Bacteriocins Nature, Function and Structure

8/18/2019 Bacteriocins Nature, Function and Structure

1/13

~ ) Pergamon

PII: S0968-4328 96)00028-5

Micron

Vol, 27, No. 6, pp. 467 479. 1996

I 1997 Published by Elsevier Science Lid

All rights reserved. Printed in Great Britain

0968 4328/96 S32.00+0,00

Bacterioc ins: N atur e F un ct ion and Structure

MOHAMED A. DAW* and FREDRICK R. FALKINER+

*D epa r tm en t q f M ed ic a l M icrob io logy , Facu l O' oJ M ed ic ine , A ( fa teh Un i t 'e r s i t v ~[ Med ic a l Sc iences , P .O. Bo .v ,~;2668, Tr ipo l i.

L ib ) 'a

+ D e p a r t me n t o f C l in i c a / Mi c r o b i o l o g y , Du b l h l U n i v e r si t y , Du b li n, R e p u b l i c q f h e h m d

b s t r a c t Bacteriocins are extracellular substances produced by different types of bacteria, including both Gram positive and

Gr am negat ive species. They can be produc ed sp onta neou sly or induced by certain chemicals such as mitomy cin C. They arc

biologically one of the importa nt substances, and have been found to be useful in membrane studies and also in typing pathogenic

microorga nisms causing serious nosocomial infections. Bacteriocins are a heterogeneous group o f particles with different

morphological and biochemical entities. They range from a simple protein to a high molecular weight complex: the active moiety

of each molecule in all cases seems to be protein in nature. The genetic determinant s of most of the bacteriocins are located on the

plasmids, ap art from few which are chrom osom al[ y encoded. The se bactericidal particles are species specific. They exert their lethal

activity through adsorbtion to specific receptors located on the external surface of sensitive bacteria, followed by metabolic,

biological and morphological changes resulting in the killing of such bacteria. This review summarises the classification,

biochemical nature, mor pholog y and mode of action o f bacleriocins as well as their genetic determinants and the microbiological

relevance of these bactericidal agents, i , 1997 Publishe d by Elsevier Science Ltd

Key words: Bacteriocins. bacte rophag e, bacterium-l ike inhibit ory substances, relaxed forms, contracte d forms, pyocins, cloacins.

bacterium typing.

CONTENTS

1. In tr oduc ti on ........................................................................................................................................................................................................... 467

A. Bac kgr ound ..................................................................................................................................................................................................... 467

B. Classi ficati on of bacter iocins ......................................................................................................................................................................... 468

C. Biochemi cal na tu re of bac terioci ns ............................................................................................................................................................... 468

D. Bac teriocinogeny and lysogeny .................................................................................................................................................................... 469

II. Ultrastructural study of bac terioci ns ................................................................................................................................................................ 470

A. General structure ...........................................................................................................................................................................................70

B. Morpho lo gy of active fo rms ....................................................................................................................................................................... 471

C. Mod e of action ................................................................................................................................................................................................ 472

D. Morpho log ica l changes associat ed with bacteriocin activity .................................................................................................................... 472

I11. Molecular bio logy of bacteri oci ns ...................................................................................................................................................................... 475

A. Genetic

determinants

of bac terioci ns ........................................................................................................................................................... 475

B. Mic robiol ogical relevance of bac terio cins ................................................................................................................................................... 476

IV. Con cl usions .......................................................................................................................................................................................................... 476

Acknowledgements .............................................................................................................................................................................................. 477

References .............................................................................................................................................................................................................. 477

I. INTRODUCTION

A . B a c k g r o u n d

Bacteriocins are bactericidal, antibiotic-like sub-

stances, apparently protein in nature, which are pro-

duced by many bacteria and have a killing action on

strains of the same or closely related species. The narrow

specificity of their action and their protein nature distin-

guish them from other classical) antibiotics Reeves,

1965; Daw, 1989: Grat ia and Grenier, 1992; Laukova

and Marekova, 1993). The first discovery was reported

by Gratia in 1925 of a highly specific antib iotic principe

V) produced by a strain of

E s c h e r i c h i a c o l i

and active

against other strains of the same species. This activity

was found to be produced by various species of Entero-

bacteriaceae and for which the generic name colicine)

was proposed. With the discovery that the production of

apparently similar agents is not limited to Enterobacteri-

aceae, Jacob

et al .

1952) proposed that the general

name bacteriocine) should be used for highly specific

antibacterial proteins, produced by certain strains of

bacteria and active mainly against strains of the same

species. This defini tion still holds, although colicin e)

and bacteriocin e) are now spelt with out the final e.

Since the discovery of the bacteriocins, most of the

studies established in this field have been descriptive

Fredericq, 1957, 1963), and the basic methods of detec-

tions, assay and the bacteriocin typing of bacterial

strains have been established. Genetic studies of coli-

cinogeny and its transfer from one cell to another were

also pionered by Fredericq and his collaborators. Fur-

ther studies on the molecular biology of the bacteriocins

followed, with emphases on biosynthesis, liberation and

the mode of action of these agents Davies and Reeves,

1975a,b; Geli and Lazdunski, 1992a,b; Lakey e t a l .

1993). These led to interesting use of the bacteriocins as

a biochemical tools in cellular physiology Libertin

e t a l . 1992; Farkas-Himsley e t a l . 1992; Becker e t a l .

1993). Bacteriocins have since gained new attention,

467


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468 M.A. Daw and F. R. Falkiner

particularly in the epidemiology of nosocomial infec-

tions; they have been found to be very useful in typing

organisms particularly those which are difficult to type

by the usual methods (Pitt, 1980; Daw et al . , 1992;

Siragusa, 1992; Unlman et a l . , 1992; Lebek et a l . , 1993).

Our knowledge on bacteriocins leans heavily on re-

sults obtained by electron microscope studies describing

the morphology and the killing process of the bacteri-

ocin particles. Due to the vast expansion of research in

this area, we will devote most of this review to the

classification, biochemical nature and mode of action of

bacteriocins as well as the molecular biology and the

microbiological relevance of these bactericidal agents.

B. C lass i f ica t ion o f bac ter ioc ins

The classification, and thus the nomenclature of

bacteriocins, has changed rapidly since early this cen-

tury. The main classification was done on colicins; they

are usually classified by the scheme devised by Fredericq

(1957), according to the specificity of their adsorption

and are further classified into subgroups according to

the specificity of their immunity. For example, colicin

El, E2 and E3 differ from each other in their immunity

pattern, but are classified as E group, because all of

them apparently adsorb to the same receptor. Similarly,

colicin Ia and Ib apparently share a common receptor

and belong to group I, but can be distinguished from

each other by their immunity specificity. Many bacterio-

cins have been studied in detail and they necessarily have

to be subdivided according to their spectrum of resist-

ance (Reeves, 1965). As a result o f this, a code of

nomenclature was established. The number of producer

strain is followed by original letter, for instance colicin

CA23 D is a type D colicin produced by

E. coli

strain

CA 23 (Fredericq, 1963; Ohno

et al . ,

1977).

Reeves (1965) listed sixteen classes of bacteriocins

named on the basis of the species that produce them.

Due, perhaps, to the high degree of specificity of bac-

teriocins, the name is the almost always based on the

specific rather than generic name of the host organism.

Therefore colicins are bacteriocins of E. coli, pyocin of

Pseudomonas aeruginosa (formerly p y o c y n ia , cloacin of

Enterobac ter c loacae ,

pestisin of

Yers in ia pes t i s ,

monocin

of Lis te r ia monocy togenes , cerecin of Bacillus cereus, and

staphylococcin of Staphy lococcus . These vernacular

names used for bacteriocins are based, not on the spec-

trum activity, but the producing organism. The fact that

they are produced by specific species of bacteria means

that their host association is immediately apparent.

Bradely (1967) designed a taxonomic criterion for the

classification of the bacteriocins, based on the natural

division of these agents. The bacteriocins were divided

into two distinct groups designated as low and high

molecular weight forms. The first is a small molecule

which is usually thermostable, cannot be sedimented

in the ultracentrifugation and cannot be resolved by

the electron microscope. The second group is a larger

molecule, easily sedimented, thermolabile, trypsin resist-

ant and can be resolved by the electron microscope. This

classification allows us to include within it bactericidal

particles such as those produced by the Bac i l lus species,

since they are unable to multiply within sensitive cells.

The only morphological difference between them and

the bacteriocin of high molecular weight is that they are

closer to phage particles (Bradley, 1966). The term

bacteriocin usually refers to the colicin-type of protein,

generally restricted to the strains of Enterobacteriaceae.

Most of the bacteriocins produced by Gram positive

bacteria do not fit into the classical definition of bac-

teriocins in that they are (1) more broadly active against

Gram positive species, (2) their action is mediated by

specific receptors and (3) their release is enhanced by

lysins. Furthermore, the absence of the outer membrane

in Gram positive bacteria and the difference in the level

of immunity of the producing strain to its own bacterio-

cins, indicates that those bacteriocins of Gram positive

bacteria should be grouped differently (Tagg

et al . ,

1976;

Strasser de-Saad and Hanea de-Nadra, 1993; Havarstein

et al . ,

1994). Tagg (1992) suggested that the term bac-

teriocin should be redefined either to take into account

the non-colicin-like characteristics of many of the more

recently described particles, or the original definition

should be retained and those particles which are broadly

similar to the colicins should be referred to as bacteriocin-

like inhibitory substances (BLIS). The prevalence of BLIS

production among Gram positive bacteria was found to

be high. A survey conducted in the 1970s concluded that

all the tested strains representing most of the Gram posi-

tive bacteria could be found to produce BLIS (Tagg

et al. ,

1976; Tagg, 1992) and the incidence may closely approach

100 .

Electron microscopic studies have been particularly

useful for the identification and classification of bac-

teriocins. Negative contrast methods, which have been

described for the purpose of studying viral structure,

were equally employed for bacteriocins (Bradely, 1966;

Govan , 1974a,b; Daw, 1989) . Uranyl acetate and

potassium phosphotungstate have usually been used as

negative stains. These were mixed with equal volumes

of bacteriocin preparations and allowed to dry onto

electron microscope specimen grids covered with carbon

film, then studied using electron microscopy. These

ultrastructure studies revealed that only the large

bacteriocin particles were visible by electron microscopy.

The mode of action and the killing process of these

bacteriocins were also studied by the negative staining

(Daw, 1989; Daw and Falkiner, 1993).

C. Biochemical na ture o f bac ter ioc ins

Bacteriocins have been erroneously considered as the

only proteins secreted by bacteria. Indeed, those intially

described were isolated and identified from the extra-

cellular state of the producing species. Nowadays, other

extracellular products have also been found. Freely

secreted bacteriocins constitute a peculiar group of

exported proteins in that their export relies on the


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Bacteriocins 469

e x p r e s s i o n o f o n l y o n e g e n e c o d i n g f o r a b a c t e r i o c i n

r e l e a se p r o t e i n ( B R P ) ; a l so c a l l e d t h e l y s is o r k i ll p r o t e i n .

I n c o m p a r i s o n , e x p o r t o f o t h e r s e c r e t e d p r o t e i n s r el ie s

o n t h e a c t i v i ty o f a t l e a s t t h r e e p r o t e i n s ( W a n d e r s m a n

a n d D e l e p e l i a r e , 1 9 9 0 ; H a v a r s t e i n e t a l . 1 9 9 4 ) . H o w -

e v e r , t h e y a r e e x p o r t e d l a t e a f t e r s y n t h e s i s a n d a c c u m u -

l a t e i n t h e c y t o p l a s m i n a s o l u b l e f o r m b e f o r e t h e y a r e

r e l e a s e d . T h e c o l i c i n l y s i s p r o t e i n s h a v e b e e n t h e m o s t

e x t e n s i v e ly s t u d i e d a n d , t o g e t h e r w i t h t h e B R P e n c o d e d

b y t h e C o l D F 1 3 p l a s m i d , t h e y s h a r e m a n y p r o p e r t i e s

i n c l u d i n g t h e i r g e n e t i c o r g a n i s a t i o n , t r a n s c r i p t i o n , s y n -

t h e s i s , a s s e m b l y a n d f u n c t i o n ( H o w a r d s e t a l . 1989;

C a v a r d a n d O u d e g a , 1 9 9 2 ) .

T h e p r o t e i n n a t u r e o f b a c t er i o c in s w a s d e t e r m i n e d

e a r ly b y t h e d e g r a d a t i o n o f c r u d e p r e p a r a t io n s b y p r o -

t e o ly t ic e n z y m e s a n d t h e ir a p p r o x i m a t e m o l e c u l a r

w e i g h t w a s d e t e r m i n e d b y t h e d i f f u s i b i li t y o f t h e l y sa t e s

t h r o u g h a g a r o r s e m i p e rm e a b l e m e m b r a n e s . S u c h

m e t h o d s w e r e f o u n d t o b e u s e f u l i n t h e i n i t i a l c h a r -

a c t e r i s a t io n o f b a c t e r i o c i n s ( L o y o l a - R o d r i g u e z

e t a l .

1 9 9 2 ) . D u e t o m o r e a d v a n c e d p u r i f i c a t i o n , s e v e r a l b a c -

t e r i o c i n s h a v e b e e n d e s c r i b e d a n d t h e i r c h e m i c a l

a n d p h y s i c a l p r o p e r t i e s a r e k n o w n ( S t i l e s e t a l . 1994;

H a s t i n g s e t a l . 1995).

D i f f e r e n t s tu d i e s h a v e s h o w n t h a t t h e b a c t e r i o c i n s a r e

p r o t e i n m o l e c u l e s w i th t r a c e s o f c a r b o h y d r a t e s ( l e ss t h a n

1 ) a n d p h o s p h o r u s ( le s s t h a n 0 . 1 ) ( K i n g s b u r y , 1 96 6;

R e e v e s , 1 9 7 2; D a w a n d F a l k i n e r , 1 9 93 ). T h e n o t a b l e

d i f fe r e n c e s b e t w e e n t h e m w e r e f o u n d t o b e a s s o c i a t ed

w i t h a m i n o a c i d c o m p o s i t i o n . D i f f e r e n t s e q u e n c e s o r

c r os s c o m p o s i t i o n o f a m i n o a c i ds w e r e fo u n d a m o n g t h e

b a c t e r i o c i n m o l e c u l e s v a r y i n g f r o m o n e b a c t e r i o c i n t o

a n o t h e r , e v e n a m o n g t h e s a m e g r o u p o f b a ct e r io c i n

( K o n i s k y a n d R i c h a r d s , 1 97 0; H o w a r d e t a L 1989:

N i e t o - L a z a n o

e t a l .

1 9 92 ; K o e b i n k a n d B r a u n , 1 9 93 ).

I n d e e d c o l i c i n E i s n o w d i v i d e d i n t o t h r e e su b c l a s se s

u s i n g S D S g e l e l e c t r o p h o r e s i s : c o l i c i n E l , E 2 a n d

E 3 . T h e m a i n d i f f e r e n c e s b e t w e e n t h e se su b c l a s se s i s

a s s o c i a t e d w i t h a m i n o a c i d t e r m i n a l s e q u e n c e .

T h e e a r l y d e t a i l e d w o r k o n t h e b i o c h e m i c a l n a t u r e o f

b a c t e r i o c i n s w a s d o n e o n c o l i c in s a n d h a s b e e n d i s c u ss e d

b y s e v e r a l i n v e s t i g a t o r s ( I v a n o v i c s , 1 9 6 2 ; K o n i sk y , 1 9 7 3 ;

L a k e y e t a l . 1 9 9 2 : B r a u n e t a l . 1 9 9 4 ) . T w o d i f f e r e n t

p u r i f i e d c o l i c i n s w e r e o b t a i n e d ; o n e i s a l i p o p o l y sa c -

c h a r i d e p r o t e i n c o m p l e x a s s o c i a t e d w i t h t h e O s o m a t i c

a n t i g e n o f t h e p r o d u c i n g E . c o l i s u c h a s c o l i ci n K - K 2 3 5 ,

a n d f o l l o w i n g i t s d i s a s so c i a t i o n t h e a c t i v i t y w a s a s so c i -

a t e d w i t h t h e c a r b o x y a l m o i e t y ( C H O ) ( V i e j o e t a l .

1 9 9 1 ; M i r a n d a

e t a L

1 9 93 ). T h e o t h e r g r o u p o f c o li c i n s

w a s f o u n d t o b e a p r o t e i n c o n t a i n i n g l i t t l e o r n o c a r b o -

h y d r a t e , s u c h a s c o l i c i n E 2 - P 9 , w h i c h w a s f o u n d t o b e a

p r o t e in w i t h a m o l e c u l a r m a s s o f a b o u t 6 0 k D a . O t h e r

r e l a t e d c o l i c i n s , E 2 - C A 4 2 a n d c o l i c i n A , w e r e a l so

p u r i f ie d a n d s h o w n t o b e a p r o t e i n w i t h l o w c a r b o -

h y d r a t e c o n t e n t ( a b o u t 1 0 ) . T h e s e t w o w e r e d i f f e re n t

f r o m t h e c o l i c i n K - K 2 3 5 ( G o o r m a g h t i g h

e t a l .

1991;

G o n z a l e z - M a n a s e t a l . 1992).

T h e b i o c h e m i ca l n a t u r e o f o t h e r b a c t e r io c i n g r o u p s

h a v e a ls o b e e n d e t e r m i n ed : t y p e - R P y o c i n s p r o d u c e d b y

P . a e r u g i n o s a w e r e p u r i f i e d b y K a g e y a m a a n d E g a m i

Fig. 1. Transmission electron micrograph of a bacteriocin

part ic les obtained from Enterobac te r c loacae negativelly

stained w ith sod ium silicotungstate 2 '7,, w/v. The bacteriocins at

field (A) are seen as bu llet-like particles possessing a base plate.

A flagellum (F) is al so see n across the field. Scale marker

indicates 0.1 nm.

( 1 9 6 2) a n d s h o w n t o b e p r o t e i n p a r t ic l e s t Ye e o f c a r b o -

h y d r a t e a n d n u c l ei c a c id . T h e s y n t h e s i s o f t h is p y o c i n i s

i n d u c i b l e a n d a c c o m p a n i e d b y c e l l l y s i s . A n o t h e r g r o u p

o f p y o c i n s , a l so c a l l e d S p y o c i n s , w a s p u r i f i e d f r o m

m i t o m y c i n C - i n d u c e d l y s a t e o f P . a e r u g i n o s a . T h e s e

w e r e d i s t i n g u i s h e d f r o m t h e R t y p e p y o c i n s b y t h e i r

d i f f u s i o n r a t e t h r o u g h a g a r , t h e i r s e n s i t i v i t y t o p r o t e a se

d i g e s t i o n a n d b y t h e f a c t t h a t t h e y a r e n o t n e u t r a l i s e d

w i t h a n t i s e r u m a g a i n s t a n y t y p e R p y o c i n s ( S a n o

e t a l .

1 9 9 3 ) . C l o a c i n C 5 , o n t h e o t h e r h a n d , i s a m a c r o m o l -

e c u l e s i m i l a r t o R p y o c i n ( D a w , 1 9 8 9 ) . A l l b a c t e r i o c i n s

a p p e a r t o r e p r e s e n t a h e t e r o g e n e o u s g r o u p o f s u b s ta n c e s

r a n g i n g f r o m a s m a l l p r o t e i n t o a h i g h m o l e c u l a r w e i g h t

p a r t ic l e w i th c o m p l e x s t r u c t u r e a n d c o m p o s i t i o n , b u t t h e

p a r t r e sp o n s i b l e f o r k i l li n g a c t i v i ty s e e m s t o b e p r o t e i n i n

e v e r y c a se .

D . B a c t e r i o c i n o g e n y a n d ly s o g e n y

T h e a n a l o g y b e t w e e n b a c t e r i o c i n p r o d u c t i o n a n d

b a c t e r i o p h a g e l i b e r a t i o n w a s n o t e d f o l l o w i n g th e d i s c o v -

e r y o f b a c t e r io c i n s , l n t i a l s t u d ie s c o n d u c t e d b y G r a t i a

( 1 9 2 5 ) e s t a b l ish e d t h e d i f f e r e n c e s b e t w e e n c o l i c i n s a n d

b a c t e r i o p h a g e s , n o t a b l y t h e a b s e n c e o f b a c t e r i o p h a g e -

l i k e m u l t i p l i c a t i o n o f c o l i c in s . L a t e r i n v e s t i g a t i o n r e -

e s t a b l i s h e d c e r t a i n c o n n e c t i o n s , p a r t i c u l a r l y b e t w e e n

t h o s e b a c t e r i o p h a g e s a n d c o l i c i n s t h a t a t t a c h t o t h e

sa m e r e c e p t o r s ( A l a t o s s a v a , 1 9 94 ). U l t r a s t r u c t u r a l

s t u d i e s s h o w e d a c e r t a i n a n a l o g y b e t w e e n t h e s e t w o

b a c t e r i c i d a l a g e n t s ( D a w , 1 9 89 ; D a w a n d F a l k i n e r ,

1 9 9 3 ) . B a c t e r i o c i n s w e r e c o n s i d e r e d t o b e i n c o m p l e t e

p h a g e s . T h i s w a s e v i d e n t i n su c h s t u d i e s , a s sh o w n i n

F i g s 1 a n d 2 . A r e l a x e d f o r m o f a b a c t e r i o c i n i so l a t e d

f r o m E . c l o a c a e i s sh o w n ( F i g . 1 ) a n d a b a c t e r i o p h a g e

i so l a t e d f r o m t h e s a m e s t r a i n ( F i g . 2 ) . T h e p h a g e i s

c o m p o s e d o f p o l y h e d r a l h e a d a n d t ai l w i t h c o n t r a c t i l e

sh e a t h a n d c o r e , a s w e l l a s t a i l f i b r e s ( D a w . 1 9 8 8 ) . B y

c o m p a r i s i o m t h e s e t w o f i g u r e s s h o w a c o n s i d e r a b l e

r e se m b l a n c e o f p h a g e t a i ls t o t h e b a c t e r i o c i n p a r t i c l e s .


4/13

4 7 0 M . A . D a w a n d F . R . F a l k i n e r

F i g . 2 . A m i c r o g r a p h o f b a c t e r i o p h a g e i s o l a te d f r o m t h e s a m e

s t r a i n o f Enterobacter cloacae f o r m e d f ro m h e a d H ) , p h a g e

ta i l T) a nd t a i l f ib re s TF) . The pha g e t a il Y) r e se m ble s the

ba c te r ioc in pa r t i c le s A) . Sc a le m a rke r ind ic a te s 0 .1 nm .

P h a g e P S 3

o

160 70 150

I n t a c t S h r u n k I n t a c t

P y o c i n R 4

180

7 0

S h r u n k

F ig . 3 . M o r p h o l o g i c a l c o m p a r i s o n o f p h a g e P S 3 a n d R - t y p e

p y o c i n o f Pseudomonas aeruginosa. The pha ge t a i l i s s l igh t ly

l a r g e r t h a n t h e b a c t e r i o c i n p a r t i c l e . D i m e n s i o n s a r e s h o w n

in A .

The bacteriocin seems to be headless phage. This empha-

sised the striking structural resemblance between bacte-

riocin and bacteriophage tail. Indeed, one of the earliest

technical problems was to make the distinction between

these two structures. This was further complicated if

these two anti-bacterial agents were produced by the

same bacterial strain. Both agents are adsorbed by

specific receptors of the cell wall and kill the sensitive

bacteria. In some cases receptors are common for certain

bacteriocins and phages, such as colicin K and phage T6

or colicin E and phage BF23 Ito e t a l . 1970). Both

bacteriocinogenic bacteria and lysogenic bacteria are

immune to the agents they produce. Bacteriocinogeny

and lysogeny are both potential characters and the

production of bacteriocin and bacteriophage is often

enhanced by some treatments, e.g. UV irridiation.

Each phenomenon, however, is a lethal process for the

bacterium. Studies on pyocins found that some had

structures very similar to phage tails, and they were

categorised as R type pyocins Ito and Kageyama, 1970;

Govan, 1974a,b). Morphologically, the tail of the phage

PS3 is similar but not identical to R type pyocins; the

phage tail is slightly bigger. The dimension of the phage

is schematically illustrated in Fig. 3. The tail of the

phage closely resembles R type, except that the size of

the tail is a little longer than that of the pyocins. The

fact that antiserum against R pyocins can neutralize

the phage activity to a considerable extent, suggests

that there are some common or very similar antigenic

component s) in their structure.

The similarity between prophage induction and of

bacteriocin synthesis suggests that a similar mechanism

is responsible for both events. Many treatments which

inhibit DNA synthesis bring about prophage induction

and also induce the synthesis of bacteriocins Hardy and

Meynell, 1972a,b; Issacson and Konisky, 1974: Hardy,

1975; Boemare e t a l . 1992). Unlike prophage induction,

induction of bacteriocin synthesis is not necessarily

accompanied by enhanced replication of the Col factor.

In P r o t e u s m i r a b i l i s the increase in ColE1-K30 after

mitomycin C treatment parallels the increase in bacterio-

cin titer, but in E . c o l i the extensive replication is not a

necessary condition for increased titers of colicins after

induction Durkacz and Sherratt, 1973; Schiess and

Goebel, 1974).

The initial studies Ito and Kageyama, 1970) estab-

lished the important differences between bacteriocins

and bacteriophages as the latter is self reproducible in

the sensitive bacteria, while the former is not. Some

studies Daw, 1989) indicated that bacteriophages, on

dilution, would show a decreasing number of discrete

phage plaques; a bacteriocin on the other hand would

show a diffuse thinning of growth becoming less marked

with increasing dilution of the supernatant. When a

culture with a high percentage of lacuna-forming cells

LFC) is diluted and maintained at a constant density,

the number of LFC remains constant for several gener-

ations. Cells releasing bacteriocin exist for several hours,

but they do not divide to produce LFC, while spon-

taneous induction of prophages varies similarly in batch

cultures. Although bacteriocins and bacteriophages lend

themselves to obvious comparisons, there is no indi-

cation for the existence of any direct relationship

between the two antibacterial agents, such as a colicin

being the product of incomplete phage development. In

particular, no case has as yet been found of antigenic

similarities between bacteriocins and bacteriophages.

Even when the same bacterial strain is lysogenic and

bacteriocinogenic, no connection between bacteriocin

and bacteriophage can be demonstrated. Neither has

any instance of conversion from the lysogenic to the

bacteriocinogenic state been observed. One must, there-

fore, conclude that in spite of many remarkable simi-

larities between lysogeny and bacteriocinogeny, there is

no compelling reason to believe that they are in any way

connected.

II. ULTRASTRUCTURAL STUDY OF

B C T E R I O C I N S

A . G e n e r a l s t r u c t u re

Little work has been done on the physical structure

of bacteriocins. Early electron microscope studies of

clocins ML, E, V and K did not produce any definitive

results concerning their morphology Konisky, 1982).


5/13

Ba c te r ioc ins 471

C o l i c i n E 3

N - t e r m i n u s C - t e r m i n u s

3 5 k D a 1 5 k D a

T r a n s l o c a t i o n c a p a c i t y R N a s e a c t i v i t y

i n t o t h e c e l l b i n d i n g o f I P

F ig . 4 . Co l ic in E 3 o f

Esherichi~l coli

u n d e r p r o t e a s e d i g e s t i o n

y i e l d s t w o f r a g m e n t s : 5 3 k D a a m i n o t e r m i n a l f r a g m e n t a n d a

1 5 k D a c a r b o x y t e r m i n a l f r a g m e n t .

F u r t h e r s t u d ie s s u g g e s t e d t h a t b a c t e r i o c i n s c a n f it

i n t o t w o g r o u p s : t h o s e t h a t c a n n o t b e r e s o l v e d b y t h e

e l e c t r o n m i c r o s c o p e s u c h a s E , V , K a n d p y o c i n S a n d

o t h e r s t h a t h a v e a d e f i n i t e s t r u c t u r e , su c h a s c l o a c i n C 5

a n d R p y o c i n . C o l i c i n E 3 r e p r e s e n t s a n e x a m p l e o f t h e

f i rs t g r o u p . P r o t e o l y t i c d i g e s t i o n o f c o l ic i n E 3 w i t h

t r y p s i n u n d e r m i l d c o n d i t i o n s y i e l d s t w o f r a g m e n t s , a

3 5 k D a a m i n o - t e r m i n a l f ra g m e n t a n d a 1 5 k D a c a r b o x y -

t e r m i n a l f r a g m e n t i n c o m p l e x w i th t h e i m m u n i t y p r o t e i n

a s s h o w n i n F i g . 4 , w h i c h s u g g e s t e d a d o m a i n s t r u c t u r e

f o r c o li c in E 3 ( O h n o et al . 1 9 7 7 ; S o h a m a n d D j e b l i ,

1992; Ya j ima et al . 1992).

M o s t o f th e s t u di es c o n c e r n i n g t h e m o r p h o l o g y

o f b a c t e r io c i n s w e r e p e r f o r m e d o n p a r t ic l e s o b t a i n e d

f r o m p y o c i n o g e n i c st ra i n s o f P. aeruginosa. E l e c t r o n

m i c r o s c o p y o f p y o c i n p r e p a r a t i o n r e v e a l e d t h e p r e s e n c e

o f n u m e r o u s s t r u c t u r e s r e s e m b l i n g t h e p h a g e t a il c o m -

p o n e n t s a s s h o w n i n F i g . 5 . C o n t r a c t e d p a r t i c l e s ( F i g .

5 a ) c o n s i s t o f a c o r e p a r t ia l l y s u r r o u n d e d b y a s h e a t h . I n

m o s t p a r t i c l e s t h e c o r e a p p e a r s e m p t y , b u t o c c a s i o n a l l y

c o r e s c a n b e s e e n t o c o n t a i n m a t e r ia l . U n c o n t r a c t e d

p a r t i c l e s ( F i g . 5 b ) r e se m b l e d b u l l e t s a n d a b a se - p l a t e

w a s v i s i b l e a t b r o a d e r e n d . H o o k - l i k e p i n s e x t e n d f r o m

t h e l o w e r e n d s o f c o n t r a c t e d s h e a t h s a n d o c c a s i o n a l l y

se v e r a l f i b r e s w e r e s e e n a t t a c h e d t o t h e b a se p l a t e .

I s o l a t e d c o n t r a c t e d s h e a t h s w e r e o b s e r v e d a n d l o n g

s h e a t h - li k e s t r u c t u r e s w e r e s e e n c o m p o s e d o f n u m e r o u s

c o n t r a c t e d s h e a t h s . H o l l o w r i n g le t s a n d m i n u t e c o g -

w h e e l s a r e a ls o o b s e r v e d . T h e m a j o r i t y o f p a r t ic l e s ,

r e g a r d le s s o t t h e p y o c i n o g e n i c s tr a i n u s e d , m e a s u r e d

a p p r o x i m a t e l y 1 00 x 1 5 n m i n t h e u n c o n t r a c t e d s t a te ;

c o n t r a c t e d p a r t ic l e s c o n s i s t o f a h o l l o w t a il p i ec e , a p -

p r o x i m a t e l y 1 0 0 x 7 n m , p a r t i a l l y e n c l o se d i n a sh e a t h

m e a s u r i n g a b o u t 4 5 x 17 n m ( G o v a n , 1 9 7 4a ). T h e m o r -

p h o l o g y o f th e s e b a c t e r i o c i n s c a n b e c h a n g e d b y f r e e z i n g

a n d t h a w i n g w h i c h m a y r e n d e r t h e s e p a r t i c l e s i n a c t i v e .

H o w e v e r , w h e n p u r i f i e d p y o c i n p r e p a r a t i o n s i n T r i s -

H C I b u f f e r ar e s u b m i t t e d t o a f a s t f r ee z e i n a n a c e t o n e -

d r y i c e b a t h , t h e p a r t i c l e s f o r m r o se t t e c l u s t e r s .

A s c h e m a t i c d i a g r a m o f t h e v a r i o u s m o r p h o l o g i c a l

f o r m s a n d s t r u c t u r e s s e e n i n e l e c t r o n m i c r o g r a p h s o f

p y o c i n s i s p r e se n t e d i n F i g . 6 : t h e se c o n s i s t o f a c o n -

t r a c t e d p a r t i c l e , a l o c k - w a sh e r r i n g l e t ( F i g . 6 a ) a n d t w o

f o r m s o f r e l a x e d p a r t i c l e s d i f f e r i n g i n s i z e a n d s t r i a t i o n

( F i g . 6 b a n d c ) . T h e s e t w o f o r m s a r e f r e q u e n t l y s e e n

t o g e t h e r i n t h e s a m e p r e p a r a t i o n ( H i g e r d et al . 1969;

G o v a n , 1 9 7 4 a: D a w , 1 9 89 ). I n g e n e r a l t h e y a r e a ll

m o r p h o l o g i c a l l y i d e n t ic a l e x c e p t f o r m i n o r d i f f er e n c e s i n

d i m e n s i o n s ; t h e c h i e f fe a t u r e b e i n g t h a t t h e y r e s e m b l e

h e a d l e s s p h a g e s .

F i g . 5. E l e c t r o n m i c r o g r a p h s o f t w o d i f f e r e n t f o r m s o f p y o c i n

o b t a i n e d f r o m s t r a i n s o f

Pseudomonas aeruginosa

C o n t r a c t e d

p y o c i n p a r t i c l e s c o n s i s t e d o f c o r e p a r t i al l y s u r r o u n d e d b y a

s h e a t h a ) a n d t h e u n c o n t r a c t e d r e l a x e d ) p y o c i n f o r m r e s e m b l e

b u l l e t s w i t h a p l a t e a t t h e b r o a d e r e n d b ) . N e g a t i v e l y st a i n e d .

Sc a le m a r ke r s ind ic a te I011 nm .

F i g . 6. A s c h e m a t i c d i a g r a m s h o w i n g b a c t e r io c i n m o r p h o l o g y .

T h e c o n t r a c t e d b a c t e r i o c i n p y o c i n ) p a r t i c l e a ) r e s e m b l e s a

l o c k - w a s h e r r i n g l et a n d t w o r e l a x e d p y o c i n p a r t i c l e s b . c) s h o w

d i f f e r e n t a p p e a r a n c e s .

B . M o r p h o l o g y o / a c ti ve j b r m s

P r e v i o u s l y , i t w a s n o t k n o w n w h e t h e r b a c t e r i c i d a l

a c t i v i t y o f t h e b a c t e r i o c i n s w a s a s so c i a t e d w i t h a d i s c r e t e

m o r p h o l o g y o r n o t , o r w h e t h e r t h e a c ti v i ty w a s c o n -

f i n e d t o t h e r e l a x e d o r c o n t r a c t e d f o r m s ( B r a d l e y a n d

R o b e r t s o n , 1 9 68 ). F u r t h e r s t u d ie s f o u n d a d i re c t c o r r e -

l a t io n b e t w e e n t h e p e r c e n t a g e o f r e la x e d p a r t i c le s a n d


6/13


t he reduc t ion in ac t iv i ty dur ing convers ion to the con-

t rac t ed s t a t e (Higerd

e t a l .

1 9 6 9 ; G o v a n , 1 97 4b ; D a w ,

1989) . I t was conc luded tha t , once con t rac t ion occurs ,

there appears to be l i t t l e o r no increase in ac t iv i ty upon

subsequen t re l axa t ion .

I n a n a t t e mp t t o u n d e r s t a n d t h e b i o l o g i c a l n a t u r e o f

c o n t r a c t e d f o r ms o f b a c t e r io c i n , H i g e r d

et al .

(1969)

adde d a so lu t ion o f pyoc ins to pur i f ied ce ll wal l p repa r -

a t ions o f sens it ive s t ra ins o f

P . a e r u g i n o s a .

A d s o r p t i o n

to the ce l l wal l o f the sens i tive s t ra ins occu r red a t t he

f l a tt e n e d e n d o f t h e b a c t e r i o c in w i t h i n o n e m i n u t e a f t e r

adso rp t ion o f the re l axed par t i c l es to the cel l wal l s and a

c o n t r a c t i o n o f th e o u t e r s h e a t h w a s o b s e r v e d . E a r l y

s tud ies on

P r o t e u s v u l g a r i s

b y C o e t z ee

et al .

(1968) also

s h o w e d t h a t a h i g h t i t e r o f b a c t e r io c i n p r e p a r a t i o n s

t rea t ed wi th

H 2 0 2

a n d e t h a n o l a n d t h e n a d s o r b e d w i t h a

s u s p e n s i o n o f s u s c e p t i b l e s t r a i n s , h a d ma n y c o n t r a c t e d

ta i l - l i ke s t ruc tu res in the superna tan t f lu id and very few

a r o u n d t h e s u s c e p t i b le o r g a n is m. T h i s s u g g e s te d t h a t t h e

r e la x e d f o r m o f t h e p a r ti c l e ma y b e t h e n a t i v e s t a te o f

the bac te r ioc in , s ince ac t iv i ty and ce l l wal l adsorp t ion

w e r e d e mo n s t r a b l e o n l y w i t h p a r t i c l e s i n t h e r e l a x e d

state.

C . M o d e o f a ct io n

M o s t o f t h e i n f o r ma t i o n r e g a r d i n g t h e mo d e o f a c t i o n

o f b a c t e r i o c i n s h a s b e e n b a s e d o n s t u di e s o f s e v e ra l

b a c t e r i o c i n s p e r f o r me d b y N o mu r a a n d h i s c o l l e a g u e s

(Nomura , 1967) . Bac ter ioc ins exer t t he i r b io log ica l

ac t ion th rough adsorp t ion to the i r spec i f i c recep to rs ,

loca ted a t t he ex te rna l su r face o f sens i tive cel ls , and a re

then t ra ns loca ted to the i r spec if i c t a rge t s w i th in these

cel ls (Parker

e t a l .

1989) . Kine t i c da ta ind ica te tha t

bac te r ioc ins behave as par t i cu la t e l e tha l agen t s , k i l l i ng

sens i t ive ce l l s i n what amount s to a s ing le h i t p rocess .

Th i s to the so ca l l ed qu an ta l k i ll i ng ra ther than

m ola r coo pera t ive k i ll i ng ac t ion o f c l as s ica l an t i -

b i o t i c s ( Ma y r - H a r t i n g

e t a l .

1972).

T h e m o d e o f a c t io n o f b a ct e ri o c in s m a y v a r y f r o m o n e

t y p e t o a n o t h e r ( B r a u n

e t a l .

1 9 9 4 ; Mo n t v i l l e a n d

Br u n o , 1 9 9 5 ) . E a r l y e x p e r i me n t s d e mo n s t r a t e d t h a t c o -

l icin E3 led to specif ic inhibi t ion of prote in syn thesis

(Schwar tz and Hel insk i , 1971) . Co l i c in E2 l eads to

s p ec i fi c i n h i bi t io n o f D N A s y n t h e si s a n d i n d u c e s D N A

d e g r a d a t i o n . O t h e r s t u d i e s i mp l i c a t e d t h e c y t o p l a s mi c

me m b r a n e a s p r i ma r y t a r g e t o f c o l ic i n A , E l , K , I a , a n d

Ib . These bac te r ioc ins and o ther re l a t ed co l i c ins c rea te

d i s r u p t i o n o f a c ti v e t r a n s p o r t a n d p r o d u c e l e a k a g e o f

i o n s b y f o r mi n g v o l t a g e - d e p e n d e n t c h a n n e l s i n p h o s -

p h o l i p i d b i l a y e r me mb r a n e s , t h e r e b y d e s t r o y i n g t h e

ce l l ' s po ten t i a l (Sche in

et al .

1978). This resul ted in the

inh ib i t ion o f p ro te in a nd nuc le i c ac id b iosyn thes i s and

u n c o u p l e d e l e c t r o n t r a n s p o r t f r o m a c t i v e t r a n s p o r t o f

t h i o me t h y l - f l - D - g a l a c t o s i d e a n d p o t a s s i u m ( L u s k a n d

Nel son , 1972) . T rea ted ce l l s l eaked po tas s ium and mag-

nes ium ions . The los s o f such ions has been imp l i ca t ed as

the p r ima ry cause o f ce ll dea th (Ko ni sky , 1982) . Neve r -

the less , each o f these co li c ins has i ts own imm uni ty

P e r i p l a s m

I m m u n i t y p r o t e i n P o r e - f o r m i n g d o m a i n

N L2 C

C y t o p l a s m

Fig . 7 . The h ydrop hi l i c a -he l i c e s of c o l i c in A o f Esherichia col l

A p o r e f o r m i n g d o m a i n a - h e li c e s 8 a n d 9 ) a re r e s p o n s i b le f o r

r e c o g n i t i o n o f t h e i m m u n i t y p r o t e i n .

p r o t e i n s ( W e a v e r

e t a l .

1981a ,b ; Van der Goo t

e t a l .

1991) . I t i s mo re l ike ly tha t imm uni ty p ro te ins inac t iva te

the i r co r respo nd ing co l i c ins by d i rec t in t e rac t ion . Di f fe r -

e n t s tu d i e s ( Ma n k o v i c h

e t a l .

1 9 8 4 ; Sh o h a m a n d D j e b li ,

1992) , us ing a cons t ruc t ion o f hybr id p ro te ins b e twee n

co l i c ins Ia , Ib , A and E1 have shown tha t t he C- t e rmina l

pore- fo rming domain was respons ib le fo r the reCog-

n i t ion o f immu ni ty p ro te in by pore fo rm ing co l ic ins .

M o s t i n v e s ti g a to r s ( T o k u d a a n d K o n i s k y , 1 9 7 8; W e a v e r

e t a l .

1981b ; Gel i and L azdunsk i , 1992a) conc luded tha t

imm uni ty p ro te ins o f pore fo rm ing co l ic ins in t e rac t

w i t h t h e p o r e - f o r mi n g d o m a i n s o f t h e ir c o r r e s p o n d i n g

co l i c ins a t t he level o f the inner me m bran e and thus the i r

a c t i o n s h o u l d p r e v e n t t h e f o r ma t i o n o f th e p o r e .

A p p a r e n t l y t h e i n se r ti o n o f p o r e f o r mi n g b a c t e r io c i n s

i s t r i g g e r e d b y s p o n t a n e o u s p e n e t r a t i o n i n t h e b i o l a y e r

o f a h y d r o p h o b i c h e l ic a l h a ir p i n n o r ma l l y b u r i e d i n

t h e w a t e r - s o l u b l e p r o t e i n s . T h e ma j o r d e t e r mi n a n t f o r

immuni ty spec i f i c i ty o f such co l i c in i s l oca ted be tween

a-he l ices 8 and 9 , as shown in F ig . 7 . Gel i and Lazdu nsk i

(1992b) , repor t ed tha t co l i c in A recogn i ses i t s immuni ty

p r o t e i n t h r o u g h i t s h y d r o p h o b i c H 2 , H 3 a n d H 4

a-he l i ces , i n add i t ion to the per ip l asmic loop . On the

o t h e r h a n d , t h e ma c r o mo l e c u l a r b a c t e r i o c i n s s u c h a s R

pyocin and c loac in C5 adsorb to sens i t ive ce l l s and exer t

the i r b io log ica l ac t iv i ty . Kazi ro and Tanaka (1965a) and

K a z i r o a n d T a n a k a ( 1 9 6 5 b ) r e p o r t e d t h a t t h e a d d i t i o n

o f R p y o c in , p r o d u c e d b y

P . a e r u g i n o s a

t o g r o w i n g

cu l tu re o f the sens i tive s tra in , caused a rap id and c om -

p le t e cessa t ion and inh ib i t ion o f the syn thes i s o f RNA,

DNA and p ro te in in the sens i t ive ce l l s . Th i s was found

due to inac t iva t ion o f the r ibosom es . The k i l l ing ac t ion

of these bac te r ioc ins i s t herefo re remin i scen t o f the

ac t ion o f v i ru l en t phages on the hos t ce ll s (Coetzee

e t a l .

1968) . The e l ec t ron microscop ic s tud ies on the

mo r p h o l o g i c a l c h a n g e s o f th e s e n s it iv e b a c t e r ia ma y l e a d

t o f u r t h e r u n d e r s t a n d i n g t o t h e mo d e o f a c t io n o f t h e s e

bac te r ioc ins .

D . M o r p h o l o g i c a l c h a n g e s a s s o c i a t e d w i t h b a c te r i o c in

a c t i v i t y

The k il li ng o f suscep t ib l e bac te r i a by a b ac te r ioc in i s a

k ine t i c p rocess . The ac t ion o f those bac te r ioc ins which


7/13

B a c t e r i o c i n s 4 7 3

a r e s h o w n t o h a v e a d e f i n i te s t r u c t u r e h a s b e e n s t u d i e d

i n a p r e l i m i n a r y m a n n e r u s i n g t h e e l e c t r o n m i c r o s c o p e .

T h e m o d e o f b a c t e r i o c i n a c t i o n i s a s s o c i a te d w i t h d i ff e r-

e n t m o r p h o l o g i c a l c h a n g e s o f b o th , t h e s h a p e o f t h e

b a c t e r i o c i n s a n d t h e m o r p h o l o g y o f th e s u s c e p t ib l e

b a c t e r i a . T h e r e h a v e b e e n r e l a ti v e l y f e w r e p o r t s o n t h e

n a t u r e o f th e m o r p h o l o g i c a l c h a n g e s p r o d u c e d i n su s -

c e p t i b le c e ll s o n t r e a t m e n t w i t h b a c t e r i o c i n s , p a r t i c u l a r l y

G r a m p o s i t i v e b a c t e r i a , d u e t o t h e f a c t t h a t m o s t o f

t h e s e b a c t e r i o c i n s a r e n o t r e s o l v a b l e b y e l e c t r o n m i c r o -

s c o p e ( V l a e m y n c k et al . 1 9 9 5 ) . E x t e n s i v e s t r u c t u r a l

c h a n g e s o c c u r r e d i n g r o u p A S t r e p t o c o c c u s s t r a i n a f t e r

e x p o s u r e t o s t a p h y l o c o c c o c i n C 55 ( C l a w s o n a n d D a j a n i ,

1970; E l l i son et al . 1 9 7 1 ) . T h e se c h a n g e s i n c l u d e t h e

c o n d e n s a t i o n o f n u c l e a r m a t e r i a l , l o ss o f r ib o s o m e s ,

m o d i f i c a t i o n o f m e s o s o m e s a n d d i s s o l u t i o n o f ce ll c o n -

t e n t s . S i m i l a r c h a n g e s w e r e a l so o b se r v e d i n s e n s i t i v e

s t r a i n o f S t r e p t o c o c c u s p y o g e n e s ( T a g g et al . 1973)

t r e a t e d w i t h s t r e p t o c i n A . T h e s e c h a n g e s r a n g e d f r o m

t h e n o r m a l a p p e a r a n c e o f t h e S t r e p t o c o c c i t o t h o s e w i th

a c o n d e n s a t i o n o f n u c l e a r m a t e r i a l w i t h a n i n c r e a s e in

t h e p e r i n u c l e a r s p a c e .

O n t h e o t h e r h a n d , m o r p h o l o g i c a l s t u di e s c o n c e r n i n g

t h e b a c t e r i o c i n s o f G r a m n e g a t i v e b a ci ll i w e re d o n e

u s i n g p y o c i n p a r t i c le s a n d m o r e r e c e n t l y w i t h c l o a ci n C 5

o f E n t e r o b a c t e r c l o a c a e ( G o v a n , 1 9 7 4 a , b ; D a w , 1 9 8 9 :

D a w a n d F a l k i n e r , 1 9 9 3) . T h e s e g r o u p s c o n d u c t e d

d e t a i l e d s t u d i e s o n a d s o r p t i o n a n d i n h i b i t o r y a c t i o n o f

p y o c i n 2 1 o n s e n s i t i v e c e l l s o f P. aeruginosa a n d c l o a c i n

C 5 o f E. c loacae u s i n g e l e c t r o n m i c r o s c o p y . T h e u n c o n -

t r a c t e d p a r t i c l e s ( t h e a c t iv e f o r m s o f b a c t e r i o c i n s , F i g s 1

a n d 5 b ) a t t a c h e d r a p i d l y b y t h e b r o a d e r o r b a s e p l a t e

e n d t o s e n s i t i v e b a c t e r i a a n d n o i m m e d i a t e a d s o r p t i o n

o f t h e b a c t e r i o c i n u s u a l l y o c c u r s a s s h o w n i n F i g . 8

( G o v a n . 1 9 7 4 a) ; w i t h a P . aeruginosa i n d i c a t o r s t r a i n

a f t e r o n e m i n u t e c o n t a c t w i t h i n d u c e d p y o c i n 2 1 . T h e

b a c t e r i a l s u r f a c e i s h e a v i l y s u r r o u n d e d b y p y o c i n

p a r t ic l e s a n d n o c o n t r a c t i o n o f a d s o r b e d p a r t ic l e s is

s h o w n . C o n s e q u e n t l y , a f t e r a d s o r p t i o n , c o n t r a c t i o n o c -

c u r s a n d t h e b a c t e r i o c i n s s t a r t t o e x e r t t h e i r a c t i o n s a s

sh o w n i n F ig . 9 ( D a w a n d F a l k i n e r , 1 99 3 ); E. c loacae

s t r a in s e n s it iv e t o c l o a c i n C 5 a f t e r f o u r m i n u t e s c o n t a c t .

T h e c o n t r a c t e d b a c t e r i o c i n s c a n b e s e e n a d s o r b e d t o t h e

b a c t e r i a l su r f a c e a n d i n t h e v i c i n i t y o f t h e b a c t e r i a l c e ll .

F i g u r e 1 0 ( G o v a n , 1 9 7 4 a) a ls o s h o w s t h e s a m e f o r a

P. aeruginosa s t r a in a f t e r f iv e m i n u t e s c o n t a c t w i t h

se n s i t i v e p y o c i n s . T h e b a c t e r i a l c e l l i s a l so su r r o u n d e d

b y c o n t r a c t e d b a c t e r i o c i n s .

A f t e r t h e a d d i t i o n o f b a c t e r i o c i n s t o s e n s i ti v e b a c te r i a

f o r a l o n g e r p e r i o d , a v a r i e t y o f c h a n g e s h a v e b e e n

o b s e r v e d . T h e e n t i r e s u r f a c e o f t h e s e n s it iv e b a c t e r i u m

a p p e a r s c o v e r e d w i t h r o d l i k e p a r t i c l e s , w i t h a n i m m i -

n e n t d i s r u p t i o n o f t h e b a c t e r i a l c e ll a s sh o w n i n F i g s 11

a n d 1 2 ( D a w a n d F a l k i n e r , 1 9 93 ; D a w , 1 9 89 ; G o v a n ,

1 9 7 4 a ) . T h e b a c t e r i a l c e l l s , a t t h i s s t a g e , w e r e d e a d a n d ,

i n d e e d , a l t e r t w o h o u r s i n c u b a t i o n w i t h t h e b a c t e r i o c i n

p r e p e r a t i o n s , m o r e t h a n 9 0 7 ,, o f t h e b a c t e r i a w e r e s e e n

t o b e c o m p l e t e l y d i s r u p t e d . T h i s , h o w e v e r , r e s u l t e d i n

t h e k i l l in g o f s e n s i t iv e b a c t e r i a w i t h o u t l y s is . O n t h e

o t h e r h a n d . w h e n b a c t e r i o c i n s w e r e m i x e d w i t h r e s i s ta n t

Fig. 8 . Pseudomonas aerughu~sa s t r a i n a f t e r o n e r a i n c o n t a c l a t

3 7 ° C w i t h p y o c i n p a r t i c l e s . T h e b a c t e r i a l s u r f a c e i s s u r r o u n d e d

b y m a n y u n c o n t r a c t e d b a c t e r i o c i n p a r ti c l es . N e g a t i v e l y s t a i n e d

w i t h p o t a s s i u m p h o s p h o t u n g s t a t e 2 , ~ , w / v . S c al e m a r k e r

i n d i c a t es 2 0 0 n m

ii L

Fig. 9 .

nterobacter cloacae

a f t e r 4 r a i n c o n t a c t w i t h c l o a c i n a t

3 1 ° C . F e w u n c o n t r a c t e d p a r t i c l e s (U ) a r e v i s ib l e c l o s e t o t h e

b a c t e ri a l s u r F ac e a n d m a n y c o n t r a c t e d ( C ) p a r t ic l e s c a n b e s e e n

a d s o r b e d t o t h e b a c t e r i a l s u r f a c e a n d i n t h e v i c i n i ty o f t h e

b a c t e r i u m . S c a l e m a r k e r i n d i c a t e s 1 0 (I n m .

s t r ai n s , n o n e o f t h e a b o v e - m e n t i o n e d k i l l in g p r o -

c e s s e s o c c u r r e d . T h e e l e c t r o n m i c r o g r a p h s s h o w n o

a t t a c h m e n t o f b a c t e r io c i n p a r t i c le s t o t h e s u r f a c e o f


8/13

474 M .A . Daw and F. R. Falkiner

Fig. 10.


after 5 min contact at 37°C

with pyocin. The bacterial cell is surrounded by contracted

particles which can be se en adsorbed to the bacterial surfaces

and in the v icinity of the bacterium. S cale marker indicates

200 nm.

t h e r e s is t a n t b a c t e r i a , a n d m o s t o f t h e p a r ti c le s r e m a i n e d

u n c o n t r a c t e d a n d s o m e d is t a n c e f r o m t h e b a c t e r i a l

c e l l, a s s h o w n i n F i g . 1 2 ; E cloacae s t a i n r e s i s t a n t t o

c l o a c i n C 5 a f t e r 3 0 m i n c o n t a c t . T h e b a c t e r i o c i n

p a r t i c l e s d i d n o t a t t a c h t o t h e s u r f a c e o f t h e r e s i s ta n t

b a c t e ri a a n d m o s t o f t h e m r e m a i n e d u n c o n t r a c t e d ( D a w ,

1989) .

D e s p i te t h e a m b i g u i t y o f th e e l e c tr o n m i c r o s c o p e

e v i d e n c e c o n c e r n i n g t h e c h a n g e s a s s o c i a t e d w i th b a c -

t e r i o c i n p a r t i c l e s d u r i n g t h e k i l l i n g p r o c e s s , i t s e e m s t h e y

a ls o g o th r o u g h m o r p h o l o g i c a l a n d c o n f o r m a t i o n a l

c h a n g e s d u r i n g t h e i r a c t i v i t y . O n l y r e l a x e d f o r m s a d s o r b

t o t h e s u s c ep t i b le s t r a in . W h e n t r ig g e r e d , a c o n t r a c t e d

t a i M i k e s t r u c t u r e c a n n o t b i n d . D i f f e r e n t a g e n t s c a n

e f f ec t t h i s a c t i v i ty . G o v a n ( 1 9 7 4 a ) f o u n d t h a t t h e a c t i v i t y

o f t h e p y o c i n w a s d e s t r o y e d a f t e r t r e a t m e n t w i t h 0 . 0 2

S D S a n d t h a t t h e p y o c i n s l o s t t h e ir o u t e r c o n t r a c t i l e

s h e a t h a n d d i d n o t a d s o r b o n t h e s e n s i t i v e b a c t e r i a l c e l l s .

T h i s a c t i v i t y w a s n o t a f f e c t e d w h e n S D S w a s u s e d a t a

c o n c e n t r a t i o n l e ss t h a n 0 . 0 1 .

D i f f e r e n t s t u d ie s ( K r i m m a n d A n d e r s o n , 1 9 67 ;

M o o d y , 1 9 6 7 a ,b ; B r a u n , 1 9 94 ), s u g g e s t e d th a t t h e

m e c h a n i s m o f c o n t r a c t io n o f th e t ai l s h ea t h o f T 4

b a c t e r i o p h a g e i n v o lv e s a c o n f o r m a t i o n a l c h a n g e i n t h e

p r o t e i n s u b u n i t , in d i c a t i n g c o n f o r m a t i o n a l c h a n g e s in

t h e d o m a i n r e q u i r e d f o r th e i n f e c ti v i ty p r o c e s s o f t h e

b a c t e r i o p h a g e . B a c t e r i o c i n s re s e m b l e c o n t r a c t i le b a c -

t e r i o p h a g e s i n m a n y r e s p ec t s , t h o u g h t h e y d o n o t

(a)

Fig. 11. Enterobacter cloacae sensitive to cloacin C5 (a) and


sensitive to pyocin (b) after 30 min

contact at 37 0C w ith the bacteriocin particles. In both con-

ditions, the bacterial surfa ce appears con voluted and c overed

with many bacteriocin part icles contracted (C) and uncon-

tracted (U). E mpty sheaths (E) were also seen in the vicini ty of

the sensitive bacterium. Scale markers indicate 200 nm.

Fig. 12. Enterobacter cloacae strain resistant to bacteriocin C5.

The bacteriocin part icles ha ve not at tached to the bacterial

surface and most o f the bacteriocins have rem ained uncon-

tracted. Scale marker indicates 200 nm.

r e p l i c a t e in s e n s i ti v e h o s t b a c t e r i a . I n m o r p h o l o g i c a l

t e r m s , t h e i r l a c k o f a n y s t r u c t u r e r e s e m b l i n g a b a c t e r i o -

p h a g e h e a d i n d i c a t e s a c o n c o m i t a n t l a c k o f t h e m a i n

r e s e r v o i r f o r n u c l e i c a c i d .


9/13


P c o l >

c e a l

P i m m > E 9

i m m l y s

D N A - 0 -

P r o t e i n s

C o l i c i n

c o m p l e x

( l l l l l ' l l l [ l l l [ l i l l i ' l l l )

C o l i c i n E 9 I m m u n i t y L y s i s

p r o t e i n p r o t e i n

F i g . 1 3 . G e n e t i c o r g a n i s a t i o n o f c o l i c i n E9 o p e r o n o f

E s h e r i c h i a

coli P c o l is a n S O S - i n d u c i b l e p r o m o t e r ceal C o l i c i n E 9

s t r u ct u r a l g e n e P i m m i m m u n i t y g en e p r o m o t e r . E 9 i m m :

i m m u n i t y p r o t e i n g e n e l v s ; l y s i s g e n e i n v o l v e d i n s e c r e t i o n o f

t h e c o l i c i n c o m p l e x .

I I I M O L E C U L A R B I O L O G Y O F B A C T E R I O C I N S

A . G e n e t i c d e t e r m i n a n t s o J b a c t e r i o c i n s

The production of bacteriocins seems to be a heredi-

tary feature associated with cytoplasmic genes (i.e

bacteriocinogenic factors). The genetic determinants of

colicins (colicinogenic factors) have been the most com-

monly studied (Reeves, 1972; Nieto-Lozano e t a l . , 1992;

Schved e t a l . , 1993; Braun et al . 1994). Most of the

genetic determinants of the bacteriocins are plasmid-

borne with few exceptions, including pneumocin and

pyocins, which are found to be chromosomally located

(Kageyama, 1975: Quirantes

e t a l . ,

1994). Bacteriocino-

genic genes may determine, not only the chemical com-

position of the bacteriocin, but also the regulation of its

biosynthesis, its release from the cell and the host cell

immunity to its own bacteriocin. Plasmid-encoded bac-

teriocins could be easily transferred from bacteriocino-

genic bacteria to compatible recipient strains either by

conjugation or transduction. The transfer of such a

plasmid emparts to the recipeint strains all the features

encoded. Transfer of bacteriocinogenic activity has

been demons trat ed with many colicins. E colicins are

plasmid-encoded proteins produced by E . c o i l , which kill

the sensitive cells by binding to the btuB-encoded cell

surface receptor (Di Masi e t a l . , 1973). The E colicin

plasmid also codes for the production of a specific

immunity protein which, upon synthesis, binds to the C

terminal domain of its cognate. James e t a l . (1992)

studied the genetic determinant of the interaction be-

tween colicin E9 and its immunity proteins. ColE9-J

plasmid encodes the colicin structural and immunity

genes in an operon (Chak and James, 1986). The pro-

moter of

E 9 i m m

genes is located within the colicin E9

structural gene. Transcription from such a promoter

allows constitutive expression of the immunity protein

and provides immunity to colicin E9. Transcription

from the inducible promotor results in the expression of

the genes of the operon, as shown in Fig. 13 and leads to

the synthesis and secretion of the colicin/immunity

protein complex.

Plasmid elimination curing can be accelerated by

exposing the host strains to certain agents such as the

intercalating dye acridine orange and ethidium bromide

or by growing the bacteriocinogenic cells at an elevated

temperature (Tolmasky

e t a l . ,

1993). Such agents could

eliminate the plasmids encoded for the bacterJocins.

Bacteriocinogenic factors from

S t a p h y h , c o c c o u s a u r e u s ,

and

C o l i s t r i d i u m p e r f e r i n g e s

have been cured by such

methods (Gagl iano and Hinsdill, 1970; Ionesco and

Bouanchaud, 1973). Dajani and Taube (1974) noted that

the curing of bacteriocin production in staphylococci

resulted in alteration the resistance of producer cells to

the action of the staphylococcin. Where staphylococcin

producer strains were unable to adsorb the bacteriocin

and were naturally resistant to its killing effect, cured

strains adsorbed the staphylococcin and were rapidly

killed by it. This makes the bacteriocinogenic strains

whose bacteriocins were plasmid-encoded readily lose

their bacteriocinogenic activity, in comparison to those

strains possessing chromosomal borne bacteriocins.

Pyocins on the other hand have peculiar features since

their genetic determinants are all located at definte sites

on the chromosome. This is in sharp contrast to the case

for many other bacteriocins such as colicins, which are

of plasmid origin. Thus, the loci of pyocins types R and

F are between alteration of t r p C D and tlT~E (Kageyama,

1975; Shinomiya e t a l . , 1983), the locus for pyocin AP41

is between @ s ' - 9 0 1 5 and a r g F and the locus for pyocin S

is close to fl a Y (Sano and Kageyama, 1984: Sano

e t a l . ,

1990) on the chromosome of the producer strains. Sano

et al .

(1990) studied the pyocin genes in detail and cloned

them on appropriate plasmids. The genes governing

pyocins AP41, S1 and $2 were found to each encode two

proteins, large and small, transcribed in the same direc-

tion; AP41, 83.6 and 10 kDa; SI, 64.6 and 10 kDa and

$2, 74 and 10 kDa. Striking homology in the amino acid

sequences found among these pyocins and colicins,

particularly the C-terminal amino acids, the central

region of the pyocins and the peptides stretching

between the N-terminal and the central parts of colicins

E2 and E3 and cloacin DF 13. Comparison of the genetic

components of these bacteriocins is depicted in Fig. 14.

The S-type pyocin has been purified and cloned from E.

c o l i cells using DEAE-cellulose and chromotography

(Ohkawa e t a l . , 1973; Sano and Kageyama, 1981). The

purified preparation is a complex composed of two

proteins, a larger component which showed essentially

the same level of killing activity as did the complex

molecule and a smaller component which made the cells

immune to pyocins. When indicator strains were trans-

formed with a cloned DNA for the smaller component,

they became insensitive to the respective pyocin (Sano

e t a l . ,

1990). In conclusion, bacteriocins differ not only

in the range of target bacteria but also in the location of

their genes, chromosomal or plasmid borne. However,

the similarity found among these agents in amino acid

sequence and in function may give clues concerning their

etiology.


10/13

4 7 6 M . A . D a w a n d F . R . F a l k i n er

P y o c i n

A P 4 1

S I

$ 2

C o l i c i n E 2

C - T e r m i n a l s T - T e r m i n a l s

0 400 800

I I I I I I I I I A m i n o a c i d s

I I I I I I V

R 6 4 0

I ' , . I . ' , I I I I I l I I I I I l I I I l I V A v 7 6

> , \ R 487

1

i ' ,l , , ; ', i il l ' il " I i l V / / / A 6 1 7

7 7 7

i l t

~ / / / / R 5 58

I [{ l

] 1 ' , I , I , 1 i l l i l i 1 1 1 : l I I ] i I V / / / A 6 8 9

~ - [ / ~ R 4 5 2

R 4 5 4

E 3 ~ / / A I I l l l l l l l l l k . + ~ \ ~ 5 5 l

: : : : : R 46 4

I i l ] l ] l l l l l l l l l l l [ I t k \ \ q 5 6 1

T r a n s l o c a t i o n R e c e p t o r

n u c l e a s e

F i g . 1 4 . A m i n o a c i d s e q u e n c e h o m o l o g y i n t h e k i ll er c o m p o -

n e n t s o f p y o c i n s A P 4 1 , S l a n d $ 2 o f Pseudomonas aeruginosa

c o l i c i n s E 2 a n d E 3 o f

Escherichia coil

a n d c l o a ci n D F 1 3 o f

Enterobacter cloacae. D o m a i n s s h o w i ng h o m o l o g y h a v e th e

s a m e s h a d i n g . L e n g t h s a re p r o p o r t i o n a l t o t h e n u m b e r o f

a m i n o a c i d s o f e a c h m o l e c u l e . A n A r g r e s i d u e n e a r t h e s t a rt

p o i n t o f e a c h n u c l e a s e d o m a i n i s f ix e d a s a r e f er e n c e p o i n t

( R 6 4 0 i n A P 4 1 ) . T h e N - t e r m i n a l M e t i s la c k i n g i n t h e p u ri f ie d

p y o c i n s .

C l o a c i n D F I 3

B. Microbiological relevance of bacteriocins

Bacteriocins have found a widespread significance in

medical microbiology, particularly in epidemiological

studies. Bacteriocin typing can be done in two ways: 1)

by determining the bacteriocin production pattern of a

strain against a set of standard indicators and 2) by

determining the bacteriocin susceptibility pattern of the

strain against a set of bacteriocins which are applied

to it. Each method has been used in epidemiology to

determine whether the isolates from different sources are

the same. If the isolates are from the same strain, their

bacteriocin production or susceptibility patterns will be

identical. The bacteriocin typing technique has been

widely used to answer the epidemiological questions in

the nosocomial infections. Although the bacteriocin

fingerprinting was found to be applicable for both Gram

positive and Gram negative bacteria, the scheme has

been most successfully used in the studies of Gram

negative bacilli.

Bacteriocin activity has been used as a marker for

typing epidemic strains of gram positive bacteria such as

S. aureus Streptococc us faeca lis

and

Colistridium per-

Jkingens. Hale and Hinsdill, 1973; Jack and Tagg, 1992;

Stiles, 1994). These studies, however, were never found

to have a wide range of application in microbiology.

More promising, is the application of bacteriocin typing

of mycobacteria, particularly atypical strains Takeya

and Tokiwa, 1972, 1974). Rapidly growing mycobac-

teria such as the Mycobacter iumfortui tum che lone i com-

plex cause serious infections in immunocompromised

patients and those with acquired immune deficiency

syndrome AIDS). My c obac te r ium species, isolated from

patients with prosthetic heart valves and hip joints, can

be classified to specific bacteriocin profiles. The myco-

bacteriocin activity can be combined and correlated with

other typing methods for further classification and

differentiation of such mycobacteria.

The role of Gram negative bacilli in a wide variety of

diseases is well known, particularly in the nosocomial

infection Du Pont and Spink, 1969; Daw and Falkiner,

1994). Hence, not only the recognition of the causative

agent is critical, but also determinat ion as to whether all

strains of a particular pathogenic organism isolated from

an outbreak o f disease originate from a common source.

Bacteriocin typing has been used to trace such organisms

and most of the definitive investigations of bacteriocin

typing have been used on Gram negative infections.

Early data on the epidemiological relevance of bacterio-

cin typing was reported on typing of E. coil and related

pathogens such as

Proteus

species and

Shigella sonnei.

A significant resurrection of interest in the bacteriocin

typing started in the early seventies. This was due the

fact that bacteriocin typing is, 1) reliable in tracing the

source of the infecting organisms, 2).discr imantory

and reproducible, 3) does not need a sophisticated

instruments and 4) can be easly used by a small labora-

tories with no need to rely on reference laboratories. The

most significant bacteriocin typing of Gram negative

bacilli concerns those groups of opportunistic bacteria

such as

Pseudomonas Klebsiella Enterobacter

and

Serratia which often cause serious infections in hospital-

ised patients Daw, 1989). The microbiological rele-

vance of bacteriocin is evident. As an epidemiological

tool, the system was found to be applicable and com-

parable to the classical typing methods. Further to

its practicability, bacteriocin typing almost always pro-

vides answers to pertinent epidemiological questions,

which make it one of the favourite typing methods,

particularly with those laboratories possessing only basic

equipment.

IV. CONCLUSIONS

Bacteriocins are protein-like antibiotics. They differ

from traditional antibiotics because of their composition

and narrow spectrum of activity, in which they kill

bacteria of the same or closely related species. The

producing strain usually shows immunity to the bac-

teriocin they produce. The classification and thus

the nomenclature of bacteriocins has gone through a

major changes since the discovery of these bactericidal

particles. Further to their classification according to the

producing bacterial strains, they have also been classi-

fied according to their electron microscopical appear-

ance. Both ultrastructure and biochemical data seem

to suggest that bacteriocin particles are heterogeneous

molecules, varying from a small unidentified structure to

a large one resolvable by electron microscopy.

The macromolecular bacteriocins exert their action by

adsorbtion to specific receptors located on the external


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s u r f a c e o f t h e se n s i t i v e s t r ai n f o l l o w e d b y m a j o r b i o l o g i -

c a l a n d m o r p h o l o g i c a l c h a n g e s o f b o t h t h e b a c t er i a l c el l

and the bac ter ioc in par t i c l e . This re s ul t s in the k i l l ing

o f t h e s e n s i t i v e s t r a i n w i t h o u t p r o d u c t i o n o f f u r t h e r

b a c t e r i o c i n p a r t ic l e s o n e o f t h e m a i n f e a t u r e s w h i c h

m a k e s t h e m c l e a r l y d i f f e r e n t t o b a c t e r i o p h a g e s .

T h e m o s t i n t e n s i v e l y s t u d i e d g r o u p s o f b a c t e r i o c i n a r e

t h e c o l i c i n s w h i c h a r e p r o d u c e d b y E . co l i a n d c l o s e l y

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T h e y a r e a r e t y p i c a l l y p l a s m i d - e n c o d e d p r o t e i n s . T h e

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