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 A₆₀₀ = 2.0 and A₆₀₀= 5.0, corresponding to late exponential and stationary phase of growth, respectively (Figure 1A).

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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 (A₆₀₀). Bands analysed by MALDI-TOF mass spectrometry are indicated by arrows, and the corresponding protein name is reported.

https://doi.org/10.1371/journal.pone.0069554.g001

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 streptavidin-conjugated 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).

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 l-arabinose-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.

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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).

https://doi.org/10.1371/journal.pone.0069554.g002

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 N-terminal 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).

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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 peroxidase-conjugated 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.

https://doi.org/10.1371/journal.pone.0069554.g003

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/3OC₁₂-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).

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Figure 4. Effect of PA3699 induction on P. aeruginosa virulence factors production.

Histogram reporting elastase, pyocyanin and proteases production measured in P. aeruginosa PAO1 carrying pHERD30T (grey bars) or pR3699 (white bars), grown in LB supplemented with 0.1% (w/v) l-arabinose. The average of three independent experiments is reported with standard deviations; statistical significance with respect to P. aeruginosa PAO1 (pHERD30T) is indicated with one asterisk (p < 0.01).

https://doi.org/10.1371/journal.pone.0069554.g004

Bacterial strains and media

The bacterial strains used in this study are listed in Table S1 (Supporting Information). All E. coli and P. aeruginosa strains were routinely grown at 37°C in Luria-Bertani broth (LB), LB supplemented with 1.5% (w/v) agar, or Pseudomonas Isolation Agar (PIA) [34]. Unless otherwise stated, antibiotics were added at the following concentrations: E. coli, 20 µg/ml gentamicin (Gm), 100 µg/ml ampicillin (Ap), 25 µg/ml kanamycin (Km), 30 µg/ml chloramphenicol (Cm), 10 µg/ml tetracycline (Tc), 10 µg/ml nalidixic acid (Nal); P. aeruginosa, 100 µg/ml Gm, 400 µg/ml Cm, 100 µg/ml Tc, 300 µg/ml carbenicillin (Cb). When required, media were supplemented with glucose, isopropyl β-D-1-thiogalactopyranoside (IPTG) or l-arabinose at the concentration reported in the text.

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 (A₆₀₀) 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 A₆₀₀ of 0.05 in fresh LB, and incubated for 18 hrs at 37°C. Promoter activity (M.u.) and cell density (A₆₀₀) 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 A₆₀₀ 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 (A₆₀₀) were determined every 1 hr for 16 hrs. Promoter activity is given as Light Counts Per Second (LCPS) divided by A₆₀₀. The strains carrying the pHERD30T-derivative plasmids were grown in LB supplemented with Gm 100 µg/ml and 0.1% l-arabinose. 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.

The pE3699-N6 plasmid was transformed in E. coli BL21 (DE3, pLysS) (Novagen). The resulting strain was grown over-night at 37°C with 200 r.p.m. shaking in LB supplemented with 25 µg/ml Km, 30 µg/ml Cm and 0.2% (w/v) glucose. The over-night culture was diluted 1:100 in 250 ml of the same medium and after 1 hr growth at 37°C with 200 r.p.m. shaking it was induced for 4 hrs with 0.5 mM IPTG.

PA3699-N6 purification was performed with the QIAexpress® Ni-NTA Fast Start Kit (Qiagen), according to manufacturer’s instructions. The PA3699-N6 recombinant protein was eluted from the Ni-NTA resin with 1 ml of Elution Buffer Native, dialyzed 1:1000 in Dialysis Buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl), and concentrated with Centricon YM-10 centrifugal filter device (Amicon). PA3699-N6 identity was verified by SDS-PAGE and Western blot analysis performed with anti-6xHis primary antibody (Qiagen) and peroxidase-conjugated anti-mouse IgG secondary antiboby (Sigma-Aldrich) on aliquots withdrawn at different steps of the purification process [34]. The 6×His tag was subsequently cleaved from 1 mg of purified PA3699-N6 by digestion with 100 µg thrombin in Thrombin Buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 3.3 mM CaCl₂). The PA3699 protein was further purified by gel permeation chromatography on a Superdex 200 HR column (Amersham Pharmacia Biotech), dialyzed 1:1000 in Storage buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 50% glycerol) and stored at -20°C.

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-³²P] dATP by fill-in with Klenow enzyme [43]. The radio-labelled DNA probe was purified with SigmaSpin post-reaction purification columns (Sigma-Aldrich) followed by phenol-chloroform (1:1) extraction and ethanol precipitation.

In the EMSA experiments, the labelled DNA probe (0.2 nM) was incubated with different protein concentrations, in Binding Buffer [20 mM Tris-HCl, 2 mM EDTA, 5 mM MgCl₂, 30 mM KCl, 5% (v/v) glycerol, 0.025% (v/v) nonidet P-40, 30 µg/ml poly(dI): (dC); pH 8.0]. A labelled aspecific probe (0.2 nM) was added to the binding mix. After 15 min of incubation at 22°C the reaction mixtures were loaded onto a 30 min pre-run 6% (w/v) polyacrylamide gel under non-denaturing conditions. The ratio acrylamide: bis-acrylamide was 37.5:1. The electrophoresis was carried out at 22°C in TBE 0.5X [34] at 5 V/cm for 4 hrs. Gel was then dried and autoradiographed.

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 A₆₀₀ 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.

Elastolytic and proteolytic activities were determined by elastin-Congo red and azocasein assays, as previously described [44–46]. Pyocyanin quantification was performed as previously described [47]. The amount of pyocyanin, in μg/ml, was calculated using the following formula: A₅₂₀/A₆₀₀ X 17.072 = μg of pyocyanin per ml [48].

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 p-values using the two-tailed Student-t test for unpaired data sets; differences with a p-value ≤ 0.01 are considered as statistically significant.