Biochimie 84 (2002) 545557

Mode of action of modified and unmodified bacteriocins

from Gram-positive bacteria
Yann Hchard a,*, Hans-Georg Sahl b
a
Laboratory of Fundamental and Applied Microbiology, University of Poitiers, 40, avenue du Recteur-Pineau, 86022 Poitiers, cedex, France
b
Institute for Medical Microbiology and Immunology, University of Bonn, Sigmund Freud Strasse 25, 53127 Bonn, Germany
Received 6 March 2002; accepted 7 June 2002

Abstract

The antibiotic activity of bacteriocins from Gram-positive bacteria, whether they are modified (class I bacteriocins, lantibiotics) or
unmodified (class II), is based on interaction with the bacterial membrane. However, recent work has demonstrated that for many
bacteriocins, generalised membrane disruption models as elaborated for amphiphilic peptides (e.g. tyriodal pore or carpet model) cannot
adequately describe the bactericidal action. Rather, specific targets seem to be involved in pore formation and other activities. For the nisin
and epidermin family of lantibiotics, the membrane-bound cell wall precursor lipid II has recently been identified as target. The duramycin
family of lantibiotics binds specifically to phosphoethanolamine which results in inhibition of phospholipase A2 and various other cellular
functions. Most of the class II bacteriocins dissipate the proton motive force (PMF) of the target cell, via pore formation. The subclass IIa
bacteriocin activity likely depends on a mannose permease of the phosphotransferase system (PTS) as specific target. The subclass IIb
bacteriocins (two-component) also induce dissipation of the PMF by forming cation- or anion-specific pores; specific targets have not yet
been identified. Finally, the subclass IIc comprises miscellaneous peptides with various modes of action such as membrane permeabilisation,
specific inhibition of septum formation and pheromone activity. 2002 Socit franaise de biochimie et biologie molculaire / ditions
scientifiques et mdicales Elsevier SAS. All rights reserved.

Keywords: Bacteriocin; Lantibiotic; Antibacterial; Pore.

1. Introduction proposed on the basis of their primary structure [1,2]. The

Antibacterial peptides or proteins ribosomally synthe-
sised by bacteria are commonly referred to as bacteriocins.
Most of those produced by Gram-positive bacteria are
synthesised by lactic acid bacteria (for a review see [1,2]).
The bacteriocins are generally secreted via a dedicated ABC
transporter but some of them are secreted via a sec-
dependent pathway. The bacteriocin gene is usually associ-
ated to a gene encoding the so-called immunity protein. The
latter protects bacteria from their own bacteriocin, although
the protection mechanism remains still unclear for most
immunity proteins. Bacteriocins classification has been
Abbreviations: Dha, didehydroalanine; Dhb, didehydrobutyrine; MIC,
minimum inhibitory concentration; PMF, proton motive force; CF, car-
boxyfluoresceine; PTS, phosphotransferase system
* Corresponding author. Tel.: +33-0-549-454-007; fax: +33-0-549-453-
503.
E-mail address: yann.hechard@univ-poitiers.fr (Y. Hchard).
2002 Socit franaise de biochimie et biologie molculaire / ditions scientifiques et mdicales Elsevier SAS. All rights reserved.
PII: S 0 3 0 0 - 9 0 8 4 ( 0 2 ) 0 1 4 1 7 - 7
class I is composed by modified peptides, named lantibiot-
ics. The class II gathers unmodified peptides. The class III
is composed by large bacteriocins whose mode of action
was poorly studied. Thus, the latter will not be described in
this review.
2. Modified bacteriocins (class I)
A substantial proportion of the peptide bacteriocins of
Gram-positive bacteria undergo extensive post-translational
modification before they are exported from the cell (class I
bacteriocins). The most prominent modifications include the
dehydration of serine and threonine residues to didehy-
droalanine (Dha) and didehydrobutyrine (Dhb), respec-
tively, and the subsequent addition of suitably located
cysteine residues via their SH groups to the C=C double
bond of Dha or Dhb. The thioether amino acids resulting
546 Y. Hchard, H.G. Sahl / Biochimie 84 (2002) 545557
from this addition reaction are, in contrast to disulfide
bridges, acid stable and can be identified in peptide hydroly-
sates as lanthionine (originating from Dha and Cys) and
3-methyllanthionine (from Dhb and Cys). It was the pres-
ence of the lanthionines that led to proposing the designa-
tion lantibiotics as an abbreviation for lanthionine-
containing peptide antibiotics. Details on the chemistry of
lantibiotics and the biosynthesis pathways can be found in
several recent reviews [36]. It should be noted that,
although the unusual residues may give the impression that
lantibiotics are related to classical peptide antibiotics such
as gramicidin or valinomycin, the ribosomal biosynthesis
clearly classify them as bacteriocins [4,7]. It appears that
only the presence of dedicated enzymes for amino acid
dehydration and thioether formation can distinguish biosyn-
thetic gene clusters of lantibiotics from those of class II
bacteriocins [4].
Ever since the structure of the most prominent lantibiotic
nisin (Fig. 1) had been elucidated [8] it was speculated that
the unusual amino acids may be important for function.
Particularly the dehydro residues were discussed to repre-
sent ideal sites for addition of thiol groups and other
nucleophiles. Such covalent modifications were thought to
result in inactivation of enzymes, coenzymes and other
compounds with functionally important groups. Indeed,
there is evidence that such a mechanism is involved in the
sporicidal activity of nisin and subtilin [9]. However, it
appeared unlikely that such a specialised mechanism could
account for the rather generalised broad-spectrum antibiotic
Fig. 1. Structure of the lantibiotics nisin (a) and mersacidin (b); residues which derive from posttranslational modification of Ser, Thr and Cys are marked
in gray; Dha, dehydroalanine; Dhb, dehydrobutyrine; Abu, aminobutyric acid; AlaSAla, lanthionine; AbuSAla, 3-methyllanthionine; for further details
on the amino acid modifications see text and [37].
activity of nisin and related lantibiotics such as epidermin,
subtilin, Pep5, mersacidin and others.
The first report on the mode of action of nisin appeared
as early as 1953 by Ramseier [10] who described a
detergent-like membrane disruption based on the observa-
tion that nisin treated bacteria rapidly leaked UV-absorbing
cellular material such as nucleotides. Later studies unrav-
elled an inhibitory role of nisin in cell wall biosynthesis [11]
which could then be attributed to interaction of the lantibi-
otic with the membrane-bound cell wall precursors lipid I
and lipid II (Fig. 2) [12]. However, subsequent investiga-
tions with growing bacteria clearly showed that a great
majority of the cells were killed rapidly along with an
instant depolarisation of the cytoplasmic membrane, leak-
age of cellular material and complete stop of biosynthetic
processes [1315]. The apparent discrepancies in the inter-
pretation of the molecular nature of the nisin killing activity
were only recently solved and united into one mode-of-
action model [1618]. It was demonstrated that nisin and
epidermin use lipid II primarily as a docking molecule for
formation of highly effective, targeted pores. Simulta-
neously, cell wall biosynthesis is inhibited through arresting
the precursor in the membrane. In addition, nisin is able to
form non-targeted pores as proposed earlier [19] and,
particularly in staphylococci, it activates cell wall hydrol-
ysing enzymes [20,21]. Thus, its antibiotic activity is based
on a multiplicity of activities which may combine differ-
ently for individual target bacteria and explain the range of
sensitivities of various bacterial species. In the following,
 Y. Hchard, H.G. Sahl / Biochimie 84 (2002) 545557
Fig. 2. Structure of the ultimate membrane-bound cell wall precursor
undecaprenylpyrophosphoryl-MurNAc(pentapeptide)-GlcNAc, lipid II.
the individual activities of lantibiotics are described in more
detail.
2.1. Amphiphilic cationic lantibiotics (type-A) can form
target-independent pores
Cationic amphiphilic peptides are wide-spread in nature.
Throughout the living world, they are produced by micro-
organisms to antagonise competitors and by plants and
insects and vertebrates as effector molecules to prevent and
combat microbial infections. The destructive effects of
amphiphilic compounds on membranes with a negative
surface charge has been known for a long time and there is
a wealth of information on the biophysical behaviour of
synthetic and natural peptides [22]. In line with the early
observation of Ramseier [10], it was initially assumed that
nisin and related lantibiotics similarly disintegrate microbial
membranes. There are numerous reports demonstrating that
these peptides kill bacterial cells by interference with
energy transduction occurring at the cytoplasmic mem-
brane. Addition of nisin and other type-A peptides immedi-
ately inhibits biosynthesis processes of macromolecules
such as DNA, RNA, proteins and polysaccharides [15].
Furthermore, bacterial cells were unable to actively take-up
amino acids and become leaky for inorganic ions and small
metabolites [13]. A concept of energisation-dependent activ-
ity was deduced from experiments with cytoplasmic
vesicles and intact cells as well as with model membranes.
Conductance measurements using artificial bilayer mem-
branes (black lipid membranes, BLM) were in good agree-
ment with the results obtained with intact cells and physi-
ological membranes [14,23]. There was no macroscopic
membrane conductivity below a certain threshold potential;
the minimum voltage ranged from 50 to 100 mV for
various peptides, and calculated pore diameters were be-
tween 1 nm for nisin and about 2 nm for subtilin with a
lifetime in the millisecond range [2325].
Initially, it was discussed that a barrel-stave model may
adequately describe the activity of these lantibiotics. A
barrel-stave model, in which -helical amphipathic peptides
547
bind via electrostatical interaction to the outer leaflet in a
parallel orientation to the membrane surface, had been
elaborated e.g. by Boheim [21] and others [22] for alame-
thicin. In order to avoid the unfavourable position of polar
residues to the lipid acyl chains several monomers of
peptides have to assemble forming a bundle of helices. After
insertion of the peptides into the membrane the non-polar
side chains of the peptides interact with the hydrophobic
lipid core of the membrane and the hydrophilic side chains
point inward, which results in the formation of a water-filled
pore. Both, the size and the stability of the barrel-stave
channel, depend on the number of peptides involved in pore
formation.
Based on conformational data derived from NMR studies
with nisin in the presence of membrane-mimicking micelles
[26,27] a wedge model for pore formation by lantibiotics
was proposed (Fig. 3a) [27,28]. This model takes into
account that type-A lantibiotics are rather flexible in aque-
ous solution and defined structural elements have only been
identified in small thioether rings [29,30]. Upon contact
with the membrane, the peptides adopt an amphiphilic
conformation with the charged residues aligned to one face
of the molecules and the hydrophobic residues aligned to
the other. The cationic peptides interact with the phospho-
lipid head groups by ionic forces causing a locally disturbed
bilayer structure, while the hydrophobic residues insert into
the membrane.
Studies with nisin demonstrated that the C-terminal
region as well as the overall negative surface charge of the
membrane [3135] were important for binding and pore
formation. It is assumed that several molecules have to
associate within the membrane in order to form a pore, since
lantibiotics are too short to transverse the membrane more
than once. On the other hand, several transmembrane
segments are necessary to form a pore with a diameter of
1 nm or more as deduced from BML studies. The formation
of pores leads to dissipation of the membrane potential and
promotes a rapid efflux of small metabolites such as amino
acids or ATP, which in turn immediately stops all cellular
biosynthetic processes as mentioned above.
2.2. Nisin and epidermin: formation of high affnity target-
mediated pores
The above model may describe the behaviour of peptides
in pure lipid bilayers where high peptide concentrations are
needed to induce effects; also, the killing activity for those
microbes which do have micromolar minimum inhibitory
concentrations (MICs) may well be primarily based on such
a mechanism. However, nisin and related bacteriocins
frequently kill bacteria in the nanomolar concentration
range, indicating that additional activities or specific targets
may be involved. Since type-A lantibiotics can act on
artificial membranes, a concept of specific targets being
involved in the membrane interaction had not been consid-
ered before. However, for nisin, a finite number of binding
548 Y. Hchard, H.G. Sahl / Biochimie 84 (2002) 545557

Fig. 3. Molecular activities of of type-A and B lantibiotics. (a) at micromolar concentrations, the cationic type-A peptides (nisin, epidermin, Pep5 and others)
from wedge-like, target-independent pores. (b) at nanomolar concentrations, nisin and epidermin form target-mediated pores using lipid II as a docking
molecule. (c) mersacidin and actagardine also bind to lipid II but only block its incorporation into peptidoglycan; such an activity is intrinsic to nisin and
epidermin as well, and becomes obvious with mutant peptides which are deficient in pore formation [18].

sites and specific antagonisation of nisin activity by the
inactive N-terminal nisin fragment 112 had been observed
[36], indicating that a defined binding site may be blocked
by the fragment. With that respect, it was important to recall
publications by Linnett and Strominger [11] who reported
that nisin inhibits peptidoglycan biosynthesis, and that it
binds to the membrane-bound peptidoglycan precursor
undecaprenylpyrophosphoryl-MurNAc(pentapeptide)-
GlcNAc, the so-called lipid II.
These reports raised the idea that lipid II may be involved
on the formation of pores. This was subsequently shown for
nisin and epidermin using lipid II supplemented liposomes
[16]. Detailed in vitro studies then demonstrated that lipid II
serves as a docking molecule for specific binding to the
bacterial membrane (Fig. 3b) [17,18]. Genetically engi-
neered nisin variants helped to identify the structural re-
quirements for the interaction of the peptide with lipid II
[18]. Mutations affecting the conformation of the
N-terminal part of nisin comprising rings A through C (e.g.
[S3T]nisin), led to reduced binding and increased the
peptide concentration necessary for pore formation; i.e. the
binding constant for the [S3T]mutant was 0.043 107 (M1)
 Y. Hchard, H.G. Sahl / Biochimie 84 (2002) 545557
as compared to 2 107 (M1) for the wild type peptide and
the minimum concentration for pore formation increased
from the 2 to 50 nM range.
Peptides with mutations in the flexible hinge region (e.g.
[N20/M21]nisin) were completely inactive in the pore
formation assay. When tested in vivo against living bacteria,
their activity was only reduced to some extent. The remain-
ing in vivo activity was shown to be due to the unaltered
capacity of the mutated peptide to bind to lipid II, and thus
to inhibit the incorporation of lipid II into the peptidoglycan
network. The N-terminal part of nisin is essential for
binding to the membrane bound cell wall precursor lipid II
and the resulting inhibition of the peptidoglycan synthesis.
In contrast to the nisin induced pore formation without a
docking-molecule, a negative surface charge of the mem-
brane is not necessary. However, the positively charged
C-terminus of nisin is still important for the initial binding
to the anionic cell wall polymer and thus for antimicrobial
activity in vivo.
Since the N-terminal double ring system of nisin is
instrumental for the interaction with lipid II, it seems likely
that subtilin and the entire group of epidermin-like lantibi-
otics [3,4,7] act similarly. All these lantibiotics share the
general setup of this ring system and, in fact, epidermin
itself has been shown to inhibit peptidoglycan synthesis and
to promote liposome leakage [16]. The combination of two
killing mechanisms, inhibition of the peptidoglycan synthe-
sis and pore formation, in one molecule obviously potenti-
ates in vivo the antibiotic activity and results in nanomolar
MIC values, a strategy that may well be worth considered
for the construction of novel antibiotics. Details on the
molecular architecture of the lipid II-mediated pores remain
to be elucidated, although it seems likely that several such
stochiometric peptide: lipid II complexes must assemble to
form a pore.
2.3. Mersacidin, actagardine, nisin and epidermin:
inhibition of peptidoglycan biosynthesis via complex
formation with lipid II
The exploration of the above described duality of nisin
and epidermin activity was greatly influenced by preceding
studies on the type-B lantibiotics mersacidin and actagar-
dine. Both are active against a variety of Gram-positive
bacteria, with actagardine being most effective against
streptococci and obligate anaerobes [37], while mersacidin
(Fig. 2) is almost equally active against staphylococci,
streptococci, bacilli, clostridia, corynebacteria, peptostrep-
tococci, and Propionibacterium acnes [38,39]. Gram-
negative bacteria are not susceptible, since peptides cannot
pass the outer membrane of bacteria. Although both lanti-
biotics in vitro have only moderate MIC values, mersacidin
has attracted recent attention due to its significant in vivo
activity. It effectively cured systemic staphylococcal infec-
tions in mice including those caused by methicillin-resistant
549
Staphylococcus aureus (MRSA strains), as well as subcuta-
neous staphylococcal abscesses in rats. In both cases the
lantibiotic equalled or even exceeded the activity of vanco-
mycin [39].
Mersacidin and actagardine were shown to interfere with
the cell wall biosynthesis by inhibiting the incorporation of
glucose and D-alanine into cell wall material of Staphylo-
coccus simulans 22, whereas DNA, RNA and protein
synthesis proceeded unhindered. Both peptides inhibit pep-
tidoglycan biosynthesis at the level of transglycosylation by
forming a complex with the membrane-bound peptidogly-
can precursor lipid II [40,41]. The binding of [14C]mersa-
cidin to growing cells as well as to isolated membranes
capable of in vitro peptidoglycan synthesis was strictly
dependent on the availability of lipid II, and antibiotic
inhibitors of lipid II formation strongly interfered with
binding of mersacidin. Furthermore, labelled mersacidin
tightly associated with micelles formed from purified and
labelled lipid II, and the addition of isolated lipid II to the
culture broth efficiently antagonised the bactericidal activity
of mersacidin. The molecular target site of mersacidin and
actagardine on lipid II differs from that of nisin (see above)
and of the glycopeptide antibiotic vancomycin, which binds
to lipid II via the C-terminal D-AlaD-Ala of the pentapep-
tide side-chain; in contrast, mersacidin and actagardine
rather interact with the disaccharide-pyrophosphate moiety
of lipid II (Fig. 3c) [41]. The two lantibiotics contain one
ring structure that has been almost completely conserved in
both molecules, indicating its importance for activity
[40,42].
2.4. Additional activities of type-A lantibiotics
In addition to pore formation and inhibition of the cell
wall biosynthesis, nisin and the related cationic lantibiotic
Pep5 have been shown to induce autolysis of susceptible
staphylococcal cells, resulting in massive cell wall degra-
dation, most markedly in the area of the septa between
dividing daughter cells. The peptides are able to release two
cell wall hydrolysing enzymes, an N-acetylmuramoyl-L-
alanine amidase and an N-acetylglucosaminidase, which are
strongly cationic proteins binding to the cell wall via
electrostatic interactions with the negatively charged
teichoic-, teichuronic-, and lipoteichoic acids; tight binding
to these polymers keeps the autolysins inactive. The cationic
peptides displace enzymes from the cell wall intrinsic
inhibitors by a cation exchange-like process, resulting in
apparent enzyme activation and rapid cell lysis [20,21].
Furthermore, nisin and subtilin inhibit the germination of
bacterial spores. This activity obviously depends on the
presence of Dha residues in position 5 of both peptides [9].
It is presumed that the double bond provides a reactive
group for interaction with a spore-associated factor that is
essential for outgrowth of spores.
550 Y. Hchard, H.G. Sahl / Biochimie 84 (2002) 545557

2.5. Cinnamycin-like lantibiotics: complex formation
with phosphatidylethanol-amine
The cinnamycin-like type-B lantibiotics display antibac-
tericidal activity against a few bacterial strains, in particular
specific Bacillus strains. Treated cells show increased mem-
brane permeability [43] as well as an impaired ATP-
dependent protein translocation [44] and calcium uptake
[45]. In addition, duramycin and cinnamycin inhibit a
number of metabolic processes in eucaryotic cells [46,47];
e.g. induction of hemolysis of erythrocytes [48] or inhibi-
tion of phospholipase A2, thereby interfering with prostag-
landin and leucotriene biosynthesis [49]. These biological
activities had usually been identified individually, e.g. in
pharmaceutical screening assays and little effort was under-
taken to deduce a common motif for molecular details.
However, it is reasonable to assume that such activities may
be explained on the molecular level by the ability of this
group of lantibiotics to form a specific complex with
phosphatidylethanol-amine [49].
2.6. Conclusions
As a general scheme it can be stated that lantibiotics act
on the cytoplasmic membrane of bacteria. Some, e.g.
mersacidin, actagardine and the cinnamycin group, bind to
specific lipid or lipid-bound targets and inhibit subsequent
enzyme reactions. The cationic amphiphilic lantibiotics, at
micromolar concentrations, disrupt the membrane in a
non-targeted fashion. Additionally, they may also bind to
specific targets and use it for high-affinity, target-mediated
pore formation. For nisin and the epidermin group, the
target has been identified and is obviously the same as for
the non-pore forming mersacidin and actagardine, i.e. lipid
II. For others such as Pep5, target-mediated poration is
likely, although the docking molecule remains to be identi-
fied. Interestingly, when a specific target is used for pore
formation, it is simultaneously blocked from its natural
function, thus yielding very effective combined antibiotic
activities by the corresponding lantibiotic. Additional anti-
biotic effects, such as induction of autolysis by nisin and
Pep5, further complicate the mode-of-action picture, but
may well explain why lantibiotics can be so potent and
should be stimulating for the construction of new generation
antibiotic drugs.
3. Unmodified bacteriocins (class II)
Class II bacteriocins are unmodified, cationic and hydro-
phobic peptides of 2060 amino acids in length (for a
review see [50]). They are divided into three subclass,
namely IIa, IIb, and IIc, on the basis of their primary
structure. Their activity (in the nanomolar range) mainly
induces membrane permeabilisation and leakage of mol-
ecules from sensitive bacteria (see Table 1). The inhibition
spectrum is rather narrow, limited to species or strains
related to the producers. Accordingly, class II bacteriocins
are mainly active against low G+C Gram-positive bacteria,
such as lactic acid bacteria, Listeria, Enterococcus and
Clostridium.
3.1. The subclass IIa bacteriocins
Among the so far described class II bacteriocins, more
than twenty share high similarities in their primary structure
(Fig. 4) as well as an anti-Listeria activity. These bacterio-
cins are gathered into the subclass IIa, for a recent review

Table 1
Mode of action of class II bacteriocins
Mode of action pH ATP Efflux References
Subclass Iia
Bavaricin MN Pore formation Dissipation Dissipation ND CF [61]
Enterocin P K+ pores No effect Dissipation Depletion of K+, CF [7375]
intracellular ATP
Mesentericin Y105 Pore formation PTS ND Dissipation ND Amino acids [6470]
permease-dependent
Pediocin PA-1 Pore formation Dissipation Dissipation Depletion of Amino acids K+ [5260]
intracellular ATP
Subclass Iib
Lactacin F Pore formation ND Dissipation Depletion of K+ and Pi efflux [76]
intracellular ATP
Lactococcin G Cation pores No effect Dissipation Depletion of Amino acids [7880]
intracellular ATP
Plantaricin EF Cation pores Dissipation Dissipation ND Cations [77]
Plantaricin JK Anion pores Dissipation Dissipation ND Anions [77]
Subclass Iic
Lactococcin A Pore formation ND Dissipation ND Amino acids [82]
Lactococcin 972 Inhibition of septum ND ND ND No efflux [8385]
formation
Plantaricin A Pore formation Dissipation Dissipation ND ND [81]
.
ND, not determined; CF, carboxyfluorescein.
 Y. Hchard, H.G. Sahl / Biochimie 84 (2002) 545557
Fig. 4. Alignment of amino acid sequences of subclass IIa bacteriocins (by Clustalw). CurA: curvacin A, EntP: enterocin P, BavMN: bavaricin MN, Div41:
divercin V41, EntA: enterocin A, Mun: muntcidin, SakP: sakacin P, PedA: pediocin PA-1, MesY: mesentericin Y105, LeuA: leucocin A, CarB2:
carnobacteriocin B2.
see [51]. In particular, they bear a highly conserved
N-terminal part including the YGNGV consensus motif, a
disulfide bridge and several conserved residues.
3.1.1. Pediocin PA-1
Pediocin PA-1 and other identical bacteriocins produced
by Pediococcus acidilactici (pediocin AcH, pediocin SJ-1,
pediocin JD) are the most extensively studied class II
bacteriocins. Bhunia et al. [52] first reported on the mode of
action of subclass IIa bacteriocins. They described that
treatment with pediocin AcH results in the leakage of K+
and some UV absorbing materials. Pediocin PA-1 was
shown to dissipate the membrane potential () of Pedio-
coccus pentosaceus and to cause release of amino acids
accumulated either in a proton motive force (PMF)-
dependent or -independent manner [53]. Furthermore, pe-
diocin PA-1 induces the release of amino acids and other
low molecular weight compounds from the vesicles of
sensitive cells while it hardly induces carboxyfluoresceine
(CF) efflux from liposomes, suggesting that a target protein
may be required for bacteriocin activity [53]. Contradictory
data have been reported by Chen et al. [54] showing that
pediocin PA-1 induces CF efflux in a concentration-
dependent manner from liposomes, suggesting that a protein
receptor is not required. They also indicate that the binding
of pediocin PA-1 to liposomes is dependent on electrostatic
interactions and not on the YGNGV consensus motif
[55,56]. The presence of a specific target at the cell surface
was also suggested by Fimland et al. [57]. In their study, a
15-mer peptide fragment (residue 2034 from pediocin
PA-1) was shown to inhibit the activity of pediocin PA-1
and other subclass IIa bacteriocins (i.e. enterocin A, leuco-
cin A and curvacin A) but to a lesser extent. These data
suggest that the 15-mer fragment interferes specifically with
the interacting bacteriocins and target cells. Pediocin JD, a
bacteriocin that may be identical to pediocin PA-1 [53], was
shown to dissipate both the and the pH of Listeria
monocytogenes sensitive cells [58]. In addition, Waite et al.
demonstrated that pediocin JD might inhibit PEP-mediated
glucose uptake via a PTS system [59,60].
It is finally proposed that pediocin PA-1 might modify
the permeability of sensitive cells likely by forming pores in
551
the cytoplasmic membrane and that it needs a specific target
molecule at the surface of the sensitive cells.
3.1.2. Bavaricin MN and divercin V41
Bavaricin MN and divercin V41 are two related bacte-
riocins (Fig. 4) produced by Lactobacillus sake [61] and
Carnobacterium divergens, respectively [62]. Bavaricin
MN was shown to dissipate both the and the pH of
energised L. monocytogenes cells and to induce CF efflux
from lipid vesicles in a concentration-dependent manner
[61]. Divercin V41 structurefunction relationships were
studied via chemical modifications and enzymatic hydroly-
sis [62]. Divercin V41 was cleaved by the endoproteinase
Asp-N in two peptides (i.e. 117 and 1843 residues). The
second peptide remains active against L. innocua, but the
activity was not quantified or compared to that of the whole
divercin V41. This result, suggesting that the N-terminal
part is not necessary for divercin V41 activity, is contradic-
tory with other data indicating that even minor modifica-
tions of the structure of subclass IIa bacteriocins lead to a
dramatic drop in the activity. In addition, chemical modifi-
cations of divercin V41, leading to net charge variation,
were performed to determine their influence on the bacte-
riocin potency. Indeed, it was proposed that electrostatic
interactions between the cationic peptide and the anionic
membrane could play a major role in bacteriocin activity. In
contrast to the previous postulate, acetylated divercin V41
still retains inhibitory activity, although this derivative is
anionic (net charge 2) [62]. The authors suggest that the
hydrophobicity of the C-terminal is more important for the
activity than its cationic nature.
3.1.3. Mesentericin Y105 and leucocin A
Mesentericin Y105 and leucocin A are two related
bacteriocins produced by Leuconostoc mesenteroides and
Leuconostoc gelidum, respectively. They display a very
similar structure with only two differences in their sequence
(Fig. 4). The three-dimensional structure of leucocin A was
reported [63], a three-strand antiparallel -sheet domain was
found in the N-terminus while the region from position 17
to 31 shows an amphiphilic -helix conformation. Mesen-
tericin Y105 was shown to exhibit a bactericidal mode of
552 Y. Hchard, H.G. Sahl / Biochimie 84 (2002) 545557
action towards L. monocytogenes [64] and to dissipate the
of whole Listeria cells [65]. Even minor modifications
either in N- or C-terminus of the mesentericin Y105
sequence impairs the activity and that the disulfide bridge is
important for the activity [66], as shown with other subclass
IIa bacteriocins.
Furthermore mesentericin Y105- and leucocin
A-resistant strains were used to investigate the molecular
mode of action of these bacteriocin. First, the inactivation of
the rpoN gene, encoding the 54 subunit of the RNA
polymerase, results in resistance of L. monocytogenes [67]
and E. faecalis [68] to mesentericin Y105. This shows that
54 directs the expression of a protein responsible for
sensitivity. Furthermore, the inactivation of the 54-
dependent mpt operon induces resistance of L. monocytoge-
nes [69] and E. faecalis [70]. The mpt operon encodes a
mannose permease of the phosphotransferase system (PTS),
EIItMan, made of three subunits IIAB, IIC and IID. In
addition, increasing glucose or mannose concentration in-
duces simultaneous expression of EIItMan and sensitivity to
mesentericin Y105 to L. monocytogenes, suggesting that the
expression level of EIItMan affects the sensitivity [69].
Besides, the lack of a mannose PTS subunit expression was
first described in a leucocin-resistant L. monocytogenes by
Ramnath et al. [71]. This protein is similar to the IIAB
subunit of EIItMan, favouring a similar (or identical) mecha-
nism for resistance to mesentericin Y105 and leucocin A.
Furthermore, a recent study strongly points out that leucocin
A actually requires an interaction with a chiral receptor at
the surface of the target cell to be active [72]. Indeed, the
D-enantiomer of leucocin A is not active against ten
different strains, whereas all of them are sensitive to the
natural leucocin A.
Taken together these results strongly indicate that EIItMan
could be the chiral receptor needed for bacteriocin interac-
tion at the surface of target cells. The membrane-associated
IIC and/or IID subunits would interact directly with the
bacteriocins. The IID subunit was finally proposed to be the
putative target molecule [69]. Alternatively, one could
hypothesise that EIItMan is not the target molecule but could
control its expression.
3.1.4. Enterocin P
Enterocin P is a 44 amino-acid bacteriocin produced by
Enterococcus faecium. Enterocin P is secreted by the
sec-dependent pathway and exhibits a broad spectrum of
activity (i.e. S. aureus, C. perfringens, C. botulinum and
L. monocytogenes) compared to most subclass IIa bacterio-
cins [73]. Enterocin P was shown to dissipate the of
previously energised cells but not of non-energised cells
[74]. In addition, enterocin P was shown to induce an efflux
of 86Rb+ (a K+ analogue) from energised cells, whereas this
efflux was not observed from either enterocin P-resistant
cells or liposomes [74]. Enterocin P was also shown to
deplete intracellular level of ATP from energised sensitive
cells even though no appearance of ATP was found in the
external medium [75]. Finally, the authors suggest that
enterocin P could form potassium ion-conducting pores in
the cytoplasmic membrane of the target cells and that a
receptor-like factor might exist in the sensitive cells despite
of the rather broad spectrum of antibacterial activity.
3.2. The subclass IIb: two-peptide bacteriocins
The subclass IIb includes bacteriocins whose activity
depends on the complementary action of two distinct
peptides. Accordingly, individual peptides hardly display
any activity. There is only one immunity gene, linked to the
two structural genes for the peptides, which encodes a
unique protein. The primary structure of the two peptides is
clearly different and no significant similarity has ever been
described between all these bacteriocins (Fig. 5).
3.2.1 . Lactacin F
Lactacin F is a two-peptide bacteriocin made from
lactacin A (57 amino acids) and LafX (48 amino acids).
Lactacin F is produced by Lactobacillus johnsonii and is
active against other Lactobacillus and Enterococcus. Lac-
tacin F dissipates , induces a leakage of K+ and
decreases the intracellular ATP concentration [76]. This ATP
depletion was shown to be likely due to an efflux of
inorganic phosphate, resulting in a shift of the ATP hydroly-
sis equilibrium. These data support that lactacin F likely
form pores into the cytoplasmic membrane.
3.2.2. Plantaricin EF and JK
Both plantaricin EF and JK are produced by Lactobacil-
lus plantarum C11 and their production is induced by the
pheromone/bacteriocin plantaricin A. These two two-
peptide bacteriocins were shown to form pores in the target
membranes but differ in their ion selectivity. Plantaricin EF
dissipates the more efficiently than plantaricin JK and
this difference could be due to the higher cation conduc-
tance of plantaricin EF [77]. In contrast, plantaricin JK
dissipates pH more efficiently than plantaricin EF and was
shown to have a higher anion conductance. Plantaricin JK is
also more efficient in growth inhibition suggesting that pH
is important for cell viability. The authors proposed that a
drop in intracellular pH leads to an inhibition of substrate-
level phosphorylation, which provides energy to form ATP.
3.2.3. Lactococcin G
Lactococcin G is a two-peptide bacteriocin produced by
Lactococcus lactis LMG 2081. This bacteriocin is com-
posed of and subunits (39 and 35 amino acids,
respectively) and is active against several lactic acid bacte-
ria and Clostridium strains [78]. The combination of both
peptides is needed to dissipate . Lactococcin G also
induces a release of amino acids, such as alanine or leucine,
accumulated through a PMF dependent mechanism [79]. In
addition, the intracellular ATP level is greatly reduced,
which results in a decrease of the ATP-driven glutamate
 Y. Hchard, H.G. Sahl / Biochimie 84 (2002) 545557 553

Fig. 5. Primary sequence of subclass IIb and IIc bacteriocins. LafA, LafX: lactacin F; PlnE, PlnF: plantaricin EF, PlnJ, PlnK: plantaricin JK; LcnG, LcnG:
lactococcin G; PlnA: plantaricin A; LcnA: lactococcin A; Lcn: lactococcin 972.

uptake. Lactococcin G has no effect on pH suggesting that
it does not form proton-conducting pores. In contrast, it
leads to a rapid efflux of potassium and other monovalent
cations probably due to high selectivity of the pores for
these ions [80]. Finally, lactococcin G is inactive against
liposomes and membrane vesicles, suggesting that other cell
wall-associated components might be involved in sensitivity
of target cells.
3.3. The subclass IIc: miscellaneous peptides
The subclass IIc and IId were previously defined but no
clear structural features support this classification (Fig. 5).
In the absence of similarities between several bacteriocins,
which could define a new class, we propose that the subclass
IIc should include miscellaneous peptides, different from
subclass IIa or subclass IIb bacteriocins.
3.3.1. Plantaricin A
Plantaricin A is a 26 amino acid peptide synthesised, as
well as plantaricin EF and JK, by L. plantarum C11. It
exhibits both bactericidal and pheromone activities. A
N-terminal truncated form (122) was shown to display
almost the same activity as the entire peptide. PlnA-22
dissipates both pH and [81]. Its enantiomer, the all-D
PlnA-22 remains bactericidal, whereas it has lost the phero-
mone activity. This shows that plantaricin A exerts its
bactericidal and pheromone activities through different
mechanisms and that plantaricin A does not require chiral
interactions for its bactericidal activity [81].
3.3.2. Lactococcin A
Lactococcin A is a hydrophobic 54 amino acid peptide
produced by L. lactis. It exhibits a very narrow spectrum of
activity, limited to other lactococci. Lactococcin A dissi-
pates and induces both the influx and the efflux of
amino acids from sensitive cells [82]. It suggests that
lactococcin A affects the permeability of the cytoplasmic
membrane. In addition, lactococcin A is active against
whole cells and membrane vesicles but not against lipo-
somes derived from lactococcal phospholipids [82], sug-
gesting that specific membrane-associated proteins are re-
quired for lactococcin A activity. This requirement is in
accordance with the very narrow activity spectrum of
lactococcin A.
3.3.3. Lactococcin 972
Lactococcin 972 is a 66 amino acid bacteriocin, produced
via the sec-dependent pathway by L. lactis IPLA 972 and
active against all lactococci tested [83]. Even though the
554 Y. Hchard, H.G. Sahl / Biochimie 84 (2002) 545557

active form of lactococcin 972 was shown to be a ho-
modimer, it could not be classified in the subclass IIb
bacteriocin that requires two different peptides. Strikingly,
lactococcin 972 is not hydrophobic, as most of the bacte-
riocins described so far. Another unique feature of lactococ-
cin 972 is that it does not induce any leakage of cytoplasmic
solutes. In addition, the effect of lactococcin 972 is not
lethal by itself since the target cells keep synthesising
macromolecules [84]. Martinez et al. reported that lactococ-
cin 972 inhibits the incorporation of a cell wall precursor,
the N-acetylglucosamine, in treated-cells. Lactococcin 972
treatment of sensitive bacteria results in cell elongation and
widening likely due to the inhibition of septum formation
[85]. This hypothesis is consistent with the observation that
lactococcin 972 hardly affect stationary-phase cells.
The bacteriocin first interacts with a target molecule, pos-
sibly the EIItMan mannose permease. This interaction is
absolutely required for in vivo activity against whole
bacterial cells, probably favouring the forthcoming interac-
tion of the bacteriocin with the cytoplasmic membrane. In
that case, the structure and the expression level of the target
molecule could influence the activity of the bacteriocin. The
possible interaction of the bacteriocin with EIItMan could
also lead to switch this permease to an open state and could
thereby participate to the permeabilisation of the target cell.
Secondly, the bacteriocin may interact with the cytoplasmic
membrane leading to both pore formation or localised
disruption of the membrane. This second step may not
require a target molecule and may be mostly dependent on

3.4. Conclusion

Subclass IIa bacteriocins tend to dissipate PMF via

dissipation of and/or pH (Table 1). It was suggested
that bacteriocins themselves could form a pore into the
cytoplasmic membrane, although no clear experimental data
support this hypothesis. Different models of pore formation
were proposed taking into account that these bacteriocins
are small amphiphilic peptides [86]. Various factors obvi-
ously influence the bacteriocin activity on the bacterial cell.
They include the structure and the amount of bacteriocin,
the composition and the potential of the cytoplasmic mem-
brane, the structure and the expression level of a protein
with an immunity function and the chemical composition of
the environment. One of the most important recent findings
is that subclass IIa bacteriocins need a target molecule at the
surface of sensitive cells to be active. The subclass IIa
bacteriocins mainly display a narrow and strain-specific
spectrum of activity. The minor differences in phospholipid
composition between strains of the same species or between
related strains could hardly explain such a high specific
spectrum. In addition, the inactivity of all-D leucocin A
strongly suggests that a chiral interaction is needed at the
bacterial surface for the bacteriocin to be active [72]. This
points towards the involvement of a surface protein as a
target molecule. Recently, a mannose PTS permease
(EIItMan) has been proposed to be a target molecule for
mesentericin Y105 [69,70] and leucocin A [71]. Besides,
Listeria strains have been shown to be cross-resistant to
various subclass IIa bacteriocins while they were still
sensitive to nisin, a class I bacteriocin [87] and Y. Hchard
(unpublished results). Accordingly, EIItMan is proposed be a
target molecule for all subclass IIa bacteriocins in L. mono-
cytogenes, E. faecalis and likely in other sensitive bacteria.
On the other hand, several subclass IIa bacteriocins are able
to permeabilise the liposomes made of lipids from target
cells, supporting the notion that a proteinaceaous target is
Fig. 6. A model for the mode of action of subclass IIa bacteriocins. IIAB,
not necessary for activity. Taking into account these seem- IIC and IID represent the subunits of the EIItMan mannose permease (see
ingly contradictory results, we can propose the following text). The bacteriocin recognises the permease and then forms pores in the
model for subclass IIa bacteriocin mode of action (Fig. 6). cytoplasmic membrane.
 Y. Hchard, H.G. Sahl / Biochimie 84 (2002) 545557
electrostatic and/or hydrophobic interactions with the mem-
brane, explaining why bacteriocins could be active against
liposomes or phospholipid vesicles.
All subclass IIb bacteriocins studied so far dissipate
(Table 1). In addition, lactococcin G dissipates pH while
plantaricin EF and JK do not. Another important feature of
lactococcin G is that it likely needs a specific receptor
molecule to be active. The subclass IIb bacteriocins are
proposed to form hydrophilic pores into the membrane of
the target cell leading to its permeabilisation, although they
could display specific ion selectivity. All these results
suggest that subclass IIb bacteriocins exhibit a quite differ-
ent mechanism of action, in accordance with the differences
found among their primary structure.
Finally, subclass IIc bacteriocins are miscellaneous pep-
tides with no structure similarity. Accordingly, they are
involved in various functions such as membrane permeabi-
lisation, pheromone activity and inhibition of septum for-
mation (Table 1).
Acknowledgements
HGS acknowledges support by the DFG (various
projects), the European Communities (Contract number
QLK2-CT-2000-00411) and by the BONFOR programme
of the Medical Faculty, University of Bonn. Anja Hoffmann
provided Fig. 1, 2 and 3. YH acknowledges Jacques Frre
for providing the Fig. 4, the Rhodia Food company and the
Ministre de lEducation Nationale, de la Recherche et de
la Technologie for their support.
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