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Bacteriocin production in Erwinia carotovora, subspecies carotovora, strain 379 Ward, Leonard John 1986

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BACTERIOCIN ERWINIA  CAROTOVORA  PRODUCTION  SUBSPECIES  IN  CAROTOVORA  STRAIN  37  By LEONARD JOHN WARD B.Sc,  The U n i v e r s i t y  of B r i t i s h  C o l u m b i a , .1981  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE  FACULTY OF GRADUATE  (Department  We a c c e p t t h i s to  of Plant  STUDIES  Science)  t h e s i s as c o n f o r m i n g  the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA '  O c t o b e r , 1986  ©Leonard John  Ward  In  presenting  requirements  this for  an  of  British  it  freely available  agree for  that  I  by  understood  that  his  that  or  be  her or  shall  of  VWV  The U n i v e r s i t y o f B r i t i s h 1956 Main M a l l Vancouver, Canada V6T 1Y3  IE-6  (3/81)  at  the  University shall  and  study.  I  copying  granted  by  the  of  publication be  allowed  Sex Columbia  of  this  It  this  without  make  further  head  representatives.  not  the  Library  permission.  Department  f u l f i l m e n t of  the  extensive  may  copying  f i n a n c i a l gain  degree  reference  for  purposes  or  partial  agree  for  permission  scholarly  in  advanced  Columbia,  department  for  thesis  thesis  of  my  is  thesis my  written  .ii  ABSTRACT  Erwinia (Ecc  carotovora  379) p r o d u c e d  subsp.  carotovora  a particulate  bacteriocin  c a r o t o v o r i c i n - 3 7 9 which resembled tail.  Carotovoricin-379,  diffuse  zones o f growth  against  several  It  inhibition,  conditions. fraction  was a c t i v e  carotovora  o f Ecc 379 u n d e r s t a n d a r d  strains.  of cultured  f o l l o w e d by c e l l  a sharp  Ecc 379 were  lysis,  production.  turbidity.  component  highly  Erwinia lysis  carotovora following  resistance  with  C  carotovoricin-  c o n d i t i o n s , a low  diffusible  bacteriocin  was d e t e c t e d w h i c h p r o d u c e d  zones o f i n h i b i t i o n  Growth o f  mitomycin  of particulate  Under t h e s e  molecular weight,  Induction  w h i c h was r e f l e c t e d b y  Ecc 379 a t 37 C w i t h o r w i t h o u t i nthe loss  i n c r e a s e d by  C (0.2 u g / m l ) .  reduction i nculture  resulted  cultural  I t s concentrations i nthe supernatant  i n d u c t i o n with mitomycin  379  c l e a r and  was d e t e c t e d i n t h e f i l t e r - s t e r i l i z e d  supernatant  was  called  a bacteriophage  producing both  o t h e r Erwinia  s t r a i n 379  large  three o f the four  indicator  strains  used.  diffuse standard Cell  i n d u c t i o n and a w i l d - t y p e  to erythromycin  and c h l o r a m p h e n i c o l  were  iii  also  temperature  sensitive.  Carotovoricin-379 by  isolating  induction. electron  intermediates These  chromatography.  central  by  the  and S e p h a c r y l  This maturation  weight, b i o a c t i v i t y particulate  staining  particulate carotovora was  cells.  appearance  By u s i n g a  protocol, c e l l carotovoricin-379 attached to  On t h e b a s i s o f t h e s e  a h y p o t h e t i c a l model  Genetic  matured  molecular  and u l t r a s t r u c t u r a l  were s e e n p h y s i c a l l y  producing  production  in  carotovoricin-379.  negative  intermediates  were  p r o c e s s was  increase  p r o j e c t i o n s which resembled  results  particles  external addition of bacteriocin  accompanied by a g e n e r a l  intact  S-300 column  EM showed a s u b u n i t - l i k e  Carotovoricin-379  gradual  modified  were a n a l y s e d b y  as f i m b r a e - l i k e p r o j e c t i o n s which  components.  of  after  o f b a c t e r i o c i n components a r o u n d a  core.  initiated  (EM)  was i n v e s t i g a t e d  a t s e v e r a l times  intermediates  microscopy  arrangement  production  for  carotovoricin-379  was o u t l i n e d . a n a l y s i s o f a temperature bacteriocin production subsp.  performed with  carotovora  strain  and w i t h o u t  sensitive,  i n Erwinia 379 {Ecc 379)  mobilization vector  iv  R68.45. E.  coli  particulate violet  transconjugants  carotovoricin-379  p e c t a t e medium  R68.45 was u s e d . frequencies  which both  and degraded  (CVP) were p r o d u c e d  crystal o n l y when  In a d d i t i o n , t h e t r a n s f e r  o b t a i n e d were  indicative of  chromosomally d e r i v e d determinants. and  produced  Erythromycin-  chloramphenicol-resistant transconjugants  were  obtained  r e g a r d l e s s o f R68.45 m e d i a t i o n .  analysis  b y a g a r o s e g e l e l e c t r o p h o r e s i s showed  Ecc  contained  with  This plasmid  and c h l o r a m p h e n i c o l  the production  carotovoricin-379 produced by heat transconjugants protuberance addition,  that  a r e s i d e n t m e g a p l a s m i d w h i c h was  self-transmissible. erythromycin  DNA  coded f o r r e s i s t a n c e along  o f t h e low m o l e c u l a r  component  similar  t r e a t e d Ecc  379.  to that Analysis of  by e l e c t r o n m i c r o s c o p y  of surface  alkaline  transconjugants.  showed a  "blebs" or v e s i c l e s .  phosphatase  enzyme) was d e t e c t e d  weight  (a periplasmic  i n supernatants  These f a c t s  In  of  implied that the  m e g a p l a s m i d may a l s o be i n v o l v e d i n t h e r e l e a s e o f many e x o - p r o t e i n s Polyclonal against  of  rabbit  particulate  Erwinia. antiserum  was  developed  carotovoricin-379.  This  V  antiserum  was t e s t e d a g a i n s t c u l t u r e supernatants  from w i l d - t y p e Erwinia coli  transconjugants  (RID),  immunosensitive  and Western b l o t t i n g .  and s e v e r a l Erwinia  using r a d i a l  x E.  immunodiffusion  e l e c t r o n microscopy  (ISEM)  The 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 r e a c t e d with supernatant  f r a c t i o n s from a l l t r a n s c o n j u g a n t s .  CVP+ t r a n s c o n j u g a n t s  c o n s i s t e n t l y showed a wider  range o f r e a c t i v e b a c t e r i o c i n components than CVP" transconjugants.  A low molecular weight  partially  a c t i v e c a r o t o v o r i c i n - 3 7 9 component was d e t e c t e d i n CVP  -  t r a n s c o n j u g a n t s by antiserum  particulate carotovoricin.  r a i s e d against  T h i s suggested  a  r e l a t e d n e s s between the two major b a c t e r i o c i n components o f c a r o t o v o r i c i n - 3 7 9 .  vi TABLE  Title  Page  OF C O N T E N T S  .  Authorization Abstract Table  i i i i i i  of Contents  List  of Tables  List  of Figures  Acknowledgements  General  Introduction  Chapter  1  v i i x . .xi xiv  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 Introduction  19  M a t e r i a l s and Methods A. C u l t u r e o f Erwinia carotovora s t r a i n s and i n d u c t i o n o f b a c t e r i o c i n product ion B. B a c t e r i o c i n p l a t e assays C. Concentration of carotovoricin-379.... D. Column c h r o m a t o g r a p h y E. E l e c t r o n microscopy F. M o d i f i e d n e g a t i v e s t a i n i n g o f whole e e l Is G. F i x a t i o n and embedding  22  Results A. B. C. D.  Mitomycin C induced c u l t u r e s Examination of the supernatant f ract ions S e r i a l subunit s t r u c t u r e o f particulate carotovoricin-379 Developmental stages o 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 by mitomycin C induced c e l l s  22 22 24 25 25 25 26 27 27 27 30  33  vii Results E.  (cont.) Extrusion of carotovoricin t h e membrane S e p h a c r y l S-300 column chromatography  F.  through  Discussion.  Chapter  38 39 44  2  Genetic Determinants P r o d u c t i o n i n Erwinia  of Carotovoricin carotovora  Intro duction  53  Materials A. B. C. D.  55 55 57 58  and Methods M e d i a and g r o w t h c o n d i t i o n s Mating p r o t o c o l s B a c t e r i o c i n p l a t e assays T o t a l DNA e x t r a c t i o n and electrophoresis  Results A.  61 S e p h a c r y l S-300 column and b i o a s s a y s DNA c o n t e n t E l e c t r o n microscopy  B. C.  chromatograpy  Discussion  Chapter  58  61 67 67 70  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 Antiserum Against P a r t i c u l a t e Carotovoricin-379 Intro duction  76  Materials A. B. C. D.  79 79 79 80  E.  F.  and Methods Bacterial strains Development o f a n t i s e r u m Radial immunodiffusion Immuno-sensitive e l e c t r o n m i c r o s c o p y (ISEM) Sodium d o d e c y l s u l p h a t e polyacrylamide gel electrophoresis (SDS-PAGE) Western b l o t t i n g  80  81 82  viii Results A. B. C.  83 Immuno-sensitive e l e c t r o n m i c r o s c o p y (ISEM) SDS-PAGE Western b l o t t i n g  85 87 90  Discussion  92  General  96  Discussion  Summary  105  References  108  ix LIST  Chapter Table  Table  1.  TABLES  1  1.  Chapter  OF  Carotovoricin p r o d u c i n g and s t r a i n s o f Erwinia carotovora  sensitive 23  2 Bacterial  strains  used  56  X LIST  OF FIGURES  Chapter 1 F i g u r e 1.  Figure 2  Figure 3  F i g u r e 4,  E l e c t r o n micrographs o f mitomycin C i n d u c e d c u l t u r e s o f 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 a t 20 C  28  Negative stained preparations o f particulate carotovoricin-379 c o n c e n t r a t e d from t h e s u p e r n a t a n t f r a c t i o n o f mitomycin C induced Erwinia carotovora subsp. carotovora s t r a i n 379 ,  31  N e g a t i v e s t a i n e d immature c a r o t o v o r i c i n - 3 7 9 s h e a r e d from p r o d u c i n g c e l l s o f Erwinia carotovora subsp. carotovora s t r a i n 379 8 h a f t e r m i t o m y c i n C i n d u c t i o n and t h e p l a q u e t y p e s they induce i n s e n s i t i v e i n d i c a t o r strains  34  Negative stained carotovoricin-379 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 o f Erwinia carotovora s u b s p . carotovora s t r a i n 379  36  F i g u r e 5.  S e p h a c r y l S-300 column chromatograms o f c a r o t o v o r i c i n - 3 7 9 a t ( a ) 44, ( b ) 52, ( c ) 58 a n d ( d ) 62 h a f t e r mitomycin C i n d u c t i o n i n c o n c e n t r a t e d s u p e r n a t a n t s o f Erwinia carotovora subsp. carotovora s t r a i n 379 40  F i g u r e 6,  S e p h a c r y l S-300 column 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 b y Erwinia carotovora subsp. carotovora s t r a i n 379 48 h a f t e r m i t o m y c i n C i n d u c t i o n and e l e c t r o n micrographs o f the corresponding n e g a t i v e s t a i n e d peak c o n t e n t s 42  xi  F i g u r e 7.  Sephacryl S-300 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 in mitomycin C induced c u l t u r e s grown under d i f f e r e n t condi t ions 45  F i g u r e 8.  H y p o t h e t i c a l model f o r the p r o d u c t i o n of c a r o t o v o r i c i n by Erwinia carotovora subsp. carotovora s t r a i n 379 49  Chapter  2  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. coli transcon jugants 62  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 S e p h a c r y l S-300 f r a c t i o n a t e d peaks from concentrated supernatants of E. coli transconjugants and E. carotovora subsp. carotovora (Ecc) s t r a i n 3 7 9 . . . 6 3  F i g u r e 3.  T o t a l DNA a n a l y s i s of w i l d - t y p e E. coli, w i l d - t y p e 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 m e d i a t i o n . . ,  68  E l e c t r o n micrographs of c e l l s and supernatants of E. coli transconjugants  69  F i g u r e 4.  Chapter  3  F i g u r e 1.  R a d i a l immunodiffusion a n a l y s i s 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 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 (Ecc 379) and Ecc 379 x E. coli transconjugants 84  xii Figure  Figure  Figure  2.  3.  4.  D e t e c t i o n o f b a c t e r i o c i n s produced 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 strain 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 microscopy employing p o l y c l o n a l antiserum r a i s e d against the large 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 supernatants ,  86  SDS-polyacrylamideg e l electrophoresis of concentrated s u p e r n a t a n t s o f Erwinia carotovora s u b s p . carotovora s t r a i n 379 and Escherichia coli  88  W e s t e r n b l o t on n i t r o c e l l u l o s e o f supernatant p r o t e i n s from Erwinia carotovora s u b s p . carotovora strain 379 and Escherichia coli t r a n s c o n j u g a n t s 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 carotovoricin-379  91  xiii  ACKNOWLEDGEMENTS  I w i s h t o t h a n k my s u p e r v i s o r Dr. R . J . Copeman for  financial  other Dr. help  and c r i t i c a l  members o f my c o m m i t t e e  R.E.W.  readings  and t h e  namely, D r . M. Shaw,  Hancock and Dr. V.C. R u n e c k l e s  for their  and s u g g e s t i o n s . I would  and  support  also like  Mr. F. S k e l t o n  microscopy. MacMillan  t o t h a n k M r s . B. V a l e n t i n e  f o r help with e l e c t r o n  Thanks a r e a l s o e x t e n d e d f o r graphics  help,  DeYoung f o r t y p i n g , L e r o y and a n a l y t i c a l  Stace-Smith  for helpful  Finally,  I would  like  Vancouver Research electron  Donna S m i t h and Robyn  Scrubb  biochemical  to Brian  f o rhelp  techniques  suggestions  t o thank  with  and D r . R. and comments.  A g r i c u l t u r e Canada  S t a t i o n f o r t h e use o f t h e i r  microscope.  1  GENERAL  Erwinia Bergey  carotovora  et  al.  i sresponsible  o f economically  crops.  These  survive  healthy  bacteria  weakened  infected  material,  enzymes  degrade  components.  separation and  o f plant  eventual  potential  rhizosphere  conceivable  the production  bacteriocin) was  means  An  loss  these  i nsoil  o f control  as n a t u r a l involves  organisms  inhabitants. type  and c e l l  by water  Because  o f these  of this  by non-  The s i m p l e s t  ofbio-control  o f an i n h i b i t o r y s u b s t a n c e  b y some  specifically  lamella  may b e f o u n d  inhibition  the  a variety of  followed  means  i n  i n a s o f t e n i n g and  death.  one p o s s i b l e  selective  pathogenic  be  cell  pathogens  microflora, the  plant  cells  Within  multiply  themiddle results  t o invade  tissues.  and secrete  This  pathogens  do n o t a t t a c k  buta r e able  thebacteria  spaces  wall  They  or o l d plant  intercellular which  for soft-rot ofa  areopportunistic  material  wounded,  (Jones)  important a g r i c u l t u r a l  as saprophytes.  plant  carotovora  subsp.  variety  and  INTRODUCTION  non-pathogenic  active  against  a l t e r n a t i v e approach  organism  Erwinia  might  would (ie. a which  carotovora.  involve the  2  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 f o r b a c t e r i o c i n p r o d u c t i o n to a non-pathogenic inhabitant.  This l a t t e r  genetic segregation other c e l l  segregation  approach r e q u i r e s a  of b a c t e r i o c i n p r o d u c t i o n from  functions.  investigated  rhizosphere  In some e x t e n s i v e l y  animal-pathogen systems,  this  has been shown to be a n a t u r a l r a t h e r  than a r t i f i c i a l l y c o n s t r u c t e d s e p a r a t i o n .  In some  such systems b a c t e r i o c i n and/or v i r u l e n c e have been shown to be segregated on g e n e t i c elements c a l l e d p l a s m i d s .  factors  independent In Erwinia  the  g e n e t i c b a s i s f o r b a c t e r i o c i n p r o d u c t i o n has not been e s t a b l i s h e d ;  however,  c e r t a i n s p e c i e s of  Erwinia  have been checked f o r plasmid  Erwinia  stewartii  content.  was shown to c o n t a i n between 11  to 13 plasmids ranging i n s i z e from 2.8 megadaltons Erwinia  ( C o p l i n er al.  carotovora  1980).  Erwinia  210  Likewise i n  both l a r g e and small molecular  weight plasmids have been noted Forbes 1981).  to  (Zink et  In a l l cases noted,  have been c l a s s i f i e d  a t t r i b u t a b l e phenotypes.  al.  1984,  the plasmids of  as c r y p t i c with no  However, the  of these plasmids suggests that  maintenance  some yet  u n i d e n t i f i e d t r a i t s may be plasmid coded i n  3  Erwinia. Plasmids replicating (Brock is  that  its  own  a r e d e f i n e d as  independently  extrachromosomal g e n e t i c  1979).  The  i t must  essential  structures  f e a t u r e of a  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  replication  and  maintenance, u t i l i z i n g  m e t a b o l i c machinery of the h a r b o u r i n g Most p l a s m i d s elimination  that to  the host  that  of organisms,  curing upon t h e  f o r the  survival  from  a wide  although  essential,  they  In e v o l u t i o n a r y terms,  o f t h e p l a s m i d must be  i n c r e a s e i n the  lethal  code f o r  isolated  f e a t u r e s a r e not  fact  i s not  suggesting that  are probably b e n e f i c i a l . the s u r v i v a l  rely  essential  They have been  plasmid-encoded  overall  to the p o i n t of  Thus, p l a s m i d s  a r e not  the  organism.  o f most p l a s m i d s  organism.  the h o s t .  variety  diluted  These approaches  the e l i m i n a t i o n  functions of  can be  using various so-called  treatments.  plasmid  fitness  due  of the  to  an  harbouring  organism. Physical hinges  evidence  upon t h e  substantially chromosome and  fact  f o r the presence that  s m a l l e r than exist  as  a l l plasmids the c i r c u l a r  a covalently  of a  plasmid  are bacterial  closed  4  supercoiled most  procedures  one o r b o t h  strand is  circle  compact f o r m w i t h i n  isolation in  or t w i s t e d  (CCC).  a cell;  however,  can i n t r o d u c e  o f t h e DNA  results  while  in a linear  DNA  DNA  a nick  molecules  organic  e x t r a c t i o n o f p r o t e i n and  shearing Plasmid  supercoiled be s e p a r a t e d ethidium  forces the  from  linear  This  which  strands  separation  lysis  followed  result  into linear  DNA  using  i n the  fragments.  intact  DNA  can  in a easily  cesium c h l o r i d e -  density  technique  by  carbohydrates.  protocols  Plasmid  of  gradient  i s b a s e d upon t h e  when a homogeneous s o l u t i o n o f c e s i u m i s subjected  to excessive g r a v i t a t i o n a l  s u c h 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  salt  s o l u t i o n i s formed. gradient  s o l u t i o n i s separated  density  i n one  (OC) DNA  largely left  conformation.  s e t s up a d e n s i t y salt  cell  bromide ( C s C l - E t B r )  that  chloride  lysis  i s however,  centrifugation. fact  involves  o f chromosomal DNA DNA  A nick  molecule.  plasmid  gentle  or break  i n both  e x t r a c t i o n and s u b s e q u e n t  Even t h e most  i s the DNA  a nick  strands.  r e s u l t s i n an open c i r c u l a r  not s u p e r c o i l e d ,  This  gradient.  This  and DNA  salt  of  gradient  d i s s o l v e d i n the  upon t h e b a s i s  The i n c o r p o r a t i o n o f  of this  ethidium  5  bromide i n t o the s a l t g r a d i e n t Firstly,  serves two  purposes.  ethidium bromide i n t e r c a l a t e s i n t o DNA and  RNA and p r o v i d e s localization  a visual,  reversible  of the n u c l e i c  acids.  ethidium bromide i n t e r c a l a t i o n  tag  for  Secondly,  differentially  lowers the d e n s i t y of the n u c l e i c  acids.  Linear  and open c i r c u l a r DNA b i n d more ethidium than h i g h l y compact s u p e r c o i l e d  DNA.  banding of plasmid s u p e r c o i l e d  This r e s u l t s i n a DNA at a higher  d e n s i t y than l i n e a r or open c i r c u l a r DNA.  All  s p e c i e s of plasmid DNA's which are s u p e r c o i l e d band at approximately Similarily,  the same  will  density.  n i c k e d plasmid molecules w i l l band i n  the same r e g i o n as l i n e a r chromosomal DNA. the s u c c e s s f u l  separation  Thus,  of plasmid from  chromosomal DNA r e l i e s upon an e f f e c t i v e lysis/extraction  p r o t o c o l which minimizes  the  amount of plasmid n i c k i n g . A plasmid molecule s h e a r i n g or enzymatic  may be n i c k e d by p h y s i c a l cleavage;  of a plasmid to n i c k i n g i s physical size. f o r enzymatic shearing.  The  susceptibility  due p r i m a r i l y to  its  Larger plasmids p r o v i d e more s i t e s cleavage and are more s u s c e p t i b l e  In a d d i t i o n ,  l a r g e r plasmids  are  in  to  6  general under a more s t r i n g e n t harbouring c e l l  and are found at a l e v e l  two c o p i e s per c e l l contrast,  c o n t r o l by the of one  (low copy number).  to  In  small plasmids are under a more r e l a x e d  c o n t r o l and are found i n much higher copy number. Furthermore, components  l a r g e r plasmids r e q u i r e c e r t a i n host  and are thus not a m p l i f i e d to  higher  l e v e l s by the a d d i t i o n of a p r o t e i n  synthesis  i n h i b i t o r such as c h l o r a m p h e n i c o l .  In s t r i k i n g  contrast,  s m a l l e r plasmids may be a m p l i f i e d  l e v e l s of one thousand c o p i e s per  to  cell.  Although l a r g e plasmids present  practical  problems with respect to t h e i r p u r i f i c a t i o n and subsequent i n v e s t i g a t i o n , conjugative throughout  they are  which f a c i l i t a t e s a population.  usually  t h e i r r a p i d spread  In a d d i t i o n ,  large  plasmids may spend a c e r t a i n p r o p o r t i o n of integrated  or c o v a l e n t l y  chromosome.  Subsequent  these i n t e g r a t e d  l i n k e d to the  elements,  bacterial  c o n j u g a t i o n and t r a n s f e r  plasmids  facilitates  both plasmid and l i n k e d chromosomal Thus p l a s m i d s ,  time  a transfer determinants.  as mobile and v a r i a b l e  p r o v i d e important t o o l s f o r  genetic the  understanding of p r o k a r y o t i c metabolism and  of of  7  regulat ion. Plasmids  are also  investigation phenomena.  of several  The b e s t  group o f p l a s m i d s factors  tools  studied  contain  and most  resistance  to a n t i b i o t i c s .  selection  Unfortunately, forbacteria  antibiotic  able  These R p l a s m i d s  came  time t o v a r i o u s  Plasmids  are also  various  virulence  disease  manifestation.  ability  human and a n i m a l involved  factors  red blood  and t o x i n s  the small  causes excessive two p l a s m i d - c o d e d enteropathogenic  cells,  secretion toxins Escherichia  were  pathogens.  i n the production  of  related to  In some e n t e r i c  to colonize  coding  in a surprisingly  pathogens,  i n t e s t i n e i s due  protein  w h i c h i s c o d e d f o r by a p l a s m i d . lyses  strong  These R f a c t o r s  the presence o f a surface  which  a  harbouring plasmids  v i a conjugation  War  to c o n t r o l  t h i s created  resistance.  to transfer  short  to  transfer  p r o m i n e n c e a t t h e end o f t h e S e c o n d W o r l d  dysentery.  the  widespread  multiple  when a n t i b i o t i c s were u s e d w i d e l y  for  f o r the  b i o l o g i c a l and e c o l o g i c a l  (R f a c t o r s ) w h i c h c o n f e r  resistance into  important  (K a n t i g e n )  Alpha-hemolysin  and e n t e r o t o x i n  which  o f w a t e r and s a l t s a r e  p r o d u c e d by coli.  8  The has  involved  their  of both plant  A n t i b i o t i c s were f i r s t  inhabitants  general  of s o i l .  a wide spectrum  pathogens  organisms).  inhibiting  The e f f e c t s o f  (killed  The compounds  (stopped  affected  t h e m s e l v e s were  o r g a n i c m o l e c u l e s which t a r g e t e d  primarily  protein-synthesizing  However as  mentioned to  above,  machinery.  provided  an e x t r e m e l y  selection  pressure  efficient  resistance  evolution  o f R plasmids which u s u a l l y  The  basis  production  factor.  Similarily  synthesis  (Broda  and were  of R factor  the s o l u t i o n control  of altered  high  o f an  contained  self-  1979).  and r e n d e r e d  a disease  on t h e  The r e s u l t was t h e  resistance  o f enzymes w h i c h m o d i f i e d  antibiotics  from  f o r the development  drug r e s i s t a n c e  transmissible  simple  the w i d e s p r e a d use o f a n t i b i o t i c s  combat d i s e a s e s  multiple  from  T h e s e s u b s t a n c e s had  were e i t h e r b a c t e r i o s t a t i c  growth) o r b a c t e r i o c i d a l  inhibit  isolated  of a c t i v i t y ,  many d i f f e r e n t o r g a n i s m s . antibiotics  and a n i m a l  t h e use o f s u b s t a n c e s w h i c h  growth.  natural in  control  was t h e the incoming  them u s e l e s s to R f a c t o r  standpoint,  (Brock  1979).  resistance, was  the organic  a n t i b i o t i c s w h i c h were n o t  9  r e c o g n i z e d by t h e a n t i b i o t i c - m o d i f y i n g This  practice  s e t up an o n g o i n g p o s i t i v e  c y c l e between c h e m i s t bacteriocins alternative In E.  research  found  strain  a certain  (Birge  1981).  a diverse  as d e f i n e d  species.  most  bacteria  (with  These  by Nomura i n 1967, f o r m e d  group o f s u b s t a n c e s  molecular weight  Further  agents which k i l l e d or  closely related  bacteriocins,  strain of  s p e c i f i c a n t i b i o t i c which  by many w o r k e r s showed t h a t  produced proteinaceous  unlike  As a r e s u l t ,  as a p o s s i b l e  that  a highly  another  inhibited  feedback  to a n t i b i o t i c therapy.  produced  inhibited  and p a t h o g e n .  were c o n s i d e r e d  1925, G r a t i a  coli  enzymes.  frequently  respect  of high  to a n t i b i o t i c s ) ,  a n t i b i o t i c s , showed a v e r y narrow  but  activity  spectrum. Bacteriocin  nomenclature r e f l e c t s attempts t o  name t h e s e u b i q u i t o u s Individual producing are  agents  bacteriocins organisms.  are usually  F o r example:  p r o d u c e d by H. influenzae^  subtilis,  p y o c i n s by P.  pyocyanea)  , c o l i c i n s by E.  by  systematically.  Erwinia  carotovora.  influenzacins  s u b t i l i n s b y B.  aeruginosa coli  named a f t e r t h e  (originally  P.  and c a r o t o v o r i c i n s  Subclasses  of bacteriocin  10  are  i d e n t i f i e d by  class  designation.  different produced  forms by  Thus,  also  colicin  from  In a d d i t i o n ,  includes  The  slightly  a colicin i s an  V  produced  by  important  of a b a c t e r i o c i n  s e n s i t i v e to  another species  Most b a c t e r i o c i n o g e n i c levels  under  bacteriocin  normal  as  a t h o u s a n d - f o l d by which 1981). light  cause  be  from  come from  low However,  several  to  of treatments (Birge  ultraviolet  (UV)  o r t r e a t m e n t w i t h DNA-damaging c h e m i c a l s s u c h  as m i t o m y c i n  C.  In some t e m p e r a t u r e  a u x o t r o p h i c mutants  o f E.  a c c o m p l i s h e d by h e a t  deprivation 1972).  increased  o f lambda p r o p h a g e  inductions  own  1981).  s t r a i n s produce  t h e same s o r t s  induction  Such  (Birge  cells  bacteriocin  cultural conditions.  y i e l d s may  be  produced  a p r o d u c i n g s t r a i n a r e immune t o t h e i r be  may  complete  nomenclature  b u t may  the  the s t r a i n of producer.  i n the d e s c r i p t i o n  bacteriocin,  be  as  of a p a r t i c u l a r b a c t e r i o c i n  V-K357 i n d i c a t e s  s t r a i n K357.  factor  numbers a f t e r  different strains, a  designation  by  l e t t e r s and/or  respectively  coli,  the f a c t  that  induction  treatment  or  induction  can  or also  nutrient  ( M a y r - H a r t i n g et  P a r a l l e l s t o phage  drawn f r o m  sensitive  can  most p r o d u c i n g  al. also cells  be  11  undergo  lysis  or q u a s i - l y s i s  bacteriocin. the  large  of these p r o t e i n s  p e r m e a b i l i t y o f most  Genetic shown t h a t  analysis  coded  m o l e c u l a r weight  f o r by  less  plasmids  (Birge  100  KD)  whereas,  (>500 KD)  coded  the b a c t e r i a l  expected, been  t h e most  analysis so  colicins  has  shown t h a t  far, colicins  (Broda  1979).  classes. than low  extensively  copy  t o be  studied  As  group.  i n a l l cases  fall  plasmids  two  are  large  c o n j u g a t i v e , and per c e l l )  are small ( l e s s  than  6 megadaltons),  conjugative  and  present  per  Colicin-producing  i n h i g h copy  major (greater  present i n while  others  nonnumbers  plasmids  Col for c o l i c i n  Genetic  into  t o two  have  investigated  f o r by  (one  by  coded  primarily  bacteriocins  number  been d e n o t e d  t o be  particulate  Some o f t h e s e p l a s m i d s  cell).  lower  colicins  chromosome.  plasmids  60 m e g a d a l t o n s ) ,  as  has  and/or  Diffusible  such  have been c o d e d  The  the  production  were f o u n d  coli  that  membranes.  were f o u n d  o r E.  fact  are plasmid  large  bacteriocins f o r by  exceeds  1981).  bacteriocins  than  to the  of b a c t e r i o c i n  these determinants  chromosomally  (usually  releasing  T h i s i s p r o b a b l y due  size  functional  in  have  f o l l o w e d by  (10-30 always an  12  appropriate  letter.  invaluable  by g e n e t i c  markers with  T h e s e p l a s m i d s have been made d e l e t i o n and i n s e r t i o n o f  forming various  colicin  plasmid  useful  cloning  replicative  vehicles  function  (col  replicons). Colicins activity  were c l a s s i f i e d  aginst  several  however c o m p l i c a t e d  E.  on t h e b a s i s o f  coli  by t h e f a c t  produced  several  colicins  have been c l a s s i f i e d  originally strains  colicins.  colicins  designated  are  resistance  i n the cross  t o l A.  by a c t i v i t y on  g r o u p B.  localized  resistance  C e l l s designated  Several  on t h e o u t e r  a particular colicin  (Broda  1979).  which a r e t o l e r a n t t o  (tonB) or e n t e r o c h e l i n  resistance  to  strains  grouping)  r e s i s t a n t to a l l c o l i c i n s  been  that  most  was  sensitive derivatives of p a r t i c u l a r  (cross  resistant  This  As an a l t e r n a t i v e ,  Thus, d e r i v a t i v e s o f c e l l s all  strains.  group A a r e phage T l  excretion  (exbB)  i n the cross colicin  receptors  have  membrane and r e s i s t a n c e  c a n i n most b u t n o t a l l  c a s e s be t r a c e d  t o an a l t e r a t i o n o r l o s s o f t h i s  receptor  1979).  (Broda  Binding sensitive  and s u b s e q u e n t  cell  usually  action  of a c o l i c i n  results in a killing  to a  o f that  13  cell.  T h e r e a r e t h r e e g e n e r a l ways i n w h i c h  colicins  a r e known t o k i l l  Some c o l i c i n s energy  a c t at the c e l l  uncouplers  are  1979).  immune  molecular  o f DNA  f o r both  others  ( c o l E2) o r RNA  As m e n t i o n e d  basis f o r this  1979).  membrane and a c t as  ( c o l E3)  above, p r o d u c i n g  to the b a c t e r i o c i n  identified  (Broda  ( c o l E l and K ) , w h i l e  cause a d e g r a d a t i o n (Broda  cells  they  cells  produce.  The  immunity has been  colicin  E2 and E3.  Cells  producing  these p r o t e i n s a l s o produce a small  molecular  weight  immunity  which complexes w i t h 1:1.  Dissociation  appropriate in  vitro The  and  results  existence  of the c o l i c i n  method o f d i s t i n c t i o n . simplest  method  bacteriophage, their  host  increased  takes  ( B i r g e 1981).  in induction protocols  together with  in a particular  b a c t e r i o c i n s and their  cell  titre.  p o s s i b l e co-  necessitates a  The o l d e s t  and p o s s i b l y  advantage o f the f a c t  unlike bacteriocins,  cells  from t h e  i n an i n c r e a s e i n t h e  c o n d i t i o n s f o r both  bacteriophage,  10,000 D)  i n a ratio of  protein  marked s i m i l a r i t i e s  assay  (about  each c o l i c i n  of this  colicin  activity  protein  resulting  multiply in  in localized  Thus, a s e r i e s  that  areas o f  of dilutions of  14  t h e sample u n d e r t e s t , of and  bacteria  bacteriocin.  decreasing  number  bacteriocin of  produces  The  d i f f e r e n t i a l results  Dilution  of discrete  phage p l a q u e s i n a gradual  et  al.  whereas thinning  1972).  d i s t i n c t i o n between b a c t e r i o c i n s  aeruginosa.  ina  i s more marked a t t h e h i g h e s t  (Mayr-Harting  bacteriophage  and  became p a r t i c u l a r l y d i f f i c u l t T h i s organism  produces  a  with  p y o c i n whose e s t i m a t e d m o l e c u l a r  was s e v e r a l  million.  a phage t a i l - l i k e  base p l a t e , assays  Electron  of producing  strains  diffusibility o f P.  of pyocin. aeruginosa  weight  of pyocin  p a r t i c l e complete  showed no z o n e s o f c l e a r i n g  strains  microscopy  f i b r e s , c o r e and s h e a t h .  P.  large  particulate  revealed  lawn  f o r phage  o f phage r e s u l t s  dilution results  growth, which  dilutions  s p o t t e d on an i n d i c a t o r  with  Routine  spot  on lawns o f i n d i c a t o r due t o t h e p o o r  In a d d i t i o n ,  almost a l l  c o n t a i n e d one o r more  bacteriophage  w i t h v a r i o u s degrees  of defectiveness  (Mayr-Harting  et  bacteriophage  tails  al.  1972).  had b a c t e r i o c i d a l  (Shinomiya  et  al,  1979).  were i n d u c e d b y s t a n d a r d mitomycin  C o r UV  light.  These  activity, similar Both  types  induction  to pyocin  of particles  regimes  using  15  A comparative pyocins cross  o f P.  study  o f the s o - c a l l e d  aeruginosa  showed t h a t a l l t y p e s  reacted immunologically  identical  u n d e r EM.  resulted  and a p p e a r e d  In a d d i t i o n  shown t o have a s i m i l a r  found  subunit  composition  of the t a i l  molecular  weight.  fiber  in this  responsible  f o r the s p e c i f i c  1980).  f o r a protein  which d i f f e r e d i n implied that  fiber  region are  binding of different  strains  (Ohsumi  et  al.  1980).  t o r e s i s t a n c e problems encountered i n  medicine,  the widespread  pathology  alternative,  phytopathogenic bacteriocins  antibacterial  use o f a n t i b i o t i c s i n  has n o t been a common p r a c t i c e . the prospect bacteria  f o r control of  (Vidaver  narrow spectrum  compounds w h i c h were l e s s  environmentally  As  by b a c t e r i o p h a g e and  was i n v e s t i g a t e d  B a c t e r i o c i n s were i d e a l  and  al.  to the l i p o p o l y s a c c h a r i d e receptors of  sensitive  an  tail  et  except  I t has been  differences  plant  which  o f v a r i o u s p y o c i n s was  identical  component  Due  were  i n a shut-down o f p r o t e i n and  t o be a l m o s t  pyocins  almost  a l l pyocins  mode o f a c t i o n  m a c r o m o l e c u l a r s y n t h e s i s (Ohsumi Protein  R-type  acceptable.  1976). specific persistent,  U n f o r t u n a t e l y few  16  bacteriocins  of phytopathogenic b a c t e r i a  characterized for  (Vidaver  the control  characterized  1976).  was t h e o n l y  tested  well  "bacteriocin".  However a g r o c i n - 8 4 by d e f i n i t i o n d i d n o t f a l l the  category  of a bacteriocin  proteinaceous In  (Vidaver  produced  a bacteriocin  carotovoricin. was  as i t was n o t  ( v a n H a l l ) Dye  which they  An a p p a r e n t l y  called  similar  bacteriocin  p u r i f i e d t o h o m o g e n e i t y and c h a r a c t e r i z e d i n  1978  (Itoh  et  al.  1978).  C a r o t o v o r i c i n - E R was  shown t o be a t h e r m o l a b i l e ,  particulate  sensitive  sulphate  unstable  t o sodium d o d e c y l  native  Synthesis  digestion  by v a r i o u s  (SDS),  conformation  (Itoh  of carotovoricin-ER  et  proteolytic al.  and was a c c o m p a n i e d by c e l l  after  induction.  Carotovoricin-ER  showed a s t r i k i n g 1978, Kamimiya Bacteriocin  resemblance et  al.  addition  enzymes  1978).  was i n d u c i b l e  light  al.  protein,  t o h i g h o r low pH, b u t s t a b l e t o  hydrolytic in  Erwinia  showed t h a t  carotovora  subspecies  into  1976).  1961 Hamon and P e r o n  carotovora  well  The u s e o f a g o c i n - 8 4  o f crown g a l l  and f i e l d  were  lysis  by UV  3-5 h o u r s  v i s u a l i z e d by EM  to pyocin  R (Itoh  et  1977) to sensitive c e l l s  caused  17  a rapid  and  inhibited 1980).  by  the  Further  mutant ER  extensive  lysis.  lysis  studies with  binding resulted  a phospholipase  showed t h a t  i n the  was  ( I t o h et  a d d i t i o n o f magnesium  o f Erwinia  strain  This  al.  A"  carotovoricin-  activation  o f a membrane  bound p h o s p h o l i p a s e  A l e a d i n g to c e l l  lysis.  lysis  inhibited  phospholipase  was  virtually  A mutant. still  However t h e  killed  by  cells  A activation  bactericidal  a c t i o n of t h i s  1981).  although  i s not  due  C a r o t o v o r i c i n - E R was  t o an  inactivation cytoplasmic and  activation o f the  ATP  energized  c a r o t o v o r i c i n - E R reduced  f o r c e (combination  proton  g r a d i e n t ) w h i c h d r i v e s ATP  membrane  by  the  of chemical  which to  was  an  the synthesis al.  the  1982). proton  potential  s y n t h e s i s most  i n t r o d u c t i o n of n o n - s p e c i f i c  t o use  agent,  a bacteriocin  as  et  (Itoh  channels.  In o r d e r control  level  ( I t o h et  that  shown t o  f o r ATP  motive  probably  later  s t a t e of  membrane n e c e s s a r y  were  primary  o f ATPase but  t r a n s p o r t o f amino a c i d s  Essentially  the  carotovoricin  cause a r e d u c t i o n i n i n t e r n a l not  intact,  carotovoricin-ER, suggesting  phospholipase  al.  i n the  This  a possible  a thorough understanding  of  the  and  18  activity  s p e c t r u m , mode o f a c t i o n and g e n e t i c and  physical  basis  activity  spectrum o f c a r o t o v o r i c i n - 3 7 9  Erwinia been  f o rproduction  carotovora  subsp.  i s n e c e s s a r y . The  carotovora  investigated previously  (Jais  p r o d u c e d by  strain 1982).  379 h a s In  r  addition, had been  t h e mode o f a c t i o n o f c a r o t o v o r i c i n - E R extensively  investigated  1980a, 1980b, 1980c,  1981, 1982).  (Itoh  et  al.  The a i m o f t h i s  t h e s i s was t o s t u d y t h e p r o d u c t i o n o f carotovoricin-379.  Specific  1)  objectives  were:  To i n v e s t i g a t e t h e s t r u c t u r a l b a s i s o f carotovoricin-379  production  using  electron  microscopy. 2)  To d e t e r m i n e i f c a r o t o v o r i c i n - 3 7 9 chromosomally  3)  and/or  plasmid  production i s  coded.  To d e t e r m i n e t h e s e r o l o g i c a l r e l a t i o n s h i p s o f the  various  components o f c a r o t o v o r i c i n - 3 7 9 .  19  CHAPTER 1  ULTRASTRUCTURAL EVIDENCE FOR ERWINIA  BY  BACTERIOCIN SECRETION  CAROTOVORA  INTRODUCTION  Bacteriocins  are  proteinaceous  antimicrobial  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 of  which are  against  (Nomura  directed  1967).  involves  The  protein  bacteriophage are  mitomycin  s u c h as  structures  tails.  i n d u c e d by C or  killing  In  by  a  which  or  and  permeability  bacteriocin  bacteriocins  irradiation. may  m o l e c u l a r weight of  the  These o b s e r v a t i o n s  production  be  as  Like  released  q u a s i - l y s i s mechanism  the  1981).  highly  resemble  f a c t , most  ultraviolet  d e p e n d i n g on  (Birge  have  prophage-inducing agents such  lysis,  inherent  to  related strains.  pyocins,  lambda p r o p h a g e , b a c t e r i o c i n s either  strains  bacteriocins  a s e l e c t i v e advantage  p r o d u c i n g s t r a i n s , by  complex  closely related  b i o l o g i c a l r o l e of  conferring  Some b a c t e r i o c i n s ,  bacteria  may  of  the  protein  producing imply  strain  that  have e v o l u t i o n a r y  links  20  to d e f e c t i v e  phage components.  Bacteriocin  p r o d u c t i o n by Erwinia  which causes potato b l a c k l e g variety crops,  of economically was f i r s t  and s o f t  shown by Hamon and P e r o n  they coined  bacteriocins  p r o d u c e d by Erwinia  research  two t y p e s One  rot of a  important a g r i c u l t u r a l  Subsequently  Further  carotovora,  t h e term c a r o t o v o r i c i n f o r carotovora  of bacteriocin  activity  by l a r g e  d i f f u s e zones o f  and t h e o t h e r by s m a l l  inhibition  ( C r o w l e y and DeBoer 1980, J a i s  characterization  preparations having thermolabile,  proteins  were  tail-like core,  base-plate  electron  release  Erwinia  (EM) ( I t o h  work w i t h  induction.  trypsin-resistant al.  1978).  et  al.  carotovoricins  However,  produce s u b s t a n t i a l  carotovoricin  established  Phage  a c o n t r a c t i l e sheath,  o f these b a c t e r i o c i n s  following  1982).  and f i b r e s were o b s e r v e d by  microscopy  Initial  et  (Itoh  zones o f  carotovoricin  activity  particulate,  p a r t i c l e s with  clear  of p u r i f i e d  the l a t t e r  involved  of  i n some s t r a i n s .  inhibition  that  (Ecc).  has d e m o n s t r a t e d t h e e x i s t e n c e  i s characterized  Physical  i n 1961.  1978). implied  i s due t o c e l l  that lysis  some s t r a i n s o f  amounts o f  c o n s t i t u t i v e l y w i t h no s i g n i f i c a n t  21  reduction  in turbidity  Moreover,  lysis  by  mitomycin  following et  al.  also  an i n c r e a s e  In t h i s  (Ecc  after  induction  i r r a d i a t i o n and  in carotovoricin titers  These o b s e r v a t i o n s  suggested  than  cell  (Itoh  that  l y s i s was  i n carotovoricin liberation.  study,  carotovora  carotovoricin-379 subspecies  production i n  carotovora  s t r a i n 379  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 various  of bacteriocin-producing  times a f t e r  made s u p p o r t component  c e l l s at  The o b s e r v a t i o n s  that  of carotovoricin-379  o f which s w e l l  heads.  induction.  the hypothesis  membrane e n c l o s e d , tips  viability.  only  some mechanism o t h e r  involved  Erwinia  was d e t e c t e d  C or u l t r a v i o l e t  1978).  possibly  or c e l l  the p a r t i c u l a t e  i s s e c r e t e d on  fimbrae-like p r o j e c t i o n s , the t o form  detachable  vesicular  22  MATERIALS AND METHODS  Culture of  Erwinia  of Bacteriocin Erwinia  carotovora  subsp.  as a b a c t e r i o c i n  constitutively  produces  T h r e e s t r a i n s o f E.  carotovora  carotovora  were u s e d as i n d i c a t o r s were grown  titres.  i n Luria  carotovora  subsp.  (Table Broth  (Maniatis  1984) pH 7.4 o r m i n i m a l M9 medium pH 7.4  (Maniatis (100  strain  subspecies  carotovora  al.  This  i t a t much h i g h e r  and one s t r a i n o f E.  et  s t r a i n 379  producer.  atroseptica  Bacteria  Induction  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 i n d u c e d t o p r o d u c e  1).  and  Production:  carotovora  was c h o s e n  Strains  et  rpm).  al.  1984) a t 20 C on a r o t a r y  Enhanced b a c t e r i o c i n  i n d u c e d by t h e a d d i t i o n 2 hours  (h) a f t e r  Bacteriocin Plate Cultures seconds  p r o d u c t i o n was  o f mitomycin  C a t 0.2 ug/ml  inoculation.  Assays:  were v o r t e x e d a t h i g h s p e e d  ( s ) a t 8, 12 and 24 h a f t e r  below.  f o r 30  i n d u c t i o n , and  s u p e r n a t a n t s were v i e w e d b y e l e c t r o n (EM) as d e s c r i b e d  shaker  Bacteriocin  microscopy a c t i v i t y was  Table  1:  strains  Carotovoricin  o f Erwinia  producing  and  sensitive  carotovora  Producer:  Erwinia  carotovora s u b s p .  serogroup Sensitive  Erwinia  carotovora s u b s p .  strain  SR8  atroseptica  strain  496  atroseptica  strain  530  XX  carotovora s u b s p .  serogroup  atroseptica  XXII  carotovora s u b s p .  serogroup  Erwinia  379  I  carotovora s u b s p .  serogroup  Erwinia  strain  strains:  serogroup  Erwinia  carotovora  XI  XVIII  carotovora  strain  504  24  a s s a y e d by p l a c i n g of  indicator  (0.85% NaCl,  5 u l of test  strains  seeded  samples  on a lawn  i n peptone s o f t  1% B a c t o - P e p t o n e ,  agar  0.45% B a c t o - A g a r pH  7.4) .  C o n c e n t r a t i o n of Aliquots  Carotovoricin-379:  of induced  10,500 x g f o r 20 min. were b r i e f l y  vortexed  filter-sterilized Sterilized  cultures  were p e l l e t e d a t  The s u p e r n a t a n t (15 s a t h i g h  using  a 0.22 um  glycol  i n the s t e r i l e  to f a c i l i t a t e  precipitates  to stand  precipitation.  were c o l l e c t e d  The  were  pH 7.4.  Insoluble  was removed by low s p e e d c e n t r i f u g a t i o n 10 min a t 4 C.  The r e s u l t i n g  p r e p a r a t i o n s were a n a l y s e d or  at  i n 1/100 t h e o r i g i n a l volume o f 5OmM  sodium phosphate b u f f e r  for  M.W.  on i c e  by c e n t r i f u g a t i o n  13,500 x g f o r 25 min and t h e p e l l e t s dissolved  (PEG)  supernatants at  22 C and t h e m i x t u r e s were a l l o w e d overnight  s p e e d ) and  filter.  18% (w/v) p o l y e t h y l e n e  8000 was d i s s o l v e d  fractions  electron  microscopy.  material  a t 8000 x g  carotovoricin-379  by column  chromatography  25  Column Chromatography: Concentrated applied packed  to the with  c a r o t o v o r i c i n - 3 7 9 (0.5  t o p o f a 35  S e p a c r y l S-300.  5OmM sodium p h o s p h a t e pH maintained monitored  a t about a t 280  bioassayed  cm  0.5  nm,  7.4  x 1.8  was  column  The  running b u f f e r  and  the flow  ml/min.  and  cm  ml)  Eluate  rate  was was  was  peaks were c o l l e c t e d  and  as d e s c r i b e d above.  E l e c t r o n Microscopy: Concentrated loaded  onto  carbon  and  min. of  c a r o t o v o r i c i n - 3 7 9 (10 u l )  copper  grids  2% p h o s p h o t u n g s t i c and  viewed  microscope  (Hill  stained acid  order  negatively protocols first  with  (PTA)  in a P h i l l i p s  10  pH  EM-300  t o 12  7.0,  drops  allowed  to  electron  Whole C e l l s :  to i n c r e a s e the r e s o l u t i o n  stained for cell  procedure,  f o r 3 to 5  1984).  M o d i f i e d Negative S t a i n i n g of In  collodion-  i n c u b a t e d a t room t e m p e r a t u r e  G r i d s were t h e n  dry,  coated with  was  whole c e l l  preparations,  pretreatment  cultures  osmium t e t r a o x i d e ( f i n a l  of  were u s e d .  were p r e f i x e d  in  c o n c e n t r a t i o n added  two In 0.2%  the  26  directly thrice pH  t o g r o w t h m e d i a ) f o r 1 h a t 20 C, washed  with  sterile  broth  and s t a i n e d w i t h  7.0. In t h e s e c o n d p r e t r e a t m e n t  grade t o l u e n e of  2% PTA  bacterial  gently  cell  before  added a t 1:2  cells.  ( v / v ) t o an a l i q u o t  allowing  complete  o f t h e two p h a s e s a f t e r  suspension  with  reagent  The t u b e s were c a p p e d and  i n v e r t e d 10 t i m e s  separation The  was  procedure,  was  then  each i n v e r s i o n .  removed and s t a i n e d as  PTA.  F i x a t i o n and Embedding: Cells  were f i x e d  Ryter-Kellenberger tetroxide with to  procedure  i n veronal  calcium  i n agar a c c o r d i n g  embedded  (1958) u s i n g  acetate buffer  \% osmium  supplemented  chloride (Kellenberger buffer),  16 h a t room t e m p e r a t u r e .  subjected  to the  to a s e r i a l i n EPON.  Blocks  of c e l l s  alcohol dehydration  Thin  f o r 14 were  and  s e c t i o n s were s t a i n e d f o r  15 t o 20 min i n 5* u r a n y l  acetate.  27  RESULTS  Mitomycin C Induced  Cultures:  Ultrastructural of  E.  carotovora  examination  o f induced  carotovora  subsp.  a f t e r 48 h a t 20  C showed b a c t e r i a l c e l l s  with  fimbrae-like  of various  1).  projections  i n close  bacterial  cells  induced  cells  structure Ic).  a proliferationof  Low l e v e l s o f p a r t i c u l a t e  were s e e n  stages  present  projections  vesicular  i n addition  supernatants  shearing retention  a  subunit  projections  to fimbral  (Fig. la-b).  sterilization  bacteriocin  with  of elongation ( F i g .  Examination o f the Supernatant Filter  intact  sections of  projections  In some p r e p a r a t i o n s ,  were a l s o  with  ( F i g . I d ) . Thin  at various  lengths ( F i g .  carotovoricin-379  association  revealed  cultures  from  (0.22 um f i l t e r ) o f  induced  activity.  non-filter-sterilized  cultures  However,  by v o r t e x b e f o r e of activity.  Fractions:  markedly  reduced  a b r i e f (15 s )  f i l t r a t i o n resulted  Microscopic preparations  i na  examination o f showed  that  28  F i g u r e 1. E l e c t r o n m i c r o g r a p h s o f 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 o f 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 a t 20 C (Bars=100nm). a-b. N e g a t i v e s t a i n e d c e l l s showing b o t h 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 o f a p r o d u c i n g c e l l showing subunit s t r u c t u r e o f fimbrae-like p r o j e c t ions at various stages o f elongation. Insert: area c o n t a i n i n g e a r l y stages o f projection; d. N e g a t i v e 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 o f 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 intact cell.  29  30  carotovoricin  particles  were a g g r e g a t e d  physically  a t t a c h e d by a c e n t r a l  (Fig.  This observation  2a)  and  core  to v e s i c l e s  explained  the loss o f  activity  upon s t e r i l i z a t i o n  attached  c a r o t o v o r i c i n - 3 7 9 w o u l d be r e t a i n e d on t h e  filter.  The b r i e f  these like  particles.  s h e a r i n g presumably  detached  V e s i c l e s o f t e n had l o n g  projections attached  exactly  as v e s i c l e s and  t h e same d i a m e t e r  thread-  t o them w h i c h were o f as c a r o t o v o r i c i n  cores  (Fig. 2b).  S e r i a l Subunit S t r u c t u r e o f  Particulate  Carotovoricin-379: Carotovoricin arranged through the head  around  particles  a central  the sheath  uncommon,  core which  ( F i g . 2c-g).  c o r e was a t t a c h e d ( F i g . 2g).  association  Although  preparations with  extended  In r a r e  instances  t e r m i n a l l y to a v e s i c u l a r attached  numerous d e t a c h e d  concentrated  consisted of subunits  the core  heads were  heads were f o u n d i n  suggesting ( F i g . 2g).  a r a t h e r weak  31  F i g u r e 2. Negative s t a i n e d preparations o f 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 t h e supernatant f r a c t i o n o f mitomycin C induced Erwinia carotovora s u b s p . 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 a t t a c h m e n t t o 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 o f c a r o t o v o r i c i n - 3 7 9 showing c o r e s u r r o u n d e d by s h e a t h . Note d i a g o n a l l y displaced, disrupted particle i l l u s t r a t i n g a c e n t r a l c o r e w i t h a modular arrangement o f 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 t h e o v a l upper terminus o f t h e 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 w i t h an i n t a c t s h e a t h s u r r o u n d i n g t h e l o w e r 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 block-like structure; f. Two s e p a r a t e d s h e a t h components e a c h 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 o f c a r o t o v o r i c i n - 3 7 9 showing a t t a c h e d head. A t t a c h m e n t i s f a c i l i t a t e d by s t e 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.  32  33  Carotovoricin-379  Developmental Stages of  P r o d u c t i o n by Mitomycin C Induced C e l l s : Particles supernatants immature and  carotovoricin  t o be  on t h e b a s i s o f a p p e a r a n c e  on known i n d i c a t o r s  strains  (Fig.  these  immature  encapsidated  while  o t h e r s had a d e f i n i t e  swelling  ( F i g . 3 b ) . Other  ends s u g g e s t i n g  bacteriocin particles a  i n d u c t i o n appeared  In some c a s e s  fully  both  i n the concentrated  8 h after  activity  f).  found  were  ( F i g . 3c and d ) .  observed  low f r e q u e n c y ,  particles  to aggregate  suggesting  were  open a t  intermediate positions  particle  were  particles  3a-  on a  These  end t o end a t  a serial  association  (Fig. 3d). Particles  found  i n 12 h p r e p a r a t i o n s  mature c a r o t o v o r i c i n - 3 7 9 .  They were  diameter  than  and more e l o n g a t e  a definite stage,  also  i n the process  observed  with  4c).  preparations  released particles,  with  subunit  e n c l o s e t h e numerous  (Fig.  8 h particles  with  o f t h i c k e n i n g were  ( F i g . 4 b ) . Whole c e l l  formed p a r t i c l e s and  larger in  b e a d e d s t r u c t u r e ( F i g . 4 a ) . At t h i s  cores  observed  resembled  addition  projecting  F u l l y mature p a r t i c l e s  were  or p a r t i a l l y to surround  core f i l a m e n t s were r e l e a s e d  34  F i g u r e 3. N e g a t i v e 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 p r o d u c i n g c e l l s o f Erwinia carotovora s u b s p . carotovora s t r a i n 379 8 h a f t e r m i t o m y c i n C i n d u c t i o n and t h e p l a q u e t y p e s 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 s h o w i n g 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 p l a q u e s p r o d u c e d by l a r g e m o l e c u l a r weight c a r o t o v o r i c i n - 3 7 9 (Sepha c r y l S-300 peak 1 ) ; f. T y p i c a l d i f f u s e p l a q u e s p r o d u c e d by low m o l e c u l a r w e i g h t immature c a r o t o v o r i c i n - 3 7 9 ( S e p h a c r y l S-300 peaks 2 t o 4 ) .  36  F i g u r e 4. Negative stained carotovoricin-379 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 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 c u l t u r e s o f Erwinia carotovora s u b s p . 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 w i t h 2% p h o s p h o t u n g s t i c a c i d pH 7.0 ( a t o d ) , o r p r e t r e a t e d w i t h 0.2* osmium t e t r o x i d e ( e ) o r an e q u a l volume o f t o l u e n e (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. Carotovoricin-379 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 w h i c h a p p e a r e l o n g a t e d and t h i c k e n e d due t o t h e a d d i t i o n o f m a t e r i a l t o the d e v e l o p i n g 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 o f thickening; c. Carotovoricin-379 p a r t i c l e s either released ( l e f t ) or i n the process o f thickening ( r i g h t ) i n a s s o c i a t i o n w i t h i n t a c t c e l l s 12 h after induction; 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 w i t h 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 particle w i t h a t t a c h e d head p r o t r u d i n g from a producing c e l l ; f. Core-like projection with terminal 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 induction; 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 p r o d u c i n g 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 carotovoricin-379 attached to and r e l e a s e d from an i n t a c t p r o d u c i n g c e l l 16 h a f t e r i n d u c t i o n ; i. Protruding p a r t i c u l a t e carotovoricin-379 w i t h a t t a c h e d h e a d s u r r o u n d e d by i n t a c t o u t e r membrane 24 h a f t e r i n d u c t i o n .  37  38  i n t o the supernatant and were r e a d i l y d e t e c t e d i n c o n t r a c t e d or extended (Fig.  forms 24 h a f t e r  induction  4d).  E x t r u s i o n o f C a r o t o v o r i c i n through the Membrane: The m o d i f i e d s t a i n i n g procedure u s i n g osmium t e t r o x i d e p r o v i d e d more d e t a i l and c l e a r l y showed 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 with attached heads extruded through the membrane ( F i g . 4e). disadvantage  of the procedure was  The  that the washings  r e s u l t e d i n a l o s s of b a c t e r i a l s u r f a c e appendages ( F i g . 4e) and p a r t i a l d i s r u p t i o n of b a c t e r i o c i n particles. 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 s at 8, 12, 24 h r e s p e c t i v e l y , a f t e r i n d u c t i o n  and  ( F i g . 4 f - i ) were  v i s u a l i z e d with the m o d i f i e d s t a i n i n g u s i n g a toluene pretreatment.  16  procedure  These p a r t i c l e s  corresponded w e l l i n appearance and dimensions the immature p a r t i c l e s i s o l a t e d  with  i n the sheared  supernatant p r e p a r a t i o n s d e s c r i b e d p r e v i o u s l y . However, these p r e p a r a t i o n s showed p h y s i c a l a s s o c i a t i o n of b a c t e r i o c i n intact c e l l s .  Pretreatment  i n t e r m e d i a t e s with with toluene presumably  p a r t i a l l y d i s r u p t e d the outer membrane a l l o w i n g  39  penetration necessary, remained  379  As  bacterial  no  washing steps  surface  S-300 Column  filtration  harvested  of concentrated  a t 44,  52,  58  showed a d e f i n i t e  molecular  weight b a c t e r i o c i n  and  weights  20,000 d a l t o n s bacteriocin equivalent had  (about  At  (peak  (peak 4)  a t 52  o f peak 3 and  peak  1 had  h  t r e n d was  another  bacteriocin  fractions  detected  at  seen  appearance of s t i l l  a t 62  peak  the  in  induction with  of these  peak  after  h after  Bioassays  peak  almost  doubled  180,000 d a l t o n s  and  weight  t o 62  to  1)  present  T h i s minor  even s m a l l e r m o l e c u l a r  This general  h,  had  (peak 3) was  peak 2 was  corresponding  44  In a d d i t i o n a m i n o r  time  f r o m 58  higher  respectively  this  concentration. shift  to  to g l o b u l a r p r o t e i n  activity.  10,000 d a l t o n s ) At  after  ( F i g . 5).  t o 15,000 d a l t o n s  bacteriocin  induction. level  (peak 2)  an  h  o f 300,000 d a l t o n s  activity.  appeared with  carotovoricin-  62  shift  major peaks c o r r e s p o n d i n g  molecular  and  ultrastructure  Chromatography:  induction  two  were  intact.  Sephacryl Gel  o f PTA.  in  the  the  peak  (lb)  h. bacteriocin  40  0.06  F i g u r e 5. S e p h a c r y l S-300 column chromatograms c a r o t o v o r i c i n - 3 7 9 a t ( a ) 44, (b) 52, ( c ) 58 and (d) 62 h a f t e r m i t o m y c i n C i n d u c t i o n i n c o n c e n t r a t e d s u p e r n a t a n t s o f Erwinia carotovora s u b s p . carotovora s t r a i n 379.  41  activity  with d i f f e r e n t  depending  on t h e i n d i c a t o r  consistently peaks.  t o peaks  peaks  strain  l a and  gave  varying  used.  Strain  produced The  degrees  low m o l e c u l a r  of  diffuse  zones  ( F i g . 3 f ) on a l l i n d i c a t o r s  strain  530  which  was  weight  carotovoricin-379.  Electron bacteriocin were added t h e r e was However, weight  micrographs components  increase  component was  showed d i f f u s e - t y p e particulate  indicated  that  particles  of a soluble  still  possible  activity  structure  t o low m o l e c u l a r  as s u b u n i t s  over time,  i n m o l e c u l a r weight  the e x i s t e n c e  except  o f the s e p a r a t e d  to b a c t e r i o c i n an  to a l l  clear  inhibitory  resistant  SR8  were most  l b and  ( F i g . 3e).  clearing  sensitivity  of i n d i c a t o r  of i n h i b i t i o n  weight  of  showed t h e g r e a t e s t  A l l strains  sensitive zones  degrees  low m o l e c u l a r as peak 4  on s t r a i n  c o u l d be  ( F i g . 6).  resolved  SR8, by  but  no  electron  microscopy. Induced diluted  C concentraion  a l l peaks  single  which  were r e - i n d u c e d and  w i t h an e q u a l volume o f medium  a mitomycin that  cultures  in Figures  scan at 8 h a f t e r  o f 0.2 5a-d  (to maintain  ug/ml) showed  were p r e s e n t i n a  the second  induction (Fig.  42  F i g u r e 6 . S e p h a c r y l S-300 column 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 48 h a f t e r m i t o m y c i n C i n d u c t i o n and e l e c t r o n m i c r o g r a p h s o f t h e c o r r e s p o n d i n g n e g a t i v e s t a i n e d peak c o n t e n t s . Bar=100nm.  44  7a).  This  initiated  suggested  another round  production  All  sulfate  the second  as s a t e l l i t e  (Fig.  supernatant  induction  of carotovoricin-379  which appeared  S-300 chromatogram  detected  that  peaks on t h e  7a).  b a c t e r i o c i n a c t i v i t y was  i n one m a j o r peak when 20mM magnesium was added t o t h e medium f o l l o w e d by  induction  ( F i g . 7b).  identical  to the p r o f i l e  concentrated  from  T h i s peak p r o f i l e was of carotovoricin-379  induced  media which a l s o c o n t a i n e d  cells  grown i n m i n i m a l M9  magnesium  (Fig.  7c).  DISCUSSION  Cell  lysis,  as s e e n by a r e d u c t i o n i n  turbidity,  occurred  carotovora  subsp.  36  induction.  h after  particles  i n induced  carotovora  were s e e n  strain  i n c u l t u r e supernatants  at 8 h after  induction.  cell  or reduction  in cell  detected  i n non-induced  production  379 (Ecc 379)  However c a r o t o v o r i c i n  bioassayed lysis  was e a s i l y  Erwinia  cultures of  and  A l t h o u g h no  v i a b i l i t y was  cultures, bacteriocin  detected  by b i o a s s a y s and  45  F i g u r e 7. S e p h a c r y l S - 3 0 0 c h r o m a t o g r a m s 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 b y 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 conditions. a. E f f e c t o f a second i n d u c t i o n a f t e r 8 h with mitomycin 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 m a g n e s i u m sulphate to Luria broth prior to induction; 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 m e d i a .  46  electron  microscopy.  Cell feature  lysis  following  induction  i n the production  m o l e c u l a r weight  of large  bacteriocins  t e m p e r a t e phage i n d u c t i o n . colicins  h a s shown t h a t  expression  of a lysis  phospholipase This  integrity  (Pugsley In to  lysis  filtration  weight,  addition  Under t h e s e  fraction. outer  was d e t e c t e d  o f 20mM magnesium  by i n d u c t i o n  conditions, gel containing molecular  A l l carotovoricin  ions  probably  membrane s u f f i c i e n t l y active bacteriocin  These o b s e r v a t i o n s  delayed  i n the large molecular  The magnesium  of p a r t i a l l y  i n t h e medium  showed a l o s s o f s m a l l e r  l e s s a c t i v e peaks.  activity  on c e l l  1984).  of carotovoricin-379  supernatants  involved i n  gene c a n be i n a c t i v a t e d by  our experiments,  by 6-8 h.  permeability.  The e f f e c t  o f 20mM magnesium  and S c h w a r t z  with  i s d e p e n d e n t on  of c o l i c i n .  t h e c u l t u r e media f o l l o w e d  lysis  the  R e c e n t work  t h e membrane  by t h e l y s i s  incorporation  and i s s i m i l a r t o  may be d i r e c t l y  membrane t r a n s p o r t  and s m a l l  gene w h i c h a c t i v a t e s a  altering  gene p r o d u c t  i s a common  suggest  that  weight  stabilized  to prevent  release  intermediates. i n addition  to c e l l  47  lysis  a n o t h e r mechanism may be i n v o l v e d i n  bacteriocin  release.  Erwinia  carotovora  subsp.  carotovora  produces a  w i d e v a r i e t y o f p e c t o l y t i c and c e l l u l o l y t i c enzymes.  T h e r e have b e e n s e v e r a l  simultaneous particulate  induction  et  Itoh  of a  l y a s e and  c a r o t o v o r i c i n or temperate  bacteriophage 1984,  of pectin  reports  i n Erwinia al.  carotovora  1980 and Kamimiya  (Chatterjee et  al.  1977).  T h e s e enzymes and c a r o t o v o r i c i n s a p p e a r e x t e r n a l l y in  the supernatant  co-induction o f a common  could  fraction  o f c u l t u r e d c e l l s and  be e x p l a i n e d  repressor  by an i n a c t i v a t i o n  o f an e x i s t i n g s e c r e t o r y  system. The provides  outer  an e f f e c t i v e  substances 1985).  membrane o f t h e E n t e r o b a c t e r i a c e a e b a r r i e r to the release o f  > 700-1000 d a l t o n s  Thus,  there  Pseudomonas  Neisseria subsequent  The s e c r e t i o n o f enzymes  and e n d o t o x i n p r o d u c t i o n  i s thought release  t o occur  of c e l l  membrane v e s i c l e s (Devoe are  and V a a r a  i s a need f o r a s e c r e t o r y  s y s t e m f o r exo-enzymes. from  (Nikaido  et  v i a t h e f o r m a t i o n and  wall al.  s i m i l a r t o those observed  by  blebs 1973).  i n this  or outer These study.  blebs In  48  conjunction  with  projections, mechanism  t h e numerous  these blebs  fimbrae-like  may f o r m  a  secretory  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 and  the basis  experimental  hypothetical and  of ultrastructural d a t a we p r o p o s e  (Fig. 8).  occurs within  or  This  produced across  an o u t e r  v e s i c l e provides  mechanism f o r s e c r e t i o n  assembly  The c o n s t r u c t i o n o f  bacteriocin  Macerating  the f o l l o w i n g  model f o r c a r o t o v o r i c i n - 3 7 9  secretion  "bleb".  observations  membrane v e s i c l e a  general  o f a l l Erwinia  exoproteins.  enzymes and c a r o t o v o r i c i n components a r e  i n the cytoplasm  and a r e t r a n s p o r t e d  t h e plasmalemma p r o b a b l y by t h e model o f  Randall  and Hardy  (1984).  proteins  is facilitated  an  membrane v e s i c l e .  outer  construction contains  The s e c r e t i o n  by t h e i r  occurs within  exocytosis  within  Carotovoricin-379 a v e s i c l e which  a fimbrial projection.  projection  of these  This  also  fimbrial  u l t i m a t e l y becomes t h e c o r e o f  carotovoricin-379.  Some b a c t e r i o c i n p r o t e i n s a r e  added e x t e r n a l l y t o t h e f i m b r i a l p r o j e c t i o n resulting  i n the formation  Other b a c t e r i o c i n p r o t e i n s  of a c o n t r a c t i l e sheath. are transported  into the  49  F i g u r e 8. Hypothetical 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 b y Erwinia carotovora subsp. carotovora 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 carotovoricin) are produced i n the cytoplasm 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 space (ps). These p r o t e i n s are subsequently secreted 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 construction occurs inducibly 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 contains a fimbrae-like projection (a). Under non-induced c o n d i t i o n s these projections e x t e n d outwards and form t h e n u m e r o u s 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 producing strains. c. U n d e r i n d u c t i o n , carotovoricin components translocated into the periplasmic space are added t o the f i m b r a e - l i k e projection. These added components e v e n t u a l l y form t h e sheath(s) of the p a r t i c u l a t e carotovoricin. During the f i n a l stages of construction, 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 the 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 at the base o f the c a r o t o v o r i c i n particle. d. Some c a r o t o v o r i c i n c o m p o n e n t s may b e translocated t h r o u g h t h e c e n t r a l c o r e t o an expanding terminal v e s i c l e o r h e a d (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 releasing intact carotovoricin with tail f i b r e s ( t f ) and base 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 from the r e s t o f the p a r t i c l e i n the 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 t w o forms e i t h e r extended (F) or c o n t r a c t e d ( G ) .  50  51  hollow  core  terminal  and s u b s e q u e n t l y  vesicle.  carotovoricin vesicle.  phospholipase bacteriocin  may  be  be f o u n d  o r may  b y some  be  induced  the  production  protein  lytic  (Glass  components.  bacteriocin with  1985) o f b o t h This  construction  the a c t i v a t i o n  phospholipase, producing In  results  components  i n an  state  of the b a c t e r i o c i n  which,  in along  of a of the  are repressed;  however,  proteins  in  a secretion  of partially  as w e l l  normally  the construction a n d some  is still  the  active  active  as t h e s e c r e t i o n  found  of  and  i t s  exocytosis resulting  bacteriocin of macerating  i n the periplasm.  a  and  increase  i n the disruption  periplasmic  enzymes  deactivates  and s e c r e t i o n  of  components  activates  cell.  the non-induced  secretion  component.  bacteriocin  or production  with  the  agent  which  results  by  with the  bacteriocin  or physical  o f recA  followed  within  damage b y c h e m i c a l  a  This  associated  activated  intact  associated  of a phospholipase. may  to a  within  of carotovoricin  DNA  repressor  o f an  i t i s released  of the v e s i c l e  activation  periplasm  completion  particle,  The r e l e a s e  disruption the  Upon  translocated  The  52  repression  however  differential bacteriocin  low  i s not  level  production.  of  complete  resulting  non-induced  in  a  particulate  53  CHAPTER  GENETIC  DETERMINANTS  IN  OF  ERWINIA  2  CAROTOVORICIN  PRODUCTION  CAROTOVORA  INTRODUCTION  Bacteriocins agents which  produced  by c e r t a i n  are active  strains common  (Nomura  only  producing  strains  production  of vegetables  described research first  b y Hamon resolved  zone.  (Birge  two  A thermolabile,  tail-like  particulate  carotovoricin-ER,  was  which  to  causes  of potato (1961).  was  clear  by a l a r g e  identified  first  The  zone o f  and  trypsin-resistant, bacteriocin,  soft  Subsequent  of bacteriocin.  by a small  and t h e s e c o n d  generally  Bacteriocin  carotovora,  types  related  advantage  1981).  and Peron  bacteria  production i s  and i s  and b l a c k l e g  i s characterized  inhibition  closely  a selective  i n Erwinia  of  Bacteriocin  of genera  to confer  antimicrobial  strains  against  1967).  i n a number  considered  rot  are proteinaceous  diffuse phage  called  as c a u s i n g  the  54  small  clear  zones  bacteriocin certain was C  resembled  Pseudomonas  inducible or other  Recent lyase  and  the pyocins  (Birge  and b a c t e r i o c i n  i n E.  carotovora  chrysanthemi  both  activity  et  of bacteriocin  pectin  are co-  e r al.  (Kamimiya  (Chatterjee  analysis  1981).  have shown t h a t  g e n e r a has shown t h a t  plasmid  This  t h e p y o c i n s p r o d u c e d by  DNA damaging a g e n t s  Genetic other  1978).  by u l t r a v i o l e t i r r a d i a t i o n , m i t o m y c i n  activity  i n E.  al.  s t r a i n s and l i k e  investigations  inducible  et  (Itoh  al.  1977) 1984).  production i n  the determinants  o r chromosomally encoded  m o l e c u l a r weight b a c t e r i o c i n s  (Birge  causing  c a n be  1981).  large  Low  diffuse  z o n e s o f i n h i b i t i o n , s u c h as t h e c o l i c i n s were found  t o be p l a s m i d  bacteriocins inhibition bacterial large  al.  causing  chromosome.  concluded  clear coded  zones o f f o r by t h e  i s o l a t e d from  s t r a i n s o f Erwinia  could  Particulate  A l t h o u g h b o t h s m a l l and  have been  1980, F o r b e s  phenotypes  small  were p r i m a r i l y  plasmids  different  encoded.  1981, Z i n k be a s s i g n e d  carotovora et  al.  several (Coplin  et  1984), no  and t h e y  were  t o be c r y p t i c .  P r e v i o u s work i n t h i s l a b o r a t o r y  has shown  that  55  both types with  of bacteriocin  Ecc s t r a i n  bacteriocin  activity  are associated  379 ( J a i s 1982) and t h a t t h e  i s produced  i n intimate  with  intact cells  1).  The p u r p o s e o f t h i s s t u d y was t o d e t e r m i n e  whether in  possibly  association  the determinants  Ecc s t r a i n  v i a extrusion  (Chapter  forbacteriocin  production  379 were c h r o m o s o m a l l y a n d / o r  plasmid  encoded.  MATERIALS AND METHODS  Media and Growth C o n d i t i o n s : All (LB)  strains  pH 7.4.  (Table  1) were grown i n L u r i a  Erwinia  carotovora  Escherichia  20 C w h i l e  Antibiotics  coli  obtained  Company were d i s s o l v e d in  plates  or l i q u i d  (Maniatis  was  i n alcohol  a final  concentration  Crystal  violet  t h e Sigma C h e m i c a l  i n water or a l c o h o l  concentration dissolved  was i n c u b a t e d a t  was grown a t 37 C.  from  culture et  Broth  and u s e d  a t t h e recommended al.  1982).  and added  Erythromycin  t o LB pH 8.0 a t  o f 30 ug/ml.  pectate  (CVP)  media was made  Table  1.  Bacterial strains  Carotovoricin  Producer:  Erwinia carotovora s e r o g r o u p XI Carotovoricin  subsp.  carotovora  strain  379  Indicators:  Erwinia carotovora serogroup I Erwinia carotovora s e r o g r o u p XX Erwinia carotovora s e r o g r o u p XXII Erwinia carotovora serogroup XVIII Other  used  s u b s p . atroseptica  strain  SR8  s u b s p . atroseptica  strain  530  s u b s p . atroseptica  strain  496  subsp.  carotovora  st r a i n  504  Strains:  Escherichia Escherichia Escherichia  coli coli coli  strain strain strain  HB101 ( v i r g i n ) HB101 + p l a s m i d HB101 + p l a s m i d  pBR322 R68.45  57  according  t o Cuppels  and Kelman  ml) p l a t e s were s p r e a d antibiotic  stock  with  solution  t o g i v e t h e recommended  concentration.  hours  (h) a t room t e m p e r a t u r e  use.  Using  prepared  CVP (20  a p p r o p r i a t e volumes o f  final  this  (1974).  Passive diffusion  f o r 24  was a l l o w e d  before  CVP + 1% LB was  same t e c h n i q u e ,  t o a c c e l e r a t e g r o w t h o f E.  coli.  Mating P r o t o c o l s : All  m a t i n g s were p e r f o r m e d  R68.45 was t r a n s f e r r e d t o Erwinia  strain  379 u s i n g a s t a n d a r d  (Puhler  LB  harbouring carotovora  p l a t e mating  technique  1984) and t r a n s c o n j u g a n t s (50 u g / m l ) .  were mated back  HB101 and t r a n s c o n j u g a n t s  + kanamycin  temperature.  coli  subsp.  on CVP + k a n a m y c i n  transconjugants strain  carotovora  and R i e s s  selected  an E.  from  strain  a t room  to virgin  were  These E.  coli  were s e l e c t e d on  (50 ug/ml) + s t r e p t o m y c i n (25  ug/ml). For w i l d - t y p e matings, of  a mid-log  carotovora  phase c u l t u r e  strain  ( w i t h and w i t h o u t sterile  LB.  (without o f E.  379 and E.  coli  R68.45), 1 ml  carotovora  subsp.  s t r a i n HB101  PBR322) were added t o 20 ml o f  These m i x t u r e s  were a l l o w e d  t o stand  58  at  room t e m p e r a t u r e  were s e l e c t e d streptomycin tested  f o r 4 h.  of c e l l s  on LB + e r y t h r o m y c i n (30 ug/ml) + (25 u g / m l ) .  for: ability  ability  Aliquots  A l l t r a n s c o n j u g a n t s were  t o produce  carotovoricin-379,  t o grow on CVP and r e s i s t a n c e t o  erythromycin  (30 ug/ml) and c h l o r a m p h e n i c o l (15  ug/ml).  Bacteriocin Plate  Assays:  A s s a y s were p e r f o r m e d sterilized peak in  from  culture  soft  Carotovoricin chromatography executed Chapter  on a lawn  agar  1% B a c t o P e p t o n e ,  5 ul filter-  supernatant, or contents of a  a column,  peptone  by p l a c i n g  of indicator  (PSA) c o n t a i n i n g  0.45% B a c t o Agar concentration,  and e l e c t r o n  seeded  0.85% N a C l ,  pH 7.4.  column  microscopy  were  according to procedures outlined i n 1.  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 : Late  l o g phase  centrifugation Tris,  cells  (10 ml) were h a r v e s t e d by  and r e s u s p e n d e d  i n 600 u l o f 0.05M  0.02M EDTA; pH 8.0 ( T E ) b u f f e r  One h u n d r e d  microliters  + 20% s u c r o s e .  o f a 5 mg/ml s t o c k  solution  59  of  lysozyme  for  (Sigma) i n TE was added and i n c u b a t e d  10 m i n u t e s  (min) a t room t e m p e r a t u r e .  hundred m i c r o l i t e r s pH  8.0 were t h e n  stock  solution  o f a 0.5M EDTA s t o c k  added and i n c u b a t e d  room t e m p e r a t u r e .  20 m i n .  f o r 20 min a t  o f SDS were then  This  added.  subsequently  chloroform,  with  until  ( M a n i a t i s et  i n t e r f a c e was s e e n  a t 50 C  al.  solution,  with  a clear 1982).  aqueous p h a s e was a d j u s t e d t o 0.IM NaCl  with  The t u b e s  l y s a t e was e x t r a c t e d t w i c e (50:50 v / v ) and t w i c e  precipitated  o f a 10%  and i n c u b a t e d  phenol:chloroform  NaCl s t o c k  solution  Two h u n d r e d m i c r o l i t e r s  were g e n t l y i n v e r t e d 10 t i m e s for  One  The  from  a 5M  and DNA and RNA were  2 volumes o f 99% e t h a n o l  a t -50 C  overnight. Precipitated  nucleic  a c i d s were p e l l e t e d a t  13,000 x g a t -20 C f o r 25 min. was  removed  was  dissolved  aliquots with 30  Residual  ethanol  i n a vacuum d e s s i c a t o r and t h e p e l l e t i n 50 u l o f T E .  o f these  Ten m i c r o l i t e r  samples were removed, t r e a t e d  2 u l o f D N a s e - f r e e RNase  (10 mg/ml s t o c k ) , f o r  min a t 37 C and a n a l y s e d by a g a r o s e g e l  electrophoresis 1984;  Glover  (AGE) ( P e r b a l l  1985).  1984; Hames et  al.  60  Electrophoresis  was p e r f o r m e d  x 20 cm submerged, h o r i z o n t a l Electrophoresis Borate, 24  h.  2.5mM EDTA  t o a depth  (U.V.  were 0.5% a g a r o s e o f 0.3 mm.  Alkaline 100 of  substrate buffer  identified soluble  (0.1M T r i s - H C l  1 mg/ml p - n i t r o p h e n y 1  at 4 C overnight.  product.  combining with  100 u l  pH 9.0)  phosphate tubes.  (Sigma)  Tubes were  P o s i t i v e r e s u l t s were  by a v i s u a l d e t e c t i o n  reaction  California).  supernatant  1.5 ml p o l y a l l o m e r E p p e n d o r f  incubated  transilluminator  was a s s a y e d by  u l of f i l t e r - s t e r i l i z e d  containing in  (Sigma) f o r 1 h and  I n c . San G a b r i e l ,  phosphatase  (w/v) ( S i g m a ) ,  DNA was s t a i n e d i n  w i t h a mid-wave u l t r a v i o l e t Products  90mM  (TBE) pH 8.2 a t 1.5 V/cm f o r 18-  1.0 ug/ml o f e t h i d i u m b r o m i d e viewed  apparatus.  was c a r r i e d o u t i n 90mM T r i s ,  Gels used  poured  on a homemade 20  of a yellow  61  RESULTS  S e p h a c r y l S-300 Column C h r o m a t o g r a p h y and  Bioassays:  Column c h r o m a t o g r a p h y o f c o n c e n t r a t e d carotovoricin strain  379  protein la).  after  was  non-induced  52  h a t 20  distributed  the  weight  two  molecular  indicators,  (peak  while  (peak  2)  1) p r o d u c e d  was  clear  diffuse  found  Bioassays  large  the s m a l l m o l e c u l a r produced  total  cultures  fraction. the  the  peaks ( F i g .  i n induced  p e a k s showed t h a t  fraction  fraction  C showed t h a t  weight  Ecc  of  1), the m a j o r i t y of the p r o t e i n  lower  of these  cultures  between two  U n l i k e the s i t u a t i o n  (Chapter in  from  molecular plaques  on  weight  plaques ( F i g .  2b) . Ecc  strain  resistance  379  showed a t e m p e r a t u r e - s e n s i t i v e  to erythromycin  (30 ug/ml)  chloramphenicol  (15 u g / m l ) .  antibiotics  expressed  lost was  was  at temperatures also  C o r 37 strain  a b l e t o grow and C.  379  Mitomycin grown a t 37  R e s i s t a n c e to  a t 20  above 35  C  C.  p i t CVP  induced  and  i n LB b u t Ecc  after  no  was  strain  cultures  C produced  these  24 of  large  379  h at Ecc  24  62  0.05  E  FRACTION F i g u r e 1. S e p h a c r y l S-300 column chromatograms o f c a r o t o v o r i c i n from Erwinia and E. c o l i transconjugants. a.  Erwinia  carotovora  subsp.  carotovora  (Ecc)  s t r a i n 379 n o n - i n d u c e d ; b. Ecc s t r a i n 379 + m i t o m y c i n (0.2 ug/ml) a t 37 C; c. R68.45 m e d i a t e d CVP~ E. c o l i t r a n s c o n j u g a n t ; d. R68.45 m e d i a t e d CVP E. c o l i t r a n s c o n j u g a n t ; e. W i l d - t y p e 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 o f c o l o n i e s and S e p h a c r y l S-300 f r a c t i o n a t e d peaks f r o m c o n c e n t r a t e d s u p e r n a t a n t s o f E. coli transconj u g a n t s and E. carotovora s u b s p . 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 a s s a y s o f 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 s m a l l (peak 2) m o l e c u l a r w e i g h t b a c t e r i o c i n components f r o m s u p e r n a t a n t s o f CVP+ and CVP" E. coli transconjugants. CVP-  colony CVP  +  peak 2 CVP  +  colony b.  CVP  +  CVP"  peak 1 CVP"  CVP  colony CVP  colony  +  peak 1 CVP  peak 2  +  +  colony  C a r o t o v o r i c i n p l a t e a s s a y s o f c o l o n i e s and s e p a r a t e d l a r g e and s m a l l m o l e c u l a r w e i g h t b a c t e r i o c i n components from s u p e r n a t a n t s o f Ecc 379. peak 2 peak 2  peak  1  producing colony  peak 2 producing colony  64  65  molecular bioassay  weight  carotovoricin  o r S-300 column c h r o m a t o g r a p h y  Column c h r o m a t o g r a p h y carotovoricin Ecc  as d e t e c t e d by  from  (Fig. lb).  of concentrated  mitomycin-induced  cultures of  s t r a i n 379 grown a t 37 C showed o n l y one peak  corresponding (Fig.  lb).  t o t h e low m o l e c u l a r  Bioassays  diffuse-type  plaques  w h i c h was r e s i s t a n t .  o f t h i s peak  In a d d i t i o n ,  or reduction i n c e l l  Column c h r o m a t o g r a p h y m e d i a t e d CVP" E. peaks  coli  (Fig. l c ) .  weight  component  activity. bacteriocin  component  produced  on a l l i n d i c a t o r s  grown a t 37 C showed no v i s i b l e lysis  weight  e x c e p t 530  Ecc s t r a i n 379  signs of c e l l s  turbidity. of supernatants  transconjugants  o f R68.45  showed two  However o n l y t h e low m o l e c u l a r had d i f f u s e - t y p e  carotovoricin  Chromatography o f c o n c e n t r a t e d from  transconjugants weight b i o a c t i v e  w i l d - t y p e mated CVP", also  showed one s m a l l  component  (Fig. Ie).  E.  coli  molecular In c o n t r a s t , +  column c h r o m a t o g r a p h y o f R68.45 m e d i a t e d C V P , coli  gave a peak p r o f i l e  induced  to that  o f non-  Ecc s t r a i n 379 ( F i g . I d ) and p r o d u c e d  carotovoricin The  similar  E.  similar  ability  t o Ecc 379 ( F i g .  2a).  t o grow on CVP was t r a n s f e r r e d  at a  66  5  frequency r  (ery )  of 1 x 10~ .  When t h e e r y t h r o m y c i n transconjugants production produced  +  produced  and p r o d u c e d  was  found  4  o f 10~ .  on t h r e e o f t h e f o u r were C V P  -  (30 ug/ml) and In a l l  phosphatase which i s  t o t h e p e r i p l a s m o f E.  phosphatase a c t i v i t y of wild-type  were  they  transconjugants  (15 u g / m l ) . alkaline  When t h e s e  i n the f i l t e r - s t e r i l i z e d  supernatants  bacteriocin  production,  to erythromycin  restricted  No a l k a l i n e  However  w i l d - t y p e m a t i n g s were  A l l o f these  transconjugants,  and  on a l l i n d i c a t o r s  wild-type  d i f f u s e - t y p e plaques  chloramphenicol  normally  from  for bacteriocin  resistant  .  indicators.  at a frequency  indicators.  - 3  resistant  most were CVP"  530 w h i c h was r e s i s t a n t .  Transconjugants  bioassayed  of 1 x 1 0  were t e s t e d f o r g r o w t h on CVP a n d  a l l four  obtained  r  ( c a m ) (15  and c h l o r a m p h e n i c o l  d i f f u s e - t y p e plaques  strain  against  at a frequency  of carotovoricin,  1.7% were C V P  and  to erythromycin  (30 ug/ml) and c h l o r a m p h e n i c o l  ug/ml) was t r a n s f e r r e d  except  Resistance  E.  supernatants.  was f o u n d  coli.  coli,  i n the  67  DNA  Content: Plasmid  profiles r  resistant  (kan )  R68.45  weight  found  These p l a s m i d s coli  as s e p a r a t e  transconjugants Plasmid  that  molecular  Ecc s t r a i n  and R68.45  of transconjugants  obtained i n  the t r a n s f e r  diffuse-type  Electron  resistance  production.  o f the r e s i s t a n t  a t 37 C i n t h e E.  with  donors,  p h e n o t y p e s was n o t coli  transconjugants.  Microscopy:  transconjugant  E. coli  bound v e s i c l e s . transconjugants  absent  and  U n l i k e t h e Erwinia  E l e c t r o n micrographs  surface  of the  (Erwp) ( F i g . 3b) a l o n g  chloramphenicol  affected  E.  ( F i g . 3a).  resistance,  expression  379.  i n these  the phenotypes o f e r y t h r o m y c i n  bacteriocin  they  i n the transconjugant  illustrated  plasmid  kanamycin  larger  c o n t a i n e d no i n s e r t  profiles  Erwinia  in wild-type  entities  w i l d - t y p e matings large  cam ,  showed  and a s e c o n d  existed  r  ery ,  transconjugants  contained both plasmid  r  o f CVP",  vesicles  of supernatants showed numerous  Electron  microscopy  showed a p r o t u b e r a n c e or "blebs"  i n non-transconjugant  of  membrane-  of intact o f many  ( F i g . 4a) w h i c h E.  coli.  CVP"  were  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 strain 379 (Ecc 3 7 9 ) a n d Ecc 3 7 9 x E. coli transconjugants w i t h a n d w i t h o u t R68.45 m e d i a t i o n . Samples were r u n o n 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 ( T B E ) a t 1 t o 1.5 V/cm f o r 18-24 h . a.  R 6 8 . 4 5 m e d i a t e d E. coli transconjugants: Lanes: 1) E. coli w i t h pBR325; 2 ) R 6 8 . 4 5 m e d i a t e d CVP" E. coli transconjugant; 3 ) E. coli w i t h pBR322; 4 ) Ecc s t r a i n 3 7 9 w i t h Erwinia plasmid (Ewrp) 10 u l s a m p l e l o a d ; 5 ) Ecc s t r a i n 3 7 9 w i t h E w r p 5 u l s a m p l e l o a d ;  b.  W i l d - t y p e m a t e d E. coli transconjugants: Lanes: 1) HB101 w i t h R 6 8 . 4 5 2 ) E. coli t r a n s c o n j u g a n t w i t h pBR322 a n d Erwp; 3 ) Ecc 3 7 9 w i t h E r w p ; 4) HB101 t r a n s c o n j u g a n t w i t h Erwp.  F i g u r e 4. E l e c t r o n m i c r o g r a p h s o f c e l l s and s u p e r n a t a n t s o f E. c o l i t r a n s c o n j u g a n t s (Bar=100nm). a. T y p i c a l n e g a t i v e s t a i n e d E. c o l i t r a n s c o n j u g a n t c e l l showing s u r f a c e v e s i c l e s ; b , c . S u p e r n a t a n t s o f CVP E. c o l i t r a n s c o n j u g a n t s showing p a r t i c u l a t e carotovoricin. Note p r e s e n c e o f t a i l f i b r e s i n c. +  70  Electron weight  micrographs  f r a c t i o n f r o m CVP  showed p a r t i c u l a t e isolated  o f the large molecular +  E.  coli  bacteriocins  Ecc p r e p a r a t i o n s  from  transconjugants i d e n t i c a l to those  (Fig.  4b-c).  DISCUSSION  Genetic Erwinia  analysis  of carotovoricin  p r o v e d t o be a s u b s t a n t i a l  convenient, However, species  used  i n t h e taxonomy o f  have c o n s i s t e n t l y  erythromycin  carotovora  f r o m o t h e r Erwinia  (Schaad  Erythromycin  with  several 379,  resistance  379 was shown  a loss  used  t o s e p a r a t e E.  and E.  strain  p r o b l e m as  w i l d - t y p e m a r k e r s were n o t a v a i l a b l e .  tests  carotovora  carotovora subsp.  o f t h i s phenotype  was a l s o  atroseptica  (30 ug/ml) i n Ecc  a t 37 C.  a chloramphenicol resistant  resistance,  subsp.  1980).  t o be t e m p e r a t u r e  was f o u n d w h i c h ,  Erwinia  resistance to  o t h e r a n t i b i o t i c s were t e s t e d  phenotype  production i n  sensitive When on Ecc s t r a i n  (15 ug/ml)  l i k e erythromycin  temperature  sensitive.  Cell  71  lysis,  as  induced  seen  cells  production  by  a reduction in turbidity  was  also  inhibited  of p a r t i c u l a t e  temperature a possible  sensitivity plasmid  curing properties temperatures  sensitive  namely E.  coli,  mobilization its  ability  suggested  to the g e n e r a l  Ecc  chromosomal  i n these  temperature  transfer strain  was  between  of genetic  379  t o a model  necessary.  v e c t o r R68.45 was  wide h o s t  The  1979) .  involvement  from  due  range,  The  chromosome  +  Holloway  1976)  and  ability  plasmids  at h i g h frequency mobilization  because  (Haas  and  to m o b i l i z e r e s i d e n t (Willetts  ability  and  The  thought  to occur v i a a c o - i n t e g r a t i o n i n the r e c i p i e n t  and  resolution  resolution  of the c o - i n t e g r a t e i s thought  Furthermore,  Crowther  o f R68.45 i s  subsequent  i n t e g r a t e maintenance  of  mobilization  1980).  ( P u h l e r and  system,  conjugative  chosen  ( c m a ) from many o r i g i n s ,  recA dependent  was  o f growth at e l e v a t e d  phenotypes,  information  as  of the phenotypes  involvement  (Brock  plasmid  a t 37 C,  bacteriocin.  In o r d e r t o d i s t i n g u i s h and/or  of  R i e s s 1984)  i n r e c A minus  cell. to  with  The be co-  recipients.  R68.45 c o n t a i n s t h e a n t i b i o t i c  markers  72  for  kanamycin,  tetracycline,  and a m p i c i l l i n ( o r  carbenicillin)  r e s i s t a n c e w h i c h make i t c o n v e n i e n t  to  plasmid  follow The  E.  coli  this  ability  A l l CVP  particulate  +  o f 10"  5  derived determinants.  r e s i s t a n c e s were t r a n s f e r r e d recipient  cell.  agarose  gel electrophoresis  Erwinia. kan  r  DNA  o f CVP  to that  found  and b a c t e r i o c i n  integrate  from  positive.  transferred t h e Erwinia  r  ery ,  DNA  by  cam  r  of a plasmid  i n wild-type  These  also  phenotypes  as an R68.45 c o chromosome.  a c o - i n t e g r a t e w o u l d be t o o l a r g e  intact  The s i z e o f to resolve  electrophoretically. analysis  transconjugants plasmids.  by AGE o f CVP", confirmed  3  suggested  analysis +  o f 10~  However, t h e s e t r a n s c o n j u g a n t s were  were p r e s u m a b l y  such  (15 ug/ml)  showed t h e p r e s e n c e  which corresponded  Erythromycin  This high frequency  derived determinants.  transconjugants  suggested  at a frequency  plasmid  of  produced  These f a c t s  (30 ug/ml) and c h l o r a m p h e n i c o l  per  transferred to  per r e c i p i e n t  transconjugants  bacteriocin.  chromosomally  a population.  t o grow on CVP was  at a frequency  R68.45.  through  r  the presence  One o f t h e p l a s m i d s  1  e r y , cam ", k a n  was  o f two  identified  by  r  73  size  as R68.45 w i t h no i n s e r t  corresponded found  to a large molecular  i n w i l d - t y p e Ecc s t r a i n  recipient  t h e Erwinia R68.45.  (E. coli  cell  visualization  Bioassays they  diffuse-type identically  transferred u s i n g these  produced  plaques. to that  Ecc and E.  transmissibility  r  molecular  weight  that coded  this  i n wild-type  -  From t h e s e  self-transmissible  f o r erythromycin  weight  transconjugants  component  R68.45  matings  the s e l f -  and p r o d u c e d  bacteriocin  by t h e CVP  transconjugants.  r  a t 37  a t 20 C.  confirmed  cam  behaved  component o f  A l lo f these  CVP , e r y ,  weight  Ecc i n d u c e d  o f the large molecular  were a l s o  that  transconjugants  weight  obtained  i n Ecc 379. -  suggested  This bacteriocin  coli  strain,  independently of  o b t a i n e d from  Transconjugants  produced  Because t h e  small molecular  c a r o t o v o r i c i n - 3 7 9 produced  plasmid  379.  plasmid  HB101) was a r e c A "  C or the small molecular  between  weight  o f separate plasmids  plasmid  showed t h a t  that  and t h e o t h e r  a small identical to  mediated  results  we  conclude  Erwinia  megaplasmid  and c h l o r a m p h e n i c o l  resistance  and a s m a l l m o l e c u l a r  component.  The p r o d u c t i o n i n E.  weight coli  bacteriocin  74  transconjugants  of particulate  bacteriocin  t o t h a t o f Ecc 379 was o n l y d e t e c t e d when  identical  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  that  t h e megaplasmid.  This fact  carotovoricin-379 production c o d e d f o r by b o t h  suggests  i n Ecc s t r a i n  379 i s  chromosomal and p l a s m i d  const i t u e n t s . Electron  microscopy  showed t h a t t h e y b l e b s not found  produced  exo-enzyme p r o d u c t i o n et  phosphatase 1973). and  suggestion coli  Erwinia  that this  secretion  1973),  aeruginosa  (Ingram and D a i n t y  of blebs  on t r a n s c o n j u g a n t s  (Chapter  1), introduces the  megaplasmid  i s involved i n  by t h e f a c t  alkaline  This  that  i n t h e E.  phosphatase which i s  i n t h e p e r i p l a s m , was f o u n d i n  filter-sterilized explain  i n Pseudomonas  i s supported  found  have  secretion in  of periplasmic proteins.  transconjugants,  normally  Blebs  (Devoe and G i l c h r i s t  i n Pseudomonas  wild-type  coli.  1985) and r e l e a s e o f a l k a l i n e  The p r e s e n c e  possibility the  al.  E.  endotoxin  meningitidis  (Thompson  many s u r f a c e v e s i c l e s o r  i n wild-type  been a s s o c i a t e d w i t h Neisseria  of a l l transconjugants  supernatants.  why i n c o s m i d  I t would  and s h o t - g u n c l o n i n g  also  75  experiments were f o u n d coli  t o accumulate  transfectants  effectively  al.  Kotoujansky  et  into  resident  i n t h e p e r i p l a s m o f E.  or transformants  1985; Z i n k al.  account  1985).  and were n o t  the supernatant  and C h a t t e r j e e 1985; These methods d i d n o t  the p o s s i b l e  megaplasmid  periplasmic  p e c t i n - d e g r a d i n g enzymes  t r a n s p o r t e d out i n t o  et  (Collmer  take  Erwinia,  with  involvement  i n the s e c r e t i o n of  proteins i n  Erwinia.  of a  76  CHAPTER  SEROLOGICAL FORMS  RELATIONSHIPS ERWINIA  OF  POLYCLONAL  3  AMONG  BACTERIOCIN  ANTISERUM  AGAINST  THE DIFFERENT DETECTED  BY  PARTICULATE  CAROTOVORICIN-379  INTRODUCTION  Particulate carotovora  bacteriocin  subsp.  carotovora  a s t r i k i n g resemblance Pseudomonas. 2) has shown pyocin  that  s t r a i n 379 b e a r s  carotovoricin-379  c a n be d i v i d e d  based  the  m o l e c u l a r weight  which produced  Erwinia  t o t h e R-type p y o c i n s o f  fractions  indicators,  (Ecc)  However, p r e v i o u s work  activity  large  p r o d u c e d by  ( C h a p t e r 1 and activity  into  several  on m o l e c u l a r w e i g h t .  Production of  (particulate)  a clear-type  was t e m p e r a t u r e  unlike  p l a q u e on  fraction,  several  s e n s i t i v e whereas t h e  s m a l l e r m o l e c u l a r weight  fractions  independent  sparse or d i f f u s e plaques.  The  addition  resulted  and p r o d u c e d  were  temperature  o f an o u t e r membrane s t a b i l i z i n g  i n the detection  of t o t a l  agent  carotovoricin  77  activity  a t 20 C i n one l a r g e m o l e c u l a r w e i g h t  which c o n t a i n e d p a r t i c l e s  resembling  when b i o a s s a y e d ,  c l e a r - t y p e plaques.  The  produced  p y o c i n s and  o f Pseudomonas  R-type p y o c i n s  peak  aeruginosa  have s t r u c t u r e s  which a r e c l o s e l y  related  morphologically  and s e r o l o g i c a l l y  to bacteriophage  tails  (Oshumi  considered  et  lies  in their  against  a range  to a d i v i s i o n  of indicator  al.  groups  noted  1980).  characteristic of  activity  strains.  o f t h e R-type p y o c i n s  showed an a l m o s t  et  for particulate  different  (Shinomiya  o f the p r o t e i n  differences  They have been  The d i s t i n g u i s h i n g  pyocins  Analysis  1980).  model s y s t e m s  bacteriocins.  specificity  al.  both  et  T h i s has  into  al.  spectra  five  1979).  subunits o f these  identical  composition  i n the t a i l  fiber  pyocins  with  region  These s m a l l d i f f e r e n c e s  were  i n cross-adsorption studies using  antisera  and has l e d t o t h e c o n c l u s i o n t h a t  a r e a major determinant  spectrum The  genetic determinants  thought and  of a given pyocin  t o be l o c a t e d  i n most  cases  small (Oshumi  detected  fibers  led  specific these  i n the a c t i v i t y  (Oshumi  et  al.  1980).  for pyocin production are  on t h e b a c t e r i a l  chromosome  pyocin p r o d u c t i o n i s not a  78  temperature  sensitive  Genetic  (Ecc  producer showed  transfer  that  erythromycin production component or  blebs  an Erwinia  from  carotovoricin  s t r a i n 379) t o Escherichia  a large  transmissible  trait.  molecular  weight  coli self-  i n Ecc s t r a i n 379 coded f o r  plasmid  and c h l o r a m p h e n i c o l  of a small molecular  resistance, the weight  and t h e p r o l i f e r a t i o n o f s u r f a c e (Chapter  carotovoricin  2).  The s m a l l m o l e c u l a r  component  produced  b y E.  t r a n s c o n j u g a n t s h a d t h e same a c t i v i t y the s m a l l m o l e c u l a r weight non-induced grown  cultures  o f t h i s study  the large  molecular  carotovoricin-379 of and  the p a r t i c u l a t e  several  coli  coli s p e c t r u m as from  a t 37 C.  was t o d e v e l o p  weight  antiserum  fraction of the relatedness  particulate carotovoricin-379 and s o l u b l e  produced  Ecc x E.  weight  isolated  Ecc 379 grown  and t o d e t e r m i n e  t h e mature i n t a c t  carotovoricin  fraction  vesicles  o f w i l d - t y p e Ecc s t r a i n 379  a t 20 C o r i n d u c e d  The p u r p o s e to  bacteriocin  forms o f  b y w i l d - t y p e Ecc 379 and transconjugants.  79  MATERIALS  Bacterial  METHODS  strains  Bacterial obtained  AND  strains  used  and t r a n s c o n j u g a n t s  as o u t l i n e d i n C h a p t e r s  1 and 2.  were  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 C h a p t e r s  and  2.  Concentration  carotovoricin  and f r a c t i o n a t i o n  were p e r f o r m e d  1  of  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 previously  concentrated Ecc  d e s c r i b e d from  strain  379 grown i n  m i n i m a l M9 media a t 20 C, 48 h a f t e r Concentrated supernatant  p r o t e i n from was  analysed  as  induction.  100 ml o f c u l t u r e by S e p h a c r y l  S-300 column  c h r o m a t o g r a p h y as p r e v i o u s l y d e s c r i b e d and  2).  The l a r g e m o l e c u l a r  and  bioassayed  to confirm  weight  activity.  (Chapter  peak was  was  injected  rabbit, the  adjuvant  intramuscularly into  an  equal  (Sigma).  the h i n d  once a week, f o r 5 c o n s e c u t i v e  6 t h week, 20 ml o f b l o o d ,  pooled  Ten m i l l i g r a m s  o f p r o t e i n was m i x e d and e m u l s i f i e d w i t h volume o f F r e u n d s c o m p l e t e  1  collected  This  leg of a  weeks.  On  from the  80  ear  vein,  was  allowed to c l o t  for  1 hour  The  r e m a i n i n g serum was  (h) f o l l o w e d  centrifugation Clarified  by  12 h  temperature  ( o v e r n i g h t ) at 4  clarified  (8000 x g)  serum was  a t room  by  low  speed  f o r 25 m i n u t e s  stored  i n 1 ml  C.  (min).  aliquots  at  -20  C.  Radial  Immunodiffusion  Clarified agar  (Difco)  serum was  i n 5OmM sodium  + 0.85%  NaCl  a depth  o f 0.3  supernatant plates  (PBS)  sequentially drop  indicated  at  22  15 min  Twenty u l o f  EM  times:  a)  and  c)  a t 22  C.  grids  carbon  of the f o l l o w i n g  f o r 15 min C;  on  used  C;  and  the  to  (ISEM) "trap" 1984)  by  colloidion  grids  on  solutions  b) PBS  7.4  dishes to  (Hill  filter-sterilized Grids  pH  C.  1/2000 d i l u t i o n  a t 22  noble  culture  Microscopy  a n t i s e r u m was  floating  buffer  to pre-cut wells  Electron  particles  i n 0.5%  into petri  i n c u b a t e d f o r 24-48 h a t 25  bacteriocin  PBS  1/100  phosphate  poured  were added  Polyclonal  in  and  cm.  Immuno-sensitive  one  diluted  pH  f o r the of antiserum 7.4  culture  were s t a i n e d  with  f o r 10  min  fluid  for  10-12  81  drops viewed  o f 2% p h o s p h o t u n g s t i c on a P h i l l i p s  Subsequent (Hill  dilution  (PTA) pH 7.0  EM-300 e l e c t r o n  decoration of trapped  1984) was  on PBS pH 7.4  acid  accomplished  and  microscope. particles  by f u r t h e r  incubations  f o r 10 min a t 22 C f o l l o w e d by 1/1000  of antiserum  G r i d s were s t a i n e d  Sodium D o d e c y l  i n PBS  and v i e w e d  Sulphate  Electrophoresis  f o r 15 min a t 22 C. as above.  Polyacrylamide Gel  (SDS-PAGE)  Electrophoresis  was  carried  o u t i n 0.5 mm  8.0%  acrylamide using Tris-glycine-SDS, e s s e n t i a l l y following Laemmili  the d i s c o n t i n u o u s system (Laemmili  Concentrated buffer  1970; L a e m m i l i  supernatants  (lOOmM T r i s - H C l  beta-mercaptoethanol), electrophoresed vertical  slab  gel unit  G e l s were s t a i n e d Western b l o t s Protein  boiled  either  1973).  in loading and 5%  f o r 1.5 h u s i n g a m i n i -  (Hoefer S c i e n t i f i c for protein  SE-200).  or used f o r  (electroblotting).  d e t e c t i o n was  methanol,  2% SDS  by  f o r 2 min and  accomplished  g e l s w i t h 0.5% C o o m a s s i e b r i l l i a n t 45%  and F a r r e  were d i s s o l v e d  pH 6.8 w i t h  a t 18 mA  outlined  10% a c e t i c  acid  by  staining  b l u e R-250 i n  (v/v) f o r 1 h  82  followed acid on  by d e s t a i n i n g  (v/v).  G e l s were p r e s e r v e d  a homemade s l a b - g e l  were s o a k e d Glycine,  dryer.  20% M e t h a n o l ;  a t 65 C  for blotting  (25mM T r i s ,  192raM  pH 8.3) f o r 1 h f o l l o w e d  and i m m u n o - d e t e c t i o n  standards  10% a c e t i c  by d r y i n g  Gels  in transfer buffer  electroblotting Protein  i n 20% m e t h a n o l ,  (Sigma) r a n g e d  by  of proteins.  from  30-200  kilodaltons.  Western B l o t t i n g The  transfer of proteins  (Western B l o t t i n g )  to n i t r o c e l l u l o s e  et  (Towbin  al.  1979; G e r s h o n i  and  Palade,  for  12 h i n a homemade b l o t t i n g a p p a r a t u s  stainless  1983) was a c c o m p l i s h e d  steel  nitrocellulose thrice  with  a mixture  Protein  i n 20mM T r i s  binding  b l o t was washed t w i c e  and  incubated  was  was washed  500mM N a C l , pH  were b l o c k e d serum  albumin  f o r 15 min e a c h  i n a 1/1000 d i l u t i o n  i n TBS  of polyclonal  + 0.1% BSA i n TBS f o r 2 h a t 37 C.  followed  with  (1 mg/ml) i n TBS f o r 2 h a t 37 C.  The  antiserum  sites  o f 2% g e l a t i n and b o v i n e  (Sigma)  with  After blotting,  transferred proteins  f o r 15 min e a c h  7.5 ( T B S ) .  (BSA)  rod electrodes.  a t 40 V, 150 mA  by two 15-minute washes  This  i n TBS and  83  incubation  i n a 1/1500 d i l u t i o n  IgG-horseradish + 0.1% twice min  BSA  f o r 2 h a t 37  f o r 15 min  each  conjugate  C.  i n TBS  hydrogen  The  blot  and  incubated  1 mg/ml d i a m i n o b e n z i d i n e  peroxide  reaction  substrate  was  (added stopped  was  i n TBS.  between p a p e r  rapid  for  pH +  1%  before  and  5-15  7.5)  dilution  B l o t s were d r i e d  TBS  washed  (DAB)  immediately by  anti-rabbit  (Sigma) i n  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  containing  The  peroxidase  o f goat  use). of  stored  towels.  RESULTS  Antiserum  a g a i n s t the  large molecular  fraction  (particulate  with  homologous a n t i g e n and  the  circular (RID)  precipitin  ( F i g . 1).  regardless medium). also  (Fig.  1).  lines  produced  in radial  g r o w t h medium u s e d  N o n - i n d u c e d c u l t u r e s o f Ecc  produced  antiserum  carotovoricin-379) reacted  These p a t t e r n s were  of the  weight  bacteriocin  several  immunodiffusion identical (LB  or  minimal  strain  379  w h i c h r e a c t e d i n RID  against p a r t i c u l a t e carotovoricin-379 CVP  +  E.  coli  transconjugants  with  84  379  i n d u c e d (M9)  1/5 d i l u t i o n of 379 i n d u c e d  CVP" w i l d - t y p e transconjugant  379  i n d u c e d (M9)  CVP E. coli transconjugant  379  induced (LB)  379  E.  coli  +  HB101  ft)  control  x  non-induced  spill  CVP" R 6 8 . 4 5 mediated transconjugant  379 n o n induced (LB)  E. R68.45  coli control  F i g u r e 1. Radial immunodiffusion analysis 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 c a r o t o v o r i c i n - 3 7 9 p r o d u c e d b y Erwinia carotovora subsp. carotovora s t r a i n 3 7 9 (Ecc 3 7 9 ) a n d Ecc 3 7 9 x E. coli transconjugants. 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 . C u t 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 f r o m Ecc 3 7 9 , Escherichia coli a n d E. coli transconjugants.  85  constitutively precipitin cultures  produced  bacteriocins  patterns similar  o f Ecc  strain  379.  t r a n s c o n j u g a n t s produced band  Antiserum bacteriocin  of induced  A l l CVP"  o n l y one m a j o r  particles  included: attached  Electron  directed  "trapped"  supernatant  from  Microscopy  against  precipitin  heads,  particulate  5 u l o f an i n d u c e d  ( F i g . 2a and 2 b ) . intact  (ISEM)  a s i g n i f i c a n t l y higher  empty s h e a t h s ,  p a r t i a l l y formed  particles  were a l s o  trapped  number  culture  Trapped  carotovoricin  addition,  2b  to those  (Fig.l).  Immuno-sensitive  of  with  particles  w i t h and  without  c o r e s and h e a d s .  or d i s r u p t e d  In  bacteriocin  on t h e same g r i d ( F i g .  arrow). When t r a p p e d p a r t i c l e s  additional Although  antiserum  they  distinct particles  appeared  particles  ruled  intact  their  (Fig. 2c).  examination  t h e y became  resembled  carotovoricin-379,  were e x p o s e d t o coated  (Fig. 2c).  particulate  outlines  In most c a s e s  appeared these  less  decorated  out o f f o c u s b u t c l o s e out t h i s  were i n f a c t  possibility.  heavily  coated  The  with  86  F i g u r e 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 b y mitomycin C i n d u c e d Erwinia carotovora subsp. carotovora s t r a i n 379 b y i m m u n o s e n s i t i v e electron microscopy employing p o l y c l o n a l antiserum raised against t h e l a r g e m o l e c u l a r weight 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 o n 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 induced culture; 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 culture applied to a grid pre-coated with polyclonal antiserum to bacteriocin (trapped bacteriocin). ( A r r o w shows p a r t i a l l y disrupted particle). 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 second incubation with polyclonal antiserum (decorat i o n ) .  87  antibodies slightly  and as a r e s u l t  distorted  (Fig.  were sometimes c u r v e d o r 2c).  SDS-PAGE Polyacrylamide gel electrophoresis the supernatant complex  protein  and dependent  temperature  of the producing  incorporation  resulted low  strain,  with mitomycin  and t h e  ( F i g . 3 a ) . At  C (0.2 ug/ml)  i n a marked i n c r e a s e i n s e v e r a l  molecular  to a g e n e r a l in  the incubation  o f an i n d u c i n g a g e n t  20 C, i n d u c t i o n  weight  subunits  increase i n a l l protein  non-induced  Incubation  protein  culture  supernatants  a t 37 C r e d u c e d  the induction  h i g h and  i n addition subunits  (Fig.  supernatant  c o n c e n t r a t i o n s by a p p r o x i m a t e l y abolished  that  o f Ecc 379 was  composition  on b o t h  showed  found  3a).  protein  5 0 - f o l d and  mechanism a c t i v e  a t 20 C  (Fig. 3a). E.  All increase  coli  transconjugants  i n protein  content  compared w i t h w i l d - t y p e E. CVP  +  E.  protein strain  coli  showed a g e n e r a l  i n supernatants coli  ( F i g . 3b and 3 c ) .  t r a n s c o n j u g a n t s produced  profiles  indistinguishable  379 ( F i g . 3b and 3 c ) .  when  supernatant  from  induced  However, u n l i k e  Ecc  Ecc  88  F i g u r e 3. S D S - p o l y a c r y l a m i d e gel electrophoresis o f c o n c e n t r a t e d s u p e r n a t a n t s o f Erwinia carotovora s u b s p . carotovora s t r a i n 379 and Escherichia coli transconjugants. a. E f f e c t o f t e m p e r a t u r e and m i t o m y c i n C on p r o d u c t i o n o f s u p e r n a t a n t p r o t e i n s by Ecc 379: Lanes: 1) I n d u c e d a t 20 C; 2,3) Peak 2 from S e p h a c r y l S-300 f r a c t i o n a t e d n o n - i n d u c e d c u l t u r e a t 20 C; 4) I n d u c e d a t 20 C; 5,6) N o n - i n d u c e d a t 20 C; 7) N o n - i n d u c e d a t 37 C; 8) I n d u c e d a t 37 C; 9,10) I n d u c e d a t 20 C. C o m p a r i s o n o f Ecc 379 w i t h E. coli transconjugants: Lanes: 1) Sigma s t a n d a r d s (30 t o 200 KD); 2) R68.45 CVP" t r a n s c o n j u g a n t s ; 3) CVP~ w i l d - t y p e mated t r a n s c o n j u g a n t ; 4) C V P R68.45 t r a n s c o n j u g a n t ; 5) Ecc 379 i n d u c e d a t 20 C; 6) Ecc 379 n o n - i n d u c e d a t 20 C; 7) E. coli HB101 c o n t r o l ; 8,9) Sigma s t a n d a r d s (30 t o 200 KD).  b.  +  c. C o m p a r i s o n o f Ecc 379 w i t h E. coli transconjugants: Lanes: 1) Sigma s t a n d a r d s (30 t o 200 KD) ; 2) E. coli HB101/R68.45 c o n t r o l ; 3) Ecc 379 n o n - i n d u c e d a t 20 C; 4) Ecc 379 i n d u c e d a t 20 C; 5) E. coli R68.45 C V P transconjugant; 6) CVP" R68.45 t r a n s c o n j u g a n t ; 7) HB101 w i l d - t y p e mated t r a n s c o n j u g a n t C V P ; 8,9) Ecc 379 a t 37 C; 10) Sigma s t a n d a r d s (30 t o 200 KD). +  -  89  90  strain  379, t h e p r o t e i n s p r o d u c e d  were i n d e p e n d e n t temperature. general, protein of  of both  CVP" E.  produced than  wild-type  p r o t e i n s produced  Erwinia, that  w i t h CVP" E.  levels  E.  coli  b y CVP coli  +  HB101. E.  coli  Western  antiserum  by CVP" t r a n s c o n j u g a n t s  to n i t r o c e l l u l o s e ,  reacted with the  against particulate carotovoricin-379 subunits  homologous a n t i g e n were t h e h i g h components  lower  absent  from  ( F i g . 4 ) . These components were  CVP" t r a n s c o n j u g a n t s . supernatant  transconjugants  illustrated  of the  and low m o l e c u l a r  c o n c e n t r a t i o n i n non-induced  immuno-reactive coli  bacteriocin  bands o f t h e homologous a n t i g e n ,  4 ) . The most r e a c t i v e  weight  protein  Blotting  when b l o t t e d  in  weight  showed  by EM.  Most p r o t e i n  (Fig.  A comparison or induced  3b and 3 c ) , and no p a r t i c u l a t e  seen  coli  of extracellular  transconjugants  s u b u n i t s were n o t p r o d u c e d  was  E.  transconjugants i n  s e v e r a l h i g h and low m o l e c u l a r  (Fig.  +  an i n d u c i n g agent o r  coli  higher  by CVP  with  A comparison o f  p r o t e i n s from  those  some d i f f e r e n c e s  c u l t u r e s and  o f induced  CVP  +  E.  Ecc  i n t h e low m o l e c u l a r  91  1  2  3  4  5  6  7  8  F i g u r e 4. W e s t e r n b l o t on n i t r o c e l l u l o s e o f s u p e r n a t a n t p r o t e i n s from Erwinia carotovora subsp. carotovora  strain  379 and Escherichia  coli  trans-  c o n j u g a n t s f o l l o w e d by i m m u n o d e t e c t i o n 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 carotovoricin-379. Lanes: 1) W i l d - t y p e mated CVP" E. coli transconjugant; 2) N o n - i n d u c e d Ecc 379 a t 20 C; 3) I n d u c e d Ecc 379 a t 20 C; 4) C V P R68.45 m e d i a t e d E. coli transconjugant; 5) C V P " R68.45 m e d i a t e d E. coli transconjugant; 6) W i l d - t y p e mated CVP" E. coli transconjugant w i t h pBR322; 7) E. coli HB101 w i t h R68.45 c o n t r o l ; 8) CVP" R68.45 m e d i a t e d E. coli transconjugant w i t h pBR322. +  92  weight r e g i o n ,  i n s p i t e of no apparent  in b a c t e r i o c i n a c t i v i t y SDS-PAGE).  CVP  +  or appearance  transconjugants  r e l a t i v e l y s m a l l e r percentage  of  differences ( v i a EM or  showed a immuno-reactive  subunits  ( F i g . 4) when compared to the  proteins  of Ecc ( F i g . 3b and 3 c ) .  reactive  DISCUSSION  The p r o t e i n subunit composition  of  c a r o t o v o r i c i n - 3 7 9 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 consisting  of over 20 subunits with a wide range of  molecular weights. supernatants  The p r o t e i n composition of  of CVP* E. coli  transconjugants  seen by SDS-PAGE was i d e n t i c a l to Erwinia.  structure  wild-type  These CVP* transconjugants  b a c t e r i o c i n at 37 C without 3c and 4 ) .  In c o n t r a s t ,  as  produced  an i n d u c i n g agent ( F i g .  an i n d u c i n g agent  d r a m a t i c a l l y i n c r e a s e d the b a c t e r i o c i n t i t r e  at 20  C i n Erwinia  4).  but had no e f f e c t  CVP~ transconjugants  released  at 37 C ( F i g . proteins,  which corresponded to bands present  some of  in wild-type  93  carotovoricin-379.  The most o b v i o u s  the  and low m o l e c u l a r  absence o f h i g h  protein  antiserum  against  demonstrated CVP  +  E.  of blotted  p r o t e i n s by  particulate  bacteriocin  that p a r t i c u l a t e  bacteriocin  coli  was v e r y  closely  related  produced to w i l d -  c a r o t o v o r i c i n - 3 7 9 p r o d u c e d b y Ecc s t r a i n 379.  type  However,  small  molecular difference of  weight  subunits.  Immuno-detection  by  d i f f e r e n c e was  d i f f e r e n c e s were n o t e d  weight  region.  The most  was t h e p r e s e n c e  i n t h e low  obvious  i n t h e Ecc  bacteriocin  a s t r o n g l y i m m u n o - r e a c t i v e band c o r r e s p o n d i n g t o  about  15,000 MW.  This  i s most  o u t e r membrane component and  due t o an  of the carotovoricin-379  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  components addition, greater their  o f t h e Ecc and E.  different  were d e t e c t e d  particulate  showed  weight  three  subunits  w h i c h was a l s o p r e s e n t  transconjugants.  producers.  T h i s was most  i n the intact  small molecular  membrane  likely  In with  due t o  immunogenic p r o p e r t i e s and  transconjugants  subunit  coli  some p r o t e i n s u b u n i t s  sensitivity.  positions CVP"  likely  CVP  +  bacteriocin.  immuno-reactive  and one l a r g e i n CVP  +  transconjugants  c o n t a i n e d an  94  additional protein  large molecular  and s e v e r a l v e r y  components  (less  than  transconjugants. bands w i t h  weight low  A comparison  i n CVP"  i n CVP  a preference  transconjugants  hypothesis  +  d i d not  transconjugants  produced  p r o t e i n s . CVP  was s u p p o r t e d  by the  of these  transconjugants  particulate  and may  -  specific  fact  i n the  (Chapter  antiserum  and t h a t  supernatant  2).  developed  against  carotovoricin-379 consistently  t h e homologous a n t i g e n . also  a l l E. coli  reacted with  In a d d i t i o n , carotovoricin  transconjugants.  This  t h a t CVP"  produce s u r f a c e v e s i c l e s  polyclonal  seem  periplasmic proteins.  p h o s p h a t a s e was f o u n d  by  This  Erwinia-Aerived  fewer  alkaline  antiserum  stain  in a hypothetical secretion  o f E. coli  transconjugants  with  i n CVP"  of protein stained  possibly allowing a less  substitution  The  present  transconjugants.  f o r Erwinia-derived  proteins  weight  i n i m m u n o r e a c t i v e p r o t e i n s does not  t o be as o b v i o u s  system  150 k d )  i m m u n o - s t a i n e d bands showed t h a t a l a r g e  immunogenically  reflect  molecular  20,000) not  number o f p r o t e i n s u b u n i t s  reduction  (about  This fact  reacted this produced suggests  95  that  the  component  low of  molecular intact  weight  carotovoricin  particulate  is  carotovoricin.  a  96  GENERAL  Ultrastructural  DISCUSSION  examination  of carotovoricin-  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 hypothesis (Chapter  of a carotovoricin  1).  hypothesis strain  Supporting  came f r o m  379 p r o d u c e d  constitutively turbidity  r e l e a s e without  evidence  the f a c t  l e d to the  for this  that  particulate  non-induced  w i t h 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  or v i a b i l i t y .  Ecc  strain  be f r a c t i o n a t e d  Examination  379 showed  into  The h i g h e r m o l e c u l a r effective  weight  of  could  (Chapter 2). was  strains  component  much more than the  (Chapters  damaging a g e n t s  of bacteriocin  o f non-  activity  component  indicator  weight  I n d u c t i o n w i t h DNA  proportion  that  two components  at i n h i b i t i n g  smaller molecular 2).  Ecc  bacteriocin  c a r o t o v o r i c i n - 3 7 9 w i t h i n the supernatant induced  lysis  1 and  skewed t h e  i n the supernatant  towards the h i g h e r m o l e c u l a r  weight  (Chapter  examination  of this  a gradual  stepwise  induction increase 379  1).  A more c a r e f u l  process  illustrated  i n the m o l e c u l a r weight  (Chapter 1).  component  of c a r o t o v o r i c i n -  97  Examination indicated these  that  cells  projections (Chapter  of  induced  a small  contained at  1).  proportion  stages  Structural  showed t h a t  resembled  particulate  carotovoricin-379  t o be  the  hypothesized represented that by  cores  the  inducible  cellular  central  cores  (Chapter  (Chapter  these  central  addition  r e s u l t i n g i n an  bioactivity.  possibility, projections after that  that  of  1).  of 1).  In  surrounding It  was  cellular  projections  cores  carotovoricin  of  components  increase order  to  were examined by (Chapter  carotovoricin-379  increased  of  be to  1).  at  The  particles  In  weight  this cellular  several  stages  r e s u l t s showed gradually  i n s i z e , molecular weight, induction.  these  investigate  EM  and  formed  in molecular  c a r o t o v o r i c i n p a r t i c l e s and  induction  In  p a r t i c l e seemed  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  an  and  the  i n a m o d u l a r manner cores  of  elongation  entire carotovoricin  arranged  these c e n t r a l  the  10%)  surface  examination  projections  addition,  of  cells  (about  subunit-like  various  carotovoricin-379  producing  and  bioactivity  after  addition,  projections  were o b s e r v e d w h i c h r e s e m b l e d  cellular these  98  different  carotovoricin particles  An e x a m i n a t i o n 379 showed contained extended  that  weight  components these  observations,  (Chapter  hypothesized  at the l e v e l  projection  or non-induced  cells. production  was  as an i n d u c i b l e a d d i t i o n o f p r o t e i n  formed  and s w e l l e d  vesicle  In t h e n o n - i n d u c e d  o r head.  This  the c e n t r a l core of  carotovoricin-379  terminally in a state  a c t i v e c a r o t o v o r i c i n components  accumulated  An  Based  o f the outer  to a fimbrae-like p r o j e c t i o n .  partially  1).  was d e v e l o p e d i n  t h e model, c a r o t o v o r i c i n - 3 7 9  secreted  separated  a h y p o t h e t i c a l model f o r  production  membrane by i n d u c e d  subunits  component  The model o u t l i n e d t h e c o n s t r u c t i o n o f  carotovoricin-379  In  contained  of carotovoricin-379  1.  weight  f o r m s , whereas t h e low  component  carotovoricin-379 Chapter  molecular  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  and c o n t r a c t e d  molecular  on  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 -  the high  intact  (Chapter 1).  i n the p e r i p l a s m i c  s p a c e and were  i n membrane v e s i c l e s o r b l e b s .  interesting  carotovoricin-379 temperature  feature  of p a r t i c u l a t e  production  was t h a t  s e n s i t i v e (Chapter  2).  i t  is  Several  other  99  phenotypes, induction  including  and a n t i b i o t i c  temperature fact DNA  transfer (Chapter  bacteriocin  was  constituents studies  379  were  also  (Chapter  involvement  coded  production  2).  This  of plasmid  showed  2).  particulate  However,  a mobilization  conjugation  v e c t o r showed  a large molecular  weight  2).  and s i z e  weight  to the p a r t i a l l y  periplasmic  bacteriocin corresponded active  (Chapter  2)  of surface v e s i c l e s  or  membrane v e s i c l e s  enzyme  alkaline  phosphatase  the supernatant  of these  cells  The external  low  phosphatase  and t h e were  found  (Chapter 2 ) .  p r o d u c t i o n o f membrane v e s i c l e s alkaline  and t h e  of carotovoricin-379.  o f t r a n s c o n j u g a n t s by EM  In a d d i t i o n ,  plasmid  resistance  T h i s component  component  that  and coded f o r  and c h l o r a m p h e n i c o l  (Chapter  by  f o r by chromosomal  a proliferation  "blebs".  mediated  o f a low m o l e c u l a r w e i g h t  activity  Analysis  coli  that  self-transmissible,  erythromycin  molecular  t o E.  2) s u g g e s t e d  379 h a r b o u r e d  component  Ecc  from  (Chapter  without  w h i c h was  in  resistance,  i n Ecc  sensitive  following  i n the 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 - 3 7 9 .  R68.45  in  lysis  introduced the p o s s i b l e  Genetic  Ecc  cell  and  has been shown i n  100  several food to  pseudomonad s p e c i e s .  spoilage  increase  substrate recently  and  the  presence  secretion  (Thompson  bacteria,  Erwinia  bacteria  of p r o t e i n s system  transconjugants  an  production component  of  involved  in  membrane o f gram  Thus a  negative release  secretory  possibly  i n Erwinia  external  (Chapter  in proteins  supernatants  a d d i t i o n a l involvement  megaplasmid  be  enzymes and  the  phosphatase  transconjugant  most  i s necessary.  o f membrane v e s i c l e s i n E.  and  increase  Unlike  a wide v a r i e t y  1985).  components  formation  alkaline  1985)  i n enzyme  a s u b s t a n t i a l b a r r i e r to  f o r macerating  The  al.  outer  (Nikaido  carotovoricin  general  et  involved  some o f w h i c h may  provides  shown  These v e s i c l e s have  produces  The  low  carotovoricin  components  along  found  In  which corresponds the  2)  coli  of  with  a  in  (Chapter  molecular  morphology to  release  3)  introduces  Erwinia  of the  in secretion. o f a low  in  of a p a r t i c u l a r s o l i d  been shown t o be  pathogenicity.  involved  membrane v e s i c l e s have been  (Wing 1984).  exoenzymes,  Some a r e  addition,  the  weight b a c t e r i o c i n in a c t i v i t y  molecular  and  plaque  weight  isolated  i n Chapter  1  101  suggests that  carotovoricin-379  p r o d u c t i o n may  i n v o l v e both plasmid and chromosomal The low molecular weight  constituents.  carotovoricin-379  component was shown i n Chapter 1 to be  dependent  upon i n d u c t i o n and the i n c o r p o r a t i o n of magnesium i n media.  The model (Chapter 1)  explained  component as a prematurely r e l e a s e d , a c t i v e b a c t e r i o c i n component.  this  partially  The e f f e c t  of  magnesium presumably s t a b i l i z e d outer membrane v e s i c l e s i n h i b i t i n g t h i s premature r e l e a s e (Chapter 1).  The r e p r e s e n t a t i o n  i n the model assumed  that  the low molecular weight component was a subset of components  physically associated  particulate carotovoricin-379. came from the f a c t developed against  with  Support f o r  this  that p o l y c l o n a l antiserum, particulate  carotovoricin-379  r e a c t e d with the low molecular weight produced by CVP" transconjugants possible  intact  explanation for this  component  (Chapter 3).  cross  One  r e a c t i o n may be  that the plasmid encoded low molecular weight component could represent  a group of p r o t e i n s which  have some b a c t e r i o c i n a c t i v i t y but are r e q u i r e d f o r the assembly of p a r t i c u l a t e  carotovoricin-379.  This i s not a novel e x p l a n a t i o n as K88 fimbrae i n  102  E.  coli  have been  encoded  the  The  the  to  requires  of  al.  indicators  of  had  effects.  These two  metabolic  death.  detectable  action  Itoh  two  a  loss  is  used  et  al.  was an  This  lysis  energized  f o r ATP 1982).  activator  type was  Ecc  of of  379,  al.  by  direct lysis  d e a t h was state  earlier  particulate  The  inactive  and  of  (Itoh  mutants,  et be  shown t o  indicators assumed  but that  not  al. due  to  membrane w h i c h  nutrient  work by  and  cells,  shown t o the  an  (1980a) f o u n d  distinct  seen.  of  carotovoricin  uptake  (Itoh  Itoh's  group, i t  carotovoricin  contained  o f membrane bound p h o s p h o l i p a s e .  was  the  nature  carotovoricin  was  synthesis In  shown t h a t  activator  It  metabolic  results in  In p h o s p h o l i p a s e A minus  cell  i n the  et  the  e f f e c t s were c e l l  however, were m e t a b o l i c a l l y 1981).  on  component  mode o f  least  conponents  further  particulate at  fimbral  1984).  consideration.  binding  plasmid-  this polypeptide  speculate  the  a  for  fimbral  m o l e c u l a r weight  examination  no  of et  (Hammond  order  low  that  absence of  accumulation  periplasm In  require  o u t e r membrane p r o t e i n  assembly. in  shown t o  cause c e l l  lysis  in  in phospholipase  this phospholipase  This  wild-  mutants.  activation  103  was  the primary  However,  mode o f a c t i o n  the phospholipase  A mutants which  carotovoricin  although  intact  metabolically  inactive  (Itoh  In v i e w o f t h e complex carotovoricin, phospholipase  fact  spectra  activator  dependent  However, t h e p r i m a r y  state  central  (pmf)  structure of  observed  a  f o r by t h e Erwinia.  in  in a differing  activity  in indicator  strains.  mode o f a c t i o n o f  i s the i n a c t i v a t i o n  o f the energized  o f t h e membrane w h i c h may be due t o t h e  formation  result  e r a i . 1982).  upon t h e amount and t y p e o f  present  carotovoricin  still  may be coded  plasmid  could result  phospholipase  were  bound  i t i s quite conceivable that  self-transmissible This  of carotovoricin.  of non-specific  i o n channels  core of the p a r t i c l e . i n a degeneration  which r e s u l t s  from  regeneration  of this  allows  to couple  cells  tendencies  by t h e  This state  o f the proton motive metabolism.  chemical  and pH g r a d i e n t  thermodynamically  o f t h e pmf w o u l d  of metabolism.  T h i s type  force  The c o n s t a n t  t o ATP s y n t h e s i s and n u t r i e n t  Degeneration  would  result  of k i l l i n g  favourable uptake.  i n a cessation a c t i v i t y has  104  been empty  reported heads  using  (T4  mutant  ghost)  T4  (Lewin  phage 1977).  which  contain  105  SUMMARY  1)  Several  forms  differed  i n appearance,  bioactivity producing carotovora absence  2)  existed  cells  i n the supernatant of carotovora  379 (Ecc  subsp.  379) grown i n t h e  o f magnesium.  The l a r g e m o l e c u l a r w e i g h t  particulate  which  zone  when b i o a s s a y e d , r e s e m b l e d a  of inhibition tail,  gave  a small  form o f  carotovoricin-379,  itive  was t e m p e r a t u r e  clear  sens-  and was i n d u c i b l e by m i t o m y c i n C.  P r o d u c t i o n o f t h e low m o l e c u l a r w e i g h t carotovoricin-379, inhibition media  4)  which  m o l e c u l a r w e i g h t and  o f Erwinia  strain  bacteriophage  3)  of carotovoricin-379  cellular  a diffuse  zone o f  magnesium.  projections  on p r o d u c i n g c e l l s . represent  gave  when b i o a s s a y e d , was e l i m i n a t e d i n  containing  Several  which  form o f  different  These  were a l s o  observed  p r o j e c t i o n s may  stages o f induced  partic-  106  ulate  carotovoricin  like  p r o j e c t i o n which s w e l l s t o a  vesicular  5)  Ecc of  6)  Ecc  particulate  production lysis  erythromycin  chloramphenicol.  379 c o n t a i n e d  weight  a self-transmissible f o r erythromycin  o f c a r o t o v o r i c i n - 3 7 9 , and  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  The s m a l l m o l e c u l a r  weight  component  coded  serologically  and  r e s i s t a n c e , a low m o l e c u l a r  component  vesicle  8)  sensitive  i n d u c t i o n , and r e s i s t a n c e t o  chloramphenicol  cin  detachable  carotovoricin-379, c e l l  megaplasmid which coded  7)  fimbrae-  head.  379 showed t e m p e r a t u r e  after and  c o n s t r u c t i o n on a  exoproteins.  carotovoricin-379  f o r by t h e Erwinia  related  and c o n t a i n s  plasmid i s  to particulate bacterio-  protein subunits  found i n  particulate  bacteriocin.  Particulate  carotovoricin-379 production i s  coded  f o r by b o t h  chromosomal and p l a s m i d  107  determinants produced ization  i n E. vector  Carotovoricin a  as w i l d - t y p e coli  production  exoproteins.  were  a chromosome  (R68.45) was  plasmid-dependent  Erwinia  when  particles  only  mobil-  used.  may  secretory  be  linked  system  to  for  a l l  108  REFERENCES  B i r g e , E. Genetics.  1981. Bacterial Springer-Verlag,  and Bacteriophage New Y o r k . 359 pp.  B r o c k , T. 1979. Biology of Microorganisms. P r e n t i c e H a l l I n c . , Englewood C l i f f s , New J e r s e y . 802 pp. B r o d a , P. 1979. Plasmids. San F r a n c i s c o . 197 pp.  W.H.  Freeman and Co.,  C o l l m e r , A., S c h a e d e l , C , Roeder, D., R e i d , J . and R i s s l e r , J . 1985. M o l e c u l a r c l o n i n g i n E. coli o f Erwinia chrysanthemi genes e n c o d i n g m u l t i p l e forms o f p e c t a t e l y a s e . Journal of Bacteriology 161: 913-920. C o p l i n , D.L., Rowan, D.G., C h i s h o l m , D.A. and Whitmoyer, R.E. 1981. C h a r a c t e r i z a t i o n o f p l a s m i d s i n Erwinia stewartii. Applied and Environmental Microbiology 42: 599-604. C r o w l e y , C F . and De B o e r , S.H. 1980. S e n s i t i v i t y o f 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. R e l e a s e o f e n d o t o x i n i n t h e form o f c e l l w a l l b l e b s d u r i n g in vitro g r o w t h o f Neisseria meningitidis. Journal of Experimental Medicine 138: 1156-1167. Endo, Y., Tsuyama, H. and N a k a t a n i , F. 1975. S t u d i e s on t h e p r o d u c t i o n o f an a n t i b a c t e r i a l a g e n t 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: 4048. E c h a n d i , E. and Moyer, J.W. 1979. P r o d u c t i o n p r o p e r t i e s and m o r p h o l o g y o f b a c t e r i o c i n s from Erwinia chrysanthemi. Phytopathology 69: 12041207.  ,  109  F o r b e s , K . J . 1981. A Genetic Study of Erwinia carotovora. Doctoral Thesis, University of Edinburgh. 121 pp. G e r s h o n i , J.M. and P a l a d e , G.E. 1983. blotting: 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.  Protein Analytical  G l a s s , R.E. 1982. London. 487 pp.  Gene Function.  G l o v e r , D.M. 1985. Press, Washington.  DNA Cloning. 190 pp.  Volume  G l o v e r , D.M. 1985 Press, Washington.  DNA Cloning. 245 pp.  Volume  Croom Helm,  I.  IRL  II.  IRL  Haas, D. and H o l l o w a y , B. 1978. Chromosome mob i 1 i z a t i o n by R p l a s m i d R68.45: A tool i n Pseudomonas genetics. Molecular and General Genetics 158: 229-237. Hames, B.D. and H i g g i n s , S . J . 1984. and Translation. IRL P r e s s , W a s h i n g t o n .  Transcription 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 Blackwell S c i e n t i f i c Publications,  Virology. London. 167 pp.  Ingram, M. and D a i n t y , R.H. 1971. Changes c a u s e d by m i c r o b e s i n s p o i l a g e o f meats. Journal of Applied Bacteriology 34: 21. I t o h , Y., I z a k i , K. and T a k a h a s h i , 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 o f a b a c t e r i o c i n from Erwinia carotovora. Journal of General and Applied Microbiology 24: 27-39. I t o h , Y., I z a k i , K. and T a k a h a s h i , H. Simultaneous synthesis o f p e c t i n lyase c a r o t o v o r i c i n i n d u c e d by m i t o m y c i n C, a c i d , o r U.V. i r r a d i a t i o n i n Erwinia Agricultural and Biological Chemistry 1140.  1980. and nalidixic carotovora. 44: 1135-  110  I t o h , Y., I z a k i , K. and T a k a h a s h i , H. 1980. Mode o f a c t i o n o f a b a c t e r i o c i n from Erwinia carotovora I. Properties of l y s i s of c e l l s infected with c a r o t o v o r i c i n E r . Journal of General and Applied Microbiology 26: 51-62. I t o h , Y., I z a k i , K. and T a k a h a s h i , H. 1980. Mode o f a c t i o n o f a b a c t e r i o c i n from Erwinia carotovora II. Degradation o f phospholipids i n c a r o t o v o r i c i n Er-treated c e l l s . Journal of General and Applied Microbiology 26: 85-95. I t o h , Y., Iwata, T., I z a k i , K. and T a k a h a s h i , H. 1981. Mode o f a c t i o n o f a b a c t e r i o c i n from Erwinia carotovora I I I . Properties of phospholipase A of Erwinia carotovora and i n v o l v e m e n t i n p h o s p h o l i p i d d e g e n e r a t i o n c a u s e d by c a r o t o v o r i c i n . Journal of General and Applied Microbiology 27: 239-251. I t o h , Y., Iwata, T., I z a k i , K. and T a k a h a s h i , H. 1982. Mode o f a c t i o n o f a b a c t e r i o c i n from Erwinia carotovora IV. E f f e c t s on m a c r o m o l e c u l a r s y n t h e s i s , ATP l e v e l , and n u t r i e n t transport. Journal of General and Applied Microbiology 28: 9599. J a i s , H. carotovora. Columbia.  1982. Bacteriocins Masters Thesis.  of Erwinia University of B r i t i s h  Kamimiya, S., I z a k i , K. and T a k a h a s h i , H. 1977. B a c t e r i o c i n s o f Erwinia ariodea with t a i l - l i k e structure of bacteriophages. Agricultural and Biological Chemistry 41: 911-912. K e l l e n b e r g e r , E. and R y t e r , A. 1958. C e l l w a l l and c y t o p l a s m i c membrane o f E. coli. Journal of Biophysical and Biochemical Cytology 4: 323. K o l o u j a n s k y , A., D i d e z , A. and B a c c a r a , M. 1985. M o l e c u l a r c l o n i n g o f Erwinia chrysanthemi pectinase 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. C l e a v a g e o f s t r u c t u r a l p r o t e i n s d u r i n g a s s e m b l y o f t h e head o f T4. 227: 680-685.  Nature  Ill  Laemmli, U.K. and F a u r e , M. 1973. M a t u r a t i o n o f t h e head o f b a c t e r i o p h a g e T4. Journal of Molecular Biology 80: 575-599. Lewin, B. 1977. Sons, New Y o r k . Maniatis, Molecular New Y o r k ,  Gene Expression.  John W i l e y and  T., F r i t s c h , E . F . and Sambrook, J . 1982. Cloning. C o l d S p r i n g Harbour L a b o r a t o r y . 545 pp.  M a y r - H a r t i n g , A., Hedges, A . J . and B e r k e l e y , 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. R i b b o n s , e d s . , 7A: 315-422. N i k a i d o , H. and V a a r a , M. 1985. M o l e c u l a r o f b a c t e r i a l o u t e r 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 bacteriocins. Annual Review of Microbiology 257-284.  basis  21:  Ohsumi, M., S h i n o m i y a , T. and Kageyama, M. 1980. C o m p a r a t i v e s t u d y on R - t y p e p y o c i n s o f Pseudomonas aeruginosa. Journal of Biochemistry 87: 1119-1126. Perbal, Cloning.  B. 1984. A Practical Guide to John W i l e y and Sons, New Y o r k .  Molecular 554 pp.  P u g s l e y , A. and S c h w a r t z , M. 1984. C o l i c i n E2 release: l y s i s , leakage, or s e c r e t i o n ? P o s s i b l e role of a phospholipase. The EMBO Journal 3: 23932397. P u h l e r , A. and R i e s s , G. i n P u h l e r 1984. Advanced Molecular Genetics. Verlag, B e r l i n . 347 pp. R a n d a l l , L. and Hardy, S. in bacteria. Microbiological  and Timmis. Springer—  1984. E x p o r t o f p r o t e i n Reviews 48: 290-298.  S c h a a d , 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 , S t . P a u l , M i n n e s o t a , 72 pp.  112  S h i n o m i y a , T., Kageyama, W., A i h a r a , Y. and K o b a y a s h i , M. 1979. C h a r a c t e r i z a t i o n o f a b a c t e r i o p h a g e r e l a t e d t o R-type p y o c i n s . Journal of Virology 32: 951-957. S h i n o m i y a , T. and S h i g a , S. 1979. B a c t e r i o c i d a l a c t i v i t y o f t h e t a i l o f Pseudomonas aeruginosa b a c t e r i o p h a g e PS-17. Journal of Virology 32: 958967. S p a r k s , R.B. and L a c y , 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 o f c r y p t i c p l a s m i d s p L S l and pLS2 f r o m Erwinia chrysanthemi. Phytopathology 70:369-372. Thompson, S., N a i d u , Y. and P e s t k a , 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 o f an e x t r a c e l l u l a r p r o t e a s e i n Pseudomonas fragi u s i n g t h e PAP reaction. 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 o f p r o t e i n s from polyacrylamide gels to n i t r o c e l l u l o s e sheets. P r o c e d u r e 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: 43504354. Tsuyumu, S. and C h a t t e r j e e , A.K. 1984. l y a s e p r o d u c t i o n i n Erwinia chrysanthemi s o f t - r o t Erwinia species. Physiological Pathology 24: 291-302.  Pectinand o t h e r Plant  V i d a v e r , A.K. 1976. P r o s p e c t s f o r c o n t r o l o f p h y t o p a t h o g e n i c b a c t e r i a by b a c t e r i o p h a g e and bacteriocins. Annual Review of Phytopathology 14: 451-465. W i l l e t t s , N.S. and C r o w t h e r , C. 1981. The i n s e r t i o n s e q u e n c e IS21 o f R68.45 and t h e m o l e c u l a r b a s i s f o r m o b i l i z a t i o n o f t h e 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 c h e m i c a l p r o p e r t i e s o f 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. Doctoral Thesis. University of B r i t i s h Columbia.  Z i n k , R.T. , Kemble, R . J . and C h a t t e r j e e , A.K. 1984. T r a n s p o s o n Tn5 m u t a g e n e s i s i n Erwinia carotovora s u b s p . carotovora and Erwinia carotovora subsp. atroseptica. Journal of Bacteriology 157: 809-814. Z i n k , R. and C h a t t e r j e e , A.K. 1985. C l o n i n g and e x p r e s s i o n i n E. coli o f p e c t i n a s e genes o f Erwinia carotovora s u b s p . carotovora. Applied and Environmental Microbiology 49: 714-717.  )  

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