Chemistry & Biology
Dioxygenase-Mediated Quenching of
Quinolone-Dependent Quorum Sensing
in Pseudomonas aeruginosa
Christian Pustelny,1Alexander Albers,2Klaudia Bu ¨ldt-Karentzopoulos,2Katja Parschat,2Siri Ram Chhabra,1
Miguel Ca ´mara,1Paul Williams,1,* and Susanne Fetzner2,*
1School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, NG7 2RD, UK
2Institute of Molecular Microbiology and Biotechnology, Westfalian Wilhelms-University Muenster, D-48149 Muenster, Germany
*Correspondence: firstname.lastname@example.org (P.W.), email@example.com (S.F.)
quorum-sensing signal molecule used by Pseudo-
monas aeruginosa. The structural similarity between
substrate for the 2,4-dioxygenase, Hod, and PQS
prompted us to investigate whether Hod quenched
PQS signaling. Hod is capable of catalyzing the con-
version of PQS to N-octanoylanthranilic acid and
carbon monoxide. In P. aeruginosa PAO1 cultures,
exogenously supplied Hod protein reduced expres-
sion of the PQS biosynthetic gene pqsA, expression
pyocyanin, and rhamnolipids, and virulence in
planta. However, the proteolytic cleavage of Hod by
extracellular proteases, competitive inhibition by the
PQS precursor 2-heptyl-4(1H)-quinolone, and PQS
binding to rhamnolipids reduced the efficiency of
Hod as a quorum-quenching agent. Nevertheless,
these data indicate that enzyme-mediated PQS inac-
tivation has potential as an antivirulence strategy
against P. aeruginosa.
Pseudomonas aeruginosa is an important opportunistic human
pathogen found in soil and water habitats. It is one of the leading
causes of nosocomial infections and the predominant respira-
tory pathogen in cystic fibrosis (CF) (Lyczak et al., 2002). In
common with many pathogenic bacteria, the production of colo-
nization, survival, and virulence factors in P. aeruginosa is coor-
dinated in a growth- and cell density-dependent manner via cell-
to-cell communication or quorum sensing (QS) (Williams et al.,
2007). The hierarchical QS system of P. aeruginosa consists of
an interdependent and overlapping regulatory network using
N-acylhomoserine lactone (AHL) and 2-alkyl-4(1H)-quinolone
(AQ) QS signal molecules (Venturi, 2006; Diggle et al., 2006;
Williams and Ca ´mara, 2009). With respect to the latter, P. aeru-
ginosa produces over 50 different AQ congeners that differ
mainly in the length of the 2-alkyl side chain (C5 to C13), which
can besaturated or unsaturated, andin the presence or absence
of a 3-position hydroxyl substituent (Le ´pine et al., 2004). AQs
are known to possess antibacterial, anti-algal, iron-chelating,
and immune modulatory activities. Among these, 2-heptyl-3-
hydroxy-4(1H)-quinolone (the P. aeruginosa quinolone signal,
PQS; Figure 1A) was identified by Pesci et al. (1999) as a QS
signal molecule. PQS is now known to be involved in biofilm
development and in the regulation of many virulence factors
and secondary metabolites including the galactophilic lectin
LecA, pyocyanin, elastase, rhamnolipids, and the MexGHI-
OpmD multidrug efflux pump (Diggle et al., 2003; Allesen-Holm
et al., 2006). A study of the global transcriptional profile of P. aer-
uginosa in response to PQS revealed a significant upregulation
of genes involved in the oxidative stress response and high-
affinity iron acquisition (Bredenbruch et al., 2006; Diggle et al.,
2007). PQS also functions as an iron trap, sequestering iron
from the growth environment and retaining it as the iron(III)-
PQS complex in association with the cell surface of P. aerugi-
nosa (Diggle et al., 2007). In addition, PQS has been suggested
to balance life and death in P. aeruginosa populations by
inducing a protective response in some cells while eliminating
and Becker, 2008).
The biosynthesis of AQs occurs via the ‘‘head-to-head’’
condensation of anthranilate and a 3-oxo-fatty acid (Ritter and
operon or the kynABU genes for providing anthranilate, together
with the pqsABCD operon and the pqsH gene (Gallagher et al.,
2002; Farrow and Pesci, 2007). The latter gene product is a
(HHQ; Figure 1A) to PQS. PqsA is an anthranilate CoA ligase,
which primes anthranilate for entry into the PQS biosynthetic
pathway (Coleman et al., 2008), whereas PqsD was identified
as a condensing enzyme that may either catalyze the head-to-
head condensation of anthranoyl-CoA with a 3-oxo acid or
may be involved in the formation of a 3-oxo acid precursor
(Zhang et al., 2008). PqsB and PqsC are highly homologous
to 3-oxoacyl-(acyl-carrier-protein) synthases and while their
precise contribution to AQ biosynthesis is not known, they are
probably involved in fatty acid recruitment and condensation
(Gallagher et al., 2002). The fifth gene in the pqs operon, PqsE,
is not required for AQ biosynthesis but instead is an effector of
the PQS response (Gallagher et al., 2002; Diggle et al., 2003;
De ´ziel et al., 2004; Farrow et al., 2008). The major AQs found in
P. aeruginosa cultures are the C7 and C9 congeners of PQS
Chemistry & Biology 16, 1259–1267, December 24, 2009 ª2009 Elsevier Ltd All rights reserved 1259
and HHQ together with the N-oxides of HHQ (Figure 1A) and
In AQ signaling, both PQS and its precursor HHQ function as
autoinducers that drive expression of the pqsABCDE operon
through an interaction with the LysR-type transcriptional acti-
vator PqsR (MvfQ) (De ´ziel et al., 2005; Diggle et al., 2007;
Wade et al., 2005). Consequently, when added exogenously to
a P. aeruginosa pqsA mutant, both PQS and HHQ effectively
drive the expression of a pqsA::lux fusion in a pqsR-dependent
manner (Fletcher et al., 2007). However, in P. aeruginosa,
PAO1, HHQ, in contrast to PQS, does not efficiently restore
the expression of key downstream virulence genes such as
lecA (Diggle et al., 2007). The N-oxides of HHQ and HNQ have
little or no activity in these assays and their signaling functions,
if any, have yet to be established.
PQS and other AQs are present in the sputum and bronchoal-
veolar lavage fluid from CF patients infected with P. aeruginosa
(Machan et al., 1992; Collier et al., 2002). Strains isolated from
infants with CFalsoshowedincreased production ofPQS(Guina
pqsE) inhibits virulence gene expression and attenuates patho-
genicity in experimental infection models (Cao et al., 2001; Dig-
gle et al., 2003; De ´ziel et al., 2005). Furthermore, halogenated
anthranilate analogsthatinhibitedAQ biosynthesisand signaling
in laboratory cultures also restricted P. aeruginosa systemic
dissemination and mortality in mice without perturbing bacterial
viability (Lesic et al., 2007). Taken together, these observations
suggest that PQS and hence AQ signaling makes an important
contribution to pathogenesis and so constitutes an attractive
antibacterial target. Quorum quenching and attenuation of
P. aeruginosa virulence by enzymes that catalyze cleavage of
signal molecules has been reported for AHL lactonases (Reim-
mann et al., 2002) and AHL acylases (Lin et al., 2003; Sio et al.,
2006). However, an enzyme that inactivates the PQS molecule
and is capable of inhibiting AQ signaling has not been described.
3-Hydroxy-2-methyl-4(1H)-quinolone 2,4-dioxygenase (Hod,
‘‘1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase’’) of Arthro-
bacter nitroguajacolicus strain Ru ¨61a is a cytoplasmic enzyme
involved in the pathway of 2-methylquinoline (quinaldine) utiliza-
tion, catalyzing the cleavage of 3-hydroxy-2-methyl-4(1H)-
quinolone (MPQS, i.e., the C1 congener of PQS) to carbon
monoxide and N-acetylanthranilic acid. Hod is a monomeric
protein with an a/b-hydrolase fold (Fischer et al., 1999; Frer-
molecular disulfide bond, which is important for stability and the
rather unique ability of Hod to re-fold to the catalytically active
native state after thermal denaturation (Beermann et al., 2007;
Boehm et al., 2008). Hod, in contrast to most other known oxy-
genases, neither contains nor requires a metal ion or organic
cofactor for catalysis (Fetzner, 2002). The hod gene together
with the other genes coding for 2-methylquinoline conversion
to anthranilate are clustered on the linear conjugative plasmid
pAL1 of strain Ru ¨61a (Overhage et al., 2005; Parschat et al.,
2007). Interestingly, linear pAL1-like plasmids have been found
in several other Arthrobacter spp. isolated from soil (Overhage
et al., 2005), suggesting that the ability to degrade 2-methylqui-
noline and MPQS, or even other AQs, is not uncommon in soil
The structural similarity of the natural substrate and PQS
prompted us to investigate whether Hod is active against PQS
and other related AQs and so capable of quenching AQ signaling
in P. aeruginosa.
Products of PQS Cleavage by Hod
Hod-catalyzed conversion of PQS resulted in formation of car-
bon monoxide, which was detected spectrophotometrically in
the form of CO hemoglobin (see Figure S1 available online), sug-
gesting that PQS, consistent with MPQS, the physiological
substrate of Hod, undergoes 2,4-dioxygenolytic ring cleavage
(Figure 1B). Electrospray mass spectrometry of the extracted
organic product from enzymatic turnover of PQS indicated
a compound with an ion at m/z of 264.159 for [M+H]+and
286.141 for [M+Na]+; analysis of authentic N-octanoylanthranilic
acid showed m/z of 264.159 for [M+H]+, consistent with the
chemical composition [C15H22O3N]+, and m/z 286.142 for
[M+Na]+. Fragmentation of [M+H]+(m/z 264.159) of the product
from PQS cleavage resulted in m/z(%) of 120.044(100) and
138.055(19) and corresponded to the fragmentation pattern of
[M+H]+of the reference compound of m/z(%) 120.044(100)
[C7H6ON]+and 138.055(20) [C7H8O2N]+(Figure S1). The product
from Hod-catalyzed PQS cleavage and authentic N-octanoylan-
thranilic acid eluted in a single peak when co-chromatographed
on a reversed-phase HPLC column.
Mass spectral analyses of the extract of the enzyme assay,
obtained by combined anion exchange/reversed-phase chro-
matography, did not indicate the additional presence of an
a-oxo acid [C16H21O4N] that would result from 2,3-dioxygeno-
lytic cleavage of PQS. To examine the possibility that an oxo
compound might have been formed as side product, but lost
Figure 1. Structures of AQs and Mode of Dioxygenase-Catalyzed
(A) Structures of MPQS, PQS, HHQ and HQNO.
(B) 2,4-Dioxygenolytic cleavage of PQS to carbon monoxide and N-octanoyl-
anthranilic acid, catalyzed by Hod from Arthrobacter nitroguajacolicus Ru ¨61a.
See also Figure S1.
Chemistry & Biology
Quinolone Quorum Quenching
1260 Chemistry & Biology 16, 1259–1267, December 24, 2009 ª2009 Elsevier Ltd All rights reserved
during extraction, the aqueous enzyme assays after complete
conversion of the substrate were directly reacted with 2,4-dini-
trophenylhydrazine. Based on calibration of the assay with pyr-
uvic acid hydrazone (detection limit of R4.3 mM), approximately
1.9% of side product would have been detected from the Hod-
catalyzed conversion of 0.23 mM PQS, if the hydrazone of an
tivity as that of pyruvic acid. However, hydrazone formation was
not observed, suggesting specificity of Hod for 2,4-dioxygeno-
lytic cleavage of PQS.
Catalytic Activity of Hodtoward AQs In Vitro, in Bacterial
Culture Media, and in the Presence of Iron(III)
To determine the activity of purified Hod toward 2-alkyl-3-
hydroxy-4(1H)-quinolones, a series of compounds with alkyl
chain lengths ranging from C1 to C11 was synthesized. Notably,
Hod-catalyzed dioxygenolysis of 2-ethyl-3-hydroxy-4(1H)-qui-
nolone was faster than that of MPQS, but further extension of
alkyl chain length impeded the reaction. Compared with the
activity of Hod [in Tris/HCl buffer (pH 8)] toward MPQS of
70 U mg?1(100%), relative activities of 126%, 60%, 23%, and
?0.009% were observed with 2-ethyl-, 2-propyl, 2-pentyl-, and
2-nonyl-3-hydroxy-4(1H)-quinolone, respectively. Conversion
of the 2-undecyl-substituted congener was not detected. The
catalytic activity of Hod toward PQS was 0.2 U mg?1, i.e., about
0.3% of MPQS activity.
Tris/HCl buffer (pH 8), were 0.16 s?1and 13.4 mM, respectively.
Since in air-saturated buffer kcat appand Km appof the enzyme for
the physiological substrate MPQS are 38.4 s?1and 2.7 mM,
respectively,thecatalyticefficiencykcat app/Km appoftheenzyme
for PQS is about 1200-fold lower than for MPQS. UV/Vis spectral
analyses indicated that HHQ, the precursor of PQS, is not con-
verted by the enzyme. Notably, HHQ acts as competitive inhib-
itor of Hod-catalyzed PQS conversion [Km´ = Km3 (1 + [I]/Kic)],
with an inhibition constant Kicof 24 mM. 2-Heptyl-4-hydroxyqui-
noline N-oxide (HQNO; Figure 1A) was neither a substrate nor an
inhibitor of Hod.
Specific Hod activities toward the physiological substrate
MPQS in bacterial culture media including sterile Luria broth
(LB) (pH 7.4), casamino acids medium (pH 7; Cornelis et al.,
1992), high-iron mineral salts medium (pH 7, with 25 mM FeSO4;
Bredenbruch et al., 2006), and QS selection medium (pH 6.7;
Diggle et al., 2007) were 42 U mg?1, 31 U mg?1, 27 U mg?1, and
10.5 and shows relative activities of 92%, 84%, and 42% at pH
7.5, 7.0, and 6.5, respectively (measured in 10 mM disodium
phosphate/borate buffer). Hence pH may contribute to the
decreased activities observed, as compared with the activity of
70 U mg?1observed in the standard biochemical assay.
MPQS and PQS were previously shown to form 2:1 and 3:1
chelate complexes with Fe(III) at physiological pH (Diggle et al.,
2007). In P. aeruginosa cultures, the PQS dissolved in the
medium and associated with the cell surface may well exist as
Fe(III) complexes (Diggle et al., 2007). Since binding of Fe(III)
ions to the 3-hydroxyl and 4-oxo groups of MPQS and PQS
might affect substrate recognition and/or turnover by the
enzyme, its activity was determined at different molar ratios of
FeCl3to MPQS or PQS. In the Tris-buffered biochemical assay,
>80% of catalytic activity was retained at up to 5-fold molar
excess of Fe(III) over organic substrate (Figure S2), suggesting
that the iron-to-PQS ratios expected to occur in P. aeruginosa
cultures in commonly used media as well as in vivo should not
significantly affect the performance of the enzyme.
When PQS (25 mM) was equilibrated in buffer with P. aerugi-
nosa rhamnolipids, the apparent activity of Hod toward PQS
was decreased to about 65% and 55% in the presence of
10 mg ml?1and 20 mg ml?1rhamnolipids, respectively. How-
ever, a further increase in rhamnolipid concentration had
comparatively minor effects, since the enzyme showed about
nolipids. The activity of Hod toward MPQS was not affected by
the presence of rhamnolipids, excluding the possibility of dena-
turation of Hod by the surfactants (data not shown).
Hod-Dependent Quenching of PQS-Dependent QS
in P. aeruginosa
To determine whether the catalytic activity of Hod was sufficient
to perturb AQ signaling in P. aeruginosa, we first determined the
impact of Hod on PQS-dependent activation of chromosomally
integrated pqsA::lux and lecA::lux fusions, in a P. aeruginosa
pqsA mutant, to circumvent any competitive inhibition due to
the presence of HHQ and in the absence of endogenously
produced PQS. Figure 2A shows that addition of 25 U Hod to
a 0.3 ml culture results in an ?4-fold reduction in pqsA::lux
expression when induced by 2 mM PQS. Similar experiments
with the lecA::lux fusion (Figure 2B) and pyocyanin (Figure 2C)
also show that Hod downregulates the production of both of
these PQS-dependent virulence determinants in a pqsA mutant.
When added to cultures of the wild-type P. aeruginosa PAO1
strain, exogenous Hod (25 U/0.3 ml) only reduced pqsA expres-
sion to 70% of the control (Figure 3A), whereas lecA expression
was reduced to 37% of the control (Figure 3B). This finding is in
Hod had little effect on pyocyanin production (Figure 3D), at 50 U
ml?1it reduced rhamnolipid levels in PAO1 wild-type cultures
by ?35% from 4.9 ± 0.4 to 3.2 ± 0.5 mg rhamnolipids ml?1
uginosa PAO1 wild-type cultures and to determine whether the
efficacy of Hod was reduced in culture, we extracted stationary
phase cultures treated with Hod and quantified PQS and HHQ
levels using LC-MS. Figure 3F shows that Hod reduced PQS
but not HHQ levels in PAO1 cultures in a concentration-depen-
dent manner. However, although the enzyme retained >80% of
catalytic activity after incubation in sterile LB for 2 days at 30?C
supernatants of P. aeruginosa PAO1. The half-life of Hod activity
in a batch culture was about 6 hr (Figure S3). In culture superna-
tant, Hod activity decreased continuously, with a similar half-life.
proteolytic degradation (Figure S3). This was confirmed by incu-
bating Hod with the P. aeruginosa PAO1 type II secretion (xcp)
mutant D40ZQ that is unable to secrete a number of exopro-
PAO1 strain, Hod reduced PQS-dependent pyocyanin produc-
tion by ?60% (data not shown).
Chemistry & Biology
Quinolone Quorum Quenching
Chemistry & Biology 16, 1259–1267, December 24, 2009 ª2009 Elsevier Ltd All rights reserved 1261
Hod Reduces P. aeruginosa Virulence in a Plant
Although P. aeruginosa is not generally considered as a plant
pathogen, plants have been used successfully as in vivo disease
models to study pseudomonas virulence, and experimental data
demonstrated conservation of virulence mechanisms between
plant and animal infection models (Starkey and Rahme, 2009).
hosts (Starkey and Rahme, 2009). Using the lettuce leaf model
we demonstrated a reduced virulence of the PQS negative
mutant PAO1 pqsA in comparison to the parent strain (data not
shown). To verify if Hod can attenuate virulence in the same
model we coinjected P. aeruginosa PAO1 with Hod, which (1)
caused much less leaf rib tissue damage than the control and
(2) reduced bacterial growth in the leaf tissues (Figure 4).
The multifactorial virulence of P. aeruginosa is tightly regulated
via a sophisticated, hierarchical QS network that incorporates
both AHL and AQ signal molecules and hence offers a number
of different potential targets for novel antibacterials. These
include the QS signal synthases (e.g., LasI, RhlI, and PqsA)
and response regulator proteins (e.g., LasR, RhlR, and PqsR)
as well as the QS signal molecules themselves. With respect to
the latter, antibodies raised against QS signal molecule conju-
gates have shown efficacy in experimental animal infection
models (Kaufmann et al., 2008) while enzymes such as lacto-
nases and acylases capable of inactivating AHL-dependent QS
reduce virulence gene expression in vitro and in planta (Dong
et al., 2007). However, to our knowledge, no enzymes capable
of quenching AQ- or PQS-dependent QS have previously been
described. Here we have reported on the potential of the dioxy-
genase Hod to inactivate PQS, downregulate the expression of
key P. aeruginosa PQS-dependent virulence genes, and reduce
in planta growth and plant tissue damage.
Whenincubated with Hod,PQSundergoes 2,4-dioxygenolytic
cleavage with concomitant formation of carbon monoxide,
consistent with the natural substrate MPQS. Carbon monoxide
scriptional regulators of P. aeruginosa; however, effective inhibi-
tion requires CO to be delivered intracellularly (Davidge et al.,
2009; Desmard et al., 2009).
The presence of the C7 alkyl chain substantially reduced the
activity of the enzyme such that its catalytic efficiency was
some 1200-fold lower than for MPQS. Interestingly, Hod was
most active against the C2 (ethyl) congener of PQS with efficacy
reducing as alkyl chain length was extended such that the
enzyme was virtually inactive against the C9 PQS congener.
Although P. aeruginosa produces PQS congeners with C5 to
C11 alkyl chains, the C7 and to a lesser extent the C9 com-
fore the primary 2-alkyl-3-hydroxy-4(1H)-quinolone QS signals
(Fletcher et al., 2007). AQ biosynthesis and pqsA expression
are not, however, exclusively dependent on PQS acting as
a coinducer of the transcriptional activator protein PqsR (Wade
et al., 2005; Xiao et al., 2006). This is because HHQ, the direct
precursor of PQS, also drives the expression of pqsA in a
PqsR-dependent manner and is more effective than PQS with
a lower EC50(0.4 mM compared with 18 mM for PQS) (Fletcher
et al., 2007). Consequently, since both HHQ and PQS accumu-
late in P. aeruginosa culture supernatants, the ability of Hod to
reduce AQ biosynthesis by downregulating pqs gene expression
in culture is therefore likely to be influenced by HHQ. P. aerugi-
nosa cultures also accumulate substantial concentrations of
Figure 2. Hod-Mediated Quorum Quenching in an AQ-Negative
P. aeruginosa pqsA Mutant
(A) pqsA expression in PAO1 pqsA CTX-lux::pqsA grown in the presence of
2 or 50 mM PQS and with or without Hod (25 U/0.3 ml).
PQS and with or without Hod (25 U/0.3 ml).
(C) Pyocyanin production in PAO1 and in PAO1 pqsA in the presence of 2 mM
PQS and with or without Hod (25 U/0.3 ml). All experiments were carried out in
triplicate at least twice. Error bars represent one standard deviation of the
mean value from three independent measurements.
Chemistry & Biology
Quinolone Quorum Quenching
1262 Chemistry & Biology 16, 1259–1267, December 24, 2009 ª2009 Elsevier Ltd All rights reserved
the N-oxide HQNO. This AQ, which does not contribute to pqsA
expression, is derived from the same substrate pool as HHQ.
While HQNO proved to be neither a substrate nor an inhibitor
of Hod, HHQ acted as a competitive inhibitor with a Kic of
24 mM (at pH 8), implying that at least at high HHQ concentra-
tions, Hod may not be effective at quenching PQS-dependent
Figure 3. Hod-Mediated Quorum Quench-
ing in P. aeruginosa PAO1
The impact of Hod on pqsA and lecA expression
(A and B), on lectin A protein (C), on pyocyanin
production (D), on rhamnolipid production (E),
and on PQS and HHQ concentrations (F). For (A),
(B) and (D), 25 U Hod/0.3 ml was used. The effect
of Hod on rhamnolipid production (E) was
analyzed with 50 U Hod/ml.
(C) Lane 1, PAO1; lane 2, PAO1 + 25 U Hod/0.3ml;
lane 3, PAO1 + 50 U Hod/0.3ml; lane 4, PAO1
pqsH (negative control); lane 5, lectin A (purified
protein control). Western
repeated three times and each experiment was
carried out in triplicate at least twice. Error bars
represent one standard deviation of the mean
value from three independent measurements.
blot analysis was
QS in P. aeruginosa cultures. In addition,
other bacterial culture medium compo-
nents, medium pH, and bacterial exo-
with Hod-mediated quorum quenching.
Therefore, we determined the activity of
Hod toward MPQS in un-inoculated
conventional rich and chemically defined
laboratory bacterial culture media and
found a reduction to between 38.5%
and 60% of that observed at pH 8 in the
standard biochemical assay. Apart from
pH, the iron content of such un-inocu-
lated growth media may also reduce the
efficacy of Hod since PQS (and MPQS)
forms 2:1 and 3:1 complexes with Fe(III). However, in the Tris-
buffered assay even at a 5-fold molar excess of iron, over 80%
of catalytic activity was retained. These data are important
since they suggest that Hod should be capable of interfering
with PQS signaling via all three pathways, i.e., (1) the pqsR
pathway in which PQS induces the expression of genes such
as pqsA but does not require pqsE; (2) the pqsR/pqsE pathway
in which PQS induces the production of lectin, pyocyanin, and
rhamnolipids; and (3) the iron-deprivation pathway in which the
iron-chelating activity of PQS induces siderophore production
(Diggle et al., 2007).
to facilitate swarming motility, promote the uptake of hydro-
phobic compounds, contribute to biofilm architecture, and
induce the rapid necrotic killing of polymorphonuclear leuco-
cytes (Jensen et al., 2007). In addition, rhamnolipids solubilize
PQS (which is very poorly water soluble) and significantly
enhance its bioavailability as revealed by the PQS-dependent
induction of lasB expression in a P. aeruginosa lasR mutant
and by the augmentation of PQS-induced apoptosis in eukary-
otic cells (Calfee et al., 2005). Given the importance of rhamno-
lipids in PQS solubilization and PQS bioactivity, it was relevant
to determine whether by sequestering PQS they reduced the
efficacy of Hod. In buffer, we noted that the apparent activity
of the enzyme toward PQS (25 mM) was reduced to ?55% in
the presence of 20 mg ml?1rhamnolipids. Further increases in
Figure 4. Hod (25 U/Lettuce Leaf) Reduces P. aeruginosa Growth
and Tissue Damage in a Plant Leaf Infection Model
Hod was co-inoculated with PAO1 into lettuce leaf ribs and incubated for
2 days. They were monitored for soft rot damage (inset) and for bacterial viable
counts (determined as cfu/mg leaf mid-rib tissue). Error bars represent the
standard error of the mean value from six independent measurements.
Chemistry & Biology
Quinolone Quorum Quenching
Chemistry & Biology 16, 1259–1267, December 24, 2009 ª2009 Elsevier Ltd All rights reserved 1263
rhamnolipid concentration had only minor affects on Hod activ-
ity, which is consistent with the observations of Calfee et al.
(2005) that the rhamnolipid-dependent augmentation of PQS
bioactivity was diminished at high rhamnolipid concentrations.
One further group of exoproducts likely to influence Hod
activity were secreted exoproteases, and we observed that in
spent stationary phase P. aeruginosa cell-free culture superna-
tants, Hod was rapidly inactivated via proteolytic cleavage with
a half-life of approximately 6 hr. Taken together, the above
data suggested that the prevailing environmental conditions
and presence of bacterial exoproducts, in particular the rhamno-
lipids and exoproteases, were likely to impact on the efficacy of
Hod as a quorum quenching enzyme. However, both exopro-
teases and rhamnolipid production are regulated via QS and
therefore the enzymatic inactivation of a key QS signal molecule
such as PQS is likely to reduce the rate of production and accu-
mulation of these exoproducts during bacterial growth.
ing enzyme, we initially examined pqsA::lux and lecA::lux
expression in a pqsA mutant supplied with exogenous PQS
since a pqsA mutant does not produce HHQ or other AQs. In
these experiments pqsA and lecA expression as well as pyocya-
nin production were substantially downregulated. In contrast,
when the parent P. aeruginosa strain was incubated with the
enzyme, pqsA expression was reduced by only 30% (as
fected. In contrast, lecA expression, lectin A protein levels, and
rhamnolipids were each substantially quenched. These data
pqsA inducer (as well as a competitive inhibitor of Hod), and by
the fact that PQS is a much more potent inducer of lectin A
and rhamnolipids than HHQ. LC-MS analysis of P. aeruginosa
wild-type cultures treated with Hod confirmed that PQS levels,
unlike HHQ, were effectively reduced in a concentration-depen-
dent manner. Because rhamnolipids are regulated via PQS (Dig-
gle et al., 2003), the Hod-mediated reduction in PQS will affect
PQS signaling directly and indirectly since lower rhamnolipid
levels will in turn reduce the bioactivity of PQS and hence its
activity as a QS signal. Thus, the quorum quenching of specific
virulence factors observed in laboratory culture could be
extended to a plant infection model where Hod reduced both
virulence and bacterial growth in leaf tissues.
Despite its relatively weak PQS-inactivating activity and sus-
ceptibility to inactivation by P. aeruginosa exoproducts, Hod
was capable of quorum quenching in vitro and in vivo. This
finding is all the more interesting considering that PQS can be
packaged into membrane vesicles that arise through the pinch-
ing off of the outer membrane and fusion with the envelope of
other bacterial cells, serving as a mechanism for trafficking
PQS within a P. aeruginosa population (Mashburn and Whiteley,
2005; Mashburn-Warren et al., 2008).
Recently, Hod has been crystallized (Steiner et al., 2007).
Although PQS is much less susceptible to cleavage by Hod
than MPQS, further insights into the three-dimensional structure
of the enzyme active site may reveal opportunities for modifica-
tion to accommodate the C7 side chain of PQS and improve
catalytic efficiency. Similarly, it may also be possible to engineer
Hod to reduce its susceptibility to P. aeruginosa exoproteases or
to encapsulate the protein such that it is delivered in a protected
environment permitting PQS substrate but not exoprotease
Pseudomonas aeruginosa produces a variety of 2-alkyl-
4(1H)-quinolones (AQ)thatwere originally discovered during
the search for novel secondary metabolites with therapeutic
potential. AQs have antibacterial, iron chelating, immune
modulatory, and signaling properties. PQS and its pre-
cursor, HHQ, both function as QS signal molecules and are
components of a sophisticated gene regulatory network
coupling cell-to-cell communication to population density
Mutation of key AQ biosynthesis or signal transduction
genes results in the attenuation of P. aeruginosa virulence
in animal and plant experimental infection models. Conse-
quently there is considerable interest in the development
of novel selective agents that prevent infection by targeting
bacterial virulence rather than growth since these are less
capable of inactivating PQS-dependent QS we discovered
that 3-hydroxy-2-methyl-4(1H)-quinolone 2,4-dioxygenase
(Hod) from Arthrobacter nitroguajacolicus strain Ru ¨61a is
capable of catalyzing the conversion of PQS to N-octanoy-
lanthranilic acid and carbon monoxide, albeit with substan-
tially less catalytic efficiency than its natural substrate
MPQS. Despite the susceptibility of Hod to pseudomonas
exoproteases and to competitive inhibition by HHQ as well
as the sequestration of PQS by rhamnolipids in bacterial
culture, Hod was capable of significantly reducing the
expression of key P. aeruginosa virulence factors and
reducing growth and tissue damage in a plant leaf infec-
tion model. These data highlight the potential of quenching
AQ-dependent QS and hence virulence through the enzy-
matic degradation of extracellular AQ signaling molecules.
Besides the ability to cleave and thus inactivate PQS, Hod
is also interesting as a carbon monoxide-forming enzyme.
Since intracellular carbon monoxide is a potent inhibitor of
the respiratory chain and also affects gene expression in
P. aeruginosa, CO release from 3-hydroxy-4(1H)-alkylquino-
lones by intracellular Hod might significantly affect bacterial
growth and metabolism.
Bacterial Strains, Plasmids, and Culture Conditions
The strains used in this study are listed in Table S1. The P. aeruginosa PAO1
rhlR and pqsH mutants were constructed by allelic exchange as described
before (Fletcher et al., 2007). Conjugal transfer was performed as described
by Kaniga et al. (1991). E. coli M15 (pREP4, pQE30-hodC69S) was grown in
LB (Sambrook et al., 1989) in the presence of ampicillin (100 mg ml?1) and
kanamycin (25 mg ml?1) at 37?C. At an optical density (OD600 nm) of 0.5, gene
expression was induced by addition of 0.5 mM isopropyl-b-D-thiogalactopyr-
anoside, and the cultivation temperature was decreased to 20?C. Cells were
harvested by centrifugation at an OD600nmof ?3.2. Unless otherwise stated,
P. aeruginosa strains were grown with shaking in LB at 37?C.
Synthesis of AQs and N-octanoylanthranilic Acid
PQS,HHQ,and related compoundswere synthesizedasdescribedbefore(Dig-
Chemistry & Biology
Quinolone Quorum Quenching
1264 Chemistry & Biology 16, 1259–1267, December 24, 2009 ª2009 Elsevier Ltd All rights reserved
thranilic acid, mp 93-94?C, was prepared as a creamy solid in 82% yield by
according to Wells et al. (1952).
Purification of Recombinant Hod
Since Hod(GenBank:CAL09864)tends toform dimers due to oxidative forma-
tion of an intermolecular disulfide bridge (Frerichs-Deeken et al., 2004), we
used protein with a substitution of cysteine-69 by serine in this study. Purifica-
tion of the Hod protein, carrying an N-terminal hexahistidine tag besides the
C69S substitution, from E. coli M15 (pREP4,pQE30-hodC69S) was performed
as described in Beermann et al. (2007). The purity of the protein preparations
was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) and Coomassie staining. Concentrations of Hod were deter-
mined by absorption measurements using an extinction coefficient (3280nm)
of 1.937 ml mg?1cm?1(Beermann et al., 2007).
Enzyme Assays and Kinetics
The catalytic activity of Hod toward MPQS was determined spectrophotomet-
rically in50 mMTris/HCl (pH 8), or in10mM disodium phosphate/boratebuffer
(pH 8) as described previously (Frerichs-Deeken et al., 2004). All assays were
2-heptyl-, and 2-nonyl-3-hydroxy-4(1H)-quinolone was determined spectro-
photometrically, using molar extinction coefficients [in 50 mM Tris/HCl buffer
(pH 8)] of 3335nm= 9.72 3 103M?1cm?1, 3337nm= 9.55 3 103M?1cm?1,
3382nm= 5.86 3 103M?1cm?1, 3377nm= 10.89 3 103M?1cm?1, and 3370nm=
10.88 3 103M?1cm?1, respectively. Enzyme-catalyzed conversion of
2-undecyl-3-hydroxy-4(1H)-quinolone was tested by recording UV/Vis-
spectra of the compound, incubated in buffer with 5 mM Hod for up to 48 hr.
Stock solutions of AQs (10 mM) were prepared in methanol. In the standard
assays, final concentrations of MPQS and PQS were 100 mM and 45 mM,
respectively. Oneunitof enzymeactivitywasdefined astheamountof enzyme
required to consume 1 mmol of substrate per minute at 30?C under the condi-
tions described. To determine the apparent kinetic constants of Hod for PQS,
final PQS concentrations of 3.5–45 mM were used in the assays and apparent
measurements were performed, using different preparations of Hod, and each
assay within a series was done at least in triplicate. Standard deviations for all
apparent Kmand kcatvalues were below ±17% of the average value within
a series of experiments (i.e., for individual protein preparations), but up to
±30% (Km app) and up to ±43% (kcat app) of the value among independent
experiments, i.e., among different protein preparations.
Hod-catalyzed conversion of HHQ was assessed by recording UV/Vis
spectra (200–400 nm) for up to 16 hr of an assay mixture that contained
5 mM of enzyme. To examine whether HHQ and HQNO act as inhibitors of
Hod, its activity toward PQS (10–50 mM) was determined in the presence of
20, 35, 40, and 50 mM HHQ and 50 mM HQNO. Two independent series of
experiments were performed, and each assay was performed in triplicate.
The activity of Hod in different culture media was also estimated in spectro-
photometric assays, after determining the corresponding molar absorption
coefficients, 3334nm, of MPQS.
To determine the effect of P. aeruginosa rhamnolipids on the activity of Hod
toward PQS, 25 mM PQS was equilibrated for 20 hr (at 37?C and 550 rpm) in
50 mM Tris/HCl buffer (pH 7.4) with different concentrations of rhamnolipids
(5–300 mg ml?1). After addition of Hod (40 mg ml?1), PQS cleavage was moni-
of PQS was determined for all PQS-rhamnolipid mixtures tested.
Identification of Products Formed from PQS
The organic product of Hod-catalyzed cleavage of PQS was purified by
combined anion exchange/reversed-phase chromatography and analyzed by
mass spectrometry. For details, see Supplemental Experimental Procedures.
To assess whether the catalytic activity of Hod possibly involves 2,3-dioxy-
genolytic cleavage of PQS, resulting in formation of an a-oxo acid, the product
from PQS conversion was reacted with 2,4-dinitrophenylhydrazine (Friede-
mann and Haugen, 1943). 39 mg of PQS (0.23 mM) was incubated for 8 hr
with 1 mg of Hod in assay buffer. Subsequent to addition of 2,4-dinitrophenyl-
hydrazine (dissolved in 2 M HCl) and incubation for 5 min, the reaction mixture
was adjusted to alkaline pH with NaOH, and 2,4-dinitrophenylhydrazones
were detected spectrophotometrically at 440 nm. The sensitivity of the assay
was determined using pyruvic acid as a reference oxo acid.
Bioluminescence Reporter Gene Assays
integrated into P. aeruginosa was determined using a combined, automated
luminometer-spectrometer (Genios Pro; TECAN Ltd). Overnight cultures of
P. aeruginosa were diluted 1:1000 in fresh LB medium, and 0.2 ml cultures
were grown in microtiter plates. Where required, AQs and/or Hod were added
at the concentrations indicated. Luminescence and turbidity were automati-
cally determined every 30 min. Luminescence is given in relative light units
Pyocyanin was extracted with chloroform from the culture supernatants of
strains grown in the presence or absence of Hod in 96 well microtiter plate
format and quantified spectrophotometrically at 520 nm (Essar et al., 1990)
using a NanoDrop spectrophotometer (Thermo Scientific). Lectin A (PA-1L)
protein levels were determined by western blot analysis of whole cell lysates
as described by Winzer et al. (2000). Rhamnolipid levels in the P. aeruginosa
wild-type with and without Hod and in the pqsA mutant were quantified indi-
rectly using the orcinol method as described in Wilhelm et al. (2007) using
a P. aeruginosa PAO1 rhlR mutant as a rhamnolipid negative control.
LC-MS Quantification of PQS and HHQ
AQs were extracted from Pseudomonas aeruginosa PAO1 culture superna-
tants grown in the presence or absence of Hod in 96 well microtiter plate
format. 150 ml of supernatant was shaken with 300 ml of ethyl acetate acidified
with acetic acid (0.1%) and centrifuged. 150 ml of the organic phase was trans-
ferred to a fresh eppendorf tube. The extraction step was repeated three times
and after drying the organic fraction (total volume 450 ml), 50 ml methanol was
used to solubilize the compounds for LC-MS analysis.
Lettuce Leaf Infection Model
Ten microliter aliquots of a P. aeruginosa culture resuspended to OD600nm0.1
in 10 mM MgSO4with or without Hod (25 U) were injected into the midribs of
fresh romaine lettuce leaves incubated for 2–5 d as described by Starkey and
Rahme (2009) and monitored for the appearance of soft-rot symptoms. In
addition, the numbers of bacterial cells (cfu) mg?1mid-rib were determined
after a defined incubation period.
Supplemental Data include three figures, one table, and Supplemental Exper-
The work undertaken in Nottingham was supported by grants from the
Biotechnology and Biological Sciences Research Council, UK, and the EU
acknowledged. S.F. gratefully acknowledges financial support by the German
Research Foundation (Deutsche Forschungsgemeinschaft; grant FE 383/
16-1). We thank H. Luftmann (Institute of Organic Chemistry, University of
Muenster) for mass spectrometrical analyses and interpretation of MS data,
H. Niewerth (University of Muenster) for performing HPLC analyses, A. Kap-
pius (University of Muenster) for expert technical assistance, M. Fletcher
(University of Nottingham) for construction of the PAO1 pqsH mutant, and
S. Bukavaz (Heinrich-Heine-University) for the purified rhamnolipids.
Received: August 25, 2009
Revised: October 29, 2009
Accepted: November 2, 2009
Published: December 23, 2009
Chemistry & Biology
Quinolone Quorum Quenching
Chemistry & Biology 16, 1259–1267, December 24, 2009 ª2009 Elsevier Ltd All rights reserved 1265
Allesen-Holm, M., Barken, K.B., Yang, L., Klausen, M., Webb, J.S., Kjelleberg,
S., Molin, S., Givskov, M., and Tolker-Nielsen, T. (2006). A characterization of
DNA release in Pseudomonas aeruginosa cultures and biofilms. Mol. Micro-
biol. 59, 1114–1128.
Ball, G., Durand, E., Lazdunski, A., and Filloux, A. (2002). A novel type II secre-
tion system in Pseudomonas aeruginosa. Mol. Microbiol. 43, 475–485.
and Hinz, H.-J. (2007). Stability, unfolding, and structural changes of cofactor-
free 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase. Biochemistry 46, 4241–
Boehm, K., Guddorf, J., Albers, A., Kamiyama, T., Fetzner, S., and Hinz, H.-J.
(2008). Thermodynamic analysisof denaturant-induced unfolding of HodC69S
protein supports a three-state mechanism. Biochemistry 47, 7116–7126.
Bredenbruch, F., Nimtz, M., Wray, V., Morr, M., Mu ¨ller, R., and Ha ¨ussler, S.
(2005). Biosynthetic pathway of Pseudomonas aeruginosa 4-hydroxy-2-alkyl-
quinolines. J. Bacteriol. 187, 3630–3635.
Bredenbruch, F., Geffers, R., Nimtz, M., Buer, J., and Ha ¨ussler, S. (2006). The
Pseudomonas aeruginosa quinolone signal (PQS) has an iron chelating
activity. Environ. Microbiol. 8, 1318–1329.
Calfee, M.W., Shelton, J.G., McCubrey, J.A., and Pesci, E.C. (2005). Solubility
and bioactivity of the Pseudomonas quinolone signal are increased by a Pseu-
domonas aeruginosa-produced surfactant. Infect. Immun. 73, 878–882.
Cao, H., Krishnan, G., Goumnerov, B., Tsongalis, J., Tompkins, R., and
Rahme, L.G. (2001). A quorum sensing-associated virulence gene of Pseudo-
monas aeruginosa encodes a LysR-like transcription regulator with a unique
self-regulatory mechanism. Proc. Natl. Acad. Sci. USA 98, 14613–14618.
Coleman, J.P., Hudson, L.L., McKnight, S.L., Farrow, J.M., 3rd, Calfee, M.W.,
Lindsey, C.A., and Pesci, E.C. (2008). Pseudomonas aeruginosa PqsA is an
anthranilate-coenzyme A ligase. J. Bacteriol. 190, 1247–1255.
Collier, D.N., Anderson, L., McKnight, S.L., Noah, T.L., Knowles, M., Boucher,
R.,Schwab, U.,Gilligan, P.,andPesci,E.C.(2002).Abacterialcelltocellsignal
in the lungs of cystic fibrosis patients. FEMS Microbiol. Lett. 215, 41–46.
Cornelis, P., Anjaiah, V., Koedam, N., Delfosse, P., Jacques, P., Thonart, P.,
and Neirinckx, L. (1992). Stability, frequency and multiplicity of transposon
insertionsinthepyoverdine region inthechromosomesofdifferent fluorescent
pseudomonads. J. Gen. Microbiol. 138, 1337–1343.
Cornforth, J.W., and James, A.T. (1956). Structure of a naturally occurring
antagonist of dihydrostreptomycin. Biochem. J. 63, 124–130.
Davidge, K.S., Sanguinetti, G., Yee, C.H., Cox, A.G., McLeod, C.W., Monk,
C.E., Mann, B.E., Motterlini, R., and Poole, R.K. (2009). Carbon monoxide-
releasing antibacterial molecules target respiration and global transcriptional
regulators. J. Biol. Chem. 284, 4516–4524.
Desmard, M., Davidge, K.S., Bouvet, O., Morin, D., Roux, D., Foresti, R.,
Ricard, J.D., Denamur, E., Poole, R.K., Montravers, P., et al. (2009). A carbon
monoxide-releasing molecule (CORM-3) exerts bactericidal activity against
Pseudomonas aeruginosa and improves survival in an animal model of bacter-
aemia. FASEB J. 23, 1023–1031.
De ´ziel, E., Le ´pine, F., Milot, S., He, J., Mindrinos, M.N., Tompkins, R.G., and
Rahme, L.G. (2004). Analysis of Pseudomonas aeruginosa 4-hydroxy-2-alkyl-
quinolines (HAQs) reveals a role for 4-hydroxy-2-heptylquinoline in cell-to-cell
communication. Proc. Natl. Acad. Sci. USA 101, 1339–1344.
De ´ziel, E., Gopalan, S., Tampakaki, A.P., Le ´pine, F., Padfield, K.E., Saucier,
M., Xiao, G., and Rahme, L.G. (2005). The contribution of MvfR to Pseudo-
monas aeruginosa pathogenesis and quorum sensing circuitry regulation:
multiple quorum sensing-regulated genes are modulated without affecting
lasRI, rhlRI or the production of N-acyl-L-homoserine lactones. Mol. Microbiol.
Diggle, S.P., Winzer, K., Chhabra, S.R., Worrall, K.E., Ca ´mara, M., and Wil-
liams, P. (2003). The Pseudomonas aeruginosa quinolone signal molecule
overcomes the cell density-dependency of the quorum sensing hierarchy,
regulates rhl-dependent genes at the onset of stationary phase and can be
produced in the absence of LasR. Mol. Microbiol. 50, 29–43.
Diggle, S.P., Lumjiaktase, P., Dipilato, F., Winzer, K., Kunakorn, M., Barrett,
D.A., Chhabra, S.R., Ca ´mara, M., and Williams, P. (2006). Functional genetic
Burkholderia pseudomallei and related bacteria. Chem. Biol. 13, 701–710.
Diggle, S.P., Matthijs, S., Wright, V.J., Fletcher, M.P., Chhabra, S.R., Lamont,
I.L., Kong, X., Hider, R.C., Cornelis, P., Ca ´mara, M., and Williams, P. (2007).
The Pseudomonas aeruginosa 4-quinolone signal molecules HHQ and PQS
play multifunctional roles in quorum sensing and iron entrapment. Chem.
Biol. 14, 87–96.
Dong, Y.-H., Wang, L.-H., and Zhang, L.-H. (2007). Quorum-quenching micro-
bial infections: mechanisms and implications. Philos. Trans. R. Soc. Lond.
B Biol. Sci. 362, 1201–1211.
Eiden, F., Wendt, R., and Fenner, H. (1978). Chinolyliden-Derivate. Archiv der
Pharmazie (Weinheim, Germany) 311, 561–568.
Essar, D.W., Eberly, L., Hadero, A., and Crawford, I.P. (1990). Identification
and characterization of genes for a second anthranilate synthase in Pseudo-
monas aeruginosa: interchangeability of the two anthranilate synthases and
evolutionary implications. J. Bacteriol. 172, 884–900.
Farrow, J.M., 3rd, and Pesci, E.C. (2007). Two distinct pathways supply
anthranilate as a precursor of the Pseudomonas quinolone signal. J. Bacteriol.
Farrow, J.M., 3rd, Sund, Z.M., Ellison, M.L., Wade, D.S., Coleman, J.P., and
Pesci, E.C. (2008). PqsE functions independently of PqsR-Pseudomonas qui-
nolone signal and enhances the rhl quorum-sensing system. J. Bacteriol. 190,
Fetzner, S. (2002). Oxygenases without requirement for cofactors or metal
ions. Appl. Microbiol. Biotechnol. 60, 243–257.
Fischer, F.,Ku ¨nne,S.,and Fetzner, S.(1999). Bacterial2,4-dioxygenases: new
membersofthea/bhydrolase-foldsuperfamilyofenzymes functionally related
to serine hydrolases. J. Bacteriol. 181, 5725–5733.
Fletcher, M.P., Diggle, S.P., Crusz, S., Chhabra, S.R., Ca ´mara, M., and
Williams, P. (2007). A dual biosensor for 2-alkyl-4-quinolone quorum-sensing
signal molecules. Environ. Microbiol. 9, 2683–2693.
Friedemann, T.E., and Haugen, G.E. (1943). Pyruvic acid. II. The determination
of keto acids in blood and urine. J. Biol. Chem. 147, 415–442.
Frerichs-Deeken, U., Ranguelova, K., Kappl, R., Hu ¨ttermann, J., and Fetzner,
S. (2004). Dioxygenases without requirement for cofactors, and their chemical
model reaction: compulsory order ternary complex mechanism of 1H-3-
hydroxy-4-oxoquinaldine 2,4-dioxygenase involving general base catalysis
by histidine 251 and single-electron oxidation of the substrate dianion.
Biochemistry 43, 14485–14499.
Gallagher, L.A., McKnight, S.L., Kuznetsova, M.S., Pesci, E.C., and Manoil, C.
(2002). Functions required for extracellular quinolone signaling by Pseudo-
monas aeruginosa. J. Bacteriol. 184, 6472–6480.
Guina, T., Purvine, S.O., Yi, E.C., Eng, J., Goodlett, D.R., Aebersold, R., and
Miller, S.I. (2003). Quantitative proteomic analysis indicates increased
synthesis of a quinolone by Pseudomonas aeruginosa isolates from cystic
fibrosis airways. Proc. Natl. Acad. Sci. USA 100, 2771–2776.
Ha ¨ussler, S., and Becker, T. (2008). The pseudomonas quinolone signal (PQS)
balances life and death in Pseudomonas aeruginosa populations. PLoS
Pathog. 4, e1000166.
Jensen, P.O., Bjarnsholt, T.,Phipps,R., Rasmussen, T.B., Calum,H., Christof-
fersen, L., Moser, C., Williams, P., Pressler, T., Givskov, M., and Hoiby, N.
(2007). Rapid necrotic killing of polymorphonuclear leukocytes is caused by
quorum-sensing-controlled production of rhamnolipid by Pseudomonas aeru-
ginosa. Microbiology 153, 1329–1338.
Kaniga, K., Delor, I., and Cornelis, R.G. (1991). A wide host range suicide
vector for improving reverse genetics in Gram-negative bacteria: inactivation
of the blaA gene of Yersinia enterocolitica. Gene 109, 137–141.
Kaufmann, G.F., Park, J., and Janda, K.D. (2008). Bacterial quorum sensing:
a new target for anti-infective immunotherapy. Expert Opin. Biol. Ther. 8,
Klendshoj, N.C., Feldstein, M., and Sprague, A.L. (1950). The spectrophoto-
metric determination of carbon monoxide. J. Biol. Chem. 183, 297–303.
Chemistry & Biology
Quinolone Quorum Quenching
1266 Chemistry & Biology 16, 1259–1267, December 24, 2009 ª2009 Elsevier Ltd All rights reserved
Le ´pine, F., Milot, S., De ´ziel, E., He, J., and Rahme, L.G. (2004). Electrospray/ Download full-text
mass spectrometric identification and analysis of 4-hydroxy-2-alkylquinolines
(HAQs) produced by Pseudomonas aeruginosa. J. Am. Soc. Mass Spectrom.
Lesic,B.,Le ´pine,F.,De ´ziel,E.,Zhang,J.,Zhang, Q.,Padfield,K., Castonguay,
M.H., Milot, S., Stachel, S., Tzika, A.A., et al. (2007). Inhibitors of pathogen
intercellular signals as selective anti-infective compounds. PLoS Pathog. 3,
Lin, Y.H., Xu, J.L., Hu, J., Wang, L.H., Ong, S.L., Leadbetter, J.R., and Zhang,
L.H. (2003). Acyl-homoserine lactone acylase from Ralstonia strain XJ12B
represents a novel and potent class of quorum-quenching enzymes. Mol.
Microbiol. 47, 849–860.
Lyczak, J.B., Cannon, C.L., and Pier, G.B. (2002). Lung infections associated
with cystic fibrosis. Clin. Microbiol. Rev. 15, 194–222.
Machan, Z.A., Taylor, G.W., Pitt, T.L., Cole, P.J., and Wilson, R. (1992). 2-Hep-
tyl-4-hydroxyquinoline N-oxide, an antistaphylococcal agent produced by
Pseudomonas aeruginosa. J. Antimicrob. Chemother. 30, 615–623.
Mashburn, L.M., and Whiteley, M. (2005). Membrane vesicles traffic signals
and facilitate group activities in a prokaryote. Nature 437, 422–425.
Mashburn-Warren,L.,Howe,J.,Garidel, P.,Richter, W.,Steiniger, F.,Roessle,
M., Brandenburg, K., and Whiteley, M. (2008). Interaction of quorum signals
tion. Mol. Microbiol. 69, 491–502.
Overhage, J., Sielker, S., Homburg, S., Parschat, K., and Fetzner, S. (2005).
Identification of large linear plasmids in Arthrobacter spp. encoding the degra-
dation of quinaldine to anthranilate. Microbiology 151, 491–500.
Parschat, K., Overhage, J., Strittmatter, A.W., Henne, A., Gottschalk, G., and
Fetzner, S. (2007). Complete nucleotide sequence of the 113-kilobase linear
catabolic plasmid pAL1 of Arthrobacter nitroguajacolicus Ru ¨61a and tran-
scriptional analysis of genes involved in quinaldine degradation. J. Bacteriol.
Pesci, E.C., Milbank, J.B., Pearson, J.P., McKnight, S., Kende, A.S., Green-
berg, E.P., and Iglewski, B.H. (1999). Quinolone signaling in the cell-to-cell
communication system of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci.
USA 96, 11229–11234.
Reimmann, C., Ginet, N., Michel, L., Keel, C., Michaux, P., Krishnapillai, V.,
Zala, M., Heurlier, K., Triandafillu, K., Harms, H., et al. (2002). Genetically pro-
grammed autoinducer destruction reduces virulence gene expression and
swarming motility in Pseudomonas aeruginosa PAO1. Microbiology 148,
Ritter, C., and Luckner, M. (1971). Zur Biosynthese der 2-n-Alkyl-4-hydroxy-
chinolinderivate (Pseudane) bei Pseudomonas aeruginosa. Eur. J. Biochem.
Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning:
A Laboratory Manual, Second Edition (Cold Spring Harbor, NY: Cold Spring
Harbor Laboratory Press).
Ca ´mara, M., Williams, P., and Quax, W.J. (2006). Quorum quenching by an
N-acyl-homoserine lactone acylase from Pseudomonas aeruginosa PAO1.
Infect. Immun. 74, 1673–1683.
Starkey, M., and Rahme, L.G. (2009). Modeling Pseudomonas aeruginosa
pathogenesis in plant hosts. Nat. Protoc. 4, 117–124.
Steiner, R.A., Frerichs-Deeken, U., and Fetzner, S. (2007). Crystallization and
preliminary X-ray analysis of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase
from Arthrobacter nitroguajacolicus Ru ¨61a: a cofactor-devoid dioxygenase of
the a/b-hydrolase-fold superfamily. Acta Crystallogr. Sect. F Struct. Biol.
Cryst. Commun. 63, 382–385.
Venturi, V. (2006). Regulation of quorum sensing in Pseudomonas. FEMS
Microbiol. Rev. 30, 274–291.
Wade, D.S., Calfee, M.W., Rocha, E.R., Ling, E.A., Engstrom, E., Coleman,
J.P., and Pesci, E.C. (2005). Regulation of Pseudomonas quinolone signal
synthesis in Pseudomonas aeruginosa. J. Bacteriol. 187, 4372–4380.
Wells, I.C., Elliott, W.H., Thayer, S.A., and Doisy, E.A. (1952). Ozonization of
some antibiotic substances produced by Pseudomonas aeruginosa. J. Biol.
Chem. 196, 321–330.
Wilhelm, S., Gdynia, A., Tielen, P., Rosenau, F., and Jaeger, K.E. (2007). The
autotransporter esterase EstA of Pseudomonas aeruginosa is required for
rhamnolipid production, motility and biofilm formation. J. Bacteriol. 189,
Williams, P., and Ca ´mara, M. (2009). Quorum sensing and environmental
adaptation in Pseudomonas aeruginosa: a tale of regulatory networks and
multifunctional signal molecules. Curr. Opin. Microbiol. 12, 182–191.
Williams, P., Winzer, K., Chan, W.C., and Ca ´mara, M. (2007). Look who’s talk-
ing: communication and quorum sensing in the bacterial world. Philos. Trans.
R. Soc. Lond. B Biol. Sci. 362, 1119–1134.
Winzer, K., Falconer, C., Garber, N.C., Diggle, S.P., Camara, M., and Williams,
P. (2000). The Pseudomonas aeruginosa lectins PA-IL and PA-IIL are
controlled by quorum sensing and by RpoS. J. Bacteriol. 182, 6401–6411.
Xiao, G., De ´ziel, E., He, J., Le ´pine, F., Lesic, B., Castonguay, M.-H., Milot, S.,
Tampakaki, A.P., Stachel, S.E., and Rahme, L.G. (2006). MvfR, a key Pseudo-
monas aeruginosa pathogenicity LTTR-class regulatory protein, has dual
ligands. Mol. Microbiol. 62, 1689–1699.
Zhang, Y.-M., Frank, M.W., Zhu, K., Mayasundari, A., and Rock, C.O. (2008).
PqsD is responsible for the synthesis of 2,4-dihydroxyquinoline, an extracel-
lular metabolite produced by Pseudomonas aeruginosa. J. Biol. Chem. 283,
Chemistry & Biology
Quinolone Quorum Quenching
Chemistry & Biology 16, 1259–1267, December 24, 2009 ª2009 Elsevier Ltd All rights reserved 1267