Laboratory of Microbial Gene Technology,
Department of Biotechnological Sciences, Agricultural University of
Norway,1 and
The Norwegian Crop Research
Institute,2 N-1432 Ås, Norway
Four class IIa bacteriocins (pediocin PA-1, enterocin A, sakacin P,
and curvacin A) were purified to homogeneity and tested for activity
toward a variety of indicator strains. Pediocin PA-1 and enterocin A
inhibited more strains and had generally lower MICs than sakacin P and
curvacin A. The antagonistic activity of pediocin-PA1 and enterocin A
was much more sensitive to reduction of disulfide bonds than the
antagonistic activity of sakacin P and curvacin A, suggesting that an
extra disulfide bond that is present in the former two may contribute
to their high levels of activity. The food pathogen Listeria
monocytogenes was among the most sensitive indicator strains for
all four bacteriocins. Enterocin A was most effective in inhibiting
Listeria, having MICs in the range of 0.1 to 1 ng/ml.
Sakacin P had the interesting property of being very active toward
Listeria but not having concomitant high levels of activity
toward lactic acid bacteria. Strains producing class IIa bacteriocins
displayed various degrees of resistance toward noncognate class IIa
bacteriocins; for the sakacin P producer, it was shown that this
resistance is correlated with the expression of immunity genes. It is
hypothesized that variation in the presence and/or expression of such
immunity genes accounts in part for the remarkably large variation in
bacteriocin sensitivity displayed by lactic acid bacteria.
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INTRODUCTION |
Many lactic acid bacteria (LAB),
including members of the genera Lactococcus,
Lactobacillus, Carnobacterium,
Enterococcus, and Pediococcus, are known to
secrete small, ribosomally synthesized antimicrobial peptides called
bacteriocins (26, 29, 34). Some of these peptides undergo
posttranslational modifications (class I bacteriocins), whereas others
are not modified (class II bacteriocins) (29, 34). Class II
bacteriocins contain between 30 and 60 residues and are usually
positively charged at a neutral pH. Studies of a large number of class
II bacteriocins have led to subgrouping of these compounds (29,
34). One of the subgroups, class IIa, contains bacteriocins that
are characterized by the presence of YGNG and CXXXXCXV sequence motifs
in their N-terminal halves as well as by their strong inhibitory effect
on Listeria (e.g., 3, 4, 22, 23, 27, 28, 31, 38,
45) (Fig. 1). Because of their
effectiveness against the food pathogen Listeria, class IIa
bacteriocins have potential as antimicrobial agents in food and feed.
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FIG. 1.
Sequence alignment of class IIa bacteriocins. Residue
numbering is according to the sequence of pediocin PA-1. Cysteine
residues are printed in boldface; the two known class IIa bacteriocins
with four cysteine residues are in the upper group. No attempt was made
to optimize the alignment in the C-terminal halves of the peptides.
Piscicolin 126 is identical to piscicocin V1a (4).
Carnobacteriocin BM1 most probably is identical to piscicocin V1b
(4). Sakacin P most probably is identical to bavaricin A
(30). Curvacin A is identical to sakacin A (2).
The consensus sequence includes residues conserved in at least 8 of the
12 sequences shown; 100% conserved residues are underlined.
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Class IIa bacteriocins act by permeabilizing the membrane of their
target cells (1, 5, 6, 9, 10, 26, 28). The most recent
studies on the mode of action of these bacteriocins indicate that
antimicrobial activity does not require a specific receptor and is
enhanced by (but not fully dependent on) a membrane potential (9,
28). Little is known about bacteriocin structure, and unravelling
the relationships between structure and function is one of the great
challenges in current bacteriocin research. A logical starting point
for structure-function studies is a thorough study of the differences
in activity and target cell specificity between naturally occurring
homologous bacteriocins. A few such studies have been described, but
these suffer from either a very limited number of tested indicator
strains or the use of culture supernatants instead of purified
bacteriocins (3, 4, 17, 45). The use of purified
bacteriocins for comparative analyses is absolutely essential, since it
is becoming increasingly evident that bacteriocin producers produce
more than one bacteriocin (4, 8, 38, 48; this
study).
In the present study, the activities of four pure class IIa
bacteriocins (pediocin PA-1, enterocin A, curvacin A, and sakacin P)
(Fig. 1) were tested against a large number of LAB as well as several
strains of the food pathogen Listeria monocytogenes. The
bacteriocins were purified from their respective producer strains by
use of an optimized purification protocol yielding highly pure samples.
The contribution of disulfide formation was assessed and found to be
important for activity. The effects of the purified bacteriocins on
(noncognate) class IIa bacteriocin-producing strains are described, and
the implications of our findings for immunity and resistance are
discussed.
| |
MATERIALS AND METHODS |
Bacterial strains and growth conditions.
Lactobacillus
spp., Pediococcus spp., Enterococcus spp.,
Leuconostoc spp., and Carnobacterium spp. were
grown in MRS broth (Oxoid, Unipath Ltd., Basingstoke, Hampshire,
England) at 30°C. Lactococci were grown in M17 medium (Difco
Laboratories, Detroit, Mich.) supplemented with 0.5% (wt/vol) glucose
at 30°C. Listeria spp. were grown on brain heart infusion
medium (Difco) at 30°C, and Clostridium spp. were grown on
both thioglycolate medium (Oxoid) and reinforced clostridial medium
(Oxoid) at 37°C. For cultivating Clostridium spp., the
Anaerogen system for anaerobic incubation (Oxoid) was used.
Bacteriocin purification and concentration determination.
Pediocin PA-1, enterocin A, sakacin P, and curvacin A were purified
from the supernatants of early-stationary-phase cultures of
Pediococcus acidilactici (35), Enterococcus
faecium CTC492 (3), Lactobacillus curvatus
LTH1174 (45), and Lactobacillus sake LTH673
(45), respectively. In terms of bacteriocin production, E. faecium CTC492 is identical to E. faecium T136
(8). The bacteriocins were purified to homogeneity
essentially as described previously (3, 35, 45). The method
consists of ammonium sulfate precipitation followed by three
chromatography steps (ion exchange, hydrophobic interaction, and
reversed phase). The final, reversed-phase chromatography step (with a
fast protein liquid chromatography system supplied by Pharmacia-LKB,
Uppsala, Sweden) was repeated two or three times to ensure maximum
purity. In contrast to the previously described method, very large
volumes of washing buffer (up to 40 times the column volume) were used
for the washing steps during ion-exchange and hydrophobic-interaction
chromatography. This small change in the protocol contributed
considerably to purity.
The purity of the bacteriocins was assessed by 10 steps of Edman
degradation with a model 477A sequencer (Applied Biosystems, Foster
City, Calif.). Purity was assessed by comparing the yields of the
expected amino acids with the yields of other amino acids (that would
result from contaminating peptides) in each degradation step. On the
basis of these analyses, purity was estimated to be >98% for sakacin
P, curvacin A, and pediocin PA-1 and about 95% for enterocin A.
Initially, the concentration of the purified bacteriocins was assessed
by two methods: measuring UV absorption at 280 nm and amino acid
composition analysis. For the former method, molar extinction
coefficients were calculated from the contributions of individual amino
acid residues by use of the program PCGENE (IntelliGenetics Inc.,
Geneva, Switzerland). The differences between the results of the two
methods were on the order of 10% for all four bacteriocins (data not
shown). The simplest of the two methods (measuring UV absorption at 280 nm) was therefore used for further routine concentration
determinations.
The purified bacteriocins were stored at 
20°C and used within 1 month of purification. The bacteriocins retained 
90% of their
activity during this period, as indicated by the results of weekly
activity assays with a standard set of three indicator strains.
Bacteriocin assay.
Bacteriocin activity was quantified with
a microtiter plate assay (21, 24). For the comparative
studies, a standardized procedure was used; indicator cells were always
derived from a fresh overnight culture and diluted 400 times before
use. The subsequent incubation time was 18 to 20 h in all assays.
The activities of the four bacteriocins toward a specific strain were
always determined in a single assay. The MICs given represent the
bacteriocin concentration needed to obtain 50% inhibition of growth.
The values are the averages of three independent measurements, which
gave standard deviations on the order of 25% of the values.
Bacteriocin activity is expressed in units, one bacteriocin unit being
the amount of bacteriocin required to reduce the growth of the
indicator strain by 50% under the conditions of the assay (200-µl
culture volume).
Disulfide bonds.
Disulfide bond formation was assessed by
determining the molecular masses of the purified bacteriocins by
electrospray ionization mass spectrometry and by determining the number
of free thiol groups as described by Ellman (15). In the
latter method, the molar extinction coefficient used for
2-nitro-5-thiobenzoate was 14,150 M
1 cm1. A
sample containing a peptide with only one cysteine was included as a
positive control.
To test the effect of the reduction of disulfide bonds on bacteriocin
activity, bacteriocin assays in which dithiothreitol (DTT; final
concentration, 10 mM) was added to the culture medium in the microtiter
plate wells were conducted. These experiments were conducted with
indicator strains whose growth was not inhibited by the presence of 10 mM DTT alone.
Induction of bacteriocin production.
Under certain
conditions, bacteriocin-negative (Bac) cultures of the
sakacin P producer L. sake LTH673 can be obtained, as described previously (7, 13). These cultures have a stable Bac phenotype, but the bacteriocin-positive
(Bac+) phenotype may be restored by adding the pheromone
needed for the expression of bacteriocin-related genes (13).
In the present study, Bac and Bac+ cultures
of L. sake LTH673 were obtained by making two identical 100-fold dilutions of a Bac overnight culture. To one of
the two dilutions, the appropriate pheromone was added to a final
concentration of 50 ng/ml (7, 13). The addition of the
pheromone led to the transcription of genes involved in bacteriocin
production, as documented previously (7).
Sequence alignments.
Sequence identities and similarities
between immunity proteins were determined with the program PALIGN,
which is part of the PCGENE software package (33). The
genetic code matrix (16) with default gap opening and
extension penalties was used. These settings kept the numbers of
insertions and deletions equal to or below five for all pairwise
sequence alignments. The following groups of amino acids were defined
as similar: A, S, and T; D and E; N and Q; R and K; I, L, M, and V; and
F, Y, and W.
| |
RESULTS AND DISCUSSION |
Class IIa bacteriocin producers produce more than one
bacteriocin.
Recent studies of bacteriocin producers suggest that
it may be common for LAB to produce more than one bacteriocin. For
example, E. faecium CTC492 not only produces the class IIa
bacteriocin enterocin A but also produces enterocin B, which does not
belong to class IIa. These two bacteriocins were shown to have
different inhibitory spectra (8, 14). Another example of the
production of multiple bacteriocins by LAB is illustrated by Table
1. The data in Table 1 show that during
purification of sakacin P, inhibitory activity toward some strains was
lost, whereas inhibitory activity toward other strains was retained
(Table 1). The lost activity could be recovered by pooling the
supernatant and the pellet obtained in the ammonium sulfate
precipitation step at the start of the purification protocol (data not
shown). Thus, in addition to sakacin P, L. sake LTH673
produces at least one other bacteriocin. This other bacteriocin(s) has
not yet been characterized, but transcription studies have shown that
at least one gene encoding a hitherto-uncharacterized bacteriocin-like
peptide is expressed in addition to the sakacin P structural gene
(7). The data in Table 1 show how erroneous results would
have been obtained if the inhibitory spectrum of sakacin P had been
assessed with culture supernatants.
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TABLE 1.
Purification of bacteriocins from L. sake
LTH673 and assessment of yields with various indicator strains
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Inhibitory spectra.
The activities of the bacteriocins toward
various indicator strains are shown in Table
2 and Fig.
2. The results show that pediocin PA-1
and enterocin A are generally more active than sakacin P and curvacin
A. Pediocin PA-1 and enterocin A also have a broader inhibitory
spectrum (Fig. 2). The magnitude of the difference in activity between
pediocin PA-1 and enterocin A on the one hand and sakacin P and
curvacin A on the other hand varies widely with the indicator strain
used. The latter represents a general phenomenon illustrated by Table
2: when LAB were used as indicator strains, the activity ratio between
the various bacteriocins differed from species to species and even from
strain to strain. More consistent results were obtained when listeriae
were used as indicator strains: enterocin A was 5 to 10 times more
active than sakacin P and pediocin PA-1, which were 5 to 10 times more
active than curvacin A.
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FIG. 2.
Sensitivity of indicator strains. Strains are
categorized according to MICs (Table 2): black, <10 ng/ml; dark grey,
10 to 100 ng/ml; light grey, 100 to 500 ng/ml; white, >500 ng/ml. Ped,
pediocin PA-1; Ent, enterocin A; Cur, curvacin A; Sak, sakacin P.
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Interestingly, sakacin P had modest activity toward LAB but was almost
as effective as enterocin A and pediocin PA-1 against listeriae.
Because of this combination of high antilisterial activity and a narrow
inhibitory spectrum (Fig. 2), sakacin P is perhaps the most promising
of the tested bacteriocins for use in LAB fermentations that are prone
to Listeria infections.
For analyzing possible synergistic inhibitory effects of class IIa
bacteriocins, the activity of every possible one-to-one combination of
two of the purified bacteriocins was measured at maximum total
concentrations of approximately 1 µg/ml. No synergistic effects were
observed (results not shown).
Role of disulfide bonds.
Mass spectrometry analysis showed
that the molecular masses of purified pediocin PA-1, enterocin A,
curvacin A, and sakacin P were 4.5, 4.7, 2.2, and 3.2 Da, respectively,
lower than those expected when cysteine residues were assumed to be in
a reduced state. Considering the standard deviation in the mass
determinations (~1 Da), these data are in accordance with the notion
that all cysteine residues are oxidized and are involved in disulfide
bonds. In accordance with this observation, the Ellman assay did not reveal any free cysteine residues in the bacteriocins, whereas it did
reveal free cysteine residues in a one-cysteine-containing control
peptide (results not shown). Thus, pediocin PA-1 and enterocin A indeed
contain two disulfide bonds, whereas sakacin P and curvacin A contain
one. It was previously shown that the two disulfide bonds in pediocin
PA-1 are formed between Cys9 and Cys14 and between Cys24 and Cys44
(23).
Interestingly, the two most active bacteriocins in this study were the
ones with two disulfide bonds, suggesting a correlation between these
two properties. To investigate the contribution of disulfide bond
formation to bacteriocin activity, three indicator strains whose growth
was not inhibited by DTT were selected for testing bacteriocin activity
under reducing conditions. As shown in Table
3, DTT dramatically reduced the
activities of pediocin PA-1 and enterocin A, whereas the activities of
sakacin P and curvacin A were only moderately reduced. Thus, in the
presence of DTT, the two-disulfide-bond-containing bacteriocins
pediocin PA-1 and enterocin A were no longer generally more potent than the one-disulfide-bond-containing bacteriocins sakacin P and curvacin A.
These results suggest that the high levels of activity of pediocin PA-1
and enterocin A may be due at least in part to the extra disulfide bond
present in the C-terminal region. The effect of DTT on the activity of
sakacin P and curvacin A suggests that the disulfide bond between fully
conserved Cys9 and Cys14 is important but not crucial for activity.
This conclusion is in accordance with previous studies on
carnobacteriocin B2 (in which the bond between Cys9 and Cys14 is not
formed) (38) and leucocin A (which retains considerable
activity after reduction and modification of the cysteines)
(22). Remarkably, quite opposite results have also been
reported. In one study, pediocin PA-1 was found to lose all of its
activity upon reduction (10). Furthermore, the activity of
mesentericin Y105 was shown to be reduced at least 2,000-fold upon
modification or mutation of Cys9 and Cys14 (18). At present, we have no explanation for the apparent inconsistencies among these
results.
Several authors have discussed possible structural models for a class
IIa bacteriocin in a membrane environment (4, 9, 17, 20,
28). The bacteriocins are unstructured in watery solutions,
whereas they adopt a partly helical structure in more hydrophobic
environments (14, 18, 20, 42). It has been suggested that
the C-terminal half of class IIa bacteriocins forms a hydrophobic or
amphiphilic transmembrane 
helix, permitting the formation of a
so-called "barrel-stave" (36) poration complex (4,
17, 29). Obviously, this simple structural model is incompatible
with the presence of a disulfide bond formed by cysteine residues
located at the beginning and end of this putative transmembrane helix
(as would be the case for pediocin PA-1 and enterocin A). Recent
studies of the three-dimensional structure of leucocin A (leucocin A
has only the Cys9-Cys14 disulfide bond) (Fig. 1) show that the most
C-terminal portion of this bacteriocin has a rather extended
(nonhelical) structure, folding back onto a preceding helical region
that comprises residues 17 to 31 (20). Extrapolating these
observations to enterocin A and pediocin PA-1, one may speculate that
the extra disulfide bond in these two bacteriocins stabilizes their
structures by covalently coupling the C-terminal residue to a cysteine
in the helical region.
Immunity.
LAB producing class IIa bacteriocins generally were
not particularly sensitive to noncognate class IIa bacteriocins, but
there was a tendency for the producers of the less potent sakacin P and
curvacin A to be sensitive to the more potent pediocin PA-1 and
enterocin A (Table 4). Remarkably,
L. sake Lb706 and L. curvatus LTH1174, which are
known to produce identical bacteriocins, displayed different behaviors
in terms of bacteriocin sensitivity.
These results do not permit discrimination between insensitivity (the
cause of which is not known) and immunity provided by a specific
immunity protein. To obtain this discrimination, we exploited the fact
that the expression of genes involved in the production of sakacin P
can be controlled (7, 13). Cultures of L. sake
LTH673 which have a stable Bac phenotype and in which the
transcription of bacteriocin-related genes (including the
spiA gene, encoding immunity for sakacin P) is switched off
can be obtained. As shown in Table 4, L. sake LTH673
Bac cells were sensitive to all four bacteriocins.
L. sake LTH673 Bac+ cells, which were obtained
after induction of a Bac culture with an appropriate
pheromone and which are known to express the spiA gene
(7), were much less sensitive. Within the time frame of the
experiments, spontaneous development of insensitivity to class IIa
bacteriocins was not observed. Thus, the low sensitivity or immunity of
Bac+ cells of L. sake LTH673 to various
(noncognate) class IIa bacteriocins is correlated with the expression
of the sakacin P gene cluster (including spiA).
Table 5 shows that sequence similarities
between proteins that (putatively) provide immunity to class IIa
bacteriocins are generally low but that most of the proteins do clearly
resemble several others. A certain similarity between the proteins was also suggested by secondary structure predictions (40, 41), which indicated that all 12 proteins shown in Table 5 were largely helical but were devoid of transmembrane helices (14).
Studies with L. sake LTH673 indicated that, despite the low
degree of sequence homology, the immunity proteins shown in Table 5
provide (partial) immunity to various class IIa bacteriocins.
Interestingly, the sakacin P producer L. sake LTH673
contains at least one more putative immunity gene (7)
(orfY in Table 5), which is transcribed in Bac+
cells only (14). This immunity gene is not coupled to a
cognate bacteriocin gene (7). In addition to L. sake LTH673, several other LAB seem to contain immunity genes that
are not directly associated with a gene encoding a cognate bacteriocin.
Copies of the sakacin P immunity gene have been detected in the
chromosome of the sakacin A producer L. sake Lb706 and, most
importantly, in the chromosome of an L. sake strain that
does not produce any bacteriocin at all (25). The producer
of carnobacteriocins BM1 and B2 contains at least three (putative)
immunity genes (38, 39). In addition to putative immunity
genes for the two class IIa bacteriocins that are produced (designated
CarBM1 and CarB2 in Table 5), a third putative immunity gene is present
downstream of the structural gene for carnobacteriocin B2 (the protein
is designated Orf-
3 in Table 5). Studies of transcription have indicated that the orf-3 gene is cotranscribed with the
genes encoding carnobacteriocin B2 and its cognate immunity protein (43).
Extrapolating the above-mentioned observations, one may speculate that
LAB from genera containing class IIa bacteriocin producers generally
possess one or more immunity genes for class IIa bacteriocins. These
genes may show various degrees of homology and may be expressed to
various extents. These suggestions could provide an explanation for the
remarkable variation in bacteriocin sensitivity that is observed for
closely related LAB strains.
We are grateful to Stephen Bayne, Novo Nordisk, Gentofte,
Denmark, for carrying out mass spectrometry analyses and to Grethe Kobro and May-Britt Selvåg Hovet for technical assistance. We thank
Tone Katla at Matforsk, The Norwegian Food Research Institute, for
assistance in work with pathogenic strains.
This work was supported in part by a grant from the Nordic Industry
Fund. M.B.B. was supported by a grant from the Norwegian Research
Council.
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Abstract
Introduction
Present address: CLB, 1006 AD Amsterdam, The Netherlands.
