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Journal of Bacteriology, May 2000, p. 2643-2648, Vol. 182, No. 9
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A C-Terminal Disulfide Bridge in Pediocin-Like
Bacteriocins Renders Bacteriocin Activity Less Temperature
Dependent and Is a Major Determinant of the Antimicrobial
Spectrum
Gunnar
Fimland,1,*
Line
Johnsen,1
Lars
Axelsson,2
May B.
Brurberg,3,4
Ingolf F.
Nes,4
Vincent G. H.
Eijsink,4 and
Jon
Nissen-Meyer1
Department of Biochemistry, University of
Oslo, Oslo,1 and MATFORSK, Norwegian
Food Research Institute,2 Norwegian Crop
Research Institute,3 and Department of
Chemistry and Biotechnology, Agricultural University of
Norway,4 Ås, Norway
Received 4 November 1999/Accepted 4 February 2000
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ABSTRACT |
Several lactic acid bacteria produce so-called pediocin-like
bacteriocins that share sequence characteristics, but differ in
activity and target cell specificity. The significance of a C-terminal
disulfide bridge present in only a few of these bacteriocins was
studied by site-directed mutagenesis of pediocin PA-1 (which naturally
contains the bridge) and sakacin P (which lacks the bridge).
Introduction of the C-terminal bridge into sakacin P broadened the
target cell specificity of this bacteriocin, as illustrated by the fact
that the mutants were 10 to 20 times more potent than the wild-type
toward certain indicator strains, whereas the potency toward other
indicator strains remained essentially unchanged. Like pediocin PA-1,
disulfide-containing sakacin P mutants had the same potency at 20 and
37°C, whereas wild-type sakacin P was approximately 10 times less
potent at 37°C than at 20°C. Reciprocal effects on target cell
specificity and the temperature dependence of potency were observed
upon studying the effect of removing the C-terminal disulfide bridge
from pediocin PA-1 by Cys
Ser mutations. These results clearly show
that a C-terminal disulfide bridge in pediocin-like bacteriocins
contributes to widening of the antimicrobial spectrum as well as to
higher potency at elevated temperatures. Interestingly, the differences
between sakacin P and pediocin PA-1 in terms of the temperature
dependency of their activities correlated well with the optimal
temperatures for bacteriocin production and growth of the
bacteriocin-producing strain.
| |
TEXT |
Many bacteria are known to produce
ribosomally synthesized antimicrobial polypeptides called bacteriocins.
Bacteriocins produced by gram-positive bacteria are usually
membrane-permeabilizing cationic peptides with less than 50 amino acid
residues (27, 29). These bacteriocins may be divided into
two classes. Class I bacteriocins, termed lantibiotics, contain
modified residues, whereas class II bacteriocins do not. One group of
class II bacteriocins (frequently called class IIa) consists of the
so-called "pediocin-like bacteriocins," produced by a variety of
lactic acid bacteria. These bacteriocins are characterized by
high antilisterial activity, by the presence of a YGNGV motif and
a disulfide bridge in their N-terminal halves, and by the fact that
they apparently kill cells by permeabilizing the target cell membrane
(9, 10). The first pediocin-like bacteriocins that were
identified and thoroughly characterized were pediocin PA-1 (7, 20,
24, 28), leucocin A-UAL 187 (16), mesentericin Y105
(19), and sakacin P and curvacin A (21, 33).
Today, at least nine other pediocin-like bacteriocins have been
isolated and characterized (4-6, 11, 22, 23, 25, 31, 32,
34). Despite similarities in their primary structures, the
pediocin-like bacteriocins have different target cell specificities
(12).
Based on their primary structures, pediocin-like bacteriocins may
roughly be divided into two regions: a hydrophilic, cationic, and
highly conserved N-terminal half and a less-conserved hydrophobic and/or amphiphilic C-terminal half (13). It has been
proposed that the well-conserved cationic N-terminal half mediates the initial binding of these bacteriocins to target cells through electrostatic interactions (8) and that the hydrophobic or amphiphilic C-terminal half penetrates into the hydrophobic part of the
target cell membrane, thereby mediating membrane leakage (13,
26). The hydrophobic or amphiphilic C-terminal half also appears
(in part) to mediate target cell specificity, since hybrid bacteriocins
containing N- and C-terminal regions from different pediocin-like
bacteriocins have antimicrobial spectra similar to that of the
bacteriocin from which the C-terminal region is derived
(13).
In addition to the conserved disulfide bridge in the N-terminal half, a
few pediocin-like bacteriocins contain a second disulfide bridge,
located in the C-terminal half (Fig. 1).
Comparative studies of natural bacteriocins have led to the suggestion
that this second disulfide bridge is an important determinant of
bacteriocin activity (12). Here, we present the results of
site-directed mutagenesis studies of pediocin PA-1 (two disulfide
bridges) and sakacin P (one disulfide bridge), aimed at analyzing the
contribution of the second disulfide bridge to the potency, target cell
specificity, and temperature dependency of activity.
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FIG. 1.
An overview of bacteriocin mutants. Disulfide bridges
are indicated. Assignment of the disulfide bridges is based on studies
by Henderson et al. (20) and on results presented in this
study. The arrows indicate the unique aspartate residue in sakacin P
and pediocin PA-1, which was used for studies with endoproteinase
Asp-N.
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Production and purification of sakacin P, pediocin PA-1, and their
mutants.
Figure 1 gives an overview of the mutants that were made
in this study. In pediocin PA-1, the C-terminal disulfide bridge was
removed by replacing cysteine residues in the C-terminal half with
serine (ped[C24S+C44S]; Fig. 1). A C-terminal disulfide bridge was
introduced in sakacin P, by introducing cysteine residues at the
positions indicated by alignment with pediocin PA-1. Thus, one cysteine
residue was added to the C terminus (position 44), whereas another
cysteine was introduced to replace an asparagine at position 24 (sak[N24C+44C]; Fig. 1). Since there is a conspicuous sequence
difference between sakacin P and pediocin PA-1 at the position
preceding residue 24 (Gly23 and Thr23, respectively), a second
disulfide variant of sakacin P was constructed in which Gly23 was
changed into Thr (sak[G23T+N24C+44C]; Fig. 1). In addition, a series
of control mutants was made, as shown in Fig. 1 (ped[C24S], ped[C44S], sak[G23T+N24S+44S], sak[44C],
sak[44S], sak[N24C], sak[G23T+N24C],
sak[G23T+N24S], and sak[G23T]).
With the exception of wild-type pediocin PA-1, which was purified from
its natural producer (
Pediococcus acidilactici LMG2351
[
28]), all bacteriocins were produced by using a
recently developed
system for bacteriocin expression in the
bacteriocin-deficient
strain
Lactobacillus sake Lb790
(
3). The system is based on
the use of two plasmids: pSAK20
and pSPP2 (for production of sakacin
P and variants) or pPED2 (for
production of pediocin PA-1 variants).
Mutations in the sakacin P and
pediocin PA-1 genes, cloned in
pSPP2 and pPED2, respectively, were made
by using the Quick Change
site-directed mutagenesis kit (Stratagene).
The DNA sequence of
the mutant plasmids was verified by automated DNA
sequence determination
with an ABI PRISM 377 DNA sequencer and the ABI
Prism dye terminator
cycle sequencing Ready Reaction kit
(Perkin-Elmer).
Epicurian Coli XL1-Blue Supercompetent cells
(Stratagene; grown at 37°C
in Luria-Bertani medium [Difco] with
vigorous agitation) were
used for the cloning of all mutated pSPP2 and
pPED2 plasmids.
pSPP2 and pPED2 derivatives containing the desired
mutations were
transformed into
L. sake Lb790/pSAK20 by
electroporation as described
previously (
1). All plasmid
isolations from
Epicurian Coli and
L. sake Lb790
were done by using the Wizard Plus SV Minipreps
DNA purification system
(Promega).
L. sake cells were treated
with lysozyme and
mutanolysin (5 mg/ml and 15 U/ml, respectively)
before lysis. The
selective antibiotic concentrations used were
150 µg of erythromycin
per ml for
E. coli, 10 µg (each) of erythromycin
and
chloramphenicol per ml for normal growth of plasmid-containing
L. sake Lb790, and 2 µg of erythromycin per ml and 5 µg of
chloramphenicol
per ml for initial selection of
L. sake
Lb790/pSAK20 transformed
with pSPP2 or pPED2
variants.
Wild-type and mutant bacteriocins were purified to homogeneity from
400- or 800-ml cultures by ammonium sulfate precipitation
followed by
cation-exchange, hydrophobic interaction, and reverse-phase
chromatography as described previously (
30). Routinely,
between
10 and 100 µg of sakacin P, pediocin PA-1, and mutant
bacteriocins
was purified from 400-ml cultures. To check that the
recombinant
lactobacilli had correctly produced and processed the
bacteriocins,
molecular masses of the isolated peptides were determined
by mass
spectrometry with a matrix-assisted laser desorption
ionization-time
of flight Voyager-DE RP mass spectrometer (Perseptive
Biosystems).
The purity of bacteriocins was verified to be greater than
90%
by analytical reverse-phase chromatography with a µRPC SC 2.1/10
C
2/C
18 column (Pharmacia Biotech) in the SMART
chromatography
system (Pharmacia Biotech). The concentration of
purified bacteriocins
was determined by measuring UV
A280, which was converted to protein
concentration with molecular extinction coefficients calculated
from
the contributions of individual amino acid
residues.
In the last reverse-phase chromatography step, sakacin P, pediocin
PA-1, and mutants that did not contain cysteine residues
in their
C-terminal region (the sakacin P mutants sak[G23T],
sak[G23T+N24S],
sak[44S], and sak[G23T+N24S+44S] and
the pediocin PA-1 mutant
ped[C24S+C44S]; Fig.
1) gave one major,
almost symmetrical absorbance
peak that contained a peptide with
bacteriocin activity and the
expected molecular weight (determined by
mass spectrometry). The
mutants that contained only one cysteine
residue in the C-terminal
region (the sakacin P mutants sak[N24C],
sak[44C], and sak[G23T+N24C]
and the pediocin PA-1 mutants
ped[C24S] and ped[C44S]; Fig.
1)
gave more complex absorbance
profiles containing several
often
asymmetrical
absorbance peaks,
presumably because of stability
problems and incorrect formation of
disulfide bridges (including
intermolecular disulfide bridges). For
each of these mutants,
the fraction with the most bacteriocin activity
was collected.
Mass spectrometry confirmed that these fractions
contained the
expected mutant
bacteriocins.
For sakacin P mutants containing two new cysteine residues
(sak[N24C+44C] and sak[G23T+N24C+44C]; Fig.
1) the final
reverse-phase
chromatography step yielded three peaks of approximately
equal
size, each containing a peptide with the expected molecular mass.
The contents of these three fractions were further studied by
mass
spectrometry analyses of the fragments obtained after treatment
with
endoproteinase Asp-N. This protease cleaves sakacin P in
front of its
unique Asp residue at position 17 that is located
between the two
N-terminal and the two C-terminal cysteine residues
(Fig.
1). The
results (not shown) revealed that the last peptide
to elute from the
column had correct disulfide bridges (that is
bridges between 9 and 14 and between 24 and 44, analogous to bridge
formation found in natural
pediocin PA-1 [
20]). The specific
activity of this
peptide was at least 100 times greater than that
of the two other
peptides. Interestingly, the activity of the
two lesser active peptides
increased greatly and became similar
to that of the initially most
active peptide, upon exposure to
small amounts of dithiothreitol (DTT)
(2 to 3 mM) during the bacteriocin
assay. Thus, the two variants that
appeared to display incorrect
disulfide bridges could be transformed
into active bacteriocins
under conditions that promoted both
structuring (i.e., the presence
of target cell membranes [
15,
17,
18]) and disulfide exchange
(the presence of low
concentrations of DTT). The activity of all
three peptides was reduced
at higher DTT concentrations (above
5
mM).
Production of pediocin PA-1 with the expression system also yielded
peptides with incorrect disulfide bridges. In contrast,
production of
pediocin PA-1 by its wild-type producer and production
of bacteriocin
variants with only two cysteine residues (e.g.,
wild-type sakacin P and
ped[C24S+C44S]) with the expression system
were unproblematic. Taken
together, the results indicate that
a protein present in the natural
producer, but not in
L. sake Lb790/pSAK20/pPED2, helps to
generate the correct disulfide bridges
in bacteriocins containing four
cysteines. The secretion machinery
used in our heterologous expression
system is derived from a strain
that produces sakacin A, a
pediocin-like bacteriocin with only
one disulfide bridge
(
2). It is thus tempting to speculate
that the secretion
machinery present in the natural pediocin PA-1
producer is to some
extent adapted to generate the correct disulfide
bridges in four
cysteine-containing pediocin-like
bacteriocins.
Mutational effects.
Six indicator strains were used for
testing the various bacteriocins. When assayed at 20 and 30°C,
pediocin PA-1 and sakacin P had similar potencies toward four of
these strains (L. coryneformis subsp.
torquens NCDO 2740, L. sake NCDO 2714, Enterococcus faecalis NCDO 581, and Carnobacterium
piscicola UI49; Table 1). Two of the
strains, P. acidilactici NCDO 1859 and
Pediococcus pentosaceus FBB63B, were at least 100 times more sensitive to pediocin PA-1 than to sakacin P (Table 1), and
these two strains were therefore useful for detecting mutations which
make sakacin P more like pediocin PA-1. Pediocin PA-1 and sakacin P
also differed in the temperature dependency of their activity. Pediocin
PA-1 had nearly the same potency at 20, 30, and 37°C, whereas sakacin
P was about 10 times more potent at 20 and 30°C than at 37°C (Table
1 and Fig. 2). Thus, measurement of the
temperature dependency of the activity of mutant bacteriocins provided
an additional way of evaluating whether a mutation had made the one
bacteriocin more like the other.
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FIG. 2.
Temperature sensitivity of bacteriocin activity. The
bars represent relative bacteriocin activities at 20°C (open bars),
30°C (shaded bars), and 37°C (solid bars). For each combination of
bacteriocin and indicator strain, the highest activity was set to 1.
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The pediocin PA-1 mutant, ped[C24S+C44S], which lacks the C-terminal
disulfide bridge, had lost much of its potency against
the two
Pediococcus strains that were sensitive to wild-type
pediocin
PA-1, but relatively insensitive to sakacin P (Table
1). Its
activity toward the four other strains was also reduced, but not
nearly
to the same extent, especially not at lower temperatures.
In contrast
to wild-type pediocin PA-1, ped[C24S+C44S] was much
less active at
37°C than at 20°C (Table
1 and Fig.
2). Thus,
the removal of the
C-terminal disulfide bridge in pediocin PA-1
rendered pediocin PA-1
more sakacin-like with respect to both
the target cell specificity and
the temperature dependence of
activity.
The complementary mutation in sakacin P, the introduction of a
C-terminal disulfide bridge, clearly rendered sakacin P more
similar to
pediocin PA-1. When tested at 20 and 30°C, sak[N24C+44C]
and
sak[G23T+N24C+44C] had approximately the same potency as sakacin
P
against the four indicator strains that were sensitive to both
sakacin
P and pediocin PA-1. However, these two mutants had become
more potent
toward the two
Pediococcus strains that were relatively
resistant to sakacin P, but sensitive to pediocin PA-1 (Table
1).
Moreover, in contrast to wild-type sakacin P, these two mutants
had
about the same potency at 37°C as at 20°C for four of the
five
strains tested (Table
1). Thus, also in terms of the temperature
dependency of activity, the introduction of the extra disulfide
bridge
had made sakacin P more similar to pediocin PA-1. Differences
in
activity between
sak[N24C+44C] and
sak[G23T+N24C+44C] were
small, the latter being slightly
more active toward most of the
strains and at most of the temperatures
tested. The control mutants,
sak[G23T+N24S+44S] and
sak[G23T+N24S], in which serine instead
of cysteine residues were
introduced into sakacin P, displayed
similar potency, target cell
specificity, and temperature dependency
of activity to wild-type
sakacin P (Table
1 and Fig.
2). Also
the mutants sak[44S] and
sak[G23T] had similar potencies to wild-type
sakacin P (Table
1).
Mutant bacteriocins with only one cysteine residue in the C-terminal
half had reduced activities and were more unstable than
those that had
either none or two cysteine residues in this region.
The sak[44C],
sak[N24C], and sak[G23T+N24C] mutants were only
about 1/10 as
active as the sakacin P (results not shown). The
two single pediocin
PA-1 mutants, ped[C24S] and ped[C44S], were
somewhat less active
than the double mutant, ped[C24S+C44S] (results
not shown). Miller et
al. (
26) previously reported that ped[C24S]
and
ped[C44S] are completely inactive. The difference between
our
observations and those of Miller et al. (
26) may be caused
by the fact that Miller et al. (
26) conducted their assays
with
unpurified mutant bacteriocins in culture supernatants of
overproducing
E. coli strains. These conditions are clearly
different from those
used in the present study and may have promoted
problems with
stability and/or incorrect disulfide formation. It is our
experience
that the three-cysteine mutants (in contrast to the
wild-type
bacteriocins or mutants with two or four cysteines) lose
activity
after a few weeks of storage at 4°C in 0.1% trifluoroacetic
acid
and 20% 2-propanol and that it is not possible to recover the
activity by exposing the mutants to small amounts (1 to 3 mM)
of DTT.
Concomitant with loss of activity, the molecular weight
increased by 48 (determined by mass spectrometry), suggesting
that the thiol group on
cysteine was oxidized to sulfonic acid.
A component with twice the
expected molecular weight was also
observed, suggesting some
dimerization through the formation of
intermolecular disulfide
bridges.
Figure
3 shows that there exists a
correlation between the temperature dependence of growth and
bacteriocin production on
the one hand and the temperature dependence
of bacteriocin activity
on the other hand. At temperatures above
30°C, the activity of
sakacin P was reduced considerably (Table
1 and
Fig.
2) and so
were the growth of the bacteriocin-producing strain and
production
levels of the bacteriocin (Fig.
3). In contrast, pediocin
PA-1
was fully active at 37°C (Table
1 and Fig.
2), a temperature
at
which both bacteriocin production and the growth of the producing
strain were at their maximum (Fig.
3). The properties of the
bacteriocins
thus seem to be well adapted to the ecological niche that
the
producer strain is likely to grow in.
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FIG. 3.
Bacteriocin production and growth of natural producer
strains. Growth of the wild-type sakacin P producer L. sake
LTH673 (A) and of the wild-type pediocin PA-1 producer P. acidilactici (B) was monitored at various temperatures while
simultaneously monitoring bacteriocin levels in the culture
supernatants. Growth is represented by the generation times that could
be calculated from the growth curves and by the maximal cell densities
that were obtained. Bacteriocin concentrations are expressed in
bacteriocin units (BU) per microliter and represent the highest levels
observed while monitoring growth. One BU was defined as the amount of
bacteriocin required to reduce the growth of the indicator strain by
50% in the bacteriocin assay described above. Bacteriocin activities
were determined with standard bacteriocin assays (see reference
28 and Table 1). The assay temperature was 30°C,
and L. sake NCDO 2714 was used as the indicator strain.
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Nuclear magnetic resonance studies of the pediocin-like bacteriocins
leucocin A-UAL 187 (
15) and carnobacteriocin B2
(
35)
have shown that the middle part of the bacteriocins is
likely
to form an alpha-helix upon interaction with a target cell
membrane.
The remaining C-terminal residues were found to be relatively
unstructured. If one extrapolates these observations to pediocin
PA-1,
the conclusion would be that the C-terminal residues must
fold back
onto the helical part, thus allowing formation of the
24-44 disulfide
bridge. Introduction of the 24-44 disulfide bridge
left the activity
of sakacin P toward a number of indicator strains
at 20 and 30°C
largely unchanged. It is therefore not likely that
formation of the
24-44 disulfide bridge in sakacin P required
major structural
rearrangements, indicating that pediocin PA-1
and sakacin P must have
quite similar structures. Interestingly,
sakacin P seems to be better
adapted to a situation without the
24-44 disulfide bridge than
pediocin PA-1 (compare ped[C24S+C44S]
with
sak[G23T+N24S+44S]; Table
1). Other structural characteristics
of
pediocin PA-1, which are adapted to the presence of the second
disulfide bridge, apparently become unfavorable in a situation
where
the C-terminal disulfide bridge is not
present.
Our results suggest that the connection between the helical part and
the following stretch of C-terminal residues provided
by the 24-44
disulfide bridge is of major importance for the target
cell specificity
of pediocin-like bacteriocins. The relative importance
of the 24-44
bridge is further illustrated by the fact that control
mutations that
did affect the C-terminal part of sakacin P, but
not disulfide
formation (sak[44S], sak[G23T], sak[G23T+N24S],
and
sak[G23T+N24S+44S]), had only marginal effects on bacteriocin
activity. The presence of the 24-44 disulfide bridge manifested
itself
in widening of the antimicrobial spectrum at lower temperatures
and
generally increased activity at higher
temperatures.
This study is one of the first examples of a site-directed mutagenesis
analysis aimed at unraveling the role of specific structural
elements
in bacteriocins produced by lactic acid bacteria. It
has been suggested
that the C-terminal half of pediocin-like bacteriocins
is an important
determinant of target cell specificity (
13,
14), and our
results pinpoint a specific C-terminal structural
element that plays a
major role in determining this specificity.
It is important to note
that the mutational effects observed in
this study are truly
strain-specific and that they do not reflect
general increases or
decreases in potency. This is especially
apparent for sakacin P mutants
tested at 20 or 30°C in that the
effect of the introduction of the
disulfide bridge on bacteriocin
activity was marginal for four of the
indicator strains, whereas
activity toward two other strains (the two
pediococci) was increased
drastically. In addition to widening the
antimicrobial spectrum,
the C-terminal disulfide bridge clearly
contributed to reducing
the temperature sensitivity, and the two
sakacin P variants that
contain the C-terminal disulfide bridge
(sak[N24C+44C] or sak[G23T+N24C+44C])
represent the first
(semi-) rationally designed variants of pediocin-like
bacteriocins with
increased
potencies.
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ACKNOWLEDGMENTS |
This work was supported by a grant from the Norwegian Research Council.
We thank Marianne Skeie for help with some of the experiments.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, University of Oslo, Post Box 1041, Blindern, 0316 Oslo, Norway. Phone: 47-22 85 66 32. Fax: 47-22 85 44 43. E-mail:
gunnar.fimland{at}biokjemi.uio.no.
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Journal of Bacteriology, May 2000, p. 2643-2648, Vol. 182, No. 9
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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