JOURNAL OF BACTERIOLOGY, May 2006, p. 3365–3370
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 188, No. 9
A Distinct QscR Regulon in the Pseudomonas aeruginosa
Yannick Lequette,1Joon-Hee Lee,1Fouzia Ledgham,2Andre ´e Lazdunski,2and E. Peter Greenberg1*
Department of Microbiology, University of Washington, Seattle, Washington 98195,1and Laboratoire d’Inge ´nierie des
Syste `mes Macromole ´culaires, IBSM/CNRS, Marseille 13402, Cedex 20, France2
Received 12 October 2005/Accepted 1 February 2006
The opportunistic pathogen Pseudomonas aeruginosa possesses two complete acyl-homoserine lactone (acyl-
HSL) signaling systems. One system consists of LasI and LasR, which generate a 3-oxododecanoyl-homoserine
lactone signal and respond to that signal, respectively. The other system is RhlI and RhlR, which generate
butanoyl-homoserine lactone and respond to butanoyl-homoserine lactone, respectively. These quorum-sensing
systems control hundreds of genes. There is also an orphan LasR-RhlR homolog, QscR, for which there is no
cognate acyl-HSL synthetic enzyme. We previously reported that a qscR mutant is hypervirulent and showed
that QscR transiently represses a few quorum-sensing-controlled genes. To better understand the role of QscR
in P. aeruginosa gene regulation and to better understand the relationship between QscR, LasR, and RhlR
control of gene expression, we used transcription profiling to identify a QscR-dependent regulon. Our analysis
revealed that QscR activates some genes and represses others. Some of the repressed genes are not regulated
by the LasR-I or RhlR-I systems, while others are. The LasI-generated 3-oxododecanoyl-homoserine lactone
serves as a signal molecule for QscR. Thus, QscR appears to be an integral component of the P. aeruginosa
quorum-sensing circuitry. QscR uses the LasI-generated acyl-homoserine lactone signal and controls a specific
regulon that overlaps with the already overlapping LasR- and RhlR-dependent regulons.
The opportunistic pathogen Pseudomonas aeruginosa can be
found free living in water and soil. This bacterium also causes
infections in a variety of animals and plants (7). Quorum-
sensing systems control hundreds of P. aeruginosa genes, in-
cluding genes that code for exoenzymes and extracellular vir-
ulence factor synthesis. Quorum sensing also plays a role in
biofilm development (4, 5, 13, 17, 22, 28). There are two well-
studied P. aeruginosa acyl-homoserine lactone (acyl-HSL) quo-
rum-sensing systems. One system is comprised of the LasR
signal receptor, which responds to the LasI-generated signal
N-3-oxododecanoyl-homoserine lactone (3OC12-HSL). The
other system is comprised of the RhlR signal receptor, which
responds to the RhlI-generated signal N-butanoyl-homoserine
lactone (C4-HSL) (5, 13, 18, 19, 26, 29). The rhlI and rhlR
genes are among the functions activated by LasR and LasI.
Both systems are also integrated in regulatory networks that
In addition to LasR and RhlR, there is a third, orphan
LasR-RhlR homolog, QscR, for which there is no cognate
acyl-HSL synthase gene (3). A qscR mutant is hypervirulent.
The influence of QscR on the expression of a few genes con-
trolled by the LasR-I and RhlR-I systems has been examined.
These genes are prematurely activated in a qscR mutant and
include genes in the phz1 and phz2 phenazine synthesis oper-
ons; hcnAB, the hydrogen cyanide synthesis operon; lasB,
which codes for elastase; rhlI; and lasI (3, 14). The mechanism
for transient repression of these genes by QscR is not clear. At
low acyl-HSL concentrations, QscR can form heterodimers
with LasR and RhlR. This might inactivate LasR and RhlR (3,
14). It is also possible that QscR sequesters acyl-HSL signals
and thereby delays the expression of LasR- and RhlR-depen-
dent genes (3, 14). To develop a better view of the role of QscR
in P. aeruginosa gene regulation, we employed microarray tech-
nology to assess the influence of QscR on the transcriptome.
We show that QscR affects transcript levels of over 400 genes,
most of which are not affected by the LasR-I or RhlR-I sys-
tems. Our microarray studies and subsequent reporter gene
experiments indicate that there is a specific QscR regulon. We
believe that QscR can directly influence specific genes in re-
sponse to the LasI-generated signal 3OC12-HSL.
MATERIALS AND METHODS
Bacterial strains and plasmids. We used the isogenic P. aeruginosa strains
PAO1 and PAO-R3 (3), and we used Escherichia coli DH5?. The P. aeruginosa
strains overexpressing qscR (YL113) and a qscR 3? deletion (YL117) were con-
structed in PAO-R3 as follows. A qscR overexpression plasmid (pJN105-QscR)
was constructed as described elsewhere previously (15). An in-frame deletion of
the qscR 3? end was created as follows. We amplified an XbaI-SacI fragment
extending from 45 bp upstream of the qscR start codon through codon 182
(PCR-1). We amplified a second SacI-XbaI fragment extending from codon 219
to 67 bases past the stop codon (PCR-2). The PCR-1 and PCR-2 products were
cloned together into XbaI-digested pJN105 to yield a plasmid coding for a QscR
DNA-binding mutant polypeptide missing amino acid residues 183 to 218
(pYL135). The SalI-SacI (with the SacI site blunt ended) fragment from pJN105-
QscR and the SalI-NotI fragment (with the NotI site blunt ended) from pYL135
were cloned into SalI-SspI-digested mini-CTX-lacZ to yield pYL129 and
pYL137. These plasmids were used to insert qscR alleles into the P. aeruginosa
chromosomal attB site by standard techniques (10, 11). The resulting P. aerugi-
nosa strains had unmarked chromosomal copies of a qscR allele. All primers used
in this study are described in Table S1 in the supplemental material.
Plasmids with point mutations in qscR were constructed with PCR products as
follows. Mutations were constructed in two PCR steps. The first PCR used one
flanking primer and an internal primer containing a point mutation. The second
PCR used the other flanking primer and a complementary internal primer with
* Corresponding author. Mailing address: Department of Microbi-
ology, HSB Room G-328, 1959 NE Pacific Street, Seattle, WA 98195-
7242. Phone: (206) 616-2881. Fax: (206) 616-2938. E-mail: epgreen@u
† Supplemental material for this article may be found at http://jb
are also influenced by the overexpressed QscR DNA-binding
domain mutant polypeptide. We believe that QscR regulates
the genes encoding these transcripts indirectly. This indirect
effect could result from heterodimer formation, by competition
of QscR with LasR for 3OC12-HSL or with RhlR for C4-HSL,
by both mechanisms, or in some unexplained nonspecific
We are left with the view that QscR affects the transcription
of well over 7% of the more than 5,500 genes in the P. aerugi-
nosa genome. Many of these genes are regulated by the LasR-
LasI and RhlR-RhlI quorum-sensing systems and may be in-
fluenced by QscR indirectly. Others appear to be regulated by
QscR together with the LasR-LasI signal 3OC12-HSL directly.
Furthermore, like rhlR, qscR activity is regulated by the LasR-
LasI quorum-sensing system. In the case of rhlR, LasR func-
tions at the level of transcription. In the case of QscR, the
LasR-LasI system dominates, because QscR requires the LasI-
generated quorum-sensing signal 3OC12-HSL for direct con-
trol of gene expression. We conclude that QscR, LasR, and
RhlR control overlapping but distinct regulons in P. aeruginosa
and that QscR is capable of direct activation of a group of P.
This study was supported by USPHS grant GM-59026 and by a grant
from the W. M. Keck Foundation.
We thank Jessica Linton from the University of Iowa DNA core
facility for microarray processing.
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3370 LEQUETTE ET AL.J. BACTERIOL.