Strain-dependent diversity in the Pseudomonas
aeruginosa quorum-sensing regulon
Sudha Chugania, Byoung Sik Kimb, Somsak Phattarasukola, Mitchell. J. Brittnachera, Sang Ho Choib,
Caroline S. Harwooda, and E. Peter Greenberga,1
aDepartment of Microbiology, University of Washington, Seattle, WA 98195; andbDepartment of Agricultural Biotechnology, Seoul National University,
Seoul 151-921, South Korea
Contributed by E. Peter Greenberg, August 17, 2012 (sent for review May 21, 2012)
Quorum sensing allows bacteria to sense and respond to changes
in population density. Acyl-homoserine lactones serve as quorum-
sensing signals for many Proteobacteria, and acyl-homoserine
lactone signaling is known to control cooperative activities.
Quorum-controlled activities vary from one species to another.
Quorum-sensing controls a constellation of genes in the opportu-
nistic pathogen Pseudomonas aeruginosa, which thrives in a num-
ber of habitats ranging from soil and water to animal hosts. We
hypothesized that there would be significant variation in quorum-
sensing regulons among strains of P. aeruginosa isolated from
different habitats and that differences in the quorum-sensing reg-
ulons might reveal insights about the ecology of P. aeruginosa.
As a test of our hypothesis we used RNA-seq to identify quorum-
controlled genes in seven P. aeruginosa isolates of diverse origins.
Although our approach certainly overlooks some quorum-sensing–
regulated genes we found a shared set of genes, i.e., a core quo-
rum-controlled gene set, and we identified distinct, strain-variable
sets of quorum-controlled genes, i.e., accessory genes. Some quorum-
controlled genes in some strains were not present in the genomes
of other strains. We detected a correlation between traits encoded
by some genes in the strain-variable subsets of the quorum reg-
ulons and the ecology of the isolates. These findings indicate a role
for quorum sensing in extension of the range of habitats in which
a species can thrive. This study also provides a framework for un-
derstanding the molecular mechanisms by which quorum-sensing
systems operate, the evolutionary pressures by which they are
maintained, and their importance in disparate ecological contexts.
bacterial communication|systems biology|transcription control
other and control gene expression in a cell density-dependent
manner. Many species of Proteobacteria use diffusible acyl-homo-
serine lactones (AHLs) as quorum-sensing signals. AHLs are
produced by signal synthase enzymes and are detected by signal-
specific transcriptional regulators. AHL quorum-sensing circuits
regulate a wide spectrum of phenotypes in a diverse array of α-,
β-, and γ-Proteobacteria (1). Interspecies differences in quorum
regulons often are a reflection of the diverse habitats that bac-
teria occupy, and quorum-controlled phenotypes often play a
crucial role in niche persistence. The classic example is quorum
control of luminescence in Vibrio fischeri, which allows this bac-
terium to discriminate between its free-living, low-population-
density seawater habitat and its high-density symbiotic habitats,
the light organs of certain fish and squid (2, 3). It is well estab-
lished that there are species-specific differences in quorum reg-
ulons, but there is little information regarding the possibility
of intraspecies strain-specific differences. We hypothesized that,
particularly for versatile species that occupy diverse niches, there
might be a shared core of quorum-controlled genes and, in
addition, strain-variable quorum-regulated genes that reflect
adaptations to the habitats from which strains are isolated. We
tested our hypothesis using isolates of the metabolically versatile
γ-Proteobacteria species Pseudomonas aeruginosa.
acteria use quorum-sensing signals to communicate with each
P. aeruginosa has been isolated from diverse environments. It
can be found in soil and water, as a member of the normal
microbiota of eukaryotes or as an opportunistic pathogen in
a wide range of hosts including plants and humans. Comparative
genomic analyses of multiple P. aeruginosa strains have identified
core (shared) and accessory (strain-variable) genome sequences
(4). Evidence indicates that accessory genes encode functions as-
sociated with adaptation and niche diversification (4). P. aeruginosa
has a quorum-sensing system comprising two AHL synthases and
three receptors. The LasI synthase produces 3OC12-HSL, for
which there are two receptors, LasR and QscR. The RhlI syn-
thase produces C4-HSL, for which the receptor is RhlR. There
are indications that, although the complete complement of syn-
thase and receptor genes is conserved among strains, there are
differences in the quorum-controlled genes (5), and some strains
from certain habitats contain LasR mutations (6–8).
Much of the existing data on genes controlled by quorum
sensing in P. aeruginosa derive from studies of a single laboratory
strain, PAO1 (9-11) an extensively passaged isolate from a
wound infection (12). Here we use RNA-seq to identify genes in
the quorum regulons of seven P. aeruginosa strains isolated from
disparate environments. Specifically we use strain PAO1 as a
reference. We generated and annotated draft genome assemblies
of the other six isolates. We generated lasI, rhlI quorum-sensing
mutants of each isolate and compared the transcriptomes of lasI,
rhlI mutants of all seven strains, with and without added AHLs,
to each other. As we predicted, there was a set of core quorum-
controlled genes in the core genome, and there were elements of
the accessory genome that showed quorum-sensing control.
There also were genes in the core genome that showed strain-to-
strain variation with respect to quorum-sensing control.
Quorum-Sensing Circuit Is Conserved Among Environmental and
Clinical P. aeruginosa Isolates. We examined intraspecific diversity
in quorum-regulated gene expression by examining seven
P. aeruginosa strains, including four environmental isolates,
two clinical isolates from chronic cystic fibrosis (CF) lung
infections, and the laboratory strain, PAO1 (Table S1). Some
information regarding genome content and assembly statistics
for the draft genomes is provided in Table 1, and annotations
are available at www.ncbi.nlm.nih.gov/genome. The draft genomes
Author contributions: S.C., B.S.K., S.H.C., C.S.H., and E.P.G. designed research; S.C. and B.S.K.
performed research; S.C., S.P., and M.J.B. contributed new reagents/analytic tools; S.C.,
S.P., M.J.B., C.S.H., and E.P.G. analyzed data; and S.C., C.S.H., and E.P.G. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The draft genome assemblies and annotations have been deposited in
the DNA Data Bank of Japan/European Molecular Biology Laboratory/GenBank database
(accession nos. AKZD00000000, AKZE00000000, AKZF00000000,AKZG00000000,
AKZH00000000, and AKBD00000000).
1To whom correspondence should be addressed. E-mail: email@example.com.
See Author Summary on page 16426 (volume 109, number 41).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| Published online September 17, 2012
show a pangenome for the seven strains consisting of 7,423 genes
and a shared core genome of 4,449 genes. To determine if all
strains in our panel produced the two P. aeruginosa AHLs, we
tested stationary-phase cultures by using bioassays. All strains
exhibited generally similar growth rates and produced both
3OC12-HSL and C4-HSL at micromolar levels (Fig. 1). These
data indicate that the prototypical P. aeruginosa quorum-sensing
circuit is conserved and operational in all examined strains. We
generated lasI, rhlI signal-generation mutants (Materials and
Methods) which did not produce detectable levels of AHLs.
Identification of Quorum-Sensing–Regulated Genes by RNA-Seq. As
described in the Materials and Methods, RNA-seq libraries were
generated by selective cDNA priming with a pool of hexamers,
none of which showed a perfect match to any of the P. aeruginosa
ribosomal RNAs (rRNAs). This approach enables enrichment of
non-rRNA transcripts without a ribosome-depletion step, lower-
ing RNA input requirements and simplifying sample preparation
(13, 14). In fact, depending on the isolate examined, the reads
mapping to rRNAs ranged from about 30% to about 80%.
Overall, the RNA-seq results (Table 2) revealed that at least
89% of the genes for a given strain had their coding sequence
covered, indicating that the overall genome coverage afforded by
this method was high. Further, sequencing read depth data in-
dicated that the numbers of non-rRNA reads were largely similar
for all samples, thus enabling valid comparisons across samples.
We identified 161 genes that were AHL-activated in strain
PAO1 (Dataset S1) and 15 genes that were AHL-repressed. For
this study, we focused only on the AHL-activated genes. We note
that quorum-sensing–dependent genes show variable activation
at different points in growth (10, 11), and we assessed AHL-
dependent gene expression only at one point in growth (OD600
2). To validate our RNA-seq method, we compared our results
with previous data generated with a microarray platform. We
identified 77 of the 93 genes shown previously to be AHL-
induced at OD6002 (15). This number includes 10 genes that
showed AHL induction but with very few reads (<10) in cells
grown with AHLs and 0 or 1 reads in samples from cells grown
without AHLs. Because of the low expression of these genes, we
did not include them in our further analysis of AHL-activated
genes. Of the 16 genes identified in the microarray analysis but
not in our RNA-seq analysis, nine are in operons that were
detected as AHL-induced by RNA-seq. Thus, the RNA-seq and
Affymetrix microarray platforms show excellent concordance.
Our RNA-seq analysis identified AHL-induced genes not detected
in the microarray analysis. The enhanced sensitivity of this tech-
nique likely led to the identification of the additional 84 genes.
In strain PAO1 many quorum-controlled genes are activated
by AND logic gates. Quorum sensing is required but not suffi-
cient for activation of specific genes (16, 17). Thus, we do not
expect our analysis of transcripts in cells from a single point in
the growth curve, in a single growth medium, at a single growth
temperature to generate a list of all genes activated by quorum
sensing. Rather it provides a base of information to allow a test
of our hypotheses about strain variability and core and accessory
As noted above, we identified 161 quorum-activated genes in
PAO1. The number of quorum-activated genes for the remaining
strains were 342 for the soil isolate BE171; 301 for the air isolate
BE173; 207 for the biofilm isolate BE177; 76 for the tomato
plant isolate PaE2; 153 for the CF isolate CI27; and 31 for the
CF isolate CIG1. A list of quorum-activated genes for all strains
is provided in Dataset S1. Overall, the quorum regulons repre-
sent ∼0.5–6.2% of the coding sequences for a given genome.
There is no obvious correlation between genome size and the
number of quorum-controlled genes detected.
Pairwise Comparisons Between the Quorum Regulon of Strain PAO1
and Other Strains. To get a sense of the variation in quorum-
activated genes from strain to strain and to lend some validity to
conclusions that previously have been drawn about the role of
quorum sensing in the regulation of cooperative activities in
P. aeruginosa using strain PAO1, we first performed pairwise
comparisons of the P. aeruginosa PAO1 quorum regulon with the
quorum regulons of the other strains. The largest set of quorum-
activated genes (342 genes in BE171) was more than twice the
size of the PAO1 set (161 genes), suggesting that P. aeruginosa
employs quorum sensing to regulate many more traits than
previously identified. With the exception of CIG1, which has
a relatively small quorum-controlled regulon of 31 genes, there
were pronounced overlaps between the strain PAO1 regulon and
the other regulons (Fig. 2). Thus, we consider the P. aeruginosa
PAO1 quorum regulon to be reasonably representative of the
species. Well-documented examples of some quorum-controlled
Table 1. Content and assembly statistics for the draft genomes and the PAO1 reference genome
StrainSource Size (bp) Contigs
CF chronic infection
CF chronic infection
*Details for strain PAO1, included for comparison, are from the Pseudomonas genome project (www.pseudomonas.com). G+C, guanine
plus adenosine mols percent.
were not detected in cultures of lasI, rhlI mutants of any isolate.
AHL concentrations in P. aeruginosa cultures at OD6003.5. AHLs
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| Published online September 17, 2012