Broad Spectrum Pro-Quorum-Sensing Molecules as
Inhibitors of Virulence in Vibrios
Wai-Leung Ng1,2, Lark Perez3, Jianping Cong1, Martin F. Semmelhack3, Bonnie L. Bassler1,2*
1Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America, 2Howard Hughes Medical Institute, Chevy Chase, Maryland,
United States of America, 3Department of Chemistry, Princeton University, Princeton, New Jersey, United States of America
Quorum sensing (QS) is a bacterial cell-cell communication process that relies on the production and detection of
extracellular signal molecules called autoinducers. QS allows bacteria to perform collective activities. Vibrio cholerae, a
pathogen that causes an acute disease, uses QS to repress virulence factor production and biofilm formation. Thus,
molecules that activate QS in V. cholerae have the potential to control pathogenicity in this globally important bacterium.
Using a whole-cell high-throughput screen, we identified eleven molecules that activate V. cholerae QS: eight molecules are
receptor agonists and three molecules are antagonists of LuxO, the central NtrC-type response regulator that controls the
global V. cholerae QS cascade. The LuxO inhibitors act by an uncompetitive mechanism by binding to the pre-formed LuxO-
ATP complex to inhibit ATP hydrolysis. Genetic analyses suggest that the inhibitors bind in close proximity to the Walker B
motif. The inhibitors display broad-spectrum capability in activation of QS in Vibrio species that employ LuxO. To the best of
our knowledge, these are the first molecules identified that inhibit the ATPase activity of a NtrC-type response regulator.
Our discovery supports the idea that exploiting pro-QS molecules is a promising strategy for the development of novel anti-
Citation: Ng W-L, Perez L, Cong J, Semmelhack MF, Bassler BL (2012) Broad Spectrum Pro-Quorum-Sensing Molecules as Inhibitors of Virulence in Vibrios. PLoS
Pathog 8(6): e1002767. doi:10.1371/journal.ppat.1002767
Editor: Michael S. Gilmore, Harvard Medical School, United States of America
Received February 16, 2012; Accepted May 7, 2012; Published June 28, 2012
Copyright: ? 2012 Ng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Howard Hughes Medical Institute, National Institutes of Health (NIH) grant 5R01GM065859, NIH grant 5R01AI054442,
National Science Foundation (NSF) grant MCB-0343821 to B.L.B., and an NIH postdoctoral fellowship GM082061 to W.L.N. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Quorum sensing (QS) is a process of bacterial cell-cell
communication that relies on the production, release, detection,
and response to extracellular signaling molecules called auto-
inducers. QS allows groups of bacteria to synchronously alter
behavior in response to changes in the population density and
species composition of the vicinal community. QS controls
collective behaviors including bioluminescence, sporulation, viru-
lence factor production, and biofilm formation (Reviewed in [1,2]).
Impairing virulence factor production or function has gained
increasing attention as a method to control bacterial pathogenicity.
The advantage of anti-virulence strategies over traditional
antibiotics is presumed to be reduced pressure on bacteria to
develop resistance [3–5]. Because QS controls virulence in many
clinically relevant pathogens, disrupting QS is viewed as a
promising possibility for this type of novel therapeutic develop-
Many pathogenic Gram-negative bacteria use acylhomoserine
lactones (HSLs) as QS autoinducers, which are detected by either
cytoplasmic LuxR-type or membrane-bound LuxN-type receptors
. To date, efforts to interfere with HSL QS in Gram-negative
bacteria have yielded several potent antagonists [10–15]. While
these strategies are exciting, some globally important Gram-
negative pathogens do not use HSLs as autoinducers. Thus,
additional strategies that target non-HSL based QS systems are
required. Here, we describe the identification and characterization
of a set of small-molecule inhibitors that act on the non-HSL QS
system of Vibrio cholerae by targeting two independent steps in the
signal transduction pathway.
V. cholerae is the etiological agent of the disease cholera and its
annual global burden is estimated to be several million cases .
V. cholerae produces and detects two QS autoinducer molecules
called CAI-1 and AI-2. CAI-1 ((S)-3-hydroxytridecan-4-one) is
produced by the CqsA synthase [17,18] and AI-2 ((2S, 4S)-2-
methyl-2,3,3,4-tetrahydroxytetrahydrofuran borate) is produced
by the LuxS synthase [19,20]. Detection of CAI-1 and AI-2 occurs
through transmembrane receptors CqsS and LuxPQ, respectively
[21,22]. CqsS and LuxPQ are two-component proteins that
possess both kinase and phosphatase activities (Figure 1 shows the
CqsA/CqsS system). At low cell density (LCD), when the receptors
are devoid of their respective ligands, their kinase activities
predominate, resulting in the phosphorylation of the response
regulator LuxO. LuxO,P is the transcriptional activator of four
genes encoding small regulatory RNAs (sRNAs), Qrr1-4 . The
Qrr sRNAs target the mRNAs encoding the quorum-sensing
master transcriptional regulators AphA and HapR. At LCD,
facilitated by the RNA chaperone Hfq, Qrr1-4 stabilize and
destabilize the aphA and hapR mRNA transcripts, respectively .
Therefore, AphA protein is made while HapR protein is not
(Figure 1). When autoinducer concentration increases above the
threshold required for detection (which occurs at high cell density
(HCD)), binding of the autoinducers to their cognate receptors
switches the receptors from kinases to phosphatases (Figure 1).
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Phosphate flow through the signal transduction pathway is
reversed, resulting in dephosphorylation and inactivation of LuxO.
Therefore, at HCD, qrr1-4 are not transcribed, resulting in
cessation of translation of aphA and derepression of translation of
hapR. This QS circuitry ensures maximal AphA production at
LCD and maximal HapR production at HCD. AphA and HapR
each control the transcription of hundreds of downstream target
genes [24,25]. Hence, reciprocal gradients of AphA and HapR
establish the QS LCD and HCD gene expression programs,
respectively (Figure 1).
In pathogens that cause persistent infections, QS commonly
activates virulence factor production at HCD. However, in V.
cholerae, which causes an acute disease, HapR production at HCD
represses genes important for biofilm formation and virulence factor
production [22,26–30]. This peculiar pattern of virulence gene
regulation can be understood in terms of the disease caused by V.
cholerae . Following successful V. cholerae infection, the ensuing
diarrhea washes huge numbers of bacteria from the human
intestine into the environment. Thus, expression of genes for
virulence and biofilm formation at LCD promotes infection, while
repression of these genes by autoinducers at HCD promotes
dissemination. Thus, molecules that activate QS have the potential
to repress virulence in V. cholerae. Moreover, QS plays an essential
role in virulence in other pathogenic vibrios including Vibrio
parahaemolyticus, Vibrio alginolyticus, and Vibrio vulnificus [32–35]. The
components of the QS circuits in these species are similar to those
of V. cholerae. Therefore, QS-activating molecules identified for V.
cholerae could be broadly useful for controlling diseases caused by
Here, we report the identification of a set of small molecules that
activate the QS system of V. cholerae. We classify the QS-activating
molecules as either QS receptor agonists or LuxO inhibitors.
Because we have already reported analyses of QS receptor
agonists, we focus here on the LuxO inhibitors. At LCD, LuxO,P
activates production of the Qrr sRNAs, which repress HapR;
inhibitors of LuxO thus activate QS due to derepression of HapR.
LuxO belongs to the NtrC protein family, s54-binding transcrip-
tional activators that rely on ATP hydrolysis to promote open
complex formation . The LuxO inhibitors identified here
function uncompetitively to perturb LuxO ATPase activity.
Genetic analysis of LuxO mutants that are insensitive to the
inhibitors suggests that the inhibitors interact with a region
adjacent to the ATP binding pocket. Finally, using a set of
phenotypic assays, we show that the inhibitors broadly activate
different vibrio QS circuits and, in turn, repress virulence factor
production and reduce cytotoxicity. Because LuxO is conserved
among vibrio QS circuits, the molecules we characterize here are
capable of inhibiting HSL-based and non-HSL-based vibrio QS
systems. Numerous NtrC-type proteins homologous to LuxO act
in two-component signaling systems and their roles in controlling
nitrogen metabolism, virulence, motility, and other important
processes have been extensively studied (Reviewed in ). To the
best of our knowledge, there exists no previous report of a
chemical probe that modulates the activity of a NtrC-family
Identification of molecules that activate QS in V. cholerae
We are interested in identifying small molecules that activate
QS in V. cholerae, in order to induce the HCD state and thus
repress virulence factor production. To do this, we developed a
whole-cell high-throughput screen that relies on QS-dependent
induction of bioluminescence (lux) in V. cholerae . We exploited
V. cholerae mutants genetically locked into the LCD state and
carrying the lux operon from V. harveyi to screen for molecules that
induce light production, indicating that they activate QS
responses. We performed the screen on two different LCD
mutants. The first mutant lacks the two autoinducer synthases,
CqsA and LuxS. Therefore, both CqsS and LuxPQ QS receptors
function as kinases and constitutively phosphorylate LuxO,
resulting in transcription of the Qrr regulatory RNAs, and
repression of translation of HapR (see INTRODUCTION). In
the absence of HapR, there is no transcription of the heterologous
lux operon, and thus, this strain is dark. The second strain carries
the luxOD47Eallele. This luxO mutation mimics LuxO,P,
rendering LuxO constitutively active [23,38]. Therefore, HapR
is repressed and the strain is dark. We anticipated identifying two
classes of molecules that could induce light production: Class 1)
Molecules that induce bioluminescence in the double synthase
mutant but not in the luxOD47Emutant. These compounds are
predicted to be QS receptor agonists; and Class 2) Molecules that
induce bioluminescence in both the double synthase mutant and
the luxOD47Emutant. Class 2 compounds likely target QS
components that lie downstream of the receptors. We screened
90,000 molecules and identified eight Class 1 compounds and
three Class 2 compounds (Figures 2A and 2B). The EC50of Class
1 compounds are comparable to that of CAI-1 and generally lower
than those of Class 2 compounds (Figure 2C). These differences
support the idea that the two classes of molecules potentiate QS
responses by distinct mechanisms. None of the compounds
affected cell growth (Figure S1).
Investigation of the targets of the QS activating
To determine which QS component each compound acts on,
we first tested the eight Class 1 compounds against V. cholerae
mutants that lack only the CqsS receptor or only the LuxPQ
receptor. All eight Class 1 compounds induced light production in
the DluxPQ strain but not the DcqsS strain; hence, these eight
molecules function as CqsS agonists (Figure S2). Interestingly,
none has structural homology to the native CAI-1 autoinducer
[17,18,39,40] (Figure 2A). The Class 1 molecules are currently
being characterized and are not discussed further here.
The three Class 2 compounds that activate QS in both of the
LCD screening strains likely act downstream of the QS receptors.
These three compounds are structurally homologous (Figure 2A);
therefore, they may function by an identical mechanism. Here, we
focused on the compound displaying the highest potency (i.e.,
compound 11, Figures 2A and 2C). Class 2 compounds could
potentially target one or more of the V. cholerae QS cytoplasmic
components that function downstream of the receptors: LuxO,
The disease cholera, caused by the pathogenic bacterium
Vibrio cholerae, is a major health concern in developing
regions. In order to be virulent, V. cholerae must precisely
control the timing of production of virulence factors. To do
this, V. cholerae uses a cell-cell communication process
called quorum sensing to regulate pathogenicity. In the
current work, we identify and characterize new classes of
small molecules that interfere with quorum-sensing-
control of virulence in multiple Vibrio species. The
molecules target the key quorum-sensing regulator LuxO.
These molecules have the potential to be developed into
new anti-infectives to combat infectious diseases of global
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s54, Hfq, and/or Qrr1-4. We reasoned that if these compounds
interfere with LuxO or s54, transcription of qrr1-4 would decrease
in the presence of the inhibitors. By contrast, if the compounds
target Hfq or act directly on Qrr1-4, they should not affect qrr1-4
transcription. GFP production from a qrr4-gfp transcriptional
fusion decreased ,3-fold when the luxOD47Estrain was treated
with compound 11 (Figure 2D). This result suggests that
compound 11 targets either LuxO or s54. If the target of
compound 11 is s54, transcription of other s54-dependent genes
should be affected when V. cholerae is treated with the compound.
We examined transcription of the s54-dependent gene vpsR 
and found that it did not change significantly in the presence of
compound 11 (data not shown). These results suggest that
compound 11 targets LuxO.
Structure-activity-relationship of Class 2 compounds
The three identified Class 2 compounds share a 5-thio-6-
azauracil core and only their side chains vary (Figure 2A). In
addition, several 5-thio-6-azauracil analogs with other modifica-
tions on their side chains displayed weak or no activity in the
screen. Therefore, differences in the hydrocarbon side chains must
be responsible for the corresponding differences in potency with
compounds harboring branched side chains displaying greater
potency (i.e., compound 11, Figure 2C). To explore the
relationship between structure and activity, we synthesized a
focused library of compounds bearing the conserved 5-thio-6-
azauracil core, and we altered the branching in the side chains.
We measured activities using bioluminescence in the V. cholerae
luxOD47Emutant. Several of the side chain modifications decreased
Figure 1. The Quorum-Sensing Circuit in Vibrio cholerae. The CqsA/CqsS signal transduction system is shown as the example for the V. cholerae
QS circuit. (Left) At low cell density (LCD), the CAI-1 autoinducer concentration is below the detection threshold, and the membrane bound CqsS
receptor functions as a kinase. The LuxO response regulator is phosphorylated and it activates the transcription of genes encoding the four Qrr sRNA
genes. Aided by the RNA chaperone Hfq, the Qrr sRNAs activate and repress translation of the AphA and HapR proteins, respectively. (Right) At high
cell density (HCD), binding of CAI-1 to CqsS inhibits its kinase activity. LuxO is not phosphorylated and transcription of the qrr genes is terminated.
Translation of AphA is inhibited and HapR is derepressed. Hundreds of genes are controlled by AphA and HapR, including genes required for biofilm
formation and virulence. HapR also functions as a transcriptional activator of the heterologous V. harveyi lux operon [22,24,26–30]. Dotted lines
denote components that are not expressed while solid lines represent those that are produced.
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potency (as shown by an increase in EC50, Figure 3). However,
increasing steric bulk by incorporation of a tert-butyl carbinol side
chain led to a 3-fold enhancement in potency (i.e., compound 12,
Figure 3). Thus, the activity of the 5-thio-6-azauracil compounds
within this series is highly sensitive to the structural features of the
alkyl side chain. In the focused group of molecules we investigated,
a bulky, hydrophobic terminal t-butyl moiety is optimal.
Class 2 compounds inhibit the LuxO ATPase activity
NtrC-type response regulators including LuxO possess three
biochemical activities: phosphoryl-group accepting activity, DNA-
binding activity, and ATP hydrolyzing activity . We investi-
gated which of these activities is inhibited by compounds 11 and
12. First, using whole-cell bioluminescence assays, we found that
both compounds activate QS in V. cholerae strains expressing either
wild type LuxO or LuxO D47E (Figures 2B and 3). Wild type
LuxO is activated by phosphorylation via the QS cascade, and the
LuxO D47E variant, which mimics LuxO,P, while not
phosphorylated is constitutively active [22,23,26,38]. Because
both wild type LuxO and LuxO D47E are vulnerable to
inhibition, it cannot be the ability of LuxO to participate in
phosphorylation or dephosphorylation that is impaired by
compounds 11 and 12.
LuxO, as a NtrC-type response regulator, binds to s54-
dependent promoters to activate transcription. Compounds 11
and 12 could prevent LuxO from binding to DNA, and in so
doing, prevent qrr transcription. To investigate this possibility, we
used electrophoretic-mobility-shift and fluorescence anisotropy
assays to probe the LuxO interaction with qrr promoter DNA.
Even in the presence of a high concentration (200 mM) of the
inhibitors, no significant change in LuxO D47E binding to qrr4
promoter DNA occurred as judged by mobility shift (Figure 4A).
Quantitative fluorescence anisotropy assays revealed that, in the
presence and absence of the LuxO inhibitors, LuxO D47E
Figure 2. Identification of QS-activating compounds in V. cholerae. (A) Chemical structures of the eleven QS-activating compounds. The
structure of CAI-1 is shown for reference. (B) Differential responses to Class 1 and Class 2 compounds by the V. cholerae DcqsA DluxS double synthase
mutant (BH1578) and the luxOD47Emutant (BH1651). The normalized light (RLU, relative light units) produced was monitored in the absence (white)
and presence of Class 1 (gray) or Class 2 (black) compounds. A representative experiment is shown using compound 1 (Class 1) and compound 11
(Class 2) from (A). (C) QS dose-response curves of V. cholerae. The normalized light (RLU, relative light units) produced by the V. cholerae DcqsA DluxS
mutant carrying the lux operon (BH1578) is plotted as a function of the concentration of the eleven QS-activating compounds shown in (A). Black
curves denote responses to Class 1 compounds. Blue curves denote responses to Class 2 compounds. The red curve denotes the response to the
native autoinducer CAI-1, which is the positive control. Error bars are present, but are too small to be observed in the plot. The bars represent
standard errors of the mean for three independent trials. (D) Effect of compound 11 on expression of qrr4. Expression of qrr4 was monitored in a V.
cholerae luxOD47Estrain carrying a qrr4-gfp transcriptional reporter (SLS353). The response is shown in the presence and absence of 50 mM compound
11. Expression of qrr4-gfp from the DluxO mutant (SLS373) is shown for reference. AU denotes arbitrary units. Error bars represent standard errors of
the mean for three independent trials.
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interacts with the qrr4 promoter DNA with an identical binding
constant (,300 nM) (Figure 4B). Thus, binding to DNA is not
altered by the inhibitors.
Finally, we examined whether compounds 11 and 12 affect
LuxO ATPase activity. To do this, we used a coupled-enzyme
assay  to assess the rate of ATP hydrolysis by LuxO in the
presence and absence of the compounds. Both compounds inhibit
ATP hydrolysis in a dose-dependent manner (Figures 5A–C).
Using traditional Michaelis-Menton enzyme kinetic analyses, we
found that both compounds decrease the Kmand the Vmaxof the
LuxO ATPase reaction (Figures 5B and 5C). The Lineweaver-
Burk plots of curves derived from control reactions and from
inhibitor-containing reactions display parallel slopes (Km/Vmax),
indicating that compounds 11 and 12 function as uncompetitive
inhibitors (Figures 5B and 5C), suggesting they bind to the pre-
formed LuxO-ATP complex to inhibit ATP hydrolysis. Indeed,
inhibition of LuxO ATPase by the analogs we identified or
synthesized (as represented by % inhibition) is correlated with their
potency (EC50) in inducing QS in the luxOD47E
(Figure 5D). We conclude that the LuxO inhibitors discovered
here activate QS in V. cholerae by specifically inhibiting the ATPase
activity of LuxO. Presumably, in the presence of the inhibitors,
LuxO is incapable of participating in open complex formation at
the qrr promoters, which prevents transcription of the Qrr sRNAs.
In turn, translation of HapR is derepressed and the QS response
A genetic screen to identify LuxO mutants resistant to
Compounds 11 and 12 likely bind to LuxO at an allosteric site
that negatively regulates ATP hydrolysis activity. To determine
where compounds 11 and 12 bind, we screened for LuxO mutants
refractory to inhibition. To do this, we engineered random
mutations into the cloned luxOD47Egene and introduced the
mutant library into a V. cholerae DluxO strain carrying the lux
operon. We screened for clones that conferred a dark phenotype in
the presence of compound 12, hypothesizing that such mutants
harbor alterations in the inhibitor binding-site. Four such mutants
were identified (Figure 6A). These LuxO D47E variants all possess
an active ATPase and are functional, as judged by their ability to
repress light production in the absence of inhibitor (Figure 6A).
Sequencing revealed that the four LuxO D47E mutants carry
I211F, L215F, L242F, or V294L alterations, implicating these
residues as important for binding of the inhibitors. We mapped
these four alterations onto the existing crystal structure of ATP-
bound Aquifex aeolicus NtrC1 (PDB:3M0E) , which has high
sequence homology to LuxO (Figure 6B). The four residues we
identified in the screen map to three regions that abut the Walker
B motif (D245, E246, L247, and C248 in LuxO) (Figure 6B). In
other NtrC-type proteins, mutations in this region have been
shown to prevent ATP hydrolysis (See DISCUSSION). These four
luxO mutations were introduced into wild type LuxO and the
resulting mutants are similarly resistant to inhibition (Figure S3).
Thus, binding of compounds 11 and 12 to this region may induce
a conformational change in the nearby ATP-binding pocket that
inhibits ATP hydrolysis.
Broad spectrum activation of vibrio QS
As mentioned, LuxO is a conserved member of vibrio QS
circuits. We therefore wondered if, similar to what we found in V.
cholerae, compounds 11 and 12 could activate QS in other Vibrio
species. To test this idea, we exploited two well-characterized
phenotypes controlled by QS: light production in V. harveyi and
colony opacity in Vibrio parahaemolyticus [44–46]. In V. harveyi, light
production is induced by QS and a V. harveyi luxOD47Emutant is
dark. Treatment of V. harveyi luxOD47Ewith compounds 11 and 12
Figure 3. Structure-Activity-Relationship of LuxO inhibitors. The core chemical structure of the LuxO inhibitors is shown at the top. All
analogs possess the identical 6-thio-5-azauracil moiety with modifications in the terminal side chains (denoted R). Variations in the side chain are
shown on the right. Normalized light (RLU, relative light units) produced by the V. cholerae luxOD47Estrain (BH1651) carrying the lux operon is plotted
as a function of concentration of the eight different analogs. Error bars are present, but are too small to be observed in the plot. The bars represent
standard errors of the mean for three independent trials.
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Figure 4. The LuxO Inhibitor does not affect DNA binding. LuxO D47E DNA binding in the presence and absence of compounds 11 and 12
was investigated by gel mobility shift assays (A) and fluorescent anisotropy assays (B). In (A), LuxO D47E was present at 1 mM. Compounds 11 and 12
were present at 200 mM. In (B), LuxO D47E was present at the indicated concentrations and compounds 11 and 12 were present at 200 mM. Error bars
are present, but are too small to be observed in the plot. The bars represent standard errors of the mean for three independent trials.
Figure 5. Enzyme kinetic analyses of LuxO ATPase inhibition. (A) Michaelis-Menton enzyme kinetic analysis of LuxO ATPase activity. The LuxO
D47E ATP hydrolysis rate is plotted as a function of the concentration of ATP in the presence of the indicated amounts of compound 11. Error bars
represent standard errors of the mean for at least three independent trials. (B) Lineweaver-Burk plot derived from the assay described in (A). (C)
Lineweaver-Burk plot derived from a LuxO D47E ATPase assay in the presence of the indicated amounts of compound 12. (D) Correlation between %
inhibition of LuxO D47E ATPase activity (2.5 mM ATP and 30 mM inhibitors) and EC50of QS-activation potency (derived from Figure 3) for the different
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induced light production 10,000-fold, indicating that these
compounds are indeed active in V. harveyi (Figure 7A). In V.
parahaemolyticus, the HapR ortholog, OpaR, controls colony
opacity. OpaR production is repressed at LCD by LuxO,P via
the V. parahaemolyticus Qrr sRNAs. V. parahaemolyticus mutants that
produce low and high levels of OpaR form translucent and opaque
colonies, respectively [32,46]. Thus, V. parahaemolyticus is naturally
translucent at LCD and opaque at HCD. McCarter et al 
recently identified a constitutively active LuxO mutant (LM4476,
luxO*) in V. parahaemolyticus that confers a constitutive translucent
colony morphology (Figure 7B, left). By contrast, an isogenic V.
parahaemolyticus DluxO strain (LM9688) forms opaque colonies
(Figure 7B, left). When the luxO*mutant is plated on medium
containing compound 11 or compound 12, the colonies switch
from translucent to opaque, a phenotype indistinguishable from
the DluxO mutant (Figure 7B, right). These results suggest that
compounds 11 and 12 inhibit V. parahaemolyticus LuxO from
repressing the OpaR-dependent QS program. We conclude that
the LuxO inhibitors identified in this study are broadly capable of
activating QS in Vibrio species that employ LuxO as the central QS
New chemical tools for controlling virulence in vibrios
In pathogenic vibrios, HapR and its homologs (e.g., V.
parahaemolyticus OpaR and V. vulnificus SmcR) function as
repressors of virulence factor production at HCD [32–34]. For
example, in V. cholerae, the genes encoding the key V. cholerae
virulence factors, the CTX toxin and the Toxin Co-regulated Pilus
(TCP), are targets of HapR repression at HCD [17,27,30]. V.
parahaemolyticus uses Type Three Secretion Systems (TTSS) for
pathogenesis, and at HCD, OpaR represses the expression of one
of the TTSS operons (TTSS-1) [32,47]. Thus, luxO mutants that
constitutively produce HapR (V. cholerae) or OpaR (V. parahaemo-
lyticus) are attenuated in virulence [22,30,32]. The previous section
Figure 6. Isolation of LuxO mutants resistant to inhibition. (A) Normalized light (RLU, relative light units) produced by the V. cholerae DluxO
strain carrying luxOD47Eand luxOD47Eharboring additional mutations in the absence (white) or presence of 100 mM of compound 11 (black) or
compound 12 (gray). Error bars represent standard errors of the mean for three independent trials. Western blot analyses demonstrate that the wild
type and all mutants produce comparable amounts of LuxO protein. (B) The locations of the resistance-conferring mutations are inferred from the
ATP-bound Aquifex aeolicus NtrC1 structure (3M0E). Two monomers of NtrC1 are shown (cyan and green). The residues predicted to form the Walker
B motif are shown in blue. The four resistance-conferring mutations (I211, L215, L242, and V294) are shown in orange. The catalytic arginine residue
and ATP are shown in magenta (with side chain) and yellow, respectively.
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shows that our LuxO inhibitors are active in multiple vibrios. To
test whether the inhibitors can disrupt the QS-controlled virulence
outputs of pathogenic vibrios, we assayed their effects on TcpA
production in V. cholerae and production and secretion of VopD, a
TTSS-1 effector protein, in V. parahaemolyticus. Western blot
analysis showed that, in a V. cholerae luxOD47Estrain, HapR and
TcpA levels increased and decreased, respectively, in the presence
of compound 12 (Figure 8A). Likewise, exposing the V. para-
haemolyticus luxO*mutant to compound 12 resulted in decreased
production and secretion of VopD (Figure 8B).
To begin to explore whether repression of these in vitro virulence
phenotype translates to repression of the in vivo phenotype, we
exploited an established V. parahaemolyticus cytotoxicity assay 
to investigate whether pathogenicity could be inhibited by
treatment with the LuxO inhibitors. We infected cultured HeLa
cells with the untreated or compound 12-treated V. parahaemolyticus
luxO*mutant and assayed HeLa cell lysis by measuring lactate
dehydrogenase released from the host cytoplasm. Consistent with
a previous report , the V. parahaemolyticus luxO* mutant is more
cytotoxic to HeLa cells than the isogenic DluxO mutant (Figure 8C).
At 2 to 3 hours post-infection, HeLa cell lysis was significantly
lower in samples infected with the luxO*mutant treated with
compound 12 than in samples infected with the luxO*mutant that
had not been treated (average cytotoxicity is ,30% and ,100%
for treated and untreated, respectively, p,0.01). At that time
point, the cytotoxic capability of the Compound 12-treated luxO*
mutant is slightly higher than that of the isogenic DluxO mutant
(Figure 8C). At 4-hour post-infection, the compound 12-treated
luxO*mutant was equally toxic (,100%) as the untreated the luxO*
mutant, while the DluxO mutant caused only ,60% HeLa cells
lysis. This residual cytotoxicity is consistent with earlier results
showing that the DluxO mutant is not completely impaired for
cytotoxicity . Thus, the level of in vitro inhibition of TTSS-1
(Figure 8B) is a good indicator of the ex vivo inhibition of
cytotoxicity (Figure 8C). The increase in cytotoxicity in Com-
pound 12-treated V. parahaemolyticus that occurred at late time
points could be due to incomplete inhibition of LuxO, uptake, or
degradation of the compound by the HeLa cells. Nonetheless, the
progression of V. parahaemolyticus killing of mammalian cells is
impaired by compound 12, consistent with the notion that
virulence factor production can be controlled by small molecule
inhibitors of LuxO.
As part of a continuing effort to identify molecules that
modulate QS in bacteria, we have identified two classes of
molecules that activate QS in V. cholerae. These newly identified
molecules serve two important purposes. First, they can be used as
novel chemical probes to study QS signal transduction mecha-
nisms. Second, from a practical standpoint, because QS represses
virulence factor production in many pathogenic Vibrio species,
Figure 7. The LuxO inhibitors activate QS in different Vibiro species. (A) Normalized light (RLU, relative light units) produced by the V.
harveyi luxOD47Estrain in the absence and presence of 50 mM of compounds 11 and 12. (B) Colony morphology of the constitutively active V.
parahaemolyticus luxO*mutant (LM4476) and the isogenic V. parahaemolyticus DluxO mutant (LM9688) in the absence and presence of 500 mM
compounds 11 and 12. Each strain was inoculated four times on the same plate.
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PLoS Pathogens | www.plospathogens.org8 June 2012 | Volume 8 | Issue 6 | e1002767
molecules that activate QS, which decreases virulence, have the
potential to be developed into anti-virulence agents to combat
infectious diseases caused by pathogenic vibrios.
The first class of molecules identified here acts on the V. cholerae
CqsS receptor. These molecules, surprisingly, do not resemble the
native CAI-1 family of ligands (Figure 2A). Previous studies
revealed that CqsS receptors from different vibrios possess distinct
ligand detection specificities. The V. cholerae receptor is promiscu-
ous in detecting a range of CAI-1-type molecules, while the V.
harveyi receptor is relatively stringent . Interestingly, none of
the Class 1 molecules identified here activates QS in V. harveyi,
lending support to the idea that CqsS receptors, although sharing
extensive homology, possess different overall stringencies for
ligands. We altered a single specificity-determining residue in the
V. cholerae CqsS receptor (Cys 170) to the corresponding amino
acid (Phe) in the V. harveyi receptor. This alteration is sufficient to
increase stringency in detection of CAI-1 type molecules [39,49],
however, it did not abolish detection of the Class 1 molecules
(Figure S4). Identification of CqsS receptor mutants with altered
selectivity to the Class 1 molecules will provide additional insight
into the molecular basis of ligand-CqsS interactions.
The second class of molecules identified, and the focus of this
work, act on LuxO, the central QS regulator that controls
transcription of the four Qrr sRNA genes. LuxO, which is a
member of the NtrC family of two-component response regulators,
possesses an N-terminal regulatory receiver domain, a central
ATPase domain (AAA+ type), and a C-terminal DNA-binding
domain. Three inhibitors have previously been identified that
target non-NtrC type response regulators, AlgR1 of Pseudomonas
aeruginosa , WalR in low-GC Gram-positive bacteria , and
DevR in Mycobacterium tuberculosis . The molecules function by
perturbing phosphorylation (AlgR1 and WalR) and DNA binding
(DevR). Our LuxO inhibitors, by contrast, function by an
uncompetitive mechanism, presumably by binding to the pre-
formed LuxO-ATP complex to prevent ATP hydrolysis. Thus,
multiple families of response regulator can be selectively inhibited
using small molecules. Furthermore, all three known response
regulator activities; phosphorylation, DNA binding, and ATPase,
are potential targets for inhibition. Analyses of LuxO inhibitor-
resistant mutants suggest that our inhibitors bind to a region close
to the predicted Walker B motif. Additional support for this idea
comes from studies of other NtrC-type proteins, which show that
Figure 8. Control of virulence factor production by LuxO inhibitors. (A) Western blot analysis of TcpA (Top), HapR (middle), and LuxS
(bottom, loading control) in a V. cholerae luxOD47Emutant in the presence of 0, 12.5, 25, 50, 100, and 200 mM compound 12. (B) Western blot analysis
of the cytoplasmic and secreted VopD in the V. parahaemolyticus constitutively active luxO*strain (LM4476) in the presence of 0, 200, and 500 mM
compound 12. An isogenic V. parahaemolyticus DluxO mutant (LM9968) is included as the control. (C) Cytotoxicity of V. parahaemolyticus LM4476
(luxO*) on cultured HeLa cells in the absence and presence of 500 mM compound 12. Cytotoxicity was measured by lactate dehydrogenase (LDH)
release from HeLa cells. 100% cytotoxicity denotes LDH activity released upon treatment with 0.45% (v/v) Triton-X100. The V. parahaemolyticus DluxO
mutant LM9968 and the no-bacteria control are included for comparison. Error bars represent standard errors of the mean for three independent
Controlling Quorum Sensing in Vibrios
PLoS Pathogens | www.plospathogens.org9 June 2012 | Volume 8 | Issue 6 | e1002767
mutations that affect ATP hydrolysis but do not interfere with
ATP binding also map to the Walker B motif and to amino acid
residues preceding the conserved GAFTGA domain [43,53,54].
Indeed, one of the LuxO inhibitor-resistant mutations identified
here (L242F) lies immediately upstream of the predicted Walker B
motif, while both the I211F and L215F mutations map to the helix
containing the GAFTGA domain. In addition, the residue
identified in the final inhibitor-resistant mutant, V294L, is
predicted to sit facing the putative catalytic arginine (R306). The
GAFTGA domain is important for interaction with the s54-RNAP
holoenzyme . Thus, it was possible that the mutations we
isolated in this region (I211F and L215F) suppress inhibition by
compounds 11 and 12 by stabilizing the LuxO-s54-RNAP
interaction without affecting inhibitor binding. If this were the
case, the ATPase activity of the purified LuxO D47E/I211F and
D47E/L215F variants would be inhibited by these compounds.
However, we purified LuxO D47E/I211F protein and found that
the ATPase activity is not inhibited (Figure S5). This result is
consistent with the idea that these mutations abolish inhibitor
binding and, in so doing, prevent ATP hydrolysis.
High sequence conservation in the ATPase domain exists
between different NtrC-type response regulator family members.
Thus, we were interested to test whether the LuxO inhibitors
could inhibit other NtrC-type response regulators. Compounds 11
and 12 only modestly inhibit (,10%) the ATPase activity of
purified E. coli NtrC at 250 mM (a concentration at which .80%
of the LuxO ATPase activity is inhibited, Figure S6). This finding
is surprising because the key residues (I211, L215, L242, and
V294) that, when mutated, confer resistance to the inhibitors in
LuxO are all present in E. coli NtrC. Thus, NtrC must possess
additional structural features that render it resistant to inhibition.
Structural comparisons between these two related RRs, coupled
with identification of inhibitor-sensitive NtrC mutants, should
allow us to understand the basis of the differences in inhibitor
Two-component signaling (TCS) proteins are widely distributed
in bacteria. In addition to their global importance in microbial
physiology, the absence of TCSs in mammalian cells makes them
attractive drug targets in pathogenic bacteria [56,57]. Even though
significant effort has been devoted to identifying novel TCS
inhibitors, to date, none has been developed into a new class of
anti-infective. Problems such as undesirable properties associated
with lead molecules have been encountered [56,57]. In particular,
inhibitors that generally target the conserved hydrophobic kinase
domains of TCS histidine kinases suffer from drawbacks such as
low cell permeability, poor selectivity, and unfavorable non-
specific off-target effects (e.g. membrane damaging) [58–60]. By
contrast, approaches to target the sensory domains of histidine
kinases have yielded a handful of promising TCS inhibitors. For
instance, LED209, an antagonist of the QseC histidine kinase,
which regulates motility and pathogenicity in enterohaemorrhagic
E. coli, reduces virulence in several pathogens both in vitro and in
vivo . In addition, in Staphylococcus aureus, inhibitory Agr peptide
analogs antagonize the AgrC histidine kinase receptors and block
abscess formation in an experimental murine model .
Targeting response regulators as a broad-spectrum anti-
infective strategy has been considered challenging because
response regulator functions, such as phosphorylation and DNA
binding, are thought to be specific. In spite of this, a handful of
molecules that inhibit particular response regulator functions have
been reported [50–52]. For example, as mentioned, Walrycins,
molecules that inhibit the phosphorylation of the essential WalR
response regulator, are active in suppressing growth in multiple
Gram-positive bacteria . In the context of our work, the
ATPase domain is highly conserved between all members of the
NtrC response regulator family. Therefore, molecules that
specifically target the ATPase domain of a single response
regulator in this family (e.g., LuxO) could potentially be developed
into general inhibitors of NtrC-family of proteins. Because NtrC-
type proteins control virulence, nitrogen metabolism, motility, and
other vital processes in bacteria , targeting the ATPase domain
offers an additional route for anti-TCS drug development.
The LuxO inhibitors identified here possess certain favorable
drug-like characteristics: potent inhibition, water-solubility, good
stability, and cell-permeability. The molecules also display low
host-cell cytotoxicity (undetectable cytotoxicity at 500 mM). These
broadly-active LuxO inhibitors are not broad-spectrum NtrC-type
inhibitors. Microarray analyses reveal that fewer than 40% of
genes affected by the inhibitors are non-LuxO targets (data not
shown). Nonetheless, our LuxO inhibitors could be used as
preliminary scaffolds for building a general NtrC-type RRs
inhibitor. Future improvements to these molecules will be focused
on the structure-activity relationships of the thio-azauracil core,
combined with simultaneously screening for molecules that inhibit
LuxO and other NtrC type response regulators.
Although NtrC is not affected by the inhibitors discovered here,
multiple LuxO response regulators from different Vibrio species are
targeted by our inhibitors. Vibrio species detect a wide array of
autoinducers (HSLs, CAI-1, and AI-2), thus, molecules that
interrupt QS in Vibrio species by targeting the cognate receptors/
synthases are likely to be autoinducer-specific and will have a
limited spectrum. By contrast, because LuxO is nearly identical in
all Vibrio species, our inhibitors can broadly activate vibrio QS
irrespective of what type of autoinducer is detected. More
importantly, we showed here that treatment of V. cholerae and V.
parahaemolyticus with the LuxO inhibitors reduces virulence factor
production and impedes cytotoxicity. Thus, our LuxO inhibitors,
upon refinement, can at a minimum be used broadly to control
virulence factor production in a variety of Vibrio species that use
QS to repress pathogenesis.
The central ATPase module of the NtrC-type RR is classified as
AAA+ type . This module is present in multiple domains of
life. For example, AAA+ ATPases are important in functions
including protein unfolding and degradation (ClpXP, FtsH, and
p97), organelle function and maintenance (PEX1 and VPS4),
replication and recombination (RuvBL1 and helicases), and
intracellular transport (Dyneins). Some eukaryotic AAA+ ATPases
have been proposed to be drug targets . Therefore, it will be
particularly fascinating to investigate whether the thio-azauracil
core discovered here can be developed into an inhibitor of AAA+
ATPases across different domains.
Antagonizing QS in bacteria represents a promising new
[8,12,14,15,61,65]. Likewise, using pro-QS agents to treat acute
infections, in which bacteria use QS to repress virulence, should be
further explored. Using the native CAI-1 ligand, we previously
showed that V. cholerae virulence factor production is repressed in
vitro . In the same vein, we show here that our synthetic pro-
QS molecules reduce virulence by inhibiting LuxO. March et al
reported that pretreatment with commensal E. coli over-producing
the V. cholerae autoinducer CAI-1 increases the survival rate of mice
following V. cholerae infection , which further supports the idea
of QS potentiators as drugs. Use of CAI-1, LuxO inhibitors, or
other QS-activating molecules as prophylactics could conceivably
prevent V. cholerae or other pathogenic vibrios from initiating the
LCD virulence gene expression program that is required for
colonization. In this scenario, inhibiting the launch of virulence
factors would provide sufficient time for the host immune system
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PLoS Pathogens | www.plospathogens.org10 June 2012 | Volume 8 | Issue 6 | e1002767
to eliminate the pathogen. In contrast to traditional antibiotics that
target essential bacterial processes, growth is not affected by
interfering with QS, so development of resistance could potentially
be minimized [8,14].
Materials and Methods
Bacterial strains and culture conditions
All V. cholerae strains are derivatives of wild type C6706str .
All V. harveyi strains are derivatives of wild type V. harveyi BB120
. V. parahaemolyticus strains were generously provided by Dr.
Linda McCarter. Escherichia coli S17-1 pir, DH5a, and Top10 were
used for cloning. The relevant genotypes of all plasmids and strains
are provided in Supporting Table S1. Unless specified, E. coli and
V. cholerae were grown in LB medium at 37uC and 30uC with
shaking, respectively. V. harveyi and V. parahaemolyticus were grown
in LM medium at 30uC with shaking. Colony opacity of V.
parahaemolyticus was monitored on LM with 2% agar. Unless
specified, antibiotic concentrations are as follows: ampicillin,
gentamicin, and kanamycin, 100 mg/L; chloramphenicol and
tetracycline, 10 mg/L; streptomycin, 5 g/L; polymyxin B, 50 U/
Screening for V. cholerae QS-activating molecules
The 90,000 molecule library was supplied by the High-
Throughput Screening Resource Center of the Rockefeller
University. The V. cholerae strains BH1578 (DcqsA DluxS pBB1)
and BH1651 (luxOD47EpBB1) were grown overnight in LB
medium with tetracycline and diluted 25-fold. The diluted cultures
were dispensed into 384-well microtiter plates containing screen-
ing molecules that were previously added to each well. The final
concentration of each compound was ,20 mM. Light production
was measured on an Envison Multilabel Reader after 6-hour
incubation at 30uC without shaking. Compounds that induced
light production .100-fold were reordered from suppliers and
Bioluminescence assays for V. cholerae and V. harveyi
Overnight cultures of reporter strains were grown in LM
medium (for V. harveyi) or LB with tetracycline (for V. cholerae
carrying pBB1) and diluted 20-fold with sterile medium. Biolumi-
nescence and OD600were measured in an Envison Multilabel
Reader following 4-hour incubation at 30uC with shaking.
Synthetic molecules were dissolved in DMSO and supplied at
varying concentrations to the reporter strains. DMSO was used as
the negative control.
The open reading frame encoding V. cholerae LuxO D47E was
amplified by PCR and cloned into plasmid pET28B that had been
previously digested with NdeI and BamHI. The resulting plasmid
was transformed into E. coli BL21 Gold (DE3) resulting in strain
WN133. Strain WN133 was grown in LB with kanamycin at 30uC
with shaking until the OD600of the culture reached ,1.0. IPTG
was added at a final concentration of 200 mM, and the culture was
incubated for an additional 4 hours at 30uC with shaking. Cells
were harvested by centrifugation, suspended in lysis buffer
(20 mM Sodium phosphate buffer pH 7.4, 0.5 M NaCl, 10%
glycerol, and 5 mM imidazole), and lysed using a Cell Cracker.
Soluble materials were loaded onto a HiTrap chelating column
charged with nickel, the column was washed extensively with lysis
buffer, and His6-tagged V. cholerae LuxO D47E enzyme was eluted
using a linear gradient of increasing concentration of imidazole
dissolved in lysis buffer. Fractions containing LuxO D47E were
pooled and concentrated with an Amicon Untra-15 filter. Protein
was snap-frozen in liquid nitrogen and stored at 280uC. Protein
concentrations were determined by UV absorbance at 280 nm. E.
coli NtrC and other LuxO D47E variants were purified using the
A modified coupled-enzyme assay was used to measure the rate
of ATP hydrolysis by LuxO D47E . Briefly, ADP released
from ATP by LuxO D47E is reacted with phosphoenolpyruvate
(PEP) to form pyruvate using pyruvate kinase (PK). Pyruvate is
reacted with NADH to form NAD and lactate using lactate
dehydrogenase (LDH). The rate of NAD production is followed at
340 nm using a spectrophotometer. ATP hydrolysis rates were
inferred from the absorbance change observed (eNADH,3402e-
NAD,340=6220 M21cm21for NADH) . The rates of ATP
hydrolysis by LuxO D47E were measured in reactions containing
100 mM Sodium phosphate buffer pH 7.4, 5 mM MgCl2,
0.2 mM NADH, 1 mM PEP, 5–20 units of PK/LDH mix
(Sigma), and 10 mM LuxO D47E. ATP and inhibitors were added
to the reactions at indicated concentrations. The rate of ATP
hydrolysis was monitored for 5 minutes. Data were fitted using
Graphpad Prism to obtain the kinetic parameters. Percent ATPase
inhibition was calculated using the following formula:
DNA binding assays
Electrophoretic mobility shift assays to study LuxO and Qrr
promoter DNA interactions were performed as described in .
Fluorescence anisotropy assays using LuxO D47E were modified
Screening for LuxO mutants resistant to inhibitors
The luxOD47Eallele was removed from plasmids harbored in
WN133 with the enzymes XbaI and BamHI and ligated into
pEVS143  that had been previously digested with AvrII and
BamHI. The luxOD47Ereading frame of the resulting plasmid
(WN2029) was randomly mutated using the GeneMorph II
Random Mutagenesis Kit. The resulting mutagenized luxOD47E
plasmid library was introduced into a V. cholerae DluxO strain by
conjugation. Individual colonies from this V. cholerae luxOD47E
mutant pool were arrayed into 96-well plates containing LB
medium with 100 mM compound 12. The V. cholerae DluxO strain
harboring non-mutated luxOD47Ewas grown in the absence of
compound 12 to provide the reference for background light
production. Following overnight static incubation at 30uC, clones
that produced light comparable to the background were selected
and re-tested in the presence and absence of compounds 11 and
12. DNA sequencing was used to determine the alterations in
luxOD47Efor inhibitor-resistant mutants. Site-directed mutageneses
were performed with the QuikChange II XL Site-Directed
Mutagenesis Kit to uncouple multiple mutations.
Western blot analysis
Overnight cultures of the V. cholerae luxOD47Estrain were diluted
1000-fold in AKI medium containing the indicated concentrations
of compound 12. The cultures were statically incubated at 37uC
for 4 hours and subsequently shaken for 4 more hours at 37uC.
Cells were collected by centrifugation, TcpA from different
Controlling Quorum Sensing in Vibrios
PLoS Pathogens | www.plospathogens.org11June 2012 | Volume 8 | Issue 6 | e1002767
samples was analyzed by Western blot as previously described
. Overnight cultures of the V. parahaemolyticus luxO* strain
(LM4476) were washed and diluted 50-fold in LM medium with
10 mM MgCl2and 10 mM sodium oxalate in the presence of the
indicated concentrations of compound 12. The cultures were
grown for 4 hours with shaking at 37uC. Viable cell count showed
that all cultures contained ,16109CFU/mL after incubation.
Cells were collected by centrifugation, and the secreted and
cytoplasmic VopD from different samples were analyzed by
Western blot as previously described .
Cytotoxicity assays were modified as previously described .
HeLa cells (26104cells/well) were cultured for 48 hours at 37uC
and 5% CO2in a 96-well plate containing DMEM with 10% fetal
bovine serum prior to infection. V. parahaemolyticus strains were
grown as described above for VopD analysis and used in the
infection assays. Immediately prior to V. parahaemolyticus infection,
DMSO or compound 12 (500 mM) was added to the HeLa.
Serially diluted bacteria were added to HeLa cells at multiplicity of
infection of 10. Lactate dehydrogenase release from HeLa cells
was assayed between 1–4 hours after infection using the CytoTox
96 nonradioactive cytotoxicity kit (Promega).
Chemical synthesis and analytical methods
All chemical syntheses and analytical methods are provided in
the Supporting Text S1.
if QS modulators affect growth, V. cholerae strain BH1578 was
incubated with 100 mM of compounds 1 to 12. Optical density at
600 nm was monitored thereafter for a total of 4 hours of
incubation at 30uC. No significant difference was observed
between the DMSO control and the treatments. Error bars
represent standard errors of the means from three independent
The effect of QS modulators on growth. To test
cholerae strains lacking each QS receptor. To determine
which QS receptor each Class 1 compound acts on, we tested the
eight Class 1 compounds against V. cholerae mutants lacking only
the CqsS receptor (black bars) or only the LuxPQ receptor (white
bars). All eight Class 1 compounds induced light production in the
DluxPQ strain but not the DcqsS strain. Normalized light
production (RLU) was measured in V. cholerae strains lacking
either the CqsS or the LuxPQ QS receptor in the presence of
50 mM of the Class 1 compounds. Error bars represent standard
errors of the means from three independent samples.
Responses to Class 1 compounds by Vibrio
resistance in the wild type LuxO protein. The luxO
mutations I211F, L215F, L242F, and V294L that confer inhibitor
resistance were individually introduced into the plasmid carrying
wild type luxO. The resulting plasmids were mobilized into a
V.cholerae DcqsA DluxS DluxO mutant carrying the heterologous V.
harveyi lux operon. In strain expressing wild type LuxO, the
inhibitors (100 mM compounds 11 and 12) were capable of
inhibiting LuxO, thus, light production was induced .5000-fold
(grey and black bars). By contrast, light production was only
induced at #300-fold in the LuxO mutants I211F, L215F, L242F,
The effect of luxO mutations on inhibitor
and V294L, suggesting these luxO mutations confer resistance to
the inhibitors in the context of the wild type protein.
cholerae CqsS mutants with altered receptor specifici-
ties. Previous studies showed that the CqsS C170Y mutation
causes increased specificity for a ligand with a C10 tail and an
overall reduction in sensitivity to CAI-1. Normalized light
production (RLU) was measured in V. cholerae strains carrying
wild type CqsS (WN1103) or the CqsSC170Yreceptor (WN1992) in
the presence of 50 mM of the Class 1 compounds. Error bars
represent standard errors of the means from three independent
samples. The results show that the C170Y mutation does not
abolish detection of some of the Class 1 compounds (e.g., cpd1,
cpd 3, and cpd 11).
Responses to Class 1 compounds by Vibrio
D47E/I211F in the presence of the LuxO inhibitors.
Mutations I211F and L215F map in close proximity to the LuxO
GAFTGA domain, which is presumed to be required for
interaction with RNA polymerase (RNAP). Therefore, it was
possible that mutations causing insensitivity to the Class 2
compounds could suppress inhibition by stabilizing the LuxO-
s54-RNAP interaction without affecting inhibitor binding. If this
were the case, the ATPase activity of LuxO D47E/I211F and
D47E/L215F variants would remain inhibited by these com-
pounds. The experiment below shows that while the ATPase
activity of LuxO D47E is inhibited by the compounds (open and
closed circles), the ATPase activity of the purified LuxO D47E/
I211F protein is not affected (open and closed squares). ATP
hydrolysis was measured using a coupled-enzyme assay that
monitors changes in absorbance at 340 nm. 100 mM of Com-
pound 12 and 2.5 mM ATP were used in the assay.
ATPase activity of LuxO D47E and LuxO
High sequence conservation in the ATPase domain exists between
different NtrC-type response regulators. To test whether the Class
2 LuxO inhibitors also inhibit other NtrC-type response
regulators, we examined E. coli NtrC. While .80% of the LuxO
ATPase activity is inhibited (open and closed circles) by 250 mM of
compound 11, the inhibitor only modestly inhibits (,10%) the
ATPase activity of purified E. coli NtrC D54E (open and closed
The effect of LuxO inhibitors on E. coli NtrC.
Bacterial strains used in this study.
Chemical Synthesis and Analytical Methods.
We thank members of the Bassler laboratory and Dr. Frederick Hughson
for insightful discussions and suggestions. We thank Dr. Linda McCarter
for generously providing Vibrio parahaemolyticus strains.
Conceived and designed the experiments: WLN LP JC MFS BLB.
Performed the experiments: WLN LP JC. Analyzed the data: WLN LP JC
MFS BLB. Contributed reagents/materials/analysis tools: WLN LP JC.
Wrote the paper: WLN LP BLB.
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