Intracellular levels and activity of PvdS, the major iron starvation sigma factor of Pseudomonas aeruginosa
Summary
In Pseudomonas aeruginosa the iron starvation sigma factor PvdS directs the transcription of pyoverdine and virulence genes under iron limitation. PvdS activity is modulated by pyoverdine through the surface signalling cascade involving the FpvA receptor and the inner membrane-spanning sensor FpvR. To gain insight into the molecular mechanisms enabling PvdS to compete with the major sigma RpoD for RNA polymerase (RNAP) binding, we determined the intracellular levels of RNAP, RpoD and PvdS in P. aeruginosa PAO1, and the effect of pyoverdine signalling on PvdS activity. Under iron limitation, P. aeruginosa contains 2221 and 933 molecules of RNAP and RpoD per cell respectively. PvdS attains 62% of RpoD levels. The high PvdS content is partly offset by retention of 30% of PvdS on the membrane, lowering the concentration of cytosolic PvdS to 45% of RpoD levels. RNAP purification from iron-starved P. aeruginosa cells demonstrated that PvdS–RNAP is poorly represented compared with RpoD–RNAP (1 and 27% of total RNAP respectively). Pyoverdine signalling does not affect the PvdS cellular content but facilitates PvdS release from the membrane, increasing its cytosolic concentration from 35% in both pvdF and fpvA signalling mutants to 70% in the wild type and 83% in the fpvR mutant.
Introduction
Transcription in bacteria is directed by RNA polymerase (RNAP), a multisubunit enzyme composed of the core fraction (RNAPc, containing the α2ββ′ω subunits) endowed with catalytic activity of RNA polymerization, and the dissociable sigma factor (σ) which accounts for promoter recognition and transcription initiation. Almost all bacteria studied so far contain multiple sigma subunits (Gruber and Gross, 2003). In addition to the primary sigma factor RpoD (σ70), which is constitutively expressed and attains the highest cellular levels, Escherichia coli possesses up to six different alternative sigma factors (Ishihama, 2000; Gruber and Gross, 2003). By virtue of their promoter specificity, alternative sigma factors provide a mean to modify the global transcription pattern in response to a multitude of stimuli, most often switching on genes involved in stress survival and adaptation to environmental conditions (Wösten, 1998; Ishihama, 2000; Gruber and Gross, 2003).
Sigma factors compete with each other for binding to RNAPc (Malik et al., 1987; Ishihama, 2000; Gruber and Gross, 2003). A central feature of the sigma factor competition model is that free RNAPc, i.e. the fraction not involved in transcription elongation and/or non-specific DNA binding, is limiting for stoichiometric binding of the sigma factor pool (Ishihama, 2000; Nyström, 2004; Grigorova et al., 2006). This is supported by the general observations that overexpression of one sigma decreases transcription of genes regulated by another sigma (Hicks and Grossman, 1996; Farewell et al., 1998) or that effectors of RNAP activity such as the ppGpp alarmone alter the relative binding affinity of individual sigmas to RNAPc (Jishage et al., 2002; Laurie et al., 2003). The composition of different holoenzymes can be adjusted in the cell by changing parameters affecting the competition between sigmas. This can take place by varying the intracellular amount of different sigmas or their activation state through post-translational modification, or by sequestration of a specific sigma by its cognate antisigma factor protein (Gruber and Gross, 2003).
In most bacteria the largest sigma factor repertoire is represented by extracytoplasmic function (ECF) sigmas (Helmann, 2002; Gruber and Gross, 2003). ECF sigma factors are small regulatory proteins belonging to the σ70 family, and are the most divergent in sequence relative to other sigmas (Lonetto et al., 1994; Helmann, 2002). The accepted view is that organisms adapted to live in diverse environments contain multiple ECF sigma factors (Helmann, 2002). This is because ECF sigmas have been evolved to regulate genes in response to a wide range of extracytoplasmic stimuli (Missiakas and Raina, 1998). The iron starvation (IS) subfamily of ECF sigma factors is a particular subgroup of ECF sigmas that bacteria express when faced with IS (Leoni et al., 2000; Visca et al., 2002). IS sigmas respond primarily to depletion of intracellular iron and, secondarily, to the presence of a specific iron chelate or siderophore in the environment (Visca et al., 2002; Braun et al., 2003). The activity of these sigmas is modulated post-translationally by cognate antisigma factor proteins through a signal transduction cascade activated by a particular siderophore or iron chelate (Visca et al., 2002; Braun and Mahren, 2005). Fourteen IS sigma factors have been identified in the opportunistic pathogen Pseudomonas aeruginosa, a feature which reflects its striking ability to utilize a variety of chelators as iron source both in the animal host and in natural environments (Visca et al., 2002).
The IS sigma factor PvdS is a central regulator of P. aeruginosa virulence genes. PvdS is expressed in lung infections of cystic fibrosis patients, and is required for biofilm formation and pathogenicity in a rabbit model of experimental endocarditis (Xiong et al., 2000; Hunt et al., 2002; Banin et al., 2005; Visca et al., 2007). PvdS directs the transcription of at least 26 genes or operons, including genes involved in the synthesis of major virulence factors such as the main endogenous siderophore pyoverdine, the PrpL and AprA proteases and (either directly or indirectly) the ADP-ribosylating exotoxin A (Cunliffe et al., 1995; Ochsner et al., 1996; Shigematsu et al., 2001; Wilderman et al., 2001; Ochsner et al., 2002). PvdS has conclusively been demonstrated to act as a sigma factor in so far as it: (i) binds RNAPc with 1:1 stoichiometry, (ii) directs the RNAP holoenzyme to a conserved DNA sequence, called IS box (consensus TAAAT-N16/17-CGT), within target promoters like those of pyoverdine biosynthetic (pvd) genes (e.g. pvdA, pvdD and pvdE/F) and (iii) causes transcription initiation at these promoters (Leoni et al., 2000; Wilson and Lamont, 2000). Expression of pvdS is regulated by the Fur-Fe(II) repressor protein which binds the pvdS promoter thereby blocking its RpoD-dependent transcription under iron-replete conditions (Barton et al., 1996; Leoni et al., 1996). Post-translational control of PvdS activity requires pyoverdine sensing, and occurs through a signalling pathway that involves the pyoverdine molecule, its outer membrane receptor FpvA and the inner membrane-spanning antisigma factor FpvR. In the absence of (ferri)pyoverdine, the activity of PvdS is antagonized by FpvR (Lamont et al., 2002). Binding of (ferri)pyoverdine to FpvA transduces a signal across the periplasm to FpvR which results in PvdS activation and consequent transcription of PvdS-controlled genes (Lamont et al., 2002; Rédly and Poole, 2005). A unique feature of pyoverdine signalling is that, beside PvdS, the antisigma factor FpvR controls the activity of a second IS sigma, FpvI, which is responsible for transcription of the sole fpvA receptor gene (Beare et al., 2003; Rédly and Poole, 2003). It has also been reported that AlgQ, the orthologue of E. coli Rsd, acts as an antisigma factor for the vegetative sigma RpoD in P. aeruginosa, eliciting RNAPc recruitment by PvdS and promoting transcription of pvd genes (Ambrosi et al., 2005). However, neither the intracellular levels of IS sigmas nor the mechanisms of competition between these proteins and other sigma factors have so far been investigated under conditions in which IS sigmas are expected to be induced.
In the present study, we explored the factors ensuring PvdS to compete with RpoD for RNAPc binding and to direct transcription of several target genes. For this purpose, we measured the intracellular concentrations of total RNAP and of the two sigma factors RpoD and PvdS under conditions of IS, and determined the in vivo concentration of the relative holoenzymes. We also investigated the effect exerted by pyoverdine signalling on PvdS activity, thus gaining further insight into the sigma competition model under varying conditions of iron availability.
Results
Iron-dependent regulation of PvdS expression and activity
The effect of iron concentration on activity and expression of PvdS was determined by measuring pyoverdine yields, expression of three pvd genes (pvdA, pvdD and pvdE; Table 1), and levels of intracellular PvdS in P. aeruginosa PAO1 at mid-exponential growth phase in low-iron medium (DCAA) supplemented with increasing iron concentrations (Fig. 1). Addition of up to 2 μM FeCl3 resulted in dose-dependent reduction of pvdA::lacZ, pvdD::lacZ and pvdE::lacZ expression, while complete repression was observed at ≥ 2 μM FeCl3. The pyoverdine production profile mirrored pvd::lacZ expression data. Moreover, Western blot analysis of PvdS expression revealed that addition of up to 2 μM FeCl3 caused a proportional decrease of PvdS concentration. These results indicate that dosage of the PvdS sigma correlates with the extent of expression of pvd genes. However, the PvdS protein is still detectable in P. aeruginosa lysates upon addition of 2 μM FeCl3 (≈ 5% of the level in the absence of added FeCl3), while pvd::lacZ expression is abrogated.
| Strain or plasmid | Genotype and/or relevant characteristics | Reference or source |
|---|---|---|
| P. aeruginosa | ||
| PAO1 (ATCC15692) | Prototroph | American type culture collection |
| K1660 | K767 ΔfpvA | Shen et al. (2002) |
| PAO1 fpvR | PAO1 fpvR::Kmr | Lamont et al. (2002) |
| PAO1 pvdF | PAO1 pvdF::Kmr | McMorran et al. (2001) |
| PAO OT11pvdS | leu-1 pro-1 pvdS::kan Kanr | Cunliffe et al. (1995) |
| PA6331 | Δpch (ΔpchDCBAΔpchRΔpchEFGHI) | Reimmann et al. (2001) |
| PA6331ΔpvdA | ΔpchΔpvdA | F. Imperi, unpublished |
| E. coli | ||
| W1485 | Prototroph | Bachmann (1987) |
| DH5αF′ | recA1 endA1 hsdR17 supE44 thi-1 gyrA96 relA1Δ(lacZYA-argF)U169 [φ80 dlacZΔM15], Nalr | Liss (1987) |
| HB101 | Δ(gpt-proA)62 leuB6 thi-1 lacY1 recA rpsL20 ara-14 galK2 xyl-5 mtl-1 supE44Δ(mcrBC-hsdRMS-mrr), Strr | Sambrook et al. (1989) |
| Plasmid | ||
| pRK2013 | Helper plasmid; ColE1 replicon, Kmr Mob+ Tra+ | Figurski and Helinski (1979) |
| pMP220 | Broad-host-range, low-copy-number promoter-probe vector; IncP replicon, lacZ Tcr Tra- | Spaink et al. (1987) |
| pMP220::PpvdA | 300-bp SphI/BglII fragment, encompassing the entire pvdA promoter, ligated to pMP220 in the same orientation as the reporter lacZ gene (formerly designated pPV51) | Leoni et al. (1996) |
| pMP190 | Broad-host-range, low-copy-number promoter-probe vector; IncQ replicon, lacZ Cmr Tra- | Spaink et al. (1987) |
| pMP190::PpvdD | Promoter of pvdD pyoverdine biosynthesis gene directionally cloned into pMP190 | Cunliffe et al. (1995) |
| pMP190::PpvdE | Promoter of pvdE pyoverdine biosynthesis gene directionally cloned into pMP190 | Cunliffe et al. (1995) |
| PUCP18/19 | E. coli-Pseudomonas shuttle vector derived from pUC18/19; pMB1, pRO1600 replicon lacZα, bla, Apr Cbr | Schweizer (1991) |
| pUCPalgQ | 896 bp PCR-generated fragment encompassing the entire algQ gene and its promoter region XhoI/BamHI-digested and the resulting 647 bp fragment ligated to the SalI–BamHI sites of pUCP19 | Ambrosi et al. (2005) |
| pUCPfpvI | 700 bp PCR-generated fragment encompassing the entire fpvI gene EcoRI/ BamHI-digested ligated to pUCP18 under the control of the Plac promoter | This study |
Iron-dependent regulation of PvdS expression and activity. P. aeruginosa cultures were grown until A600 ≈ 0.4 in DCAA supplemented with increasing concentration of FeCl3, as indicated.
A. Pyoverdine levels (◆ right ordinate) were determined by measuring the A405 of cell-free culture supernatants diluted in 100 mM Tris-HCl, pH 8.0, normalized by the A600 of the culture (A405 × A600−1 ± SD). The histograms show the LacZ (left ordinate) activity of pvdA::lacZ (black) pvdE::lacZ (white) and pvdD::lacZ (grey) transcriptional fusions, expressed in Miller units (Miller, 1972). Values are the averages from four independent assays (± SD).
B. Whole-cell lysates (30 μg) from each culture were probed with a mouse polyclonal anti-PvdS antibody. Purified PvdS was used as positive control (Ctrl).
Intracellular concentration of RNAP and RpoD in P. aeruginosa
The observation that pyoverdine production and pvd gene expression correlate with the intracellular levels of PvdS, combined with previous evidence of decreased transcription of pvd genes upon RpoD overexpression in P. aeruginosa (Ambrosi et al., 2005), suggest that PvdS is subjected to competition with RpoD for RNAPc binding. Therefore, we determined the intracellular content of RNAPc and RpoD in P. aeruginosa by means of quantitative immunoblot assays of RNAP subunits and cell counting. To set up our system, the copy number of RNAPc and RpoD was preliminarily quantified in E. coli W1485 (Bachmann, 1987; Table 1), a direct ancestor of the reference strain W3110 previously used for similar determinations (Jishage et al., 1996). In exponentially growing E. coli W1485 cells (generation time ≈ 46 min in LB at 37°C), the relative concentrations of RpoA (α subunit of RNAP) and RpoD were 1.9 and 0.7 ng μg−1 of total proteins respectively (Fig. S1; Table 2). Assuming that the whole RpoA pool is present as a complex with RNAP, our estimates of the cellular content of RNAP (calculated as ½ RpoA) and RpoD were 26.4 and 10.1 fmol μg−1 of total proteins, equivalent to 3089 and 1181 molecules per cell respectively. These values are fairly consistent with those reported for E. coli W3110 under similar growth conditions (2000 and 700 molecules of RNAP and RpoD, respectively, calculated on a per genome basis) (Jishage et al., 1996).
| Strain | Culture medium | Generation time (min ± SD) | A600 | cfu × 108 ml−1 (± SD) | Protein concn (μg ml−1 ± SD) | Specific protein concn (ng μg−1)a | Protein copy number per cfu | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| RpoA | RpoD | PvdS | RNAPb | RpoD | PvdS | ||||||
| W1485 | LB | 46 ± 6 | 1.1 | 6.8 ± 0.9 | 132 ± 16 | 1.93 | 0.71 | ND | 3089 | 1181 | ND |
| PAO1 | LB | 43 ± 6 | 1.1 | 6.3 ± 1.0 | 161 ± 12 | 2.04 | 0.72 | ND | 4282 | 1591 | ND |
| DCAA | 62 ± 8 | 0.4 | 6.5 ± 0.6 | 58 ± 9 | 3.03 | 1.21 | 0.23 | 2221 | 933 | 582 | |
- a. The reported values correspond to means of three independent quantitative immunoblot determinations, with SD < 12% of each value.
- b. Deduced from the determination of RpoA concentration, assuming the typical α2ββ′ω composition of RNAPc.
- ND, not determined.
As the intracellular concentration of RNAPc depends on the bacterial growth rate and, consequently, on the culture medium (Bremer and Dennis, 1996), analysis was performed in P. aeruginosa cells grown both in LB medium and in the iron-poor DCAA medium. Generation times, biomass (A600), viable cell counts (cfu) and total protein concentrations at exponential phase were determined (Table 2). As expected, growth in low-iron medium increased the generation time calculated from A600 readings (≈ 62 min in DCAA versus ≈ 43 min in LB). Although the cfu recovered after 4 h growth were similar for all cultures, the protein concentration for DCAA-grown cells was only 36% of that for LB-grown ones, reflecting the overall reduced size of cells grown under nutrient starvation (Higgs et al., 2002; Weart et al., 2007 and references therein; Table 2). In both media, the expression of RpoA and RpoD was maintained at a nearly constant level from the exponential to the stationary phase, i.e. between 4 and 9 h (Fig. 2). Interestingly, the relative concentration of RpoA and RpoD at the exponential phase was lower for LB than for DCAA cultures, ranging from 55.7 and 10.3 fmol μg−1 of total proteins in LB to 82.7 and 17.4 fmol μg−1 of total proteins in DCAA respectively (Fig. 2 and Table 2). When these data were considered on a per cell basis, the intracellular levels of RNAP and RpoD were 4282 and 1591 molecules in LB-grown cells and 2221 and 933 molecules in DCAA-grown cells respectively. The apparent decrease in the RNAP and RpoD copy number per cell under iron-limiting conditions (DCAA) was the result of the dramatic reduction of absolute protein content, compared with LB cultures.
Quantitative immunoblot analysis of RpoA and RpoD expression in P. aeruginosa PAO1. Nitrocellulose filters containing twofold serial dilutions of whole-cell lysates from P. aeruginosa cells grown to exponential (Exp; 4 h) and stationary (Stat; 9 h) phase in LB (A) and DCAA (B) and standard amounts of purified RpoD and RpoA were probed with commercial monoclonal anti-RpoD and anti-RpoA antibodies, as indicated. The amounts of whole-cell lysates (μg) and reference proteins (ng) are shown.
Intracellular concentration of PvdS in P. aeruginosa
The intracellular concentration of PvdS was calculated in the iron-starved P. aeruginosa cell extracts previously used for RpoA and RpoD determinations. To rule out any cross-hybridization signal due to sequence similarity between P. aeruginosa PAO1 IS sigmas (≤ 35.5% sequence identity; Visca et al., 2002), the specificity of anti-PvdS polyclonal antibodies was first confirmed by the absence of any signal relative to PvdS in immunoblot analysis of total bacterial proteins from the P. aeruginosa pvdS mutant PAO OT11pvdS (Fig. 3A). Quantitative immunoblot analysis showed that the relative concentration of PvdS was maximum in exponential growth-phase, attaining ≈ 10.8 fmol μg−1 of total proteins, while it drastically decreased below the sensitivity limit of antibody detection (≈ 0.6 ng of purified PvdS; Fig. 3B) in stationary phase. Therefore, PvdS attained 582 copies per cell during its maximal expression at the exponential phase in iron-poor medium, corresponding to ≈ 62% of RpoD levels. No PvdS expression was observed in DCAA supplemented with 100 μM FeCl3 (Fig. 3A) as well as in LB medium (data not shown).
Quantitative immunoblot analysis of PvdS expression in P. aeruginosa PAO1.
A. Specificity of the mouse polyclonal antibody against PvdS. Whole-cell lysates (25 μg) from P. aeruginosa strains PAO1 and PAO OT11pvdS grown to the mid-exponential phase in iron-poor [DCAA; (−FeCl3)] and iron-rich [DCAA plus 100 μM FeCl3; (+FeCl3)] media were probed with the anti-PvdS antibody. Purified PvdS (20 ng) was used as positive control.
B. PvdS levels in P. aeruginosa PAO1. Twofold serial dilutions of whole-cell lysates from exponential (Exp; 4 h) and stationary (Stat; 9 h) phase cultures in DCAA were probed with the anti-PvdS antibody. Definite amounts of purified PvdS from a serial twofold dilution were used for protein estimation by densitometric analysis. The amounts of whole-cell lysates (μg) and purified PvdS (ng) are indicated.
Intracellular concentration of RpoD- and PvdS-dependent RNAP holoenzymes in P. aeruginosa
In order to determine the RNAPc fraction engaged with PvdS, total RNAP was partially purified from iron-starved P. aeruginosa cells by heparin affinity chromatography. This resulted in enrichment of a protein fraction containing a mixture of RNAPc, various holoenzymes that were present in the cells during exponential growth in DCAA. Each purification step was analysed by immunoblot for the presence of RpoA (as a marker of total RNAP), RpoD and PvdS (Fig. 4A). Following passage of the crude cell extract through the heparin matrix, no RpoA signal was detected in the flow-through (Fig. 4A, lane 4), suggesting that the whole RNAP pool was retained by the column. On the other hand, the flow-through contained traces of RpoD and a remarkably high concentration of PvdS. These proteins represent the pool of free sigmas (i.e. the fraction uncomplexed with RNAPc) which is not retained by heparin. In contrast to the flow-through fraction, the level of RpoD in the eluate (i.e. the fraction complexed with RNAPc) was remarkably higher than the level of PvdS (Fig. 4A, lane 7).
Quantification of RpoD- and PvdS-dependent RNAP holoenzymes in P. aeruginosa PAO1.
A. Heparin-affinity purification of RpoD- and PvdS-dependent RNAP holoenzymes from lysates of iron-starved P. aeruginosa PAO1. Equal volumes (10 μl) of samples at different stages of purification (see Experimental procedures) were subjected to SDS-PAGE. Proteins were stained with Coomassie brilliant blue (lower panels) or electrotransferred onto nitrocellulose filters and probed with commercial monoclonal anti-RpoD and anti-RpoA antibodies, and with polyclonal anti-PvdS antibody (down). Samples in each lanes are: 1, soluble cell extract; 2, polymin P precipitate; 3, (NH4)2SO4 precipitate; 4, flow-through; 5, wash with 0.1 M NaCl; 6, wash with 0.2 M NaCl; 7, eluate with 0.6 M NaCl; 8, commercial vegetative RNAP holoenzyme (250 ng; Epicentre) supplemented with purified PvdS (30 ng); M, protein size standards of 116.0, 66.2, 45.0, 35.0, 25.0 kDa (Fermentas). The specificity of antibodies used for immunodetection is indicated.
B. Quantitative immunoblot analysis of RpoD, RpoA and PvdS in the soluble cell extract of iron-starved P. aeruginosa PAO1 (fraction 1) and in the RNAP-enriched eluate (fraction 7). Definite protein amounts (μg) from fractions 1 and 7, and standard amounts of purified RpoD, RpoA and PvdS (ng) were probed with antibodies against RpoD, RpoA and PvdS. The specificity of antibodies used for protein immunodetection is indicated.
The percentage of RpoD and PvdS holoenzymes in the eluate, relative to total RNAP, was determined by quantitative immunoblot analysis, and compared with the concentration of sigmas and RNAP in the soluble cell extract (Fig. 4B). The yields of RNAP, RpoD and PvdS in the soluble cell extract and in the heparin-enriched eluate are shown in Table 3. Based on RpoA quantification, RNAP was 13.6-fold concentrated in the eluate fraction relative to the soluble fraction. Trapping RNAP onto heparin led to an 8.4-fold increase in the concentration of RpoD in the eluate, relative to the soluble cell extract, while PvdS concentration was similar in the two fractions. This indicates that, under the experimental conditions used and different from RpoD, a substantial amount of PvdS is not engaged as RNAP holoenzyme. Thus, assuming that the whole amount of RpoD and PvdS recovered in the eluate is in complex with RNAP, we determined that RpoD- and PvdS-dependent RNAP holoenzymes account for 27.3% and 1.3% of total RNAP.
| Sample | RpoA (total RNAPb) | RpoD | PvdS | |||
|---|---|---|---|---|---|---|
| Concn (fmol μg−1)c | Purification fold | Concn (fmol μg−1) | Purification fold | Concn (fmol μg−1) | Purification fold | |
| Clear lysate | 123.0 (61.5) | 1.0 | 27.2 | 1.0 | 13.2 | 1.0 |
| Eluate | 1680.2 (840.1) | 13.6 | 229.0 | 8.4 | 11.0 | 0.8 |
- a. Cells were exponentially grown in DCAA.
- b. Deduced from the determination of RpoA concentration, assuming the typical α2ββ′ω composition of RNAPc.
- c. Indicated as fmol of immunodetectable protein per μg of total proteins in sample. The reported values correspond to means of three independent quantitative immunoblot determinations, with SD < 11% of each value.
To rule out that the low yields of PvdS–RNAP holoenzyme from heparin-affinity chromatography were consequent to destabilization of this complex during partial purification, an attempt was made to recover total RNAP from the cell lysate by immunoprecipitation with anti-RpoA antibodies. The immunoprecipitates were analysed by immunoblot for RpoA, RpoD and PvdS content. Because of a low efficiency of immunoprecipitation, only 1.7% of RNAP was recovered from the soluble cell lysate by an excess of anti-RpoA antibodies (i.e. 1096 fmol mg−1 of clear lysate proteins). Of this immunoprecipitable fraction, ≈ 13% was recovered as RpoD–RNAP complex, while the PvdS–RNAP complex was undetectable. The same results were observed also for P. aeruginosa PAO1(pUCPalgQ), overexpressing the RpoD-antisigma AlgQ (Ambrosi et al., 2005), in spite of the moderate increase of PvdS-dependent transcription observed in this strain (Table 4; Fig. S2). Based on the lower sensitivity limit of PvdS immunodetection (≈ 0.6 ng), we deduce that the PvdS–RNAP complex accounts for < 2.5% of the RNAP holoenzyme pool of iron-starved P. aeruginosa cells, in spite of the overall abundance and transcriptional activity of the PvdS sigma.
| Strain | LacZ activity (%)a | ||
|---|---|---|---|
| pvdA::lacZ | pvdD::lacZ | pvdE::lacZ | |
| PAO1b | 17 392 (100) | 9334 (100) | 10 920 (100) |
| K1660 ΔfpvAb | 10 435 (60) | 5787 (62) | 5 897 (54) |
| PAO1 fpvR | 18 262 (105) | 9521 (102) | 10 265 (94) |
| PAO1 pvdF | 11 130 (64) | 4928 (53) | 6 268 (57) |
| PAO1 pvdF+ PVDc | 17 670 (102) | 9377 (100) | 10 822 (99) |
| K1660 ΔfpvA(pUCPfpvI) | 17 632 (101) | 9521 (102) | 11 073 (101) |
| PAO1(pUCPalgQ) | 21 218 (122) | NT | NT |
- a. LacZ activity was determined in lysates of P. aeruginosa cultures exponentially grown in DCAA, and expressed in Miller units (Miller, 1972). Values are means of four independent determinations. The standard deviation is < 13% of each value. In parentheses are given the percentages of LacZ activity relative to P. aeruginosa PAO1.
- b. Transformation of PAO1 and K1660 ΔfpvA with the control vectors pUCP18 and pUCP19 had no significant effect on the activity of different pvd::LacZ fusions expressed by DCAA-grown cells.
- c. Pyoverdine-conditioned medium (PVD) was added to achieve 30 μM final pyoverdine concentration (for details, see Experimental procedures).
- NT, not tested.
Effect of pyoverdine signalling on activity and intracellular concentration of PvdS in P. aeruginosa
In addition to iron, the activity of PvdS is regulated by the inner membrane-spanning antisigma factor FpvR through the pyoverdine signalling cascade (Lamont et al., 2002). In the absence of efficient pyoverdine-mediated iron uptake, a portion of PvdS molecules is inactivated by the FpvR antisigma factor by a still undefined mechanism. In order to verify if FpvR controls PvdS activity by influencing its stability, the intracellular concentration of PvdS was compared in wild-type P. aeruginosa PAO1 and in the fpvR, ΔfpvA and pvdF mutants, blocked at different steps of the pyoverdine signalling pathway (Fig. 5A). In parallel, PvdS activity was monitored by comparing in the same strains the pyoverdine yields, the LacZ levels expressed by pvdA::lacZ, pvdD::lacZ and pvdE::lacZ transcriptional fusion, and the expression levels of PvdA by immunoblot (Table 4; Fig. 5B). A remarkable reduction of reporter gene expression was observed for all pvd::lacZ promoter fusions in the pvdF (36–47%) and in the ΔfpvA (38–46%) mutants, relative to PAO1, while expression in the fpvR mutant was comparable to wild type (94–105%). Similar results were observed for pyoverdine production in the ΔfpvA and fpvR mutants (Fig. 5A). To more closely examine the role of pyoverdine as signal, PAO1 and PAO pvdF cultures were treated with a pyoverdine-conditioned medium to obtain a final concentration of 30 μM pyoverdine (for details, see Experimental procedures). Addition of exogenous pyoverdine restored wild-type levels of reporter gene expression in the pvdF mutant (99–102% relative to PAO1), and had no significant effect on PvdS-dependent transcription in the pyoverdine-proficient PAO1 strain (Table 4, data not shown). Notably, no differences in PvdS activity were observed upon addition of medium conditioned by the siderophore-defective mutant (data not shown). Immunoblot analysis of PvdA expression mirrored the transcriptional response observed for the pvdA::lacZ fusion in the different genetic backgrounds (Fig. 5). These results are in line with those previously reported for pvd genes (Lamont et al., 2002; Shen et al., 2002). However, here we show that there are no significant differences in PvdS levels between the wild type and signalling mutants (Fig. 5B), suggesting that pyoverdine signalling is likely to control PvdS activity rather than the PvdS intracellular levels. To corroborate this hypothesis, PvdS stability was investigated under conditions of active and inactive pyoverdine signalling. Following addition of protein synthesis inhibitors, PAO pvdF cultures were treated or not with exogenous pyoverdine, and intracellular PvdS levels were compared between induced and uninduced cells at 5, 10 and 20 min after treatment. PvdS showed a similar stability profile under both conditions (Fig. 5C), indicating that pyoverdine signalling has no appreciable effect on PvdS stability.
Effect of pyoverdine signalling on PvdS levels, activity and stability.
A. Schematic representation of the pyoverdine (black circle) signalling pathway in the P. aeruginosa strains used for this experiment. Relative pyoverdine levels produced by each strain grown in DCAA to mid-exponential phase are expressed as A405 × A600−1. Values are the averages from three independent experiments with a SD ≤ 10%. ND, not detectable.
B. Twofold serial dilutions of whole-cell lysates (10–2.5 μg of total proteins for PvdA detection; 20–5 μg of total proteins for PvdS detection) from wild-type P. aeruginosa (WT), and ΔfpvA, fpvR and pvdF mutants grown to the mid-exponential phase in DCAA or DCAA supplemented with pyoverdine-conditioned medium (PVD), were probed with anti-PvdA or anti-PvdS antibodies, as indicated.
C. Immunoblot analysis of PvdS levels in PAO pvdF cells induced (+) or uninduced (−) with PVD. Protein synthesis was inhibited by addition of chloramphenicol, kanamycin and tetracycline (300, 200 and 300 μg × ml−1 respectively). Samples were collected at indicated time points. Whole-cell lysates (10 μg of total proteins) were probed with anti-PvdS antibody. Purified PvdA and PvdS proteins (20 ng each) were used as internal controls (Ctrl).
Subcellular localization of PvdS in P. aeruginosa
FpvR has recently been shown to interact with PvdS in vivo (Rédly and Poole, 2005), raising the possibility that part of the PvdS pool is sequestered by FpvR at the cytoplasmic membrane. As the membrane association of PvdS has not yet been investigated, we analysed the subcellular localization of PvdS in wild type and fpvRΔfpvA and pvdF mutant strains. Membranes were separated from soluble cell extracts, and the levels of PvdS in membrane and soluble fractions were determined by quantitative immunoblot analysis (Fig. 6A).
Subcellular localization of PvdS in P. aeruginosa.
A. Partition of PvdS between the soluble (S) and the membrane (M) protein fractions from wild type (WT) and ΔfpvA, ΔfpvA(pUCPfpvI), fpvR and pvdF mutants of P. aeruginosa grown to the mid-exponential phase in DCAA supplemented or not with pyoverdine-conditioned medium (PVD). For each strain, equivalent volumes of subcellular fractions were separated by SDS-PAGE, electrotransferred onto a nitrocellulose membrane, and probed with the anti-PvdS antibody to determine the relative levels of PvdS by densitometric analysis (for details, see Experimental procedures). The histogram summarizes the results obtained from three independent experiments (±SD), with a representative experiment shown in the upper part of the panel. FpvR-independent and FpvR-dependent retention of PvdS on the membrane, and the effect of pyoverdine signalling, are schematized on the right.
B. Membrane samples from P. aeruginosa PAO1 were treated or not (NT) with 0.1 M NaOH, 2% sarcosyl and 5 M urea. After resedimentation of the membranes, equal volumes of the supernatant (S) and membrane-retained proteins (R) were resolved by SDS-PAGE, electroblotted and probed for PvdS immunodetection.
A different subcellular distribution of PvdS was reproducibly observed between strains (Fig. 6A). In particular, 63% and 67% of total PvdS was associated with the membrane fraction in the ΔfpvA and pvdF mutants respectively. Conversely, the fpvR mutant and, to a lesser extent, the wild type showed a prevalent distribution of PvdS in the cytosolic fraction (83% and 70% of total PvdS respectively). Addition of exogenous pyoverdine to PAO pvdF cultures caused the release of a substantial amount of membrane-associated PvdS (≈ 42% of total PvdS), resulting in a PvdS subcellular distribution similar to that observed in PAO1 (Fig. 6A). Thus, pyoverdine acts as a signal which facilitates PvdS release from the membrane. Interestingly, overexpression of FpvI compensated the ΔfpvA mutation, in so far as it restored the wild-type profile of PvdS subcellular distribution and transcription activity (Table 4, Fig. 6A), in line with the dichotomical control of the FpvR antisigma on both FpvI and PvdS sigma factors.
Intriguingly, a minor fraction of PvdS was retained by the membrane in the fpvR-defective background, suggesting that PvdS is either endowed with an intrinsic membrane binding capacity, or that other molecules serve as an anchor for specific membrane retention of PvdS.
In order to investigate the biochemical nature of PvdS–membrane association, membranes from wild type and fpvR and ΔfpvA mutant strains were treated with chemical agents that disrupt different types of protein bonding with membranes. PvdS was released from P. aeruginosa membranes by sarcosyl (2%), arguing for its localization at the inner membrane level, and by alkali (0.1 M NaOH) and denaturant (5 M urea), suggesting that PvdS behaves as a peripheral protein and ruling out any unspecific cosedimentation of PvdS with the membranes. (Fig. 6B). Lastly, there were no differences in the susceptibility of PvdS to chemical extraction from the membrane between wild type, the fpvR, the pvdF and the fpvA mutant (data not shown).
Discussion
In the last two decades, great effort has been spent on understanding the competition strategies between vegetative and alternative sigma factors (reviewed in Ishihama, 2000; Gruber and Gross, 2003; Gourse et al., 2006). However, limited information is available on the ECF sigma factors, in spite of their central role in adaptation and/or survival of bacteria under diverse environmental conditions (Gruber and Gross, 2003). Within the ECF family, IS sigmas constitute a group of iron repressible transcription factors whose intracellular levels and molecular mechanisms of post-translational regulation remain ill defined. The vast majority of IS sigmas direct transcription of a single gene, namely the receptor gene for the uptake of an iron chelate (Visca et al., 2002). However, the range of PvdS regulation is broader than the sole control of pyoverdine synthesis. Indeed, PvdS controls a large regulon, recognizing the promoters of many genes or operons primarily implicated in siderophore biogenesis and synthesis of extracellular factors in P. aeruginosa (Leoni et al., 2000; Visca et al., 2002). Hence, PvdS should be regarded as the major IS sigma factor in P. aeruginosa.
Knowledge of the intracellular levels of RNAP and individual sigma factors is a prerequisite for establishing a competition model between sigmas. While estimates of RNAP and sigma(s) copy numbers have been obtained for a variety of reference species (e.g. E. coli, Bacillus subtilis) and strains, under different physiological conditions and using various techniques (Iwakura et al., 1974; Bremer and Dennis, 1996; Jishage et al., 1996; Ju et al., 1999; Fujita, 2000), such information is missing for P. aeruginosa. Therefore, we determined the intracellular content of RNAP and RpoD in P. aeruginosa cells from standard LB medium and iron-poor (DCAA) medium (Fig. 2), and showed that the concentration of total RNAP varies from ≈ 4200 to ≈ 2200 molecules per cell, depending on the culture medium, and remains constant from the exponential to the stationary phase. Notably, the cellular content of RNAP preliminarily determined by us for the E. coli W1485 control strain (Table 2 and Fig. S1) was consistent with that previously reported for similar E. coli strains grown at a comparable rates (Iwakura et al., 1974; Bremer and Dennis, 1996; Ishihama, 2000). In P. aeruginosa PAO1, estimates of the vegetative RpoD sigma were ≈ 1500 and 900 molecules per cell in LB and DCAA respectively. Comparison between P. aeruginosa PAO1 and E. coli W1485 grown under identical conditions revealed higher (≈ 27%) intracellular levels of both total RNAP and RpoD in P. aeruginosa (summarized in Table 2). These differences are likely to reflect the need for a more abundant transcriptional machinery to meet with the higher gene content of P. aeruginosa compared with E. coli (5570 versus 4400 ORFs respectively).
It has been reported that about one-third of E. coli RNAP molecules are not involved in transcription elongation and/or non-specific DNA binding (Ishihama, 2000). As this is the only RNAP fraction available for sigma binding in the cytosol, our counts for P. aeruginosa would imply that RpoD alone could be sufficient to fully saturate the free RNAPc pool, as it is for E. coli. Indeed, the RNAP : RpoD ratio is similar in P. aeruginosa and E. coli (2.7 and 2.6 respectively; data derived from Table 2). Then, how does PvdS successfully compete with RpoD for RNAP holoenzyme formation in P. aeruginosa? We found that PvdS is maximally expressed during the exponential phase (Fig. 3), consistent with the notion that pyoverdine is produced at maximum rate during this growth stage (Meyer and Abdallah, 1978; Putignani et al., 2004). Furthermore, PvdS expression is very sensitive to extracellular iron concentration, as the result of Fur repression on the pvdS promoter (Fig. 1; Cunliffe et al., 1995; Leoni et al., 1996). Addition of Fe(III) up to 2 μM caused dose-dependent reduction of the PvdS intracellular content and, consequently, of transcription from pvd promoters (Fig. 1). Notably, pvd promoters were not transcribed when intracellular PvdS levels were less than ≈ 5% of maximum expression, i.e. for less than ≈ 30 PvdS molecules per cell (Fig. 1 and Table 2). This highlights the importance of PvdS copy number for pvd gene expression.
PvdS attains a maximum of ≈ 600 molecules per cell, reaching 62% of the RpoD levels (Fig. 3 and Table 2), which is a very high concentration for an alternative sigma factor. None of the six alternative sigmas of E. coli attains such high levels (Maeda et al., 2000a), even though estimates for the two ECF sigma factors RpoE and FecI (3–9 and 0.4–0.9 molecules per cell respectively) were previously obtained under conditions in which these sigma factors were not expected to be induced (Maeda et al., 2000b). While the impressively high PvdS levels are likely to reflect the high PvdS–RNAP demand for recognition of multiple promoters (≈ 26), the observed PvdS : RpoD ratio can only in part explain the change in transcription pattern under IS, as the major determinant for such change is the ratio between different RNAP holoenzymes in the cytoplasm.
Mutational and structural studies revealed a similar way of interaction between different sigma factors of the σ70 family and RNAPc (reviewed by Burgess and Anthony, 2001; Gruber and Gross, 2003; Wilson and Lamont, 2006). The primary RNAPc-binding determinants are contained in domains 2, 4 and partly 3 relative to the σ70 structure. It is noteworthy that ECF sigma factors are a ‘stripped-down’ version containing only smaller variant of domains 2 and 4 (Lonetto et al., 1992, 1994). Therefore, the minor extension of the overall ECF sigma structures may significantly reduce the interaction with RNAPc, and hence the binding affinity. In a mixed reconstitution experiment Maeda et al. (2000a) estimated an approximately ninefold and sevenfold lower affinity for RNAPc binding for the E. coli ECF sigmas RpoE and FecI, relative to RpoD. Wilson and Lamont (2000) reported biochemical evidence for a lower affinity of purified PvdS for RNAPc with respect to the vegetative sigma RpoD. Rather than performing in vitro studies, we focused on the in vivo analysis of the RNAP pool under condition (low-iron) which is conducive to PvdS expression. Our results show that only 1% of total RNAPc is complexed with PvdS, while 27% is complexed with RpoD (Fig. 4 and Table 3). Interestingly, our determination of the RpoD-dependent holoenzyme fraction in P. aeruginosa is fully consistent with that reported for E. coli (Maeda et al., 2000a). The limited fraction of recoverable PvdS–RNAP complex could reasonably be explained by the low affinity of PvdS for RNAPc (Wilson and Lamont, 2000) and/or by an intrinsic instability of the PvdS–RNAP complex. However, the low amount of PvdS–RNAP holoenzyme may not be that surprising, given that PvdS is predicted to activate < 1% of all P. aeruginosa genes. It is evident that such concentration of PvdS–RNAP holoenzyme (≈ 29 molecules per cell) is enough for efficient transcription initiation at target promoters (Fig. 1).
We provide evidence that the vast majority (≈ 95%) of PvdS is not associated with RNAPc (Fig. 4), suggesting that expression of PvdS at high levels is required to direct the RNAP equilibrium towards formation of the PvdS-containing holoenzyme. We also observed that overexpression of the AlgQ protein, the RpoD antisigma, moderately increases PvdS activity (Table 4) without causing detectable changes in the levels of both PvdS and PvdS-dependent RNAP holoenzyme (Ambrosi et al., 2005; Fig. S2 and data not shown). This could mean that unappreciable changes in levels of PvdS–RNAP may have an appreciable effect on the expression of pvd genes, and/or that AlgQ increases PvdS activity by mechanisms additional to RpoD binding (Dove and Hochschild, 2001; Ambrosi et al., 2005).
Lastly, the regulation of PvdS activity by pyoverdine signalling through the FpvA-FpvR receptor-antisigma pair (Lamont et al., 2002; Fig. 4A) raises the possibility that the actual amount of PvdS available for holoenzyme formation could be lower than that determined in the whole-cell extract. Our results reveal that pyoverdine signalling modulates PvdS activity by mechanism(s) other than controlled proteolysis (Fig. 5). Indeed, pyoverdine signalling seems to control PvdS activity by modulating its cytosolic concentration through FpvR-dependent sequestration on the inner membrane. In wild-type P. aeruginosa PAO1 grown under iron-limiting conditions, ≈ 30% of PvdS is associated with the inner membrane (Fig. 6A), and plausibly unavailable for RNAP holoenzyme formation. On this basis, the predictable number of soluble PvdS molecules in competition with the cytoplasmic pool of sigma factors is ≈ 420 per cell (47% of RpoD). We also show that in the pyoverdine signalling negative background (ΔfpvA and pvdF mutants) a substantial amount of soluble PvdS (47% and 52% of the wild-type levels, i.e. ≈ 200 and 220 of 420 molecules for ΔfpvA and pvdF mutants respectively) is sequestered on the inner membrane by FpvR (Fig. 6A). This redistribution of PvdS results in a marked reduction of pvd gene expression (Lamont et al., 2002; Table 4), indicating that the membrane-associated PvdS pool is transcriptionally inactive. As a consequence of the dual control of FpvR on both PvdS and FpvI (Beare et al., 2003; Rédly and Poole, 2005), overexpression of FpvI in the ΔfpvA mutant caused a substantial release of FpvR-bound PvdS from the membrane and restored wild-type level of PvdS activity (Table 4, Fig. 6A). Hence, FpvR behaves as an antisigma sensu strictu, sequestering PvdS on the membrane and preventing its activity. Conversely, forcing the signalling cascade from active (wild-type PAO1) to hyperactive state (fpvR mutant) results in a slight increase in the number of cytosolic PvdS (19%, i.e. ≈ 420 versus 500 molecules), which has no apparent effect on PvdS-dependent transcription (Lamont et al., 2002; Fig. 6A, Table 4). On the other hand, the fpvR mutation does increase PvdS activity in a signalling defective background, as previously reported for the fpvA mutant (Lamont et al., 2002). We also show that the signalling activity of exogenously added pyoverdine can be detected only in a pyoverdine-deficient mutant, but not in the wild type (Table 4, 5, 6). These observations suggest that in wild-type P. aeruginosa PAO1 grown under conditions of strong iron limitation pyoverdine signalling does actually take place and the cytosolic PvdS pool reaches sufficient levels for promoting maximal expression of PvdS-dependent genes, thus masking any minor increase in its cytosolic concentration. We also noticed that a minor amount of PvdS specifically associates with the inner membrane even in the absence of FpvR (Fig. 6A). At present we cannot provide an explanation for such FpvR-independent PvdS retention on the membrane. However, the presence of 12 additional FpvR-like IS antisigmas in P. aeruginosa PAO1 (Visca et al., 2002), raises the possibility that cross-talk between signalling systems could occur, eventually expanding the number of molecular partners involved in post-translational control of PvdS activity.
In conclusion, we provide novel information on the intracellular levels of RNAP, vegetative sigma RpoD and IS sigma PvdS in P. aeruginosa. The high levels of PvdS appear to be compensated by a disadvantageous competition with RpoD for RNAP holoenzymes formation, and by a PvdS post-translational control through FpvR-dependent retention on the inner membrane. This equilibrium is influenced by extracytoplasmic pyoverdine signalling through the FpvA receptor protein.
Experimental procedures
Strains, plasmids and media
Bacterial strains and plasmids used in this study are listed in Table 1. E. coli and P. aeruginosa were routinely grown at 37°C in LB medium (Sambrook et al., 1989). DCAA was used as the low-iron medium for P. aeruginosa (Visca et al., 1993). To decrease iron availability and improve experimental reproducibility, Chelex-100 treatment of DCAA was repeated twice, and a single preparation of this medium was used for all experiments. Media were solidified with 1.2% agar N.1 (Unipath). To assess the role of pyoverdine as inducer, a pyoverdine-conditioned medium was prepared by growing the pyochelin-deficient P. aeruginosa PAO6331 strain in DCAA for 12 h at 37°C. The cells were removed by centrifugation and filtration through a 0.2-μm filter. Pyoverdine concentration was calculated as previously described (Wilderman et al., 2001). Pyoverdine-conditioned medium was added to obtain 30 μm final pyoverdine concentration. As control, an equal volume of a medium conditioned by the PvdA mutant PA6331ΔpvdA was used. Unless otherwise stated, antibiotics were used in selective media at the following concentrations: tetracycline (Tc) 12.5 μg ml−1 for E. coli and 150 μg ml−1 for P. aeruginosa; kanamycin (Km) 25 μg ml−1 for E. coli and 300 μg ml−1 for P. aeruginosa; carbenicillin 500 μg ml−1 for P. aeruginosa; chloramphenicol (Cm) 30 μg ml−1 for E. coli and 100 μg ml−1 for P. aeruginosa; nalidixic acid 20 μg ml−1; ampicillin 25 μg ml−1 and streptomycin 25 μg ml−1 for E. coli.
DNA manipulations and genetic techniques
Recombinant DNA was manipulated as described elsewhere (Sambrook et al., 1989). Transfer of plasmids from E. coli to P. aeruginosa was performed by triparental mating with the helper plasmid pRK2013 (Figurski and Helinski, 1979). For construction of the pUCPfpvI plasmid, a 700 bp fragment encompassing the fpvI gene was obtained by PCR amplification with primers FwfpvI (5′-GGG AAT TCG GAT TGC GCT GCG AGC-3′) and RvfpvI (5′-GGG ATC CTG GAG GGC CAT CTG TTG-3′) corresponding to nucleotides −144 to −125 and +540 to +556, relative to the TTG translation start codon of the fpvI gene, using PAO1 genomic DNA as the template. The EcoRI and BamHI restriction sites (underlined) were included to make directional cloning of the amplicon into the corresponding restriction sites of pUCP18, which yielded pUCPfpvI.
Growth conditions
To determine the intracellular content of RpoA and RpoD, P. aeruginosa PAO1 and E. coli W1485 were grown at 37°C in LB under vigorous aeration (250 revolutions per minute in a New Brunswick 25 orbital shaker). RpoA, RpoD and PvdS intracellular levels were also determined in P. aeruginosa PAO1 cells grown in DCAA medium under the same conditions. In particular, overnight P. aeruginosa and E. coli cultures in LB or DCCA were 100-fold diluted in the same media to an A600 ≈ 0.01, followed by periodic A600 measurements to determine the generation time. Samples from mid-exponential (4 h) and stationary (9 h) growth phase were collected and cellular protein concentration was determined by the DC protein assay kit (Bio-Rad) with bovine serum albumin as the standard. Viable cell counts (expressed as cfu ml−1) obtained by the plate dilution method were also performed for mid-exponential samples, and compared with the corresponding A600 readings.
Pyoverdine determinations and β-galactosidase activity assays
P. aeruginosa cultures were grown in DCAA at 37°C for 4 h with vigorous aeration until A600 ≈ 0.4. Pyoverdine was quantified by measuring the A405 of culture supernatants diluted in 100 mM Tris-HCl, pH 8.0 (Visca et al., 1992). The LacZ activity from P. aeruginosa cells carrying the different promoter-probe plasmids (Table 1) was determined spectrophotometrically using o-nitrophenyl-β-d-galactopyranoside as the substrate, normalized to the A600 of the bacterial culture and expressed in Miller units (Miller, 1972). Pyoverdine determinations and β-galactosidase assays were expressed as means of at least four independent experiments, each performed in duplicate.
Preparation of protein extracts and cell fractionation
Appropriate volumes of bacterial cultures at a definite cell concentration were centrifuged, and pellets suspended in SDS-PAGE loading buffer (0.25 M Tris-HCl, pH 6.8; 2% SDS; 10% 2-mercaptoethanol; 20% glycerol) for SDS-PAGE analysis of whole-cell extracts. For separation of soluble from membrane proteins, cell pellets from 100 ml of DCAA cultures were suspended in 1 ml of ice-cold extract buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 2 mM phenylmethylsulphonil fluoride) and disrupted by sonication. Unbroken cells and cellular debris were removed by low-speed centrifugation (3000 g for 10 min at 4°C) before separation of soluble from membrane fraction by ultracentrifugation (50 000 g for 90 min at 4°C). The supernatant containing the soluble protein fraction was removed, and the pellet containing membrane proteins was suspended in the original volume of ice-cold extract buffer. Fractionation efficiency was assessed by measuring isocitrate dehydrogenase activity (a cytoplasmic enzyme marker), and lactate dehydrogenase activity (an inner membrane enzyme marker), as previously described (Goldberg and Ellis, 1983; Vassault, 1983). Separation was considered acceptable when subcellular fractions showed less than 2% cross-contamination between enzyme markers of the different fractions.
The association type of PvdS with the membranes of P. aeruginosa was assessed by means of different chemical treatments. Membrane samples were treated with 0.1 M NaOH, or 2% sarcosyl (N-lauryl-sarcosine), or 5 M urea for 30 min at 4°C, and then centrifuged at 50 000 g for 90 min at 4°C. Proteins retained in the pellet were solubilized in SDS-PAGE-loading buffer. Proteins in the supernatants were precipitated with 10% trichloroacetic acid and suspended in SDS-PAGE-loading buffer. All protein samples were heated at 100°C for 5 min prior to SDS-PAGE analysis. Experiments were performed in triplicate.
PvdS stability assay
For in vivo assessment of PvdS stability, PAO pvdF was grown in DCAA to the mid-exponential phase. Protein synthesis was inhibited by the addition of Cm (300 μg ml−1), Km (200 μg ml−1) and Tc (300 μg ml−1). The culture was divided into two aliquots and treated with either pyoverdine-conditioned medium (30 mM final pyoverdine concentration) or with the same volume of a medium conditioned by the PvdA mutant PA6331ΔpvdA. Cells were collected at 0, 5, 10 and 20 min after treatment, and whole-cell extracts were subjected to Western blot analysis.
Partial purification of the RNA polymerase holoenzyme
RNA polymerase was purified from mid-exponential P. aeruginosa cells grown in DCAA by minor modifications of a previously described protocol (Burgess and Jendrisak, 1975). All purification steps were performed in cold room at 4°C. The frozen cell pellet from 1 l culture was mechanically disrupted with alumina (aluminum oxide, type A-5, Sigma). The cell paste was recovered with 10 ml of TGED buffer (20 mM Tris-HCl, pH 8.0, 5% glycerol, 0.05 mM EDTA, 0.3 mM dithiothreitol, 2 mM phenylmethylsulphonil fluoride) containing 0.1 M NaCl, and centrifuged at 18 000 g for 20 min. Polymin P (polyethyleneimine, Sigma) was added to the supernatant to a final concentration of 0.15%. The mixture was stirred for 30 min and the precipitate was collected by centrifugation at 35 000 g for 10 min. The pellet was dissolved in 10 ml of TGED buffer containing 1 M NaCl and stirred for 1 h with 50% saturation of (NH4)2SO4. The precipitate obtained by centrifugation at 35 000 g for 10 min was dissolved in 10 ml of TGED buffer containing 0.1 M NaCl and dialysed against 100 volumes of the same buffer. The dialysate was loaded on a HiTrap Heparin HP column (1 ml, Amersham Biosciences). The column was washed first with 5 ml of TGED buffer containing 0.1 M NaCl, and then with 5 ml of the same buffer containing 0.2 M NaCl. RNAP was eluted with 4 ml of TGED buffer containing 0.6 M NaCl. For quantification of RNAP subunits, the eluate was concentrated to a final volume of 1.3 ml by ultrafiltration through a Microcon YM-10 (10 kDa molecular weight cut-off) filter.
Immunoprecipitation
Pseudomonas aeruginosa cells from mid-exponential phase cultures in DCAA medium were collected by centrifugation and suspended in cold lysis buffer (150 mM NaCl, 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM phenylmethylsulphonyl fluoride). Cells were then treated with 2 mg ml−1 lysozyme for 30 min at 25°C, and the clear lysates were obtained by centrifugation for 20 min at 18 000 g at 4°C. After protein quantification, an appropriate volume equivalent to 1 mg of whole proteins was incubated with 3 μg of monoclonal anti-RpoA antibodies (Neoclone) for 2 h at 4°C. For immunoprecipitation, Protein G-Sepharose (Gibco) was added to the protein solution at 0.5%, and incubated for 12 h at 4°C. The Protein G-Sepharose-bound immunoprecipitate was washed twice in cold lysis buffer and then collected by centrifugation, suspended in SDS-PAGE-loading buffer, and heated at 100°C for 5 min prior to SDS-PAGE analysis.
SDS-PAGE, immunoblotting and densitometric analyses
Protein samples were analysed by SDS-PAGE in duplicate (Laemmli, 1970). Gels were either stained with Coomassie brilliant blue to visualize resolved proteins, or electrotransferred onto a nitrocellulose filter (Hybond C extra, Amersham), and probed for PvdS, PvdA, RpoD or RpoA using mouse polyclonal anti-PvdS antibody or mouse monoclonal anti-PvdA, anti-RpoD and anti-RpoA antibodies (Putignani et al., 2004; Ambrosi et al., 2005; Neoclone). The last two antibodies recognize common epitopes in the RpoD and RpoA orthologues from E. coli and P. aeruginosa (http://www.neoclone.com). Immune complexes were detected with secondary anti-mouse antibodies conjugated with either alkaline phosphatase (Promega) or horseradish peroxidase (Calbiochem). Filters were developed with 5-bromo-4-chloro-3-indoyl-phosphate and nitro blue tetrazolium chloride reagents for colorimetric alkaline phosphatase detection (Promega), or with the Amersham ECL chemiluminescent reagents (Amersham Biosciences) followed by exposure to an X-ray film (Kodak) for autoradiography.
Quantitative Western blot analysis of different RNAP subunits in protein samples was performed by direct densitometric comparison with a standard curve obtained from known quantities of purified RpoA or RpoD or PvdS, which were run in the same gel. The amount of RpoA, RpoD and PvdS was calculated using at least two points that fell within the segment of the standard curve for which densitometric intensity was a linear function of protein amount. The number of RNAP subunits per cell was determined by multiplying the relative concentration of the protein of interest (mol μg−1 of total protein) by the amount of total protein per cell (μg per cell) and Avogadro's number (Table 2). All quantification experiments were repeated at least three times. Densitometric measurements of band intensities were obtained by the Quantity One software and a Gel Doc 2000 CCD camera (Bio-Rad). Purified E. coli RNAP (Epicentre), PvdS and PvdA (laboratory stock; Leoni et al., 2000; Putignani et al., 2004) were quantified by the DC protein assay (Bio-Rad), and used as positive controls in Western blot analysis.
Acknowledgements
This work was supported by grants from the Ministry for Health of Italy (‘Ricerca Corrente 2006’ to the National Institute for Infectious Diseases ‘L. Spallanzani’), the Ministry of University and Research of Italy (PRIN-2006) and the Fondazione per la Ricerca sulla Fibrosi Cistica (Grant FFC#5/2007) to P.V.
