Pseudomonas aeruginosa AlgR represses the Rhl quorum-sensing system in a biofilm-specific manner.
ABSTRACT AlgR controls numerous virulence factors in Pseudomonas aeruginosa, including alginate, hydrogen cyanide production, and type IV pilus-mediated twitching motility. In this study, the role of AlgR in biofilms was examined in continuous-flow and static biofilm assays. Strain PSL317 (DeltaalgR) produced one-third the biofilm biomass of wild-type strain PAO1. Complementation with algR, but not fimTU-pilVWXY1Y2E, restored PSL317 to the wild-type biofilm phenotype. Comparisons of the transcriptional profiles of biofilm-grown PAO1 and PSL317 revealed that a number of quorum-sensing genes were upregulated in the algR deletion strain. Measurement of rhlA::lacZ and rhlI::lacZ promoter fusions confirmed the transcriptional profiling data when PSL317 was grown as a biofilm, but not planktonically. Increased amounts of rhamnolipids and N-butyryl homoserine lactone were detected in the biofilm effluent but not the planktonic supernatants of the algR mutant. Additionally, AlgR specifically bound to the rhlA and rhlI promoters in mobility shift assays. Moreover, PAO1 containing a chromosomal mutated AlgR binding site in its rhlI promoter formed biofilms and produced increased amounts of rhamnolipids similarly to the algR deletion strain. These observations indicate that AlgR specifically represses the Rhl quorum-sensing system during biofilm growth and that such repression is necessary for normal biofilm development. These data also suggest that AlgR may control transcription in a contact-dependent or biofilm-specific manner.
- SourceAvailable from: Huw D Williams[Show abstract] [Hide abstract]
ABSTRACT: The opportunistic pathogen Pseudomonas aeruginosa chronically infects the airways of Cystic Fibrosis (CF) patients during which it adapts and undergoes clonal expansion within the lung. It commonly acquires inactivating mutations of the anti-sigma factor MucA leading to a mucoid phenotype, caused by excessive production of the extracellular polysaccharide alginate that is associated with a decline in lung function. Alginate production is believed to be the key benefit of mucA mutations to the bacterium in the CF lung. A phenotypic and gene expression characterisation of the stationary phase physiology of mucA22 mutants demonstrated complex and subtle changes in virulence factor production, including cyanide and pyocyanin, that results in their down-regulation upon entry into stationary phase but, (and in contrast to wildtype strains) continued production in prolonged stationary phase. These findings may have consequences for chronic infection if mucoid P. aeruginosa were to continue to make virulence factors under non-growing conditions during infection. These changes resulted in part from a severe down-regulation of both AHL-and AQ (PQS)-dependent quorum sensing systems. In trans expression of the cAMP-dependent transcription factor Vfr restored both quorum sensing defects and virulence factor production in early stationary phase. Our findings have implications for understanding the evolution of P. aeruginosa during CF lung infection and it demonstrates that mucA22 mutation provides a second mechanism, in addition to the commonly occurring lasR mutations, of down-regulating quorum sensing during chronic infection this may provide a selection pressure for the mucoid switch in the CF lung.PLoS ONE 01/2014; 9(5):e96166. · 3.53 Impact Factor
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ABSTRACT: AlgR is a key Pseudomonas aeruginosa transcriptional response regulator required for virulence. AlgR activates alginate production and twitching motility, but represses the Rhl quorum sensing (QS) system, including rhamnolipid production. The role of AlgR phosphorylation is enigmatic as phosphorylated AlgR (AlgR-P) is required for twitching motility through the fimU promoter, but is not required to activate alginate production. In order to examine the role of AlgR phosphorylation in vivo, a PAO1 algRD54E strain was created that constitutively activated fimU transcription and exhibited twitching motility. A corresponding PAO1 algRD54N strain that does not activate fimU or twitching motility was compared to PAO1, PAO1 algRD54E, PAO1 ΔalgZ (deletion of the algZ (fimS), encoding a putative histidine kinase) and PAO1 ΔalgR deletion strains for swarming motility, rhamnolipid production and rhlA transcription. PAO1 and PAO1 algRD54E produced approximately two-fold higher levels of rhamnolipids than PAO1 algRD54N and PAO1 ΔalgZ strains, thereby indicating that phosphorylated AlgR is required for normal rhamnolipid production. Examination of purified AlgR, AlgR-P, AlgRD54N and AlgRD54E showed that AlgR-P and AlgRD54E bound preferentially to the fimU and rhlA promoters. Additionally, AlgR-P bound specifically to two sites within the rhlA promoter, not bound by unphosphorylated AlgR. Taken together, these results indicate that phosphorylated AlgR-P has increased affinity for the rhlA promoter and is required for the coordinate activation of twitching motility, rhamnolipid production and swarming motility in P. aeruginosa.Journal of bacteriology 10/2013; · 3.94 Impact Factor
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ABSTRACT: A defining characteristic of biofilms is antibiotic tolerance that can be up to 1,000-fold greater than that of planktonic cells. In P. aeruginosa, biofilm tolerance to antimicrobial agents requires the biofilm-specific MerR-type transcriptional regulator BrlR. However, the mechanism by which BrlR mediates biofilm tolerance has not been elucidated. Genome-wide transcriptional profiling indicated that brlR was required for maximal expression of genes associated with antibiotic resistance, in particular those encoding the multidrug efflux pumps MexAB-OprM and MexEF-OprN. Chromatin immunoprecipitation (ChIP) analysis revealed a direct regulation of these genes by BrlR with DNA binding assays confirming BrlR binding to the promoter regions of the mexAB-oprM and mexEF-oprN operons. qRT-PCR analysis further indicated BrlR to be an activator of mexAB-oprM and mexEF-oprN gene expression. Moreover, immunoblot analysis confirmed increased MexA abundance in cells overexpressing brlR. Inactivation of both efflux pumps rendered biofilms significantly more susceptible to five different classes of antibiotics by affecting MIC but not the recalcitrance of biofilms to killing by cidal antimicrobial agents. Overexpression of either efflux pump in a ΔbrlR strain partly restored tolerance of ΔbrlR biofilms to antibiotics. Expression of brlR in mutant biofilms lacking both efflux pumps partly restored antimicrobial tolerance of biofilms to wild type levels. Our results indicate that BrlR acts as an activator of multidrug efflux pumps to confer tolerance to P. aeruginosa biofilms and to resist the action of antimicrobial agents.Journal of bacteriology 05/2013; · 3.94 Impact Factor
JOURNAL OF BACTERIOLOGY, Nov. 2007, p. 7752–7764
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 189, No. 21
Pseudomonas aeruginosa AlgR Represses the Rhl Quorum-Sensing
System in a Biofilm-Specific Manner?†
Lisa A. Morici,1Alexander J. Carterson,1Victoria E. Wagner,2Anders Frisk,1Jill R. Schurr,3
Kerstin Ho ¨ner zu Bentrup,1Daniel J. Hassett,4Barbara H. Iglewski,2
Karin Sauer,5and Michael J. Schurr6*
Department of Microbiology and Immunology, Program in Molecular Pathogenesis and Immunity, Tulane University Health Sciences
Center, New Orleans, Louisiana 701121; Department of Microbiology and Immunology, University of Rochester, Rochester,
New York 146422; Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 701123;
University of Cincinnati College of Medicine, Department of Molecular Genetics, Biochemistry, and Microbiology,
Cincinnati, Ohio 452674; Department of Biological Sciences, State University of New York at Binghamton,
Binghamton, New York 139025; and University of Colorado at Denver and Health Sciences Center,
Department of Microbiology, Aurora, Colorado 800456
Received 28 November 2006/Accepted 22 August 2007
AlgR controls numerous virulence factors in Pseudomonas aeruginosa, including alginate, hydrogen cyanide
production, and type IV pilus-mediated twitching motility. In this study, the role of AlgR in biofilms was examined
in continuous-flow and static biofilm assays. Strain PSL317 (?algR) produced one-third the biofilm biomass of
wild-type strain PAO1. Complementation with algR, but not fimTU-pilVWXY1Y2E, restored PSL317 to the wild-type
biofilm phenotype. Comparisons of the transcriptional profiles of biofilm-grown PAO1 and PSL317 revealed that a
number of quorum-sensing genes were upregulated in the algR deletion strain. Measurement of rhlA::lacZ and
rhlI::lacZ promoter fusions confirmed the transcriptional profiling data when PSL317 was grown as a biofilm, but
not planktonically. Increased amounts of rhamnolipids and N-butyryl homoserine lactone were detected in the
biofilm effluent but not the planktonic supernatants of the algR mutant. Additionally, AlgR specifically bound to the
rhlA and rhlI promoters in mobility shift assays. Moreover, PAO1 containing a chromosomal mutated AlgR binding
site in its rhlI promoter formed biofilms and produced increased amounts of rhamnolipids similarly to the algR
deletion strain. These observations indicate that AlgR specifically represses the Rhl quorum-sensing system during
biofilm growth and that such repression is necessary for normal biofilm development. These data also suggest that
AlgR may control transcription in a contact-dependent or biofilm-specific manner.
The opportunistic pathogen Pseudomonas aeruginosa is the
major cause of morbidity and mortality in patients with cystic
fibrosis (CF) (28). The factors that enable P. aeruginosa to
predominate and persist in the CF lung despite aggressive
antimicrobial therapy are numerous and include alginate pro-
duction (27), antimicrobial resistance mechanisms (20, 66),
and secreted factors (41, 56). Furthermore, several studies
suggest that P. aeruginosa persists in the CF lung as organized
communities known as biofilms (14, 75). Biofilms are com-
posed of many individual bacteria in various stages of devel-
opment and contain self-generating diversity to produce insur-
ance effects (4, 37). Bacterial biofilms are encased in an
extracellular polymeric substance (40) and are intrinsically
more resistant than planktonic organisms to innate immune
defense mechanisms and antimicrobial therapy (8, 20, 46).
To date, three exopolysaccharides associated with P. aerugi-
nosa biofilms, alginate (12), the product of psl genes (85), and
the product of pel genes (25), have been identified. Biofilms
formed by mucoid P. aeruginosa contain significant amounts of
alginate, and alginate production in mucoid strains influences
biofilm architecture (29, 54). However, others have shown that
alginate is not the predominant polysaccharide present in non-
mucoid P. aeruginosa biofilms cultured in vitro (85) and is not
required for biofilm development (76). Evidence from the ex-
isting literature indicates that alginate is most likely an exopo-
lysaccharide produced under stress by P. aeruginosa (5, 79, 84).
The conversion of nonmucoid P. aeruginosa to the alginate-
overproducing mucoid phenotype is a critical step in the patho-
genesis of CF disease and coincides with a worsening prognosis
for the CF patient (28). Thus, the activation of the alginate
biosynthetic pathway and biofilm development in P. aeruginosa
both represent a critical juncture in CF pathology.
One of the molecular mechanisms for the constitutive ex-
pression of the exopolysaccharide alginate has been discovered
and involves the alternative sigma factor, AlgU (47, 48) (also
known as AlgT ). Upon activation through mutations ac-
quired in mucA (48), alginate is produced in copious amounts
by transcriptional activation of the regulatory protein AlgR
and its subsequent upregulation of the 12 alginate biosynthetic
genes (algD through algA) (28, 49, 68–70). The transcriptional
regulator AlgR is required for algD transcription by binding to
three sites within the algD promoter (RB1, RB2, and RB3)
(51–53). Mucoid P. aeruginosa strains in which algR is dis-
rupted are no longer able to produce alginate (18).
AlgR has been shown to regulate several other P. aeruginosa
processes, including hydrogen cyanide (HCN) production (7)
* Corresponding author. Mailing address: University of Colorado at
Denver and Health Sciences Center, Department of Microbiology,
12800 E. 19th Avenue, Aurora, CO 80045. Phone: (303) 724-4224. Fax:
(303) 724-4226. E-mail: Michael.firstname.lastname@example.org.
† Supplemental material for this article may be found at http://jb
?Published ahead of print on 31 August 2007.
and twitching motility (44, 82, 83), suggesting a more global
role for AlgR in P. aeruginosa pathogenesis. In support of this,
AlgR is required for full virulence in both the acute septicemia
and pneumonia murine infection models (43). However, the
genes involved in the global affects observed in the virulence
studies have not yet been identified.
In this study, the AlgR regulon of the nonmucoid laboratory
strain PAO1 was examined during biofilm growth using flow
chamber and static biofilm technology. These findings expand
the role of AlgR as a regulator of virulence in P. aeruginosa by
demonstrating that AlgR directly represses the Rhl quorum-
sensing circuit in a biofilm-specific manner. Furthermore,
these findings support the hypothesis that AlgR may utilize
contact-dependent or biofilm-specific mechanisms of gene reg-
ulation that may account for its differential regulation of al-
ginate production, twitching motility, and biofilm maturation.
MATERIALS AND METHODS
Bacterial strains and plasmids. The bacterial strains and plasmids used in this
study are listed in Table 1. Plasmid CTXlacZ490ScaI was constructed using
oligonucleotides lacZhinD and CTXXho (Table 2) to amplify the first 490 nu-
cleotides of lacZ from plasmid pRS415 (74). The PCR product was digested with
HinDIII and XhoI and ligated into plasmid mini-CTX-1 (34) digested with the
same restriction enzymes. The resulting plasmid, CTXlacZ490, was subjected to
site-directed mutagenesis using the Stratagene Quick Change II protocol with
oligonucleotides lacZScaF’ and lacZScaR’ (Table 2) to introduce an in-frame
ScaI restriction endonuclease site into the lacZ open reading frame to create
translational fusions. Plasmid pCR2.1 rhlI was constructed by ligation of the
PCR product of oligonucleotides rhlIgsF and rhlIgsR (Table 2) into Invitrogen’s
pCR2.1 vector. Plasmid pCR2.1 rhlA was constructed by cloning the PCR prod-
uct of oligonucleotides rhlAgsF and rhlAgsR into pCR2.1.
Continuous culture biofilm growth. Flowthrough biofilms were grown in a
one-flowthrough model using Pseudomonas putida minimal medium supple-
mented with glutamate (1.6 mM) as sole carbon source as described previously
(67). Briefly, 3 ml of an overnight culture of the same medium used for the
biofilm was inoculated into a flowthrough biofilm system with a flow rate of
approximately 0.4 ml/min and grown for 6 days in minimal medium at room
temperature (?26°C). The resulting biofilm was collected in RNALater (Am-
bion) for RNA isolation.
Swarming motility assay. P. aeruginosa strains were grown in FAB (73) with
1.6 mM glucose, glutamate, or succinate medium solidified with 0.5% Noble
agar. Plates were inoculated by using a sterilized platinum wire with log-phase
cells (optical density at 600 nm [OD600] of 0.6) grown in the respective carbon
source overnight and incubated at 30°C for 24 h. The zones of migration from the
point of inoculation were measured in triplicate for each condition.
RNA isolation and preparation for Affymetrix GeneChip analysis. P. aerugi-
nosa strains PAO1 and PSL317 were grown as biofilms using the flowthrough
model described above. RNA was isolated using a CsCl gradient as previously
described (71) and analyzed with an Agilent 2100 Bioanalyzer to determine the
RNA integrity (see Fig. S1 in the supplemental material). Ten micrograms of
total RNA was used for cDNA synthesis, fragmentation, and labeling according
to the Affymetrix GeneChip P. aeruginosa genome array expression protocol.
Briefly, random hexamers (Invitrogen) were added to 10 ?g of RNA along with
in vitro-transcribed Bacillus subtilis control spikes. cDNA was synthesized using
Superscript III (Invitrogen) and the following conditions: 25°C for 10 min, 37°C
for 60 min, and 70°C for 10 min. RNA was removed by alkaline treatment and
subsequent neutralization. The cDNA was purified by using a QIAquick PCR
purification kit (QIAGEN) and eluted in 40 ?l of elution buffer (QIAGEN). The
cDNA was then fragmented by using 0.6 U DNase I (Amersham) per ?g cDNA
at 37°C for 10 min. The fragmented cDNA was end labeled with biotin-ddUTP
by using a BioArray terminal labeling kit (Enzo) per the manufacturer’s instruc-
tions. A gel shift mobility assay was performed using NeutrAvadin (Pierce) on a
TABLE 1. List of strains and plasmids used in this study
Strain or plasmid Relevant properties Reference or origin
P. aeruginosa strains
AlgR binding site mutated in rhlI promoter from CCGTTCATCC to
rhlA::lacZ promoter fusion in attB site of PAO1 chromosome
PAO1 rhlI::lacZD. Hassett;
rhlA::lacZ promoter fusion in attB site of PSL317 (?algR) chromosome
rhlI::lacZ promoter fusion in attB site of PAO1 chromosome
rhlI::lacZ promoter fusion in attB site of PSL317 (?algR) chromosome
E. coli strains
DH5??80?lacZ?M15 ?(lacZYA-argF)U169 recA1 endA1 hsdR17(rK
supE44 ??thi-1 gyrA96 relA1
thi-1 thr leu tonA lacY supE recA::RP4-2-Tc::Mu
Self-proficient integration vector with tet, ?-FRT-attP-MCS, ori, int, and oriT
Mini-CTX1; promoterless lacZ cloned between HindIII and XhoI
Mini-CTX1lacZ with ScaI site inserted at 5? end of lacZ
Translational fusion containing ?1864 to ?9 of rhlA gene
Source of Flp recombinase
pVDtac39 fimTU pilVWXY1Y2E
pCR2.1 rhlA wild-type promoter (?850 to ?1032)
pCR2.1 rhlI wild-type promoter (?19 to ?196)
pCR2.1 rhlA promoter containing CCGT to TTAC mutation in RB1
pCR2.1 rhlA promoter containing CCGT to TTAC mutation in RB2
pCR2.1 rhlA promoter containing CCGT to TTAC mutation in RB1 and RB2
pCR2.1 rhlI promoter containing CCGT to TTAC mutation in RB1
ori RSK mob sacB Apr
pCVD442 rhlI promoter containing CCGT to TTAC mutation in RB1
VOL. 189, 2007AlgR REGULATES QUORUM SENSING IN P. AERUGINOSA BIOFILMS7753
5% polyacrylamide gel stained with SYBR green (Roche) to ensure complete
fragmentation and labeling. Samples were hybridized, washed, stained, and
scanned as described in the Affymetrix GeneChip P. aeruginosa genome array
expression analysis protocol.
Microarray data analysis. The absolute expression transcript levels were nor-
malized for each chip by globally scaling all probe sets to a target signal intensity
of 500. Three statistical algorithms (detection, change call, and signal log ratio)
were used to identify differential gene expression in experimental and control
samples. The decision of a present, absent, or marginal identification for each
gene was determined by using MicroArray Suite software (version 5.0; Af-
fymetrix). Those transcripts that received an “absent” designation were removed
from further analysis. A t test was used to isolate those genes whose transcrip-
tional profile was statistically significant (P ? 0.05) between the control and
experimental conditions. Pair-wise comparisons between the individual experi-
mental and control chips were done by batch analyses using MicroArray Suite to
generate a change call and signal log ratio for each transcript. A positive change
was defined as a call whereby more than 50% of the transcripts increased or
marginally increased for up-regulated genes or decreased or marginally de-
creased for down-regulated genes. Lastly, the median value of the signal log
ratios for each comparison was calculated and only transcripts that had a value
greater than or equal to 1 for up-regulated and less than or equal to 1 for
down-regulated genes were placed on the final list of transcripts whose profile
had changed. The signal-log ratio was converted and expressed as the change
Biofilm imaging. P. aeruginosa PAO1, PSL317, PSL317 (pVDtacPIL), PSL317
(pVDZ’2R), and PAO1rhlImut were grown in a flowthrough biofilm as described
above and imaged with the aid of an image chamber (Stovall, Inc.). An overnight
culture of 3 ml of the individual strains grown in Pseudomonas minimal medium
(see above) was inoculated into a flowthrough biofilm system with a flow rate of
approximately 0.4 ml/min. The biofilms were grown for 1, 3, or 6 days in minimal
medium supplemented with 130 mg/liter of glutamate as a carbon source. The
bacteria were stained with LIVE/DEAD BacLight (Molecular Probes). Z-section
images were collected on a Zeiss Axioplan II microscope (step size, 0.1 to 0.2
?m; magnification, ?630) using Slidebook 4.0 as the imaging software (Intelli-
gent Imaging Inc., Denver, CO). Postacquisition images were processed using
Volocity software (Improvision, Ltd., Lexington, MA). Quantitative analysis of
the flow cell-grown biofilms was performed with the COMSTAT image analysis
software package (31).
Ninety-six-well-plate biofilm assay. The 96-well biofilm assay was performed
as previously described (23) with the following modifications. Briefly, biofilm
formation was assayed by the ability of cells to adhere to the wells of 96-well
microtiter plates (Becton Dickinson Labware). Overnight cultures grown in the
minimal medium supplemented with glutamate (1.6 mM) used for the continu-
ous-flow biofilm of PAO1, PSL317, PSL317 (pVDtacPIL), and PSL317
(pVDZ’2R) were diluted 1:100 in fresh minimal medium and inoculated into the
96-well plate. The plates were incubated at 25°C for 24 h to allow for biofilm
formation. After 24 h, the plates were washed once in ddH2O and then a solution
of 1% crystal violet was added to stain the cells. The plates were set aside for 10
min and washed three times to remove any residual crystal violet. A solution of
33% acetic acid was added to each well to lyse the bacterial cells and solubilize
the crystal violet. The absorbance was determined at 580 nm in a ?Quant
microtiter plate reader (Biotek Instruments, Inc.). The assays were performed in
triplicate with five technical replicates (wells) for each replicate.
Promoter fusion assays. A PCR product generated using oligonucleotides
rhlAF1 and rhlAR1 (Table 2) encompassing ?1864 to ?9 bp relative to the
translational start of rhlA was amplified with SmaI and SacI restriction sites and
directionally cloned into CTXscaI-lacZ to generate a translational fusion. The
final construct, CTXrhlA::lacZ, was sequenced before use in P. aeruginosa. Esch-
erichia coli DH5? harboring the plasmid CTXrhlA::lacZ was used in triparental
conjugations with either PAO1, PSL317, or PSL317 (pVDZ’2R) and the helper
strain, DH5? pRK2013 (22). Conjugants were screened by PCR for each pro-
moter and the plasmid backbone was removed by pFlp2-mediated excision (33).
Integration at the attB site was confirmed by PCR using attB-specific primers and
Southern blotting. PAO1 containing the rhlI::lacZ chromosomal fusions was
generously provided by Daniel Hassett. The deletion of algR in PAO1 containing
the chromosomal fusion genes was performed as previously described (44) and
confirmed by Southern blotting.
?-Galactosidase assays were performed as described by Miller (50), with slight
modification. All assays were performed on P. aeruginosa strains grown for 6 days
as continuous culture biofilms in the flow cell model or to stationary phase in P.
putida minimal medium broth at room temperature at 250 rpm. After the indi-
cated incubation period, bacteria were removed by scraping and resuspended in
200 ?l phosphate-buffered saline. An amount of 800 ?l Z-buffer (60
mMNa2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, and 50 mM
?-mercaptoethanol) was added, and the OD600of each suspension was mea-
sured. Amounts of 20 ?l chloroform and 20 ?l 0.1% sodium dodecyl sulfate were
added to each tube and vortexed for 10 s. An amount of 200 ?l of orthonitro-
phenyl galactoside (4 mg/ml in H2O) was added, and the reaction was allowed to
proceed for 1 to 3 min (determined empirically for each promoter). The reaction
was stopped by the addition of 250 ?l 1 M NaHCO4. Samples were centrifuged
to pellet the cells and chloroform, and the supernatant was measured at 420 nm.
P. aeruginosa strains harboring a promoterless lacZ gene in the attB site were
used as negative controls in every experiment to determine background ?-galac-
TABLE 2. Oligonucleotides used in this study
Name Oligonucleotide sequence (5? to 3?) Gene Location (strand)
SDM of RB1 and RB2
SDM of RB1 and RB2
?873 to ?850 (?)
?1032 to ?1008 (?)
?196 to ?172 (?)
?19 to ?2 (?)
?976 to ?952 (?)
?763 to ?787 (?)
?912 to ?955 (?)
?955 to ?912 (?)
?143 to ?110 (?)
?110 to ?143 (?)
?634 to ?610 (?)
?390 to ?500 (?)
?149 to ?122 (?)
?126 to ?151 (?)
?634 to ?610 (?)
?9 to ?11 (?)
?1864 to ?1844 (?)
aEMSA, electrophoretic mobility shift assay; SDM, site-directed mutagenesis; PAO1rhlImut, PAO1rhlImut strain construction; plasmid, complementation or fusion
bUnderlined bases changed to alter AlgR binding site in rhlI promoter.
cSmaI restriction site underlined.
dXbaI restriction site underlined.
eHindIII restriction site underlined.
fXhoI restriction site underlined.
gScaI restriction site underlined.
7754MORICI ET AL.J. BACTERIOL.
Autoinducer quantitation. The concentrations of N-3-oxododecanoyl-L-homo-
serine lactone (3-oxo-C12-HSL) and N-butyryl homoserine lactone (C4-HSL)
were assayed in each sample in triplicate using previously described bioassays
Rhamnolipid quantitation. The concentrations of rhamnolipids in the efflu-
ents of biofilm-grown or supernatants of broth-cultured P. aeruginosa PAO1,
PSL317 (?algR), or PSL317 harboring pVDZ’2R were determined by the orcinol
method (78). Briefly, 500 ?l of three independently grown biofilm or broth
cultures was extracted twice with 1 ml of diethyl ether. The ether fractions were
pooled and evaporated to dryness. One milliliter of a 0.19% orcinol (in 53%
H2SO4) solution was added to each sample. The samples were heated to 80°C for
30 min and cooled at room temperature for 15 min, and the absorption was
measured at 421 nm by UV spectrophotometer. The concentration of rhamno-
lipids was calculated by comparing the data with 0 to 50 ?g/ml rhamnose stan-
dards. Standards, blanks, and unknowns were analyzed in triplicate from three
AlgR gel mobility shift assay. The binding of AlgR to the rhlA and rhlI
promoter regions was examined by using recombinant AlgR, expressed as pre-
viously described (51). A 175-bp DNA fragment of the rhlA promoter (?847 to
?1022 in relation to the translational start site) was excised from pCRrhlA by
using EcoRI and gel purified. A 174-bp DNA fragment of the rhlI promoter (?19
to ?196 in relation to the translational start site) was excised from pCRrhlI by
using EcoRI and gel purified. The RB1 promoter fragment of algD (52) was
utilized as a positive control. The fragments were end labeled with [?-32P]ATP
(6,000 Ci/mmol; NEN Dupont) using T4 polynucleotide kinase (Invitrogen,
Carlsbad, CA). The probes were purified by being passed through a G-25 Seph-
adex microspin column (Amersham Pharmacia Biotech, Inc., Piscataway, NJ).
Binding reactions were carried out as described previously, with some modifica-
tions (51). Briefly, the probes were mixed with 200 pmol of purified AlgR
containing 25 mM Tris-HCl (pH 8.0), 0.5 mM dithiothreitol, 20 mM KCl, 0.5 mM
EDTA, 5% glycerol, 10 ?g salmon sperm DNA, and an additional 0.25 ?g of
poly(dI-dC) per ml as nonspecific competitor DNA. Competition assays were
performed by the addition of 1, 5, and 10 ?g of unlabeled rhlA or rhlI fragments
or 10 ?g of poly(dI-dC) to determine the specificity of AlgR. In addition,
mutagenesis of the AlgR consensus sequence (CCGTTCGTCC) in rhlA RB1
(?920 to ?939 relative to the translational start), rhlA RB2 (?936 to ?955), and
rhlI RB1 (?134 to ?115) was performed according to Mohr et al. (52) using a
QuikChange II mutagenesis system (Stratagene) and oligonucleotides rhlaNBFor
and rhlaNBRev (Table 2) for rhlA and rhlINBFor and rhlINBRev (Table 2) for
rhlI to generate plasmids pCRrhlARB1M, pCRrhlARB2M, pCRrhlARB1&2M,
and pCRrhlIRB1M (Table 1). The mutation of the rhlA AlgR consensus se-
quence CCGTTCGTCC to TTACTCGTCC was confirmed by DNA sequencing
of the gel shift fragments. After incubation for 10 min at room temperature, the
samples were separated by electrophoresis on a 5% native polyacrylamide gel
with Sharp’s buffer (6.7 mM Tris-HCl [pH 8.0], 3.3 mM sodium acetate, 1.0 mM
EDTA) used as running buffer for approximately 1.5 h at 30 mA. Subsequently,
the gel was dried and bands were visualized by autoradiography.
Mutagenesis of the rhlI promoter. The construct for mutation of the rhlI
chromosomal AlgR binding site was constructed in vitro by crossover PCR (32).
Two initial PCRs were performed, the first using forward primer RP1 and
reverse primer RP2 and the second using forward primer RP3 and reverse
primer RP4 (Table 2). The products of these reactions have complementary
sequences (the 5? end of primer RP2 is complementary to primer RP3) and
contain the desired mutation TTAC. These products were used as the template
for a subsequent crossover PCR using primers RP1 and RP4. This resulting
product was digested with SacI and XbaI and cloned directionally into the suicide
vector pCVD442 (19) to create plasmid pCVD442rhlImut. The plasmid
pCVD442rhlImut was introduced into PAO1 by triparental conjugation. Single
recombinants were selected by screening for carbenicillin resistance. The allelic
exchange (second) recombination event was induced by selection for sucrose
resistance. Several clones were selected for DNA sequencing to confirm the
Statistical analyses. Statistics on the lacZ reporter assays, autoinducer quan-
titations, rhamnolipid determinations, and elastolytic assays were performed
with one-way analysis of variance (ANOVA) with Tukey’s correction. Statistics
on biofilm key variables were done with COMSTAT (31).
Biofilm maturation is dependent on AlgR. Previous work
indicated that an algR mutant was deficient in biofilm forma-
tion in a static biofilm model up to the 8-h time point (83). In
order to further examine this phenotype, wild-type PAO1 and
its isogenic algR deletion strain PSL317 (Table 1) were grown
for 6 days in a continuous-flow system and the biofilm devel-
opment was imaged at days 1, 3, and 6. In the flow chamber
biofilm system, P. aeruginosa was grown under hydrodynamic
conditions with a continuous nutrient supply and glutamate as
the carbon source (31, 38, 83). As shown in Fig. 1A and B,
wild-type PAO1 and the algR mutant strain (PSL317) formed
similar biofilms after 24 h of culture. However, by day 3, the
PSL317 biofilm was greatly reduced in biomass and thickness
compared to those of wild-type PAO1 (Fig. 1C, D, E, and F) as
measured by COMSTAT (Table 3). By day 6, PAO1 formed
the characteristic column-like macrocolonies surrounded by
fluid-filled channels (Fig. 2A and B). In contrast, the algR
mutant contained sparsely distributed microcolonies (Fig. 2C
and D). Furthermore, day 3 and day 6 ?algR biofilms were
significantly decreased in total biomass (P ? 0.001), average
FIG. 1. Effects of algR deletion on 1- and 3-day continuous-flow
biofilms. Three-dimensional reconstructions of Z-section images taken
at 1 day (A and B) or 3 days (C to F). PAO1 and PSL317 (?algR) were
grown in an imaging flow chamber as continuous-culture biofilms.
Postacquisition deconvolution and three-dimensional rendering were
performed with Volocity (Improvision, Lexington, MA). XY, top view
(A to D); XZ, side view (E to F); magnification, ?630. The biofilms
were stained with LIVE/DEAD BacLight (Molecular Probes) for vi-
TABLE 3. COMSTAT analyses of P. aeruginosa strains grown as
no. of days
11.74 ? 0.92
15.35 ? 1.76
12.18 ? 1.1
15.08 ? 1.96
14.9 ? 0.86
28.20 ? 1.75
0.31 ? 0.03
0.70 ? 0.12
5.32 ? 0.99b
4.44 ? 1.01b
6.28 ? 1.01b
6.43 ? 1.06b
10.9 ? 0.95
11.39 ? 1.05b
0.78 ? 0.03b
0.67 ? 0.05
64.24 ? 0.58b
5.44 ? 1.51b
10.62 ? 1.67b
0.95 ? 0.15
614.41 ? 1.69 14.93 ? 1.7024.08 ? 3.62 0.74 ? 0.04
6 1.50 ? 0.11b
1.60 ? 0.24b
9.0 ? 0.86b
1.40 ? 0.18b
aAll values are means ? standard deviations.
bP ? 0.001; one-way ANOVA with Tukey-Kramer posttest.
VOL. 189, 2007AlgR REGULATES QUORUM SENSING IN P. AERUGINOSA BIOFILMS7755
thickness (P ? 0.001), and maximum thickness (P ? 0.001; day
6 only) compared to those of PAO1 biofilms as determined by
COMSTAT analysis (Table 3).
It has been previously demonstrated that algR mutants are
defective in type IV pilus-mediated twitching motility (82, 83)
and that AlgR activates twitching motility via the fimTU-
pilVWXY1Y2E operon (44). In order to determine if the defect
in twitching motility in the algR mutant could account for the
defects observed in biofilm formation, we complemented
PSL317 in trans with the fimTU-pilVWXY1Y2E operon which
restored twitching motility (44). It has also been shown that
twitching and flagellar motility are required for swarming mo-
tility (39) and proper biofilm formation (73). The ?algR mu-
tant is defective for swarming motility, and complementation
with the plasmid pVDtacPIL harboring the fimTU and
pilVWXY1Y2E genes restored normal swarming activity (Table
4). However, complementation with pVDtacPIL did not re-
store the ?algR static or flowthrough biofilms to the wild-type
phenotype (Fig. 2E and F and 3; Table 3), whereas com-
plementation with algR did (Fig. 2G and H and 3A and B;
Table 3). It has also been shown that biofilm phenotypes are af-
fected by the composition of nutrients used to grow the biofilm (38,
58, 73). However, at least for the static biofilm assay, the same
phenotype for the algR biofilm was observed in LB as in minimal
medium with glutamate (Fig. 3B). This suggests that AlgR controls
genes in addition to those involved in twitching motility and alginate
production which may be responsible for the defects in biofilm for-
mation in algR-deficient strains.
Transcriptional profile of the AlgR regulon during contin-
uous culture biofilm growth. In order to determine which
AlgR-dependent genes were responsible for the defective bio-
film phenotype observed, global transcriptional analyses were
performed on wild-type (PAO1) and algR deletion (PSL317)
strains in the day 6 biofilms. Microarray analysis of PAO1 and
PSL317 grown as continuous culture biofilms identified 765
genes that were differentially regulated by at least twofold and
whose differences were statistically significant (P ? 0.05; see
the supplemental material). The most highly up-regulated
genes in the ?algR biofilm were classified as phage/transposon
genes (42 genes), putative enzymes (33 genes), and secreted
factors (24 genes). In contrast, genes involved in motility and
attachment (18 genes) and transcriptional regulation (36
genes) were largely down-regulated (Fig. 4). Interestingly, of
the 76 genes represented in the quorum-sensing regulon by
FIG. 2. Effects of algR deletion on 6-day continuous-flow biofilm. Three-dimensional reconstructions of Z-section images taken at 6 days.
PAO1, PSL317, PSL317 (pVDtacPIL), and PSL317 (pVDZ’2R) were grown in an imaging flow chamber as continuous-culture biofilms.
Postacquisition deconvolution and three-dimensional rendering were performed with Volocity (Improvision, Lexington, MA). The biofilms were
stained as described in the Fig. 1 legend. (A, C, E, and G) Side view. (B, D, F, and H) Top view. Magnification, ?630. pVDtacPIL, plasmid
pVDtac39 containing the genes fimTU and pilVWXY1Y2E; pVDZ’2R, plasmid pVDZ’2 with algR.
FIG. 3. Effects of algR deletion on static biofilm formation. Static
biofilm assay in minimal medium supplemented with 1.6 mM gluta-
mate (A) or LB (B) of PAO1, PSL317, PSL317 (pVDtacPIL), PSL317
(pVDZ’2R), and PAO1 mutrhlI cultured for 24 h. The assays were
performed in triplicate with five technical replicates (wells) for each
replicate.*, P ? 0.001 (one-way ANOVA with Tukey’s correction).
Error bars show the standard errors of the means.
TABLE 4. Swarming motility assays on various carbon sources
Zone of migration ona:
3.8 ? 0.03
1.0 ? 0.1b
3.4 ? 0.1
3.7 ? 0.1
2.5 ? 0.3
3.5 ? 0.2
0.9 ? 0.03b
2.4 ? 0.1
3.1 ? 0.4
2.5 ? 0.3
3.4 ? 0.1
0.9 ? 0.0b
2.3 ? 0.1
3.7 ? 0.1
2.4 ? 0.1
aThe concentration of the carbon source in the swarming plates was 1.6 mM.
bP ? 0.05 compared to the results for PAO1; one-way ANOVA with Tukey-
7756 MORICI ET AL. J. BACTERIOL.
three independent studies (30, 72, 81), 44 (58%) were regu-
lated by AlgR during biofilm growth (Fig. 5 and Table 5).
Moreover, with the exception of three genes, the quorum-
sensing genes identified in the ?algR biofilm transcriptional
profile were not present in three separate AlgR-profiling ex-
periments that utilized planktonic growth conditions (44), sug-
gesting that AlgR regulation of these quorum-sensing genes
may be biofilm specific (Fig. 5). Most of the genes belonging to
the AlgR and quorum-sensing regulons remain to be charac-
terized, while 23 of the genes have been well described (Table
5)—for example, the two most highly regulated quorum-sens-
ing genes identified, rhlA and rhlB, which were up-regulated
111- and 79-fold, respectively, in the algR deletion strain and
are tightly regulated by the RhlR-RhlI tandem (55). The LasR-
LasI-dependent genes lasA and lasB (80) were also signifi-
cantly elevated in PSL317 (4- and 10-fold, respectively). In
addition, the HCN synthesis genes hcnABC (65) were signifi-
cantly upregulated (three-, seven-, and fourfold, respectively)
in the ?algR strain. rsaL, the negative regulator of lasI (15),
was significantly down-regulated, by fivefold, in the algR dele-
tion mutant (Table 5). The vast number of quorum-sensing
genes that were differentially regulated in the ?algR biofilm, but
not in algR mutants grown planktonically, indicated that AlgR
may regulate quorum sensing in a biofilm-specific manner.
Quorum sensing is regulated by AlgR. Table 5 lists 44 genes
identified in the array analysis that also belong to the quorum-
sensing regulon according to three independent analyses (30,
72, 81). Of the 23 genes that are well characterized, three genes
(pqsA, rsaL, and phzB) and two operons (hcnABC and rhlAB)
contain putative AlgR binding sites in their promoter regions.
Interestingly, the genes involved in rhamnolipid biosynthesis
(rhlA and rhlB) demonstrated the highest severalfold changes
in the transcriptome analysis (111- and 79-fold, respectively).
Several studies have demonstrated that the levels of rhamno-
lipids can influence biofilm architecture and composition (3,
10, 36). Therefore, it was hypothesized that AlgR repression of
rhlAB may be required for normal biofilm development in the
continuous-culture model. In order to confirm the array data,
an rhlA::lacZ translational fusion was constructed and inte-
grated as a single copy on the chromosome of the ?algR and
wild-type strains. No difference in ?-galactosidase activity was
observed when PAO1 and PSL317 were grown in minimal
medium broth (Fig. 6A). However, rhlA expression was signif-
icantly increased in PSL317 compared to its expression in
PAO1 when the strains were cultured for 6 days in continuous
culture biofilms (P ? 0.05) (Fig. 6A). Both LasR-3-oxo-C12-
HSL and RhlR-C4-HSL can regulate rhlAB expression. There-
fore, the ability of AlgR to regulate either of these regulatory
genes was also explored. Although rhlI was not identified in the
transcriptome analysis, its promoter region contains a perfect
AlgR consensus sequence located at ?133 to ?117 relative to
the translational start site (Fig. 7E). Therefore, the expression
of a rhlI::lacZ transcriptional fusion (as well as lasI::lacZ ex-
pression, for comparison) in PAO1 and PSL317 was examined
to determine the effect of AlgR on the autoinducer synthases.
The expression of rhlI in PSL317 was significantly increased
(P ? 0.01) under biofilm growth conditions but not in broth
(Fig. 6B). In contrast, no difference between PAO1 and
PSL317 in lasI transcription was observed when grown as a
biofilm or planktonically (data not shown). These data suggest
that AlgR represses Rhl but not Las quorum sensing in a
Production of C4-HSL is repressed by AlgR in a biofilm. In
order to confirm the results of the lacZ promoter fusion ex-
pression studies, the levels of C4-HSL and 3-oxo-C12-HSL pro-
duced by PAO1 and PSL317 grown in aerobically shaken min-
imal medium broth and in continuous-flow biofilms were
compared. The amount of C4-HSL was increased nearly five-
FIG. 4. Functional classification analysis of P. aeruginosa PSL317 versus PAO1 biofilm Affymetrix transcriptional profiling. Functional classi-
fication was performed on genes that had twofold or higher expression in the PSL317 (?algR) than PAO1 continuous-flow biofilm culture grown
for 6 days. All genes that had a significant difference in expression (P ? 0.05, as determined by one-way ANOVA) were included. Functional classes
were determined using the Pseudomonas Genome Project website (www.pseudomonas.com).
VOL. 189, 2007 AlgR REGULATES QUORUM SENSING IN P. AERUGINOSA BIOFILMS 7757
fold (P ? 0.01) in PSL317 compared to the amount in PAO1
in 6-day biofilm effluents (Fig. 6D). These differences were less
apparent in planktonically grown P. aeruginosa. PSL317 pro-
duced approximately twofold more C4-HSL (P ? 0.05) than
PAO1 in minimal medium broth. No significant difference in
3-oxo-C12-HSL levels was observed between strains PAO1 and
PSL317 during biofilm or planktonic growth (data not shown).
These results indicate that AlgR represses the production of
the Rhl autoinducer C4-HSL, but not 3-oxo-C12-HSL, during
Rhamnolipid production was repressed by AlgR in a bio-
film. Since rhlA and rhlB were the most significantly increased
quorum-sensing genes in ?algR biofilms, the concentrations of
rhamnolipids in day 6 biofilm effluents and in supernatants of
broth-grown P. aeruginosa were measured. Rhamnolipid con-
centrations were significantly higher (P ? 0.001) in PSL317
than in PAO1 when grown as biofilms. In contrast, no differ-
ences in rhamnolipid production were observed when PAO1
and PSL317 were cultured in minimal medium broth (Fig. 6C).
These results were in agreement with the elevated levels of
C4-HSL observed in ?algR biofilms and indicate that AlgR
FIG. 5. AlgR regulates quorum-sensing (QS) genes in a biofilm.
Venn diagram of differentially expressed (at least twofold; P ? 0.05)
genes in an ?algR (PSL317; lightest gray) biofilm. The transcriptomes
of three previous quorum-sensing analyses (30, 72, 81) contained 76
genes in common. The PSL317 biofilm global transcriptional analyses
showed that 44 of these genes were within the AlgR regulon. Previous
analyses of the AlgR regulon using planktonic growth conditions iden-
tified only two QS genes when PSL317 was grown to mid-log phase
(A), one QS gene when grown to stationary phase (B), and two QS
genes when AlgR was overexpressed (C) (44).
TABLE 5. Quorum-sensing genes regulated during biofilm
growth by AlgR
Planktonic fold change
2 (SP), 5 (OE)
aNC, no change in gene expression. Change (n-fold) was determined by
comparing the transcription of the algR deletion strain PSL317 to that of wild-
type PAO1 during planktonic growth to either mid-logarithmic phase (ML;
OD600? 0.4) or stationary phase (SP; OD600? 0.6) or by comparing PAO1
overexpressing (OE) AlgR from the plasmid pCMR7 to wild-type PAO1 grown
to mid-log phase (OD600? 0.4) (Lizewski et al. ?44?).
bChange (n-fold) was determined by comparing the transcription of the algR
deletion strain PSL317 to that of wild-type PAO1 during continuous culture
biofilm growth for 6 days (this study).
7758MORICI ET AL. J. BACTERIOL.
represses the Rhl quorum-sensing cascade in continuous-cul-
The gene encoding elastase, lasB, was also upregulated (10-
fold) in the ?algR mutant in a biofilm. This suggested that
AlgR either directly or indirectly (i.e., via Rhl) regulated elas-
tase production. Therefore, the amounts of elastolytic activity
in effluents from PAO1 and PSL317 continuous-culture bio-
films were measured after 6 days of growth. Elastolytic activity
was twofold higher in the ?algR mutant (P ? 0.001; data not
shown). The elastolytic activity was also tested in the superna-
tants of PAO1 and PSL317 grown in minimal medium broth
overnight. Supernatants from planktonic PSL317 had 1.5-fold-
higher elastolytic activity than the wild-type or complemented
strains, but the differences were less significant than those from
biofilms (P ? 0.01; data not shown). These results suggest that
AlgR represses lasB gene transcription and downstream elas-
tase production, possibly via its repression of the Rhl quorum-
sensing circuit (62).
AlgR binds to the rhlA and rhlI promoters. Since rhlA and
rhlI transcription was repressed by AlgR in the biofilms, the
promoter regions of each gene were examined for putative
AlgR binding sites. The rhlI promoter region contains a nearly
perfect AlgR recognition sequence located at ?129 to ?120
relative to the translational start site (Fig. 7E). The rhlA pro-
moter region contains two putative overlapping AlgR binding
sites, RB1 located at ?925 to ?934 and RB2 at ?940 to ?949
relative to the translational start. Therefore, the ability of AlgR
to bind each of these sites in an in vitro gel mobility shift assay
was tested. As shown in Fig. 7A and B, specific complexes were
formed when AlgR was added to the radioactive rhlA and rhlI
DNA fragments. The addition of nonradioactive specific com-
petitor reduced the amount of probe shifted by AlgR in a
dose-dependent manner (Fig. 7A and B; lanes 2 to 4), indicat-
ing the specificity of AlgR for both the rhlA and rhlI promoter
region. Furthermore, mutation of the AlgR consensus se-
quence of conserved nucleotides CCGT (52) to TTAC within
the rhlI promoter completely abolished AlgR binding (Fig.
7D). When the same site-directed mutagenesis was performed
on the two putative AlgR binding sites in the rhlA promoter,
mutation of RB1 but not RB2 resulted in a loss of mobility
shift (Fig. 7C). These results provide in vitro evidence that
AlgR binds specifically to the rhlI and rhlA promoters and lend
support to the hypothesis that AlgR directly controls rhlA and
Mutation of the rhlI promoter AlgR binding site on the
PAO1 chromosome inhibits biofilm formation. In order to
determine if AlgR repression of the Rhl quorum-sensing sys-
tem was required for normal biofilm development under the
conditions examined, the AlgR binding site within the rhlI
promoter was mutated in vitro and allelic exchange was used
FIG. 6. AlgR represses Rhl quorum sensing in a biofilm. ?-Galactosidase assay of rhlA-lacZ (A) and rhlI-lacZ (B) single-copy chromosomal
promoter fusions in PAO1, PSL317, and PSL317 (pVDZ’2R) cultured in broth or as biofilms. Measurement of rhamnolipids (C) and autoinducer
C4-HSL (D) in biofilm effluent and broth supernatants of PAO1, PSL317, and PSL317 (pVDZ’2R). The assays were all performed in triplicate.
*, P ? 0.05;**, P ? 0.01;***, P ? 0.001 (one-way ANOVA with Tukey’s correction). Error bars show the standard errors of the means.
VOL. 189, 2007AlgR REGULATES QUORUM SENSING IN P. AERUGINOSA BIOFILMS7759
to place it on the chromosome of PAO1. Mutation of the
wild-type sequence CCGT to TTCA was confirmed by DNA
sequencing. Gel mobility shift assays confirmed that AlgR
did not bind this mutated sequence (Fig. 7D), and therefore,
AlgR repression of rhlI should be abolished in the mutated
strain. When PAO1 containing the mutated rhlI promoter
(PAO1rhlImut) was grown for 6 days as a continuous-cul-
ture biofilm, the resulting phenotype was similar to that of
the algR mutant strain. PAO1rhlImut biofilms were signifi-
cantly reduced in total biomass, average thickness, and max-
imum thickness and were significantly rougher than wild-
type biofilms (Fig. 8 and Table 3). In addition, this strain
produced elevated amounts of rhamnolipids compared to
the levels in PAO1, similar to the algR mutant (Fig. 6C).
These results demonstrate that AlgR regulation of rhlI is
direct and confirm that AlgR repression of the Rhl quorum-
sensing circuit during biofilm growth is essential for proper
In this report, evidence is presented demonstrating that the
P. aeruginosa virulence regulator AlgR controls biofilm matu-
ration by repressing the Rhl quorum-sensing system in P.
aeruginosa PAO1. An indication that AlgR may be required for
biofilm initiation in the static biofilm model was previously
demonstrated when an algR mutant formed one-third the bio-
mass of wild-type P. aeruginosa after 8 h of static culture (83).
In contrast to the reported observations in the 8-h static bio-
FIG. 7. AlgR binds to the rhlA and the rhlI promoters. (A and B) Gel mobility shift assays with 200 pmol of recombinant AlgR incubated with labeled
rhlA (A) and rhlI (B) promoter fragments containing putative AlgR binding sites (sequences shown in panel E). ?, probe alone; ?, probe plus AlgR.
Lane 1, 10 ?g nonspecific competitor; lanes 2 to 4, 1, 5, and 10 ?g specific competitor; lane 5, cell extract of empty vector. (C and D) Gel mobility shift
assay of rhlA wild-type (RB1&2) and rhlA mutant promoter fragments (RB1M, RB2M, and RB1&2M) (C) and rhlI wild-type (RB1) and rhlI mutant
(RB1M) (D) promoter fragments. Lane 1, probe alone; lane 2, 200 pmol of purified AlgR; lane 3, 200 pmol of empty vector extract. (E) Alignment of
the AlgR binding sites within the algD, algC, hcnA, rhlA, and rhlI promoters. Numbering is from the translational start of each gene.
FIG. 8. Effect of mutation of the AlgR binding site in the rhlI
promoter of PAO1. Three-dimensional reconstructions of Z-section
images taken at 6 days. PAO1 and PAO1rhlImut were grown in an
imaging flow chamber as continuous-culture biofilms. Postacquisition
deconvolution and three-dimensional rendering were performed wth
Volocity (Improvision, Lexington, MA). (A and C) Top view. (B and
D) Side view. Magnification, ?630. The biofilms were stained as de-
scribed in the Fig. 1 legend.
7760MORICI ET AL.J. BACTERIOL.
film system (83), we report that the P. aeruginosa algR deletion
mutant displayed adherence and initial colonization of the
flowthrough cell similar to that of the wild type up to 24 h,
but abnormal biofilm formation was observed after 3 days of
Possible explanations for the altered biofilm phenotype ob-
served for the algR mutant include a twitching motility defect,
altered swarming motility, and increased rhamnolipid secre-
tion. Type IV pilus-mediated twitching plays a role in biofilm
development by enabling P. aeruginosa microcolonies to spread
over the substratum (38, 59). Mutants that were defective in
twitching motility were not impaired in the early stages of
biofilm development but eventually formed biofilms distin-
guishable from those of wild-type organisms in a flow chamber
system like the one used in this study (31, 38, 59). AlgR con-
trols twitching motility, and this control is likely dependent
upon phosphorylation (82, 83). In addition, the expression of
the fimTU-pilVWXY1Y2E operon alone can complement the
algR twitching motility defect, strongly indicating that AlgR
controls this operon and that lack of its expression results in
loss of twitching motility (1, 44). Under the conditions tested,
the biofilm formed by the algR deletion strain displayed one-
third less biomass and an inability to form the columnar architec-
ture typical of mature wild-type biofilms (11). When twitching
motility was restored by the introduction of the fimTU-
pilVWXY1Y2E operon in trans, normal biofilm maturation was
not restored after 6 days of culture. Therefore, twitching mo-
tility likely did not play a significant role in the biofilm defect
observed for the algR mutant. It has also been shown that
quorum sensing affects biofilm formation in a nutritionally
dependent fashion through swarming motility (73). Since the
algR biofilm phenotype was not dependent upon nutrition, as
rich and minimal medium biofilms resulted in the same phe-
notype (Fig. 3), and complementation of swarming motility
with the fimTU-pilVWXY1Y2E operon (Table 4) did not com-
plement the algR biofilm defect (Fig. 2E and F), the deletion of
algR increased rhamnolipid production (Fig. 6C) which re-
sulted in the altered biofilm phenotype observed.
Microarray analyses were utilized to determine which AlgR-
controlled genes did affect biofilm formation, and a large num-
ber of known quorum-sensing genes were identified. Based
upon these results, the possibility that coordinate regulation of
quorum-sensing circuits and the AlgR regulatory network is
necessary for successful biofilm development was explored.
Interestingly, one previous report has indicated that there may
be a connection between AlgR and the Rhl quorum-sensing
system, when the culturing of an rhlI mutant resulted in com-
pensatory mutations in algR (2). P. aeruginosa currently has
three identified types of quorum-sensing systems: (i) the Las
system which produces 3-oxo-C12-HSL (60); (ii) the Rhl system
which produces C4-HSL (61); and (iii) the PQS system which
produces 2-heptyl-3-hydroxy-4-quinolone (63). Disruption or
alteration of the quorum-sensing regulatory cascade in P.
aeruginosa has been shown to interfere with normal biofilm
architecture and development (10, 14, 36). However, others
have reported no difference between wild-type biofilms and
biofilms defective in the Las quorum-sensing system (31, 77).
These inconsistencies may be attributed to differences in P.
aeruginosa strains and culture conditions. One recent report
indicates that the expression of the Las and Rhl quorum-
sensing systems is clearly dependent upon growth conditions
(21). The Rhl quorum-sensing system has also been studied
extensively in biofilms (10, 42). Rhamnolipid surfactants under
the control of the Rhl quorum-sensing system are essential for
proper maintenance of water channels and biofilm architecture
(10), and there is evidence that rhamnolipid expression occurs
after stalks have formed but before the capping in of the
mushroom-like structures (42).
The ability of AlgR to directly regulate Rhl quorum sensing
most likely explains the biological properties of the ?algR
strain during biofilm growth. Such dysregulation of quorum
sensing could also explain the attenuated phenotypes of both
algR deletion and AlgR overexpression strains during murine
infection (43), as disruption of all three quorum-sensing sys-
tems has resulted in loss of virulence (6, 16, 26). Interestingly,
the overall atypical characteristics of ?algR biofilms resembled
those of P. aeruginosa biofilms overexpressing rhamnolipids
(10). AlgR regulation of rhamnolipid production has been
previously suggested by the involvement of AlgC in rhamno-
lipid synthesis (57). The product of the algC gene, which is
involved in alginate production through its phosphomanno-
mutase activity (86, 87) and in lipopolysaccharide synthesis
through its phosphoglucomutase activity (9), also participates
in rhamnolipid production (57). In addition, the overproduc-
tion of rhamnolipids by P. aeruginosa, as observed in ?algR
biofilms, inhibits the maintenance of the biofilm infrastructure
(10). Furthermore, the addition of exogenous rhamnolipids to
established P. aeruginosa and Burkholderia biofilms causes de-
tachment and dispersion of bacteria from the biofilm (3, 36).
Thus, numerous investigators have established that excess lev-
els of rhamnolipids interfere with normal biofilm architecture.
The inability of AlgR to repress rhlAB transcription or the
upstream positive regulator rhlI can account for the increased
levels of rhamnolipids observed in ?algR biofilms. Moreover,
the mutation of the chromosomal rhlI promoter AlgR binding
site in strain PAO1rhlImut resulted in elevated rhamnolipid
production and a biofilm phenotype identical to that of the
algR deletion strain. Taken together, these results strongly
support the notion that dysregulation of the Rhl quorum-
sensing system by deletion of algR leads to overproduction of
rhamnolipids and the altered biofilm phenotype. These studies
also suggest that the signal to which AlgR responds in vivo may
be involved in the dispersion of the biofilm. It is relatively easy
to imagine that some physiologically relevant molecule binds
to AlgR or AlgZ and causes the derepression of rhlI, resulting
in increased rhamnolipid production and, hence, dispersion of
The regulatory mechanisms of AlgR are complex in that
both phosphorylation-dependent and -independent mecha-
nisms of activation have been demonstrated (45, 83). When the
predicted phosphorylated residue of AlgR, aspartate 54, was
mutated to asparagine, alginate production was still activated
(45). However, the same mutation abolished AlgR activation
of twitching motility (83). Furthermore, AlgR can switch from
a repressor of HCN production in the nonmucoid background
to an activator of HCN production in mucoid strains (7). In the
current study, it appears that AlgR only controls the Rhl quo-
rum-sensing system when it is attached in a biofilm, further
complicating the sensory input required for AlgR regulation. A
similar contact dependence has been reported for another
VOL. 189, 2007AlgR REGULATES QUORUM SENSING IN P. AERUGINOSA BIOFILMS7761
AlgR-controlled gene, algC, when Davies and Geesey (13)
showed that bacterial attachment induces the transcription of
specific genes, including algC. This would imply that attach-
ment to a surface may provide a signal to the bacterium to
stimulate the AlgR regulon. The signaling requirements for
AlgR activation and repression of target genes such as those
involved in alginate production, HCN production, and twitch-
ing motility may parallel changes in the bacterium’s environ-
In addition to rhlAB and rhlI, a significant number of tran-
scripts that encode secreted factors also appear to be repressed
by AlgR in nonmucoid PAO1 biofilms (Fig. 4). In earlier work,
17 proteins uniquely expressed in an algR mutant were iden-
tified by two-dimensional sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis (43). Furthermore, AlgR represses
hcnA in nonmucoid P. aeruginosa and activates its expression
in mucoid strains (7). Taken together, these findings further
support the idea that AlgR plays a previously unrecognized
role as a repressor of gene expression in nonmucoid P. aerugi-
nosa. In addition, the concept that AlgR switches from being a
repressor to an activator of virulence products during mucoidy
is intriguing and is under further investigation.
In conclusion, our results indicate that AlgR represses the
Rhl quorum-sensing system in nonmucoid P. aeruginosa during
continuous culture biofilm growth and that such repression is
necessary for proper biofilm maturation. Furthermore, the
ability of AlgR to repress rhlI and rhlAB during biofilm growth,
but not during planktonic culture, suggests that AlgR may
utilize a contact-dependent or biofilm-specific mode of regu-
lation. Further insight into the coordinate regulation of the
AlgR- and Rhl-dependent pathways during biofilm growth will
enhance our understanding of P. aeruginosa pathogenesis in
The work was supported by NIH grants RO1AI50812 to M.J.S. and
R37AI37713 to B.H.I., NIH training grant 5T32AI07285 to V.E.W.,
and NIH grant R01-40541 and a Cystic Fibrosis Foundation grant to
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