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Journal of Bacteriology, December 1998, p. 6565-6570, Vol. 180, No. 24
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Lantibiotic Nisin Induces Transmembrane
Movement of a Fluorescent Phospholipid
Gert N.
Moll,
Wil N.
Konings, and
Arnold J. M.
Driessen*
Department of Microbiology and Groningen
Biomolecular Sciences and Biotechnology Institute, University of
Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands
Received 13 July 1998/Accepted 15 October 1998
 |
ABSTRACT |
Nisin is a pore-forming antimicrobial peptide. The capacity of
nisin to induce transmembrane movement of a fluorescent phospholipid in
lipid vesicles was investigated. Unilamellar phospholipid vesicles that
contained a fluorescent phospholipid
(1-acyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl}-sn-glycero-3-phosphocholine) in the inner leaflet of the bilayer were used. Nisin-induced movement of the fluorescent phospholipid from the inner leaflet to the outer
leaflet of the membrane reached stable levels, which were dependent on
the concentration of nisin added. The rate constant k of
this nisin-induced transmembrane movement increased with the nisin
concentration but was not dependent on temperature within the range of
5 to 30°C. In contrast, the rate constant of movement of fluorescent
phospholipid from vesicle to vesicle strongly depended on temperature.
The data indicate that nisin transiently disturbs the phospholipid
organization of the target membrane.
| |
INTRODUCTION |
Asymmetric distribution of
phospholipids over the inner and outer leaflets of biological membranes
is essential for membrane function (28, 29). In eukaryotic
membranes, the phospholipid distribution is maintained by proteins
termed "flippases" and by interactions with cytoskeletal proteins
(12). For instance, the ATP-dependent flippase in
erythrocytes translocates amino phospholipids from the outer
leaflet to the inner leaflet of the membrane (36).
Also, MDR P-glycoprotein translocates phospholipid (43). In bacterial membranes, little is known about the
distribution of phospholipids over the inner and outer leaflets. Rapid
movement of fluorescent phospholipids across the inner membrane of
Escherichia coli has been demonstrated and is probably a
protein-mediated process (14). Movement of a fluorescent
phospholipid from the outer leaflet to the inner leaflet was
demonstrated in the membrane of Bacillus megaterium
(13).
Channel-forming cytotoxins (from Staphylococcus aureus
[alpha-toxin] and Pseudomonas aeruginosa) in addition to
relatively small "pore-forming" antimicrobial peptides, such
as melittin, the synthetic peptide GALA (10), and
mastoparan X (24), are known to cause movement of membrane
phospholipids from one leaflet to the other (35). In the
cases mentioned above, the peptide-induced phospholipid movement might
reflect an important mechanistic aspect of pore formation. For example,
fusion of the inner and outer leaflets of the target membrane may play
a role such as that proposed for magainin-induced "toroidal" pores
(22).
Nisin is a cationic peptide antibiotic of 34 amino acids that contains
five intramolecular rings. Its genetics (18), synthesis regulation (19), and structure (21, 39-42) are
known, and several mutants have been generated (16-18, 20).
Because of its antimicrobial activities, of which permeabilization of
the membranes of the target cells is the most important
(25), nisin is used as a food preservative. Nisin
preferentially interacts with anionic phospholipid-containing (3,
8, 9) membranes, transiently enhancing the permeability for small
ions and solutes (5, 11, 15, 27, 31-34). We previously
proposed a "wedge-like" model for pore formation by nisin (9,
26). The positively charged carboxyl terminus of nisin, together
with the bound lipids, enter into the membrane. Both the transmembrane
electrical potential (
) (9, 32, 33) and the pH
(cis acid) stimulate nisin-mediated membrane
permeabilization (26). In this study, we demonstrate that
nisin induces rapid movement of a fluorescent phospholipid from the
inner leaflet to the outer leaflet of unilamellar phospholipid vesicles. This provides support for nisin-induced disturbance of the
phospholipid organization in the membrane, as proposed previously in
the wedge-like model for nisin-induced pore formation.
| |
MATERIALS AND METHODS |
Materials.
Nisin was obtained as a gift from Aplin & Barrett. Dioleoylphosphatidylcholine (DOPC),
dioleoylphosphatidylglycerol (DOPG), L-
-phosphatidylethanolamine-N-(lissamine
rhodamine B sulfonyl) (Rh-PE),
1-acyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl}-sn-glycero-3-phosphocholine (C6-NBD-PC), and 1-acyl-2{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]cap-royl}-sn-glycero-3-[phospho-rac-(1-glycerol)] (C6-NBD-PG) were obtained from Avanti Polar Lipids. The
1-acyl chains of C6-NBD-PC and C6-NBD-PG
represent the fatty acid content of egg lysophosphatidylcholine.
Preparation of small unilamellar phospholipid vesicles.
Phospholipids dissolved in organic solvent were dried under nitrogen.
Subsequently, a small volume of ethanol was added, and the lipids were
dried again. Multilamellar vesicles were formed by vortexing in the
presence of buffer and glass beads. Small unilamellar vesicles were
obtained by sonication (tip diameter, 3 mm) (Soniprep 150; Beun de
Ronde, Abcoude NL) of the multilamellar vesicles under the following
conditions: 30 min, 10-µm amplitude, pulses of 30 s on and
30 s off, incubation on ice under a stream of nitrogen. Metal
particles and large vesicles were removed by centrifugation for 15 min
at 80,000 × g at 4°C. Vesicles composed of
DOPC-Rh-PE (98:2 [wt/wt]) were prepared by ethanol injection (2), followed by dialysis to remove ethanol.
Preparation of large unilamellar phospholipid vesicles with
symmetric transmembrane distribution of
C6-NBD-phospholipids.
Phospholipid vesicles labelled
with 2% (wt/wt) C6-NBD-phospholipid were prepared by
reverse-phase evaporation (38), followed by extrusion
performed 11 times through 400-nm-pore-size polycarbonate filters
(Avestin) in either
piperazine-N,N'-bis(2-ethanesulfonic acid)
(PIPES), or phosphate buffer (pH 7.0) containing 0.21 M sucrose and 50 mM K+ or 50 mM Na+. Extravesicular sucrose was
removed by dialysis against the same buffer without sucrose.
Preparation of large unilamellar phospholipid vesicles with
C6-NBD-lipids exclusively in the inner leaflet.
Vesicles prepared as in the preceding paragraph, which contain
C6-NBD-phospholipid in both inner and outer membrane
leaflets, were incubated four times at 20°C with sonicated small DOPG
vesicles that do not contain C6-NBD-phospholipid,
essentially as described previously (10). Each incubation
was followed by centrifugation for 15 min at 80,000 × g and at 4°C, after which the supernatant with the sonicated
small DOPG vesicles was discarded and the
DOPG-C6-NBD-phospholipid vesicles were resuspended. The
incubation with sonicated small DOPG vesicles was at a 5- to 10-fold
(first incubation) or one- to twofold (subsequent three incubations)
excess quantity over the amount of total phospholipid present in the
DOPG-C6-NBD-phospholipid vesicles.
Preparation of large unilamellar phospholipid vesicles with
C6-NBD-phospholipids exclusively in the outer leaflet.
DOPG vesicles without C6-NBD-phospholipids were prepared as
described above by reverse-phase evaporation (38) followed
by extrusion. These acceptor vesicles were incubated twice during 1 h at 20°C with twice the amount (on a phospholipid basis)
small unilamellar donor vesicles, prepared by sonication as described above in "Preparation of small unilamellar phospholipid vesicles" but containing 6% (wt/wt) C6-NBD-phospholipid.
Centrifugation for 15 min at 80,000 × g and at 4°C
followed each incubation. The supernatant with small unilamellar
vesicles was discarded, and the vesicles with
C6-NBD-phospholipid in the outer leaflet were resuspended.
Transmembrane movement of C6-NBD-phospholipids.
Transmembrane movement of C6-NBD-phospholipids was measured
in two ways. In both assays, nisin causes transmembrane movement of
C6-NBD-phospholipids from the inner leaflet of the
unilamellar DOPG (58 µM) vesicles to the outer leaflet, which
initially does not contain C6-NBD-phospholipids.
The first method makes use of the decrease of measurable NBD emission
due to energy transfer to Rh-PE. Nisin-induced transmembrane movement
of C6-NBD-phospholipids to the outer leaflet of DOPG (58 µM) vesicles is followed by the diffusion of
C6-NBD-phospholipid to DOPC-Rh-PE (98:2 [wt/wt]) (1 mM
DOPC) vesicles, resulting in a decrease of the NBD emission, which was
measured as a percentage at the point at which a stable level of
fluorescence decrease was reached. The NBD fluorescence measured when
vesicles composed of C6-NBD-phospholipid-DOPC-Rh-PE were
prepared was taken as the 100% control of fluorescence decrease.
In the second assay, transmembrane movement of
C
6-NBD-phospholipids from the inner leaflet to the outer
leaflet of DOPG vesicles
is measured in the absence of quenching
DOPC-Rh-PE vesicles. The
NBD fluorescence increase due to shielding of
NBD groups in the
outer leaflet from the aqueous phase by
membrane-bound nisin is
measured (
9). Analysis of the
kinetics of transmembrane movement
was performed by assuming
second-order kinetics and by using the
table curve program (Jandel).
The NBD fluorescence increase was
fitted according to the equation that
gave the best fit:
y =
a +
b(1
e
kx) +
d(1
eex). The value of
the second rate constant
e was very small (generally
between
10
2 and 10
5 times smaller than
k) compared to the first rate constant k.
In parallel
experiments, the NBD fluorescence increase was measured
for DOPG
vesicles with symmetric transbilayer distribution of
C
6-NBD-phospholipid.
All fluorescence measurements were performed at 30°C, unless
otherwise indicated, using a Perkin Elmer LS-50 spectrofluorimeter.
For
the NBD fluorescence, excitation and emission wavelengths
were 475 and
530 nm, respectively. The slit widths were set at
4
nm.
Transfer of C6-NBD-phospholipids between phospholipid
vesicles.
Interbilayer movement of lipids was measured as follows:
DOPG-Rh-PE vesicles (98:2 [wt/wt]) containing either 2% (wt/wt)
C6-NBD-PG or C6-NBD-PC were incubated with DOPG
or DOPC vesicles without fluorescent probe. Rate constants of
phospholipid transfer were derived from the NBD fluorescence increase.
Calcein leakage.
Liposomes were prepared by reverse-phase
evaporation (38) in a solution containing 0.21 M sucrose, 8 mM calcein, and 50 mM K-PIPES, pH 7.0. After dialysis against 50 mM
K-PIPES (pH 7.0), external calcein was removed by centrifugation for 15 min at 80,000 × g and 4°C, followed by two washes
(each wash done by resuspension and then centrifugation). The integrity
of the phospholipid vesicles was assayed by measuring the fluorescence
increase after the addition of nisin and after subsequent addition of
0.2% Triton X-100 to calcein-loaded phospholipid vesicles. Excitation
and emission wavelengths were 470 and 520 nm, respectively, and the
slit widths were 2.5 nm.
Phosphate determinations.
Phosphate determinations were
performed by the method of Rouser et al. (30).
| |
RESULTS |
In the present study, we investigated the capacity of nisin to
induce transmembrane movement of the C6-NBD-phospholipid.
In a first assay, unilamellar DOPG vesicles which contained
C6-NBD-phospholipids in the inner leaflet were incubated
with an excess of DOPC-Rh-PE vesicles. Nisin is known to bind to
negatively charged phospholipids, like DOPG, but not to zwitterionic
DOPC phospholipids (9). After nisin-mediated movement to the
outer leaflet of the membrane, the C6-NBD-phospholipids
diffused to the Rh-PE-containing DOPC vesicles. Once inserted into
these vesicles, the NBD emission was largely used for the excitation of
rhodamine, which gave rise to a decrease in the emission of
C6-NBD-PC (Fig. 1A and C) and C6-NBD-PG (Fig. 1B and C). In the absence of nisin or when
DOPC vesicles (Fig. 1C) were used instead of DOPG vesicles, no decrease in the NBD fluorescence was observed. This implied that the acidic phospholipid DOPG was needed for the nisin-induced transmembrane movement of the C6-NBD-phospholipid. Interestingly, the
decrease in NBD fluorescence was much greater for C6-NBD-PC
than for C6-NBD-PG (Fig. 1C). Control experiments showed
that nisin did not inhibit the transfer of either C6-NBD-PG
or C6-NBD-PC from DOPG vesicles towards DOPC vesicles.
These control experiments were performed by following the increase in
NBD fluorescence due to transfer of C6-NBD-phospholipid
from DOPG (58 µM)-C6-NBD-phospholipid (2% [wt/wt] in
both leaflets)-Rh-PE (2% [wt/wt] in both leaflets) vesicles, in
which the NBD fluorescence was quenched by the Rh-PE, towards DOPC (1 mM) vesicles (data not shown).
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FIG. 1.
Nisin-induced transmembrane movement of
C6-NBD-PC in DOPG vesicles. (A and B) DOPG (58 µM)
vesicles, which contained either C6-NBD-PC (arrow 1 in
panel A) or C6-NBD-PG (arrow 2 in panel B) exclusively in
the inner leaflet, were added to DOPC (1 mM) vesicles that contained
Rh-PE (2% [wt/wt]). Addition of 166 nM (b), 500 nM (c), 1,000 nM
(d), and 1,500 nM (e) nisin (arrows 3 in panels A and B) caused a
decrease in C6-NBD-PC (A) and C6-NBD-PG (B)
fluorescence. Lines a were obtained after addition of buffer. In a
separate measurement, C6-NBD-PC (A) and
C6-NBD-PG (B) were incorporated in DOPC-Rh-PE vesicles:
fluorescence values of 20.7 (A) and 18.4 (B) correspond to 100%
transfer (horizontal broken lines) of C6-NBD-phospholipid
to DOPC-Rh-PE vesicles. a.u., arbitrary units. (C) DOPG (58 µM)
vesicles with either C6-NBD-PC ( ) or
C6-NBD-PG ( ) exclusively in the inner leaflet or with
DOPC (58 µM) vesicles with C6-NBD-PC ( ) exclusively in
the inner leaflet. Nisin-induced NBD fluorescence decrease followed
migration of the C6-NBD-phospholipids to DOPC (1 mM)
vesicles with Rh-PE (2% [wt/wt]) and was measured as a percentage at
the point at which a stable level of fluorescence decrease was reached.
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|
To exclude the possibility that the C6-NBD-phospholipids in
the inner leaflet diffused to the acceptor vesicles through an aqueous
pore formed by nisin instead of via a transmembrane movement, the
integrity of the vesicles was determined by following the nisin-induced leakage of the fluorophore calcein. Under
conditions where extensive transmembrane movement of
C6-NBD-PC took place, nisin-induced calcein leakage was
only moderate and limited to around 10% at the highest nisin
concentration (Fig. 2). This demonstrated that the decrease in C6-NBD-PC fluorescence, as
observed in Fig. 1, was the result of a transmembrane movement of the
C6-NBD-PC. The small decrease in C6-NBD-PG
fluorescence (Fig. 1C) was, however, only slightly larger than that
observed for calcein leakage. Therefore, no conclusion could be drawn
on the nature of the nisin-induced C6-NBD-PG movement. As
observed previously (10), control experiments with
phospholipid vesicles containing the
C6-NBD-phospholipids exclusively in the outer leaflet
showed in the absence of nisin a rapid maximal decrease in fluorescence
due to diffusion of the C6-NBD-phospholipid to the
quenching Rh-PE-DOPC acceptor vesicles.
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FIG. 2.
Nisin-induced release of calcein from phospholipid
vesicles. DOPG (58 µM) vesicles loaded with calcein were incubated
with nisin. At arrow 1, nisin at either 166 (a), 500 (b), 1,000 (c), or
1,500 (d) nM was added, followed by addition of 0.2% Triton X-100
(arrows 2). a.u., arbitrary units.
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|
In order to enable a more direct analysis of the transmembrane
movement, experiments were performed with DOPG vesicles containing the
C6-NBD-phospholipid in the inner leaflet, without the
addition of Rh-PE-DOPC acceptor vesicles. In this case, nisin-induced
movement of C6-NBD-phospholipid to the outer leaflet caused
an increase in NBD fluorescence. This is due to shielding of the NBD
group from the aqueous environment by the membrane-bound nisin
(9). The NBD group in C6-NBD-phospholipids loops
back to the phospholipid head group (1, 6, 7). Indeed,
addition of nisin to DOPG vesicles with C6-NBD-PC (Fig.
3A and B) or with C6-NBD-PG
(data not shown) exclusively in the inner leaflet caused an increase in
the NBD fluorescence. Analyses of the kinetics of the fluorescence increase revealed that at all nisin concentrations, the rate constant k was lower for C6-NBD-PC (Fig. 3C) than for
C6-NBD-PG; in the latter case, the rate constant ranged
from 0.046 ± 0.007 s1 at 166 nM nisin to 0.194 ± 0.032 s1 at 1.5 µM nisin (data not shown). In
contrast, addition of the nisin to vesicles with symmetric distribution
of C6-NBD-PC or C6-NBD-PG resulted in a very
rapid increase in fluorescence, which reached a higher plateau within
less than a second. This was observed previously for vesicles with
symmetric distribution of head group-labelled NBD-phosphatidylethanolamine (9).
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FIG. 3.
Nisin induced a transmembrane movement of
C6-NBD-PC. In panel A, at the arrow, 500 nM nisin (line 1)
or buffer (line 2) was added to DOPG (58 µM) vesicles with
C6-NBD-PC exclusively in the inner leaflet, and the
increase in the NBD fluorescence was recorded. The same experiment was
performed with various concentrations of nisin. At each nisin
concentration a different plateau level of fluorescence increase (B)
and a different rate constant (C) were measured.
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|
Nisin pore formation is known to depend on the proton motive force. We
investigated the effect of a valinomycin-induced membrane potential
() on transmembrane movement of C6-NBD-PC. Nisin was added to DOPG vesicles with C6-NBD-phospholipid exclusively
in the inner leaflet, and the fluorescence increase was recorded. Neither for C6-NBD-PC nor for C6-NBD-PG was any
effect of the on the extent or rate of fluorescence increase
observed (data not shown). Under the conditions used, nisin-induced
dissipation of the was much faster than the rate of
nisin-induced NBD fluorescence increase (data not shown). The rapid
dissipation of the therefore probably prevented the detection of
a possible effect of the on nisin-induced transmembrane movement
of C6-NBD-phospholipid.
The effect of temperature on the transmembrane movement of
C6-NBD-phospholipid was investigated by using the assay
with the C6-NBD-phospholipid exclusively in the inner
leaflet. The nisin-induced transmembrane movement of
C6-NBD-PC was not affected by temperature (Fig.
4). As a control, the temperature
dependence of the transfer of C6-NBD-PC from
C6-NBD-PC-Rh-PE-DOPG vesicles to DOPG vesicles was
measured. In contrast to the rate constant of transmembrane movement,
the rate constant of the movement of C6-NBD-PC from vesicle
to vesicle was strongly affected by temperature (Fig. 4). Also, the
nisin-induced C6-NBD-PG movement observed when nisin is
added to vesicles in which the inner leaflet contains
C6-NBD-PG was dependent on temperature. At 500 nM nisin,
k increased from 0.017 ± 0.003 s1 at
5°C to 0.066 ± 0.012 s1 at 30°C (data not
shown), which further concurs with the suggestion that the
C6-NBD-PG movement occurs via a mechanism that is different from that of the C6-NBD-PC transmembrane movement.
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FIG. 4.
Temperature dependence of the rate constants of
transmembrane movement and intervesicle movement of
C6-NBD-PC. Nisin (500 nM) was added to DOPG (58 µM)
vesicles, with C6-NBD-PC ( ) exclusively in the inner
leaflet. Intervesicle movement was measured by incubating DOPG (58 µM-Rh-PE-C6-NBD-PC vesicles with pure DOPG (174 µM)
vesicles ( ). Lines were obtained by regression. Standard deviations
of the absolute values of the rate constants were around 17% for
transmembrane movement and around 2% for intervesicle movement.
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|
| |
DISCUSSION |
The present work shows that nisin induces movement of a
fluorescent phospholipid, C6-NBD-PC, from the inner leaflet
of unilamellar phospholipid vesicles to the outer leaflet. Nisin
is a pore-forming antimicrobial peptide. However, various lines
of evidence indicate that C6-NBD-PC undergoes
transmembrane movement and does not pass through an aqueous pore formed
by nisin. First, the extent of calcein leakage was very low compared to
the transmembrane movement of C6-NBD-PC. Nisin Z
forms an anion-selective pore in DOPG vesicles (3). This
excludes the theoretical possibility that the negative charge of
calcein (and/or C6-NBD-PG) might limit its passage through a nisin pore due to electrostatic repulsion between DOPG and
calcein (and/or C6-NBD-PG). The calcein leakage
experiments also demonstrated that the vesicles remained intact under
the conditions employed. Second, movement of
C6-NBD-PG was much more restricted than that of
C6-NBD-PC (Fig. 1C), whereas the rate constant of
C6-NBD-PG movement was higher than that of
C6-NBD-PC. Third, the rate constant of transmembrane
movement of C6-NBD-PC markedly increased with the
nisin concentration (Fig. 3C). Similarly, another study (35) showed that the (low) rate constant of transmembrane movement of
lysophospholipid in erythrocytes increased with cytotoxin (of S. aureus and of P. aeruginosa)
concentration. Finally, the rate constant of intervesicle movement of
C6-NBD-PC increased strongly with the temperature, while
the rate constant of transmembrane movement of C6-NBD-PC
was not affected by temperature. Similar results were found in
experiments on flippase activity in B. megaterium membrane
vesicles. In that study, the rate constant of transmembrane movement of phospholipid was largely independent of temperature, whereas the rate constant of intervesicle transport was dependent on
temperature (13).
Why was the nisin-induced transbilayer movement of lipids
restricted to C6-NBD-PC without having an equal
effect on the movement of C6-NBD-PG? The lack of the
C6-NBD-PG movement was possibly due to the
immobilization of the anionic PG by the cationic nisin as previously
suggested by 31P nuclear magnetic resonance experiments
(9). The relative nisin-induced C6-NBD-PG
movement hardly exceeded the calcein leakage. Therefore, the nature of
this limited movement is not clear at this time.
What is the mechanism of nisin-induced transmembrane movement of
C6-NBD-PC? In all likelihood, transmembrane movement of
C6-NBD-PC is the consequence of insertion of part(s) of the
nisin molecule into the membrane with concomitant disturbance of the
membrane (Fig. 5). Insertion of nisin
into lipid monolayers (3, 8) has been demonstrated, and the
C terminus of nisin is known to insert deeply into the membrane
(23). A study in which a His-tagged nisin was used indicated
that the C-terminal part of nisin had the ability to translocate across
the membrane (44). Physicochemical measurements on the
interaction of nisin with Listeria lipid vesicles also
indicated an insertion of the C-terminal part of nisin into the
membrane (45). In equilibrium, however, nisin seemed to be
oriented parallel with the membrane surface, assuming a surface-bound state (4). The wedge-like model (Fig. 5) invokes a proton
motive force-driven insertion of the C-terminal part of nisin (9, 26). It could well be that the insertion of the C-terminal region of nisin induced temporary fusion joints that permit transmembrane movements.
In the experiments in which C6-NBD-PC moves to the outer
leaflet and subsequently to Rh-PE-containing vesicles, nisin
concentration-dependent stable levels of movement were reached
after 5 min (data not shown). Supposing a surface area of 0.716 nm2/DOPG molecule (37) and using a
phospholipid/nisin ratio that ranges from 39 to 438, the number of
molecules per vesicle was between 1,602 and 18,000. Therefore, the
observed saturation of the fluorescence decrease was probably not due
to a low nisin/phospholipid ratio. Apparently, nisin only transiently
induced transmembrane movement of C6-NBD-phospholipid.
Within the range used, the rate constant of transmembrane movement
increased with the nisin concentration (Fig. 3C). This confirms that
nisin indeed bound to all vesicles and that binding of additional nisin
cooperatively enhanced transmembrane movement of C6-NBD-PC.
This phenomenon might be the result of aggregation of nisin molecules.
In conclusion, the present study shows for the first time that nisin
transiently induces rapid transmembrane movement of
C6-NBD-PC. This nisin-induced transmembrane movement
indicates that a transient disturbance of the phospholipid organization
of the membrane takes place locally during nisin-induced membrane permeabilization.
| |
ACKNOWLEDGMENT |
Margaret Mullaly is gratefully acknowledged for reading the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands. Phone: (31) (50) 3632164. Fax: (31) (50) 3632154. E-mail:
A.J.M.DRIESSEN{at}BIOL.RUG.NL.
| |
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Journal of Bacteriology, December 1998, p. 6565-6570, Vol. 180, No. 24
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
