~ )

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

Bacteriocins: Nature, Function and Structure

MOHAMED A. DAW* and FREDRICK R. FALKINER+
*Department qf Medical Microbiology, Facul O' oJ" Medicine, A(fateh Unit'ersitv ~[ Medical Sciences, P.O. Bo.v ,~;2668, Tripoli.
Lib)'a
+Department of Clinica/ Microbiology, Dublhl University, Dublin, Republic qf hehmd

Abstract Bacteriocins are extracellular substances produced by different types of bacteria, including both Gram positive and
Gram negative species. They can be produced spontaneously or induced by certain chemicals such as mitomycin C. They arc
biologically one of the important substances, and have been found to be useful in membrane studies and also in typing pathogenic
microorganisms causing serious nosocomial infections. Bacteriocins are a heterogeneous group of 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 determinants of most of the bacteriocins are located on the
plasmids, apart from few which are chromosomal[y encoded. These 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, morphology and mode of action of bacleriocins as well as their genetic determinants and the microbiological
relevance of these bactericidal agents, i', 1997 Published by Elsevier Science Ltd
Key words: Bacteriocins. bacterophage, bacterium-like inhibitory substances, relaxed forms, contracted forms, pyocins, cloacins.
bacterium typing.

CONTENTS
1.

II.

Introduction ...........................................................................................................................................................................................................
A. Background .....................................................................................................................................................................................................
B. Classification of bacteriocins .........................................................................................................................................................................
C. Biochemical nature of bacteriocins ...............................................................................................................................................................
D. Bacteriocinogeny and lysogeny .....................................................................................................................................................................
Ultrastructural study of bacteriocins ................................................................................................................................................................

467
467
468
468
469
470

A. General structure............................................................................................................................................................................................
470

I11.

IV.

B. Morphology of active forms ........................................................................................................................................................................

C. Mode of action ................................................................................................................................................................................................
D. Morphological changes associated with bacteriocin activity ....................................................................................................................
Molecular biology of bacteriocins ......................................................................................................................................................................
A. Genetic determinants of bacteriocins ...........................................................................................................................................................
B. Microbiological relevance of bacteriocins ...................................................................................................................................................
Conclusions ...........................................................................................................................................................................................................
Acknowledgements ...............................................................................................................................................................................................
References ..............................................................................................................................................................................................................

I. INTRODUCTION
A. Background

Bacteriocins are bactericidal, antibiotic-like substances, apparently protein in nature, which are produced 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 distinguish them from other (classical) antibiotics (Reeves,
1965; Daw, 1989: Gratia and Grenier, 1992; Laukova
and Marekova, 1993). The first discovery was reported
by Gratia in 1925 of a highly specific antibiotic (principe
V) produced by a strain of Escherichia coli and active
against other strains of the same species. This activity
was found to be produced by various species of Enterobacteriaceae and for which the generic name (colicine)
was proposed. With the discovery that the production of
apparently similar agents is not limited to Enterobacteriaceae, Jacob et al. (1952) proposed that the general
467

471
472
472
475
475
476
476
477
477

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 definition 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 detections, assay and the bacteriocin typing of bacterial
strains have been established. Genetic studies of colicinogeny and its transfer from one cell to another were
also pionered by Fredericq and his collaborators. Further 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 et al.,
1993). These led to interesting use of the bacteriocins as
a biochemical tools in cellular physiology (Libertin
et al., 1992; Farkas-Himsley et al., 1992; Becker et al.,
1993). Bacteriocins have since gained new attention,

468

M.A. Daw and F. R. Falkiner

particularly in the epidemiology of nosocomial infections; 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 al., 1992; Lebek et al., 1993).
Our knowledge on bacteriocins leans heavily on results obtained by electron microscope studies describing
the morphology and the killing process of the bacteriocin 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. Classification of bacteriocins
The classification, and thus the nomenclature of
bacteriocins, has changed rapidly since early this century. 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 bacteriocins have been studied in detail and they necessarily have
to be subdivided according to their spectrum of resistance (Reeves, 1965). As a result of 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 bacteriocins, 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 pyocynia), cloacin of
Enterobacter cloacae, pestisin of Yersinia pestis, monocin
of Listeria monocytogenes, cerecin of Bacillus cereus, and
staphylococcin of Staphylococcus. These vernacular
names used for bacteriocins are based, not on the spectrum 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 Bacillus 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 bacteriocins 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 bacteriocins, 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 bacteriocin 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 bacteriocinlike 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 positive 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 bacteriocins. 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 nature of bacteriocins

Bacteriocins have been erroneously considered as the
only proteins secreted by bacteria. Indeed, those intially
described were isolated and identified from the extracellular 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

Bacteriocins
expression of only one gene coding for a bacteriocin
release protein (BRP); also called the lysis or kill protein.
In comparison, export of other secreted proteins relies
on the activity of at least three proteins (Wandersman
and Delepeliare, 1990; Havarstein et al., 1994). However, they are exported late after synthesis and accumulate in the cytoplasm in a soluble form before they are
released. The colicin lysis proteins have been the most
extensively studied and, together with the BRP encoded
by the ColDF13 plasmid, they share many properties
including their genetic organisation, transcription, synthesis, assembly and function (Howards et al., 1989;
Cavard and Oudega, 1992).
The protein nature of bacteriocins was determined
early by the degradation of crude preparations by proteolytic enzymes and their approximate molecular
weight was determined by the diffusibility of the lysates
through agar or semipermeable membranes. Such
methods were found to be useful in the initial characterisation of bacteriocins (Loyola-Rodriguez et al.,
1992). Due to more advanced purification, several bacteriocins have been described and their chemical
and physical properties are known (Stiles et al., 1994;
Hastings et al., 1995).
Different studies have shown that the bacteriocins are
protein molecules with traces of carbohydrates (less than
1%) and phosphorus (less than 0.1%) (Kingsbury, 1966;
Reeves, 1972; Daw and Falkiner, 1993). The notable
differences between them were found to be associated
with amino acid composition. Different sequences or
cross composition of amino acids were found among the
bacteriocin molecules varying from one bacteriocin to
another, even among the same group of bacteriocin
(Konisky and Richards, 1970; Howard et aL, 1989:
Nieto-Lazano et al., 1992; Koebink and Braun, 1993).
Indeed colicin E is now divided into three subclasses
using SDS gel electrophoresis: colicin El, E2 and
E3. The main differences between these subclasses is
associated with amino acid terminal sequence.
The early detailed work on the biochemical nature of
bacteriocins was done on colicins and has been discussed
by several investigators (Ivanovics, 1962; Konisky, 1973;
Lakey et al., 1992: Braun et al., 1994). Two different
purified colicins were obtained; one is a lipopolysaccharide protein complex associated with the O somatic
antigen of the producing E. coli such as colicin K-K235,
and following its disassociation the activity was associated with the carboxyal moiety (CHO) (Viejo et al.,
1991; Miranda et aL, 1993). The other group of colicins
was found to be a protein containing little or no carbohydrate, such as colicin E2-P9, which was found to be a
protein with a molecular mass of about 60 kDa. Other
related colicins, E2-CA42 and colicin A, were also
purified and shown to be a protein with low carbohydrate content (about 10 %). These two were different
from the colicin K-K235 (Goormaghtigh et al., 1991;
Gonzalez-Manas et al., 1992).
The biochemical nature of other bacteriocin groups
have also been determined: type-R Pyocins produced by
P. aeruginosa were purified by Kageyama and Egami

469

Fig. 1. Transmission electron micrograph of a bacteriocin

particles obtained from Enterobacter cloacae, negativelly
stained with sodium silicotungstate2 '7,,w/v. The bacteriocinsat
field (A) are seen as bullet-like particles possessing a base plate.
A flagellum (F) is also seen across the field. Scale marker
indicates 0.1 nm.

(1962) and shown to be protein particles tYee of carbohydrate and nucleic acid. The synthesis of this pyocin is
inducible and accompanied by cell lysis. Another group
of pyocins, also called S pyocins, was purified from
mitomycin C-induced lysate of P. aeruginosa. These
were distinguished from the R type pyocins by their
diffusion rate through agar, their sensitivity to protease
digestion and by the fact that they are not neutralised
with antiserum against any type R pyocins (Sano et al.,
1993). Cloacin C5, on the other hand, is a macromolecule similar to R pyocin (Daw, 1989). All bacteriocins
appear to represent a heterogeneous group of substances
ranging from a small protein to a high molecular weight
particle with complex structure and composition, but the
part responsible for killing activity seems to be protein in
every case.

D. Bacteriocinogeny and lysogeny

The analogy between bacteriocin production and

bacteriophage liberation was noted following the discovery of bacteriocins, lntial studies conducted by Gratia
(1925) established the differences between colicins and
bacteriophages, notably the absence of bacteriophagelike multiplication of colicins. Later investigation reestablished certain connections, particularly between
those bacteriophages and colicins that attach to the
same receptors (Alatossava, 1994). Ultrastructural
studies showed a certain analogy between these two
bactericidal agents (Daw, 1989; Daw and Falkiner,
1993). Bacteriocins were considered to be incomplete
phages. This was evident in such studies, as shown in
Figs 1 and 2. A relaxed form of a bacteriocin isolated
from E. cloacae is shown (Fig. 1) and a bacteriophage
isolated from the same strain (Fig. 2). The phage is
composed of polyhedral head and tail with contractile
sheath and core, as well as tail fibres (Daw. 1988). By
comparisiom these two figures show a considerable
resemblance of phage tails to the bacteriocin particles.

470

M.A. Daw and F. R. Falkiner

Fig. 2. A micrograph of bacteriophage isolated from the same

strain of Enterobacter cloacae, formed from head (H), phage
tail (T) and tail fibres (TF). The phage tail (Y) resembles the
bacteriocin particles (A). Scale marker indicates 0.1 nm.
Pyocin R4

Phage PS3

180
9o

160

70

150

70

Shrunk
Intact
Shrunk
Intact
Fig. 3. Morphological comparison of phage PS3 and R-type
pyocin of Pseudomonas aeruginosa. The phage tail is slightly
larger than the bacteriocin particle. Dimensions are shown
in A.

The bacteriocin seems to be headless phage. This emphasised the striking structural resemblance between bacteriocin 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 et al., 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 et al., 1992). Unlike prophage induction,
induction of bacteriocin synthesis is not necessarily
accompanied by enhanced replication of the Col factor.
In Proteus mirabilis, the increase in ColE1-K30 after
mitomycin C treatment parallels the increase in bacteriocin titer, but in E. coli 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) established 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 generations. Cells releasing bacteriocin exist for several hours,
but they do not divide to produce LFC, while spontaneous induction of prophages varies similarly in batch
cultures. Although bacteriocins and bacteriophages lend
themselves to obvious comparisons, there is no indication 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, therefore, conclude that in spite of many remarkable similarities between lysogeny and bacteriocinogeny, there is
no compelling reason to believe that they are in any way
connected.

II. ULTRASTRUCTURAL STUDY OF

BACTERIOCINS
A. General structure

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).

Bacteriocins

471

Colicin E3
N-terminus
35kDa
Translocation capacity
into the cell

C-terminus
15kDa
RNase activity
binding of IP

Fig. 4. Colicin E3 of Esherichi~l coli under protease digestion

yields two fragments: 53 kDa amino terminal fragment and a
15 kDa carboxyterminal fragment.

Further studies suggested that bacteriocins can fit

into two groups: those that cannot be resolved by the
electron microscope such as E, V, K and pyocin S and
others that have a definite structure, such as cloacin C5
and R pyocin. Colicin E3 represents an example of the
first group. Proteolytic digestion of colicin E3 with
trypsin under mild conditions yields two fragments, a
35 kDa amino-terminal fragment and a 15 kDa carboxyterminal fragment in complex with the immunity protein
as shown in Fig. 4, which suggested a domain structure
for colicin E3 (Ohno et al., 1977; Soham and Djebli,
1992; Yajima et al., 1992).
Most of the studies concerning the morphology
of bacteriocins were performed on particles obtained
from pyocinogenic strains of P. aeruginosa. Electron
microscopy of pyocin preparation revealed the presence
of numerous structures resembling the phage tail components as shown in Fig. 5. Contracted particles (Fig.
5a) consist of a core partially surrounded by a sheath. In
most particles the core appears empty, but occasionally
cores can be seen to contain material. Uncontracted
particles (Fig. 5b) resembled bullets and a base-plate
was visible at broader end. Hook-like pins extend from
the lower ends of contracted sheaths and occasionally
several fibres were seen attached to the base plate.
Isolated contracted sheaths were observed and long
sheath-like structures were seen composed of numerous
contracted sheaths. Hollow ringlets and minute cogwheels are also observed. The majority of particles,
regardless ot" the pyocinogenic strain used, measured
approximately 100 x 15 nm in the uncontracted state;
contracted particles consist of a hollow tailpiece, approximately 100 x 7 nm, partially enclosed in a sheath
measuring about 45 x 17 nm (Govan, 1974a). The morphology of these bacteriocins can be changed by freezing
and thawing which may render these particles inactive.
However, when purified pyocin preparations in TrisHCI buffer are submitted to a fast freeze in an acetone dry ice bath, the particles form rosette clusters.
A schematic diagram of the various morphological
forms and structures seen in electron micrographs of
pyocins is presented in Fig. 6: these consist of a contracted particle, a lock-washer ringlet (Fig. 6a) and two
forms of relaxed particles differing in size and striation
(Fig. 6b and c). These two forms are frequently seen
together in the same preparation (Higerd et al., 1969;
Govan, 1974a: Daw, 1989). In general they are all
morphologically identical except for minor differences in
dimensions; the chief feature being that they resemble
headless phages.

Fig. 5. Electron micrographs of two different forms of pyocin

obtained from strains of Pseudomonas aeruginosa. Contracted
pyocin particles consisted of core partially surrounded by a
sheath (a) and the uncontracted (relaxed) pyocin form resemble
bullets with a plate at the broader end (b). Negatively stained.
Scale markers indicate I011 nm.

Fig. 6. A schematic diagram showing bacteriocin morphology.

The contracted bacteriocin (pyocin) particle (a) resembles a
lock-washer ringlet and two relaxed pyocin particles (b. c) show
different appearances.

B. Morphology o/ active jbrms

Previously, it was not known whether bactericidal
activity of the bacteriocins was associated with a discrete
morphology or not, or whether the activity was confined to the relaxed or contracted forms (Bradley and
Robertson, 1968). Further studies found a direct correlation between the percentage of relaxed particles and

472

M . A . Daw and F. R. Falkiner

the reduction in activity during conversion to the contracted state (Higerd et al., 1969; Govan, 1974b; Daw,
1989). It was concluded that, once contraction occurs,
there appears to be little or no increase in activity upon
subsequent relaxation.
In an attempt to understand the biological nature of
contracted forms of bacteriocin, Higerd et al. (1969)
added a solution of pyocins to purified cell wall preparations of sensitive strains of P. aeruginosa. Adsorption
to the cell wall of the sensitive strains occurred at the
flattened end of the bacteriocin within one minute after
adsorption of the relaxed particles to the cell walls and a
contraction of the outer sheath was observed. Early
studies on Proteus vulgaris by Coetzee et al. (1968) also
showed that a high titer of bacteriocin preparations
treated with H 2 0 2 and ethanol and then adsorbed with a
suspension of susceptible strains, had many contracted
tail-like structures in the supernatant fluid and very few
around the susceptible organism. This suggested that the
relaxed form of the particle may be the native state of
the bacteriocin, since activity and cell wall adsorption
were demonstrable only with particles in the relaxed
state.

C. Mode o f action

Most of the information regarding the mode of action

of bacteriocins has been based on studies of several
bacteriocins performed by Nomura and his colleagues
(Nomura, 1967). Bacteriocins exert their biological
action through adsorption to their specific receptors,
located at the external surface of sensitive cells, and are
then translocated to their specific targets within these
cells (Parker et al., 1989). Kinetic data indicate that
bacteriocins behave as particulate lethal agents, killing
sensitive cells in what amounts to a single hit process.
This to the so called "quantal" killing rather than
"molar" cooperative killing action of classical antibiotics (Mayr-Harting et al., 1972).
The mode of action of bacteriocins may vary from one
type to another (Braun et al., 1994; Montville and
Bruno, 1995). Early experiments demonstrated that colicin E3 led to specific inhibition of protein synthesis
(Schwartz and Helinski, 1971). Colicin E2 leads to
specific inhibition of DNA synthesis and induces DNA
degradation. Other studies implicated the cytoplasmic
membrane as primary target of colicin A, El, K, Ia, and
Ib. These bacteriocins and other related colicins create
disruption of active transport and produce leakage of
ions by forming voltage-dependent channels in phospholipid bilayer membranes, thereby destroying the
cell's potential (Schein et al. 1978). This resulted in the
inhibition of protein and nucleic acid biosynthesis and
uncoupled electron transport from active transport of
thiomethyl-fl-D-galactoside and potassium (Lusk and
Nelson, 1972). Treated cells leaked potassium and magnesium ions. The loss of such ions has been implicated as
the primary cause of cell death (Konisky, 1982). Nevertheless, each of these colicins has its own immunity

Periplasm
Immunity protein

L2

Pore-forming domain

C
Cytoplasm

Fig. 7. The hydrophilic a-helices of colicin A of Esherichia coll.

A pore forming domain (a-helices 8 and 9) are responsible for
recognition of the immunity protein.

proteins (Weaver et al., 1981a,b; Van der Goot et al.,

1991). It is more likely that immunity proteins inactivate
their corresponding colicins by direct interaction. Different studies (Mankovich et al., 1984; Shoham and Djebli,
1992), using a construction of hybrid proteins between
colicins Ia, Ib, A and E1 have shown that the C-terminal
pore-forming domain was responsible for the reCognition of immunity protein by pore forming colicins.
Most investigators (Tokuda and Konisky, 1978; Weaver
et al., 1981b; Geli and Lazdunski, 1992a) concluded that
immunity proteins of pore forming colicins interact
with the pore-forming domains of their corresponding
colicins at the level of the inner membrane and thus their
action should prevent the formation of the pore.
Apparently the insertion of pore forming bacteriocins
is triggered by spontaneous penetration in the biolayer
of a hydrophobic helical hairpin normally buried in
the water-soluble proteins. The major determinant for
immunity specificity of such colicin is located between
a-helices 8 and 9, as shown in Fig. 7. Geli and Lazdunski
(1992b), reported that colicin A recognises its immunity
protein through its hydrophobic H2, H3 and H4
a-helices, in addition to the periplasmic loop. On the
other hand, the macromolecular bacteriocins such as R
pyocin and cloacin C5 adsorb to sensitive cells and exert
their biological activity. Kaziro and Tanaka (1965a) and
Kaziro and Tanaka (1965b) reported that the addition
of R pyocin, produced by P. aeruginosa to growing
culture of the sensitive strain, caused a rapid and complete cessation and inhibition of the synthesis of RNA,
DNA and protein in the sensitive cells. This was found
due to inactivation of the ribosomes. The killing action
of these bacteriocins is therefore reminiscent of the
action of virulent phages on the host cells (Coetzee
et al., 1968). The electron microscopic studies on the
morphological changes of the sensitive bacteria may lead
to further understanding to the mode of action of these
bacteriocins.
D. Morphological changes associated with bacteriocin
activity

The killing of susceptible bacteria by a bacteriocin is a

kinetic process. The action of those bacteriocins which

Bacteriocins

are shown to have a definite structure has been studied

in a preliminary manner using the electron microscope.
The mode of bacteriocin action is associated with different morphological changes of both, the shape of the
bacteriocins and the morphology of the susceptible
bacteria. There have been relatively few reports on the
nature of the morphological changes produced in susceptible cells on treatment with bacteriocins, particularly
Gram positive bacteria, due to the fact that most of
these bacteriocins are not resolvable by electron microscope (Vlaemynck et al., 1995). Extensive structural
changes occurred in group A Streptococcus strain after
exposure to staphylococcocin C55 (Clawson and Dajani,
1970; Ellison et al., 1971). These changes include the
condensation of nuclear material, loss of ribosomes,
modification of mesosomes and dissolution of cell contents. Similar changes were also observed in sensitive
strain of Streptococcus pyogenes (Tagg et al., 1973)
treated with streptocin A. These changes ranged from
the normal appearance of the Streptococci to those with
a condensation of nuclear material with an increase in
the perinuclear space.
On the other hand, morphological studies concerning
the bacteriocins of Gram negative bacilli were done
using pyocin particles and more recently with cloacin C5
of Enterobacter cloacae (Govan, 1974a,b; Daw, 1989:
Daw and Falkiner, 1993). These groups conducted
detailed studies on adsorption and inhibitory action of
pyocin 21 on sensitive cells of P. aeruginosa and cloacin
C5 of E. cloacae using electron microscopy. The uncontracted particles (the active forms of bacteriocins, Figs 1
and 5b) attached rapidly by the broader or base plate
end to sensitive bacteria and no immediate adsorption
of the bacteriocin usually occurs as shown in Fig. 8
(Govan. 1974a); with a P. aeruginosa indicator strain
after one minute contact with induced pyocin 21. The
bacterial surface is heavily surrounded by pyocin
particles and no contraction of adsorbed particles is
shown. Consequently, after adsorption, contraction occurs and the bacteriocins start to exert their actions as
shown in Fig. 9 (Daw and Falkiner, 1993); E. cloacae
strain sensitive to cloacin C5 after four minutes contact.
The contracted bacteriocins can be seen adsorbed to the
bacterial surface and in the vicinity of the bacterial cell.
Figure 10 (Govan, 1974a) also shows the same for a
P. aeruginosa strain after five minutes contact with
sensitive pyocins. The bacterial cell is also surrounded
by contracted bacteriocins.
After the addition of bacteriocins to sensitive bacteria
for a longer period, a variety of changes have been
observed. The entire surface of the sensitive bacterium
appears covered with rod like particles, with an imminent disruption of the bacterial cell as shown in Figs 11
and 12 (Daw and Falkiner, 1993; Daw, 1989; Govan,
1974a). The bacterial cells, at this stage, were dead and,
indeed, alter two hours incubation with the bacteriocin
preperations, more than 90 '7,, of the bacteria were seen
to be completely disrupted. This, however, resulted in
the killing of sensitive bacteria without lysis. On the
other hand. when bacteriocins were mixed with resistant

473

Fig. 8. Pseudomonas aerughu~sa strain after one rain contacl at

37C with pyocin particles. The bacterial surface is surrounded
by many uncontracted bacteriocin particles. Negatively stained
with potassium phosphotungstate 2",~, w/v. Scale marker
indicates 200 nm

+ii L

Fig. 9. Enterobacter cloacae after 4 rain contact with cloacin at

31C. Few uncontracted particles (U) are visible close to the
bacterial surFace and m a n y contracted (C) particles can be seen
adsorbed to the bacterial surface and in the vicinity of the
bacterium. Scale marker indicates 10(I nm.

strains, none of the above-mentioned killing processes occurred. The electron micrographs show no
attachment of bacteriocin particles to the surface of

474

M.A. Daw and F. R. Falkiner

(a)

Fig. 10. Pseudomonas aeruginosa after 5 min contact at 37C

with pyocin. The bacterial cell is surrounded by contracted
particles which can be seen adsorbed to the bacterial surfaces
and in the vicinity of the bacterium. Scale marker indicates
200 nm.

the resistant bacteria, and most o f the particles remained

uncontracted and some distance from the bacterial
cell, as shown in Fig. 12; E. cloacae stain resistant to
cloacin C5 after 3 0 m i n contact. The bacteriocin
particles did not attach to the surface o f the resistant
bacteria and m o s t o f them remained uncontracted (Daw,
1989).
Despite the ambiguity o f the electron microscope
evidence concerning the changes associated with bacteriocin particles during the killing process, it seems they
also go t h r o u g h m o r p h o l o g i c a l and c o n f o r m a t i o n a l
changes during their activity. Only relaxed forms adsorb
to the susceptible strain. W h e n triggered, a contracted
taiMike structure c a n n o t bind. Different agents can
effect this activity. G o v a n (1974a) f o u n d that the activity
o f the pyocin was destroyed after treatment with 0.02 %
SDS and that the pyocins lost their outer contractile
sheath and did not adsorb on the sensitive bacterial cells.
This activity was not affected when SDS was used at a
concentration less than 0.01%.
Different studies ( K r i m m and Anderson, 1967;
M o o d y , 1967a,b; Braun, 1994), suggested that the
mechanism o f contraction o f the tail sheath o f T4
bacteriophage involves a c o n f o r m a t i o n a l change in the
protein subunit, indicating c o n f o r m a t i o n a l changes in
the d o m a i n required for the infectivity process o f the
bacteriophage. Bacteriocins resemble contractile bacteriophages in m a n y respects, t h o u g h they do not

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

Pseudomonas aeruginosa sensitive to pyocin (b) after 30 min
contact at 370C with the bacteriocin particles. In both conditions, the bacterial surface appears convoluted and covered
with many bacteriocin particles contracted (C) and uncontracted (U). Empty sheaths (E) were also seen in the vicinity of
the sensitive bacterium. Scale markers indicate 200 nm.

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

The bacteriocin particles have not attached to the bacterial
surface and most of the bacteriocins have remained uncontracted. Scale marker indicates 200 nm.

replicate in sensitive host bacteria. In morphological

terms, their lack o f any structure resembling a bacteriophage head indicates a c o n c o m i t a n t lack o f the main
reservoir for nucleic acid.

Bacteriocins
Pcol >

ceal

Pimm >

E9 imm

lys

DNA -0-(
Proteins

(lllll '!lll[lll[lilli'lll)
Colicin E9

Immunity
protein

Lysis
protein

Colicin
complex

Fig. 13. Genetic organisation of colicin E9 operon of Esherichia

coli. Pcol is an SOS-inducible promoter, ceal. Colicin E9
structural gene Pimm, immunity gene promoter. E9imm:
immunity protein gene lvs; lysis gene involved in secretion of
the colicin complex.

III. M O L E C U L A R B I O L O G Y OF B A C T E R I O C I N S
A. Genetic determinants oJ" bacteriocins

The production of bacteriocins seems to be a hereditary feature associated with cytoplasmic genes (i.e
bacteriocinogenic factors). The genetic determinants of
colicins (colicinogenic factors) have been the most commonly studied (Reeves, 1972; Nieto-Lozano et al., 1992;
Schved et al., 1993; Braun et al. 1994). Most of the
genetic determinants of the bacteriocins are plasmidborne with few exceptions, including pneumocin and
pyocins, which are found to be chromosomally located
(Kageyama, 1975: Quirantes et al., 1994). Bacteriocinogenic genes may determine, not only the chemical composition 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 bacteriocins could be easily transferred from bacteriocinogenic 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 demonstrated with many colicins. E colicins are
plasmid-encoded proteins produced by E. coil, which kill
the sensitive cells by binding to the btuB-encoded cell
surface receptor (Di Masi et al., 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 et al. (1992)
studied the genetic determinant of the interaction between colicin E9 and its immunity proteins. ColE9-J
plasmid encodes the colicin structural and immunity
genes in an operon (Chak and James, 1986). The promoter of E9imm 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

475

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 et al., 1993). Such agents could
eliminate the plasmids encoded for the bacterJocins.
Bacteriocinogenic factors from Staphyh,coccous aureus,
and Colistridium perferinges have been "cured" by such
methods (Gagliano 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 trpCD and tlT~E(Kageyama,
1975; Shinomiya et al., 1983), the locus for pyocin AP41
is between @s'-9015 and argF and the locus for pyocin S
is close tofla Y (Sano and Kageyama, 1984: Sano et al.,
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 direction; 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.
coli cells using DEAE-cellulose and chromotography
(Ohkawa et al., 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 transformed with a cloned DNA for the smaller component,
they became insensitive to the respective pyocin (Sano
et al., 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.

476

M . A . Daw and F. R. Falkiner

C-Terminals
0
I

T-Terminals

400
I

II

800
I Amino acids

III

IV

R640
Pyocin
AP41

I',.I.

IIIIlIIIIIlIIIlIV//Av76

',I
">,\

',l ,,''1;',

li

SI

il

R487

iill'il!!"

,7 ,7,7
II

IilV///A617

It

~" [{// l ///

R558

II

$2

1', I', I',

/

1illili111! :lII]iIV///A689
/

~" - [ /

R452

Colicin E2
R454

E3

~//AIIlllllllllk

Cloacin DFI3

I il]l]lllllllllll[I

:
Translocation

.+~\~ 55l

: :

: :R464

tk\\q 561
Receptor
nuclease

Fig. 14. A m i n o acid sequence homology in the killer components of pyocins AP41, S l a n d $2 of Pseudomonas aeruginosa,
colicins E2 and E3 of Escherichia coil and cloacin DF13 of
Enterobacter cloacae. D o m a i n s showing homology have the
same shading. Lengths are proportional to the n u m b e r of
amino acids of each molecule. An Arg residue near the start
point of each nuclease domain is fixed as a reference point
(R640 in AP41). The N-terminal Met is lacking in the purified
pyocins.

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, Streptococcus faecalis and Colistridium perJkingens. (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 mycobacteria such as the Mycobacteriumfortuitum chelonei complex cause serious infections in immunocompromised
patients and those with acquired immune deficiency

syndrome (AIDS). Mycobacterium species, isolated from

patients with prosthetic heart valves and hip joints, can
be classified to specific bacteriocin profiles. The mycobacteriocin 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 determination as to whether all
strains of a particular pathogenic organism isolated from
an outbreak of 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 bacteriocin 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).discrimantory
and reproducible, (3) does not need a sophisticated
instruments and (4) can be easly used by a small laboratories 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 hospitalised patients (Daw, 1989). The microbiological relevance of bacteriocin is evident. As an epidemiological
tool, the system was found to be applicable and comparable to the classical typing methods. Further to
its practicability, bacteriocin typing almost always provides 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 bacteriocin 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 classified according to their electron microscopical appearance. 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

Bacteriocins

surface of the sensitive strain, followed by major biological and morphological changes of both the bacterial cell
and the bacteriocin particle. This results in the killing
of the sensitive strain without production of further
bacteriocin particles, one of the main features which
makes them clearly different to bacteriophages.
The most intensively studied groups of bacteriocin are
the colicins which are produced by E. coli and closely
related enterobacteria.
They are are typically plasmid-encoded proteins. The
other major group is the pyocin of P. aeruginosa, which
is controlled by the presence of specific genetic determinants encoded on the chromosome of the producing
bacteria.
In addition to the intensive studies on these bacteriocins and their contribution to modern biology, they have
gained new attention in medicine, particularly in the
epidemiology of nosocomial infections. Due to the
simple production of the bacteriocins, it makes them a
favourite typing assay for pathogenic organisms. This
allows control of the cross-infections which occur in
the hospitals that may result in serious consequences,
particularly among immunocomromised patients.
Acknowledgements--The authors would like to thank Drs Mohamed J.
Mabruk, K. S. Genghish, M. I. Mahmoud, Naget Saied, Mustafa
Hajaji, M. Farouki, S. Tloba and A. Rahoma, departments of Medical
Microbiology and Parasitology, Faculty of Medicine, Tripoli, Libya
for their helpful comments and suggestions.

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