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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The quorum sensing (QS) signalling system of Pseudomonas aeruginosa controls many important functions, including virulence. Although the production of the QS signal molecule N-3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) is positively autoregulated, its concentration reaches a steady level long before stationary phase. The RsaL protein represses transcription of the lasI signal synthase gene, and thus reduces QS signal production. We show that RsaL binds simultaneously with LasR to the rsaL-lasI bidirectional promoter thereby preventing the LasR-dependent activation of both genes. In an rsaL mutant, 3OC12-HSL production continues to increase throughout growth. Thus RsaL provides homeostasis by functioning in opposition to LasR and limiting 3OC12-HSL production to a physiological concentration. Furthermore, transcription profiling revealed that RsaL regulates 130 genes independent of its effect on QS signal molecule production, including genes involved in virulence. We show that RsaL can repress pyocyanin and hydrogen cyanide virulence genes in two ways: directly, by binding to their promoters, and indirectly, by decreasing levels of the signals for their QS signal-dependent transcription. These investigations highlight the importance of RsaL as a global regulator of P. aeruginosa physiology that provides a counterbalance to 3OC12-HSL-dependent gene activation via multiple mechanisms.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Pseudomonas aeruginosa is a common inhabitant of soil and water and an opportunistic human pathogen that can cause both acute and chronic infections in hospitalized and immunocompromised hosts. It is the major cause of death in cystic fibrosis patients and a main cause of chronic wounds (Lyczak et al., 2002; Gjødsbøl et al., 2006). P. aeruginosa infections are difficult to eradicate as a consequence of intrinsic antibiotic resistance and growth in bacterial communities referred to as biofilms (Smith and Iglewski, 2003; Parsek and Greenberg, 2005).

Quorum sensing (QS) is a social behaviour, which enables a bacterial population to co-ordinate gene expression in response to cell density. The QS response is achieved when the concentration of a diffusible signal molecule reaches a threshold level (Fuqua et al., 1994; Miller and Bassler, 2001; Williams et al., 2007).

The P. aeruginosa QS circuitry is complex and hierarchical. The key QS signal N-3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) is required for the function of all other elements of the circuit. This signal is synthesized by LasI, which is encoded by lasI, and its cognate signal receptor is the transcriptional regulator LasR, encoded by the lasI-linked lasR. LasR activates transcription of hundreds of genes, including those coding for the other QS systems, which involve N-butanoyl-homoserine lactone (C4-HSL) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) signal molecules (reviewed in Diggle et al., 2006; Schuster and Greenberg, 2006). In addition there is a second 3OC12-HSL-dependent transcription factor, QscR that controls expression of additional genes (Lequette et al., 2006). As a whole, the QS circuit plays a key role in pathogenesis, regulating the production of virulence factors, including LasA and LasB elastases, alkaline protease, phospholipase C, lipase, exotoxin A, lectins, hydrogen cyanide and pyocyanin, the formation of biofilms, and the expression of antibiotic efflux pumps (reviewed in Smith and Iglewski, 2003; Schuster and Greenberg, 2006). Moreover, the QS signal molecules themselves may play a role in the cross-talk between host and parasite (Wagner et al., 2006).

The lasI gene is among those activated by LasR-3OC12-HSL, and this constitutes a positive feedback loop, which leads to amplification of 3OC12-HSL production. Positive autoregulation is common to many but not all acyl-HSL QS systems. It amplifies the rate of signal production and it may provide hysteresis to population density-dependent gene expression; once a critical concentration of signal is reached, signal production per cell will increase and small decreases in population density will not reverse the response. Although lasI is positively autoregulated, experimental data show that in P. aeruginosa the 3OC12-HSL concentration reaches a steady level long before stationary phase (Chugani et al., 2001; Ward et al., 2004). This indicates that an unidentified homeostatic mechanism must be devoted to capping 3OC12-HSL production.

The P. aeruginosa QS response is influenced by many regulators, which affect to various extent the production of the QS signal molecules (Schuster and Greenberg, 2006; Venturi, 2006). Some of these factors are negative regulators of 3OC12-HSL production and therefore could be involved in the signal homeostasis. The RsaL transcriptional regulator, encoded by the rsaL gene, represses LasI expression by binding the promoter of lasI (PlasI) (de Kievit et al., 1999; Rampioni et al., 2006). Besides LasR, RsaL is the only regulator known to bind PlasI, and among the QS repressors characterized to date it displays the most dramatic effect on 3OC12-HSL production (Schuster et al., 2004; Rampioni et al., 2006). Moreover, RsaL is the only QS repressor directly dependent on LasR-3OC12-HSL for its expression (de Kievit et al., 1999). These features place RsaL as a key regulator in the complex QS circuit and lead us to question a possible role for this protein in QS signal homeostasis.

Here, we show how the interplay between RsaL and LasR regulates the rsaL-lasI bidirectional promoter to function in 3OC12-HSL homeostasis. We also demonstrate that RsaL is a global regulator that dramatically affects transcript levels of many genes in two ways, either by repressing 3OC12-HSL production or by acting as a transcriptional regulator that interacts with promoters directly.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

RsaL is involved in 3OC12-HSL homeostasis

To investigate the role of RsaL in 3OC12-HSL homeostasis, we measured production of 3OC12-HSL and we monitored PlasI-lacZ transcription in cultures of P. aeruginosa PAO1 and in a rsaL mutant of this strain (Fig. 1A). The rsaL mutation had no effect on 3OC12-HSL production and lasI transcription until the late logarithmic phase (A600 about 2.0) at which time 3OC12-HSL concentration and PlasI activity were maintained at a constant level in the wild-type strain, while continuing to increase in the rsaL mutant.

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Figure 1. Effect of RsaL on 3OC12-HSL production and PlasI activity. A. 3OC12-HSL production (open symbols) and β-galactosidase activities (Miller units, M.u.; filled symbols) of P. aeruginosa PAO1 (circles) and its rsaL mutant derivative (triangles) carrying the pPlasI190 plasmid. Standard deviations (vertical lines) are based on the mean values for three independent experiments. B. Western analysis performed with anti-RsaL antiserum on the cellular soluble fractions derived from P. aeruginosa PAO1 culture samples withdrawn at the first eight points of the growth curve as in (A); P, purified RsaL protein as positive control; N, cellular soluble fraction derived from PAO1-DM1 culture at an A600 of 2.0 as negative control.

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Western immunoblotting showed that LasR levels were comparable in the rsaL mutant strain and its parent, thus RsaL does not influence lasR expression (data not shown). The level of RsaL in the wild-type strain increased between culture densities (A600) of 2 and 3, and subsequently fell back to a constant level similar to that disclosed at A600 of 2 (Fig. 1B). The increase in RsaL levels coincides with the point at which the production of 3OC12-HSL diverges in the parent and mutant strains. This is consistent with the conclusion that RsaL induction by 3OC12-HSL results in sufficient RsaL to keep 3OC12-HSL production at a steady level, balancing the positive feedback acting on lasI expression. These results show that RsaL is a key effector of 3OC12-HSL homeostasis.

RsaL and LasR can bind PlasI simultaneously

DNase I protection assays demonstrated that RsaL and LasR bind two distinct but partially overlapping sites on PlasI, suggesting that the two proteins might compete for binding to this promoter (Schuster et al., 2004; Rampioni et al., 2006). To clarify this point, we performed electrophoretic mobility shift assays (EMSAs) with a DNA-probe encompassing PlasI and purified LasR and RsaL. To assure maximum binding of LasR, we included 3OC12-HSL in the reaction mixtures (Schuster et al., 2004). Control experiments without 3OC12-HSL showed that RsaL binding was not influenced by the signal (data not shown). With both RsaL and LasR we observed a super-shifted band (Fig. 2A and B) that did not appear in the presence of RsaL or LasR alone. This supports the view that RsaL and LasR can bind PlasI simultaneously. Furthermore, LasR bound to pre-formed RsaL-PlasI and to free probe similarly (Fig. 2A), and RsaL bound to pre-formed LasR-PlasI and to free probe similarly (Fig. 2B). This suggests that binding of one protein to PlasI does not alter the affinity of the other protein. These data were confirmed by a DNase I protection assay showing the simultaneous binding of RsaL and LasR to PlasI, without an appreciable change in the protection pattern of the two regulators irrespective of their independent or simultaneous binding (Fig. 2C and D).

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Figure 2. RsaL and LasR binding to the rsaL-lasI bidirectional promoter. A and B. EMSA assays. RsaL and LasR concentrations are indicated above the lanes. The probe concentration for each sample was 2 nM. The arrows indicate the free probe (a), the RsaL/DNA complex (b), the LasR/DNA complex (c), and the RsaL/LasR/DNA complex (d). C. DNase I protection assay of RsaL and LasR on the rsaL-lasI intergenic region. Solid line indicates RsaL protection; dotted line indicates LasR protection; dashed line indicates simultaneous protection of both RsaL and LasR; M, Maxam and Gilbert sequencing reactions (A+G). The plus symbol above the lanes indicates the presence of RsaL and/or LasR at a final concentration of 5 μM and 1 μM respectively. D. Map of the rsaL-lasI bidirectional promoter; only the intergenic region is represented in scale. The putative rsaL transcription start point and the lasI transcription start point are indicated by bent arrows. The white and black boxes indicate the RsaL and LasR binding sites respectively (Schuster et al., 2004; Rampioni et al., 2006).

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RsaL is a negative autoregulator

The binding of LasR at a unique site located in the lasI-rsaL intergenic region triggers the divergent transcription of both genes (Whiteley and Greenberg, 2001; Schuster et al., 2004). We refer to this intergenic region as PlasI or as PrsaL depending on whether lasI or rsaL transcription is considered. We asked whether RsaL was an autoregulator by comparing PrsaL activity in the parent and in the rsaL mutant strains (Fig. 3A). The rsaL mutation led to elevated PrsaL activity. This is consistent with the hypothesis that RsaL is a negative autoregulator. However, PrsaL is activated by LasR-3OC12-HSL and the observed increase of PrsaL activity in the rsaL mutant could be due to the increased 3OC12-HSL production in this strain (Fig. 1). We tested the second hypothesis by using a set of plasmids allowing inducible expression of RsaL, LasR, or both in Escherichia coli carrying the transcriptional fusion PrsaL-lacZ. We found that PrsaL was activated by LasR and repressed by the concurrent presence of RsaL (Fig. 3B). As shown previously, RsaL also represses the LasR-dependent transcription of PlasI in E. coli (de Kievit et al., 1999). Therefore, the binding of RsaL to a unique site on the intergenic region between lasI and rsaL directly represses the transcription of both genes simultaneously.

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Figure 3. Effect of RsaL on PrsaL activity. A. β-Galactosidase activities (Miller units, M.u.) of P. aeruginosa PAO1 (full circles) and its rsaL mutant derivative (open circles) carrying the pPrsaL190 plasmid. B. β-Galactosidase activities (Miller units, M.u.) of exponential phase-grown E. coli (pPrsaL190) strains carrying the pJL42 expression vector or its derivatives expressing LasR (pJLLasR) or RsaL (pJLRsaL) or both proteins (pJLRsaL-LasR). For both (A) and (B) standard deviations (vertical lines) are based on the mean values for three independent experiments.

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RsaL is a global regulator

Our results show that RsaL is a DNA binding protein that can interact with the lasI-rsaL intergenic region and influence activity of the two flanking promoters directly. We wanted to test the hypothesis that it can also influence other promoters independently of its effect on lasI. Thus we employed microarray technology to compare the expression profiles of the lasI rsaL mutant strain (PAO-DM1) containing the RsaL expression vector pPSRsaL to the same strain with the empty vector pBBR1MCS-5. To compensate for the lasI mutation we included 5 μM 3OC12-HSL in the growth medium of both cultures. This experimental strategy uncoupled the regulatory relationship between RsaL and QS; the only difference between the compared strains being the presence or the absence of RsaL. We found that 120 genes were repressed and 10 genes were activated by RsaL (Table S1). Many of the repressed genes code for virulence or antibiotic resistance, including phzA1, phzD2, phzS and phzM involved in pyocyanin production (Mavrodi et al., 2001), the hcnABC operon involved in hydrogen cyanide biosynthesis (Pessi and Haas, 2000), the mexEF-oprN operon involved in antibiotic resistance (Köhler et al., 1997), and the cupA1-cupA2 genes involved in biofilm formation (Vallet et al., 2001). Moreover, RsaL repressed three characterized (PA0294-AguR, PA2492-MexT and PA4227-PchR) and three putative (PA2115, PA2196 and PA3174) transcriptional regulators, indicating that many genes could be controlled by RsaL indirectly. Many but not all of the RsaL-regulated genes are inversely controlled by LasR or RhlR (Hentzer et al., 2003; Schuster et al., 2003; Wagner et al., 2003). This suggests that RsaL might have an additional QS homeostatic effect of regulating QS-controlled genes inversely to QS.

The transcriptome analysis indicated that pyocyanin and hydrogen cyanide biosynthesis genes (phz1 and phz2 operons, phzM, phzS and hcnABC respectively), were repressed by RsaL. To validate the transcriptome analysis we measured pyocyanin and cyanide production in PAO-DM1(pBBR1MCS-5) and PAO-DM1(pPSRsaL) strains. In agreement with the transcriptome results, pyocyanin and hydrogen cyanide were higher in PAO-DM1(pBBR1MCS-5) than in PAO-DM1(pPSRsaL) (Fig. 4A and B).

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Figure 4. Effect of RsaL on virulence factors production. A. Cell density-dependent pyocyanin production in PAO1-DM1(pPSRsaL) (full circles) and PAO1-DM1(pBBR1MCS-5) (open circles) strains. B. Hydrogen cyanide production measured in stationary-phase cultures of the indicated strains. For (A) and (B) standard deviations (vertical lines) are based on the mean values for three independent experiments.

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RsaL binds to the promoters of pyocyanin and hydrogen cyanide genes

We wanted to know whether RsaL could bind to promoters of genes it repressed independently of its effect on LasR activity. Thus we performed EMSA with purified RsaL and DNA probes encompassing the promoters of phzA1, phzA2, phzM, phzS and hcnA. The EMSA showed that RsaL is capable of forming stable complexes with the phzA1, phzM and hcnA promoters (Fig. 5A), but not with the phzA2, and phzS promoters (data not shown). This is consistent with the hypothesis that some but not all of the genes identified in the transcriptome analysis are regulated by RsaL directly. An analysis of the DNA corresponding to all the EMSA probes revealed sequences similar to the RsaL binding site on the lasI promoter (TATGnAAnTTnCATA; Rampioni et al., 2007) in the shifted probes but not in the unshifted probes. Although the level of identity is quite low, the nucleotides found to be important for RsaL binding on PlasI are reasonably well conserved (Fig. 5B). Interestingly, the putative RsaL binding sites identified in the phzA1, phzM and hcnA promoters were located downstream of putative LasR binding sites (las-box), suggesting that repression of these promoters by RsaL could occur by the same mechanism as described for PlasI (Rampioni et al., 2006).

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Figure 5. RsaL binding to the phzA1, phzM and hcnA promoters. A. EMSA of RsaL binding to probes (2 nM for each lane) corresponding to the indicated promoters. RsaL concentrations (nM) are indicated above the lanes. As specific and unspecific competitors, unlabelled probe (200 nM) and calf thymus DNA (1 μg) were, respectively, added to the second last and to the last lane of each EMSA. Arrows indicate the RsaL/DNA complexes. B. Alignment of the RsaL binding site on PlasI with putative RsaL binding sites found in the indicated promoters. Identical residues are grey-shadowed, purine-pyrimidine conservation is indicated by capital letters; nucleotides the replacement of which was demonstrated to strongly (full triangles) or weakly (open triangles) affect RsaL binding to PlasI are also indicated (Rampioni et al., 2007).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

When P. aeruginosa is cultured in complex media, levels of 3OC12-HSL attain a steady-state level in the range of a few μM prior to the onset of stationary phase, despite the fact that expression of the 3OC12-HSL synthase gene is positively autoregulated; this implies that there is another regulatory element that balances the positive autoregulation to provide 3OC12-HSL homeostasis. In fact, a previously published mathematical model predicted such a homeostatic mechanism (Ward et al., 2004). By studying 3OC12-HSL production in a rsaL mutant of strain PAO1 (Fig. 1) we show that RsaL plays a pivotal role in 3OC12-HSL homeostasis. The level of 3OC12-HSL in rsaL mutant cultures continues to rise in late logarithmic and stationary phase to a level about 10-fold the level reached in a wild-type culture (Fig. 1).

Transcription of rsaL is dependent upon 3OC12-HSL. This generates a negative feedback loop, which counteracts the positive feedback loop mediated by LasR-3OC12-HSL. This constitutes a homeostatic system. Moreover, RsaL directly represses its own expression, so that the levels of this negative regulator are maintained within confined limits (Fig. 6). Homeostasis would allow a population of P. aeruginosa cells to maintain 3OC12-HSL at an appropriate level in a given environment and to change steady-state levels of 3OC12-HSL as environmental conditions change. However, other mechanisms can play roles in controlling 3OC12-HSL homeostasis, including for instance, signal-molecule-degrading enzymes and import–export pumps (Köhler et al., 2001; Aendekerk et al., 2002; Huang et al., 2006; Sio et al., 2006).

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Figure 6. Schematic model of the mechanism underlying 3OC12-HSL homeostasis in P. aeruginosa. Once the quorum has been reached, the LasR/3OC12-HSL complex triggers the transcription of both rsaL and lasI genes (A). The consequent increase of 3OC12-HSL levels and thus of activated LasR generates a positive feedback loop also responsible for the increase of RsaL levels (B). RsaL binding to the rsaL-lasI bidirectional promoter represses the expression of both rsaL and lasI genes, thus counteracting the positive feedback (C). The dynamic equilibrium between (B) and (C) provides 3OC12-HSL homeostasis.

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Our previous studies showed that both LasR, which is required for activation of the 3OC12-HSL synthase gene lasI, and RsaL, which represses lasI, can bind to contiguous sites in the rsaL-lasI intergenic region. Thus one possible mechanism for their antagonistic effects is that they can occlude each other from binding. However, we show that LasR and RsaL are capable of simultaneous binding to the intergenic regulatory region (Fig. 2). Apparently, when both LasR and RsaL are bound to this DNA RsaL repressor activity is dominant over the LasR activator function. Regulators belonging to the LuxR family, like LasR, make contacts with RNA polymerase (RNAP) both upstream of their binding site, through the α-CTD subunit, and downstream, through other regions of RNAP bound to the −10 promoter region (Nasser and Reverchon, 2007). In this view, the repression of both lasI and rsaL genes by RsaL could be explained postulating that RsaL inhibits transcription in the lasI direction by binding at the −10 region of PlasI, and simultaneously interferes with transcription in the rsaL direction by impairing the contacts between LasR and the RNAP α-CTD subunit. However, alternative mechanisms for RsaL autoregulation cannot be ruled out.

The QS circuits in P. aeruginosa are complex and it is possible that RsaL exerts a homeostatic effect on QS-controlled genes via direct interactions with their promoters. To begin to test this possibility we performed a transcriptome analysis, which revealed that RsaL controls 130 genes independently of its effect on 3OC12-HSL production, including genes that could be important in human infection such as those involved in pyocyanin and hydrogen cyanide production (Fig. 4). Most of the 130 genes are repressed and many of the repressed genes are activated by the LasR or RhlR QS systems (Hentzer et al., 2003; Schuster et al., 2003; Wagner et al., 2003). Among these, pyocyanin and hydrogen cyanide virulence genes possess promoters to which RsaL is capable of binding (Fig. 5). Therefore, RsaL can repress these virulence genes both directly, by binding to their promoters, and indirectly, by hampering their 3OC12-HSL-dependent transcription.

As discussed above, 3OC12-HSL levels reach a plateau in late logarithmic phase of growth raising the question of how P. aeruginosa at this stage of growth could sense further increases in cell density. This bacterium possesses a second C4-HSL QS system and C4-HSL levels continue to increase in late-logarithmic growth (data not shown; Chugani et al., 2001; Köhler et al., 2001). Interestingly, many genes are regulated by only one of the two QS systems, while others respond to both (Schuster and Greenberg, 2006). Considering that pyocyanin and hydrogen cyanide genes respond to both 3OC12-HSL and C4-HSL (Pessi and Haas, 2000; Whiteley and Greenberg, 2001), the direct repression exerted by RsaL on these genes could reflect the need for keeping the concentration of their products at a steady level, even if the population density continues to rise. The overproduction of virulence factors could in some circumstances be counter-productive, for instance by eliciting an effective host immune response. Moreover, at high concentrations hydrogen cyanide and pyocyanin could be toxic for P. aeruginosa itself.

At low cell densities the P. aeruginosa QS negative regulators RsmA and QscR repress acyl-HSL synthesis, and consequently virulence factors production. However, their action is negligible when the population size increases (Chugani et al., 2001; Pessi et al., 2001). Here we show that RsaL plays a major role in the control of virulence genes at high cell densities. In this respect, the homeostatic control exerted by RsaL could be particularly important during chronic P. aeruginosa infections where RsaL could function to maintain production of virulence factors at optimal levels despite high microbial cell densities.

This study demonstrates that a major function of RsaL in P. aeruginosa physiology is to govern the homeostasis of 3OC12-HSL by controlling, in concert with LasR, the expression of rsaL and lasI (Fig. 6). Besides this, RsaL is an integral part of the QS signalling network that controls gene expression through different and not mutually exclusive mechanisms: (i) repression of 3OC12-HSL signalmolecule production, (ii) direct binding of target genes and (iii) indirect control of some genes via auxiliary regulators. The broad range of RsaL functions expands the QS regulon and increases its complexity.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Bacterial strains and culture conditions

Pseudomonas aeruginosa PAO1 (wild type) and its rsaL derivative strain (rsaL::ISlacZ/hah) were obtained from the University of Washington Genome Center P. aeruginosa mutant library (http://www.genome.washington.edu/UWGC/pseudomonas). All E. coli and P. aeruginosa PAO1 strains were routinely grown at 37°C in Luria–Bertani (LB; Sambrook et al., 1989) broth with aeration. When required, antibiotics were added at the following concentrations (μg ml−1): E. coli, chloramphenicol (Cm, 30) or gentamicin (Gm, 10); P. aeruginosa PAO1, gentamicin (Gm, 100), tetracycline (Tc, 50), kanamycin (Km, 200), or chloramphenicol (Cm, 200).

Construction of PAO-DM1 strain and of plasmids

The P. aeruginosa lasI rsaL double mutant strain (PAO-DM1) was constructed as follows. We generated the plasmid pMARL1 by cloning in pMosblue (Amersham-Pharmacia) a 1079 bp polymerase chain reaction (PCR) fragment with engineered BamHI and XbaI sites flanking bp +149 to +1228 relative to the rsaL translational start, and the plasmid pBMRAL2 by cloning in pBluescript (Stratagene) a 1072 bp PCR fragment with engineered BamHI and KpnI sites flanking bp +319 to +1393 relative to the lasI translational start. A BamHI Km-resistance cassette (derived from pUC4K, Amersham-Pharmacia) was inserted in pMRAL1 yielding pMRAL1Km, and then a BamHI-XbaI fragment from pMRAL1Km was cloned in the corresponding sites of pBMRAL2 to generate pBluQSDM. The recombinant DNA fragment encompassing the Km cassette flanked by the 5′-truncated rsaL and lasI genes was excised from pBluQSDM and cloned in pEXGM19 (Hoang et al., 1998) as a XbaI-KpnI fragment, originating pEXQSDM. This plasmid was then used in a marker exchange experiment in P. aeruginosa PAO1. The resulting rsaL lasI double mutant strain (PAO-DM1) was verified by Southern analysis. We also checked that the mutant does not produce 3OC12-HSL (data not shown) and RsaL protein (Fig. 1B).

Plasmids pBBR1MCS-5 and pPSRsaL were previously described (Kovach et al., 1995; Rampioni et al., 2006).

A DNA fragment encompassing the entire rsaL-lasI intergenic regions was amplified by PCR and cloned in both orientations upstream of the promoterless lacZ in the pMP190 promoter probe vector (Spaink et al., 1987) to yield pPlasI190 and pPrsaL190.

Plasmids pJLLasR, pJLRsaL and pJLRsaL-LasR allow the inducible expression of lasR or rsaL or both genes respectively. l-arabinose is the inducer for lasR and isopropyl-β-d-thiogalactopyranoside is the rsaL inducer. To generate pJLRsaL, a 310 bp PCR fragment with engineered HindIII sites flanking bp −32 to +278 relative to the rsaL translational start was ligated to HindIII-digested pJL42. To generate the plasmid for the expression of both RsaL and LasR, pJLRsaL-LasR, the same fragment was ligated to HindIII-digested pJLLasR. We obtained pJL42 and pJLLasR from Professor J.-H. Lee, Pusan National University, Busan, South Korea.

Polyclonal antiserum production

Purified RsaL (200 μg) was emulsified with complete Freund's adjuvant (Sigma) and used to immunize a rabbit by intramuscular injection. After 3 weeks, a second boost of RsaL (150 μg) in complete Freund's adjuvant was given, followed by a third boost (100 μg) at the sixth week. The animal was bled 2 weeks later and the serum was stored at 4°C. Animal experiments were performed according to the legislative decree 116/92 by the Italian Ministry of Health.

Western immunoblotting, EMSAs and DNase I protection assays

Western immunoblotting (Sambrook et al., 1989) was with anti-RsaL serum (1:500) and horseradish peroxidase-conjugate anti-rabbit IgG as secondary antibody (1:5000; Promega). Final development was performed with the Amersham ECL chemiluminescent reagents (Amersham Biosciences).

The RsaL and LasR proteins were previously purified (Schuster et al., 2004; Rampioni et al., 2006). The EMSAs and DNase I protection assays procedures and the generation of DNA probes encompassing the rsaL-lasI intergenic region were previously described (Rampioni et al., 2006). Other DNA probes were generated by PCR amplification of the non-coding DNA regions upstream of phzA1 (371 bp), phzA2 (333 bp), phzM (326 bp), phzS (239 bp) and hcnA (400 bp) and labelled at both ends using [γ-32P]-ATP and T4 nucleotide kinase as previously described (Schuster et al., 2004).

Expression profiling experiments

PAO-DM1(pBBR1MCS-5) and PAO-DM1(pPSRsaL) were grown at 37°C in 100 ml of LB medium supplemented with 5 μM 3OC12-HSL and 50 mM MOPS (pH 7.0), in 500 ml culture flasks. RNA was extracted from each culture at an A600 of 2.0. Total RNA extraction, further sample preparation and processing of the P. aeruginosa GeneChip genome arrays were done in duplicate as previously described (Schuster et al., 2003). The Affymetrix Microarray Suite version 5.0 was used for initial data processing. Transcript data were further analysed with the web-based program cyber-t (http://cybert.ics.uci.edu./help/index.html). This program uses a Bayesian statistical approach that incorporates prior information of within-treatment measurement in addition to the experimentally observed variance. The Bayesian prior is based on the observation that genes of similar expression level have similar measurement errors, amounting to pseudoreplication of the experiment. The variance of any given gene can therefore be estimated from the variance from a number of genes with similar expression level, which improves confidence in the microarray data obtained from a low number of replicates. For the data analysed in this study, the Bayesian prior estimate was 10 and the sliding window size was 100. The P-value threshold was 10−4. There was no cut-off for fold changes in gene expression.

β-Galactosidase, 3OC12-HSL, pyocyanin, and hydrogen cyanide assays

β-Galactosidase activities were measured as previously described (Miller, 1972). 3OC12-HSL concentration was measured using the Pseudomonas putida SM17 (prsal220) lacZ-based reporter strain as previously described (Rampioni et al., 2007). For pyocyanin and hydrogen cyanide assays PAO-DM1(pBBR1MCS-5) and PAO-DM1(pPSRsaL) cultures were supplemented with 5 μM 3OC12-HSL and 50 mM MOPS (pH 7.0). For the pyocyanin assay, cells were grown with strong aeration in LB medium at 37°C and pyocyanin levels were measured along the growth curve as previously described (Xu et al., 2005). For hydrogen cyanide assay, cells were grown at 37°C in Castric-minimal medium under microaerophilic conditions (Castric, 1975). Hydrogen cyanide was measured as previously described (Gewitz et al., 1976).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

G.R. was in part supported by a Short-term Fellowship from EMBO (ASTF No. 2-2006). I.B. was supported by a fellowship from the Italian Cystic Fibrosis Research Foundation (Project FFC9/2007). This work was supported by a grant from the Italian Ministry of University and Research PRIN-2006 entitled ‘Basi genetiche e molecolari della patogenicità batterica’, and by a grant from Italian Cystic Fibrosis Research Foundation (Project FFC10/2007). We thank Professor J.-H. Lee (Pusan National University, Busan, South Korea) for providing pJL42 and pJLLasR plasmids, and to the ICGEB staff of the animal house in their assistance in raising polyclonal RsaL antibodies.

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
MMI_6029_sm_Table_S1.pdf163KSupporting info item
MMI_table_s1.pdf163KSupporting info item

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