From the Department of Cell and Molecular
Biology/Microbiology and the ¶ Department of Oral Biochemistry,
Faculty of Odontology, Göteborg University,
Göteborg SE-405 30, Sweden
Received for publication, October 10, 2000, and in revised form, November 28, 2000
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ABSTRACT |
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Quorum sensing in Gram-negative bacteria involves
acylated homoserine lactones (AHLs) and a transcription factor,
activated by the AHLs. In this study, a possible involvement of
intracellular Ca2+ as second messenger and/or protein
kinase activity during signal transduction is analyzed. When
N-hexanoyl-L-homoserine lactone was
added to a suspension of Fura-2-loaded Serratia
liquefaciens, there was a decline in
[Ca2+]i, measured as a decrease in the Fura-2
fluorescence ratio. As controls, the addition of the signal molecule
N-3-oxohexanoyl-L-homoserine lactone, which is
not produced by S. liquefaciens, did not induce changes in
[Ca2+]i. Using a protein kinase activity assay on
AHL-stimulated cells, an increase in kinase activity after
N-butanoyl-L-homoserine lactone stimulation of
S. liquefaciens cells was detected, whereas the kinase
activity induced by
N-3-oxohexanoyl-L-homoserine lactone was not
statistically significant. The conclusion from this study is that
changes in [Ca2+]i are involved in quorum sensing
signal transduction in the Gram-negative bacteria S. liquefaciens. We also conclude that kinase activity is induced in
S. liquefaciens upon AHL stimulation. We suggest that the
transient intracellular [Ca2+] changes and kinase
activity, activated by the AHL signal, are critical for the
quorum-sensing signal transduction.
Many species of Gram-negative bacteria use quorum sensing to
regulate important physiological functions, e.g. expression
of virulence factors and biofilm formation. Transcription of specific sets of genes is activated at a certain cell density, which the bacteria sense as concentrations of small diffusible signal molecules, produced by the bacteria. Quorum sensing in Gram-negative bacteria involves acylated homoserine lactones
(AHLs)1 as signal molecules,
AHL-synthesizing protein, (LuxI homologue), and the transcriptional
activator protein (LuxR homologue) (1, 2). It is proposed that the AHL
molecule binds to the LuxR, allowing it to function as a transcription
activator, and most data are consistent with this hypothesis, although
most data may also be explained by a signal transduction mechanism, an
indirect effect of AHLs, involving other factors beside the LuxR
homologues and the AHL signal molecules. LuxR, the Vibrio
fischeri luminescence (lux) gene activator, is the best
studied member of the LuxR family of bacterial transcription regulators
required for cell density-dependent gene expression. LuxR
homologues occur in a number of different Gram-negative bacteria
(e.g. TraR in Agrobacterium tumefaciens and LasR
and RhlR in Pseudomonas aeruginosa) regulating Ti-plasmid transfer and production of virulence determinants, respectively. Direct
interaction between LuxR and its most studied AHL signal, N-3-oxohexanoyl-L-homoserine lactone (OHHL), has
not been clearly demonstrated in vitro (1-5). However,
recently Zhu and Winans (6) purified TraR from A. tumefaciens, after overexpression in Escherichia coli,
as a complex with OHHL, confirming a direct interaction between a LuxR
homologue and its AHL signal molecule. Interestingly, Bassler et
al. (7, 8) have found, in the Vibrio harveyi
luminescence quorum sensing system, a potential AHL binding receptor
LuxN, necessary for luminescence, with a membrane-spanning domain. They
suggest a two-component signal transduction; i.e. the signal
binds to a receptor, which then transfers information (e.g.
by a phosphorylation step), leading to transcription. Also, Sitnikov
et al. (3) hypothesized involvement of another factor beside
the LuxR homologue, which may be species-specific in its AHL response
in one strain of bacteria and more unspecific in another strain.
Protein kinases play a key role in signal transduction pathways in both
eukaryotic and prokaryotic cells (9, 10). Protein kinase C (PKC) is a
heterogeneous group of serine/threonine protein kinases often involved
in cellular responses to hormones and neurotransmitters in eukaryotes.
Recently, several studies have reported on discoveries of
serine/threonine protein kinases also in prokaryotes (10, 11). Protein
kinase C requires membrane binding for maximal activity, and this
binding requires Ca2+. Ca2+ decreases the
amount of negatively charged phospholipids, thereby increasing PKC
membrane binding (12, 13), activity (14, 15), and regulation (16).
In eukaryotic cells, intracellular Ca2+ is thoroughly
studied. The level of [Ca2+]i is tightly
controlled, and changes in [Ca2+]i are known to
regulate a variety of processes such as secretion, cell cycle
transition, fertilization, and chemotaxis. In prokaryotes, however, the
role for [Ca2+]i is not well studied. As more
data continuously are being revealed, it has become evident that many
Ca2+-controlled processes in eukaryotes have parallels in
prokaryotes. As for eukaryotic cells, the [Ca2+]i
in bacteria lies in the submicromolar range and is regulated by cell
membrane- and organelle-associated Ca2+ channels and pumps
(9, 17, 18). In bacteria, the expression of certain genes has been
demonstrated to be, at least in part, both extra- and intracellular
Ca2+-dependent. Extracellular Ca2+
can act as an environmental signal, sensed by outer membrane receptors
possibly influencing gene transcription (17). Sensitivity to low
Ca2+ concentrations is suggested to involve a high affinity
sensor protein, having either an extra- or intracellular localization (17). Intracellular Ca2+ as a second messenger has been
studied in bacterial chemotaxis, and it has been shown for E. coli that stimulation with chemotactic attractants and repellants
induce changes in [Ca2+]i (19, 20).
In the present study, involvement of factors, such as changes in
intracellular calcium concentration and protein phoshorylation, during
AHL activation of LuxR homologues in quorum sensing was investigated.
As models for AHL-induced quorum sensing, the Gram-negative bacteria
Serratia liquefaciens and V. fischeri were used.
Reagents--
The potassium salt of Fura-2 was obtained from
Molecular Probes, Inc. (Leiden, The Netherlands). Stock solutions of
Fura-2 (5 mM) in water were prepared, and light exposure
during cell loading and fluorescence measurements were kept to a
minimum. OHHL was purchased from Sigma. N-Hexanoyl
homoserine lactone (HHL) was purchased from Quorum Science Inc.
(Coralville, IA). N-Butanoyl homoserine lactone (BHL) was a
generous gift from Prof. P. Williams (School of Pharmaceutical
Sciences/Institute of Infections and Immunity, University of
Nottingham, United Kingdom). Stock solutions, 2.0 mM in
phosphate-buffered saline (PBS), of the AHLs were prepared 30 min prior
to the experiments. BiotrakTM PKC assay system (RPN 77) and
[ Bacterial Strains and Culture Procedures--
As a model for
quorum sensing, a mutant S. liquefaciens strain,
SwrI Fura-2 Loading--
The potassium salt of Fura-2 was loaded into
bacteria essentially as described by Tisa and Adler (19). Briefly, 100 ml of cell culture grown overnight to an optical density of about 1.0 were collected at 6000 × g for 5 min. The cell pellet was
resolved in 2-3 ml of electroporation buffer (1 mM Hepes
(pH 7.2) in 10% glycerol). This procedure was followed by two more
washes in electroporation buffer, and finally the cell pellet was
resuspended in 2 ml of electroporation buffer. The washed cells were
stored on ice until electroporation. Fura-2 (50 µM) was
introduced into the cells (200-µl suspension in a cuvette, 0.2-cm
electrode gap) by the use of a single pulse of electricity at a
capacitance of 25 microfarads with a field intensity of 2.5 kV at 200 ohms for 4-6 ms in a Bio-Rad gene pulser. Immediately after
electroporation cells were diluted in 2 ml filtered used LB
medium and conditioned at room temperature for 15 min. After
three washes in the same medium, the cell pellet was resuspended in 2 ml of a Ca2+ buffer (120 mM NaCl, 20 mM Hepes-Tris (pH 7.2), 10 mM glucose, 10 mM CaCl2, 4.7 mM KCl, 1.2 mM KH2PO4, and 1.2 mM
MgSO4) and kept at room temperature until use, within
2 h.
Fura-2 Ratio Fluorescence Spectrophotometry--
Intracellular
calcium measurements were performed in a double beam luminescence
spectrometer at 340- and 380-nm excitation and 510-nm emission
wavelengths in a quartz cuvette at 20 °C with slow magnetic
stirring. For every measurement, 200 µl of the Fura-2-loaded cell
suspension was added to a quartz cuvette filled with 3 ml of
Ca2+ buffer. The cell suspension was allowed to equilibrate
for 10 min before measurement. All additions to the cuvette had a
volume of 30 µl, corresponding to 1% of the total cuvette volume.
Typically, fluorescence intensity data were collected every 1.9 s
for 4 min, and the ratio of light emission at the two excitation
wavelengths (340 and 380 nm) were recorded. The addition of PBS as
control and the addition of AHL in PBS were performed after 120 and
180 s, respectively. In the Fura-2 ratio fluorescence images (see below), fluctuations in fluorescence intensity sometimes were seen for
30 s after the addition of AHL. Therefore, the average fluorescence ratio value during 60 s following the addition of AHL
or PBS was calculated in each sample. AHLs were also added to Fura-2
loaded E. coli cells (strain MG 1655) to study possible activation of a Gram-negative bacteria without this BHL- and
HHL-dependent quorum sensing system. Results are presented
as the mean value ± S.E. (n = 8) for each AHL.
Fura-2 Ratio Fluorescence Imaging--
Images of intracellular
Ca2+ transients was monitored using Fura-2-loaded cells.
The recordings were made with a Rainbow excitation wavelength filter
wheel (Life Science Resources Ltd., Cambridge, UK) carrying 340- and
380-nm filters, attached to an inverted Diaphot 300 microscope (Nikon,
Tokyo, Japan) equipped with oil immersion, fluorescence objectives. The
emitted light was recorded at 510 nm using a digital CCD video camera
(Optronics, Goleta, CA) at 400× primary magnification. The photon
sampling time was typically 2-5 s. Data obtained was processed by
means of a calcium image software, MiraCal (Life Science Resources
Ltd.). The calculated fluorescence ratios, proportional to
[Ca2+]i, were displayed as a series of images,
where a decrease in light intensity corresponds to a decrease in
[Ca2+]i. Background fluorescence was measured and
subtracted in all measurements. For each measurement, 40 µl of cell
suspension was transferred to and spread out on a methylsilanized
coverglass. After settlement, an attached cell was focused on, and
image recording was initiated. After 30-60 s of base-line measurement,
5 µl of AHL stock or PBS were added. After an observed shift in
fluorescence ratio, the recordings were continued for an additional
120 s.
Protein Kinase C Assay--
An overnight culture of S. liquefaciens was stimulated by BHL or OHHL (20 µM)
for 90 s prior to centrifugation at 6000 × g for
5 min, resuspension in homogenization buffer, and cell disintegration (8 × 1-min ultrasonication). Protein kinase C activity was
assayed by measuring the rate of 33PO incorporation from
[
To compare several unmatched groups, statistical analyses were
performed using a computerized analysis of variance (StatView 5.01, Abacus Concepts Inc., Berkeley, CA).
Measurements of [Ca2+]i in Fura-2-loaded
Bacteria in Suspension--
Upon the addition of PBS to a suspension
of Fura-2-loaded S. liquefaciens, no change in fluorescence
ratio was observed; i.e. there was no change in
[Ca2+]i. When 20 µM of HHL was
added, however, there was a decline in [Ca2+]i,
here demonstrated as the mean fluorescence ratio value during 60 s
after the addition of HHL (Fig.
1A). The change in
[Ca2+]i differed significantly from the resting
level (p < 0.05, n = 8). The addition
of 20 µM OHHL (Fig. 1B) did not induce changes
in [Ca2+]i that differed significantly from the
resting level (n = 8). Fura-2-loaded E. coli, treated with OHHL or HHL as described, did not demonstrate
any change in fluorescence ratio (data not shown).
Imaging of [Ca2+]i in Fura-2-loaded Single
Bacteria--
The addition of 200 µM HHL to S. liquefaciens settled to silanized glass induced a transient
decline in [Ca2+]i, observed as a decrease in
light intensity in the ratio fluorescence images (Fig.
2A). Similarly, 200 µM OHHL added to V. fischeri induced a
transient decrease in observed [Ca2+]i (Fig.
2B). Typically, the cells recovered after 5 s, but most
often they exhibited short bursts of [Ca2+]i
fluctuations after recovery.
Detection of Kinase Activity in AHL-stimulated Bacteria--
When
S. liquefaciens had been stimulated by BHL for 90 s
prior to homogenization, the This is the first study of the involvement of intracellular
[Ca2+] and protein phosphorylation in bacterial
quorum-sensing signal transduction. We demonstrate that both
intracellular [Ca2+] transients and kinase activity are
induced by stimulation of the quorum-sensing bacteria with either of
its specific AHL signal molecules, BHL or HHL. These substances are
produced by S. liquefaciens, where HHL has the same
effect as BHL if used at a higher concentration (21).
The V. fischeri LuxR protein is a 250-amino acid polypeptide
(23, 24). Based on mutational analysis, it has been shown that LuxR
consists of two functional domains where the C-terminal together with
RNA polymerase, can interact with the lux-regulatory DNA,
and the N-terminal contains an AHL binding domain that inhibits the
activity of the C-terminal domain in the absence of the AHL signal
molecule (5, 25). A LuxR mutant with the N terminus deleted is
independent of cell density and shows the same or higher activity as
LuxR in the presence of AHL molecules (3, 26). Zhu and Winans (6)
observed for the A. tumefaciens TraR protein that the AHL
binding increases the affinity of TraR for the DNA binding site and
that the binding might stabilize the TraR protein against cytoplasmic
proteases. This stabilization is hypothesized to be a conversion from
unbound TraR monomers to AHL-bound oligomers of the TraR protein (6).
LuxR has been found to be associated with the cytoplasmic membrane, but
it has no transmembrane structures (27). When trying to purify LuxR
from a cell membrane fraction of V. fischeri, it has been
shown that LuxR remains associated to membranes in the presence of a
number of detergents as well as KCl, but LuxR can be solubilized from
membranes by EDTA treatment (27). Solubilization of LuxR from membranes
by EDTA might indicate the involvement of Ca2+ as a factor
for membrane attachment. It is known that Ca2+ may have a
general role in the lateral distribution of membrane lipids (9).
Accordingly, Ca2+ might have a role in an eventual
multimerization of the membrane-attached LuxR monomers.
We assume that the transcription factor "SwrR" regulated by BHL and
HHL in S. liquefaciens is a homologue to the LuxR protein in
V. fischeri. In S. liquefaciens, DNA sequencing
has revealed the presence of an open reading frame next to the
swrI gene (coding for the SwrI protein, which synthesizes
BHL and HHL signal molecules), denoted swrR, with homology
to the LuxR family of AHL-binding transcriptional regulators
(unpublished results referred to by Givskov et al.
(28)).
In trying to elucidate the role of intracellular Ca2+
transients in quorum-sensing signaling, the total amino acid sequence of LuxR protein, as determined by Devine et al. (23, 24), was analyzed for calcium binding motifs, active sites of enzymes, and
other functional regions, in the Prosite data
base2 (29). In the LuxR
sequence, the data base search revealed identity with an
ATP/GTP-binding site motif (residues 194-201) and predicted several
sites for phosphorylation by protein kinase C and casein kinase II. The
data base analysis also predicted a site for myristoylation (residues
119-124). No identity for EF-hand calcium binding motifs was found in
the LuxR sequence. However, when the requirement for similarity was set
to 72%, an EF-hand motif was suggested (residues 74-86). The Prosite
analysis thus provides support for more than one alternative hypothesis
for how Ca2+ transients and/or protein phosphorylation
might be involved in quorum sensing signaling.
In both detection approaches used to study
[Ca2+]i mobilization, ratio fluorescence
spectrometry of cell populations and ratio fluorescence imaging for
single bacteria analysis, we observed a decrease in intracellular
[Ca2+] upon stimulation with a specific AHL molecule.
When HHL was added to a suspension of S. liquefaciens, there
was a decline in [Ca2+]i observed as a decrease
in the Fura-2 fluorescence ratio (Fig. 1A). On the other
hand, the addition of a nonspecific AHL signal, OHHL (Fig.
1B) or the addition of PBS buffer only did not induce
significant changes in [Ca2+]i. The cells thus
respond with a mobilization of [Ca2+]i to HHL but
not to OHHL, an AHL molecule that is not produced by S. liquefaciens. As a comparison, E. coli was not activated by either HHL or OHHL. During fluorescence experiment treatment with salt-free electroporation buffer, V. fischeri
rapidly and extensively formed mucus affecting the homogeneity and
translucency of the cell suspensions, thereby invalidating the results.
In Fura-2 ratio fluorescence imaging experiments, the addition of HHL
and OHHL to settled S. liquefaciens and V. fischeri, respectively, induced rapid declines in observed
[Ca2+]i. The cells recovered after a short series
of repeated [Ca2+]i fluctuations (Fig. 2). The
ratio imaging of settled single cells, where, roughly, a pixel
corresponds to the size of a single bacterial cell, showed
[Ca2+]i transients already after a few seconds,
followed by [Ca2+]i fluctuations. Upon initial
recovery, however, the cell [Ca2+]i continued to
fluctuate for ~15 s, sometimes even longer. To be sure to detect all
of these fluctuations in the conventional Fura-2 fluorescence
measurements, the average fluorescence over a longer time period,
60 s, was measured. Within these 60 s, the rapid
[Ca2+]i responses demonstrated in the single cell
measurements are well included. These [Ca2+]i
fluctuations might explain the "noisy" appearance of fluorescence
data from cell suspensions (data not shown). In eukaryotic cells, most
often transient increases in calcium concentration have been coupled to
signal transduction. The data presented here, showing a transient
decrease in [Ca2+]i, are consistent with
data from a bacterial chemotaxis study presented by Tisa and Adler
(19), where stimulation of Fura-2-loaded E. coli cells with
a chemotactic attractant (L-serine) also led to a transient
decrease in [Ca2+]i. Tisa and Adler present a
theory for the coupling between an observed decrease in
[Ca2+]i and signal transduction. They suggest
that Ca2+ might help stabilize and maintain a chemotactic
protein in its phosphorylated state, stimulating tumbling. A decrease
in [Ca2+]i would result in an unphosphorylated
protein, stimulating running.
Possible roles for the demonstrated Ca2+ transients in
AHL-dependent quorum sensing may be involvement in the
attachment of LuxR (or LuxR homologue) to the membrane or exposure of
its AHL-binding site. A myristoylation site in LuxR, suggested by the
Prosite data matching (i.e. an acyl-binding domain) is a
possible site for membrane association or may be an AHL-binding site.
Both of these alternatives might in turn be dependent on changes in
intracellular [Ca2+], by a "calcium-myristoylation
switch," in which an acyl-binding site is exposed upon
Ca2+ stimulation (30).
Many bacterial regulators employ a signal transduction system, where a
protein is activated by kinase-dependent phosphorylation. The AHL-induced [Ca2+]i response observed in the
fluorescence experiments might indicate a possible activation of LuxR
homologues by a Ca2+-dependent phosphorylation
upon AHL stimulation. Kinase stimulation by Ca2+ activated
calmodulin-like proteins have been described in bacteria (31). The data
base search predicted an ATP/GTP-binding site motif (residues 194-201)
in the LuxR sequence, and several sites for phosphorylation by protein
kinase C and casein kinase II. We therefore investigated possible
protein kinase activity upon AHL stimulation of S. liquefaciens cell suspensions. We found an increase in kinase
activity in cell homogenates from bacteria that had been stimulated
with BHL. However, when a In Fig. 4 alternative roles for
intracellular [Ca2+] and kinase activity in LuxR
homologue activation is considered. We suggest a model where AHL
interaction with the LuxR homologue is dependent on or synergistic with
AHL interaction with a "non-LuxR AHL receptor" situated,
presumably, in the periplasmic region. A direct binding of OHHL to a
LuxR homologue, TraR, has been demonstrated (6). Assuming the
involvement of [Ca2+]i in this interaction,
possible mechanisms for this may be exposure of the AHL binding site on
the LuxR homologue upon [Ca2+]i changes or
exposure of the site for membrane attachment, which in turn may affect
the AHL binding site. In parallel to the suggested Ca2+
effects, LuxR may be activated for transcription through
phosphorylation by an AHL-activated, or possibly
Ca2+-activated, kinase. Both direct AHL interaction with
LuxR and AHL binding to a not yet identified "non-LuxR AHL
receptor" may be essential for transcription activation by the LuxR
homologue. However, one AHL interaction in this pathway may be more
species-specific than the other.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES
-33PO]ATP were obtained from Amersham Pharmacia Biotech.
, from wild type strain MG44 was used. This mutant
strain does not synthesize BHL and HHL, two AHLs known to be produced
by wild type S. liquefaciens (21). In addition, a V. fischeri strain, MJ-215, was used. This strain is unable to
produce OHHL and HHL, two of three known AHLs produced by V. fischeri (22). As a non-AHL-producing or -responding Gram-negative
control bacteria, we used the E. coli strain MG 1655. Cultures for experiments were grown in LB media, pH 7.5, at room
temperature with moderate shaking overnight (16-17 h).
SwrI
were grown in LB5 medium (5 g of yeast, 10 g of
Tryptone, and 5 g of NaCl per liter) and MJ-215 in LB20 medium (5 g of yeast, 10 g of Tryptone, and 20 g of NaCl per liter).
All AHLs were used at higher concentrations than what is considered
optimal, since the AHLs are most probably not completely dissolved in
the stock solution, and the concentration was consequently
overestimated. V. fischeri (MN215) was tested for light
production during these culture conditions. No decrease in light
production was observed at the [OHHL] used in fluorescence
experiments, 20 µM and 0.2 mM compared with 2 µM. Instead a slight increase in light intensity was
observed with increased [OHHL], indicating an overestimation of
[AHLs] in these experiments. BHL, which is the AHL signal most specific for S. liquefaciens, was used in the PKC assay,
while HHL, which is also produced by S. liquefaciens wild
type and known to have the same effect as BHL if used at higher
concentrations (21), was used in Fura-2 ratio fluorescence
spectrophotometry and fluorescence imaging. Since the bacterial strains
used in these experiments are AHL-synthetase mutants, unable to produce the AHL signal molecules, we know that the receptors for the AHL molecules are not activated by their ligands before the onset of the experiments.
-33PO]ATP in the cell homogenate in the presence or
absence of a PKC-selective peptide substrate with serine/threonine
residues (specific for the
-
-
-isoforms of PKC) using the
BiotrakTM protein kinase C assay system (RPN 77) as
described. Data are presented as means ± S.E., and n
ranges from 12 to 23.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Fig. 1.
[Ca2+]i
measured as fluorescence ratio, in Fura-2-loaded bacteria before and
after stimulation with AHLs. A, HHL; B,
OHHL. The Gram-negative S. liquefaciens SwrI
mutant strain, unable to produce AHL signal molecules, was
Fura-2-loaded by means of electroporation. Intracellular calcium was
measured in a double beam fluorescence spectrophotometer at 340- and
380-nm excitation and 510-nm emission wavelengths at 20 °C.
Fluorescence intensity data were measured every 1.9 s, and the
ratio of light emission at the two excitation wavelengths were recorded
for 4 min. The addition of PBS as control and the addition of AHL in
PBS were performed after 120 and 180 s, respectively. For
each sample, the average fluorescence ratio value during 60 s
following the addition of AHL or PBS was calculated. The change in
fluorescence ratio after the addition of HHL differs significantly from
the resting level (p < 0.05, n = 8).
Results are presented as the mean value ± S.E.,
n = 8 for each AHL.
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Fig. 2.
Fluorescence ratio images of Fura-2-loaded
bacteria before and after the addition of AHLs. A,
S. liquefaciens and HHL; B, V. fischeri and OHHL. The Gram-negative S. liquefaciens
SwrI mutant strain, unable to produce AHL signal
molecules, and V. fischeri strain MJ-215, unable to produce
OHHL and HHL, were Fura-2-loaded by means of electroporation.
Fluorescence images were monitored with an excitation wavelength filter
wheel carrying 340- and 380-nm filters, attached to an inverted
microscope. Emitted light was recorded at 510 nm using a digital CCD
video camera. The photon sampling time was typically 2-5 s. Calculated
fluorescence ratios, proportional to [Ca2+]i, are
displayed as a series of images where a decrease in light intensity
corresponds to a decrease in [Ca2+]i.
-phosphate group transfer from ATP to a
peptide or protein substrate in the cell homogenate was significantly higher compared with unstimulated cell samples (p < 0.05, n = 5). Stimulation with OHHL, prior to sample
preparation, also seemed to increase the amount of transferred
phosphate groups; however, this increase was not statistically
significant. We assume that the measured phosphate transfer is due to
an induction of kinase activity (Fig.
3A). When a PKC-specific
substrate, a peptide with a serine/threonine residue of the
-
-
type, was added during sample analysis, the kinase activity of the
BHL-stimulated samples were decreased about 20%. Interestingly, when
no AHL signal had been added to the bacteria, the samples exhibited PKC
activity, although not significantly different from stimulated cells
(Fig. 3B).
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Fig. 3.
Kinase activity, measured as 33PO
incorporation upon AHL stimulation in homogenates of S. liquefaciens. A, kinase activity without
added PKC-substrate; B, kinase activity in the presence of a
PKC-specific pseudosubstrate. An overnight culture of the Gram-negative
S. liquefaciens SwrI mutant strain, unable to
produce AHL signal molecules, was stimulated by BHL or OHHL (20 µM) for 90 s prior to cell disintegration. Kinase
activity was assayed by measuring the rate of 33PO
incorporation from [
-33PO]ATP added to the cell
homogenates, in the presence or absence of a PKC-selective peptide
substrate with serine/threonine residues specific for the
-
-
isoforms of PKC. When no substrate was added, bacterial samples
stimulated by BHL (A), demonstrated
-phosphate group
transfer from ATP to a substrate in the cell homogenate, being
significantly higher compared with unstimulated cell samples
(p < 0.05, n = 5). Stimulation with
OHHL also seemed to increase the amount of transferred phosphate
groups. In the presence of a PKC-specific substrate, the kinase
activity of BHL-stimulated samples was decreased by 20%.
(B).
DISCUSSION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-
-
PKC isoenzyme substrate was
present during measurements the kinase activity in BHL-treated samples
was dampened about 20%. The finding may indicate a competitive
inhibition of the BHL-induced kinase activity by the PKC-specific
-
-
substrate. If so, a possible "non-
-
-
substrate" is most probably synthezised by the cells and harbored in
the homogenate. In contrast, when cell samples were not stimulated with
AHLs prior to homogenization, kinase activity was measurable in the
presence of the
-
-
-PKC substrate type only, indicating a
specific PKC activity.
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Fig. 4.
A model demonstrating alternative mechanisms
for transcription activation of LuxR. Both direct AHL interaction
with LuxR and AHL binding to a not yet identified non-LuxR AHL-receptor
may be essential for transcription activation by LuxR. Direct binding
of AHL to LuxR (A), as a one-step event, requires free
diffusion of AHL through the periplasmic region. Assuming the
involvement of a non-LuxR AHL-receptor (B), an additional,
possibly synergistic, pathway is suggested. Binding of AHL to a
non-LuxR AHL receptor triggers a change of
[Ca2+]i, in addition to activation of an AHL- or
Ca2+-activated kinase, leading to exposure of an AHL
binding site on LuxR or exposure of a membrane attachment site
affecting the AHL binding site. Many bacterial regulators are activated
by a kinase-dependent phosphorylation. Observed AHL-induced
[Ca2+]i responses might indicate a possible
activation of LuxR by a Ca2+-dependent
phosphorylation, upon AHL stimulation. Activation of the LuxR initiates
transcription. A LuxR-associated myristoyl site, i.e. an
acyl-binding domain, possibly being the site for membrane association
or the AHL-binding site, might initiate the transcription factor as it
anchors to the membrane or exposes the AHL binding site.
The conclusion of this study is that changes in
[Ca2+]i are induced by quorum-sensing signals in
the Gram-negative bacteria S. liquefaciens and V. fischeri. We also conclude that kinase activity is induced in
S. liquefaciens upon AHL stimulation. We therefore suggest
that transient intracellular [Ca2+] changes and kinase
activity following AHL stimulation are critical for the quorum-sensing
signal transduction in S. liquefaciens.
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ACKNOWLEDGEMENTS |
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The S. liquefaciens SwrI mutant strain (MG44) was a generous gift from Dr. M. Givskov (Department of Microbiology, The Technical University of Denmark). The V. fischeri strain MJ215 was a generous gift from Professor P. Dunlap (Center of Marine Technology, Baltimore, MD). BHL was a generous gift from Prof. P. Williams. Prof. P. Williams and Prof. T. Nyström are acknowledged for valuable comments on the manuscript.
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FOOTNOTES |
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* This work was supported by the Carl Trygger Research Fund and MASTEC (Research Program on Marine Biofouling, Göteborg university and Chalmers University of Technology).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Cell and Molecular Biology/Microbiology, Göteborg University, POB 462, SE 405 30 Göteborg, Sweden. Tel.: 46-31-773-2566; Fax: 46-31-773-2599; E-mail: maria.werthen@gmm.gu.se.
Published, JBC Papers in Press, December 1, 2000, DOI 10.1074/jbc.M009223200
2 The Prosite data base is available on the World Wide Web.
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ABBREVIATIONS |
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The abbreviations used are: AHL, acylated homoserine lactone; BHL, N-butanoyl-L- homoserine lactone; HHL, N-hexanoyl-L-homoserine lactone; OHHL, N-3-oxohexanoyl-L-homoserine lactone; PBS, phosphate-buffered saline; PKC, protein kinase C.
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