A New Transcriptional Repressor of the Pseudomonas aeruginosa Quorum Sensing Receptor Gene lasR

Pseudomonas aeruginosa pathogenic potential is controlled via multiple regulatory pathways, including three quorum sensing (QS) systems. LasR is a key QS signal receptor since it acts as a global transcriptional regulator required for optimal expression of main virulence factors. P. aeruginosa modulates the QS response by integrating this cell density-dependent circuit to environmental and metabolic cues. Hence, QS also controls the adaptation to challenging environmental niches, such as infection sites. However, little is known about the molecular mechanisms connecting QS and other signalling pathways. In this work, DNA-affinity chromatography was used to identify new lasR transcriptional regulators. This approach led to the identification and functional characterization of the TetR-like transcriptional repressor PA3699. This protein was purified and shown to directly bind to the lasR promoter region in vitro. The induction of PA3699 expression in P. aeruginosa PAO1 cultures repressed lasR promoter activity and the production of LasR-dependent virulence factors, such as elastase, pyocyanin, and proteases. These findings suggest a role for PA3699 in P. aeruginosa pathogenicity. P. aeruginosa genome encodes at least 38 TetR-family proteins, and PA3699 is the eighth member of this group functionally characterized so far and the first one shown to bind the lasR promoter in vitro.


Introduction
The definition of quorum sensing (QS) was coined about 20 years ago to describe a communication system based on the production, detection, and response to signal molecules, allowing bacterial populations to trigger a coordinated response at a threshold cell density [1].Today it is well known that QS regulation plays a key role in a number of relevant bacterial processes, including colonization of plants and animal tissues, and production of antibiotics [2,3].
In many bacteria, the QS regulatory device is interwoven with other global regulatory networks responsive to different environmental cues (e.g., temperature, pH, osmolarity, oxidative stress, nutrient starvation).These networks cross-talk in the bacterial cell in order to determine the optimal survival strategy [4].The advantage of integrating QS with other regulatory pathways becomes a compelling necessity when the QS response controls multiple functions and its activation commits bacteria to a strong reorganization of the whole cellular metabolism [5].
One of the most studied model organisms in QS research is Pseudomonas aeruginosa.This versatile bacterium is able to thrive in a wide range of environmental niches, including the human body.The high adaptability of P. aeruginosa is reflected by its behaviour as a pathogen.In humans, P. aeruginosa causes community-and hospital-acquired infections, colonizing different body districts like lungs, eyes, ears, urinary tract, injured skin (burns and wounds).Such infections are often difficult to eradicate as a consequence of antibiotic resistance and biofilm formation [6].In particular, P. aeruginosa chronic lung infection is the major cause of death in cystic fibrosis patients, a genetic disease affecting about 1/3,000 newborns in the Caucasian population [6].
P. aeruginosa pathogenicity strongly depends on the fine and coordinated regulation of a wide array of virulence factors [7,8].This is achieved thanks to a complex network of regulatory and signalling pathways controlling virulence-related phenotypes, in response to environmental cues and bacterial population structure [9].Among these pathways, a preeminent role in P. aeruginosa virulence is played by three interconnected QS systems, based on the production of different signal molecules.The QS signal molecule N-3-oxododecanoyl-homoserine lactone (3OC 12 -HSL) is synthesized by LasI, encoded by the lasI gene, and its cognate receptor is the cytoplasmic transcriptional regulator LasR, encoded by the lasR gene.3OC 12 -HSL progressively accumulates in the bacterial culture and at a threshold concentration it binds LasR.The LasR/3OC 12 -HSL complex, in turn, activates transcription of hundreds of genes, including the genes coding for the receptors of the other two QS systems, based on Nbutanoylhomoserine lactone (C 4 -HSL) and 2-heptyl-3hydroxy-4-quinolone (PQS) signal molecules.As a whole, the P. aeruginosa QS circuit has a key role in pathogenesis, regulating the production of virulence factors, the formation of biofilm and the expression of antibiotic efflux pumps [10].
The three QS systems of P. aeruginosa are interwoven and connected to other regulatory pathways [10][11][12].Indeed the expression of genes involved in the synthesis and perception of QS signal molecules is finely regulated at the transcriptional and post-transcriptional level in response to various metabolic and environmental stimuli [12][13][14][15].However, the proteins involved in this regulation and their mechanisms of action are largely unknown [11,12].To date only few transcriptional regulators have been shown to directly bind the promoters of genes involved in P. aeruginosa QS signal synthesis and perception.In particular, RsaL directly represses lasI transcription [16,17], while Vfr directly activates the lasR promoter region [18].Moreover, AlgR2 (AlgQ), besides acting as an anti-sigma factor for the vegetative sigma RpoD [19], can also bind to and downregulate the lasR promoter [20].However, the stimuli controlling the transcriptional activity of Vfr, AlgR2 (AlgQ) and RsaL are still unknown.
The objective of this work has been the identification of novel transcriptional regulators of LasR expression.To this aim, P. aeruginosa cytoplasmic proteins able to bind the lasR promoter region have been picked-up by DNA-affinity chromatography and identified by mass spectrometry.This led to the functional characterization of the TetR-like protein PA3699, which acts as a novel repressor of lasR transcription and of P. aeruginosa virulence factors production.

Identification of P. aeruginosa proteins interacting with the lasR promoter
To increase the probability of fishing proteins interacting with the lasR promoter (PlasR), the activity of PlasR was preliminarily monitored along the P. aeruginosa PAO1 growth curve by means of a PlasR::lacZ transcriptional fusion (Figure 1A Table S2).PlasR activity increased steadily during the exponential phase and reached a plateau at the onset of the stationary phase.The maximal promoter activity was maintained for about four hours, and then declined (Figure 1A), suggesting that at the end of the exponential phase and during the stationary phase of growth, one or more transcriptional regulator(s) bind to PlasR and affect its promoter activity.Hence, crude protein extracts were prepared from P. aeruginosa PAO1 cultures grown to A 600 = 2.0 and A 600 = 5.0, corresponding to late exponential and stationary phase of growth, respectively (Figure 1A).
To prepare the DNA-affinity chromatography matrix, a biotinylated double-stranded DNA fragment, corresponding to the lasR promoter region cloned in the PlasR::lacZ transcriptional fusion, was immobilized on a streptavidinconjugated chromatography resin.Protein crude extracts were independently incubated with the matrix and, after extensive washing, proteins specifically bound to PlasR were eluted, separated by SDS-PAGE, and identified by MALDI-TOF mass spectrometry (Table 1).The experiment was performed in duplicate for each crude extract.Examples of SDS-PAGE gels are shown in Figure 1B.Overall, twelve protein bands were reproducibly detected on the SDS-PAGE gels (Table 1), while no protein bands were visible in control experiments with beads uncoupled to DNA (data not shown).Among these, RpoA, Ssb and TufB are involved in general DNA processing, while the PhaF phasin is involved in polyhydroxyalkanoate segregation during cell division [21].Moreover three proteins not involved in DNA processing (i.e., Lon, PepA and GroEL) were also retrieved (Table 1).
Of the two transcription factors previously known to bind PlasR (i.e., Vfr and AlgR2) [18,20], only Vfr was picked-up (Table 1), indicating that the DNA-affinity chromatography approach here described allows identification of some, but not all, the transcription factors specific for PlasR.Therefore, besides AlgR2, other regulators of this promoter may have escaped our analysis.
Last, but of primary importance with respect to our aims, among the picked-up proteins PA0123, PA0448, PA3699 and PA4135 were annotated in the P. aeruginosa genome as putative transcription factors with unknown function (Table 1).On the basis of their sequence, PA0123 and PA0448 are annotated as putative LysR-like family transcriprional regulators [22].Interestingly, the three-dimensional structure of PA3699 and PA4135 has been recently solved (Protein Data Bank accession numbers, 3KKD and 2FBI, respectively).On the basis of primary sequence and structural features, PA3699 and PA4135 can be assigned to the TetR-like and MarR-like family of transcriptional regulators, respectively [22].In this view, the above results provide the first experimental evidence that PA0123, PA0448, PA3699 and PA4135 are expressed in P. aeruginosa cultures and able to bind DNA in vitro.On the basis of the above considerations, these four factors were selected for further analysis as novel putative lasR regulators (Table 1).

In vivo characterization of putative lasR transcriptional regulators
In order to study the in vivo effect of PA0123, PA0448, PA3699 and PA4135 on PlasR activity, a set of P. aeruginosa PAO1 in frame deletion mutants in the corresponding genes was generated.As a control, also a mutation in the vfr gene was introduced in P. aeruginosa PAO1.A genetic cassette carrying the PlasR::lux transcriptional fusion was introduced in a chromosomal neutral site of the five mutant strains and of P. aeruginosa wild type to conveniently measure PlasR activity as a function of light emission along the growth curve.The DNA region cloned in this genetic cassette was identical to that used as a bait in the DNA-affinity chromatography experiment.
In accordance with literature data, PlasR activity was strongly decreased as a consequence of vfr mutation (Figure S1) [18], while no significant differences with respect to the wild type activity were observed in the PA0123, PA0448, PA3699 and PA4135 mutant strains (Figure S1).Similar results were obtained by using the PlasR::lacZ transcriptional fusion as reporter system (data not shown).b Proteins identified only when using PlasR as DNA bait are in bold characters.
c.The protein score is according to the Mascot programme (scores ≥ 76 correspond to an error probability p < 0.05 in our data set) [37].
It was surprising that inactivation of the four new putative regulators able to bind PlasR in vitro did not affect the activity of this promoter in vivo.However, the DNA-affinity chromatography allows the purification of proteins present in a synthetic binding buffer, a condition that is unlikely to mimic the intracellular milieu.Thus, some of the factors picked-up in vitro using PlasR as bait could be not sufficiently expressed and/or active in vivo under the experimental conditions used for the promoter activity assay.To overcome this problem, the PA0123, PA0448, PA3699, and PA4135 genes were cloned in the expression vector pHERD30T, under the control of an Larabinose-inducible promoter (Table S2) [23].The resulting plasmids were independently introduced in the wild type P. aeruginosa PAO1 strain carrying the PlasR::lux transcriptional fusion, and promoter activity was measured along the growth curve in the presence of L-arabinose; the pHERD30T empty vector was used as control.With the only exception of PA0448, L-arabinose-induced expression of the tested proteins reduced the activity of the PlasR::lux fusion.However, induction of PA0123 and PA4135 also resulted in a strong inhibition of P. aeruginosa growth (Figure 2A).Since bioluminescence emission is an energy demanding process, we hypothesized that PA0123 and PA4135 induction might affect growth only in a lux proficient background.However, PA0123 and PA4135 induction had a negative effect on P. aeruginosa growth also in the PAO1 wild type strain lacking the PlasR::lux fusion in the chromosome (data not shown).Overall, these results indicate that PA0123 and PA4135 are toxic for P. aeruginosa when their expression is induced, at least in the growth conditions used in our experimental setting.
Since an effect on bacterial growth is particularly meaningful when studying a cell density-dependent pathway like QS, further studies were focused on PA3699, the only protein that, once induced, strongly inhibited PlasR activity without affecting P. aeruginosa growth (Figure 2A).
It could be reasonably argued that the procedure described in the above paragraphs could isolate DNA binding proteins that do not play a role in vivo on the regulation of the target promoter and that the overexpression of transcriptional regulators could lead to pleiotropic effects/phenotypes.These causalities likely occurred in the case of PA0448, PA0123 and PA4135, since the first protein has no effect on PlasR activity and the other two are toxic to the cell when overexpressed.Conversely, PA3699 induction did not affect growth, while it repressed PlasR activity during the whole growth curve and proportionally to the amount of L-arabinose present in the growth medium (Figure 2B and 2C).All these features argue for a bona fide repressor activity of PA3699 on PlasR.

PA3699 binds the lasR promoter region in EMSA assays
In order to confirm DNA-affinity chromatography results, electrophoretic mobility shift assay (EMSA) of a DNA probe (PlasRp) encompassing the promoter region of lasR were conducted using the purified PA3699 protein.To this purpose, PA3699 was fused to a six-histidine tag (6xHis) at the Nterminal domain and purified by nickel-nitrilotriacetic acid (Ni-NTA)-affinity chromatography.The 6×His tag was subsequently cleaved by thrombin digestion, yielding the native PA3699 protein in soluble and highly pure form (Figure 3A and 3B).
As shown in Figure 3C, incubation of the PlasRp probe with purified PA3699 led to the formation of a complex endowed with lower electrophoretic motility with respect to the free probe, and the amount of the shifted complex was proportional to the concentration of purified PA3699.Since the electrophoretic motility of an unspecific DNA probe did not change in the presence of PA3699, the binding of this protein to PlasRp is specific (Figure 3C).
Altogether, these data provide an unequivocal proof of the direct molecular interaction between PA3699 and the lasR promoter region.

PA3699 affects the production of QS-controlled virulence factors
The LasR/3OC 12 -HSL complex is required for full expression of most P. aeruginosa virulence factors, including elastase, pyocyanin, and proteases [24].
In order to assess whether the repression exerted by PA3699 on LasR expression may affect P. aeruginosa pathogenic potential, the expression of LasR-dependent virulence phenotypes was compared in P. aeruginosa carrying either the pHERD30T empty vector or its derivative plasmid for L-arabinose-dependent induction of PA3699 (pR3699).Consistent with the strong repressive effect exerted by PA3699 induction on PlasR activity, results showed that also the production of LasR-dependent phenotypes such as elastase, pyocyanin, and proteases was strongly decreased in P. aeruginosa upon PA3699 induction (Figure 4).However, pyocyanin, elastase and protease production remained unaffected in the P. aeruginosa PA3699 mutant strain with respect to the wild type, consistently with what observed for the PlasR promoter activity (data not shown).

CONCLUDING REMARKS
The masterly management of an enormous metabolic potential via multiple regulative networks and signalling systems allows P. aeruginosa adaptation to the most challenging environments, including the human host [9,11,12].
The P. aeruginosa las QS system is among the most studied and best characterized bacterial regulatory pathways, also because it is considered a promising target for anti-virulence therapies [25][26][27].Nevertheless, its interconnections with other P. aeruginosa regulative networks and signalling systems are still poorly understood [9].
So far, in vivo (genetic) approaches led to the identification of a number of genes involved in the modulation of the las system activity/response, highlighting the multiplicity of pathways connecting cell-density and environmental/metabolic signalling.However, the transcriptional factors directly controlling the expression of the las genes in response to environmental/ metabolic stimuli are mostly unknown [11,12,15].In this work, DNA-affinity chromatography has been used for the first time for the identification of transcriptional regulators of the lasR gene, encoding the 3OC 12 -HSL receptor.
Unexpectedly, the genetic inactivation of the four putative PlasR transcription factors identified by in vitro DNA-affinity chromatography (i.e., PA0123, PA0448, PA3699, and PA4135) did not affect the activity of PlasR in vivo.A possible explanation for this result is that at least some of these factors are not sufficiently expressed and/or active in vivo under the conditions used in the promoter activity assay.This hypothesis is supported by the observation that a progressive increase of PA3699 induction caused a parallel decrease in PlasR activity, indicating this factor as a novel transcriptional repressor of PlasR.
According to its structural features, PA3699 belongs to the TetR-family, a preeminent group of transcriptional regulators in bacteria [28].Transcriptional regulators of the TetR-family typically activate upon binding of a specific ligand to an allosteric site [28].Thus, it is likely that PA3699 regulates lasR transcription only in the presence of a specific ligand when expressed at a physiological level.Since the interaction of a transcriptional regulator with its DNA target sequence is governed by a thermodynamic equilibrium between the bound and unbound form, the induction of PA3699 expression could shift this equilibrium to the bound state, resulting in PlasR regulation also in the absence of the actual stimulus/ligand.Phenotypic microarray experiments are in progress to identify the growth conditions leading to PA3699 full activity and/or expression.
P. aeruginosa genome encodes at least 38 TetR-family proteins, and PA3699 is the eighth member of this group functionally characterized so far and the first one shown to bind the lasR promoter in vitro [28][29][30][31][32][33].
Overall, DNA-affinity chromatography combined with induction of selected factors has been proven as a useful approach for the identification of regulators that are not highly expressed or active under standard laboratory conditions, but that might be relevant in specific environmental niches, like for instance infection sites.
Although further studies are required to assess the involvement of PA3699 in P. aeruginosa physiology and pathogenesis, this work represents a further step toward the disclosure of the complex network controlling cell-cell communication, social behaviours and host adaptability of P.

Recombinant DNA techniques
Plasmids used or generated in this study and details on their construction are reported in Table S2 (Supporting Information).Preparation of plasmid DNA, purification of DNA fragments, restrictions, ligations, and transformations of E. coli were carried out by standard procedures [34].PCR amplifications were performed using Bio Red-Taq DNA polymerase (Bioline) or Accuzyme DNA polymerase (Bioline).The oligonucleotides used in this study are listed in Table S3 (Supporting Information).Automated sequencing was performed by Genechron sequence service (Genechron).
When required, plasmids were transferred from E. coli S17.1 λpir to P. aeruginosa PAO1 by bi-parental conjugation, and from E. coli DH5α to P. aeruginosa PAO1 by tri-parental conjugation with the helper strain E. coli HB101 pRK2013 [35].

DNA-affinity chromatography
DNA-affinity purification was performed as previously described [36].A biotinylated DNA fragment encompassing the lasR promoter region (from nucleotide -359 to +13 relative to the lasR start codon) was PCR amplified with primers FW381 and RV334 (Table S3); primer FW381 was biotinylated.
P. aeruginosa PAO1 protein crude extracts were obtained from cells grown in 300 ml of LB at 37°C to an absorbance at 600 nm wavelength (A 600 ) of 2.0 or 5.0, with 200 r.p.m. shaking.Bacterial cells were harvested by centrifugation and suspended in 2 ml of Sonication Buffer [10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.05% (v/v) Triton X-100] supplemented with 1 mg/ml lysozyme.After 30 min of incubation at 37°C, cells were disrupted by sonication.Cellular debris were removed by centrifugation and subsequent filtration.Protein concentration in the supernatant was determined with the Bradford Protein Assay Kit (Bio-Rad) according to manufacturer's instruction.
One mg of paramagnetic streptavidin-conjugated resin (Dynabeads M-280, Invitrogen) was equilibrated in 200 µl of Wash Buffer 2X [10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl, 0.05% (v/v) Triton X-100], and then incubated with 20 µg of the biotin-labelled PCR product for 25 min at 22°C.Unbound DNA was removed by magnetic separation in a magnet particle concentrator (Dynal, Invitrogen).Dynabeads were washed three times with 1 ml Wash Buffer 1X, and then incubated with 50 mg of P. aeruginosa protein crude extract for 2 hours at 22°C.Unbound proteins were removed by magnetic separation in a magnet particle concentrator, and the Dynabeads were washed six times with Sonication Buffer.Proteins specifically bound to the DNA-bead complexes were eluted in 20 µl of Elution Buffer [1.2 M NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.05% (v/v) Triton X100].Eluted proteins were separated by Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis (SDS-PAGE) on a 12% (v/v) polyacrylamide gel and stained with Coomassie Brilliant Blue [34].Protein identity was determined by Matrix-Assisted Laser Desorption/ Ionization-Time Of Flight (MALDI-TOF) mass spectrometry.MALDI-TOF analyses were performed as previously described [37].

Construction of P. aeruginosa mutants
P. aeruginosa in frame deletion mutants were generated using the pDM4 plasmid as previously described [38].Briefly, the flanking DNA regions (about 650 bp each) of the selected genes were PCR amplified with primers listed in Table S3 and sequentially cloned in pDM4.The resulting pDM4-derivative plasmids, listed in Table S2, were independently introduced in P. aeruginosa PAO1 by conjugation.Clones with a chromosomal insertion of the pDM4-derivative plasmids were selected on LB agar plates supplemented with 400 µg/ml Cm e 10 µg/ml Nal.Plasmid excision from the chromosome was subsequently selected on LB agar plates supplemented with 10% (w/v) sucrose.Mutant strains were checked by PCR analysis.

Construction of PlasR transcriptional fusions
The DNA region encompassing the lasR promoter (spanning from nucleotide -359 to +13 relative to the lasR start codon) was PCR amplified with the primers FW333 and RV334 (Table S3) and cloned by EcoRI-PstI restriction in the pMP220 and mini-CTX-lux plasmids [39,40].The resulting pMPlasR::lacZ and mini-CTX-PlasR::lux plasmids (Table S2) were conjugated in selected P. aeruginosa strains.The mini-CTX plasmid backbone was removed from the P. aeruginosa strains carrying the PlasR::lux cassette by using the pFLP2 plasmid (Table S2).pFLP2 was subsequently cured by sucrose counterselection as previously described [41].

Over-expression of putative lasR regulators
The PA0123, PA0448, PA3699 and PA4135 genes were PCR amplified using the primers listed in Table S3 and cloned in the pHERD30T vector [23], generating the plasmids pR0123, pR0448, pR3699 and pR4135, respectively (Table S2).These plasmids were introduced in P. aeruginosa PAO1 carrying the PlasR::lux transcriptional fusion by conjugation.Positive clones were selected on PIA plates supplemented with 100 µg/ml Gm.

Promoter activity assay
PlasR activity in P. aeruginosa PAO1 carrying the pMPlasR::lacZ plasmid was measured by standard Miller assay [42].An over-night culture was diluted to an A 600 of 0.05 in fresh LB, and incubated for 18 hrs at 37°C.Promoter activity (M.u.) and cell density (A 600 ) were determined every 2 hr for 18 hrs.
PlasR activity in P. aeruginosa strains carrying the PlasR::lux chromosomally-inserted transcriptional fusion was determined as bioluminescence emission per cell by using the automated luminometer-spectrometer Wallac 1420 VICTOR 3V (PerkinElmer).Over-night cultures were diluted to an A 600 of 0.05 in fresh LB, and 0.2 ml of these cultures were grown at 37°C in microtiter plates.Luminescence and cell density (A 600 ) were determined every 1 hr for 16 hrs.Promoter activity is given as Light Counts Per Second (LCPS) divided by A 600 .The strains carrying the pHERD30T-derivative plasmids were grown in LB supplemented with Gm 100 µg/ml and 0.1% Larabinose.For both the lacZ-and lux-based promoter assays average values and standard deviations were calculated from three independent experiments.

PA3699 purification
The PA3699 gene was cloned in the pET-28b(+) vector (Novagen), generating the pE3699-N6 plasmid (for more details see Table S2).In this plasmid the PA3699 gene is transcriptionally-coupled in frame with a sequence coding for a 6 histidine tag (6xHis) at the N-terminus.
Protein concentration was determined with the Bradford protein assay kit (Bio-Rad) according to manufacturer's instruction.

Electrophoretic Mobility Shift Assay (EMSA)
A 210 bp DNA fragment encompassing the lasR promoter region (from nucleotides -359 to -150 relative to the lasR start codon) was PCR amplified with primers FW 333 and RV 535 (Table S3) from P. aeruginosa PAO1 genome and T/A cloned in the pDRIVE-T-EASY vector (Qiagen), generating pDPlasR (for more details see Table S2).pDPlasR was digested with EcoRI, and the resulting 218 bp DNA fragment was labeled with [α-32 P] dATP by fill-in with Klenow enzyme [43].The radiolabelled DNA probe was purified with SigmaSpin post-reaction purification columns (Sigma-Aldrich) followed by phenolchloroform (1:1) extraction and ethanol precipitation.

Phenotypic assays
P. aeruginosa PAO1 wild type carrying the pHERD30T empty vector or the pR3699 plasmid (see Table S2) were grown over night at 37°C in LB supplemented with 100 µg/ml Gm.Cultures were diluted to an A 600 of 0.02 in LB supplemented with 0.1% L-arabinose, and supernatants were collected for elastolytic, proteolytic and pyocyanin assays after 6 hrs incubation at 37°C with 200 r.p.m shaking.
The average data and standard deviations of the phenotypic assays were calculated from three independent experiments.

Statistical analysis
Statistical significance was determined by calculating the pvalues using the two-tailed Student-t test for unpaired data sets; differences with a p-value ≤ 0.01 are considered as statistically significant.
Figure 1.PlasR activity and PlasR-affinity chromatography.(A) Growth curve of the P. aeruginosa PAO1 wild type strain carrying the pMPlasR::lacZ plasmid (dashed line) and corresponding PlasR promoter activity (solid line).The points of the growth curve at which the protein crude extracts for DNA-affinity chromatography were prepared are indicated by arrows.(B) SDS-PAGE analysis of proteins bound to the PlasR promoter region.Protein crude extracts were prepared from P. aeruginosa PAO1 cultures grown in LB broth to the indicated cell densities (A 600 ).Bands analysed by MALDI-TOF mass spectrometry are indicated by arrows, and the corresponding protein name is reported.doi: 10.1371/journal.pone.0069554.g001

Figure 2 .
Figure 2. Effect of the induction of PlasR-bound proteins on PlasR promoter activity. .(A) Histogram reporting PlasR maximal promoter activity (grey bars) and the corresponding cell density (white bars) measured in P. aeruginosa PAO1 PlasR::lux strains carrying the plasmids indicated below the graph, grown in LB supplemented with 0.1% (w/v) L-arabinose.(B) Graph reporting PlasR promoter activity (filled symbols) and cell density (open symbols) measured during the growth curve in P. aeruginosa PAO1 PlasR::lux carrying pHERD30T (triangles) or pR3699 (circles), grown in LB supplemented with 0.1% (w/v) L-arabinose.(C) Histogram reporting PlasR maximal promoter activity measured in P. aeruginosa PAO1 PlasR::lux strains carrying pHERD30T (grey bars) or pR3699 (white bars) grown in LB supplemented with different L-arabinose concentrations (%, w/v), indicated below the graph.In (A), (B) and (C) the average of three independent experiments is reported with standard deviations; in (A) and (C) statistical significance with respect to P. aeruginosa PAO1 PlasR::lux (pHERD30T) is indicated with one asterisk (p < 0.01).doi: 10.1371/journal.pone.0069554.g002

Figure 3 .
Figure 3. PA3699 purification and PlasR-binding assay. .(A) SDS-PAGE analysis of samples withdrawn at different steps of PA3699 purification.Lane L, PageRuler Unstained Protein Ladder (Fermentas); lane 1, non-induced protein crude extract; lane 2, induced protein crude extract; lane 3, soluble fraction of the induced protein crude extract; lane 4, purified protein; line 5, purified protein after thrombin cleavage.(B) Western blot analysis performed with mouse anti-6xHis primary antibody and anti-mouse peroxidaseconjugated secondary antibody on a gel identical to the one shown in (A).(C) Autoradiography of an EMSA showing direct interaction between a DNA probe encompassing the lasR promoter region and purified PA3699.PA3699 concentration (μM) is indicated above each lane.An unspecific probe was added in the reaction mixture as control.The PA3699-PlasR complex and the free DNA probes are indicated.doi: 10.1371/journal.pone.0069554.g003

Table 1 .
List of PlasR-binding proteins identified by MALDI-TOF analysis from the SDS-PAGE gels shown in Figure1B.
[22]A number, gene name and functional class refer to the Pseudomonas Genome Database annotation[22].