Appl Environ Microbiol, February 1998, p. 439-445, Vol. 64, No. 2
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Lacticin 3147, a Broad-Spectrum Bacteriocin Which
Selectively Dissipates the Membrane Potential
Olivia
McAuliffe,1,2
Maire
P.
Ryan,3
R. Paul
Ross,3
Colin
Hill,2,*
Pieter
Breeuwer,1 and
Tjakko
Abee1
Department of Food Science, Wageningen
Agricultural University, 6708HD Wageningen, The
Netherlands,1 and
Department of
Microbiology, University College Cork,
Cork,2 and
National Dairy Products
Research Centre, Moorepark, Fermoy,3 County
Cork, Republic of Ireland
Received 22 July 1997/Accepted 10 November 1997
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ABSTRACT |
Lacticin 3147 is a broad-spectrum bacteriocin produced by
Lactococcus lactis subsp. lactis DPC3147
(M. P. Ryan, M. C. Rea, C. Hill, and R. P. Ross, Appl.
Environ. Microbiol. 62:612-619, 1996). Partial purification of the
bacteriocin by hydrophobic interaction chromatography and reverse-phase
fast protein liquid chromatography revealed that two components are
required for full activity. Lacticin 3147 is bactericidal against
L. lactis, Listeria monocytogenes, and
Bacillus subtilis; at low concentrations of the
bacteriocin, bactericidal activity is enhanced when target cells are
energized. This finding suggests that the presence of a proton motive
force promotes the interaction of the bacteriocin with the cytoplasmic
membrane, leading to the formation of pores at these low lacticin 3147 concentrations. These pores were shown to be selective for
K+ ions and inorganic phosphate. The loss of these ions
resulted in immediate dissipation of the membrane potential and
hydrolysis of internal ATP, leading to an eventual collapse of the pH
gradient at the membrane and ultimately to cell death. Our results
suggest that lacticin 3147 is a pore-forming bacteriocin which acts on a broad range of gram-positive bacteria.
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INTRODUCTION |
Bacteriocins produced by lactic acid
bacteria (LAB) are typically defined as proteinaceous compounds with
activity against related species, including organisms involved in
food-borne disease and food spoilage (17, 18). The
commercial potential for these inhibitory compounds has fueled an
intensive research effort into their exploitation as food
preservatives. To date, nisin remains the only purified bacteriocin
approved for use in food products (11). However, it is
believed that the inclusion of bacteriocin-producing starter cultures
in food fermentations will be more widely accepted than the use of pure
bacteriocin preparations, which could be considered food additives.
Lacticin 3147 is a bacteriocin produced by Lactococcus
lactis subsp. lactis DPC3147, a strain isolated from an
Irish kefir grain. The bacteriocin has a broad inhibition spectrum,
similar to that of nisin, but is both genetically and biologically
distinct from nisin (29). It is a heat-stable compound which
is active at physiological pH. The genetic determinants for production
and immunity are encoded on pMRC01, a 60.2-kb plasmid which can be conjugally transferred to strains with industrially important characteristics. A pMRC01 transconjugant of a commercial cheese-making strain, L. lactis subsp. cremoris DPC4268, was
used as a single-strain starter to manufacture cheddar cheese. The
presence of lacticin 3147 in the cheese could be detected throughout
the 6-month ripening period and resulted in significantly lower levels
of nonstarter LAB (29). Lacticin 3147 has also been used in
the veterinary field, where a prophylactic role in the prevention of
mastitis in cattle was recently developed (30).
Most of the characterized LAB bacteriocins appear to have a common
mechanism of action in that they dissipate the proton motive force
(PMF), i.e., the membrane potential (
) and the pH gradient (pH), in target organisms through the formation of pores in the cytoplasmic membrane (1, 2, 7, 14, 15, 24, 31, 32). Nisin, a
broad-spectrum lantibiotic, acts at the membrane without the
requirement for a specific receptor protein and results in the efflux
of ions, amino acids, and ATP (3, 12-14). Class II
bacteriocins, such as lactococcin A (16, 31), lactococcin B
(32), and the pediocin-like bacteriocins (7, 9,
10), have narrow host ranges and appear to have receptor-mediated
action which leads to leakage of ions, PMF dissipation, and ATP
depletion. While the antimicrobial activity of these bacteriocins is
due to the action of a single peptide, others require the complementary action of two peptides to inhibit target organisms. For example, lactococcin G (26) is a two-component bacteriocin which acts by forming small pores which allow K+ efflux, resulting in
ATP hydrolysis and the dissipation of the membrane potential. A model
presented for the action of lactococcin G suggested that the rapid
hydrolysis of internal ATP was due to K+ uptake by a
K+ ATPase in the cell membrane in an attempt to compensate
for the loss of this ion through the bacteriocin pore (24).
Another two-component bacteriocin, lacticin F, causes efflux of
phosphate and K+ ions as well as PMF dissipation (2,
25). Like other class II bacteriocins, lactococcin G and lacticin
F have narrow host ranges (25, 26). Recently, a
broad-host-range, two-component bacteriocin, thermophilin 13, was
isolated from Streptococcus thermophilus; this bacteriocin
acts on target organisms through dissipation of the PMF
(23).
Here, we present evidence that the broad-host-range bacteriocin
lacticin 3147 is a membrane-active, two-component bacteriocin and
report its effects on the energetic parameters of cells of L. lactis subsp. cremoris, Listeria
monocytogenes, and Bacillus subtilis, including ATP
levels, , pH, and K+ and phosphate levels. These
species vary in their sensitivity to the bacteriocin but, strikingly,
the membrane potential is selectively dissipated at the same low levels
of the bacteriocin. However, the killing efficiency is associated with
a reduction in cellular ATP levels, and these correlate with the
amounts of K+ and phosphate retained in the cells.
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MATERIALS AND METHODS |
Bacterial strains and culture conditions.
The bacteriocin
producer L. lactis subsp. lactis DPC3147 was
grown at 30°C without aeration in M17 (Oxoid Ltd., Basingstoke, Hampshire, England) supplemented with 0.5% (wt/vol) glucose (GM17 broth). L. lactis subsp. cremoris HP was used as
the standard sensitive strain and was cultured in the same manner.
Other sensitive strains used included L. monocytogenes Scott
A, which was grown in tryptic soy broth (Difco Laboratories, Detroit,
Mich.) with 0.6% (wt/vol) yeast extract (Oxoid) at 37°C, and
B. subtilis ATCC 6051, which was grown in brain heart
infusion broth (Difco) containing 1% (wt/vol) glucose at 37°C with
shaking.
Bacteriocin assay.
Bacteriocin activity was estimated by the
agar well diffusion assay as described by Parente and Hill
(27). Molten agar was cooled to 48°C and seeded with the
sensitive strain (approximately 2 × 107 fresh
overnight-grown cells). The inoculated medium was dispensed into
sterile petri plates, allowed to solidify, and dried. Wells (approximately 4.6 mm in diameter) were made in the seeded agar plates.
Aliquots (50 µl) of a twofold serial dilution of the bacteriocin preparation were dispensed into the wells, and plates were incubated overnight at 30°C. The arbitrary units (AU) per milliliter were obtained as described by Ryan et al. (29).
Preparation of lacticin 3147.
For the purposes of
purification, L. lactis subsp. lactis DPC3147 was
propagated in 8 liters of tryptone-yeast broth (containing, per liter,
tryptone at 2.5 g, yeast extract at 5 g, glucose at 10 g, 
-glycerol phosphate at 19 g, MgSO4 · 7H2O at 0.25 g, and MnSO4 · 4H2O at 0.05 g; pH 6.75) that had been passed through a column containing 50 g of XAD-16 beads to clear contaminating hydrophobic peptides from the broth medium. Following autoclaving, the
resulting medium was inoculated with 1% L. lactis subsp.
lactis DPC3147 and incubated at 30°C for 16 h. The
cells were removed by centrifugation at 10,000 × g for
15 min, and the supernatant was applied to a column (2 by 30 cm)
containing 50 g of XAD-16 beads at a flow rate of approximately 15 ml/min. The column was washed with 40% ethanol (15 ml/min), and the
bacteriocin was eluted with 70% isopropanol-10 mM acetic acid (pH 2)
in 20-ml fractions. The isopropanol was removed by evaporation with
oxygen-free nitrogen gas (BOC Gases, Cork, Ireland). The bacteriocin
was assayed after each step as described above with L. lactis subsp. cremoris HP as the sensitive indicator.
The most active bacteriocin fractions were pooled (approximately seven
fractions) and applied to a C18 reverse-phase Varian Bond
Elut cartridge (JVA Analytical Ltd., Dublin, Ireland), which was
activated by being rinsed with 1 volume of methanol and 1 volume of 5 mM sodium phosphate buffer (pH 7). The column was washed with 30%
ethanol (approximately 4 ml), and the active bacteriocin was eluted
with approximately 2 ml of 70% isopropanol-10 mM acetic acid (pH 2).
Active fractions thus obtained were concentrated by evaporation with
N2 gas, pooled, and applied to a fast protein liquid
chromatography (FPLC) C18 reverse-phase column (Pharmacia). The column was equilibrated with 0.1% trifluoroacetic acid in water
and eluted with a linear gradient of 0 to 90% acetonitrile containing
0.1% trifluoroacetic acid at a flow rate of 1 ml/min, and 1-ml
fractions were collected. The fractions thus obtained were assayed, and
the bacteriocin peptides were isolated.
Effect of lacticin 3147 on sensitive cells.
Cells were grown
to an optical density at 620 nm (OD620) of 0.7, harvested,
washed, and resuspended to approximately 107 cells per ml
in 2.5 mM sodium phosphate buffer (pH 7.0), in 2.5 mM sodium phosphate
buffer (pH 7.0) supplemented with 10 mM glucose, or in GM17 broth. The
bacteriocin was added at different concentrations, and samples were
taken at appropriate times to determine the viable cell count and the
OD620.
Measurement of ATP levels.
Intracellular and extracellular
ATP levels were determined as described previously (6) with
some modifications. Cells were suspended in 2.5 mM sodium phosphate
buffer (pH 7.0) with 10 mM glucose and 1,200 AU of lacticin 3147 per
ml. At various times, 20- and 50-µl samples were taken to determine
the total and extracellular ATP concentrations, respectively. The
20-µl samples were immediately mixed with 80 µl of dimethyl
sulfoxide. The 50-µl samples were spun down immediately for 2 min,
and 20 µl of the supernatant was removed and mixed with 80 µl of
dimethyl sulfoxide. All samples were diluted with 5 ml of water
filtered by nanopure filtration. ATP concentrations were determined
with a Lumac/3M biocounter M2010 by use of the Lumac
luciferin-luciferase enzyme assay. Enzyme (100 µl) was added to 200 µl of sample, and luminescence was measured.
Measurement of the .
The transmembrane electrical
potential (inside negative) was determined by the quenching of the
potential-sensitive fluorescent probe 3,3'-dipropylthiacarbocyanine
[diSC3(5); Molecular Probes Inc., Eugene, Oreg.].
Fluorescence was measured with a Perkin-Elmer LS50 spectrofluorometer
at 30°C with continuous stirring. An excitation wavelength of 643 nm
and an emission wavelength of 666 nm were used. Cells were suspended in
50 mM potassium HEPES buffer (pH 7.0) with 10 mM glucose. On addition
of the K+/H+ exchanger nigericin (Sigma
Chemical Co., St. Louis, Mo.), the pH was completely dissipated.
Measurement of the pH.
The transmembrane pH gradient
(inside alkaline) was measured by loading cells with the pH-sensitive
fluorescent probe 5 (and 6-)-carboxyfluorescein diacetate succinimidyl
ester (cFDASE; Molecular Probes) as described previously
(5). The cells were concentrated threefold in 1 ml of
potassium HEPES buffer (pH 8.0). The cells were then incubated at
30°C for 10 min in the presence of 1.0 µM cFDASE, washed, and
resuspended in the same volume of 50 mM potassium phosphate buffer (pH
7.0). Nonconjugated probe was eliminated by incubating the cells with
10 mM glucose at 30°C for 30 min. The cells were washed twice,
resuspended in the same volume of potassium phosphate buffer, and
placed on ice until used. The intracellular pH was determined by
diluting the loaded cells to a concentration of 107 cells
per ml in a 3-ml glass cuvette, and fluorescence was measured with a
spectrofluorometer.
Determination of K+ and inorganic phosphate contents
of cells.
Intracellular and extracellular K+ contents
were determined as follows. Cells were suspended in 2.5 mM sodium HEPES
buffer (pH 7.0) to an OD620 of 1.0. Glucose was added to a
final concentration of 10 mM, lacticin 3147 was added to a
concentration of 1,200 AU/ml, and valinomycin (Sigma) was added to a
concentration of 1.0 µM. Samples (1 ml) were taken at intervals and
immediately chilled on ice. The samples were centrifuged at 10,000 × g at 0°C for 7 min. The supernatant was removed and stored
for the determination of extracellular K+. The cell pellet
was resuspended in 1 ml of 5% trichloroacetic acid and frozen
overnight at 
20°C. The samples were thawed and incubated at 95°C
for 10 min. Demineralized water (4 ml) was added to each sample, which
was then centrifuged for 15 min at 10,000 × g. The
supernatant was retained for intracellular K+
determination. The K+ concentration in the samples was
determined by flame photometry (Jenway PFP7).
To determine inorganic phosphate concentrations, the supernatants were
assayed for phosphate as described by Chen et al. (8).
Protein determination.
Protein concentrations were
determined by the bicinchoninic acid method (Sigma procedure TRPO-562)
with bovine serum albumin as a standard.
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RESULTS |
Lacticin 3147 is a two-component bacteriocin.
Application of
an active, partially purified lacticin 3147 preparation to a
C18 reverse-phase column with an acetonitrile gradient
resulted in the FPLC chromatogram shown in Fig.
1. Agar well diffusion assays of each of
the eluted fractions showed negligible inhibition of the sensitive
indicator, L. lactis subsp. cremoris HP. When the
fractions corresponding to lac 1 and lac 2 (Fig. 1) were combined and
assayed, bacteriocin activity was restored. Therefore, it appears that
the FPLC purification step separated two components required for
activity, and it may be concluded that lacticin 3147 requires the
complementary action of two peptides, lac 1 and lac 2, for full
activity.
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FIG. 1.
FPLC chromatogram (C18 reverse-phase column)
of partially purified lacticin 3147. Also shown are the results of an
agar well diffusion assay of fractions (lac 1 and lac 2) which combined
to form an active bacteriocin.
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Lacticin 3147 is bactericidal.
Lacticin 3147 has been shown to
have a broad inhibition spectrum (29), inhibiting all
gram-positive bacteria which have been tested thus far. To investigate
the mode of action of lacticin 3147 on target organisms, the
bacteriocin was added to suspensions of sensitive cells, and the viable
count and optical density were determined over time.
L. lactis subsp. cremoris HP cells in buffer were
incubated with increasing concentrations of lacticin 3147. In a
concentration-dependent manner, an initial rapid decline in viability
was observed (Fig. 2A). However,
prolonged incubation did not result in further killing. A similar
experiment was performed with cells incubated with the bacteriocin in
the presence of glucose, i.e., buffer containing 10 mM glucose (Fig.
2B) and GM17 broth (Fig. 2C). Under these conditions, the killing
efficiency of the bacteriocin was dramatically increased; even at the
lowest concentration, no viable cells could be detected after 2 h.
In all cases, the optical density of the bacteriocin-treated cultures
did not change during the experiments (data not shown). These results
indicate that lacticin 3147 has bactericidal, rather than bacteriolytic
or bacteriostatic, activity and that, at low lacticin 3147 concentrations, energized cells are more sensitive to the bacteriocin.
Energized cells have a PMF which may favor the insertion of the
bacteriocin molecules into the membrane, as is the case with the
pore-forming lantibiotic nisin (3, 12, 14).
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FIG. 2.
Effect of lacticin 3147 addition on the viability of
L. lactis subsp. cremoris HP in buffer (A),
buffer supplemented with 10 mM glucose (B), and GM17 broth (C).
Symbols:  , no addition;  , addition of 75 AU/ml;  , addition of
300 AU/ml;  , addition of 600 AU/ml;  , addition of 1,200 AU/ml.
Data points along the horizontal axis represent 0% survival. The
initial cell number was 107 CFU/ml. Data points represent
the average of at least three experiments done in duplicate.
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Effect of lacticin 3147 on ATP levels.
The impact of lacticin
3147 on the energetic condition of sensitive cells was determined by
measuring the cellular ATP levels. On the addition of glucose, the
intracellular ATP concentration in L. lactis subsp.
cremoris HP cells increased approximately 10-fold; the
addition of 1,200 AU of lacticin 3147 per ml resulted in a rapid
decrease in the cellular ATP levels. After 10 min of incubation with
the bacteriocin, no ATP could be detected inside the cells (Fig.
3). However, an increase in the
extracellular ATP concentration was not observed (data not shown),
indicating that the bacteriocin-induced depletion of ATP was not due to
the release of ATP from cells.
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FIG. 3.
Intracellular ATP levels in cells of L. lactis subsp. cremoris HP (), L. monocytogenes Scott A (), and B. subtilis ATCC 6051 () treated with lacticin 3147. At arrow 1, 10 mM glucose was added;
at arrow 2, 1,200 AU of lacticin 3147 per ml was added. Data points
represent the average of at least three experiments done in
duplicate.
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These results suggest that the pores formed by the bacteriocin are not
large enough to allow the leakage of large compounds, such as ATP, and
that the rapid decrease in intracellular ATP levels is probably due to
hydrolysis. Since lacticin 3147 is a broad-host-range bacteriocin
(29), this experiment was also performed with two different
gram-positive bacteria, L. monocytogenes Scott A and
B. subtilis ATCC 6051. As with L. lactis subsp.
cremoris HP, intracellular ATP levels decreased (Fig. 3),
while no leakage of ATP was observed (data not shown). The different
levels of sensitivity of the three strains to the bacteriocin (Fig.
4) were reflected in the different rates
of ATP hydrolysis.
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FIG. 4.
Inhibitory action of lacticin 3147 against the
gram-positive species L. lactis subsp.
cremoris HP (A), L. monocytogenes Scott A (B),
and B. subtilis ATCC 6051 (C) illustrating the difference in
sensitivity of the test strains. A twofold serial dilution of the
bacteriocin was used as described in Materials and Methods.
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Lacticin 3147 selectively dissipates the component of the
PMF.
To determine if lacticin 3147 acts on the cytoplasmic
membrane of target organisms, the effect of the bacteriocin on the
components of the PMF, i.e., and pH, was examined.
The was measured qualitatively with the potential-sensitive
fluorescent cyanine dye diSC3(5). Generation of a
(inside negative) on addition of glucose to the cells resulted in
quenching of the fluorescent signal. In the presence of the
K+/H+ exchanger nigericin (1.0 µM), the cells
were able to maintain a maximum , which could be completely
dissipated by the addition of the K+ ionophore valinomycin
(1.0 µM) (data not shown). The addition of lacticin 3147 to cells
maintaining a maximum led to immediate depolarization of the
cytoplasmic membrane (Fig. 5A). No
further dissipation was observed when valinomycin was added to cells
treated with the bacteriocin, indicating that complete dissipation had occurred as a result of bacteriocin action. In fact, lacticin 3147 was
as effective at dissipating the as valinomycin. Figure 5B
demonstrates that lacticin 3147 may require an energized membrane for
insertion. The bacteriocin was added to cells prior to glucose addition; when glucose was added, a transient was rapidly
dissipated. Apparently, upon creation of a potential difference across
the membrane, the insertion of the bacteriocin molecules is enhanced. When the experiment was performed with L. monocytogenes
Scott A and B. subtilis ATCC 6051, similar results were
obtained; the was completely dissipated at the same lacticin
3147 concentration (data not shown). Regardless of the different
sensitivities of the strains, the depolarization of the membrane was
immediate in all cases. This result is in contrast to the results
obtained for internal ATP levels, where the rate of decrease on
addition of lacticin 347 was strain dependent.
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FIG. 5.
Effect of lacticin 3147 on the of L. lactis subsp. cremoris HP cells. The following
additions were made as indicated by the arrows: (A) arrow 1, L. lactis subsp. cremoris HP cells; arrow 2, 10 mM glucose; arrow 3, 1.0 µM nigericin; arrow 4, 1,200 AU of
lacticin 3147 per ml; arrow 5, 1.0 µM valinomycin; and (B) arrow 1, L. lactis subsp. cremoris HP cells; arrow 2, 1,200 AU of lacticin 3147 per ml; arrow 3, 10 mM glucose; arrow 4, 1.0 µM valinomycin; arrow 5, 1.0 µM nigericin. The fluorescence of the
diSC3(5) probe is depicted inversely.
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The generation of a pH in L. lactis subsp.
cremoris HP was monitored by measuring the changes in the
intracellular pH with the pH-sensitive fluorescent probe cFDASE
(5). The fluorescence of the probe was rapidly
increased on addition of glucose, indicating an increase in the
internal pH due to the extrusion of H+ ions. The addition
of nigericin caused a rapid and complete collapse of the pH, leading
to an equilibration of the internal pH with the outside environment.
The addition of lacticin 3147 resulted in an increase in the internal
pH and not a collapse of the pH (Fig.
6A). This increase in the internal pH was
maintained for approximately 4 min before dissipation of the pH
occurred. The gradual dissipation of the pH in the presence of
lacticin 3147 is best explained by the depletion of the ATP pool (Fig.
3). Therefore, lacticin 3147 does not cause immediate dissipation of
the pH of sensitive cells, as the membrane does not become permeable to H+ ions; therefore, the bacteriocin is selective for the
component of the PMF. A similar increase in the intracellular pH
can be seen when valinomycin is added to cells that have generated a pH; however, valinomycin-treated cells can maintain this internal pH
until nigericin is added (Fig. 6B).
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FIG. 6.
Effect of lacticin 3147 (A) and valinomycin (B) on the
pH of glucose-energized cells of L. lactis subsp.
cremoris HP. Glucose (10 mM) was added at arrow 1; 1,200 AU
of lacticin 3147 per ml (A) and 1.0 µM valinomycin (B) were added at
arrow 2; and 1.0 µM nigericin was added at arrow 3 to completely
dissipate the pH.
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Efflux of K+ ions and inorganic phosphate is the result
of bacteriocin action.
The bactericidal action of lactococcin G
(24), lactostrepcin 5 (33), and lacticin F
(2) results in the leakage of K+ ions from
susceptible cells. To determine whether lacticin 3147 has an impact on
the internal K+ pool of L. lactis subsp.
cremoris HP, control cells and cells treated with the
bacteriocin were permeabilized with trichloroacetic acid, and the
intracellular and extracellular K+ concentrations were
measured by flame photometry.
In the absence of the bacteriocin, energized cells of L. lactis subsp. cremoris HP maintained an intracellular
concentration of K+ of approximately 130 mM. The subsequent
addition of lacticin 3147 caused a dramatic loss of cellular
K+ (Fig. 7A). Measurement of
the K+ content outside the cells indicated that the
bacteriocin had induced massive leakage of K+ from the
cells, as the concentration outside had increased dramatically (Fig.
7A). The efflux of K+ was immediate, and after 15 min of
treatment with 1,200 AU of lacticin 3147 per ml, no trace could be
detected inside the cells. Similar results were observed for L. monocytogenes Scott A (Fig. 7B), whereas the efflux of
K+ from lacticin 3147-treated cells of B. subtilis ATCC 6051 was somewhat slower (Fig. 7C). The same
experiments were carried out with cells which were not energized; the
concentrations of intracellular K+ were similar to those in
cells in the presence of glucose, and the rates of efflux of
K+ were comparable to those shown in Fig. 7 for all three
organisms (data not shown). Nisin and the K+ ionophore
valinomycin also induced K+ leakage (data not shown).
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FIG. 7.
Lacticin 3147-induced K+ efflux from
energized L. lactis subsp. cremoris HP (A),
L. monocytogenes Scott A (B), and B. subtilis
ATCC 6051 (C) cells. Symbols: , intracellular K+
concentration; , extracellular K+ concentration. Data
points represent the average of at least two experiments done in
duplicate.
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Lacticin F was shown to make cells permeable to inorganic phosphate as
well as K+ ions (2); therefore, the samples were
assayed for internal and external inorganic phosphate contents.
Lacticin 3147 induced the loss of intracellular phosphate from L. lactis subsp. cremoris HP at a concentration of 1,200 AU/ml. In unenergized cells, the release was very rapid, as was found
with K+ (Fig. 8A). A reduced
rate of efflux was observed in energized cells, but the intracellular
levels of phosphate were much lower here than in unenergized cells,
approximately 20 mM versus 75 mM. In both cases, no trace of phosphate
could be detected intracellularly after 15 min of incubation with the
bacteriocin (Fig. 8A). When L. monocytogenes Scott A was
treated with lacticin 3147, a lower initial level of phosphate and a
reduced efflux rate were measured for unenergized cells, but the cells
retained a low level of free phosphate internally (Fig. 8B). However,
the difference between the unenergized and energized cells was not as
obvious in this case. For B. subtilis ATCC 6051, there was
little difference in the effect of lacticin 3147 on phosphate levels in
the unenergized and energized cells (Fig. 8C). The levels of
intracellular phosphate were comparable, as were the rates of efflux.
Experiments with valinomycin revealed that no phosphate was released
when cells were treated with this ionophore (data not shown), since
valinomycin is K+ specific.
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FIG. 8.
Inorganic phosphate (Pi) efflux from cells of
L. lactis subsp. cremoris HP (A),
L. monocytogenes Scott A (B), and B. subtilis ATCC 6051 (C). Symbols: and  , intracellular Pi
concentrations in unenergized and energized cells, respectively; and , extracellular Pi concentrations in unenergized and energized
cells, respectively. Data points represent the average of at least two
experiments done in duplicate.
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DISCUSSION |
The results presented in this paper suggest that the
broad-host-range lacticin 3147 is a membrane-active, two-component
bacteriocin that is hydrophobic in nature. The data suggest that the
bacteriocin induces cell death by making sensitive cell membranes
permeable, allowing for the efflux of K+ ions and
phosphate. This action results in the dissipation of the
component of the PMF, the hydrolysis of intracellular ATP, and
ultimately cell death.
Two peptides, lac 1 and lac 2, were separated by FPLC in the final
purification step. In isolation, these components have negligible
activity; the complementary action of both peptides is required for
full activity. The combined action of two peptides has been
demonstrated for other bacteriocins, including lacticin F (1,
25), lactococcin G (24, 26), and thermophilin 13 (23). It has been observed for these bacteriocins that
equivalent amounts of both peptides are required for an interaction
with target cells. Therefore, a preparation of lacticin 3147 purified to the stage prior to the separation of the individual components by
FPLC was used in this study to mimic the natural composition of the
bacteriocin secreted by the producing organism.
As with other LAB bacteriocins, the primary site of action of lacticin
3147 appears to be the cytoplasmic membrane (1, 2, 7, 14, 15, 19,
23, 24, 31, 32). Results from killing assays performed with
lacticin 3147 suggest that the action of the bacteriocin is enhanced
when target cells are energized, leading to the conclusion that the
presence of a PMF may favor insertion into the membrane of the
bacteriocin at low concentrations. Lacticin 3147 can also induce cell
death in the absence of an energized membrane, although somewhat less
effectively. However, at high concentrations the bacteriocin appears to
be as effective against unenergized cells; this result suggests that saturating amounts of the bacteriocin can overcome the requirement for
an energized membrane.
It was observed that lacticin 3147 makes the membranes of sensitive
cells permeable, allowing the efflux of K+ ions and
phosphate but not larger compounds, such as ATP. This movement of ions
results in a collapse of the ; however, the pH of
L. lactis subsp. cremoris HP is not
immediately dissipated, suggesting that lacticin 3147 does not induce
proton leakage and that the pores formed by the bacteriocin are
selective. Over time, however, the pH is dissipated, possibly a
secondary effect resulting from the inability of the cells to maintain
the activity of the F0F1-ATPase (the
membrane-bound enzyme responsible for generating the pH by pumping
protons across the membrane in L. lactis [4, 20]) in the absence of ATP, which is rapidly hydrolyzed on
addition of lacticin 3147. This mechanism of action resembles that of
the narrow-spectrum, two-component bacteriocins, e.g., lactococcin G
(24) and lacticin F (2), which form selective ion
pores, but differs from that of the broad-spectrum bacteriocin nisin, which forms much larger pores as a result of the aggregation of several bacteriocin molecules; the latter activity makes the
membrane permeable to larger compounds, such as ATP and amino acids
(14, 21, 22).
All three species tested in this study, L. lactis
subsp. cremoris HP, L. monocytogenes Scott
A, and B. subtilis ATCC 6051, lost a large percentage of
intracellular K+ within minutes of treatment with lacticin
3147. Treatment of L. lactis subsp. cremoris
HP with lacticin 3147 also resulted in the release of all detectable
internal phosphate; as with K+, this release occurred
almost immediately. The level of free phosphate in energized cells of
L. lactis subsp. cremoris HP was much lower
than that in unenergized cells, presumably due to the formation of
phosphorylated sugar intermediates of the glycolytic pathway. Also, the
rate of efflux of phosphate was somewhat reduced under these
conditions, perhaps due to the lower initial levels. It is known that
for L. lactis ML3 the uptake of phosphate is driven by
a unidirectional ATP-dependent transport system (28). Therefore, reaccumulation of phosphate under conditions where this
transport system is active, i.e., in the presence of an energy source,
may explain the reduction in the rate of efflux. The loss of these
essential ions may also explain the ATP hydrolysis that occurs as a
result of lacticin 3147 action; the cells utilize the available ATP in
a futile attempt to reaccumulate K+ and phosphate by
ATP-dependent uptake systems, resulting in an eventual collapse of the
pH. Similar transport mechanisms in Listeria and
Bacillus species are, however, not well understood. Both
organisms have the ability to retain a certain level of intracellular phosphate, even after prolonged incubation with lacticin 3147. It is
possible that these organisms also have phosphate bond-dependent uptake
systems for K+ and phosphate and that the various rates of
ATP hydrolysis can be explained by the differences in the retention or
reaccumulation of these ions by the organisms. Overall, this results in
a lower rate of ATP hydrolysis and therefore less efficient killing by the bacteriocin. These differences in ion retention and reaccumulation may explain why L. monocytogenes Scott A and B. subtilis ATCC 6051 are more resistant to lacticin 3147 than
L. lactis subsp. cremoris HP.
There are other possible explanations for the observed variations in
the levels of sensitivity to lacticin 3147. For example, the ability of
the bacteriocin to interact with the cytoplasmic membrane is influenced
by factors such as the composition of the cell envelope, including the
peptidoglycan layer, and the lipid composition of the membrane, as has
been demonstrated with nisin (13, 14). The presence of
K+ and/or phosphate ATPases may also affect the efficiency
of the bacteriocin, since these systems appear to be involved in ATP hydrolysis. Furthermore, the type of ATP-generating pathway, i.e., via
substrate-level phosphorylation in the glycolysis of L. lactis subsp. cremoris and via oxidative
phosphorylation in the electron transfer chain of B. subtilis, may affect the capacity to synthesize ATP, resulting in
differences in the killing efficiency of lacticin 3147 among the three
organisms studied here.
On the basis of these results, we propose that lacticin 3147 forms
pores which allow K+ and phosphate to leak; the resulting
change in electrical charge across the membrane causes the immediate
dissipation of the . In an attempt to recover these ions, the
cells use phosphate bond-dependent transport, resulting in rapid ATP
hydrolysis and leading to cell death. We have shown that this
two-component bacteriocin acts against L. lactis subsp.
cremoris HP, L. monocytogenes Scott A, and
B. subtilis ATCC 6051 despite the differences in the
lipid compositions of the cytoplasmic membranes of these organisms. The
broad range of lacticin 3147, as well as its activity at a neutral pH,
paves the way for the use of this bacteriocin in food systems for which
no natural alternative to artificial preservatives is presently
available.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, University College Cork, Cork, Republic of Ireland.
Phone: 353-21-902397. Fax: 353-21-903101. E-mail:
c.hill{at}ucc.ie.
| |
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Appl Environ Microbiol, February 1998, p. 439-445, Vol. 64, No. 2
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
