Article

Quinolones: from antibiotics to autoinducers. FEMS Microbiol Rev

School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham, UK.
FEMS microbiology reviews (Impact Factor: 13.24). 03/2011; 35(2):247-74. DOI: 10.1111/j.1574-6976.2010.00247.x
Source: PubMed

ABSTRACT

Since quinine was first isolated, animals, plants and microorganisms producing a wide variety of quinolone compounds have been discovered, several of which possess medicinally interesting properties ranging from antiallergenic and anticancer to antimicrobial activities. Over the years, these have served in the development of many synthetic drugs, including the successful fluoroquinolone antibiotics. Pseudomonas aeruginosa and related bacteria produce a number of 2-alkyl-4(1H)-quinolones, some of which exhibit antimicrobial activity. However, quinolones such as the Pseudomonas quinolone signal and 2-heptyl-4-hydroxyquinoline act as quorum-sensing signal molecules, controlling the expression of many virulence genes as a function of cell population density. Here, we review selectively this extensive family of bicyclic compounds, from natural and synthetic antimicrobials to signalling molecules, with a special emphasis on the biology of P. aeruginosa. In particular, we review their nomenclature and biochemistry, their multiple properties as membrane-interacting compounds, inhibitors of the cytochrome bc(1) complex and iron chelators, as well as the regulation of their biosynthesis and their integration into the intricate quorum-sensing regulatory networks governing virulence and secondary metabolite gene expression.

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REVIEW ARTICLE
Quinolones: from antibiotics to autoinducers
Stephan Heeb, Matthew P. Fletcher, Siri Ram Chhabra, Stephen P. Diggle, Paul Williams &
Miguel C
´
amara
School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham, UK
Correspondence: Miguel C
´
amara, School of
Molecular Medical Sciences, Centre for
Biomolecular Sciences, University Park,
University of Nottingham, Nottingham NG7
2RD, UK. Tel.: 144 115 951 5036; fax: 144
115 846 7951; e-mail:
miguel.camara@nottingham.ac.uk
Received 8 June 2010; revised 25 June 2010;
accepted 16 July 2010.
Final version published online 25 August 2010.
DOI:10.1111/j.1574-6976.2010.00247.x
Editor: Dieter Haas
Keywords
quorum sensing; quinolone; quinoline;
Pseudomonas
;
Burkholderia
; virulence.
Abstract
Since quinine was first isolated, animals, plants and microorganisms producing a
wide variety of quinolone compounds have been discovered, several of which
possess medicinally interesting properties ranging from antiallergenic and
anticancer to antimicrobial activities. Over the years, these have served in the
development of many synthetic drugs, including the successful fluoroquinolone
antibiotics. Pseudomonas aerug inosa and related bacteria produce a number of 2-
alkyl-4(1H)-quinolones, some of which exhibit antimicrobial activity. However,
quinolones such as the Pseudomonas quinolone signal and 2-heptyl-4-hydroxyqui-
noline act as quorum-sensing signal molecules, controlling the expression of many
virulence genes as a function of cell population density. Here, we review selectively
this extensive family of bicyclic compounds, from natural and synthetic antimi-
crobials to signalling molecules, with a special emphasis on the biology of
P. aeruginosa. In particular, we review their nomenclature and biochemistry, their
multiple properties as membrane-interacting compounds, inhibitors of the
cytochrome bc
1
complex and iron chelators, as well as the regulation of their
biosynthesis and their integration into the intricate quorum-sensing regulatory
networks governing virulence and secondary metabolite gene expression.
Introduction
Quinolones are molecules structurally derived from the
heterobicyclic aromatic compound quinoline, the name of
which originated from the oily substance obtained after the
alkaline distillation of quinine (Gerhardt, 1842). Since the
isolation of quinine from Cinchona bark in 1811, many
other quinoline derivatives have been isolated from natural
sources (Fig. 1). In particular, 2-hydroxyquinoline and
4-hydroxyquinoline, which predominantly exist as 2(1H)-
quinolone and 4(1H)-quinolone, respectively, and form the
core structure of many alkaloids, were isolated from plant
sources. Several different animal and bacterial species also
produce compounds of the quinolone class. These differ not
only in the varied substitutions in the carbocyclic and
heteroaromatic rings but also have other rings fused to the
quinolone nucleus. These have been reviewed on a yearly
basis by J.P. Michael in Natural Product Reports (Michael,
2008). Some of these naturally occurring quinolones have
profound medicinal properties while others have served as
lead structures and provided inspiration for the design of
synthetic quinolones as useful drugs. For example (Fig. 1),
among 2-quinolones, rebamipide is an antiulcer agent and
repirinast has antihistamine properties useful in the treat-
ment of allergic asthma (Uchida et al., 1987). While screen-
ing compounds for potential cancer chemopreventive
properties, casimiroine, isolated from the seeds of Casimiroa
edulis, was found to have antimutagenic activity (Ito et al.,
1998). Several 4-quinolone alkaloids, mainly isolated from
plant and microbial sources, have antimicrobial activity.
For example, 2-alkyl-4(1H)-quinolones (AQs) (Fig. 1, com-
pounds 1–4) and 1-methyl-2-[(4Z)-tridecenyl]-4(1H)-
quinolone, evocarpine, its structural isomers and unsaturated
homologues (Fig. 1, compounds 5–9) isolated from the
extracts of Evodia rutaecarpa show antibacterial activity
against Helicobacter pylori, which is implicated in the
pathogenesis of chronic gastritis, peptic ulcers and gastric
cancers (Rho et al., 1999; Hamasaki et al., 2000). The
alkaloid 1 shown in Fig. 1 is rather rare as it bears n-decyl,
an even number of carbons in the 2-position. Also, no fewer
than eight further 4-quinolones (Fig. 1, compounds 10–17)
isolated from the fermentation broth of the actinomycete
FEMS Microbiol Rev 35 (2011) 247–274
c
2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
MICROBIOLOGY REVIEWS
Page 1
Pseudonocardia spp. CL38489 are active in inhibiting the
growth of H. pylori. The most potent compound is the
epoxide (Fig. 1, compound 16), which has a potent bacter-
icidal [minimal inhibitory concentration (MIC)
10 ng mL
1
] and an even more pronounced bacteriostatic
effect (MIC 0.1 ng mL
1
) (Dekker et al., 1998). These
quinolones are characterized by the presence of a geranyl or
oxidized geranyl side chain at C-2 in place of the usual fatty
acid-derived alkyl or alkenyl chain normally found in
microbial quinolones. The screening of synthetic analogues
of quinine for novel antiplasmodial drugs led to the seren-
dipitous discovery of a precursor used in the synthesis of
chloroquine, 7-chloroquinoline, which exhibited antimicro-
bial activities in vitro. Further investigation of this and
similar compounds such as the structurally related 1,8-
naphthyridones (which are quinolones with a nitrogen atom
substituting C-8) resulted in the discovery of nalidixic acid
(1-ethyl-7-meth yl-4-o x o-1,8-naphth yridine-3-carboxylic acid),
N
O
HO
N
Quinine
N
H
O
COOH
N
H
Cl
O
Rebamipide
N
H
O
O
O
O
O
Repirinast Casimiroine
N O
O
O
Me
OMe
n = 9
n = 10
n = 12
n = 14
1,
2,
3,
4,
m = 3, n = 4
m = 5, n = 4
8,
9,
10,
11,
12,
13,
14,
15,
Evocarpine, m = 7, n = 3
m = 3, n = 7
m = 6, n = 4
5,
6,
7,
N
O
Me
n
N
O
Me
m
n
N
O
Me
m
n
N
O
R
R
R
N
O
Me
Me
O
16
17
N
O
Me
OH
Flumequine
N
O
COOH
F
Natural and synthetic quinolones of medicinal interest
Natural quinolones having antimicrobial properties
Synthetic quinolone antibiotics
Cinoxacin
O
O
N
N
O
COOH
Et
Oxolinic acid
N
O
COOH
O
O
Et
Nalidixic acid
N
N
O
COOH
Et
Norfloxacin
N
O
COOH
F
N
HN
Et
Ciprofloxacin
N
O
COOH
F
N
HN
Chloroquine
N
N
HN
Cl
Fig. 1. Natural and synthetic quinolones of
medicinal interest, quinolone antibiotics. Several
plant, animal and microbial species produce
quinolone compounds of medicinal interest
such as the antimalarial quinine extracted from
Cinchona spp., or the 2-quinolone casimiroine,
an antimutagen extracted from Casimiroa edulis.
Among the synthetic 2-quinolones are the
antiulcer agent rebamipide and the
antihistamine, repirinast. Naturally occurring
quinolones having antimicrobial activities such as
evocarpine and related compounds (nos 1–17)
produced by Evodia rutaecarpa are active against
Helicobacter pylori, a causative agent of peptic
ulcers and gastric cancer. The quest for synthetic
analogues of quinine led to the discovery of
nalidixic acid, oxolinic acid and cinoxacin, and
then to the development of an extensive family of
fluoroquinolone antibiotics such as flumequine,
norfloxacin and ciprofloxacin. The heteroaro-
matic ring atom numbering common to all
quinolones is indicated for quinine.
FEMS Microbiol Rev 35 (2011) 247–274
c
2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
248 S. Heeb et al.
Page 2
which was to become the first practical synthetic quinolone
antibiotic (Lesher et al., 1962). This rapidly led to the develop-
ment of several other 4-quinolone-based antibiotics such as
oxolinic acid, cinoxacin and flumequine (Fig. 1), used
clinically to treat Gram-negative bacterial infections, and
later on to second-generation drugs such as norfloxacin and
ciprofloxacin (Fig. 1), also effective against some Gram-
positive bacteria. All of the quinolone antibiotics are char-
acterized by the presence of a carboxylic acid function at C-3
(Rohlfing et al., 1976; M
˚
ardh et al., 1977; Barry et al., 1984;
Galante et al., 1985; Aboul-Fadl & Fouad, 1996).
Interest in the antipathogenic properties of common
bacteria started with the pioneering work of Louis Pasteur.
Notably, in 1877, Pasteur reported that the coinoculation of
Bacillus anthracis with other common living bacteria in
animals prevented the development of anthrax, when septi-
caemia could be avoided. This was interpreted, following the
idea that ‘life can prevent life’, as being the result of a
competition for oxygen (Pasteur & Joubert, 1877). After
Emmerich and Pawlowsky were able to prevent the develop-
ment of anthrax in preinfected rabbits and guinea-pigs by
the inoculation of Streptococcus spp., Charles Bouchard
reproduced this effect in 1889 with pure cultures of Bacillus
pyocyaneus (Pseudomonas aeruginosa) (Bouchard, 1889).
Ten years later, in 1899, Rudolf Emmerich and Oscar L
¨
ow
concluded that P. aeruginosa released an active antibacterial
substance into the medium after cell-free preparations from
this organism were found to be sufficient to prevent the
development of anthrax, and as it was thought to be the
result of an enzymatic process, they called it pyocyanase
(Emmerich & L
¨
ow, 1899). In 1945, this preparation was
determined to consist of a mixture of heat-stable com-
pounds that were separated, partially characterized and
named the Pyo compounds (Hays et al., 1945). Pyo I–IV,
which later were found to be AQs (Fig. 2), presented strong
antibacterial activities against Gram-positive organisms,
although much less against Gram-negative bacteria, with
Pyo II being 10 times more potent compared with the
others. However, Pyo II was toxic and ineffective at protect-
ing mice at subtoxic doses against Streptococcus pneumoniae
or Mycobacterium tuberculosis infections (Wells et al., 1947).
Natural antimicrobial q uinol ones
In addition to having antimicrobial activity in vitro, the Pyo
compounds produced by P. aeruginosa were found to
antagonize, under certain conditions, the action of strepto-
mycin and dihydrostreptomycin against Gram-positive bac-
teria (Lightbown, 1950, 1954). This inhibitory activity was
rapidly attributed to Pyo II, which is a mixture of 2-alkyl-4-
hydroxyquinoline N-oxides (AQNOs) (Cornforth & James,
1954, 1956; Lightbown & Jackson, 1954). The inhibitory
activity of these N-oxides (at concentration ratios with
respect to streptomycin of the order of 1 : 100) was found
to correlate with the potent inhibition of electron transport
in both heart-muscle and bacterial cells through the cyto-
chrome bc
1
segment (ubiquinol:cytochrome c oxidoreduc-
tase) of the respiratory chain (Lightbown & Jackson, 1954,
1956). This is in line with the need for respiration and the
transmembrane potential required for bacteria to take up
aminoglycoside antibiotics (Hancock, 1962; Damper &
Epstein, 1981; Arrow & Taber, 1986; Taber et al., 1987).
2-Heptyl-4-hydroxyquinoline N-oxide (HQNO) acts as a
ubiquinone and menaquinone analogue on quinone-
reactive cytochrome b enzymes in various organisms (Van
Ark & Berden, 1977; Smirnova et al., 1995; Rothery &
Weiner, 1996). The antimicrobial effect of the N-oxides
appears to be limited to Gram-positive bacteria and offers
an explanation as to how P. aeruginosa becomes the
dominant species over Staphylococcus aureus in cystic
fibrosis (CF) lung infections (Machan et al., 1992), although
additional factors such as pyocyanin, cyanide and
N-(3-oxododecanoyl)-
L-homoserine lactone have been
shown to play a similar role (Qazi et al., 2006; Voggu et al.,
2006). Furthermore, long-term exposure of S. aureus to
physiological concentrations of HQNO selects for amino-
glycoside-resistant, small-colony variants that are typically
found in chronic lung infections (Hoffman et al., 2006;
Biswas et al., 2009). Interestingly, the formation of S. aureus
small-colony variants is the first step towards the develop-
ment of dual target resistance against fluoroquinolones (Pan
et al., 2002). The low in vivo efficacy, combined with the
strong toxicity on mitochondrial respiration, prevented the
development of AQNOs as therapeutic antibiotics. How-
ever, due to its interference with quinone-dependent cyto-
chromes, HQNO became an invaluable reagent for the study
of electron transport chains.
A number of quinolones with interesting antimicrobial
properties are also produced by various pseudomonads and
other microorganisms. For example, under iron limitation,
Pseudomonas fluorescens ATCC 17400 produces quinolobac-
tin (8-hydroxy-4-methoxyquinaldic acid, Fig. 3), which acts
as a siderophore (Mossialos et al., 2000). Quinolobactin
results from the rapid hydrolysis of the precursor
molecule 8-hydroxy-4-methoxy-2-quinolinethiocarboxylic
acid (thioquinolobactin), which, as opposed to quinolobac-
tin, has strong antifungal activity against the plant pathogen
Pythium debaryanum (Matthijs et al., 2007). Thioquinolo-
bactin is synthesized via a unique pathway from
L-trypto-
phan via xanthurenic acid and sulphurylation by QbsE, a
small sulphur carrier protein (Matthijs et al., 2004; Godert
et al., 2007). Pseudomonas fluorescens G308, a potential
biocontrol strain, produces N-mercapto-4-formylcarbostyril
[Cbs, 4-formyl-1-sulphanyl-2(1H)-quinolone, Fig. 3], a
quinolone that contains an unusual N-mercaptoamide
functional group and that has strong antifungal properties
FEMS Microbiol Rev 35 (2011) 247–274
c
2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
249Quinolones: antibiotics and autoinducers
Page 3
N
H
O
R
N
OH
R
R= n-C H ; 2-pentyl-4(1H )-quinolone
R= n-C
H ; 2-heptyl-4(1H )-quinolone
R= n-C
H ;2-nonyl-4(1H )-quinolone
R= n-C
H ;2-undecyl-4(1H )-quinolone
R= n-C
H ; 2-pentyl-4-hydroxyquinoline (PHQ)
2-pentyl-4-quinolinol (IUPAC)
R= n-C
H ; 2-heptyl-4-hydroxyquinoline (HHQ)
2-heptyl-4-quinolinol (IUPAC)
R= n-C
H ; 2-nonyl-4-hydroxyquinoline (NHQ)
2-nonyl-4-quinolinol (IUPAC)
R= n-C
H ; 2-undecyl-4-hydroxyquinoline (UHQ)
2-undecyl-4-quinolinol (IUPAC)
AQs
N
OH
R
OH
N
H
O
R
OH
R= CH ; 3-hydroxy-2-methyl-4(1H)-quinolone (C1-PQS)
R= n-C
H ; 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS)
R= n-C
H ; 3-hydroxy-2-nonyl-4(1H )-quinolone (C9-PQS)
R= CH
; 2-methyl-3,4-dihydroxyquinoline
R= n-C
H ; 2-heptyl-3,4-dihydroxyquinoline
R= n-C
H ; 2-nonyl-3,4-dihydroxyquinoline
2-methyl-3,4-quinolinediol (IUPAC)
2-heptyl-3,4-quinolinediol (IUPAC)
2-nonyl-3,4-quinolinediol (IUPAC)
PQS and its analogues
N
OH
R
O
2-nonyl-1-hydroxy-4(1H)-quinolone
2-undecyl-1-hydroxy-4(1H )-quinolone
R= n-C
H ;
R= n-C
H ;
2-heptyl-1-hydroxy-4(1H )-quinolone
R= n-C
H ;
R= n-C
H ;
2-heptyl-4-hydroxyquinoline N-oxide (HQNO)
4-hydroxy-2-nonylquinoline N-oxide (IUPAC)
R= n-C
H ; 2-nonyl-4-hydroxyquinoline N-oxide (NQNO)
R= n-C
H ; 2-undecyl-4-hydroxyquinoline N-oxide (UQNO)
4-hydroxy-2-undecylquinoline N-oxide (IUPAC)
AQNOs
NR
O
OH
N
OH
OH
2-Hydroxy-4(1H)-quinolone 4-Hydroxy-2(1H)-quinolone
2
2,4-Quinolinediol
(
IUPAC
)
,4-Dihydroxyquinoline (DHQ)
N
H
OH
O
DHQ
H
N
OH
O
Fig. 2. Structure, IUPAC names and abbreviations of AQ molecules synthesized by Pseudomonas aeruginosa and a synthetic analogue. Both the
tautomeric lactam and the phenolic forms of each molecule are shown. Arrows indicate the equilibrium of these molecules as would exist under
physiological conditions. Where more than one name exists for a molecule, the IUPAC designation is indicated, although this may not be the
nomenclature used most frequently. The compound C1-PQS is a synthetic analogue that is not produced by P. aeruginosa.
FEMS Microbiol Rev 35 (2011) 247–274
c
2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
250 S. Heeb et al.
Page 4
against plant pathogens such as Fusarium spp., Cladosporium
cucumerinum and Colletotrichum lagenarium (Fakhouri
et al., 2001). Although the biosynthetic pathway for Cbs
formation has not yet been elucidated, it was suggested that
this compound may be derived from AQs produced via a
similar biochemical pathway as that in P. aeruginosa. The
marine bacteria Pseudomonas bromoutilis (Wratten et al.,
1977) and Alteromonas strain SWAT5 (Long et al., 2003)
both synthesize 2-pentyl-4-quinolone (PHQ, also called 2-n-
pentyl-4-quinolinol) and 2-heptyl-4-hydroxyquinoline
(HHQ, also called 2-n-heptyl-4-quinolinol), which were
identified as a consequence of their antibacterial activities.
PHQ inhibits the growth of cyanobacteria (Synechococcus),
algae (Chaetoceros simplex, Cylindotheca fusiformis and Tha-
lassiosira weissflogii) and impacts on particle-associated
marine bacterial communities (Long et al., 2003). HHQ and
PHQ have antibacterial activity against Vibrio anguillarum,
S. aureus, Candida albicans and Vibrio harveyi (Wratten
et al., 1977). From sponge-associated marine pseudomonads,
several other AQs with various substitutions have been
identified and appear to have antibacterial, antiplasmodial,
antiviral or cytotoxic properties (Debitus et al., 1998; Bultel-
Ponc
´
e et al., 1999). The obligate aerobic yeast Yarrowia
lipolytica produces 1-hydroxy-2-dodecyl-4(1H)-quinolone,
a potent inhibitor of the alternative NADH:ubiquinone
oxidoreductases, which acts as a ubiquinone analogue (Es-
chemann et al., 2005), an activity reminiscent of the quino-
lone N-oxides produced by P. aeruginosa, which act on the
cytochrome bc
1
complex. In addition, a large number of
additional quinoline alkaloids produced by a variety of other
microorganisms, plants and animals have been discovered
every year (annually reviewed by J.P. Michael), of which the
majority still have to be studied with respect to their
biological properties. However, as all the natural quinolones
described so far lack the 3-carboxy group, which is essential
for the binding and blocking of DNA-type IIA topoisomerase
complexes, the antibacterial mechanism of action of these
compounds remains to be elucidated.
Synthetic quinolone antibiotics
The practical applications of nalidixic acid (Fig. 1) as an
antimicrobial of therapeutic interest became evident soon
after it was discovered (Lesher et al., 1962; Ward-Mcquaid
et al., 1963). Because it is a polar molecule that avidly
conjugates to serum proteins, and therefore presents a large
volume of distribution, it is inadequate for the systemic
treatment of infections. However, both nalidixic acid and its
principal 7-hydroxymethyl metabolite that remains active
undergo rapid renal excretion and readily accumulate in the
urinary tract (Rollo, 1966; Van Bambeke et al., 2005). As
nalidixic acid is notably efficient at arresting the growth of
common enterobacteria, its principal indication was in the
treatment of uncomplicated urinary tract infections (Ward-
Mcquaid et al., 1963). It is, however, of little use against
infections occurring outside of the urinary tract or those
that are caused by organisms such as P. aeruginosa and
Gram-positive pathogens that are intrinsically resistant to
the practical therapeutic concentrations of the antibiotic.
From the 1980s onwards, there appeared successive genera-
tions of antibiotics related to nalidixic acid such as the
fluoroquinolones, which, due to substitutions in the mole-
cule, more specifically the addition of a 6-fluoro group, have
extended therapeutic spectra and enhanced pharmacoki-
netic properties. The development of fluoroquinolones
(Fig. 1) such as flumequine, norfloxacin and ciprofloxacin
(one of the most consumed antibiotic worldwide; Ruiz,
2003) extended the spectrum of activity of quinolone
antibiotics against infections caused by a variety of
otherwise resistant organisms such as P. aeruginosa and
both aerobic and anaerobic Gram-positive pathogens,
and enabled the treatment or the prevention of more
severe conditions such as renal, respiratory, abdominal and
sexually transmitted bacterial infections (for an extensive
review on quinolone antibiotics, see Van Bambeke et al.,
2005).
Quinolone antibiotics act by inhibiting the two type IIA
bacterial topoisomerases: DNA gyrase and topoisomerase IV
(bacterial type IIA topoisomerases have been reviewed
recently by Sissi & Palumbo, 2010). DNA gyrase is a
heterotetramer formed by two subunits encoded by gyrA
(nalA) and gyrB (nalC). GyrA together with GyrB acts by
creating DNA gates or double-stranded gaps in the DNA
through which the strands are passed, introducing negative
supercoils into DNA and relaxing the positive supercoil-
ing resulting from replication as the strands unwind
N
OCH
C
O
OH
OH
8-Hydroxy-4-methoxy-
quinaldic acid
(quinolobactin)
N
OCH
C
O
SH
OH
8-Hydroxy-4-methoxy-
2-quinolinethiocarboxylic acid
(thioquinolobactin)
4-Formyl-1-sulfanyl-2(1H)-quinolone
(N-mercapto-4-formylcarbostyril, Cbs)
H OHS
SH
N
O
C
O
H
Fig. 3. Sulphur-containing quinolones produced by some pseudomo-
nads. Thioquinolobactin, a compound exhibiting strong antifungal
properties, is produced by P. fluorescens ATCC 17400. Upon sponta-
neous hydrolysis, thioquinolobactin is rapidly converted into quinolobac-
tin, which then acts as a siderophore. Pseudomonas fluorescens G308
produces Cbs, which also exhibits potent fungicidal properties.
FEMS Microbiol Rev 35 (2011) 247–274
c
2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
251Quinolones: antibiotics and autoinducers
Page 5
(Champoux, 2001; Wang, 2002; Corbett & Berger, 2004; Leo
et al., 2005; Drlica et al., 2009). Nalidixic and oxolinic acid, a
more potent, but structurally similar quinolone antibiotic
(Staudenbauer, 1976), were initially found to inhibit in vitro
the supercoiling activity of purified DNA gyrase (Gellert
et al., 1977; Sugino et al., 1977). Whereas only gyrase is able
to introduce negative supercoiling, the function of DNA
topoisomerase IV, a heterotetramer formed by two ParC-
ParE subunits similar to the GyrA-GyrB subunits of DNA
gyrase, is essential for the relaxation of supercoiled DNA and
the resolution of catenated DNA molecules after replication
(Kato et al., 1990, 1992; Adams et al., 1992; Peng & Marians,
1993; Hoshino et al., 1994; Chen et al., 1996). The precise
mode of action of the quinolone antibiotics on type IIA
topoisomerases has long been debated. However, crystal-
lographic studies strongly suggest that these molecules
essentially act by blocking the DNA–topoisomerase com-
plexes when the nucleic acid is cleaved (Chen et al ., 1996;
Laponogov et al., 2009). Thus, in addition to sharing
structural and functional similarities, both DNA gyrase and
topoisomerase IV can be inhibited by quinolone antibiotics,
leading to bacterial cell death due to chromosome fragmen-
tation. Whereas the target of quinolone antibiotics in
Escherichia coli and other Gram-negative bacteria is mainly
DNA gyrase, in Gram-positive species such as S. aureus or
S. pneumoniae, their principal mode of action lies mainly in
the inhibition of topoisomerase IV, with exceptions depend-
ing on the particular fluoroquinolone compound and
bacterial species (Higgins et al., 2003; Eliopoulos, 2004;
Laponogov et al., 2009).
Resistance to quinolone antibiotics (reviewed by Jacoby,
2005 and by Martinez et al., 2009a) can be achieved by three
nonexclusive mechanisms: (1) by the acquisition of point
mutations in the genes encoding either of the two type IIA
topoisomerases targeted, DNA gyrase and DNA topoisome-
rase IV, (2) by reducing the effective concentrations of the
drugs in the cytoplasm, either passively by alterations in the
membrane permeability or actively by overexpressing efflux
systems, and (3) by acquisition of mobile quinolone resis-
tance determinants (Ruiz, 2003; Jacoby, 2005). While only
target modification confers high-level resistance to quino-
lone antibiotics, the low-level resistance (less than a 10-fold
increase in the minimum inhibitory concentration) con-
ferred by the other mechanisms augments the probability of
developing such mutations. Mutants that exhibit more than
a 100-fold decrease in quinolone sensitivity have been found
to carry single point mutations in the chromosomally
encoded DNA gyrase subunit gene gyrA, with additional
enhanced resistance when certain point mutations are
simultaneously present in the distantly located gyrB gene
(Yamagishi et al., 1986; Yoshida et al., 1988; Cullen et al.,
1989; Yoshida et al., 1990, 1991; Oram & Fisher, 1991;
Cambau et al., 1993; Ruiz, 2003). Molecular details of these
mutations and the binding of quinolones to DNA–topoi-
somerase complexes have been described elsewhere exten-
sively (Ng et al., 1996; Cabral et al., 1997; Hiasa & Shea,
2000; Friedman et al., 2001; Jacoby, 2005).
In Gram-negative bacteria, a reduction in antibiotic
permeability can be achieved by altering the cell envelope.
For example, E. coli or P. aeruginosa clinical isolates that
produce altered lipopolysaccharides differ in the accumula-
tion of quinolones compared with wild-type strains, a
nonspecific mechanism thought to involve changes in sur-
face hydrophobicity and consequently affecting passive drug
diffusion (Cohen et al., 1989; Leying et al., 1992; Everett
et al., 1996; Chenia et al., 2006). However, a reduction in
permeability to quinolones is most often achieved by
disrupting or downregulating a number of outer-membrane
proteins that form channels through which quinolones enter
the bacterial cell or by the activation of tripartite multidrug
efflux systems belonging to the AcrAB-TolC resistance-
nodulation-division (RND) family that can prevent quino-
lone antibiotics reaching effective concentrations in the
cytoplasm (reviewed by Martinez et al., 2009b). For in-
stance, P. aeruginosa encodes at least 12 RND systems
(Stover et al., 2000; Schweizer, 2003), eight of which
(MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-
OprM, MexJK-OprM, MexHI-OpmD, MexVW-OprM and
MexPQ-OpmE) have been reported to export fluoroquino-
lones and other antibiotics (Chuanchuen et al., 2002; Li
et al., 2003; Sekiya et al., 2003; Van Bambeke et al., 2003;
Mima et al., 2005), although antimicrobial resistance does
not appear to be their primary biological function (Aes-
chlimann, 2003; Van Bambeke et al., 2003; Poole, 2004,
2008).
Resistance to quinolone antibiotics can also result from
the acquisition of plasmid-borne determinants. The MFS-
type efflux pump QepA and the Aac(6
0
)-Ib-cr enzyme that
confers decreased susceptibility to piperazinyl fluoroquino-
lones such as ciprofloxacin and norfloxacin by acetylation
are examples of recently discovered resistance genes carried
by plasmids (Robicsek et al., 2006; P
´
erichon et al., 2007;
Yamane et al., 2007). More frequent, however, are the
plasmids carrying Qnr quinolone resistance loci, of which
at least five families have been identified, mostly in Enter-
obacteriaceae (Jacoby et al., 2008; Cavaco et al., 2009; Wang
et al., 2009). These genes encode proteins of the pentapep-
tide repeat family that interact with DNA gyrase and
topoisomerase IV, preventing quinolone inhibition by mi-
micking DNA, which probably reduces the availability of
holoenzyme–DNA targets for quinolone inhibition (Tran &
Jacoby, 2002; Hegde et al., 2005; Tran et al., 2005a, b). As
with the other quinolone resistance determinants, this
function may be considered biologically fortuitous because
natural quinolones inhibiting type IIA topoisomerases have
not been discovered.
FEMS Microbiol Rev 35 (2011) 247–274
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2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
252 S. Heeb et al.
Page 6
Quinolones produced by
P. aerug inosa
Besides Pyo II, which is a 2 : 1 mixture of HQNO and
2-nonyl-4-hydroxyquinoline N -oxides (NQNO) (Light-
bown, 1950, 1954), with small quantities of 2-undecyl-4-
hydroxyquinoline N-oxide (UQNO) (Cornforth & James,
1956), P. aeruginosa also releases a large number of related
molecules. Using ozonolysis and UV absorption spectra in
comparison with synthetic standards, Wells et al. (1952)
identified Pyo Ib as 2-heptyl-4(1H)-quinolone (HHQ), Pyo
Ic as 2-nonyl-4(1H)-quinolone (NHQ) and Pyo III as a
monounsaturated alkyl side chain variant of NHQ (Wells,
1952; Wells et al., 1952).
The AQ biosynthetic enzymes of P. aeruginosa enable this
organism to generate a diverse range of related AQ mole-
cules (Fig. 2 and Box 1). An early study using GC and
electron capture MS identified over 20 different AQs (Taylor
et al., 1995), with HHQ being the most prevalent, followed
by NHQ. Variations of these compounds containing satu-
rated and monounsaturated alkyl side chains varying from
one to 13 carbons in length, and the two major N-oxides,
HQNO and NQNO, were also found. Two subsequent
studies used electrospray ionization and LCMS to obtain
the mass spectra of over 50 different AQs. These mainly
consisted of 2-heptyl-3-hydroxy-4(1H)-quinolone [termed
the Pseudomonas quinolone signal (PQS)], HHQ, HQNO
and NHQ, with several other saturated and monounsatu-
rated alkyl side chains of various lengths (L
´
epine et al., 2003,
2004). Additional AQs that have been found in significant
amounts are 2-nonyl-3-hydroxy-4(1H)-quinolone (C9-PQS),
2-undecyl-4-hydroxyquinoline (UHQ), NQNO and UQNO
(Taylor et al., 1995; D
´
eziel et al., 2004; L
´
epine et al., 2004).
Several variations of these compounds are produced (Fig. 2
and Box 1), but many at seemingly biologically insignificant
levels, perhaps as a consequence of a lack of specificity of the
AQ biosynthetic enzymes for b-keto fatty acids of different
chain lengths rather than for any particular biological
function. In addition, a metabolite identified as 2,4-dihy-
droxyquinoline (DHQ) was found in cultures of both
P. aeruginosa and Burkholderia thailandensis (L
´
epine et al.,
2007; Zhang et al., 2008). DHQ, although structurally
related, is technically not an AQ as it lacks a 2-alkyl chain.
It is neither a degradation product nor a precursor of AQs
and the precise function of this molecule remains unknown.
However, DHQ inhibits the growth and cell viability of
mouse lung epithelial MLE-12 cells (Zhang et al., 2008) and
therefore this molecule may play a role in pathogenicity in
respiratory tract infections.
Properties of AQs
Generally, AQs have a low aqueous solubility. For example,
the solubility of PQS is around 1 mg L
1
(5 mM) in water
(L
´
epine et al., 2003). Because of this hydrophobic nature, a
Box 1. Nomenclature and abbreviations of AQs used in this review
The structures, IUPAC-based nomenclature and abbreviations of all
the major AQs produced by Pseudomonas aeruginosa are
summarized in Fig. 2. Some non-IUPAC names that have been used
to describe some of these same molecules in the scientific literature
have been included for clarity. AQs, 2-alkyl-4(1H)-quinolones (lactam
form) are tautomeric with 2-alkyl-4-hydroxyquinolines (phenolic
form), of which the predominance of one form over the other is
determined by the pH (Katritzky & Lagowski, 1963; Katritzky et al.,
1991; Larsen, 2005). For example, it has been demonstrated using
pK
a
values for 2-methyl-3-hydroxy-4(1H)-quinolone (C1-PQS) that
over physiological pH ranges, the neutral 4-quinolone form is the
predominant tautomer (Diggle et al., 2007). These tautomeric forms
are shown in Fig. 2, with their relative ratios indicated by the arrows.
Ideally, for consistency and structural accuracy, nomenclature and
abbreviations based on only one tautomeric form should have been
uniformly adopted. However, this causes some difficulties because in
the available scientific literature individual research groups have
subjectively referred to these molecules in either one form or the
other. For example, even the names used for the two main AQ
molecules involved in signalling, PQS and HHQ, are inconsistent with
each other with regard to tautomerism: Pseudomonas quinol
one
signal and 2-heptyl-4-hydroxyquinol
ine. The nomenclature and
abbreviations used in this review therefore amount to a compromise
between what is technically correct, taking into account IUPAC
designations and structural predominance due to physiological pH,
and also what has been the prevalent terminology used in the
scientific literature for each molecule. Hence, the abbreviation PQS to
designate 2-heptyl-3-hydroxy-4(1H)-quinolone has been maintained
and the alkyl side chain variants of this molecule abbreviated by the
number of carbon atoms in the side chain, for example C1-PQS, C9-
PQS. The designation HHQ for 2-heptyl-4-hydroxyquinoline has also
been maintained, and the other alkyl side-chain derivatives have
been abbreviated accordingly (e.g. PHQ for 2-pentyl-4-
hydroxyquinoline, etc.). It should be noted that the N-oxide series of
compounds (AQNOs, e.g. HQNO, 2-heptyl-4-hydroxyquinoline
N-oxide; NQNO, 2-nonyl-4-hydroxyquinoline N-oxide) can adopt the
2-alkyl-1-hydroxy-4(1H)-quinolone form (Fig. 2) but not at
physiological pH. DHQ or 2,4-dihydroxyquinoline can exist in both,
4-hydroxy-2(1H)-quinolone (predominant at physiological pH) and
2-hydroxy-4(1H)-quinolone tautomeric forms (Fig. 2), but to avoid
confusion and to conform to the literature citations, DHQ is used to
denote this molecule in this review. To further help the reader, below
is a list of the proposed nomenclature of the AQs that are mentioned
along with their abbreviations. Included in this table are the
associated synonyms that have been used to describe these same
molecules elsewhere in the scientific literature.
Suggested nomenclature Synonyms
2-alkyl-4(1H)-quinolone (AQ) 2-alkyl-4-hydroxyquinoline (AHQ)
4-hydroxy-2-alkylquinoline (HAQ)
2-alkyl-4-hydroxyquinoline
N-oxide (AQNO)
4-hydroxy-2-alkylquinoline
N-oxide
2-alkyl-1-hydroxy-4(1H)-quinolone
2-heptyl-3-hydroxy-4(1H)-
quinolone
2-heptyl-3,4-dihydroxyquinoline
Pseudomonas quinolone signal
(PQS)
2-heptyl-3,4-quinolinediol
FEMS Microbiol Rev 35 (2011) 247–274
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253Quinolones: antibiotics and autoinducers
Page 7
high proportion of the AQs are associated with the bacterial
outer membrane and with membrane vesicles (MVs)
(Mashburn-Warren et al., 2008). Of the total amount of
PQS produced by P. aeruginosa PA14, around 80% appears
to be contained within vesicles, in contrast with o 1% of
either of the P. aeruginosa N-acyl-homoserine lactone
(AHL)-based signal molecules N-(3-oxododecanoyl)-
L-
homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-
L-
homoserine lactone (C4-HSL) (Mashburn & Whiteley,
2005). The PQS contained within these MVs is seemingly
both bioactive and bioavailable because the addition of MVs
containing PQS restored the production of pyocyanin in a
PQS-negative mutant (PQS being indispensable for the
production of pyocyanin in P. aeruginosa). The MVs them-
selves do not seem to have any direct effect on the produc-
tion of pyocyanin and PQS does not need to be packaged
into MVs to exert its effects. MV formation in P. aeruginosa
PA14 would not seem to be an active process as it occurs
independent of growth or of protein synthesis (Mashburn &
Whiteley, 2005). Instead, PQS appears to initiate the forma-
tion of MVs, into which it is then packaged due to its
lipophilic nature (Mashburn & Whiteley, 2005). A mechan-
ism for MV formation has been proposed via the interaction
of PQS with the 4
0
-phosphate and acyl chain of bacterial
lipopolysaccharide (Mashburn-Warren et al., 2008). Because
HHQ is much less efficient in inducing vesicle formation,
this activity seems to be dependent on the 3-hydroxy group
of PQS and its analogues (Mashburn-Warren et al., 2008,
2009). A pqsH mutant, deficient in the conversion of HHQ
to PQS, is defective in vesicle formation. Of PQS and its
analogues, MV formation appears to be optimal when a C7
2-alkyl side chain moiety is present, although C5 and C3
alkyl side chain variants also exhibit some activity and MVs
can still be induced to some extent by PQS analogues lacking
a 2-alkyl side chain, indicating that this group is dispensable.
Additionally, compounds that can inhibit PQS production
such as indole and its derivatives reduce MV formation,
presumably as there is less PQS available to induce vesicle
formation (Tashiro et al., 2010). It has been suggested that
packaging into MVs could protect PQS from degradation by
surrounding cells or competing microbial communities.
Arthrobacter nitroguajacolicus R
¨
u61 produces the cytoplas-
mic enzyme Hod [3-hydroxy-2-methyl-4(1H)-quinolone
2,4-dioxygenase], which catalyses the 2,4-dioxygenolytic
ring cleavage of PQS with the concomitant formation of
carbon monoxide and N-octanoyl-anthranilic acid (Pus-
telny et al., 2009). As purified Hod is capable of inhibiting
AQ signalling when added to cultures of P. aerug inosa,at
present, the extent of protection conferred by MVs to AQs
against enzymatic degradation is unclear.
Rhamnolipids are produced by P. aeruginosa and act as
biosurfactants, facilitating swarming motility (Caiazza et al.,
2005). However, rhamnolipids also enhance the aqueous
solubility and activity of AQs in vitro. The addition of
increasing amounts of rhamnolipids enhanced the ability of
PQS at a range of concentrations to induce the expression of a
lasB
0
-
0
lacZ translational reporter, suggesting that in P. aerugi-
nosa the induction of elastase production by PQS is enhanced
in the presence of rhamnolipids (Calfee et al., 2005). Whether
rhamnolipids are indeed effectively utilized to solubilize PQS
in vivo is, however, not known at present, and with respect to
the above reporter system, an excess of rhamnolipids even
seems to be detrimental to its expression. A reason for this
may be that above a certain threshold concentration, PQS is
sequestered into rhamnolipid micelles and therefore becomes
less available to the cells (Calfee et al., 2005).
AQNOssuchasHQNOarepotentinhibitorsofthe
cytochrome bc
1
complex and an interesting, but as yet
undescribed facet is the mechanism by which P. aeruginosa
avoids self-poisoning as a consequence of the endogenous
production of these molecules. Gram-negative bacteria, as
opposed to Gram-positive species such as Bacillus subtilis or
S. aureus, are normally resistant to these compounds, and
possible explanations for this have been (1) a reduced cell wall
permeability , (2) enzymatic inactivation or (3) an active efflux
system to transport the molecule out of the cells (Machan
Suggested nomenclature Synonyms
3-hydroxy-2-nonyl-4(1H)
-quinolone (C9-PQS)
3,4-dihydroxy-2-nonylquinoline
2-nonyl-3,4-quinolinediol
2-pentyl-4-hydroxyquinoline
(PHQ)
2-pentyl-4(1H)-quinolone
4-hydroxy-2-pentylquinoline
2-pentyl-4-quinolinol
2-heptyl-4-hydroxyquinoline
(HHQ)
2-heptyl-4(1H)-quinolone
4-hydroxy-2-heptylquinoline
2-heptyl-4-quinolinol
2-nonyl-4-hydroxyquinoline
(NHQ)
2-nonyl-4(1H)-quinolone
4-hydroxy-2-nonylquinoline
2-nonyl-4-quinolinol
2-undecyl-4-hydroxyquinoline
(UHQ)
2-undecyl-4(1H)-quinolone
4-hydroxy-2-undecylquinoline
2-undecyl-4-quinolinol
2-heptyl-4-hydroxyquinoline
N-oxide (HQNO)
4-hydroxy-2-heptylquinoline N-oxide
2-heptyl-1-hydroxy-4(1H)-quinolone
2-nonyl-4-hydroxyquinoline
N-oxide (NQNO)
4-hydroxy-2-nonylquinoline N-oxide
2-nonyl-1-hydroxy-4(1H)-quinolone
2-undecyl-4-hydroxyquinoline
N-oxide (UQNO)
4-hydroxy-2-undecylquinoline N-oxide
2-undecyl-1-hydroxy-4(1H)-quinolone
2,4-dihydroxyquinoline
(DHQ)
4-hydroxy-2(1H)-quinolone
2-hydroxy-4(1H)-quinolone
2,4-quinolinediol
Box 1. Continued.
FEMS Microbiol Rev 35 (2011) 247–274
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254 S. Heeb et al.
Page 8
et al., 1992). However, none of these mechanisms can account
for the resistanc e of P. aeruginosa towards endogenously
produced HQNO . Aerobic respiration in P. aeruginosa is
achieved by a branched electron transport chain ending in
five different terminal oxidases, three of which (cytochrome
oxidases cbb
3
-1, cbb
3
-2 and aa
3
) rec eiv e electrons from ubiqui-
none via the cytochr ome bc
1
complex and cytochromes c,
whereas the remaining two are the cytochro me bo
3
and the
cyanide-insensitive cytochrome bd quinol ox idases, which
bypass the cytochromes bc
1
c electron transfer pathway
and get their electrons directly from ubiquinone (Williams
et al., 2007a). Thus, if the cytochrome bc
1
complexes of
P. aeruginosa were sensitive to HQNO, this compound alone
would have the potential to inhibit three out of five electron
transport chains, and in combination with the production of
cyanide, 80% of aerobic respiration would be inhibited.
Pseudomonas aeruginosa is also able to perform anaerobic
respiration using nitrogen oxides as terminal electron accep-
tors. Nitrite, nitric oxide (NOR) and nitrous oxide terminal
reductases rec eiv e electrons from the cytochrome bc
1
complex,
while nitrate reductase (NAR) obtains electrons directly from
ubiquinone and also in part via a dedicated membrane-bound
formate deh ydr ogenase (W illiams et al., 2007a). In this case,
HQNO could potentially block all the nitrogen oxide anaero-
bic respiration, ex c ept for the N AR respiratory chain involving
formate oxidation. A recent study reported that abolishing AQ
production in P. aeruginosa enhanced anaerobic growth on
nitrate, and that addition of PQS appeared to repress the
growth of the wild type and inhibited denitrifying enzymes
(Toyofuku et al., 2008). Although this effect was attributed to
the iron-chelating properties of PQS, the involv ement of
increased HQNO production cannot be excluded. Whether
P. aeruginosa prevents self-poisoning with HQNO under
aerobic conditions by favouring the cytochrome bo
3
and/or
the cyanide-insensitive cytochrome bd oxidase pathways or
whether there are mechanisms to prevent competitive inhibi-
tion of ubiquinone-dependent enzymes under aerobic and
anaerobic growth on nitrate remains to be determined.
Biosynthesis of AQs in
P. aeruginosa
AQ biosynthesis requires multiple genes, which were initi-
ally identified by screening a P. aeruginosa transposon
mutant library for clones displaying reduced pyocyanin
production and were termed pqsABCDE, pqsR (mvfR), pqsH
and pqsL (Cao et al., 2001; D’Argenio et al., 2002; Gallagher
et al., 2002; L
´
epine et al., 2002). The pqsABCDE (PA0996-
PA1000) genes are arranged in an operon, and adjacent to
these are the anthranilate synthase genes phnAB (PA1001-
PA1002) and pqsR (mvfR, PA1003). Two other genes are also
involved in AQ biosynthesis, pqsH (PA2587) and pqsL
(PA4190), but both of these are located separately elsewhere
on the chromosome.
The pqsR gene encodes a LysR-type transcriptional reg-
ulator that has a helix-turn-helix motif at the N-terminus
with the first 280 amino acids sharing high similarity
(62–71%) with other LysR-ty pe regulators (Cao et al., 2001;
Maddocks & Oyston, 2008). PqsR is the transcriptional
regulator of both the pqsABCDE and the phnAB operons
and is of crucial importance for AQ production. A mutation
in the gene coding for this regulator in P. aeruginosa strain
PA14 resulted in the abolition of phnAB and pqsABCDE
transcription along with PQS and AQ biosynthesis and had
corresponding effects on other virulence determinants in-
cluding pyocyanin, elastase, exoprotein and 3-oxo-C12-HSL
production and consequently the reduced ability to cause
disease in plants and animals (Cao et al., 2001; D
´
eziel et al.,
2004).
The pqsABCD genes are involved in the biosynthesis of all
AQs (D
´
eziel et al., 2004). The first step in this biosynthesis
involves the activation of anthranilate by PqsA, an anthra-
nilate coenzyme A ligase (Coleman et al., 2008). PqsB and
PqsC, which are similar to b-keto-acyl-ACP (acyl carrier
protein) synthases involved in fatty acid metabolism, are
predicted to elongate acyl side chains of AQ precursors.
However, little is currently known about the enzymatic
functions of these proteins. PqsD shares some sequence
similarity with the Cys-His-Asn active site of the E. coli
initiation condensing enzyme FabH (Luckner & Ritter,
1965; Ritter & Luckner, 1971; Bredenbruch et al., 2005;
Zhang et al., 2008). The crystal structure of PqsD with and
without a potential covalently bound anthranilate-AQ inter-
mediate product has been resolved recently (Bera et al.,
2009). Nonpolar mutations in either the
pqsA, pqsB or pqsD
genes completely abolish the production of AQs (Digg le
et al., 2003; Zhang et al., 2008).
In addition to these four AQ biosynthesis genes, the
pqsABCDE operon also encodes PqsE, which has sequence
similarities to proteins of the metallo-b-hydrolase super-
family. This extensive family of hydrolytic enzymes mediates
a wide range of functions, such as b-lactamases, glyoxalases,
AHL-lactonases and arylsulphatases. These enzymes are
usually characterized by a conserved metal ion-binding
HXHXDH amino-acid motif, which is also found in PqsE
and that forms an active site able to bind two iron atoms (Yu
et al., 2009). However, although the PqsE the crystal
structure is available, little knowledge has been gained about
its exact function, its natural substrate remaining unknown.
The deletion of pqsE reduces the production of several
virulence factors including pyocyanin, lectin and hydrogen
cyanide (HCN), while overexpression of pqsE has the
opposite effect. A transcriptomic analysis has recently re-
vealed that the abundances in the mRNAs of 400 genes
depend on the level of expression of pqsE, and that virulence
in plant and animal infection models in the absence of AQ
depends on this gene (Rampioni et al., 2010). The activity of
FEMS Microbiol Rev 35 (2011) 247–274
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2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
255Quinolones: antibiotics and autoinducers
Page 9
PqsE has also been reported to be dependent on RhlR, which
acts downstream, but in synergy with PqsE (Hazan et al.,
2010). Interestingly, PqsE is not involved in AQ biosynthe-
sis, and appears to be a crucial element mediating the
cellular response to PQS to achieve full virulence (Gallagher
et al., 2002; Diggle et al., 2003; D
´
eziel et al., 2005; Farrow
et al., 2008).
The pqsH gene encodes a predicted FAD-dependent
monooxygenase that hydroxylates the 3
0
carbon atom of
HHQ in the final step of PQS biosynthesis. As such, pqsH
mutants do not produce 3-hydroxylated quinolones, but
continue to produce other AQs (D
´
eziel et al., 2004). Because
pqsH is regulated by LasR, but not by PqsR, it is therefore
conceivable that under certain circumstances, a differential
regulation of these two elements may lead to the over-
production of HHQ with respect to PQS, due to a lack of
PqsH (D
´
eziel et al., 2004).
The pqsL gene encodes a second, distinct monooxygenase
that is required for the synthesis of HQNO and related
N-oxides via oxidation of the quinolone ring nitrogen atom.
The detailed mechanism of AQNO biosynthesis is still
unknown, but interestingly, HHQ does not appear to be a
precursor of HQNO, as the addition of deuterated HHQ to a
culture of P. aeruginosa resulted in the biosynthesis of
deuterated PQS, but not of deuterated HQNO (D
´
eziel
et al., 2004). Additionally, a pqsL mutant overproduces PQS
compared with its isogenic wild-type parent, suggesting that
PqsL interacts with and diverts a fraction of the HHQ
precursor products towards AQNO biosynthesis and away
from HHQ and PQS biosynthesis (D’Argenio et al., 2002).
A simplified scheme for the biosynthesis of AQs is
detailed in Fig. 4. Before the role of these molecules in
signalling was discovered, an AQ biosynthesis pathway had
already been proposed. This was based on radiolabelled
precursor feeding experiments, which indicated condensa-
tion of anthranilic acid with b-keto fatty acids, releasing
CO
2
and H
2
O (Cornforth & James, 1956; Luckner & Ritter,
1965; Ritter & Luckner, 1971). This was confirmed more
recently by MS and nuclear magnetic resonance (NMR)
analysis of the AQs produced after feeding
13
C and
15
N
isotope-labelled precursors to P. aeruginosa (Bredenbruch
et al., 2005). This study also ruled out a second possible
pathway of AQ biosynthesis that involved the formation of a
kynurenic acid precursor resulting from a reaction between
N
OH
HQNO
O
PqsA
TrpGDF KynU
KynB
PqsBCD?
PqsD
KynA
NH
O
CO H
NH
Kynurenine
NH
O
OH
Anthranilic acid
S
ACP
OO
+
+
NH
S
O
CoA
Activated anthranilate
Activated -ketodecanoate
N
H
O
CO H
NH
CHO
N-Formylkynurenine
N
H
CO H
NH
Tryptophan
N
H
O
OH
PQSDHQ
N
H
OH
O
N
H
O
HHQ
HO
OH
OH
CO H
OH
O
CO H
CO H
OH
O
O
AroCK
PhnAB TrpEG
Pyruvic acid
Shikimic acid Chorismic acid
H
O
H
PO
Erythrose 4-phosphate
OH
OH
PqsH
PqsL
Fig. 4. Proposed biosynthetic pathway of PQS, HHQ, HQNO and DHQ in
Pseudomonas aeruginosa. AQs are derived from a condensation reaction
between anthranilate and b-keto fatty acids. Anthranilate is derived from
either the PhnAB/TrpEG or the KynABU metabolic pathways using either
chorismate or tryptophan as precursors, respectively. Anthranilate is first
activated with coenzyme A (CoA) by PqsA. Anthranilate-CoA and an
activated b-ketodecanoate are condensed, possibly via the PqsBCD
enzymes to HHQ, releasing CO
2
and H
2
O. The monooxygenase PqsH
converts HHQ to PQS. HQNO is derived from the same starting products
as HHQ, but utilizes the additional monooxygenase PqsL. HHQ is not a
precursor for HQNO. DHQ, which technically is not an AQ, is produced by
PqsD independent of PqsB and PqsC.
FEMS Microbiol Rev 35 (2011) 247–274
c
2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
256 S. Heeb et al.
Page 10
orotic acid and anthranilate. The heterologous expression of
AQ biosynthesis genes in E. coli revealed that the production
of DHQ (Fig. 2) only requires PqsA and PqsD (Zhang et al.,
2008). Activated anthraniloyl-CoA, generated by PqsA, is
transferred to the cysteine residue in the active site of PqsD
(unactivated anthranilate does not transfer). Here, it reacts
with either malonyl-CoA or malonyl-ACP to form 3-(2-
aminophenyl)-3-oxopropanoyl-CoA, a short-lived inter-
mediate that undergoes an internal rearrangement to form
DHQ. Some variation of this biosynthesis, utilizing longer
chain b-keto fatty acids in place of malonyl-CoA or
malonyl-ACP, is possibly the mechanism by which AQ
molecules such as HHQ are produced. However, because
the above process only utilizes PqsA and PqsD, AQ bio-
synthesis is likely to be more complex as the functions of
PqsB and PqsC are still unclear (Zhang et al., 2008; Bera
et al., 2009).
The substrates required for AQ biosynthesis have also
been investigated. Two pairs of genes responsible for anthra-
nilate biosynthesis had been described previously: phnAB
(Essar et al., 1990a), located adjacent to the pqs operon
(Gallagher et al., 2002), and trpEG, which encode enzymes
involved in tryptophan biosynthesis (Essar et al., 1990b).
The phnAB genes code for proteins resembling the E. coli
anthranilate synthase subunits TrpE and TrpG (Essar et al.,
1990a) and are cotranscribed by PqsR (Cao et al., 2001). It
was initially thought that these would provide anthranilate
as a precursor for phenazine biosynthesis as inactivation of
these genes reduced pyocyanin production. However, it was
subsequently shown that PhnA and PhnB do not appear to
be involved in this function (Mavrodi et al., 2001) and
therefore the reduction in pyocyanin production in a phnAB
mutant is instead most likely to be the consequence of the
reduced availability of AQs. In addition to PhnA and PhnB,
TrpE and TrpG also direct the synthesis of anthranilate from
chorismate, which can then be utilized either for tryptophan
or for AQ biosynthesis. A third source of anthranilate comes
from the homologues of the tryptophan 2,3-dioxygenase
KynA, the kynurenine formamidase KynB and the kynur-
eninase KynU to produce anthranilate from tryptophan
(Kurnasov et al., 2003; Farrow & Pesci, 2007). Detailed
analyses indicate that in rich growth media containing the
aromatic amino acid tr yptophan, the kynurenine pathway is
the main source of anthranilate for AQ production, whereas
the phnAB genes supply anthranilate in minimal media in
the absence of exogenous tryptophan (Farrow & Pesci,
2007). Interestingly, increased PQS production by P. aerugi-
nosa strains isolated from infected CF lungs has been
correlated with the presence of aromatic amino acids in the
growth medium (Palmer et al., 2005). In this case, the
transcription of pqsA was found to be induced by trypto-
phan, phenylalanine and tyrosine, while the nonaromatic
amino acid serine had little effect. The kynurenine pathway
may therefore be the principal source of anthranilate in a
lung infection context.
The requirement of anthranilate for PQS production has
been demonstrated. When P. aeruginosa PAO1 parent and
isogenic las QS mutants unable to produce PQS were grown
in the presence of anthranilate labelled with
14
C in the
heteroaromatic ring, most of the radioactivity was found in
the AQ extracts for those strains able to generate PQS,
whereas very little was found in the supernatant extracts of
the QS mutants (Calfee et al., 2001). This suggested that the
strains not producing PQS would incorporate anthranilate,
but not convert it into AQs. Additionally, when P. aeruginosa
was grown with increasing amounts of methyl-anthranilate,
PQS biosynthesis levels were reduced as this compound
acted as a competitor of anthranilate (Calfee et al., 2001).
The production of elastase, which is dependent on PQS
signalling, was also inhibited by methyl-anthranilate in a
concentration-dependent manner. At 1.5 mM, methyl-
anthranilate practically abolished elastase production, the
suggested consequence of a much reduced level of PQS
production.
Feeding experiments with isotope-labelled AQ precursors
such as
15
N-anthranilate coupled with GC–MS analysis
resulted in the production of AQs having incorporated
around 66% of
15
N, further demonstrating that anthranilate
serves as a common precursor for AQs and that the hetero-
aromatic nitrogen in the quinolone ring originates from this
molecule (Bredenbruch et al., 2005). Similarly, feeding
labelled
13
C-acetate to P. aeruginosa PAO1 demonstrated
that the heteroaromatic ring of the quinolone moiety was
formed from acetate. The resulting GC–MS fragmentation
pattern, together with confirmation by NMR spectroscopy,
indicated that the mechanism of this reaction was via a
direct head-to-head reaction involving anthranilate and
b-keto fatty acids derived from acetate (Bredenbruch et al.,
2005). b-Keto fatty acids are therefore essential precursors in
the biosynthesis of AQs. Some studies had suggested that
there is a link between rhamnolipid biosynthesis and AQ
production, which was interesting because rhamnolipids are
composed of a rhamnose moiety and fatty acids of the same
chain lengths as those involved in AQ biosynthesis. Rham-
nolipids have also been shown to increase PQS solubility
and may mediate this function in vivo (Calfee et al., 2005).
It was initially thought that rhlG coded a potential
b-ketoacyl-ACP reductase that could participate in the
provision of fatty acids utilized as a substrate for AQ
biosynthesis (Bredenbruch et al., 2005) as RhlG was as-
sumed to direct the incorporation of these fatty acids into
rhamnolipids (Campos-Garc
´
ıa et al., 1998; D
´
eziel et al.,
2003; Sober
´
on-Ch
´
avez et al., 2005). However, recent studies
have contradicted this, as an rhlG mutant was unaltered in
rhamnolipid production compared with the corresponding
wild type (Zhu & Rock, 2008). Furthermore, the crystal
FEMS Microbiol Rev 35 (2011) 247–274
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257Quinolones: antibiotics and autoinducers
Page 11
structure of RhlG revealed that its function was inconsistent
with the proposed fatty acid biosynthetic pathway (Miller
et al., 2006). Therefore, it appears that RhlG is not involved
in rhamnolipid or AQ biosynthesis.
In P. aeruginosa, PQS is likely to be the end product of the
AQ synthetic pathway or is not substantially converted into
other molecules, as when labelled PQS was added to wild-
type cultures, no additional compounds could be identified
(D
´
eziel et al., 2004).
Quorum sensing (QS) and AQ production
in
P. aerug inosa
When favourable nutritional conditions are encountered,
bacteria will proliferate to form established multicellular
communities that have the potential to adapt to and modify
their environment. This allows further exploitation of
nutrient resources that would otherwise be restricted for
individual cells. The mechanism by which a bacterium
adapts from the lifestyle of an individual cell to a commu-
nity capable of modify ing their environment has been
termed QS and it is defined as a mechanism by which
bacteria regulate specific target genes in response to a critical
concentration of endogenously produced signal molecules
dedicated to the probing of the cell population density
(Venturi, 2006; Williams et al., 2007b). This process is
mediated by the production and sensing of autoinducers,
small signalling molecules, whose concentration in the
extracellular medium reflects cell population density.
Pseudomonas aeruginosa produces two AHLs as QS signal
molecules, each acting as the autoinducer of a specific
sensing and responding system: 3-oxo-C12-HSL acts on the
las system and C4-HSL acts on the rhl system. The core of
each system is composed of a synthase producing an AHL
for the activation of a specific transcriptional regulator: LasI
produces 3-oxo-C12-HSL for the activation of LasR (Gam-
bello & Iglewski, 1991; Passador et al., 1993; Pearson et al.,
1994) and RhlI produces C4-HSL for the activation of RhlR
(Latifi et al., 1995; Pearson et al., 1995; Winson et al., 1995).
Initially, each synthase gene is expressed at basal levels and
the AHLs produced diffuse into the surrounding medium.
Autoinduction is achieved when the accumulation of an
AHL reaches a threshold concentration and the activated
transcriptional regulators LasR and RhlR further enhance
the expression of the synthase genes lasI and rhlI, respec-
tively, generating positive feedback loops (Seed et al., 1995).
When the transcriptional regulators are activated they will
induce the transcription of overlapping subsets of genes. For
example, LasR will induce the production of virulence
factors such as elastase (Passador et al., 1993) and pyoverdin
(Stintzi et al., 1998), while RhlR will increase the production
of rhamnolipid biosurfactants (Ochsner & Reiser, 1995),
cytotoxic lectins, pyocyanin and elastase, among other
virulence factors (Pearson et al., 1997). In addition to some
overlap between the genes targeted by both AHL QS systems
due to the similarities of the palindromic las/rhl boxes
recognized by LasR and RhlR (Schuster et al., 2004; Schuster
& Greenberg, 2006, 2007), activated LasR will also induce
the rhl system (Latifi et al., 1996), creating a hierarchical
regulatory network, which in turn is further modulated by
additional regulatory elements (reviewed in von Bodman
et al., 2008 and in Williams & C
´
amara, 2009).
Besides the AHL-based QS systems, P. aeruginosa utilizes
an autoinducer regulatory system based on the AQs. This
system relies on the PQS and its precursor molecule HHQ to
control global gene expression (Pesci et al., 1999; D
´
eziel
et al., 2004). The transcriptional regulator PqsR controls the
expression of the pqsABCDE and phnAB biosynthetic oper-
ons and therefore pqsR is essential for the production of AQs
(Gallagher et al., 2002; D
´
eziel et al., 2004; McGrath et al.,
2004).
The pqsR gene is convergently transcribed with respect to
the pqsABCDE-phnAB
operons and two transcriptional start
sites have been mapped 190 and 278 bp upstream of its start
codon (Wade et al., 2005). The distant promoter appears to
have a typical s
70
-binding site signature, indicative of basal
transcription, and a putative las/rhl box operator sequence is
found centred 239–258 bp upstream of this transcriptional
start site (517–536 bp upstream of the start codon). In vitro,
PqsR binds at two different locations upstream of pqsA, and
the strength and position of the binding depend on the
presence of PQS (Wade et al., 2005). The pqsA transcrip-
tional starting point has been mapped 71 bp upstream of the
start codon (McGrath et al., 2004). Alterations of a LysR-
type box located at 45 in the pqsA promoter can result in
the loss of PqsR-binding capacity and in the reduction of
transcription initiation, suggesting that this element plays a
central role in the regulation of the pqsABCDE operon by
PqsR and PQS (Xiao et al., 2006b). Overexpression of pqsR
strongly repressed the transcription of antA, which encodes
an anthranilate 1,2-dioxygenase. This is thought to ensure
an adequate supply of anthranilate for the biosynthesis of
AQs by reducing its metabolic degradation (Oglesby et al.,
2008).
When the pqsABCDE operon and pqsR were cloned in
E. coli and expressed from their native promoters, HHQ and
NHQ were produced, but not PQS because E. coli lacks a
pqsH homologue. Similarly, compared with the wild type,
the activity of the pqsA promoter and AQ production levels
(except for PQS) remained comparable when pqsH was
disrupted. This indicates that in addition to PQS, other
AQs can also act as autoinducers (Xiao et al., 2006a). It has
been suggested that HHQ induces a conformational change
in PqsR, as binding of PqsR to the pqsA promoter in vitro is
enhanced by HHQ, although not as much as with PQS. In
an AQ-negative double pqsA pqsH mutant derived from
FEMS Microbiol Rev 35 (2011) 247–274
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258 S. Heeb et al.
Page 12
strains PAO1 or PA14, PQS was found to be 100 times more
potent at inducing the pqsA promoter than HHQ (Xiao
et al., 2006a; Diggle et al., 2007). In strain PA14, the deletion
of pqsH reduced the overall expression of the pqsR regulome
by less than twofold, and the addition of exogenous PQS to
this mutant did not revert the expression levels of this
regulome substantially above wild-type levels, further im-
plying a role for HHQ in inducing many of the genes. An
exception to this was phzA1, as PQS appears to be essential
for the transcription of this gene and for the production of
pyocyanin (Xiao et al., 2006a). Altogether, these studies
indicate that HHQ acts as an autoinducer independent of
PQS. Other AQs such as NHQ can also activate PqsR and as
such could potentially be considered as autoinducers,
although not as potent as PQS (Xiao et al., 2006a; Fletcher
et al., 2007).
The las and rhl QS systems are linked to AQ production
and regulation, forming an incoherent feed-forward loop
likely to produce accelerated pulse-like responses (Alon,
2007): the las system positively controls AQ production by
inducing the pqsR and pqsA promoters and the rhl system
downregulates its effects (Pesci et al., 1999; McKnight et al.,
2000; McGrath et al., 2004; Wade et al., 2005; Xiao et al.,
2006b) (Fig. 5). In a lasR mutant, transcription of pqsR is
reduced about fourfold compared with the wild type (Wade
et al., 2005) and LasR appears to induce pqsR transcription
by binding to a conserved las/rhl box situated 517–536 bp
upstream of its translational start site (McGrath et al., 2004;
Xiao et al., 2006b; Gilbert et al., 2009). In line with this, a
transcriptional pqsR-lacZ fusion can be significantly induced
in E. coli expressing lasR by the addition of 3-oxo-C12-HSL,
indicating that the LasR/3-oxo-C12-HSL system acts as an
inducer of pqsR (Wade et al., 2005). A lasR mutant accumu-
lates the HHQ series of AQs, but produces very little PQS
early in growth, a consequence of LasR also positively
controlling the expression of pqsH, which encodes the
monooxygenase required for the conversion of HHQ to
PQS (Whiteley et al., 1999; Gallagher
et al., 2002; D
´
eziel
et al., 2004). The transcription of pqsA is considerably
reduced in a lasI mutant (McGrath et al., 2004). However, a
functional las QS system is not required for AQ biosynthesis,
as a lasR mutant still produces PQS in the late stationary
phase and expressions of pqsR and pqsH in a lasR mutant are
delayed, but not abolished during growth (Diggle et al.,
2003; Xiao et al., 2006b). As rhlR overexpressed from a
plasmid partially overcomes the delay in PQS production
caused by a lasR mutation in strain PA14 (Dekimpe &
D
´
eziel, 2009), it appears that RhlR could replace some of
the functions of LasR with respect to the pqsA and pqsH
promoters to induce the production of PQS, although this is
somewhat paradoxical because RhlR is generally considered
to be a repressor of AQ production and indicates that the
current LasR-RhlR-AQ QS hierarchy model in P. aeruginosa
may be somewhat more sophisticated than currently
thought.
While the las QS system positively regulates AQ and PQS
production, the rhl system acts as a negative modulator of
their regulatory effects (Fig. 5). A 50% increase in pqsR
transcription has been observed in an rhlR mutant, suggest-
ing in this case that RhlR has a repressive effect (Wade et al.,
2005). Similarly, transcription from the pqsA promoter is
enhanced in an rhlI mutant and addition of C4-HSL to
antagonize the induction of pqsA by 3-oxo-C12-HSL, with
the consequence of reducing the production of PQS
(McGrath et al., 2004). Two las/rhl boxes are found at 311
and 151 bp upstream of the pqsA transcriptional start site
(Xiao et al., 2006b). Deletion of the distal las/rhl box in this
promoter increases transcription, while additional deletion
of the proximal box does not further increase pqsA promoter
activity. The deletion of rhlR causes an increase in the
transcription of pqsA independent of the presence of the
311 box, suggesting that RhlR binds to this box and causes
a downregulation of the pqsA promoter, whose mechanism
is still unclear. In vitro electrophoretic mobility shift assays
carried out on a 253-bp DNA fragment containing part of
the pqsA promoter using lysates of E. coli producing RhlR in
the presence or absence of C4-HSL-RhlR did not indicate
binding to this region; however, the fragment used did
not include the las/rhl box situated 311 bp upstream of
the transcriptional starting point (Wade et al., 2005).
Identification of LasR targets in vivo using chromatin
immunoprecipitation coupled to DNA microarray hybridi-
zation (ChIP-chip) identified this distal las/rhl box as a
LasR-binding site (Gilbert et al., 2009). As the rhl system is
itself driven by the production of PQS, a negative auto-
regulatory feedback loop is formed (Diggle et al., 2003). The
simultaneous provision of exogenous C4-HSL and PQS
restores rhlI transcription levels in a lasR mutant compar-
able with the wild type. However, under the same condi-
tions, the addition of these molecules separately did not
cause increased rhlI transcription, suggesting a synergistic
mechanism involving the two signalling molecules
(McKnight et al., 2000).
Thus, in P. aeruginosa, the autoinducible AQ system is
upregulated by the las and downregulated by the rhl QS
systems. AQ production is furthermore indirectly self-
limited by the positive regulatory effects it exerts on the rhl
QS system (Fig. 5).
Regulation of virulence factor expression
by AQs
The first demonstration that AQs regulate virulence factor
production in P. aeruginosa was that PQS positively con-
trolled the expression of the lasB (elastase) gene (Pesci et al.,
1999). It was later shown that this effect was considerably
FEMS Microbiol Rev 35 (2011) 247–274
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2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
259Quinolones: antibiotics and autoinducers
Page 13
enhanced when PQS and C4-HSL acted synergistically to
upregulate lasB expression (McKnight et al., 2000). The
regulation of virulence factor production by AQs is not
restricted to elastase. Addition of PQS upregulates the
expression of lecA and pyocyanin production in a concen-
tration-dependent manner (Diggle et al., 2003). However,
PAO1 cultures growing in the presence of PQS above a
concentration of 100 mM had an extended lag phase and
reached reduced ODs at the stationary phase. Despite this,
the expression of lecA occurred at lower population densities
and therefore the maximal expression was still observed
during the early stationary phase, although the addition of
PQS still resulted in an advancement of lecA expression and
elastase and pyocyanin production into the logarithmic
phase (Diggle et al., 2003). It worth noting that these effects
were not seen when either HHQ or 3-formyl-HHQ instead
of PQS was added to the cultures. Previous studies found
that both RhlR and RpoS are essential for lecA expression
(Winzer et al., 2000) and addition of PQS failed to restore
lecA transcription in rhlR or rpoS mutants, confirming the
importance of these two regulators for lecA promoter
activity. However, PQS was able to overcome the repression
of lecA by the H-NS-type protein MvaT and the post-
transcriptional regulator RsmA (Diggle et al., 2003). We
have recently found that PQS, but not HHQ, induces the
transcription of the small regulatory RNA RsmZ (Fig . 5), a
mechanism that explains how post-transcriptional regula-
tion by RsmA can be overcome by PQS and reveals that this
PQS
Post-transcriptional
regulation
O
N
H
HHQ
3-oxo-C12-HSL
O
OH
N
H
pqsA pqsB pqsC pqsD pqsE
lasR
rsaL
lasI
rhlRrhlI
pqsRphnA phnB
1 kb
O
N
H
O
O
O
C4-HSL
H
O
N
O
O
?
pqsH
rsmZ
RsmZ
RsmA
Fig. 5. Regulation of AQ production in
Pseudomonas aeruginosa. The las QS system
positively regulates the transcription of pqsR,
pqsABCDE and pqsH. The PqsABCD proteins
synthesize HHQ, which is converted to PQS by
PqsH. Autoinduction occurs when either HHQ or
PQS binds to PqsR and enhances the expression
of the pqs operon. The rhl QS system, also
positively controlled by the las system, exerts a
negative effect on the AQ system, although it is
itself positively regulated by AQs. The terminal
output of this regulatory network is the PqsE
protein of still unknown enzymatic function. In
addition, PQS, via an unknown mechanism, posi-
tively controls the transcription of the small RNA
RsmZ, which in turns has a negative effect on the
RNA-binding protein RsmA involved in
post-transcriptional regulation. Biosynthetic
enzymes are represented by globular shapes,
while transcriptional regulators are shown as
cubes. Filled arrows and blunted lines represent
positive and negative regulation, respectively.
FEMS Microbiol Rev 35 (2011) 247–274
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2010 Federation of European Microbiological Societies
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260 S. Heeb et al.
Page 14
molecule can act on the expression of virulence genes at
both the transcriptional and the post-transcriptional levels
(S. Heeb et al., unpublished data).
Virulence factor production is also affected when AQ
production is inhibited. Addition of the anthranilate analo-
gue, methyl-anthranilate, to P. aeruginosa caused a decrease
in the production of PQS and a subsequent reduction in
elastase produced (Calfee et al., 2001). The effects observed
with methyl-anthranilate are not restricted to elastase, with
concentrations of 500 mM completely inhibiting the expres-
sion of lecA and pyocyanin production, but with no adverse
effect on growth. This effect could partially be restored by
the provision of exogenous PQS (Diggle et al., 2003).
The subset of genes regulated by AQs has now been
examined in greater detail using transcriptomic analysis. It
has been found that PqsR, through the induction of the
pqsABCDE operon and the action of PqsE, positively reg-
ulates a subset of LasR- and RhlR-dependent genes. A pqsR
mutant of strain PA14 displayed the upregulation of 121 and
the repression of 22 mRNAs when compared with the
corresponding wild type (D
´
eziel et al., 2005). In this pqsR
mutant, the transcription of the pqsABCDE and phnAB
operons was abolished and that of pqsR itself was reduced.
The transcription of the phz1 operon, hcnABC, chiC (chit-
inase), mexGHI-opmD, lecA and lecB was also found to be
reduced in the absence of PqsR.
However, the role of AQs in the regulation of virulence
gene expression is now the subject of some debate. It had
been demonstrated previously that in both pqsR and pqsE
mutants, pyocyanin production, phzA1 expression, LecA,
elastase and rhamnolipid production levels were consider-
ably reduced compared with the wild type (Cao et al., 2001;
Gallagher et al., 2002; Diggle et al., 2003; D
´
eziel et al., 2005)
and that the addition of PQS, HHQ or HQNO to these
mutants could not restore these phenotypes (Gallagher
et al., 2002; Diggle et al., 2003; D
´
eziel et al., 2005).
Altogether, these studies suggested that AQ production
may not be indispensable for the regulation of these
phenotypes. A subsequent study shed new light on the
mechanisms by which AQs induce gene transcription by
revealing that PqsE alone can drive the expression of the
target genes, through the rhl QS system (Farrow et al., 2008).
By expressing PqsE in AQ-negative pqsA or pqsR mutants, it
was demonstrated that pyocyanin, rhamnolipid and elastase
production could be restored in the absence of AQs. This
restoration of exoproducts was not observed in an rhlR
mutant, which suggests that PqsE may exert its effects
through the rhl
system (Farrow et al., 2008). These findings
raise a number of intriguing questions as to the function of
AQs in P. aeruginosa. For example, is the primary function
of both, PQS and HHQ, to bind PqsR and to upregulate the
pqsABCDE operon, thereby forming, on the one hand, an
autoinduction loop and ultimately, on the other, producing
as the major output, an increase in the levels of PqsE?
Another intriguing question raised by the data is about the
function of PQS itself. There are conflicting reports on the
necessity of PQS for virulence in different P. aeruginosa wild-
type strains, although different hosts have been used: PQS
has been shown to be necessar y for the virulence of strain
PAO1 in nematodes (Gallagher et al., 2002), but unnecessary
for PA14 in a burned mouse model (Xiao et al., 2006a).
There are also conflicting reports as to the efficacy of PQS at
inducing the pqsA promoter via PqsR. One study found that
in PA14, PQS was more effective than HHQ at upregulating
pqsA (Xiao et al., 2006a), but conversely, another study
demonstrated that in strain PAO1, PQS was the less effective
molecule (Fletcher et al., 2007). This contradiction may be
due to differences in strain-specific mechanisms, but taken
together with new research on the role of PqsE, PQS may
not be as important to the direct regulation of virulence
factors in P. aeruginosa as first envisioned and may have
evolved as a fortuitous byproduct with other functions
(Bredenbruch et al., 2006; Digg le et al., 2007). Furthermore,
the primary role of PqsR requires some further clarification.
It is probable that the loss of v irulence noted in pqsR
mutants (Cao et al., 2001; D
´
eziel et al., 2005) is primarily
due to the corresponding loss of PqsE production, seen in
the fact that mice mortality in strain PA14 was much
decreased from the wild type and was equivalent in both
pqsA and pqsE mutants (D
´
eziel et al., 2005). Therefore, the
primary role of PqsR may be that it is responsible for
the expression of pqsE via the production of AQs and the
corresponding autoinduction of the pqsABCDE operon, at
least as far as the production of pyocyanin and expression of
lecA are concerned. The induction of pyocyanin production
by the AQ QS system further leads to the regulation of the
PYO stimulon, a set of around 50 genes whose expression is
affected, primarily via the transcriptional regulator SoxR, by
this phenazine (Dietrich et al., 2006).
Role of AQs in iron metabolism
In addition to its role as a cell-to-cell signalling molecule,
PQS is also able to chelate ferric iron (Fe
31
). The presence of
the 3
0
-hydroxy group on the molecule mediates this and
allows two or three PQS molecules to bind Fe
31
at physio-
logical pH ranges of 6–8. Compounds similar to PQS (such
as C9-PQS) also possess iron-binding capabilities, but
molecules lacking the 3-hydroxy group such as HHQ are
unable to do so (Bredenbruch et al., 2005; Diggle et al .,
2007).
Addition of PQS to P. aeruginosa cultures upregulates the
genes involved in the production of the siderophores
pyoverdine and pyochelin, which are produced in response
to iron starvation, as indicated by the upregulation of
siderophore-mediated iron transport systems such as the
FEMS Microbiol Rev 35 (2011) 247–274
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261Quinolones: antibiotics and autoinducers
Page 15
pyochelin biosynthetic clusters (pchDCBA and pchEGF), the
iron pyochelin outer-membrane receptor fptA and the
pyoverdine genes pvdE and pvdS (Bredenbruch et al., 2005;
Diggle et al., 2007). The pch genes were upregulated at 5, 11
and 20 h after inoculation between 3- and 25-fold. Also, the
genes pvdJAD encoding pyoverdine synthetases were upre-
gulated between 2- and 10-fold at 11 and 20 h (Bredenbruch
et al., 2005). Quantitative real-time PCR showed that pvdA
and pchE are upregulated by 6- and 17-fold, respectively,
upon addition of 20 mM PQS (Digg le et al., 2007). Also, in
strain PAO1 wild type as well as in pqsA, pqsE or pqsR
mutants, the addition of PQS, but not of HHQ, strongly
induced pyoverdine production (Diggle et al., 2007).
PQS, with its effect on free iron levels, also affects the
transcription of other genes. During growth in iron-replete
media, both lecA and pqsA were strongly induced by the
addition of 50 mM PQS in a PAO1 pqsA mutant. However,
the induction of pqsA was not due to the iron-chelating
properties of PQS because when grown in an iron-deficient
casamino acid (CAA) medium, PQS, PQS–Fe
31
(3 : 1) and
HHQ all induced the pqsA promoter, but methyl-PQS did
not (Diggle et al., 2007).
Around 60% of the PQS produced by P. aeruginosa is
associated with the cell envelope (L
´
epine et al., 2003; Diggle
et al., 2007), and the membranes of cells grown in iron-rich
media are visibly pink due to complexed Fe
31
, possibly
stored in AQ-containing inclusion bodies (Royt et al., 2001,
2007). Therefore, there is the possibility that PQS could act
as an iron trap and storage molecule in the cell membrane
and that it may be able to deliver iron directly to the cells.
However, experiments carried out with a P. aeruginosa
pvdD/pchEF double mutant, which lacks any iron acquisi-
tion systems, revealed that it was unable to grow in an iron-
deficient CAA medium in the presence of added PQS. In
contrast, this mutant had a similar growth compared w ith
the parental PAO1 strain when exogenous PQS was not
added to the medium. These data suggest that although PQS
may trap iron in the cell membrane, it is unlikely that it can
act as a siderophore per se (Diggle et al., 2007).
Iron-dependent regulation of AQ production appears to
be controlled by the availability of one of their precursors,
anthranilate. Under iron-limiting conditions, the ferric
uptake regulator Fur does not repress the transcription of
two genes prrF1 and prrF2, encoding small regulatory RNAs
(Wilderman et al., 2004), which post-transcriptionally re-
press the expression of the antABC and catBCA operons
specifying enzymes for the degradation of anthranilate.
Hence, in a prrF1 prrF2 double mutant, PQS production is
abolished under iron-limiting conditions, probably as a
consequence of anthranilate depletion (Oglesby et al.,
2008). Therefore, under iron-limiting conditions, the supply
of anthranilate for the biosynthesis of AQs is controlled by
Fur and the PrrF sRNAs, an effect that was further
reinforced by the iron starvation response resulting from
the iron-chelating property of PQS (Bredenbruch et al.,
2006; Diggle et al., 2007).
Because HHQ performs functions similar to those of
PQS, such as the induction of the pqsA promoter (Xiao
et al., 2006a; Diggle et al., 2007), many of the specific effects
observed upon addition of PQS may be due to its iron-
chelating properties. It is also probable that the production
of PQS and its chelating effects could confer a survival
advantage when P. aeruginosa is growing with other compet-
ing microorganisms in iron-limited environments. The red-
coloured PQS–Fe
31
complex can also be toxic to other
organisms. For example, its production has been found to
confer the ‘red death lethal phenotype to P. aeruginosa in a
Caenorhabditis elegans infection model (Zaborin et al.,
2009).
Iron availability also influences the levels at which AQs
induce the activity of PqsR as a transcriptional activator, and
therefore, iron also acts directly as a modulator of the AQ
signalling system in P. aeruginosa (Hazan et al., 2010).
Interestingly, iron has also been found bound to PqsE,
although without knowledge of the function of this enzyme,
the biological significance of this remains unclear (Yu et al.,
2009).
Additional regulators of AQ production in
P. aeruginosa
Besides autoinduction by PQS and its precursor HHQ,
modulation by the las and rhl QS systems, and metabolic
and regulatory adjustments following iron availability, AQ
production is regulated by additional factors (Table 1). For
example, AQ production is enhanced under phosphate-
limiting conditions. In P. aeruginosa, the transcriptional
regulator PhoB mediates responses to phosphate limitation
(Anba et al., 1990). As a PHO box has been found over-
lapping the distal transcriptional starting point of pqsR and
as AQ production is no longer enhanced in a phoB mutant,
these elements may mediate the increased AQ production
observed following phosphate limitation (Jensen et al.,
2006).
PtxR is a transcriptional regulator that positively affects
the production of exotoxin A and negatively affects the
production of pyocyanin. PtxR reduced the expression of
the pqsABCDE operon, probably indirectly and not via the
repression of pqsR (Carty et al., 2006). However, PtxR also
regulated the las positively and the rhl QS systems negatively,
which paradoxically should have resulted in an induction of
the pqsA promoter. Therefore, it appears that PtxR could be
part of an intricate network of feed-forward loops (Alon,
2007) that connect the las, rhl and pqs QS systems.
The gene pmpR (pqsR-mediated PQS regulator, PA0964)
was found by screening transposon mutants for clones in
FEMS Microbiol Rev 35 (2011) 247–274
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262 S. Heeb et al.
Page 16
which the phzA1 promoter had altered expression profiles.
PmpR, a protein of the YebC-like superfamily, binds to the
pqsR promoter and affects its transcription negatively. A
pmpR mutant was therefore found to have increased mRNA
levels of pqsR, pqsA and pqsH, which was suggested to result
in the observed induction of the phzA1 promoter and of
pyocyanin production, and enhanced swarming motility
and biofilm formation (Liang et al., 2008).
The gene ppyR (psl and pyoverdine operon regulator,
PA2663) appears to encode a membrane sensor that posi-
tively regulates exopolysaccharide and pyoverdine produc-
tion, perhaps in response to the presence of NOR (Attila
et al., 2008). The deletion of ppyR caused the downregula-
tion of several genes including the pqsABCDE operon and
the psqH gene for AQ and PQS biosynthesis, as well as the
antABC operon for anthranilate degradation. As a conse-
quence, a ppyR mutant produced no detectable PQS. How-
ever, the mechanism by which PpyR exerts its effects or the
signals that it senses are unknown.
Impact of AQs on microbial interactions
The prominence of P. aeruginosa as a major opportunistic
pathogen in nosocomial infections and in lung infections in
Table 1. Factors influencing AQ production
Factors Mechanisms References
PqsR and AQs Some AQs positively regulate AQ biosynthesis by
autoinduction. PqsR binding to the pqsA promoter is
enhanced and the transcription of the pqsABCDE operon is
induced by PQS, and to different levels by other AQs such as
HHQ and NHQ
Pesci et al. (1999), Gallagher et al. (2002), D
´
eziel et al . (2004),
McGrath et al. (2004), Wade et al. (2005), Xiao et al.
(2006a, b), Diggle et al. (2007), Fletcher et al. (2007), Oglesby
et al. (2008)
las QS system LasR and 3-oxo-C12-HSL positively regulate AQ production
by inducing the transcription of pqsR and pqsABCDE,and
further enhances the production of PQS by inducing pqsH
Pesci et al. (1999), Whiteley et al. (1999), McKnight et al.
(2000), Gallagher et al. (2002), Diggle et al. (2003), D
´
eziel
et al. (2004), McGrath et al. (2004), Wade et al. (2005), Xiao
et al. (2006a, b), Dekimpe & D
´
eziel (2009), Gilbert et al.
(2009)
rhl QS system RhlR negatively regulates AQ production by repressing pqsR
transcription. The expression of rhlI and the biosynthesis of
C4-HSL also negatively affect the activity of the pqsA
promoter
McKnight et al. (2000), Diggle et al. (2003), McGrath et al.
(2004), Wade et al. (2005), Xiao et al. (2006a, b)
Fur and Fe
31
Under low iron conditions, the metabolism of anthranilate is
adjusted by Fur and the PrrF sRNAs, maintaining AQ
production. Iron saturation increases AQ production,
probably by inducing the kynurenine pathway leading to
anthranilate. Iron levels also affect the activities of AQs as
inducers of PqsR
Oglesby et al. (2008), Hazan et al. (2010)
PhoB and PO
4
3
AQ production is enhanced by phosphate limitation. PhoB
could be mediating this by binding to a PHO box present in
the pqsR promoter
Jensen et al. (2006)
PtxR Reduces the expression of the pqsABCDE operon
independently of pqsR. PtxR also acts positively on the las and
negatively on the rhl QS systems
Carty et al. (2006)
PmpR PmpR negatively affects the transcription of pqsR by binding
to its promoter
Liang et al . (2008)
PpyR PpyR appears to be essential for the transcription of
pqsABCDE and psqH
Attila et al. (2008)
Dynorphin k-opioid receptor agonists dynorphin and U-50,488 enhance
AQ production by inducing the pqsA promoter
Zaborina et al. (2007)
Farnesol Reduces pqsA transcription and AQ production by interfering
with PqsR
Cugini et al. (2007)
Indole and
derivatives
Indole, its oxidation products and other bicyclic compounds,
including some naphthalene analogues and 8-quinolinol,
inhibit MV formation and PQS synthesis by unknown
mechanisms
Tashiro et al. (2010)
Sputum Growth in sputum, rich in aromatic amino acids such as
tryptophan, induces the pqsA promoter and increases AQ
production
Palmer et al . (2005), Farrow & Pesci (2007)
FEMS Microbiol Rev 35 (2011) 247–274
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263Quinolones: antibiotics and autoinducers
Page 17
CF patients (Govan & Deretic, 1996) led to the investigation
of the role of AQs in the regulation of virulence factor
production and the establishment and severity of infection.
The initial studies using clinical isolates from sputum in CF
patients showed that they all produced PQS (Collier et al.,
2002) and also HHQ, HQNO, NQNO and UQNO (Machan
et al., 1992). In addition, there was a correlation between the
levels of PQS and the bacterial sample load. Furthermore,
PQS was also found in isolates from paediatric CF patients
and from patients at early stages of P. aeruginosa infection
(Guina et al., 2003). The regulation of PQS production in
some of these isolates was irregular as this molecule was
detected early in growth, during the log phase. The use of a
simulated CF sputum medium has been shown to support
the growth of P. aeruginosa to high population densities
(Palmer et al., 2005) and also the differential regulation of
PQS production. In particular, the expression of the phnAB
genes is induced 14–22-fold, in line with the upregulation of
the pqsABCDE operon (17–19-fold) compared with the
expression of these genes in a morpholinepropanesulphonic
acid-buffered glucose medium. This upregulation results in
a fivefold increase in PQS production and presumably the
other AQs, and is not triggered by changes in AHL levels. It
is possibly linked to the presence of aromatic amino acids in
the sputum medium such as tryptophan, which is used by
P. aeruginosa for anthranilate biosynthesis (Farrow & Pesci,
2007). A recent study revealed that although PhhR is an
aromatic amino acid-responsive transcriptional regulator
that controls genes involved in phenylalanine and tyrosine
catabolism in P. aeruginosa, the biosynthetic genes for AQs
are not differentially expressed according to the presence of
this regulator (Palmer et al., 2010).
Pseudomonas aeruginosa forms biofilms to protect itself
from the harsh environmental conditions generated by the
host immune system and antimicrobials. AQs play an
important role in the establishment and maintenance of the
biofilm lifestyle by a number of different mechanisms. The
exogenous addition of 60 mM PQS to growing cultures of
P. aeruginosa PAO1 resulted in a significant enhancement in
biofilm formation partly due to the induction of expression
of the lectin gene lecA (Diggle et al., 2003) as this gene plays
a role in maintaining biofilm architecture in this organism
(Diggle et al., 2006b). Pseudomonas aeruginosa can also
release, possibly through lysis of cell subpopulations, extra-
cellular DNA, which acts as an interconnecting matrix in
bacterial biofilms (Whitchurch et al., 2002). DNA has
cation-chelating and antimicrobial properties and can cause
the disruption of the bacterial outer membrane by chelating
Mg
21
, which is essential for membrane stability. This in turn
could result in more DNA release (Mulcahy et al., 2008). In
addition, Mg
21
chelation induces the expression of the
PhoPQ two-component system, increasing the resistance of
P. aeruginosa towards aminoglycosides such as gentamicin
and cationic antimicrobial peptides. These broad-spectrum
antimicrobial peptides are released from host immune cells
and can disrupt the bacterial outer membrane, causing cell
death. Maximum DNA release takes place in the late log
phase when PQS production is at its highest (Diggle et al.,
2003; L
´
epine et al., 2003). Similarly, a pqsA mutant releases
low levels of extracellular DNA and forms flat, thin unstruc-
tured biofilms with increased sensitivity to detergents. The
detergent sensitivity may be due to the loss of this extra-
cellular DNA as a wild-type biofilm treated with DNase
retains this sensitivity (Allesen-Holm et al., 2006; H
¨
aussler &
Becker, 2008). A correlation between bacterial cell lysis and
PQS levels has been established, which may explain the
release of the extracellular DNA observed in biofilms (D’Ar-
genio et al., 2002). A mutation in the pqsL gene (which
results in PQS overproduction) resulted in pronounced lysis
in bacterial colonies, whereas those from pqsA and pqsR
mutants displayed no lysis, but this could be restored upon
addition of exogenous PQS. It has been proposed that PQS
induces prophage-mediated lysis and that this is responsible
for the DNA release (D’Argenio et al., 2002). The chromo-
some of P. aeruginosa harbours the filamentous Pf4 proph-
age, whose deletion results in the loss of bacterial autolysis
and aberrant biofilm formation (Rice et al., 2009). PQS also
acts as a pro-oxidant, which can increase the sensitivity of P.
aeruginosa to peroxide and ciprofloxacin (H
¨
aussler & Beck-
er, 2008), possibly resulting in cell lysis and DNA release.
AQs inhibit the growth of S. aureus and the yeast
C. albicans, suggesting that they may be used as antibiotics
by P. aeruginosa, during the early stages of infection,
enabling it to eradicate any competing organisms (Machan
et al., 1992). This idea is further supported by the fact that
AQs packaged in MVs inhibited the growth of S. epidermidis
(Mashburn & Whiteley, 2005), whereas mutants in kynAU
were unable to kill S. aureus and a kynB mutant displayed
reduced killing, presumably due to the lack of AQ produc-
tion (Farrow & Pesci, 2007). As mentioned earlier (Natural
antimicrobial quinolones), both HHQ and PHQ have
antibacterial activities, while PHQ additionally presents
antialgal properties (Wratten et al., 1977; Long et al., 2003).
HHQ and HQNO produced by a clinical isolate of
P. aeruginosa inhibited the growth of metronidazole-
resistant H. pylori in a cross-streak assay (Lacey et al.,
1995). These findings may explain why early colonizers of
the CF lung such as S. aureus are sometimes absent upon
P. aeruginosa colonization, which outcompetes other organ-
isms sharing the same niche (Machan et al., 1992). Conse-
quently, the combined iron-chelating properties and the
impact on virulence factor production of AQs help
P. aeruginosa to generate a highly favourable environment
in which to thrive.
Interestingly, farnesol, a sesquiterpene signal molecule
produced by C. albicans, reduces the transcription of pqsA
FEMS Microbiol Rev 35 (2011) 247–274
c
2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
264 S. Heeb et al.
Page 18
by interacting with PqsR and probably interfering with the
normal binding of this transcriptional regulator to the pqsA
promoter (Cugini et al., 2007). This results in a decrease in
both PQS and pyocyanin production and suggests that this
type of interspecies competition can be reciprocal. In addi-
tion, HQNO has been shown to induce the formation of
persistent small-colony variants of S. aureus that may resist
P. aeruginosa niche colonization and possibly explain the
coexistence of these two organisms in some infections
(Hoffman et al., 2006).
Roles of AQs in infection
The role of AQs in virulence and the severity of infection has
been demonstrated using several disease models. A muta-
tion in phnAB resulted in a fourfold decrease in virulence
compared with the wild type in a wax moth (Galleria
mellonella) larvae model (Jander et al., 2000). In addition,
mutations in pqsC, pqsD, pqsE, pqsR, pqsH and phnA
resulted in severely reduced killing of the nematode
(C. elegans)byP. aerug inosa to between 37 and 39% of the
wild-type levels (Gallagher et al., 2002). Using a burned
mouse model, mutants in pqsA and pqsE also exhibit
reduced virulence (D
´
eziel et al., 2005; Rampioni et al.,
2010). In the same disease model, a pqsR mutant showed
an 35% reduced mortality rate compared with the wild
type. This mutant also showed reduced PQS, 3-oxo-C12-
HSL, pyocyanin, elastase and exoprotein production
(Cao et al., 2001). Most interestingly, a pqsH mutation in
P. aeruginosa PA14 was not attenuated, suggesting that PQS
may not be essential for virulence and that virulence may be
regulated via the biosynthetic precursor, HHQ (Xiao et al.,
2006a).
Virulence factor and AQ production are upregulated in
response to host stress responses to P. aeruginosa. The
synthetic opioid U-50,488 and the endogenous k-opioid
receptor agonist dynorphin, which is released into the
human small intestine during inflammation and appears
to bind and enter bacterial cells, have been tested in
P. aeruginosa PAO1 and were found to enhance virulence
factor production (Zaborina et al., 2007). Furthermore, the
addition of U-50,488 or dynorphin to a PAO1 culture
induced a dose-dependent increase in pyocyanin produc-
tion and enhanced pqsA and lecA (but not pqsR ) expression,
with a corresponding increase in PQS, HHQ and HQNO
production. These opioid agonists also enhanced P. aerugi-
nosa virulence against Lactobacillus and C. elegans, probably
as a result of the above increases in virulence determinant
production.
AQs may also interfere with host responses by acting as
immune modulators. PQS suppresses T-cell proliferation
and interleukin-2 release in concanavalin A-activated hu-
man peripheral blood mononuclear cells (hPBMCs). PQS
also induces tumour necrosis factor-a release from lipopo-
lysaccharide-activated hPBMCs, at concentrations around
10
mM (Hooi et al., 2004). In vitro, PQS reduces the release
of interleukin-12 from lipopolysaccharide-stimulated bone
marrow-derived dendritic cells, preventing the development
of naı
¨
ve T cells into T-helper type 1 cells, which promote
cell-mediated immunity. The concentration of PQS required
to lower the cytokine release to 50% in this case was below
20 mM (Skindersoe et al., 2009). Additionally, both HHQ
and PQS appear to suppress host innate immune systems by
interfering with the nuclear transcription factor-kB signal-
ling pathway. This effect can be achieved with cell-free
extracts from cultures of wild-type P. aeruginosa, but not
from the cultures of a corresponding pqsA mutant (Kim
et al., 2010). It therefore seems possible that AQs play a role
in the dysregulation of the host immune response during
infection.
Production of AQs by other bacteria
DNA database analysis has revealed the presence of pqs gene
homologues in 4 40 species and strains that are more or
less related to P. aeruginosa. In particular, Burkholderia
pseudomallei and B. thailandensis appear to have the com-
plete putative pqsABCDE operons in their chromosomes,
sharing 31–53% identity to that of P. aeruginosa (Diggle
et al., 2006a). These were named hhqABCDE as no PQS had
been detected in these organisms. The hhqA and hhqE genes
are functionally conserved with their P. aeruginosa homo-
logues as they were able to complement PAO1 pqsA and pqsE
mutants, respectively, and restore PQS, HHQ, lectin and
pyocyanin production in the pqsA mutant and pyocyanin
and lectin production in the pqsE mutant. Using a combina-
tion of a novel AQ bioreporter and LCMS/MS, HHQ was
detected in culture supernatants of Pseudomonas putida and
Burkholderia cenocepacia and HHQ, NHQ, UHQ and
HQNO in B. pseudomallei (Diggle et al., 2003). Although a
mutant unable to generate AQs in B. pseudomallei presented
altered colony morphology and increased elastase produc-
tion, the actual role of these molecules in the biology of this
organism remains to be unravelled. AQs have also been
identified in a number of species of Burkholderia such as
Burkholderia ambifaria, B. thailandensis, B. pseudomallei and
Pseudomonas cepacia (probably an unclassified Burkholder-
ia). The main AQs produced by these organisms are 3-
methyl derivatives of PHQ, HHQ and NHQ termed
4-hydroxy-3-methyl-2-alkylquinolines (Moon et al., 1996;
Vial et al., 2008). Consequently, the operon responsible for
their synthesis has been renamed hmqABCDEFG (formerly
hhqABCDE), with the predicted methyltransferase hmqG
being involved in the biosynthesis of these AQs. None of
the above bacteria has pqsH orthologues nor produces PQS,
and previous efforts to detect PQS in other pseudomonads
FEMS Microbiol Rev 35 (2011) 247–274
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2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
265Quinolones: antibiotics and autoinducers
Page 19
such as P. fluorescens, Pseudomonas syringae and Pseudomo-
nas fragi have been unsuccessful (L
´
epine et al., 2004).
Furthermore, in B. thailandensis, B. ambifaria and B. pseu-
domallei, the 3
0
position is largely methylated (Vial et al.,
2008), which would presumably preclude any pqsH analogue
hydroxylating in these molecules and hence the production
of PQS. These findings suggest that these organisms may
lack the complexity of PQS signalling found in P. aeruginosa.
Concluding remarks
The discovery of quinine and related natural antiplasmodial
alkaloids, combined with the advances of synthetic chem-
istry, spurred significant research in the field and resulted in
the development and evaluation of thousands of novel
synthetic compounds. Among these were nalidixic acid and
the extensive family of synthetic quinolone antibiotics. In
parallel, the quest for antimicrobials of natural origin lead to
the discovery of the AQNOs and of the extensive family of
AQ compounds mostly produced by P. aeruginosa and
related bacteria. Synthetic and natural quinolone antimicro-
bials, however, appear to share little in common with respect
to their mode of action. The biological roles of the natural
quinolones of bacterial origin are diverse and include
intercellular signalling. The discovery of a non-AHL-based
QS system in P. aeruginosa mediated via AQs and linked to
the las and rhl QS systems provides a major insight into a
complex regulatory network that plays key roles in infection
via the regulation of v irulence and biofilm maturation.
Some AQs, such as PQS, are also able to sequester iron and
have multiple functionalities. AQ biosynthesis requires
several proteins and occurs via a condensation reaction
between anthranilate and b-keto fatty acids. Their produc-
tion is upregulated by both the las QS system and by AQs
themselves and downregulated by the rhl QS system. AQs
are present in bacteria other than P. aeruginosa, mainly other
pseudomonads and Burkholderiaceae, although the role in
these organisms is not, at present, very well defined. It is
possible that AQs could be produced by many other
bacterial genera, as the number of studies where these
compounds have been specifically screened has been small
and yet several AQs-producing species have been discovered
(L
´
epine et al., 2004; Diggle et al., 2006a; Vial et al., 2008). In
most of the species that produce AQs, the role of these
compounds or their biosynthesis remains unclear, althoug h
for many where genomic sequences are available, ortholo-
gues of the pqsABCDE operon extensively studied in
P. aeruginosa can often be identified.
Quorum quenching is the process by which the signalling
mediating QS is interfered with, leading to the disruption of
the normal means by which bacteria coordinate their
behaviour according to their population density and
preventing colonization (Raina et al., 2009). Quorum
quenching can be exerted naturally by microorganisms to
prevent the establishment of competing species and offers a
strategy for the development of novel antimicrobial drugs
(Rasmussen & Givskov, 2006). Hence, quinolone quenching
offers the possibility to interfere with AQ signalling in
pathogens such as P. aeruginosa, which rely on it to control
virulence. As a primary target to interfere with AQ-
mediated signalling, binding of PQS and HHQ to PqsR
could be blocked. This would not only interfere with the
production of AQs and their associated properties beneficial
for the bacterial cell by preventing the expression of the
pqsABCDE operon, but would also downregulate all the
other AQ-regulated genes, including those essential for
virulence. Compounds such as farnesol inhibit the induc-
tion capacity of PqsR on the pqsA promoter (Cugini et al.,
2007). However, as these compounds were found to act only
at relatively high concentrations (in the mM range), other,
more potent inhibitors are needed to stimulate clinical
interest. Another possibility would be to inhibit the action
of PqsE, whose function is still unclear, but that is required
for the production of several virulence factors. Analogues of
AQ precursors such as methyl-anthranilate or halogenated
derivatives of anthranilate have been found to inhibit AQ
synthesis, thus interfering with the signalling system prob-
ably by acting as competitive inhibitors of PqsA (Calfee
et al., 2001; Lesic et al., 2007; Coleman et al., 2008). This
approach has recently shown promising results in limiting
the systemic proliferation of P. aeruginosa infection in mice
(Lesic et al., 2007).
To complete our understanding of the AQ signalling
system in P. aeruginosa, the roles of some components still
remain to be fully unravelled. PqsB, PqsC and PqsL are likely
to be involved in the biosynthesis of AQs as revealed by the
structural domains they share with other known enzymes
and by mutational analysis (Gallagher et al., 2002; Diggle
et al., 2003; Bredenbruch et al., 2005; Farrow et al., 2008).
However, the exact role of PqsB and PqsC in the biosynthesis
of HHQ and PQS still remains unclear. Similarly, little is
known about the interaction of PqsL with precursor and
other biosynthetic proteins to generate the N-oxide AQNOs,
because the otherwise common precursor molecule HHQ
does not appear to be required for their production (D
´
eziel
et al., 2004). Even more critically, the role played by PqsE,
which appears to be an enzyme of the metallo- b-hydrolase
superfamily mediating the signal transduction that upregu-
lates swarming motility, the production of pyocyanin, lectin,
HCN, the transcription of many genes and ultimately
virulence (Rampioni et al., 2010) remain to be elucidated.
The biology of AQ biosynthesis, its regulation and the
signalling functions of the AQs control the behaviour and
virulence of P. aeruginosa. AQ signalling is proving to be
complex, leading to many open questions that still remain
unanswered. For example, the biological roles of AQs such
FEMS Microbiol Rev 35 (2011) 247–274
c
2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
266 S. Heeb et al.
Page 20
as DHQ or the N-oxide derivatives in P. aerug inosa remain
to be elucidated, as are the functions of AQs in other
pathogenic and beneficial microorganisms producing them.
From their inconspicuous discovery as potentially useful
compounds having weak antimicrobial properties, quino-
lones in general and AQs in particular are highly versatile
molecules that play central roles in the biology of the
producer organisms.
Acknowledgements
We are very grateful to the Biological and Biotechnological
Sciences Research Council UK (BBF0143921) and the Eur-
opean Union (FP6 Marie Curie EST ANTIBIOTARGET
MST-CT-2005-020278 and FP7 NABATIVI HEALTH-F3-
2009-2009-223670) for kindly supporting the research per-
formed by the authors in the topic of this review. Box 1, Figs
2 and 4 have been adapted from Fletcher et al. (2010), with
special permission from Springer Science1Business Media,
Dordrecht, the Netherlands, to whom we are also grateful.
Statement
Re-use of this article is permitted in accordance with the
Terms and Conditions set out at http://wileyonlinelibrary.
com/onlineopen#OnlineOpen_Terms
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