l i n g and p a r t i a l e n c a p s i d a t i o n (Bar=100nm); c. S h e a t h - l i k e p a r t i c l e without p r o j e c t i n g core (Bar=10nm); d. Two s h e a t h - l i k e p a r t i c l e s as seen i n 3c l i n k e d end-on (Bar=10nm); e. T y p i c a l c l e a r plaques produced by l a r g e molecular weight c a r o t o v o r i c i n - 3 7 9 (Seph- a c r y l S-300 peak 1); f. T y p i c a l d i f f u s e plaques produced by low molecular weight immature c a r o t o v o r i c i n - 3 7 9 (Sephacryl S-300 peaks 2 to 4).  36 F i g u r e 4. Negative s t a i n e d c a r o t o v o r i c i n - 3 7 9 p a r t i c l e s and c e l l u l a r p r o j e c t i o n s produced by mitomycin C induced c u l t u r e s of Erwinia carotovora subsp. carotovora s t r a i n 379. C u l t u r e f l u i d s were e i t h e r n e g a t i v e l y s t a i n e d with 2% phosphotungstic a c i d pH 7.0 (a to d), or p r e t r e a t e d with 0.2* osmium t e t r o x i d e (e) or an equal volume of toluene ( f to i ) p r i o r to negative s t a i n i n g . A l l bars = 100 nm. a. C a r o t o v o r i c i n - 3 7 9 p a r t i c l e s 12 h a f t e r i n d u c t i o n which appear elongated and thick e n e d due to the a d d i t i o n of m a t e r i a l to the developing c e n t r a l core; b. I s o l a t e d c e n t r a l core i n the process of t h i c k e n i n g ; c. C a r o t o v o r i c i n - 3 7 9 p a r t i c l e s e i t h e r r e l e a s e d ( l e f t ) or i n the process of t h i c k e n i n g ( r i g h t ) i n a s s o c i a t i o n with i n t a c t c e l l s 12 h a f t e r i n d u c t i o n ; d. F u l l y mature c a r o t o v o r i c i n - 3 7 9 p a r t i c l e s r e l e a s e d 24 h a f t e r i n d u c t i o n with c o n t r a c t e d ( l e f t ) or extended ( r i g h t ) sheaths; e. I n t a c t mature c a r o t o v o r i c i n - 3 7 9 p a r t i c l e w ith attached head p r o t r u d i n g from a producing c e l l ; f . C o r e - l i k e p r o j e c t i o n with t e r m i n a l v e s i c l e attached to b a c t e r i a l c e l l 8 h a f t e r i n d u c t i o n ; g. B a c t e r i o c i n - l i k e p r o j e c t i o n attached to a producing c e l l 12 h a f t e r i n d u c t i o n ; h. P a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 attached to and r e l e a s e d from an i n t a c t producing c e l l 16 h a f t e r i n d u c t i o n ; i . P r o t r u d i n g p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 with attached head surrounded by i n t a c t outer membrane 24 h a f t e r i n d u c t i o n . 37 38 into the supernatant and were readily detected in contracted or extended forms 24 h after induction (Fig. 4d). Extrusion of Carotovoricin through the Membrane: The modified staining procedure using osmium tetroxide provided more d e t a i l and c l e a r l y showed carotovoricin-379 p a r t i c l e s with attached heads extruded through the membrane (Fig. 4e). The disadvantage of the procedure was that the washings resulted in a loss of b a c t e r i a l surface appendages (Fig. 4e) and p a r t i a l disruption of bacteriocin p a r t i c l e s . B a c t e r i o c i n - l i k e projections at 8, 12, 16 and 24 h respectively, after induction (Fig. 4 f - i ) were vi s u a l i z e d with the modified staining procedure using a toluene pretreatment. These p a r t i c l e s corresponded well in appearance and dimensions with the immature p a r t i c l e s i s o l a t e d in the sheared supernatant preparations described previously. However, these preparations showed physical association of bacteriocin intermediates with intact c e l l s . Pretreatment with toluene presumably p a r t i a l l y disrupted the outer membrane allowing 39 p e n e t r a t i o n of PTA. As no washing steps were necessary, b a c t e r i a l s u r f a c e u l t r a s t r u c t u r e remained i n t a c t . S e p h a c r y l S - 3 0 0 C o l u m n C h r o m a t o g r a p h y : Gel f i l t r a t i o n of concentrated c a r o t o v o r i c i n - 379 harvested at 44, 52, 58 and 62 h a f t e r i n d u c t i o n showed a d e f i n i t e s h i f t to higher molecular weight b a c t e r i o c i n ( F i g . 5). At 44 h, two major peaks corresponding to g l o b u l a r p r o t e i n molecular weights of 300,000 da l t o n s (peak 1) and 20,000 da l t o n s (peak 2) r e s p e c t i v e l y had b a c t e r i o c i n a c t i v i t y . In a d d i t i o n a minor peak e q u i v a l e n t to 15,000 da l t o n s (peak 3) was present and had b a c t e r i o c i n a c t i v i t y . This minor peak appeared with an even s m a l l e r molecular weight peak (about 10,000 da l t o n s ) (peak 4) at 52 h a f t e r i n d u c t i o n . At t h i s time peak 2 was almost at the l e v e l of peak 3 and peak 1 had doubled i n c o n c e n t r a t i o n . This general trend was seen i n the s h i f t from 58 to 62 h a f t e r i n d u c t i o n with the appearance of s t i l l another b a c t e r i o c i n peak ( l b ) corresponding to 180,000 daltons at 62 h. Bioassays of these f r a c t i o n s d e t e c t e d b a c t e r i o c i n 40 0.06 F i g u r e 5. Sephacryl S-300 column chromatograms c a r o t o v o r i c i n - 3 7 9 at (a) 44, (b) 52, (c) 58 and (d) 62 h a f t e r mitomycin C i n d u c t i o n i n concentrated supernatants of Erwinia carotovora subsp. carotovora s t r a i n 379. 41 a c t i v i t y with d i f f e r e n t degrees of c l e a r i n g depending on the i n d i c a t o r s t r a i n used. S t r a i n SR8 c o n s i s t e n t l y showed the g r e a t e s t s e n s i t i v i t y to a l l peaks. A l l s t r a i n s of i n d i c a t o r were most s e n s i t i v e to peaks l a and lb and produced c l e a r zones of i n h i b i t i o n ( F i g . 3e). The low molecular weight peaks gave v a r y i n g degrees of d i f f u s e i n h i b i t o r y zones ( F i g . 3f) on a l l i n d i c a t o r s except s t r a i n 530 which was r e s i s t a n t to low molecular weight c a r o t o v o r i c i n - 3 7 9 . E l e c t r o n micrographs of the separated b a c t e r i o c i n components i n d i c a t e d that as subunits were added to b a c t e r i o c i n p a r t i c l e s over time, there was an i n c r e a s e i n molecular weight ( F i g . 6). However, the e x i s t e n c e of a s o l u b l e low molecular weight component was s t i l l p o s s i b l e as peak 4 showed d i f f u s e - t y p e a c t i v i t y on s t r a i n SR8, but no p a r t i c u l a t e s t r u c t u r e c o u l d be r e s o l v e d by e l e c t r o n microscopy. Induced c u l t u r e s which were re-induced and d i l u t e d with an equal volume of medium (to maintain a mitomycin C c o n c e n t r a i o n of 0.2 ug/ml) showed that a l l peaks i n Figures 5a-d were present i n a s i n g l e scan at 8 h a f t e r the second i n d u c t i o n ( F i g . 42 F i g u r e 6 . Sephacryl S-300 column chromatograms of c a r o t o v o r i c i n - 3 7 9 produced by Erwinia carotovora subsp. carotovora s t r a i n 379 48 h a f t e r mitomycin C i n d u c t i o n and e l e c t r o n micrographs of the corresponding negative s t a i n e d peak contents. Bar=100nm.  44 7a). T h i s suggested that the second i n d u c t i o n i n i t i a t e d another round of c a r o t o v o r i c i n - 3 7 9 p r o d u c t i o n which appeared as s a t e l l i t e peaks on the S-300 chromatogram ( F i g . 7a). A l l supernatant b a c t e r i o c i n a c t i v i t y was detected i n one major peak when 20mM magnesium s u l f a t e was added to the medium f o l l o w e d by i n d u c t i o n ( F i g . 7b). T h i s peak p r o f i l e was i d e n t i c a l to the p r o f i l e of c a r o t o v o r i c i n - 3 7 9 concentrated from induced c e l l s grown i n minimal M9 media which a l s o contained magnesium ( F i g . 7c). D I S C U S S I O N C e l l l y s i s , as seen by a r e d u c t i o n i n t u r b i d i t y , o c c u r r e d i n induced c u l t u r e s of Erwinia carotovora subsp. carotovora s t r a i n 379 (Ecc 379) 36 h a f t e r i n d u c t i o n . However c a r o t o v o r i c i n p a r t i c l e s were seen i n c u l t u r e supernatants and bioas s a y e d at 8 h a f t e r i n d u c t i o n . Although no c e l l l y s i s or r e d u c t i o n i n c e l l v i a b i l i t y was detec t e d i n non-induced c u l t u r e s , b a c t e r i o c i n p r o d u c t i o n was e a s i l y d e t e c t e d by bioas s a y s and 45 F i g u r e 7. S e p h a c r y l S-300 chromatograms o f c a r o t o v o r i c i n - 3 7 9 p r o d u c e d by Erwinia carotovora s u b s p . carotovora s t r a i n 379 i n m i t o m y c i n C i n d u c e d c u l t u r e s grown u n d e r d i f f e r e n t c o n d i t i o n s . a. E f f e c t o f a s e c o n d i n d u c t i o n a f t e r 8 h w i t h m i t o m y c i n C i n L u r i a b r o t h (no added magnesium); b. E f f e c t o f t h e a d d i t i o n o f 20mM magnesium s u l p h a t e t o L u r i a b r o t h p r i o r t o i n d u c t i o n ; c. E f f e c t o f magnesium p r e s e n t as a component o f m i n i m a l M9 media. 46 e l e c t r o n microscopy. C e l l l y s i s f o l l o w i n g i n d u c t i o n i s a common f e a t u r e i n the prod u c t i o n of l a r g e and small molecular weight b a c t e r i o c i n s and i s s i m i l a r to temperate phage i n d u c t i o n . Recent work with c o l i c i n s has shown that l y s i s i s dependent on expr e s s i o n of a l y s i s gene which a c t i v a t e s a phospholipase a l t e r i n g the membrane p e r m e a b i l i t y . This gene product may be d i r e c t l y i n v o l v e d i n membrane t r a n s p o r t of c o l i c i n . The e f f e c t on c e l l i n t e g r i t y by the l y s i s gene can be i n a c t i v a t e d by i n c o r p o r a t i o n of 20mM magnesium i n the medium (Pugsley and Schwartz 1984). In our experiments, a d d i t i o n of 20mM magnesium to the c u l t u r e media f o l l o w e d by i n d u c t i o n delayed l y s i s by 6-8 h. Under these c o n d i t i o n s , g e l f i l t r a t i o n of c a r o t o v o r i c i n - 3 7 9 c o n t a i n i n g supernatants showed a l o s s of s m a l l e r molecular weight, l e s s a c t i v e peaks. A l l c a r o t o v o r i c i n a c t i v i t y was detected i n the l a r g e molecular weight f r a c t i o n . The magnesium ions probably s t a b i l i z e d the outer membrane s u f f i c i e n t l y to prevent r e l e a s e of p a r t i a l l y a c t i v e b a c t e r i o c i n i n t e r m e d i a t e s . These o b s e r v a t i o n s suggest that i n a d d i t i o n to c e l l 47 l y s i s another mechanism may be i n v o l v e d i n b a c t e r i o c i n r e l e a s e . Erwinia carotovora subsp. carotovora produces a wide v a r i e t y of p e c t o l y t i c and c e l l u l o l y t i c enzymes. There have been s e v e r a l r e p o r t s of a simultaneous i n d u c t i o n of p e c t i n l y a s e and p a r t i c u l a t e c a r o t o v o r i c i n or temperate bacteriophage i n Erwinia carotovora ( C h a t t e r j e e 1984, Itoh et al. 1980 and Kamimiya et al. 1977). These enzymes and c a r o t o v o r i c i n s appear e x t e r n a l l y i n the supernatant f r a c t i o n of c u l t u r e d c e l l s and c o - i n d u c t i o n c o u l d be e x p l a i n e d by an i n a c t i v a t i o n of a common r e p r e s s o r of an e x i s t i n g s e c r e t o r y system. The outer membrane of the E n t e r o b a c t e r i a c e a e p r o v i d e s an e f f e c t i v e b a r r i e r to the r e l e a s e of substances > 700-1000 d a l t o n s (Nikaido and Vaara 1985). Thus, there i s a need f o r a s e c r e t o r y system f o r exo-enzymes. The s e c r e t i o n of enzymes from Pseudomonas and endotoxin p r o d u c t i o n by Neisseria i s thought to occur v i a the formation and subsequent r e l e a s e of c e l l w a l l blebs or outer membrane v e s i c l e s (Devoe et al. 1973). These b l e b s are s i m i l a r to those observed i n t h i s study. In 48 c o n j u n c t i o n with the numerous f i m b r a e - l i k e p r o j e c t i o n s , these blebs may form a s e c r e t o r y mechanism f o r b a c t e r i o c i n and p o s s i b l y macerating enzymes. On the b a s i s of u l t r a s t r u c t u r a l o b s e r v a t i o n s and experimental data we propose the f o l l o w i n g h y p o t h e t i c a l model f o r c a r o t o v o r i c i n - 3 7 9 assembly and s e c r e t i o n ( F i g . 8). The c o n s t r u c t i o n of b a c t e r i o c i n occurs w i t h i n an outer membrane v e s i c l e or "bleb". This v e s i c l e p r o v i d e s a general mechanism f o r s e c r e t i o n of a l l Erwinia e x o p r o t e i n s . Macerating enzymes and c a r o t o v o r i c i n components are produced i n the cytoplasm and are t r a n s p o r t e d across the plasmalemma probably by the model of Randal l and Hardy (1984). The s e c r e t i o n of these p r o t e i n s i s f a c i l i t a t e d by t h e i r e x o c y t o s i s w i t h i n an outer membrane v e s i c l e . C a r o t o v o r i c i n - 3 7 9 c o n s t r u c t i o n occurs w i t h i n a v e s i c l e which a l s o c o n t a i n s a f i m b r i a l p r o j e c t i o n . This f i m b r i a l p r o j e c t i o n u l t i m a t e l y becomes the core of c a r o t o v o r i c i n - 3 7 9 . Some b a c t e r i o c i n p r o t e i n s are added e x t e r n a l l y to the f i m b r i a l p r o j e c t i o n r e s u l t i n g i n the formation of a c o n t r a c t i l e sheath. Other b a c t e r i o c i n p r o t e i n s are t r a n s p o r t e d i n t o the 49 F i g u r e 8. H y p o t h e t i c a l model f o r t h e p r o d u c t i o n o f c a r o t o v o r i c i n by Erwinia carotovora s u b s p . caroto- vora s t r a i n 379. a. Erwinia e x o p r o t e i n s ( m a c e r a t i n g enzymes and c a r o t o v o r i c i n ) a r e p r o d u c e d i n t h e c y t o p l a s m and a r e t r a n s p o r t e d a c r o s s t h e c y t o p l a s m i c membrane (cm) i n t o t h e p e r i p l a s m i c s p a c e ( p s ) . T hese p r o t e i n s a r e s u b s e q u e n t l y s e c r e t e d w i t h i n an o u t e r membrane v e s i c l e (omv). b. C a r o t o v o r i c i n c o n s t r u c t i o n o c c u r s i n d u c i b l y w i t h i n an o u t e r membrane v e s i c l e w h i c h a l s o c o n t a i n s a f i m b r a e - l i k e p r o j e c t i o n ( a ) . Under n o n - i n d u c e d c o n d i t i o n s t h e s e p r o j e c t i o n s e x t e n d o u twards and f o r m t h e numerous f i m b r a e p r e s e n t on c a r o t o v o r i c i n p r o d u c i n g s t r a i n s . c. Under i n d u c t i o n , c a r o t o v o r i c i n components t r a n s l o c a t e d i n t o t h e p e r i p l a s m i c s p a c e a r e added t o t h e f i m b r a e - l i k e p r o j e c t i o n . T h e s e added components e v e n t u a l l y f o r m t h e s h e a t h ( s ) o f t h e p a r t i c u l a t e c a r o t o v o r i c i n . D u r i n g t h e f i n a l s t a g e s o f c o n s t r u c t i o n , c a r o t o v o r i c i n components r e s p o n s i b l e f o r t h e f o r m a t i o n o f a b a s e p l a t e (bp) a r e added a t th e b a s e o f t h e c a r o t o v o r i c i n p a r t i c l e . d. Some c a r o t o v o r i c i n components may be t r a n s l o c a t e d t h r o u g h t h e c e n t r a l c o r e t o an e x p a n d i n g t e r m i n a l v e s i c l e o r head (h) . e. R e l e a s e o f t h e c a r o t o v o r i c i n i s f a c i l i t a t e d by t h e a c t i v a t i o n o f a p h o s p h o l i p a s e w h i c h b r e a k s open t h e e n c l o s i n g o u t e r membrane r e l e a s i n g i n t a c t c a r o t o v o r i c i n w i t h t a i l f i b r e s ( t f ) and b a s e p l a t e ( b p ) . f , g . The head (h) o f t h e c a r o t o v o r i c i n may d i s s o c i a t e f r o m t h e r e s t o f t h e p a r t i c l e i n t h e s u p e r n a t a n t o f a p r o d u c i n g s t r a i n . The r e s t o f t h e c a r o t o v o r i c i n may e x i s t i n two forms e i t h e r e x t e n d e d (F) o r c o n t r a c t e d ( G ) . 50 51 h o l l o w c o r e and s u b s e q u e n t l y t r a n s l o c a t e d t o a t e r m i n a l v e s i c l e . Upon c o m p l e t i o n o f an i n t a c t c a r o t o v o r i c i n p a r t i c l e , i t i s r e l e a s e d w i t h i n a v e s i c l e . The r e l e a s e o f c a r o t o v o r i c i n f o l l o w e d by d i s r u p t i o n o f t h e v e s i c l e may be a s s o c i a t e d w i t h t h e a c t i v a t i o n o f a p h o s p h o l i p a s e . T h i s p h o s p h o l i p a s e may be f o u n d a s s o c i a t e d w i t h t h e b a c t e r i o c i n o r may be a c t i v a t e d w i t h i n t h e p e r i p l a s m by some i n d u c e d b a c t e r i o c i n component. DNA damage by c h e m i c a l o r p h y s i c a l a g e n t a c t i v a t e s t h e p r o d u c t i o n o f r e c A p r o t e i n w h i c h d e a c t i v a t e s a r e p r e s s o r ( G l a s s 1985) o f b o t h b a c t e r i o c i n and l y t i c components. T h i s r e s u l t s i n an i n c r e a s e i n b a c t e r i o c i n c o n s t r u c t i o n and s e c r e t i o n w h i c h , a l o n g w i t h t h e a c t i v a t i o n o r p r o d u c t i o n o f a p h o s p h o l i p a s e , r e s u l t s i n t h e d i s r u p t i o n o f t h e p r o d u c i n g c e l l . In t h e n o n - i n d u c e d s t a t e t h e c o n s t r u c t i o n and s e c r e t i o n o f t h e b a c t e r i o c i n and some o f i t s components a r e r e p r e s s e d ; however, t h e e x o c y t o s i s o f p e r i p l a s m i c p r o t e i n s i s s t i l l a c t i v e r e s u l t i n g i n a s e c r e t i o n o f p a r t i a l l y a c t i v e b a c t e r i o c i n components as w e l l as t h e s e c r e t i o n o f m a c e r a t i n g enzymes n o r m a l l y f o u n d i n t h e p e r i p l a s m . The 52 r e p r e s s i o n however i s n o t c o m p l e t e r e s u l t i n g i n a d i f f e r e n t i a l low l e v e l o f n o n - i n d u c e d p a r t i c u l a t e b a c t e r i o c i n p r o d u c t i o n . 53 C H A P T E R 2 G E N E T I C DETERMINANTS OF C A R O T O V O R I C I N P R O D U C T I O N I N 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 a r e p r o t e i n a c e o u s a n t i m i c r o b i a l a g e n t s p r o d u c e d by c e r t a i n s t r a i n s o f b a c t e r i a w h i c h a r e a c t i v e o n l y a g a i n s t c l o s e l y r e l a t e d s t r a i n s (Nomura 1967). B a c t e r i o c i n p r o d u c t i o n i s common i n a number o f g e n e r a and i s g e n e r a l l y c o n s i d e r e d t o c o n f e r a s e l e c t i v e a d v a n t a g e t o p r o d u c i n g s t r a i n s ( B i r g e 1981). B a c t e r i o c i n p r o d u c t i o n i n Erwinia carotovora, w h i c h c a u s e s s o f t r o t o f v e g e t a b l e s and b l a c k l e g o f p o t a t o was f i r s t d e s c r i b e d by Hamon and P e r o n ( 1 9 6 1 ) . S u b s e q u e n t r e s e a r c h r e s o l v e d two t y p e s o f b a c t e r i o c i n . The f i r s t i s c h a r a c t e r i z e d by a s m a l l c l e a r zone o f i n h i b i t i o n and t h e s e c o n d by a l a r g e and d i f f u s e z o n e. A t h e r m o l a b i l e , t r y p s i n - r e s i s t a n t , phage t a i l - l i k e p a r t i c u l a t e 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 - E R , was i d e n t i f i e d as c a u s i n g t h e 54 s m a l l c l e a r zones ( I t o h et al. 1978). This b a c t e r i o c i n resembled the pyocins produced by c e r t a i n Pseudomonas s t r a i n s and l i k e the pyocins was i n d u c i b l e by u l t r a v i o l e t i r r a d i a t i o n , mitomycin C or other DNA damaging agents (Birge 1981). Recent i n v e s t i g a t i o n s have shown that both p e c t i n l y a s e a c t i v i t y and b a c t e r i o c i n a c t i v i t y are co- i n d u c i b l e i n E. carotovora (Kamimiya er al. 1977) and i n E. chrysanthemi ( C h a t t e r j e e et al. 1984). Genetic a n a l y s i s of b a c t e r i o c i n p r o d u c t i o n i n other genera has shown that the determinants can be plasmid or chromosomally encoded (B i r g e 1981). Low molecular weight b a c t e r i o c i n s causing l a r g e d i f f u s e zones of i n h i b i t i o n , such as the c o l i c i n s were found to be plasmid encoded. P a r t i c u l a t e b a c t e r i o c i n s causing sma l l c l e a r zones of i n h i b i t i o n were p r i m a r i l y coded f o r by the b a c t e r i a l chromosome. Although both small and la r g e plasmids have been i s o l a t e d from s e v e r a l d i f f e r e n t s t r a i n s of Erwinia carotovora ( C o p l i n et al. 1980, Forbes 1981, Zink et al. 1984), no phenotypes could be assigned and they were concluded to be c r y p t i c . Previous work i n t h i s l a b o r a t o r y has shown that 55 both types of b a c t e r i o c i n a c t i v i t y are a s s o c i a t e d with Ecc s t r a i n 379 ( J a i s 1982) and that the b a c t e r i o c i n i s produced i n i n t i m a t e a s s o c i a t i o n with i n t a c t c e l l s p o s s i b l y v i a e x t r u s i o n (Chapter 1). The purpose of t h i s study was to determine whether the determinants f o r b a c t e r i o c i n p r o d u c t i o n i n Ecc s t r a i n 379 were chromosomally and/or plasmid encoded. MATERIALS AND METHODS Media and Growth Condit ions: A l l s t r a i n s (Table 1) were grown i n L u r i a Broth (LB) pH 7.4. Erwinia carotovora was incubated at 20 C while Escherichia coli was grown at 37 C. A n t i b i o t i c s obtained from the Sigma Chemical Company were d i s s o l v e d i n water or a l c o h o l and used i n p l a t e s or l i q u i d c u l t u r e at the recommended c o n c e n t r a t i o n ( M a n i a t i s et al. 1982). Erythromycin was d i s s o l v e d i n a l c o h o l and added to LB pH 8.0 at a f i n a l c o n c e n t r a t i o n of 30 ug/ml. C r y s t a l v i o l e t pectate (CVP) media was made Table 1 . B a c t e r i a l s t r a i n s used C a r o t o v o r i c i n Producer: Erwinia carotovora subsp. serogroup XI C a r o t o v o r i c i n I n d i c a t o r s : Erwinia carotovora subsp. serogroup I Erwinia carotovora subsp. serogroup XX Erwinia carotovora subsp. serogroup XXII Erwinia carotovora subsp. serogroup XVIII Other S t r a i n s : carotovora s t r a i n 379 atroseptica atroseptica atroseptica carotovora s s t r a i n SR8 s t r a i n 530 s t r a i n 496 t r a i n 504 Escherichia coli s t r a i n HB101 ( v i r g i n ) Escherichia coli s t r a i n HB101 + plasmid pBR322 Escherichia coli s t r a i n HB101 + plasmid R68.45 57 a c c o r d i n g to Cuppels and Kelman (1974). CVP (20 ml) p l a t e s were spread with a p p r o p r i a t e volumes of a n t i b i o t i c stock s o l u t i o n to give the recommended f i n a l c o n c e n t r a t i o n . P a s s i v e d i f f u s i o n f o r 24 hours (h) at room temperature was allowed before use. Using t h i s same technique, CVP + 1% LB was prepared to a c c e l e r a t e growth of E. coli. Mating P r o t o c o l s : A l l matings were performed at room temperature. R68.45 was t r a n s f e r r e d from an E. coli harbouring s t r a i n to Erwinia carotovora subsp. carotovora s t r a i n 379 u s i n g a standard p l a t e mating technique (Puhler and Riess 1984) and transconjugants were s e l e c t e d on CVP + kanamycin (50 ug/ml). These transconjugants were mated back to v i r g i n E. coli s t r a i n HB101 and transconjugants were s e l e c t e d on LB + kanamycin (50 ug/ml) + streptomycin (25 ug/ml). For w i l d - t y p e matings, (without R68.45), 1 ml of a mid-log phase c u l t u r e of E. carotovora subsp. carotovora s t r a i n 379 and E. coli s t r a i n HB101 (with and without PBR322) were added to 20 ml of s t e r i l e LB. These mixtures were allowed to stand 58 at room temperature f o r 4 h. A l i q u o t s of c e l l s were s e l e c t e d on LB + erythromycin (30 ug/ml) + streptomycin (25 ug/ml). A l l transconjugants were t e s t e d f o r : a b i l i t y to produce c a r o t o v o r i c i n - 3 7 9 , a b i l i t y to grow on CVP and r e s i s t a n c e to erythromycin (30 ug/ml) and chloramphenicol (15 ug/ml). Bacter ioc in Plate Assays: Assays were performed by p l a c i n g 5 u l f i l t e r - s t e r i l i z e d c u l t u r e supernatant, or contents of a peak from a column, on a lawn of i n d i c a t o r seeded i n peptone s o f t agar (PSA) c o n t a i n i n g 0.85% NaCl, 1% Bacto Peptone, 0.45% Bacto Agar pH 7.4. C a r o t o v o r i c i n c o n c e n t r a t i o n , column chromatography and e l e c t r o n microscopy were executed a c c o r d i n g to procedures o u t l i n e d i n Chapter 1. Tota l DNA Extrac t ion and Elec trophores i s : Late l o g phase c e l l s (10 ml) were harvested by c e n t r i f u g a t i o n and resuspended i n 600 u l of 0.05M T r i s , 0.02M EDTA; pH 8.0 (TE) b u f f e r + 20% sucrose. One hundred m i c r o l i t e r s of a 5 mg/ml stock s o l u t i o n 59 of lysozyme (Sigma) i n TE was added and incubated f o r 10 minutes (min) at room temperature. One hundred m i c r o l i t e r s of a 0.5M EDTA stock s o l u t i o n pH 8.0 were then added and incubated f o r 20 min at room temperature. Two hundred m i c r o l i t e r s of a 10% stock s o l u t i o n of SDS were then added. The tubes were g e n t l y i n v e r t e d 10 times and incubated at 50 C f o r 20 min. This l y s a t e was e x t r a c t e d twice with phenol:chloroform (50:50 v/v) and twice subsequently with chloroform, u n t i l a c l e a r i n t e r f a c e was seen ( M a n i a t i s et al. 1982). The aqueous phase was ad j u s t e d to 0.IM NaCl from a 5M NaCl stock s o l u t i o n , and DNA and RNA were p r e c i p i t a t e d with 2 volumes of 99% ethanol at -50 C ov e r n i g h t . P r e c i p i t a t e d n u c l e i c a c i d s were p e l l e t e d at 13,000 x g at -20 C f o r 25 min. Re s i d u a l ethanol was removed i n a vacuum d e s s i c a t o r and the p e l l e t was d i s s o l v e d i n 50 u l of TE. Ten m i c r o l i t e r a l i q u o t s of these samples were removed, t r e a t e d with 2 u l of DNase-free RNase (10 mg/ml s t o c k ) , f o r 30 min at 37 C and analysed by agarose g e l e l e c t r o p h o r e s i s (AGE) ( P e r b a l l 1984; Hames et al. 1984; Glover 1985). 60 E l e c t r o p h o r e s i s was performed on a homemade 20 x 20 cm submerged, h o r i z o n t a l apparatus. E l e c t r o p h o r e s i s was c a r r i e d out i n 90mM T r i s , 90mM Borate, 2.5mM EDTA (TBE) pH 8.2 at 1.5 V/cm f o r 18- 24 h. Gels used were 0.5% agarose (w/v) (Sigma), poured to a depth of 0.3 mm. DNA was s t a i n e d i n 1.0 ug/ml of ethidium bromide (Sigma) f o r 1 h and viewed with a mid-wave u l t r a v i o l e t t r a n s i l l u m i n a t o r (U.V. Products Inc. San G a b r i e l , C a l i f o r n i a ) . A l k a l i n e phosphatase was assayed by combining 100 u l of f i l t e r - s t e r i l i z e d supernatant with 100 u l of s u b s t r a t e b u f f e r (0.1M T r i s - H C l pH 9.0) c o n t a i n i n g 1 mg/ml p-nitropheny1 phosphate (Sigma) i n 1.5 ml p o l y a l l o m e r Eppendorf tubes. Tubes were incubated at 4 C ov e r n i g h t . P o s i t i v e r e s u l t s were i d e n t i f i e d by a v i s u a l d e t e c t i o n of a yellow s o l u b l e r e a c t i o n product. 61 RESULTS Sephacryl S-300 Column Chromatography and Bioassays: Column chromatography of concentrated c a r o t o v o r i c i n from non-induced c u l t u r e s of Ecc s t r a i n 379 a f t e r 52 h at 20 C showed that the t o t a l p r o t e i n was d i s t r i b u t e d between two peaks ( F i g . l a ) . U n l i k e the s i t u a t i o n i n induced c u l t u r e s (Chapter 1), the m a j o r i t y of the p r o t e i n was found i n the lower molecular weight f r a c t i o n . Bioassays of these two peaks showed that the l a r g e molecular weight f r a c t i o n (peak 1) produced c l e a r plaques on i n d i c a t o r s , while the s m a l l molecular weight f r a c t i o n (peak 2) produced d i f f u s e plaques ( F i g . 2b) . Ecc s t r a i n 379 showed a t e m p e r a t u r e - s e n s i t i v e r e s i s t a n c e to erythromycin (30 ug/ml) and chloramphenicol (15 ug/ml). R e s i s t a n c e to these a n t i b i o t i c s was expressed at 20 C i n LB but was l o s t at temperatures above 35 C. Ecc s t r a i n 379 was a l s o able to grow and p i t CVP a f t e r 24 h at 24 C or 37 C. Mitomycin induced c u l t u r e s of Ecc s t r a i n 379 grown at 37 C produced no l a r g e 62 0.05 E FRACTION F i g u r e 1. Sephacryl S-300 column chromatograms of c a r o t o v o r i c i n from Erwinia and E. c o l i t r a n s c o n j u g a n t s . a. Erwinia carotovora subsp. carotovora (Ecc) s t r a i n 379 non-induced; b. Ecc s t r a i n 379 + mitomycin (0.2 ug/ml) at 37 C; c. R68.45 mediated CVP~ E. c o l i transconjugant; d. R68.45 mediated CVP + E. c o l i transconjugant; e. Wild-type mated CVP~ E. c o l i t r a n s c o n j u g a t e s . 63 F i g u r e 2 . C a r o t o v o r i c i n p l a t e assays of c o l o n i e s and Sephacryl S-300 f r a c t i o n a t e d peaks from con c e n t r a t e d supernatants of E. coli t r a n s c o n - jugants and E. carotovora subsp. carotovora (Ecc) s t r a i n 379. a. C a r o t o v o r i c i n p l a t e assays of c o l o n i e s and f r a c t i o n a t e d l a r g e (peak 1) and small (peak 2) molecular weight b a c t e r i o c i n components from supernatants of CVP+ and CVP" E. coli t r a n s c o n j u g a n t s . CVP- C V P + CVP" colony peak 1 colony C V P + CVP" C V P + peak 2 colony peak 1 C V P + C V P + C V P + colony peak 2 colony b. C a r o t o v o r i c i n p l a t e assays of c o l o n i e s and separated l a r g e and small molecular weight b a c t e r i o c i n components from supernatants of Ecc 379. peak 2 peak 1 peak 2 peak 2 producing producing colony colony 64 65 molecular weight c a r o t o v o r i c i n as detected by bi o a s s a y or S-300 column chromatography ( F i g . l b ) . Column chromatography of concentrated c a r o t o v o r i c i n from mitomycin-induced c u l t u r e s of Ecc s t r a i n 379 grown at 37 C showed only one peak corresponding to the low molecular weight component ( F i g . l b ) . Bioassays of t h i s peak produced d i f f u s e - t y p e plaques on a l l i n d i c a t o r s except 530 which was r e s i s t a n t . In a d d i t i o n , Ecc s t r a i n 379 grown at 37 C showed no v i s i b l e s i g n s of c e l l s l y s i s or r e d u c t i o n i n c e l l t u r b i d i t y . Column chromatography of supernatants of R68.45 mediated CVP" E. coli transconjugants showed two peaks ( F i g . l c ) . However only the low molecular weight component had d i f f u s e - t y p e c a r o t o v o r i c i n a c t i v i t y . Chromatography of concentrated b a c t e r i o c i n from w i l d - t y p e mated CVP", E. coli transconjugants a l s o showed one small molecular weight b i o a c t i v e component ( F i g . I e ) . In c o n t r a s t , column chromatography of R68.45 mediated CVP +, E. coli gave a peak p r o f i l e s i m i l a r to that of non- induced Ecc s t r a i n 379 ( F i g . Id) and produced c a r o t o v o r i c i n s i m i l a r to Ecc 379 ( F i g . 2a). The a b i l i t y to grow on CVP was t r a n s f e r r e d at a 66 frequency of 1 x 10~ 5. Re s i s t a n c e to erythromycin ( e r y r ) (30 ug/ml) and chloramphenicol (cam r) (15 ug/ml) was t r a n s f e r r e d at a frequency of 1 x 1 0 - 3 . When the erythromycin and chloramphenicol r e s i s t a n t t ransconjugants were t e s t e d f o r growth on CVP and produ c t i o n of c a r o t o v o r i c i n , most were CVP" and produced d i f f u s e - t y p e plaques on a l l i n d i c a t o r s except s t r a i n 530 which was r e s i s t a n t . However 1.7% were CVP + and produced w i l d - t y p e b a c t e r i o c i n a g a i n s t a l l four i n d i c a t o r s . Transconjugants from w i l d - t y p e matings were obtained at a frequency o f 10~ 4. When these were bioassayed f o r b a c t e r i o c i n p r o d u c t i o n , they produced d i f f u s e - t y p e plaques on three of the fou r i n d i c a t o r s . A l l of these transconjugants were CVP - and r e s i s t a n t to erythromycin (30 ug/ml) and chloramphenicol (15 ug/ml). In a l l transconjugants, a l k a l i n e phosphatase which i s normally r e s t r i c t e d to the per i p l a s m of E. coli, was found i n the f i l t e r - s t e r i l i z e d supernatants. No a l k a l i n e phosphatase a c t i v i t y was found i n the supernatants of wi l d - t y p e E. coli. 67 DNA Content: Plasmid p r o f i l e s of CVP", e r y r , cam r, kanamycin r e s i s t a n t (kan r) transconjugants showed that they contained both R68.45 and a second l a r g e r molecular weight plasmid found i n w i l d - t y p e Ecc s t r a i n 379. These plasmids e x i s t e d i n the transconjugant E. coli as separate e n t i t i e s and R68.45 i n these transconjugants contained no i n s e r t ( F i g . 3a). Plasmid p r o f i l e s of transconjugants obtained i n wi l d - t y p e matings i l l u s t r a t e d the t r a n s f e r of the l a r g e Erwinia plasmid (Erwp) ( F i g . 3b) along with the phenotypes of erythromycin r e s i s t a n c e , chloramphenicol r e s i s t a n c e and d i f f u s e - t y p e b a c t e r i o c i n p r o d u c t i o n . U n l i k e the Erwinia donors, e x p r e s s i o n of the r e s i s t a n t phenotypes was not a f f e c t e d at 37 C i n the E. coli t r a n s c o n j u g a n t s . E l e c t r o n Microscopy: E l e c t r o n micrographs of supernatants of transconjugant E. coli showed numerous membrane- bound v e s i c l e s . E l e c t r o n microscopy of i n t a c t CVP" transconjugants showed a protuberance of many s u r f a c e v e s i c l e s or " b l e b s " ( F i g . 4a) which were absent i n non-transconjugant E. coli. 68 F i g u r e 3. T o t a l DNA a n a l y s i s o f w i l d - t y p e E. coli, w i l d - t y p e E. carotovora s u b s p . carotovora s t r a i n 379 (Ecc 379) and Ecc 379 x E. coli t r a n s c o n j u g a n t s w i t h and w i t h o u t R68.45 m e d i a t i o n . Samples were run on a 0.5% a g a r o s e g e l u s i n g T r i s - B o r a t e EDTA (TBE) a t 1 t o 1.5 V/cm f o r 18-24 h. a. R68.45 m e d i a t e d E. coli t r a n s c o n j u g a n t s : Lanes: 1) E. coli w i t h pBR325; 2) R68.45 m e d i a t e d CVP" E. coli t r a n s c o n j u g a n t ; 3) E. coli w i t h pBR322; 4) Ecc s t r a i n 379 w i t h Erwinia p l a s m i d (Ewrp) 10 u l sample l o a d ; 5) Ecc s t r a i n 379 w i t h Ewrp 5 u l sample l o a d ; b. W i l d - t y p e mated E. coli t r a n s c o n j u g a n t s : Lanes: 1) HB101 w i t h R68.45 2) E. coli t r a n s c o n j u g a n t w i t h pBR322 and Erwp; 3) Ecc 379 w i t h Erwp; 4) HB101 t r a n s c o n j u g a n t w i t h Erwp. Figure 4. E l e c t r o n micrographs of c e l l s and supernatants of E. c o l i transconjugants (Bar=100nm). a. T y p i c a l negative s t a i n e d E. c o l i transconjugant c e l l showing s u r f a c e v e s i c l e s ; b,c. Supernatants of CVP + E. c o l i transconjugants showing p a r t i c u l a t e c a r o t o v o r i c i n . Note presence of t a i l f i b r e s i n c. 70 E l e c t r o n micrographs of the l a r g e molecular weight f r a c t i o n from CVP + E. coli transconjugants showed p a r t i c u l a t e b a c t e r i o c i n s i d e n t i c a l to those i s o l a t e d from Ecc p r e p a r a t i o n s ( F i g . 4b-c). DISCUSSION Genetic a n a l y s i s of c a r o t o v o r i c i n p r o d u c t i o n i n Erwinia proved to be a s u b s t a n t i a l problem as convenient, w i l d - t y p e markers were not a v a i l a b l e . However, t e s t s used i n the taxonomy of Erwinia s p e c i e s have c o n s i s t e n t l y used r e s i s t a n c e to erythromycin to separate E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica from other Erwinia (Schaad 1980). Erythromycin r e s i s t a n c e (30 ug/ml) i n Ecc s t r a i n 379 was shown to be temperature s e n s i t i v e with a l o s s of t h i s phenotype at 37 C. When s e v e r a l other a n t i b i o t i c s were t e s t e d on Ecc s t r a i n 379, a chloramphenicol r e s i s t a n t (15 ug/ml) phenotype was found which, l i k e erythromycin r e s i s t a n c e , was a l s o temperature s e n s i t i v e . C e l l 71 l y s i s , as seen by a r e d u c t i o n i n t u r b i d i t y of induced c e l l s was a l s o i n h i b i t e d at 37 C, as was p r o d u c t i o n of p a r t i c u l a t e b a c t e r i o c i n . The temperature s e n s i t i v i t y of the phenotypes suggested a p o s s i b l e plasmid involvement due to the general c u r i n g p r o p e r t i e s of growth at e l e v a t e d temperatures (Brock 1979) . In order to d i s t i n g u i s h between chromosomal and/or plasmid involvement i n these temperature s e n s i t i v e phenotypes, t r a n s f e r of g e n e t i c i n f o r m a t i o n from Ecc s t r a i n 379 to a model system, namely E. coli, was necessary. The c o n j u g a t i v e m o b i l i z a t i o n v e c t o r R68.45 was chosen because of i t s wide host range, chromosome m o b i l i z a t i o n a b i l i t y (cma +) from many o r i g i n s , (Haas and Holloway 1976) and a b i l i t y to m o b i l i z e r e s i d e n t plasmids at high frequency ( W i l l e t t s and Crowther 1980). The m o b i l i z a t i o n a b i l i t y of R68.45 i s thought to occur v i a a c o - i n t e g r a t i o n and subsequent r e s o l u t i o n i n the r e c i p i e n t c e l l . The r e s o l u t i o n of the c o - i n t e g r a t e i s thought to be recA dependent (Puhler and Riess 1984) with co- i n t e g r a t e maintenance i n recA minus r e c i p i e n t s . Furthermore, R68.45 con t a i n s the a n t i b i o t i c markers 72 f o r kanamycin, t e t r a c y c l i n e , and a m p i c i l l i n (or c a r b e n i c i l l i n ) r e s i s t a n c e which make i t convenient to f o l l o w t h i s plasmid through a p o p u l a t i o n . The a b i l i t y to grow on CVP was t r a n s f e r r e d to E. c o l i at a frequency of 10" 5 per r e c i p i e n t of R68.45. A l l CVP + transconjugants produced p a r t i c u l a t e b a c t e r i o c i n . These f a c t s suggested chromosomally d e r i v e d determinants. Erythromycin (30 ug/ml) and chloramphenicol (15 ug/ml) r e s i s t a n c e s were t r a n s f e r r e d at a frequency of 10~ 3 per r e c i p i e n t c e l l . This high frequency suggested plasmid d e r i v e d determinants. DNA a n a l y s i s by agarose g e l e l e c t r o p h o r e s i s of CVP + e r y r , cam r transconjugants showed the presence of a plasmid which corresponded to that found i n w i l d - t y p e Erwinia. However, these transconjugants were a l s o kan r and b a c t e r i o c i n p o s i t i v e . These phenotypes were presumably t r a n s f e r r e d as an R68.45 co- i n t e g r a t e from the Erwinia chromosome. The s i z e of such a c o - i n t e g r a t e would be too l a r g e to r e s o l v e i n t a c t e l e c t r o p h o r e t i c a l l y . DNA a n a l y s i s by AGE of CVP", e r y r , cam1", kan r transconjugants confirmed the presence of two plasmids. One of the plasmids was i d e n t i f i e d by 73 s i z e as R68.45 with no i n s e r t and the other corresponded to a l a r g e molecular weight plasmid found i n wil d - t y p e Ecc s t r a i n 379. Because the r e c i p i e n t c e l l (E. coli HB101) was a recA" s t r a i n , v i s u a l i z a t i o n of separate plasmids suggested that the Erwinia plasmid t r a n s f e r r e d independently of R68.45. Bioassays u s i n g these transconjugants showed that they produced small molecular weight d i f f u s e - t y p e plaques. T h i s b a c t e r i o c i n behaved i d e n t i c a l l y to that obtained from Ecc induced at 37 C or the small molecular weight component of c a r o t o v o r i c i n - 3 7 9 produced at 20 C. Transconjugants obtained i n wi l d - t y p e matings between Ecc and E. coli confirmed the s e l f - t r a n s m i s s i b i l i t y of the l a r g e molecular weight plasmid i n Ecc 379. A l l of these transconjugants were a l s o CVP -, e r y r , cam r and produced a small molecular weight b a c t e r i o c i n component i d e n t i c a l to that produced by the CVP - R68.45 mediated tr a n s c o n j u g a n t s . From these r e s u l t s we conclude that t h i s s e l f - t r a n s m i s s i b l e Erwinia megaplasmid coded f o r erythromycin and chloramphenicol r e s i s t a n c e and a small molecular weight b a c t e r i o c i n component. The pr o d u c t i o n i n E. coli 74 transconjugants of p a r t i c u l a t e b a c t e r i o c i n i d e n t i c a l to that of Ecc 379 was only d e t e c t e d when chromosomal c o n s t i t u e n t s were t r a n s f e r r e d along with the megaplasmid. This f a c t suggests that c a r o t o v o r i c i n - 3 7 9 p r o d u c t i o n i n Ecc s t r a i n 379 i s coded f o r by both chromosomal and plasmid const i t u e n t s . E l e c t r o n microscopy of a l l transconjugants showed that they produced many s u r f a c e v e s i c l e s or blebs not found i n wi l d - t y p e E. coli. Blebs have been a s s o c i a t e d with endotoxin s e c r e t i o n i n Neisseria meningitidis (Devoe and G i l c h r i s t 1973), exo-enzyme p r o d u c t i o n i n Pseudomonas aeruginosa (Thompson et al. 1985) and r e l e a s e of a l k a l i n e phosphatase i n Pseudomonas (Ingram and Dainty 1973). The presence of blebs on transconjugants and w i l d - t y p e Erwinia (Chapter 1), intro d u c e s the p o s s i b i l i t y that t h i s megaplasmid i s i n v o l v e d i n the s e c r e t i o n of p e r i p l a s m i c p r o t e i n s . This suggestion i s supported by the f a c t that i n the E. coli transconjugants, a l k a l i n e phosphatase which i s normally found i n the periplasm, was found i n f i l t e r - s t e r i l i z e d supernatants. It would a l s o e x p l a i n why i n cosmid and shot-gun c l o n i n g 75 experiments with Erwinia, p e c t i n - d e g r a d i n g enzymes were found to accumulate i n the perip l a s m of E. coli t r a n s f e c t a n t s or transformants and were not e f f e c t i v e l y t r a n s p o r t e d out i n t o the supernatant (Collmer et al. 1985; Zink and C h a t t e r j e e 1985; Kotoujansky et al. 1985). These methods d i d not take i n t o account the p o s s i b l e involvement of a r e s i d e n t megaplasmid i n the s e c r e t i o n of p e r i p l a s m i c p r o t e i n s i n Erwinia. 76 C H A P T E R 3 S E R O L O G I C A L R E L A T I O N S H I P S AMONG T H E D I F F E R E N T FORMS O F ERWINIA B A C T E R I O C I N D E T E C T E D BY P O L Y C L O N A L A N T I S E R U M A G A I N S T P A R T I C U L A T E CAROTOVORICIN - 3 7 9 I N T R O D U C T I O N P a r t i c u l a t e b a c t e r i o c i n produced by Erwinia carotovora subsp. carotovora (Ecc) s t r a i n 379 bears a s t r i k i n g resemblance to the R-type pyocins of Pseudomonas. However, previous work (Chapter 1 and 2) has shown that 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 n l i k e pyocin a c t i v i t y can be d i v i d e d i n t o s e v e r a l f r a c t i o n s based on molecular weight. P r o d u c t i o n of the l a r g e molecular weight ( p a r t i c u l a t e ) f r a c t i o n , which produced a c l e a r - t y p e plaque on s e v e r a l i n d i c a t o r s , was temperature s e n s i t i v e whereas the sm a l l e r molecular weight f r a c t i o n s were temperature independent and produced sparse or d i f f u s e plaques. The a d d i t i o n of an outer membrane s t a b i l i z i n g agent r e s u l t e d i n the d e t e c t i o n of t o t a l c a r o t o v o r i c i n 77 a c t i v i t y at 20 C i n one l a r g e molecular weight peak which contained p a r t i c l e s resembling pyocins and when bioassayed, produced c l e a r - t y p e plaques. The R-type pyocins of Pseudomonas aeruginosa have s t r u c t u r e s which are c l o s e l y r e l a t e d both m o r p h o l o g i c a l l y and s e r o l o g i c a l l y to bacteriophage t a i l s (Oshumi et al. 1980). They have been con s i d e r e d model systems 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 . The d i s t i n g u i s h i n g c h a r a c t e r i s t i c of pyocins l i e s i n t h e i r d i f f e r e n t a c t i v i t y s p e c t r a against a range of i n d i c a t o r s t r a i n s . T h i s has l e d to a d i v i s i o n of the R-type pyocins i n t o f i v e s p e c i f i c i t y groups (Shinomiya et al. 1979). A n a l y s i s of the p r o t e i n subunits of these pyocins showed an almost i d e n t i c a l composition with s m a l l d i f f e r e n c e s noted i n the t a i l f i b e r r e g i o n (Oshumi et al. 1980). These small d i f f e r e n c e s were detected i n c r o s s - a d s o r p t i o n s t u d i e s u s i n g s p e c i f i c a n t i s e r a and has l e d to the c o n c l u s i o n that these f i b e r s are a major determinant i n the a c t i v i t y spectrum of a given pyocin (Oshumi et al. 1980). The g e n e t i c determinants f o r pyocin p r o d u c t i o n are thought to be l o c a t e d on the b a c t e r i a l chromosome and i n most cases pyocin p r o d u c t i o n i s not a 78 temperature s e n s i t i v e t r a i t . G enetic t r a n s f e r from an Erwinia c a r o t o v o r i c i n producer (Ecc s t r a i n 379) to Escherichia coli showed that a l a r g e molecular weight s e l f - t r a n s m i s s i b l e plasmid i n Ecc s t r a i n 379 coded f o r erythromycin and chloramphenicol r e s i s t a n c e , the pro d u c t i o n of a small molecular weight b a c t e r i o c i n component and the p r o l i f e r a t i o n of s u r f a c e v e s i c l e s or blebs (Chapter 2). The small molecular weight c a r o t o v o r i c i n component produced by E. coli transconjugants had the same a c t i v i t y spectrum as the small molecular weight f r a c t i o n i s o l a t e d from non-induced c u l t u r e s of wi l d - t y p e Ecc s t r a i n 379 grown at 20 C or induced Ecc 379 grown at 37 C. The purpose of t h i s study was to develop antiserum to the l a r g e molecular weight f r a c t i o n of c a r o t o v o r i c i n - 3 7 9 and to determine the r e l a t e d n e s s of the mature i n t a c t p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 and the p a r t i c u l a t e and s o l u b l e forms of c a r o t o v o r i c i n produced by wi l d - t y p e Ecc 379 and s e v e r a l Ecc x E. coli t r a n s c o n j u g a n t s . 79 MATERIALS AND METHODS B a c t e r i a l s t r a i n s B a c t e r i a l s t r a i n s used and transconjugants were obtained as o u t l i n e d i n Chapters 1 and 2. Media and growth c o n d i t i o n s were d e s c r i b e d i n Chapters 1 and 2. C o n c e n t r a t i o n and f r a c t i o n a t i o n of c a r o t o v o r i c i n were performed as o u t l i n e d i n Chapter 1. Development of Antiserum C a r o t o v o r i c i n - 3 7 9 was concentrated as p r e v i o u s l y d e s c r i b e d from Ecc s t r a i n 379 grown i n minimal M9 media at 20 C, 48 h a f t e r i n d u c t i o n . Concentrated p r o t e i n from 100 ml of c u l t u r e supernatant was analysed by Sephacryl S-300 column chromatography as p r e v i o u s l y d e s c r i b e d (Chapter 1 and 2). The l a r g e molecular weight peak was pooled and bioassayed to confirm a c t i v i t y . Ten m i l l i g r a m s of p r o t e i n was mixed and e m u l s i f i e d with an equal volume of Freunds complete adjuvant (Sigma). This was i n j e c t e d i n t r a m u s c u l a r l y i n t o the hind l e g of a r a b b i t , once a week, f o r 5 cons e c u t i v e weeks. On the 6th week, 20 ml of blood, c o l l e c t e d from the 80 ear v e i n , was allowed to c l o t at room temperature f o r 1 hour (h) f o l l o w e d by 12 h ( o v e r n i g h t ) at 4 C. The remaining serum was c l a r i f i e d by low speed c e n t r i f u g a t i o n (8000 x g) f o r 25 minutes (min). C l a r i f i e d serum was s t o r e d i n 1 ml a l i q u o t s at -20 C. R a d i a l Immunodiffusion C l a r i f i e d serum was d i l u t e d 1/100 i n 0.5% noble agar ( D i f c o ) i n 5OmM sodium phosphate b u f f e r pH 7.4 + 0.85% NaCl (PBS) and poured i n t o p e t r i dishes to a depth of 0.3 cm. Twenty u l of c u l t u r e supernatant were added to pre-cut w e l l s and the p l a t e s incubated f o r 24-48 h at 25 C. Immuno-sensitive E l e c t r o n Microscopy (ISEM) P o l y c l o n a l antiserum was used to " t r a p " b a c t e r i o c i n p a r t i c l e s on EM g r i d s ( H i l l 1984) by s e q u e n t i a l l y f l o a t i n g carbon c o l l o i d i o n g r i d s on one drop of the f o l l o w i n g s o l u t i o n s f o r the i n d i c a t e d times: a) 1/2000 d i l u t i o n of antiserum i n PBS f o r 15 min at 22 C; b) PBS pH 7.4 f o r 10 min at 22 C; and c) f i l t e r - s t e r i l i z e d c u l t u r e f l u i d f o r 15 min at 22 C. Grids were s t a i n e d with 10-12 81 drops of 2% phosphotungstic a c i d (PTA) pH 7.0 and viewed on a P h i l l i p s EM-300 e l e c t r o n microscope. Subsequent d e c o r a t i o n of trapped p a r t i c l e s ( H i l l 1984) was accomplished by f u r t h e r i n c u b a t i o n s on PBS pH 7.4 f o r 10 min at 22 C f o l l o w e d by 1/1000 d i l u t i o n of antiserum i n PBS f o r 15 min at 22 C. Gr i d s were s t a i n e d and viewed as above. Sodium Dodecyl Sulphate 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 (SDS-PAGE) E l e c t r o p h o r e s i s was c a r r i e d out i n 0.5 mm 8.0% acrylamide u s i n g T r i s - g l y c i n e - S D S , e s s e n t i a l l y f o l l o w i n g the d i s c o n t i n u o u s system o u t l i n e d by Laemmili (Laemmili 1970; Laemmili and F a r r e 1973). Concentrated supernatants were d i s s o l v e d i n l o a d i n g b u f f e r (lOOmM T r i s - H C l pH 6.8 with 2% SDS and 5% beta-mercaptoethanol), b o i l e d f o r 2 min and e l e c t r o p h o r e s e d at 18 mA f o r 1.5 h u s i n g a m i n i - v e r t i c a l s l a b g e l u n i t (Hoefer S c i e n t i f i c SE-200). Gels were s t a i n e d e i t h e r f o r p r o t e i n or used f o r Western b l o t s ( e l e c t r o b l o t t i n g ) . P r o t e i n d e t e c t i o n was accomplished by s t a i n i n g g e l s with 0.5% Coomassie b r i l l i a n t blue R-250 i n 45% methanol, 10% a c e t i c a c i d (v/v) f o r 1 h 82 f o l l o w e d by d e s t a i n i n g i n 20% methanol, 10% a c e t i c a c i d ( v / v ) . Gels were preserved by d r y i n g at 65 C on a homemade s l a b - g e l dryer. Gels f o r b l o t t i n g were soaked i n t r a n s f e r b u f f e r (25mM T r i s , 192raM G l y c i n e , 20% Methanol; pH 8.3) f o r 1 h f o l l o w e d by e l e c t r o b l o t t i n g and immuno-detection of p r o t e i n s . P r o t e i n standards (Sigma) ranged from 30-200 k i l o d a l t o n s . Western B l o t t i n g The t r a n s f e r of p r o t e i n s to n i t r o c e l l u l o s e (Western B l o t t i n g ) (Towbin et al. 1979; Gershoni and Palade, 1983) was accomplished at 40 V, 150 mA f o r 12 h i n a homemade b l o t t i n g apparatus with s t a i n l e s s s t e e l rod e l e c t r o d e s . A f t e r b l o t t i n g , n i t r o c e l l u l o s e with t r a n s f e r r e d p r o t e i n s was washed t h r i c e f o r 15 min each i n 20mM T r i s 500mM NaCl, pH 7.5 (TBS). P r o t e i n b i n d i n g s i t e s were blocked with a mixture of 2% g e l a t i n and bovine serum albumin (BSA) (Sigma) (1 mg/ml) i n TBS f o r 2 h at 37 C. The b l o t was washed twice f o r 15 min each i n TBS and incubated i n a 1/1000 d i l u t i o n of p o l y c l o n a l antiserum + 0.1% BSA i n TBS f o r 2 h at 37 C. This was f o l l o w e d by two 15-minute washes i n TBS and 83 i n c u b a t i o n i n a 1/1500 d i l u t i o n of goat a n t i - r a b b i t IgG-horseradish peroxidase conjugate (Sigma) i n TBS + 0.1% BSA f o r 2 h at 37 C. The b l o t was washed twice f o r 15 min each i n TBS and incubated f o r 5-15 min i n s u b s t r a t e b u f f e r (50mM T r i s - H C l pH 7.5) c o n t a i n i n g 1 mg/ml diaminobenzidine (DAB) + 1% hydrogen peroxide (added immediately before use). The r e a c t i o n was stopped by r a p i d d i l u t i o n of s u b s t r a t e i n TBS. B l o t s were d r i e d and s t o r e d between paper towels. RESULTS Antiserum a g a i n s t the l a r g e molecular weight f r a c t i o n ( p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 ) r e a c t e d with the homologous antigen and produced s e v e r a l c i r c u l a r p r e c i p i t i n l i n e s i n r a d i a l immunodiffusion (RID) ( F i g . 1). These p a t t e r n s were i d e n t i c a l r e g a r d l e s s of the growth medium used (LB or minimal medium). Non-induced c u l t u r e s of Ecc s t r a i n 379 a l s o produced b a c t e r i o c i n which r e a c t e d i n RID with antiserum against p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 ( F i g . 1). CVP + E. coli transconjugants 84 379 i n d u c e d (M9) CVP" w i l d - t y p e t r a n s c o n j u g a n t 1/5 d i l u t i o n o f 379 i n d u c e d 379 i n d u c e d (M9) CVP + E. coli t r a n s c o n j u g a n t 379 i n d u c e d (LB) 379 n o n - i n d u c e d E. coli HB101 c o n t r o l 379 n on- i n d u c e d (LB) ft) x s p i l l CVP" R68.45 m e d i a t e d t r a n s c o n j u g a n t E. coli R68.45 c o n t r o l F i g u r e 1. R a d i a l i m m u n o d i f f u s i o n a n a l y s i s o f t h e r e l a t i o n s h i p between p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 p r o d u c e d by Erwinia carotovora s u b s p . carotovora s t r a i n 379 (Ecc 379) and Ecc 379 x E. coli t r a n s - c o n j u g a n t s . Agar c o n t a i n i n g a f i n a l d i l u t i o n o f 1/100 o f p o l y c l o n a l a n t i s e r u m a g a i n s t p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 was p o u r e d . Cut w e l l s were f i l l e d w i t h s u p e r n a t a n t s from Ecc 379, Escherichia coli and E. coli t r a n s c o n j u g a n t s . 85 c o n s t i t u t i v e l y produced b a c t e r i o c i n s with p r e c i p i t i n p a t t e r n s s i m i l a r to those of induced c u l t u r e s of Ecc s t r a i n 379. A l l CVP" transconjugants produced only one major p r e c i p i t i n band ( F i g . l ) . Immuno-sensitive E l e c t r o n Microscopy (ISEM) Antiserum d i r e c t e d a g a i n s t p a r t i c u l a t e b a c t e r i o c i n "trapped" a s i g n i f i c a n t l y h i g h e r number of p a r t i c l e s from 5 u l of an induced c u l t u r e supernatant ( F i g . 2a and 2b). Trapped p a r t i c l e s i n c l u d e d : i n t a c t c a r o t o v o r i c i n with and without attached heads, empty sheaths, cores and heads. In a d d i t i o n , p a r t i a l l y formed or d i s r u p t e d b a c t e r i o c i n p a r t i c l e s were a l s o trapped on the same g r i d ( F i g . 2b arrow). When trapped p a r t i c l e s were exposed to a d d i t i o n a l antiserum they became coated ( F i g . 2c). Although they resembled i n t a c t p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 , t h e i r o u t l i n e s appeared l e s s d i s t i n c t ( F i g . 2 c ) . In most cases these decorated p a r t i c l e s appeared out of focus but c l o s e examination r u l e d out t h i s p o s s i b i l i t y . The p a r t i c l e s were i n f a c t h e a v i l y coated with 8 6 Figure 2. D e t e c t i o n o f b a c t e r i o c i n s p r o d u c e d by m i t o m y c i n C i n d u c e d Erwinia carotovora s u b s p . carotovora s t r a i n 379 by i m m u n o s e n s i t i v e e l e c t r o n m i c r o s c o p y e m p l o y i n g p o l y c l o n a l a n t i s e r u m r a i s e d a g a i n s t t h e l a r g e m o l e c u l a r w e i g h t peak o b t a i n e d by S e p h a c r y l S-300 f r a c t i o n a t i o n o f s u p e r n a t a n t s (Bars=100nm). a. B a c t e r i o c i n p a r t i c l e s r e t a i n e d on u n c o a t e d g r i d r e c e i v i n g 5 u l o f s u p e r n a t a n t f r o m an i n d u c e d c u l t u r e ; b. 5 u l o f s u p e r n a t a n t from an i n d u c e d c u l t u r e a p p l i e d t o a g r i d p r e - c o a 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 t o b a c t e r i o c i n ( t r a p p e d b a c t e r - i o c i n ) . (Arrow shows p a r t i a l l y d i s r u p t e d p a r t i c l e ) . c. T r a p p e d b a c t e r i o c i n as i n b, e x p o s e d t o a s e c o n d i n c u b a t i o n w i t h p o l y c l o n a l a n t i s e r u m ( d e c o r a t i o n ) . 87 a n t i b o d i e s and as a r e s u l t were sometimes curved or s l i g h t l y d i s t o r t e d ( F i g . 2 c). SDS-PAGE Poly 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 showed that the supernatant p r o t e i n composition of Ecc 379 was complex and dependent on both the i n c u b a t i o n temperature of the producing s t r a i n , and the i n c o r p o r a t i o n of an i n d u c i n g agent ( F i g . 3a). At 20 C, i n d u c t i o n with mitomycin C (0.2 ug/ml) r e s u l t e d i n a marked i n c r e a s e i n s e v e r a l high and low molecular weight p r o t e i n subunits i n a d d i t i o n to a general i n c r e a s e i n a l l p r o t e i n subunits found i n non-induced c u l t u r e supernatants ( F i g . 3a). Incubation at 37 C reduced supernatant p r o t e i n c o n c e n t r a t i o n s by approximately 5 0 - f o l d and a b o l i s h e d the i n d u c t i o n mechanism a c t i v e at 20 C ( F i g . 3a). A l l E. coli transconjugants showed a general i n c r e a s e i n p r o t e i n content i n supernatants when compared with w i l d - t y p e E. coli ( F i g . 3b and 3c). CVP + E. coli transconjugants produced supernatant p r o t e i n p r o f i l e s i n d i s t i n g u i s h a b l e from induced Ecc s t r a i n 379 ( F i g . 3b and 3c). However, u n l i k e Ecc 88 F i g u r e 3. SDS-polyacrylamide g e l e l e c t r o p h o r e s i s of concentrated supernatants of Erwinia carotovora subsp. carotovora s t r a i n 379 and Escherichia coli t r a n s c o n j u g a n t s . a. E f f e c t of temperature and mitomycin C on pro d u c t i o n of supernatant p r o t e i n s by Ecc 379: Lanes: 1) Induced at 20 C; 2,3) Peak 2 from Sephacryl S-300 f r a c t i o n a t e d non-induced c u l t u r e at 20 C; 4) Induced at 20 C; 5,6) Non-induced at 20 C; 7) Non-induced at 37 C; 8) Induced at 37 C; 9,10) Induced at 20 C. b. Comparison of Ecc 379 with E. coli t r a n s c o n j u g a n t s : Lanes: 1) Sigma standards (30 to 200 KD); 2) R68.45 CVP" transconjugants; 3) CVP~ wi l d - t y p e mated transconjugant; 4) CVP + R68.45 transconjugant; 5) Ecc 379 induced at 20 C; 6) Ecc 379 non-induced at 20 C; 7) E. coli HB101 c o n t r o l ; 8,9) Sigma standards (30 to 200 KD). c. Comparison of Ecc 379 with E. coli transconjugants: Lanes: 1) Sigma standards (30 to 200 KD) ; 2) E. coli HB101/R68.45 c o n t r o l ; 3) Ecc 379 non-induced at 20 C; 4) Ecc 379 induced at 20 C; 5) E. coli R68.45 CVP + transconjugant; 6) CVP" R68.45 transconjugant; 7) HB101 wi l d - t y p e mated transconjugant CVP -; 8,9) Ecc 379 at 37 C; 10) Sigma standards (30 to 200 KD). 89 90 s t r a i n 379, the p r o t e i n s produced by CVP + E. coli were independent of both an in d u c i n g agent or temperature. CVP" E. coli transconjugants i n genera l , produced higher l e v e l s of e x t r a c e l l u l a r p r o t e i n than w i l d - t y p e E. coli HB101. A comparison of p r o t e i n s produced by CVP + E. coli or induced Erwinia, with CVP" E. coli transconjugants showed that s e v e r a l h i g h and low molecular weight p r o t e i n subunits were not produced by CVP" transconjugants ( F i g . 3b and 3c), and no p a r t i c u l a t e b a c t e r i o c i n was seen by EM. Western B l o t t i n g Most p r o t e i n bands of the homologous antigen, when b l o t t e d to n i t r o c e l l u l o s e , r e a c t e d with the antiserum a g a i n s t p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 ( F i g . 4). The most r e a c t i v e subunits of the homologous antigen were the high and low molecular weight components ( F i g . 4 ) . These components were i n lower c o n c e n t r a t i o n i n non-induced c u l t u r e s and absent from CVP" transc o n j u g a n t s . A comparison of immuno-reactive supernatant p r o t e i n s from CVP + E. c o l i transconjugants with those of induced Ecc i l l u s t r a t e d some d i f f e r e n c e s i n the low molecular 91 1 2 3 4 5 6 7 8 Fi g u r e 4. Western b l o t on n i t r o c e l l u l o s e of supernatant p r o t e i n s from Erwinia carotovora subsp. carotovora s t r a i n 379 and Escherichia coli t r a n s - conjugants f o l l o w e d by immunodetection u s i n g p o l y c l o n a l antiserum against p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 . Lanes: 1) Wild-type mated CVP" E. coli transconjugant; 2) Non-induced Ecc 379 at 20 C; 3) Induced Ecc 379 at 20 C; 4) CVP + R68.45 mediated E. coli transconjugant; 5) CVP" R68.45 mediated E. coli transconjugant; 6) Wild-type mated CVP" E. coli transconjugant with pBR322; 7) E. coli HB101 with R68.45 c o n t r o l ; 8) CVP" R68.45 mediated E. coli transconjugant with pBR322. 92 weight region, in sp i te of no apparent dif ferences in bac ter ioc in a c t i v i t y or appearance (v ia EM or SDS-PAGE). CVP+ transconjugants showed a r e l a t i v e l y smaller percentage of immuno-reactive subunits ( F i g . 4) when compared to the react ive proteins of Ecc ( F i g . 3b and 3c). D I S C U S S I O N The prote in subunit composition of carotovoric in-379 as analysed by SDS-PAGE and Western b l o t t i n g i l l u s t r a t e d a complex s tructure cons i s t ing of over 20 subunits with a wide range of molecular weights. The prote in composition of supernatants of CVP* E. coli transconjugants as seen by SDS-PAGE was i d e n t i c a l to wi ld-type Erwinia. These CVP* transconjugants produced b a c t e r i o c i n at 37 C without an inducing agent ( F i g . 3c and 4) . In contrast , an inducing agent dramat ica l ly increased the bac ter ioc in t i t r e at 20 C in Erwinia but had no effect at 37 C ( F i g . 4). CVP~ transconjugants released prote ins , some of which corresponded to bands present in wi ld-type 93 c a r o t o v o r i c i n - 3 7 9 . The most obvious d i f f e r e n c e was the absence of high and low molecular weight p r o t e i n s u b u n i t s . Immuno-detection of b l o t t e d p r o t e i n s by antiserum against p a r t i c u l a t e b a c t e r i o c i n demonstrated that p a r t i c u l a t e b a c t e r i o c i n produced by CVP + E. coli was very c l o s e l y r e l a t e d to w i l d - type 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. However, small d i f f e r e n c e s were noted i n the low molecular weight r e g i o n . The most obvious d i f f e r e n c e was the presence i n the Ecc b a c t e r i o c i n of a s t r o n g l y immuno-reactive band corresponding to about 15,000 MW. This i s most l i k e l y due to an outer membrane component of the c a r o t o v o r i c i n - 3 7 9 and the a n t i g e n i c d i f f e r e n c e s i n the outer membrane components of the Ecc and E. coli producers. In a d d i t i o n , some p r o t e i n subunits were detected with g r e a t e r s e n s i t i v i t y . This was most l i k e l y due to t h e i r d i f f e r e n t immunogenic p r o p e r t i e s and p o s i t i o n s i n the i n t a c t p a r t i c u l a t e b a c t e r i o c i n . CVP" transconjugants showed three immuno-reactive small molecular weight subunits and one l a r g e subunit which was a l s o present i n CVP + t r a n s c o n j u g a n t s . CVP + transconjugants contained an 94 a d d i t i o n a l l a r g e molecular weight (about 150 kd) p r o t e i n and s e v e r a l very low molecular weight components ( l e s s than 20,000) not present i n CVP" trans c o n j u g a n t s . A comparison of p r o t e i n s t a i n e d bands with immuno-stained bands showed that a l a r g e number of p r o t e i n subunits d i d not s t a i n immunogenically i n CVP" t r a n s c o n j u g a n t s . This r e d u c t i o n i n immunoreactive p r o t e i n s does not seem to be as obvious i n CVP + transconjugants and may r e f l e c t a pre f e r e n c e i n a h y p o t h e t i c a l s e c r e t i o n system f o r Erwinia-derived p r o t e i n s . CVP - transconjugants produced fewer Erwinia-Aerived p r o t e i n s p o s s i b l y a l l o w i n g a l e s s s p e c i f i c s u b s t i t u t i o n of E. coli p e r i p l a s m i c p r o t e i n s . This hypothesis was supported by the f a c t that CVP" transconjugants produce s u r f a c e v e s i c l e s and that a l k a l i n e phosphatase was found i n the supernatant of these transconjugants (Chapter 2). The p o l y c l o n a l antiserum developed a g a i n s t p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 c o n s i s t e n t l y r e a c t e d with the homologous an t i g e n . In a d d i t i o n , t h i s antiserum a l s o r e a c t e d with c a r o t o v o r i c i n produced by a l l E. coli t r a n s c o n j u g a n t s . This f a c t suggests 95 t h a t t h e low m o l e c u l a r w e i g h t c a r o t o v o r i c i n i s a component o f i n t a c t p a r t i c u l a t e c a r o t o v o r i c i n . 96 G E N E R A L D I S C U S S I O N U l t r a s t r u c t u r a l examination of c a r o t o v o r i c i n - 379 and c a r o t o v o r i c i n - p r o d u c i n g c e l l s l e d to the hypothesis of a c a r o t o v o r i c i n r e l e a s e without l y s i s (Chapter 1). Supporting evidence f o r t h i s h y pothesis came from the f a c t t h at non-induced Ecc s t r a i n 379 produced p a r t i c u l a t e b a c t e r i o c i n c o n s t i t u t i v e l y with no d e t e c t a b l e r e d u c t i o n i n c e l l t u r b i d i t y or v i a b i l i t y . Examination of c a r o t o v o r i c i n - 3 7 9 w i t h i n the supernatant of non- induced Ecc s t r a i n 379 showed that a c t i v i t y c o u l d be f r a c t i o n a t e d i n t o two components (Chapter 2). The higher molecular weight component was much more e f f e c t i v e at i n h i b i t i n g i n d i c a t o r s t r a i n s than the s m a l l e r molecular weight component (Chapters 1 and 2). Induction with DNA damaging agents skewed the p r o p o r t i o n of b a c t e r i o c i n i n the supernatant towards the higher molecular weight component (Chapter 1). A more c a r e f u l examination of t h i s i n d u c t i o n process i l l u s t r a t e d a gradual stepwise i n c r e a s e i n the molecular weight of c a r o t o v o r i c i n - 379 (Chapter 1). 97 Examination of induced producing c e l l s i n d i c a t e d that a small p r o p o r t i o n (about 10%) of these c e l l s contained s u b u n i t - l i k e s u r f a c e p r o j e c t i o n s at v a r i o u s stages of e l o n g a t i o n (Chapter 1). S t r u c t u r a l examination of c a r o t o v o r i c i n - 3 7 9 showed that the c e l l u l a r p r o j e c t i o n s resembled the c e n t r a l cores of p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 (Chapter 1). In a d d i t i o n , the e n t i r e c a r o t o v o r i c i n p a r t i c l e seemed to be arranged i n a modular manner surrounding these c e n t r a l cores (Chapter 1). It was hypothesized that these c e l l u l a r p r o j e c t i o n s represented the c e n t r a l cores of c a r o t o v o r i c i n and that i n t a c t p a r t i c u l a t e b a c t e r i o c i n may be formed by an i n d u c i b l e a d d i t i o n of components to these cores r e s u l t i n g i n an i n c r e a s e i n molecular weight and b i o a c t i v i t y . In order to i n v e s t i g a t e t h i s p o s s i b i l i t y , c a r o t o v o r i c i n p a r t i c l e s and c e l l u l a r p r o j e c t i o n s were examined by EM at s e v e r a l stages a f t e r i n d u c t i o n (Chapter 1). The r e s u l t s showed that c a r o t o v o r i c i n - 3 7 9 p a r t i c l e s g r a d u a l l y i n c r e a s e d i n s i z e , molecular weight, and b i o a c t i v i t y a f t e r i n d u c t i o n . In a d d i t i o n , c e l l u l a r p r o j e c t i o n s were observed which resembled these 98 d i f f e r e n t c a r o t o v o r i c i n p a r t i c l e s (Chapter 1). An examination of f r a c t i o n a t e d c a r o t o v o r i c i n - 379 showed that the high molecular weight component contained i n t a c t p a r t i c u l a t e c a r o t o v o r i c i n i n both extended and c o n t r a c t e d forms, whereas the low molecular weight component contained separated components of c a r o t o v o r i c i n - 3 7 9 (Chapter 1). Based on these o b s e r v a t i o n s , a h y p o t h e t i c a l model f o r c a r o t o v o r i c i n - 3 7 9 p r o d u c t i o n was developed i n Chapter 1. The model o u t l i n e d the c o n s t r u c t i o n of c a r o t o v o r i c i n - 3 7 9 at the l e v e l of the outer membrane by induced or non-induced c e l l s . In the model, c a r o t o v o r i c i n - 3 7 9 p r o d u c t i o n was hypothesized as an i n d u c i b l e a d d i t i o n of p r o t e i n subunits to a f i m b r a e - l i k e p r o j e c t i o n . This p r o j e c t i o n formed 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 and swe l l e d t e r m i n a l l y i n a v e s i c l e or head. In the non-induced s t a t e p a r t i a l l y a c t i v e c a r o t o v o r i c i n components accumulated i n the p e r i p l a s m i c space and were s e c r e t e d i n membrane v e s i c l e s or b l e b s . An i n t e r e s t i n g f e a t u r e of p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 p r o d u c t i o n was that i t i s temperature s e n s i t i v e (Chapter 2). Sev e r a l other 99 phenotypes, i n c l u d i n g c e l l l y s i s f o l l o w i n g i n d u c t i o n and a n t i b i o t i c r e s i s t a n c e , were a l s o temperature s e n s i t i v e i n Ecc 379 (Chapter 2). This f a c t i n t r o d u c e d the p o s s i b l e involvement of plasmid DNA i n the pr 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 . Genetic t r a n s f e r from Ecc to E. coli mediated by R68.45 (Chapter 2) suggested that p a r t i c u l a t e b a c t e r i o c i n was coded f o r by chromosomal c o n s t i t u e n t s (Chapter 2). However, conjugation s t u d i e s without a m o b i l i z a t i o n v e c t o r showed that Ecc 379 harboured a l a r g e molecular weight plasmid which was s e l f - t r a n s m i s s i b l e , and coded f o r erythromycin and chloramphenicol r e s i s t a n c e and the pr o d u c t i o n of a low molecular weight b a c t e r i o c i n component (Chapter 2). This component corresponded i n a c t i v i t y and s i z e to the p a r t i a l l y a c t i v e low molecular weight component of c a r o t o v o r i c i n - 3 7 9 . A n a l y s i s of transconjugants by EM (Chapter 2) showed a p r o l i f e r a t i o n of s u r f a c e v e s i c l e s or "bl e b s " . In a d d i t i o n , membrane v e s i c l e s and the p e r i p l a s m i c enzyme a l k a l i n e phosphatase were found i n the supernatant of these c e l l s (Chapter 2). The p r o d u c t i o n of membrane v e s i c l e s and e x t e r n a l a l k a l i n e phosphatase has been shown i n 100 s e v e r a l pseudomonad s p e c i e s . Some are i n v o l v e d i n food s p o i l a g e and membrane v e s i c l e s have been shown to i n c r e a s e the presence of a p a r t i c u l a r s o l i d s u b s t r a t e (Wing 1984). These v e s i c l e s have r e c e n t l y been shown to be i n v o l v e d i n enzyme s e c r e t i o n (Thompson et al. 1985) U n l i k e most b a c t e r i a , Erwinia produces a wide v a r i e t y of exoenzymes, some of which may be i n v o l v e d i n p a t h o g e n i c i t y . The outer membrane of gram negative b a c t e r i a provides a s u b s t a n t i a l b a r r i e r to r e l e a s e of p r o t e i n s (Nikaido 1985). Thus a s e c r e t o r y system f o r macerating enzymes and p o s s i b l y c a r o t o v o r i c i n components i n Erwinia i s necessary. The formation of membrane v e s i c l e s i n E. coli transconjugants and the e x t e r n a l r e l e a s e of a l k a l i n e phosphatase (Chapter 2) along with a general i n c r e a s e i n p r o t e i n s found i n transconjugant supernatants (Chapter 3) i n t r o d u c e s an a d d i t i o n a l involvement of the Erwinia megaplasmid i n s e c r e t i o n . In a d d i t i o n , the p r o d u c t i o n of a low molecular weight b a c t e r i o c i n component which corresponds in a c t i v i t y and plaque morphology to the low molecular weight c a r o t o v o r i c i n components i s o l a t e d i n Chapter 1 101 suggests that carotovoricin-379 production may involve both plasmid and chromosomal const i tuents . The low molecular weight carotovoricin-379 component was shown in Chapter 1 to be dependent upon induction and the incorporat ion of magnesium in media. The model (Chapter 1) explained th i s component as a prematurely released, p a r t i a l l y act ive b a c t e r i o c i n component. The effect of magnesium presumably s t a b i l i z e d outer membrane ves i c l e s i n h i b i t i n g th i s premature release (Chapter 1). The representation in the model assumed that the low molecular weight component was a subset of components phys i ca l l y associated with intact p a r t i c u l a t e carotovoricin-379. Support for th i s came from the fact that po lyc lonal antiserum, developed against p a r t i c u l a t e carotovoricin-379 reacted with the low molecular weight component produced by CVP" transconjugants (Chapter 3). One poss ible explanation for th i s cross react ion may be that the plasmid encoded low molecular weight component could represent a group of proteins which have some bac ter ioc in a c t i v i t y but are required for the assembly of p a r t i c u l a t e carotovoricin-379. This i s not a novel explanation as K88 fimbrae in 102 E. coli have been shown to r e q u i r e a plasmid- encoded outer membrane p r o t e i n f o r f i m b r a l assembly. The absence of t h i s p o l y p e p t i d e r e s u l t s i n the accumulation of f i m b r a l conponents i n the p e r i p l a s m (Hammond et al. 1984). In order to s p e c u l a t e f u r t h e r on the nature of the low molecular weight component of Ecc 379, an examination of the mode of a c t i o n of c a r o t o v o r i c i n r e q u i r e s c o n s i d e r a t i o n . Itoh et al. (1980a) found that b i n d i n g of p a r t i c u l a t e c a r o t o v o r i c i n by i n d i c a t o r s had at l e a s t two d i s t i n c t d i r e c t e f f e c t s . These two e f f e c t s were c e l l l y s i s and metabolic death. In phospholipase A minus mutants, no d e t e c t a b l e c e l l l y s i s was seen. The c e l l s , however, were m e t a b o l i c a l l y i n a c t i v e ( I t o h et al. 1981). This metabolic death was shown to be due to a l o s s i n the e n e r g i z e d s t a t e of the membrane which i s used f o r ATP s y n t h e s i s and n u t r i e n t uptake ( I t o h et al. 1982). In e a r l i e r work by Itoh's group, i t was shown that p a r t i c u l a t e c a r o t o v o r i c i n c o ntained an a c t i v a t o r of membrane bound phospholipase. This a c t i v a t o r was shown to cause c e l l l y s i s i n w i l d - type i n d i c a t o r s but not i n phospholipase mutants. It was assumed that t h i s phospholipase a c t i v a t i o n 103 was the primary mode of a c t i o n of c a r o t o v o r i c i n . However, the phospholipase A mutants which bound c a r o t o v o r i c i n although i n t a c t were s t i l l m e t a b o l i c a l l y i n a c t i v e ( I t o h er a i . 1982). In view of the complex s t r u c t u r e of c a r o t o v o r i c i n , i t i s q u i t e c o n c e i v a b l e that a phospholipase a c t i v a t o r may be coded f o r by the s e l f - t r a n s m i s s i b l e plasmid observed i n Erwinia. T h i s f a c t c o u l d r e s u l t i n a d i f f e r i n g a c t i v i t y s p e c t r a dependent upon the amount and type of phospholipase present i n i n d i c a t o r s t r a i n s . However, the primary mode of a c t i o n of c a r o t o v o r i c i n i s the i n a c t i v a t i o n of the energized s t a t e of the membrane which may be due to the formation of n o n - s p e c i f i c ion channels by the c e n t r a l core of the p a r t i c l e . This s t a t e would r e s u l t i n a degeneration of the proton motive f o r c e (pmf) which r e s u l t s from metabolism. The constant r e g e n e r a t i o n of t h i s chemical and pH gr a d i e n t allows c e l l s to couple thermodynamically f a v o u r a b l e tendencies to ATP s y n t h e s i s and n u t r i e n t uptake. Degeneration of the pmf would r e s u l t i n a c e s s a t i o n of metabolism. This type of k i l l i n g a c t i v i t y has 104 been r e p o r t e d u s i n g mutant T4 phage w h i c h c o n t a i n empty heads (T4 g h o s t ) ( L e w i n 1977). 105 S U M M A R Y 1) S e v e r a l forms of c a r o t o v o r i c i n - 3 7 9 which d i f f e r e d i n appearance, molecular weight and b i o a c t i v i t y e x i s t e d i n the supernatant of producing c e l l s of Erwinia carotovora subsp. carotovora s t r a i n 379 (Ecc 379) grown i n the absence of magnesium. 2) The l a r g e molecular weight p a r t i c u l a t e form of c a r o t o v o r i c i n - 3 7 9 , which gave a small c l e a r zone of i n h i b i t i o n when bioassayed, resembled a bacteriophage t a i l , was temperature sens- i t i v e and was i n d u c i b l e by mitomycin C. 3) Prod u c t i o n of the low molecular weight form of c a r o t o v o r i c i n - 3 7 9 , which gave a d i f f u s e zone of i n h i b i t i o n when bioassayed, was e l i m i n a t e d i n media c o n t a i n i n g magnesium. 4) S e v e r a l c e l l u l a r p r o j e c t i o n s were a l s o observed on producing c e l l s . These p r o j e c t i o n s may represent d i f f e r e n t stages of induced p a r t i c - 106 u l a t e c a r o t o v o r i c i n c o n s t r u c t i o n on a fimbrae- l i k e p r o j e c t i o n which s w e l l s to a detachable v e s i c u l a r head. 5) Ecc 379 showed temperature s e n s i t i v e p r o d u c t i o n of p a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 , c e l l l y s i s a f t e r i n d u c t i o n , and r e s i s t a n c e to erythromycin and chloramphenicol. 6) Ecc 379 contained a s e l f - t r a n s m i s s i b l e megaplasmid which coded f o r erythromycin and chloramphenicol r e s i s t a n c e , a low molecular weight component of c a r o t o v o r i c i n - 3 7 9 , and v e s i c l e formation which p o s s i b l y r e p r e s e n t s a s e c r e t o r y mechanism f o r Erwinia e x o p r o t e i n s . 7) The smal l molecular weight c a r o t o v o r i c i n - 3 7 9 component coded f o r by the Erwinia plasmid i s s e r o l o g i c a l l y r e l a t e d to p a r t i c u l a t e b a c t e r i o - c i n and con t a i n s p r o t e i n subunits found i n p a r t i c u l a t e b a c t e r i o c i n . 8) P a r t i c u l a t e c a r o t o v o r i c i n - 3 7 9 p r o d u c t i o n i s coded f o r by both chromosomal and plasmid 107 d e t e r m i n a n t s as w i l d - t y p e p a r t i c l e s were o n l y p r o d u c e d i n E. coli when a chromosome m o b i l - i z a t i o n v e c t o r (R68.45) was u s e d . C a r o t o v o r i c i n p r o d u c t i o n may be l i n k e d t o a p l a s m i d - d e p e n d e n t s e c r e t o r y s y s t e m f o r a l l Erwinia e x o p r o t e i n s . 108 REFERENCES Bi r g e , E. 1981. Bacterial and Bacteriophage Genetics. S p r i n g e r - V e r l a g , New York. 359 pp. Brock, T. 1979. Biology of Microorganisms. P r e n t i c e H a l l Inc., Englewood C l i f f s , New Je r s e y . 802 pp. Broda, P. 1979. Plasmids. W.H. Freeman and Co., San F r a n c i s c o . 197 pp. Collmer, A., Schaedel, C , Roeder, D., Reid, J . and R i s s l e r , J . 1985. Molecular c l o n i n g i n E. coli of Erwinia chrysanthemi genes encoding m u l t i p l e forms of pecta t e l y a s e . Journal of Bacteriology 161: 913-920. C o p l i n , D.L., Rowan, D.G., Chisholm, D.A. and Whitmoyer, R.E. 1981. C h a r a c t e r i z a t i o n of plasmids i n Erwinia stewartii. Applied and Environmental Microbiology 42: 599-604. Crowley, C F . and De Boer, S.H. 1980. S e n s i t i v i t y of some Erwinia carotovora serogroups to macromolecular b a c t e r i o c i n s . Canadian Journal of Microbiology 26: 1023-1028. Devoe, I.W. and G i l c h r i s t , J.E. 1973. Release of endotoxin i n the form of c e l l w a l l blebs d u r i n g in vitro growth of Neisseria meningitidis. Journal of Experimental Medicine 138: 1156-1167. Endo, Y., Tsuyama, H. and Nakatani, F. 1975. Studies on the p r o d u c t i o n of an a n t i b a c t e r i a l agent by Erwinia carotovora and i t s p r o p e r t i e s . Annals of the Phytopathological Society of Japan 41: 40- 48. Echandi, E. and Moyer, J.W. 1979. Produ c t i o n , p r o p e r t i e s and morphology of b a c t e r i o c i n s from Erwinia chrysanthemi. Phytopathology 69: 1204- 1207. 109 Forbes, K.J. 1981. A Genetic Study of Erwinia carotovora. D o c t o r a l T h e s i s , U n i v e r s i t y of Edinburgh. 121 pp. Gershoni, J.M. and Palade, G.E. 1983. b l o t t i n g : p r i n c i p l e s and a p p l i c a t i o n s , Biochemistry 131: 1-15. P r o t e i n Analytical G l a s s , R.E. 1982. Gene Function. Croom Helm, London. 487 pp. Glover, D.M. 1985. Press, Washington. DNA Cloning. 190 pp. Volume I. IRL Glover, D.M. 1985 Press, Washington. DNA Cloning. 245 pp. Volume II. IRL Haas, D. and Holloway, B. 1978. Chromosome mob i 1 i z a t ion by R plasmid R68.45: A t o o l i n Pseudomonas g e n e t i c s . Molecular and General Genetics 158: 229-237. Hames, B.D. and Higgins, S.J. 1984. Transcription and Translation. IRL Press, Washington. 328 pp. Hammond, S.M., Lambert, P.A. and R y c r o f t , A.N. 1984. The Bacterial Cell Surface. Croom Helm, London. 226 pp. H i l l , S.A. 1984. Methods in Plant Virology. B l a c k w e l l S c i e n t i f i c P u b l i c a t i o n s , London. 167 pp. Ingram, M. and Dainty, R.H. 1971. Changes caused by microbes i n s p o i l a g e of meats. Journal of Applied Bacteriology 34: 21. Itoh, Y., I z a k i , K. and Takahashi, H. 1978. P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of a b a c t e r i o c i n from Erwinia carotovora. Journal of General and Applied Microbiology 24: 27-39. Itoh, Y., I z a k i , K. and Takahashi, H. 1980. Simultaneous s y n t h e s i s of p e c t i n l y a s e and c a r o t o v o r i c i n induced by mitomycin C, n a l i d i x i c a c i d , or U.V. i r r a d i a t i o n i n Erwinia carotovora. Agricultural and Biological Chemistry 44: 1135- 1140. 110 Itoh, Y., I z a k i , K. and Takahashi, H. 1980. Mode of a c t i o n of a b a c t e r i o c i n from Erwinia carotovora I. P r o p e r t i e s of l y s i s of c e l l s i n f e c t e d with c a r o t o v o r i c i n Er. Journal of General and Applied Microbiology 26: 51-62. Itoh, Y., I z a k i , K. and Takahashi, H. 1980. Mode of a c t i o n of a b a c t e r i o c i n from Erwinia carotovora I I . Degradation of p h o s p h o l i p i d s i n c a r o t o v o r i c i n E r - t r e a t e d c e l l s . Journal of General and Applied Microbiology 26: 85-95. Itoh, Y., Iwata, T., I z a k i , K. and Takahashi, H. 1981. Mode of a c t i o n of a b a c t e r i o c i n from Erwinia carotovora I I I . P r o p e r t i e s of phospholipase A of Erwinia carotovora and involvement i n p h o s p h o l i p i d degeneration caused by c a r o t o v o r i c i n . Journal of General and Applied Microbiology 27: 239-251. Itoh, Y., Iwata, T., I z a k i , K. and Takahashi, H. 1982. Mode of a c t i o n of a b a c t e r i o c i n from Erwinia carotovora IV. E f f e c t s on macromolecular s y n t h e s i s , ATP l e v e l , and n u t r i e n t t r a n s p o r t . Journal of General and Applied Microbiology 28: 95- 99. J a i s , H. 1982. Bacteriocins of Erwinia carotovora. Masters T h e s i s . U n i v e r s i t y of B r i t i s h Columbia. Kamimiya, S., I z a k i , K. and Takahashi, H. 1977. B a c t e r i o c i n s of Erwinia ariodea with t a i l - l i k e s t r u c t u r e of bacteriophages. Agricultural and Biological Chemistry 41: 911-912. K e l l e n b e r g e r , E. and Ryter, A. 1958. C e l l w a l l and c y t o p l a s m i c membrane of E. coli. Journal of Biophysical and Biochemical Cytology 4: 323. Koloujansky, A., Didez, A. and Baccara, M. 1985. Mole c u l a r c l o n i n g of Erwinia chrysanthemi p e c t i n a s e and c e l l u l a s e s t r u c t u r a l genes. The EMBO Journal 4: 781-785. Laemmli, U.K. 1970. Cleavage of s t r u c t u r a l p r o t e i n s d u r i n g assembly of the head of T4. Nature 227: 680-685. I l l Laemmli, U.K. and Faure, M. 1973. Maturation of the head of bacteriophage T4. Journal of Molecular Biology 80: 575-599. Lewin, B. 1977. Gene Expression. John Wiley and Sons, New York. M a n i a t i s , T., F r i t s c h , E.F. and Sambrook, J . 1982. Molecular Cloning. Cold S p r i n g Harbour Laboratory. New York, 545 pp. Mayr-Harting, A., Hedges, A.J. and Berkeley, R.C.W. 1972. Methods f o r s t u d y i n g b a c t e r i o c i n s . Methods in Microbiology. J.N. N o r r i s , D.W. Ribbons, eds., 7A: 315-422. Nikaido, H. and Vaara, M. 1985. Molecular b a s i s of b a c t e r i a l outer membrane p e r m e a b i l i t y . Microbiological Reviews 49: 1-32. Nomura, M. 1967. C o l i c i n s and r e l a t e d b a c t e r i o c i n s . Annual Review of Microbiology 21: 257-284. Ohsumi, M., Shinomiya, T. and Kageyama, M. 1980. Comparative study on R-type pyocins of Pseudomonas aeruginosa. Journal of Biochemistry 87: 1119-1126. P e r b a l , B. 1984. A Practical Guide to Molecular Cloning. John Wiley and Sons, New York. 554 pp. Pugsley, A. and Schwartz, M. 1984. C o l i c i n E2 r e l e a s e : l y s i s , leakage, or s e c r e t i o n ? P o s s i b l e r o l e of a phospholipase. The EMBO Journal 3: 2393- 2397. Puhler, A. and Rie s s , G. i n Puhler and Timmis. 1984. Advanced Molecular Genetics. S p r i n g e r — V e r l a g , B e r l i n . 347 pp. Rand a l l , L. and Hardy, S. 1984. Export of p r o t e i n i n b a c t e r i a . Microbiological Reviews 48: 290-298. Schaad, N.W. 1980. Identification of Plant Pathogenic Bacteria. American P h y t o p a t h o l o g i c a l S o c i e t y , St. Paul, Minnesota, 72 pp. 112 Shinomiya, T., Kageyama, W., Aihara, Y. and Kobayashi, M. 1979. C h a r a c t e r i z a t i o n of a bacteriophage r e l a t e d to R-type pyocins. Journal of Virology 32: 951-957. Shinomiya, T. and Shiga, S. 1979. B a c t e r i o c i d a l a c t i v i t y of the t a i l of Pseudomonas aeruginosa bacteriophage PS-17. Journal of Virology 32: 958- 967. Sparks, R.B. and Lacy, G.H. 1980. P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of c r y p t i c plasmids pLSl and pLS2 from Erwinia chrysanthemi. Phytopathology 70:369-372. Thompson, S., Naidu, Y. and Pestka, J . 1985. U l t r a s t r u c t u r a l l o c a l i z a t i o n of an e x t r a c e l l u l a r protease i n Pseudomonas fragi u s i n g the PAP r e a c t i o n . Journal of Applied and Environmental Microbiology 50: 1038-1042. Towbin, H., S t a e h l i n , T. and Gordon, J . 1979. E l e c t r o p h o r e t i c t r a n s f e r of p r o t e i n s from p o l y a c r y l a m i d e g e l s to n i t r o c e l l u l o s e sheets. Procedure and some a p p l i c a t i o n s . Proceedings of the National Academy of Sciences, U.S.A. 76: 4350- 4354. Tsuyumu, S. and C h a t t e r j e e , A.K. 1984. P e c t i n - l y a s e p r o d u c t i o n i n Erwinia chrysanthemi and other s o f t - r o t Erwinia s p e c i e s . Physiological Plant Pathology 24: 291-302. Vidaver, A.K. 1976. Prospects f o r c o n t r o l of phytopathogenic b a c t e r i a by bacteriophage and b a c t e r i o c i n s . Annual Review of Phytopathology 14: 451-465. W i l l e t t s , N.S. and Crowther, C. 1981. The i n s e r t i o n sequence IS21 of R68.45 and the molecular b a s i s f o r m o b i l i z a t i o n of the b a c t e r i a l chromosome. Plasmid 6: 30-52. Wing, P.L. 1984. P h y s i c a l and chemical p r o p e r t i e s of e x o p o l y s a c c h a r i d e i s o l a t e d from Pseudomonas fragi ATCC 4973. D o c t o r a l T h e s i s . U n i v e r s i t y of B r i t i s h Columbia. Zink, R.T. , Kemble, R.J. and C h a t t e r j e e , A.K. 1984. Transposon Tn5 mutagenesis i n Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. atroseptica. Journal of Bacteriology 157: 809-814. Zink, R. and C h a t t e r j e e , A.K. 1985. C l o n i n g and expr e s s i o n i n E. coli of p e c t i n a s e genes of Erwinia carotovora subsp. carotovora. Applied and Environmental Microbiology 49: 714-717. )

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