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Negative control of quorum sensing by RpoN (sigma54) in Pseudomonas aeruginosa PAO1.

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  • 1. Laboratoire de Biologie Microbienne, Université de Lausanne, CH-1015 Lausanne, Switzerland.
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    • Heurlier K 1
    (1 author)

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Journal of Bacteriology, 01 Apr 2003, 185(7):2227-2235
https://doi.org/10.1128/jb.185.7.2227-2235.2003 PMID: 12644493 PMCID: PMC151487

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Abstract 

In Pseudomonas aeruginosa PAO1, the expression of several virulence factors such as elastase, rhamnolipids, and hydrogen cyanide depends on quorum-sensing regulation, which involves the lasRI and rhlRI systems controlled by N-(3-oxododecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone, respectively, as signal molecules. In rpoN mutants lacking the transcription factor sigma(54), the expression of the lasR and lasI genes was elevated at low cell densities, whereas expression of the rhlR and rhlI genes was markedly enhanced throughout growth by comparison with the wild type and the complemented mutant strains. As a consequence, the rpoN mutants had elevated levels of both signal molecules and overexpressed the biosynthetic genes for elastase, rhamnolipids, and hydrogen cyanide. The quorum-sensing regulatory protein QscR was not involved in the negative control exerted by RpoN. By contrast, in an rpoN mutant, the expression of the gacA global regulatory gene was significantly increased during the entire growth cycle, whereas another global regulatory gene, vfr, was downregulated at high cell densities. In conclusion, it appears that GacA levels play an important role, probably indirectly, in the RpoN-dependent modulation of the quorum-sensing machinery of P. aeruginosa.

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J Bacteriol. 2003 Apr; 185(7): 2227–2235.
PMCID: PMC151487
PMID: 12644493

Negative Control of Quorum Sensing by RpoN (σ54) in Pseudomonas aeruginosa PAO1

Karin Heurlier, Valerie Dénervaud, Gabriella Pessi,† Cornelia Reimmann, and Dieter Haas*
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Abstract

In Pseudomonas aeruginosa PAO1, the expression of several virulence factors such as elastase, rhamnolipids, and hydrogen cyanide depends on quorum-sensing regulation, which involves the lasRI and rhlRI systems controlled by N-(3-oxododecanoyl)-l-homoserine lactone and N-butyryl-l-homoserine lactone, respectively, as signal molecules. In rpoN mutants lacking the transcription factor σ54, the expression of the lasR and lasI genes was elevated at low cell densities, whereas expression of the rhlR and rhlI genes was markedly enhanced throughout growth by comparison with the wild type and the complemented mutant strains. As a consequence, the rpoN mutants had elevated levels of both signal molecules and overexpressed the biosynthetic genes for elastase, rhamnolipids, and hydrogen cyanide. The quorum-sensing regulatory protein QscR was not involved in the negative control exerted by RpoN. By contrast, in an rpoN mutant, the expression of the gacA global regulatory gene was significantly increased during the entire growth cycle, whereas another global regulatory gene, vfr, was downregulated at high cell densities. In conclusion, it appears that GacA levels play an important role, probably indirectly, in the RpoN-dependent modulation of the quorum-sensing machinery of P. aeruginosa.

Intercellular communication systems allow bacteria to monitor environmental conditions and to coordinate the expression of several genes, particularly those specifying extracellular products and virulence factors, in a cell density-dependent manner (44). This communication, termed quorum sensing, is based on the production of diffusible signal molecules (autoinducers) which accumulate in the environment during bacterial growth. Upon reaching a threshold concentration at high cellular densities, they can bind to and activate specific transcriptional regulators. In many gram-negative bacteria, quorum-sensing systems consist of pairs of genes in which a luxI-type gene encodes an N-acylhomoserine lactone (AHL; autoinducer) synthase and a luxR-type gene encodes a transcriptional regulator activated by the cognate autoinducer (15, 60, 67).

In the pathogen Pseudomonas aeruginosa, which is responsible for nosocomial infections (63) as well as for serious infections in patients suffering from cystic fibrosis, cancer, or burn wounds (6, 62), pathogenicity is due to the production of both cell-associated and extracellular virulence factors, most of which are regulated by quorum sensing (15, 60, 67). P. aeruginosa contains two interdependent quorum-sensing systems (26, 32, 45, 70). In the lasRI system, the LasI synthase catalyzes the biosynthesis of N-(3-oxododecanoyl)-l-homoserine lactone (OdDHL). The LasR-OdDHL complex positively regulates the expression of virulence factors such as elastases (LasB and LasA), exotoxin A, alkaline protease, hydrogen cyanide (HCN), and pyocyanin and, moreover, induces lasI itself, forming an autoinduction loop. LasR activated by OdDHL also positively regulates the expression of the rhlR gene. RhlR is the transcriptional regulator of the second autoinducer system and functions with N-butyryl-l-homoserine lactone (BHL), whose biosynthesis requires the RhlI synthase. The rhlRI system enhances the expression of multiple exoproducts such as LasB elastase, HCN, pyocyanin, and rhamnolipids (14). Thus, there is a quorum-sensing hierarchy in which the las system is dominant.

Both quorum-sensing systems are positively regulated by the global regulator GacA; in particular, the response regulator GacA has a marked enhancing effect on BHL formation and BHL-dependent virulence factor production (48, 52). In addition, the CRP (cyclic AMP receptor protein) homologue Vfr specifically activates the lasR promoter (1, 66). Recently, the QscR protein, a homolog of LasR and RhlR, has been shown to act as a repressor of lasI and rhlI, leading to a downregulation of quorum-sensing-dependent virulence factors (8). Thus, LasR- and RhlR-controlled quorum-sensing mechanisms are embedded in global regulatory networks. Recent work on sigma factor σ54 (RpoN) in P. aeruginosa revealed that this alternative sigma factor is important for virulence in several models, e.g., infection of the respiratory epithelium in cystic fibrosis xenografts and of burned mice (9, 21). Reduced virulence of P. aeruginosa rpoN mutants may be explained, in part, by the fact that rpoN function is necessary for the expression of pili and flagella, two important adherence factors (25, 42, 61). However, considering the pleiotropic effects of rpoN mutations on metabolic functions in P. aeruginosa (5, 27, 40, 61), it has been postulated that the role of RpoN in the regulation of virulence factors could be quite complex, extending beyond positive control of pilin and flagellin synthesis (9). Here we show that RpoN exerts global negative control on the quorum-sensing machinery of P. aeruginosa. At least part of this effect appears to be mediated by GacA.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

The strains and plasmids used in this study are listed in Table ​Table1.1. Both Escherichia coli and P. aeruginosa strains were routinely grown in nutrient yeast broth (NYB) or on nutrient agar plates at 37°C (58). l-Glutamine was added at a concentration of 1 mM for growing the rpoN mutant strains in NYB or in minimal medium E (64). For β-galactosidase and autoinducer extraction experiments, P. aeruginosa strains were grown with aeration in NYB in 50-ml Erlenmeyer flasks. When required, antibiotics were added to media at the following concentrations: tetracycline (Tc), 25 μg ml−1 (E. coli) or 125 μg ml−1 (P. aeruginosa); ampicillin (Ap), 100 μg ml−1 (E. coli); gentamicin (Gm), 10 μg ml−1; kanamycin (Km), 400 μg ml−1; and spectinomycin (Sp), 1,500 μg ml−1 (P. aeruginosa).

TABLE 1.

Bacterial strains and plasmids

Strain or plasmidGenotype or phenotypeaReference or origin
Pseudomonas aeruginosa
PAO1Wild type23
PAO4460rpoN::Ω-Km40
PAO6281PAO1 gacA::Ω-Sp/Sm52
PAO6304PAO1 att Tn7::vfr′-′lacZ GmrThis study
PAO6320PAO1 att Tn7::gacA′-′lacZ GmrThis study
PAO6358PAO1 ΔrpoNThis study
PAO6359PAO1 rpoN::Ω-KmThis study
PAO6360ΔrpoN att Tn7::rpoN+ GmrThis study
PAO6361ΔrpoN att Tn7::gacA′-′lacZ GmrThis study
PAO6362ΔrpoN att Tn7::vfr′-′lacZ GmrThis study
PAO6363ΔrpoN gacA::Ω-Sp/SmThis study
PAO6366PAO1 qscR::Ω-Sp/SmThis study
PAK-SRSpontaneous Smr mutant of PAK25
PAK-N1rpoN::Ω-Tc in PAK-SR25
Escherichia coli
DH5αF−endA1 hsdR17 supE44 thi-1 recA1 gyrA96 relA1 Δ(lacZYA-argF)U169 deoR λ([var phi]80dlacZΔM15)55
S17-1pro thi hsdR+ Tpr Smr; chromosome::RP4-2 Tc::Mu-Km::Tn757
SM10/λpirthi-1 thr-1 leuB26 tonA21 lacYI supE44 recA integrated RP4-2 Tcr::Mu Kmr/λpir37
Plasmids
pBBR1MCSBroad-host-range cloning vector; Cmr30
pBluescript-II KS, SKCloning vectors; ColE1 replicon; AprStratagene
pECP60rhlA′-′lacZ translational fusion on pSW205; Apr46
pHP45ΩColE1 replicon carrying a Ω-Sp/Sm cassette; Spr Smr Apr50
pME3087Suicide vector, with EcoRI-SstI-KpnI-BamHI-XbaI-PstI-SphI-HindIII MCS, ColE1 replicon; Tcr65
pME3280bMini-Tn7 gene delivery vector based on pUX-BF5 with HindIII-PstI-MluI-SpeI MCS; Gmr AprS. Zuber and D. Haas, unpublished
pME3338pME3280b with 2.1-kb EcoRI-HindIII fragment of pME3829 containing rpoN; Gmr AprThis study
pME3827lasR Gmr47
pME3828pME3087 carrying qscR::Ω-Sp/Sm on 3.15-kb fragment; TcrThis study
pME3829pBBR1MCS with 3.1-kb BamHI-HindIII insert including genes PA4461 and rpoN from pRP1-rpoN; CmrThis study
pME3834pME3087 carrying 1.2-kb KpnI-BamHI insert with a deletion in rpoN; TcrThis study
pME3836qscR′-′lacZ translational fusion in pME6010, with 700 bp upstream of start codon and 8 codons of qscR fused to ′lacZ gene from pNM482; TcrThis study
pME3840rhlRI′ Cmr47
pME3846rhlI′-′lacZ translational fusion; Tcr48
pME3850.1hcnA-lacZ transcriptional fusion; Tcr47
pME3851rhlR′-′lacZ, translational fusion; TcrThis study
pME3853lasI′-′lacZ, translational fusion; Tcr48
pME3858lasR′-′lacZ translational fusion in pME6010, with 0.4-kb SmaI-PstI fragment from pME3827 fused to ′lacZ gene from pNM482; TcrThis study
pME6010Cloning vector; Tcr20
pME6118ColE1 replicon, with 1.4-kb EcoRI-BamHI fragment containing gacA′-′lacZ translational fusion; Apr52
pME6157pBLS-II SK carrying vfr gene with its promoter region, AprThis study
pME6165pME6313 with 0.6-kb blunt end XhoI-PvuI fragment from pME6157, containing promoter region and 5′ end of vfr fused with ′lacZ gene, giving vfr′-′lacZ translational fusion; GmrThis study
pME6166pME6313, with 1.4-kb EcoRI-BamHI fragment from pME6118 (gacA′-′lacZ) preceded by 0.35-kb SphI-EcoRI fragment from pHP45Ω and containing transcription and translation termination signals of Ω-Sp/Sm; GmrThis study
pME6186rhlI′-′lacZ translational fusion in which rpoN potential box GGCAG-N5-CTGCC at positions −43/−31 was replaced with GGCAG-N5-CAAAC; TcrThis study
pME6313Mini-Tn7 gene delivery vector based on pUX-BF5 with HindIII-PstI-SmaI-SpeI MCS; Gmr AprH. Winteler and D. Haas, unpublished
pNM481, 482Cloning vectors for translational ′lacZ fusions; ColE1 replicon; Apr38
pRP1-rpoNpUC119 with SalI-HincII insert of 3.1 kb including genes PA4461 and rpoN; AprY. Itoh, unpublished
pTS400lasB′-′lacZ translational fusion on pSW205; Apr44
pUX-BF5“Carrier” plasmid containing att Tn7::mini-Tn7-Km system; Kmr3
pUX-BF13“Helper” plasmid containing Tn7 transposition functions; R6K replicon; Apr3
aMCS, multiple cloning site; Tp, trimethoprim.

To counterselect E. coli S17-1 donor cells in matings with P. aeruginosa for gene replacement, chloramphenicol (Cm) was used at a concentration of 10 μg ml−1; mutant enrichment experiments were performed with tetracycline at a final concentration of 20 μg ml−1 and carbenicillin at a final concentration of 2,000 μg ml−1. Flagellar swimming was tested as described by Rashid and Kornberg (51) on NYB containing 1 mM l-glutamine solidified with 0.3% (wt/vol) agar. Swarming was tested on plates containing 0.5% (wt/vol) agar, 8 g of nutrient broth per liter (Oxoid), 5 g of glucose per liter, and 1 mM l-glutamine (51). Twitching motility was assayed on 1% (wt/vol) agar supplemented with Luria broth and 1 mM l-glutamine (29).

DNA manipulation and cloning procedures.

Small- and large-scale preparations of plasmid DNA were made by the cetyltrimethylammonium bromide method (13) and with JetStar columns (Genomed, Basel, Switzerland), respectively. Chromosomal DNA was purified from P. aeruginosa as described elsewhere (16). Restriction enzyme digestions, PCRs, ligation, electrophoresis, and electroporation were performed with standard procedures (52) and as described elsewhere (17, 46). Nucleotide sequences of PCR-derived constructs were determined on both strands with a dye terminator kit (Perkin-Elmer product 402080) and an ABI Prism 373 sequencer. Comparison of nucleotide and deduced amino acid sequences was performed with the Genetics Computer Group program GAP.

Plasmid constructions.

Plasmid pME3829 was obtained by subcloning a 3.1-kb BamHI-HindIII fragment from pRP1-rpoN (kindly provided by Y. Itoh), containing genes PA4461 and rpoN, into pBBR1MCS (Fig. ​(Fig.1).1). Plasmid pME3836 was constructed by PCR-amplifying a 744-bp fragment (3 min at 95°C; 25 cycles of 1 min at 95°C, 30 s at 58°C, 1 min at 72°C; and 3 min at 72°C) with PAO1 chromosomal DNA as the template, with the primers qscR-3 (5′-AGGCCAGGATCCTGTTTATTGTCT-3′) and qscR-4 (5′-GACAAAATCTGCAGATATCCCTCT-3′). Artificial restriction sites (italic) for BamHI and PstI, respectively, were incorporated into these primers. The resulting 730-bp BamHI-PstI fragment, including a potential Lux box located 486 bp upstream of the ATG and the first eight codons of qscR, was fused in-frame with the ′lacZ reporter gene from pNM482 in vector pME6010.

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Chromosomal region of P. aeruginosa PAO1 surrounding the rpoN gene (59). The construction of strains PAO6358 and PAO6359 and of plasmids pME3338 and pME3829 is described in Materials and Methods. KH4, KH5, and KH10 indicate primers used in PCR. The assignment of pstN to open reading frame PA4464 is based on sequence comparison with the rpoN gene cluster in E. coli (28).

To obtain plasmid pME3338, a 2.1-kb EcoRI-HindIII fragment containing the rpoN gene (Fig. ​(Fig.1)1) was excised from pME3829 and inserted into the SpeI site of pME3280b (S. Zuber and D. Haas, unpublished data). To construct plasmid pME3851, the rhlR promoter region and first nine rhlR codons were amplified by PCR as above with the primers RR1 (5′-CGCTTGCTCGAGACCCGGC-3′) and RR2 (5′-AAAACTGCAGCAGCAAAAAGCCTCCGTC-3′), which are XhoI and PstI tagged, respectively (italic); plasmid pME3840 was used as the template. The 0.3-kb PCR product was cut with XhoI, blunted, cut with PstI, fused in frame with the ′lacZ reporter gene of pNM482, and cloned into pME6010. To generate plasmid pME3858, a 0.4-kb SmaI-PstI fragment from pME3827, containing the lasR promoter region and the first 23 codons of lasR, was fused in frame with the ′lacZ reporter gene from pNM482 in vector pME6010.

A putative rpoN recognition site overlapping the 3′ end of the lasR (lux) box in the rhlI promoter was mutated as follows. Primer rhlR-1 (5′-CCGTGGATCCGGCGATCCTC-3′), which anneals around the BamHI site (italic) in the rhlR coding sequence, and primer Acl-2 (5′-GATGAACGTTTGGCAACCTGCCAGATCTGGT-3′), which is complementary to part of the lasR box in the rhlI promoter and carries an artificial AclI site (italic), were used to PCR-amplify a 551-bp fragment from pME3846. A second PCR fragment of 183 bp was amplified from pME3846 with primer Acl-3 (5′-GCCAAACGTTCATCCTCCTTTAGTCT-3′), which is tagged with an artificial AclI site (italic) and anneals immediately downstream of the putative rpoN box, and primer rhlI-4 (5′-AAAACTGCAGCGGAAAGCCCTTCCAGCG-3′), which is complementary to codons 8 to 13 of rhlI and carries an artificial PstI site (italic). The two PCR fragments were ligated to each other at their AclI sites, fused as a 0.71-kb BamHI-PstI fragment to the ′lacZ gene of pNM481, and cloned into pME6010. Sequence analysis of the resulting construct, pME6186, confirmed that in the putative rpoN recognition sequence GGCAGGTTGCCTGC, the last three bases were replaced by AAA, destroying the second half-site of the GG-N10-GC motif.

P. aeruginosa mutant constructions.

Care was taken to construct all mutants and chromosomal reporter fusions in the same PAO1 background. In PAO6358, a 0.9-kb fragment was deleted in-frame in the rpoN gene. The deletion includes the helix-turn-helix motif and the rpoN box (Fig. ​(Fig.1)1) and was obtained as follows. A 604-bp KpnI-SacI fragment from pME3829, including the first 190 codons of rpoN, was linked to a 620-bp SacI-BamHI fragment containing the last six codons of rpoN, gene PA4463, and the beginning of pstN (Fig. ​(Fig.1),1), which had been amplified by PCR as above with primers KH4 (5′-AAAAGAGCTCCGCAAGCGACTGGTGTGA-3′) and KH5 (5′-AAAAGGATCCGGCGATGCCATTGCCGAA-3′) and cut at the artificial restriction sites for SacI and BamHI (italic). The resulting 1.22-kb fragment was cloned into the suicide plasmid pME3087 digested with KpnI and BamHI, giving plasmid pME3834. In conjugation with PAO1 as the recipient and S17-1/pME3834 as the donor, tetracycline-resistant transconjugants having a chromosomally integrated pME3834 plasmid were selected. After carbenicillin enrichment, glutamine-auxotrophic colonies were obtained and verified by PCR for their 0.9-kb deletion in rpoN with primers KH5 and KH10 (5′-TCCAGCAGGAAATCCAGGAAG-3′) (Fig. ​(Fig.11).

In strain PAO6359, the rpoN gene was interrupted by the insertion of a kanamycin resistance cassette (Ω-Km). We obtained this mutant by transducing the mutation from PAO4460 (40) into PAO1 with the temperate phage E79tv-2 (39) as described before (17). Kanamycin-resistant transductants were auxotrophic for glutamine. The double rpoN gacA mutant PAO6363 was obtained by transduction of the gacA mutation of PAO6281 into PAO6358 with phage E79tv-2.

In strain PAO6366, the qscR gene was interrupted by the insertion of a spectinomycin-streptomycin resistance cassette (Ω-Sp/Sm). The qscR gene was amplified by PCR with PAO1 chromosomal DNA as the template and primers QSCR1 (5′-AAAAGAGCTCATGGAGCGTGCGAGAAGAAC-3′) and QSCR2 (5′-TAAAGGATCCTATCCGGCCATTCGGTGAAT-3′), which contain artificial restriction sites for SacI and BamHI (italics). The 2-kb Ω-Sp/Sm resistance cassette from pHP45Ω was inserted as an EcoRI fragment into the EcoRI site located 30 codons downstream of the qscR ATG start codon. The resulting 3.15-kb fragment was cloned into the suicide plasmid pME3087, giving pME3828. This plasmid, carrying qscR::Ω-Sp/Sm, was mobilized into PAO1 and chromosomally integrated, with selection for tetracycline resistance. The chromosomal insertion in a tetracycline-sensitive, spectinomycin-resistant clone was verified by Southern blotting.

Construction of chromosomal insertions in the Tn7 attachment site.

Complementation by a single copy of the rpoN+ gene was carried out with a Tn7-based system developed for gram-negative bacteria (3, 22). Chromosomal insertion of the mini-Tn7 construct pME3338 carrying rpoN+ (Fig. ​(Fig.1)1) was obtained via a triparental mating between the recipient PAO6358 (grown overnight at 43°C), E. coli SM10/λpir carrying the pUX-BF13 helper plasmid, and E. coli S17-1/pME3338, with selection for gentamicin and chloramphenicol resistance. The rpoN+ insertion in the resulting strain, PAO6360, was verified by the loss of the auxotrophy for glutamine and by Southern blotting analysis.

Chromosomal vfr′-′lacZ strains were constructed as follows. First, the vfr gene was amplified by PCR from chromosomal DNA of PAO1 with the XhoI (italic)-tagged primer pVFR1 (5′-CATCCTCGAGGAAGGCTTCGC-3′) and the EcoRI (italic)-tagged primer pVFR2 (5′-GGAATTCATGGGTGCTGTTCA-3′). The resulting 1.15-kb PCR fragment was cleaved with EcoRI and XhoI and inserted into pBluescriptII-SK to give pME6157. The blunted 0.6-kb XhoI-PvuI fragment of pME6157 was fused to ′lacZ in the SmaI site of pNM482, and the resulting vfr′-′lacZ fusion on a 3.7-kb EcoRI-DraI fragment was ligated to a 0.35-kb SphI (T4 DNA polymerase treated)-EcoRI fragment carrying the transcription stop signal of the Ω-Sp/Sm cassette, and cloned into the blunted HindIII site of the Tn7 delivery vector pME6313. The transcription stop signal upstream of the vfr′-′lacZ fusion in this construct, named pME6165, prevents potential readthrough from the gentamicin resistance gene. The vfr′-′lacZ fusion was delivered to the chromosome of PAO1 and PAO6358 by triparental mating with E. coli S17-1/pME6165 and E. coli SM10λpir/pUX-BF13 as donors. Gentamicin-resistant transconjugants were checked by Southern analysis.

A translational gacA′-′lacZ fusion excised from plasmid pME6118 as a 4.5-kb EcoRI-DraI fragment was ligated to the SphI (T4 DNA polymerase treated)-EcoRI fragment carrying the transcription stop signal of Ω-Sp/Sm and inserted into the blunted HindIII site of pME6313. The mini-Tn7 gacA′-′lacZ construct of the resulting plasmid, pME6166, was delivered to the chromosome of PAO1 and PAO6358 as described above. The resulting strains, PAO6320 and PAO6361, were checked by Southern analysis.

Semiquantitative determination of autoinducer concentrations by thin-layer chromatography.

P. aeruginosa strains were cultivated with shaking in 20 ml of NYB amended with 1 mM glutamine in 50-ml Erlenmeyer flasks at 37°C to obtain an optical density at 600 nm (OD600) of 0.6 or 1.5. Cells were removed by centrifugation, and the pH of supernatants was adjusted to 5.0 prior to extraction with 3 volumes of dichloromethane in a separating funnel. Water was removed from the solvent phase with anhydrous Na2SO4, and dichloromethane was evaporated with a rotary evaporator. The extracts were concentrated 200-fold by dissolving them in aqueous 50% (vol/vol) acetonitrile. The presence of AHLs was tested by C18 reverse-phase (Merck) thin-layer chromatography, developed by elution in methanol-water (60:40, vol/vol), and revealed by overlaying either Chromobacterium violaceum CV026 (36), for BHL, or Agrobacterium tumefaciens NTL4/pZLR4 (7), for OdDHL. The amounts of BHL and OdDHL were estimated by comparison with standards, i.e., 4, 6, or 8 nmol of BHL and 50, 100, or 150 pmol of OdDHL.

β-Galactosidase assay.

P. aeruginosa strains were cultivated with shaking in 20 ml of NYB with 1 mM glutamine in 50-ml Erlenmeyer flasks at 37°C. β-Galactosidase specific activities were determined by the method of Miller (55).

RESULTS

Growth characteristics of P. aeruginosa rpoN mutants.

As the rpoN gene is the first gene in a cluster of five (27, 59), we constructed an in-frame deletion mutation in rpoN to avoid potential polar effects on the expression of the downstream genes. The resulting rpoN mutant, PAO6358, was complemented by a single rpoN+ copy inserted into the unique chromosomal Tn7 attachment site in strain PAO6360 (Fig. ​(Fig.1).1). An rpoN::Ω-Km insertion mutant, PAO6359, was also constructed (Fig. ​(Fig.1).1). Both rpoN-negative strains were auxotrophic for glutamine and unable to swim, swarm, and twitch, in agreement with previous studies showing that P. aeruginosa rpoN mutants are defective for flagella and type IV pili (21, 25, 29, 40, 61). In rich medium (NYB) amended with 1 mM l-glutamine, both rpoN mutants had a longer doubling time (about 70 min) than the wild-type PAO1 and the complemented mutant PAO6360 (about 40 min), indicating that substrate utilization was somewhat impeded by the loss of RpoN function. It is known that the utilization of several amino acids as C and N sources depends on RpoN (40, 61). However, both the wild type and the rpoN mutant reached stationary phase at similar levels (2.5 × 109 to 3 × 109 cells/ml). Under anaerobic conditions in a GasPak jar, the rpoN mutant PAO6358 and the wild-type PAO1 grew similarly on nutrient agar amended with 1 mM l-glutamine and either 100 mM KNO3 or 5 mM KNO2, suggesting that RpoN is not essential for denitrification.

Effects of rpoN null mutations on AHL production in P. aeruginosa.

To investigate whether RpoN influences the production of the quorum-sensing signals OdDHL and BHL, we quantified AHL levels at cell densities corresponding to early exponential phase (OD600 = 0.6, i.e., about 6 × 108 cells/ml) and to late exponential phase (OD600 = 1.5) (Table ​(Table2).2). The rpoN mutants PAO6358 and PAO6359 produced about two times more OdDHL and about five times more BHL than did the parent strain during both early and late exponential phases of growth. As the rpoN deletion mutant was phenotypically similar to the insertion mutant, only the former was complemented. In the complemented rpoN mutant, PAO6360, autoinducer levels were close to those of the wild type (Table ​(Table2).2). A similar regulation phenomenon was also detected in overnight cultures of an rpoN mutant of strain PAK-N1 (PAK-SR rpoN::Ω-Tc [25]), which produced six to seven times more AHL than did the parental wild type, PAK-SR (data not shown). By comparison with strain PAO1, strain PAK-SR yielded fivefold-lower autoinducer levels (data not shown).

TABLE 2.

Negative control of autoinducer production by RpoN in P. aeruginosaa

StrainGenotypeOdDHL (μM)
BHL (μM)
OD600 = 0.6OD600 = 1.5OD600 = 0.6OD600 = 1.5
PAO1Wild type0.15 ± 0.030.38 ± 0.020.13 ± 0.030.67 ± 0.34
PAO6358ΔrpoN0.38 ± 0.030.76 ± 0.040.50 ± 0.103.11 ± 0.38
PAO6359rpoN::Ω-Km0.28 ± 0.030.82 ± 0.150.55 ± 0.103.33 ± 0.34
PAO6360ΔrpoN attTn7::rpoN+0.05 ± 0.030.41 ± 0.090.06 ± 0.050.95 ± 0.25
aConcentrations of OdDHL and BHL were estimated for P. aeruginosa strains grown aerobically in NYB amended with 1 mM glutamine (see Materials and Methods). Data shown are mean values ± standard deviations for three independent experiments.

Effects of rpoN null mutations on expression of lasRI and rhlRI quorum-sensing genes.

To confirm the regulatory effects of RpoN on AHL synthesis, we studied the expression of the lasR, lasI, rhlR, and rhlI genes with translational lacZ fusions in the wild-type PAO1, in both rpoN mutants PAO6358 and PAO6359, and in the complemented mutant PAO6360. Null mutation of the rpoN gene resulted in an approximately threefold derepression of lasR on pME3858 and lasI on pME3853 at cell densities below an OD600 of 1.0, but these effects were reversed at high cell densities (Fig. ​(Fig.2A2A and B). The complemented mutant PAO6360 showed lasR and lasI expression similar to that in the parent (Fig. ​(Fig.2A2A and B). RpoN was also found to control the second quorum-sensing circuit in that translational rhlR′-′lacZ and rhlI′-′lacZ fusions (on pME3851 and pME3846, respectively) were induced four- to fivefold at low and high cell densities (Fig. ​(Fig.2C2C and D). We additionally tested transcriptional lasR-lacZ and rhlR-lacZ fusions in an rpoN and in a wild-type background. The derepressing effect of the rpoN mutation was similar to that found with the analogous translational fusions (data not shown). In conclusion, the β-galactosidase activities of the lasRI and rhlRI fusion constructs closely paralleled AHL levels (Table ​(Table22).

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RpoN control of lasR, lasI, rhlR, and rhlI expression. β-Galactosidase activities resulting from the translational fusions lasR′-′lacZ carried by pME3858 (A), lasI′-′lacZ on pME3853 (B), rhlR′-′lacZ on pME3851 (C), and rhlI′-′lacZ on pME3846 (D) were determined in P. aeruginosa wild-type PAO1 (□), in the rpoN deletion mutant PAO6358 ([open diamond]), in the rpoN insertion mutant PAO6359 (○), and in the rpoN deletion mutant complemented with monocopy rpoN+ PAO6360 ([open triangle]), grown in NYB with 1 mM glutamine. Each point is the mean ± standard deviation for three cultures.

Mutation in rpoN leads to increased expression of exoproduct genes.

Quorum sensing controls the production of numerous extracellular enzymes and metabolites in P. aeruginosa; we chose to measure three of them, elastase, rhamnolipids, and HCN (67), by following the expression of some representative structural genes: lasB (for elastase), rhlA (the first of two genes for rhamnolipid synthesis), and hcnA (the first of three genes encoding HCN synthase). All three genes are most strongly induced by a synergistic action of both the las and rhl quorum-sensing systems (47, 68). Translational lacZ fusions of the lasB and rhlA genes on plasmids pTS400 and pECP60, respectively, showed a two- to fourfold increase of expression in the rpoN mutant PAO6358 compared to the levels in the wild type and in the complemented rpoN mutant. An hcnA-lacZ transcriptional fusion on pME3850.1 gave a similar result (data not shown). These experiments as well as the autoinducer determinations (Table ​(Table2)2) and the quorum-sensing expression studies (Fig. ​(Fig.2)2) were all conducted in the same growth medium, NYB amended with 1 mM glutamine. Growth reached a plateau at an OD600 of 2.5 to 3.0 under these conditions. The results obtained with the lasB, rhlA, and hcnA fusions are consistent with an overall negative effect of RpoN acting transiently on lasRI and constantly on rhlRI expression.

Mechanisms of quorum-sensing control by RpoN.

We considered four hypotheses, which are not mutually exclusive, to explain the effects of RpoN on quorum-sensing in strain PAO1. (i) RpoN could directly repress the lasR, lasI, rhlR, and rhlI genes at their promoters, similar to the negative effect that σ54 can exert on alginate synthesis by binding directly to the algD promoter in P. aeruginosa (5). (ii) RpoN could repress Vfr, a positive regulator of lasR gene expression (1). (iii) RpoN could activate QscR, a negative effector of quorum sensing (8, 33). (iv) RpoN could repress GacA, a positive regulator of the expression of the lasR, rhlR, and rhlI genes (48, 52).

(i) RpoN recognizes a TGGCAC-N5-TTGCA consensus sequence in which the TGGC-N9-GC motif is most strongly conserved (4, 53). We searched for the presence of such a motif in the lasR, lasI, rhlR, and rhlI promoter regions; note that the transcription start sites have been determined experimentally for the lasR, lasI, and rhlI genes (1, 41, 48, 56). We did not find evidence for a conserved RpoN motif in the lasR, lasI, and rhlR promoter regions at positions that would be compatible with a repressive effect of RpoN, nor did we detect such a motif in the promoter of the rsaL gene, which exerts negative control on the lasI gene (11). By contrast, in the rhlI promoter, a potential RpoN recognition sequence, TGGCAG-N5-CTGCC, was found at positions −43 to −31 relative to the +1 site. This sequence overlaps a lasR (lux) box placed at −57 to −38. A 3-bp mutation replacing TGC at −33 to −31 with AAA was constructed in the rhlI promoter region. This mutation leaves the lasR box intact but would be expected to interfere with recognition of RpoN. However, the expression of an rhlI′-′lacZ fusion was not influenced by this mutation in the wild type or the rpoN mutant PAO6358 (data not shown).

(ii) In order to determine if the influence of RpoN on quorum sensing was mediated by Vfr, we measured the expression of a chromosomal translational vfr′-′lacZ fusion in a wild-type (PAO6304) and an rpoN mutant background (PAO6362). The expression of vfr was low throughout growth and significantly reduced in the rpoN background at high cell densities (Fig. ​(Fig.3).3). Inspection of the vfr promoter (54), however, did not reveal the presence of an RpoN recognition sequence, suggesting that the positive effect of RpoN on vfr expression may be indirect. The consequence of this regulation will be considered in the Discussion.

An external file that holds a picture, illustration, etc. Object name is jb0730916003.jpg

Influence of RpoN on vfr expression. β-Galactosidase activities from the chromosomally located translational fusion vfr′-′lacZ were determined in the wild-type PAO6304 (□) and in the rpoN mutant background strain PAO6362 ([open diamond]). Each result is the mean ± standard deviation for three cultures. Bacteria were grown in NYB with 1 mM glutamine.

(iii) A qscR::Ω-Sp/Sm insertion mutant, PAO6366, was constructed and tested for expression of the lasR, lasI, rhlR, and rhlI genes with the lacZ fusion constructs pME3858, pME3853, pME3851, and pME3846, respectively. However, each fusion gave a similar expression profile in the wild-type PAO1 and in the qscR mutant PAO6366 (data not shown). A translational qscR′-′lacZ fusion (constructed as detailed in Materials and Methods) was expressed at a low level of 3 to 4 Miller units in the wild-type PAO1 as well as in the rpoN mutant PAO6358, at an OD600 of 2.0. These results suggest that RpoN does not modulate the quorum-sensing machinery via QscR.

(iv) In order to check if the influence of RpoN on quorum sensing was a result of RpoN-mediated control of gacA expression, we assayed a chromosomal translational gacA′-′lacZ fusion in the wild type (PAO6320) and the rpoN deletion mutant (PAO6361). A marked negative effect of RpoN on gacA expression occurred throughout growth (Fig. ​(Fig.4).4). In the wild type as well as in the complemented rpoN mutant, gacA expression was about three times lower than in the rpoN mutant (Fig. ​(Fig.4).4). In agreement with previous data (52), the expression levels of the lasB′-′lacZ, rhlA′-′lacZ, and hcnA-lacZ fusions were very low in a gacA mutant compared with those in the wild type and the rpoN mutant (Fig. ​(Fig.5A,5A, B, and C).

An external file that holds a picture, illustration, etc. Object name is jb0730916004.jpg

Influence of RpoN on gacA expression. β-Galactosidase activities from the chromosomally located translational fusion gacA′-′lacZ were determined in the wild-type PAO6320 (□), in the rpoN mutant PAO6361 ([open diamond]), and in the rpoN mutant PAO6361 complemented with pME3829 ([open triangle]). Each result is the mean ± standard deviation for three cultures. Bacteria were grown in NYB with 1 mM glutamine.

An external file that holds a picture, illustration, etc. Object name is jb0730916005.jpg

Influence of RpoN and GacA on lasB, rhlA, and hcnA expression. β-Galactosidase activities from the translational fusions lasB′-′lacZ on pTS400 (A) and rhlA′-′lacZ on pECP60 (B) and from the transcriptional fusion hcnA-lacZ on pME3850.1 (C) were determined in the wild-type PAO1 (□), the rpoN mutant PAO6358 ([open diamond]), the rpoN gacA double mutant PAO6363 (○), and the gacA mutant PAO6281 ([open triangle]). Each result is the mean ± standard deviation for three cultures. Bacteria were grown in NYB with 1 mM glutamine.

If the repressive effects of RpoN on the quorum-sensing machinery were essentially a consequence of repression of GacA, then we might expect that a gacA rpoN double mutant would not differ substantially from a gacA mutant in terms of quorum-sensing-dependent expression. In the case of the lasB′-′lacZ fusion, this was indeed observed (Fig. ​(Fig.5A).5A). However, in the case of the rhlA′-′lacZ and hcnA-lacZ fusions, the gacA rpoN mutant gave intermediate expression levels (Fig. ​(Fig.5B5B and C), indicating that a simple linear model (RpoN —| GacA → LasRI/RhlRI → RhlA/HcnA) would incompletely describe the situation and that RpoN may act on the expression of the rhlA and hcnA genes via GacA-independent pathways. The hcnA-lacZ fusion of pME3850.1 used is controlled tightly and probably exclusively by LasR and RhlR (47). It is likely that RpoN also acts on quorum sensing via regulators other than GacA, but these remain to be identified.

DISCUSSION

In P. aeruginosa, the alternative sigma factor RpoN has several roles. As in enteric bacteria, it is a vital component of nitrogen assimilation. rpoN mutants of strains PAO1 and PAK but not strain PA14 are auxotrophic for glutamine (21, 40, 61). In strain PAO, RpoN cooperates with the two-component systems NtrB-NtrC and CbrA-CbrB in the utilization of various carbon and nitrogen sources, e.g., arginine, histidine, and polyamines (40, 61). Moreover, PAO and PAK mutants that are defective for rpoN do not produce flagella and pili and therefore do not show swimming and twitching mobility (25, 61; this study). For the transcription of flagellar and pilus genes, σ54 needs the transcriptional regulators FleQ, FleR, and PilR (2, 35). Remarkably, the genomic sequence of strain PAO predicts >20 transcriptional regulators activating σ54; however, the function of most of these has not yet been discovered (59). Whether any of these is involved in the modulation of quorum-sensing activity reported here is also unknown. Although a direct repressive effect of RpoN on some quorum-sensing gene promoter has not been rigorously excluded, it seems more likely that RpoN, together with some transcriptional regulator(s), would transcribe one or several regulatory elements that control quorum sensing.

Certain transcriptional regulators that interact with σ54, e.g., NtrC and CbrB, are response regulators of two-component systems in P. aeruginosa. Since the cognate sensor kinases NtrB and CbrA are involved in the utilization of various N sources (40), we considered the possibility that the production of AHLs by P. aeruginosa might be influenced by the N source. However, we did not observe any significant differences in AHL levels produced aerobically by P. aeruginosa PAO1 cells when the growth medium contained either ammonium (a good N source) or nitrate (a poor N source) (data not shown).

One role of the postulated RpoN-dependent control element(s) is to downregulate the expression of the global regulator GacA. This downregulation occurs at both low and high cell densities (Fig. ​(Fig.4).4). By contrast, RpoN has a positive effect on the expression of another quorum-sensing regulator, Vfr, especially during late growth phases (Fig. ​(Fig.3).3). Vfr positively controls the expression of the lasR regulator (1). It therefore appears that the derepressing effect of an rpoN mutation on lasR expression (Fig. ​(Fig.2A2A and B) could be the result of the enhanced expression of GacA during early growth phases and at low cell densities. During later stages of growth and at higher cell densities, the downregulation of vfr may compensate for the upregulation of gacA in an rpoN mutant. This would explain the observation that lasR and, indirectly, lasI are not overexpressed in an rpoN mutant background at an OD600 of ≥1.5 (Fig. ​(Fig.2A2A and B).

The signal transduction pathways by which GacA acts on the expression of target genes, many of which are involved in the synthesis of exoproducts and biofilm formation (43), are incompletely understood at present (19). In P. aeruginosa, a detailed analysis of hcnABC expression revealed the existence of two GacA-dependent pathways (47, 48, 52). In the first pathway, GacA exerts a positive effect on the expression of the lasR and rhlRI genes and, as a consequence, also on the transcription of LasR- and RhlR-dependent genes, including hcnABC. In the second, AHL-independent pathway, GacA exerts a positive effect on target gene expression at a posttranscriptional level; in the case of the hcnABC genes, this effect requires a sequence surrounding the hcnA ribosome-binding site (48) and the RNA-binding protein RsmA (49). In both pathways, the DNA sequences directly recognized by the GacA protein, presumably in its phosphorylated form, have remained elusive. We considered the possibility that RpoN might regulate the expression of RsmA. However, we found no evidence for such an effect in experiments with an rsmA′-′lacZ fusion and Western blotting (our unpublished data).

It is interesting that another alternative sigma factor, the stress and stationary-phase sigma factor RpoS (σ38), is also involved in quorum-sensing regulation in P. aeruginosa. In an rpoS null mutant, BHL levels are elevated throughout growth, essentially due to derepression of rhlI expression, by comparison with the wild-type strain PAO1. As a consequence, expression of exoproduct genes, e.g., hcnB, is increased in an rpoS mutant (69). There is also evidence that the rhlRI system can positively control the RpoS level (31). Environmental conditions greatly influence the relative amounts of sigma factors in bacterial cells (24). These considerations led us to propose an empirical model in which GacA and some sigma factors (such as RpoN and RpoS) globally exert opposite effects on the quorum-sensing machinery in P. aeruginosa.

Interactions between quorum sensing and RpoN have also been proposed in E. coli. Addition of a signal molecule termed autoinducer 2 (a furanone compound) to E. coli cultures induces, among a large number of genes, the ybhH gene (corresponding to the gene PA4463 lying downstream of rpoN; Fig. ​Fig.1)1) as well as the ygeV gene, encoding a σ54-dependent regulator homologous to LuxO, which is a component of the quorum-sensing cascade regulating bioluminescence in Vibrio harveyi (12, 34).

Considering the fact that inactivation of rpoN leads to the loss of two important adherence factors (pili and flagella) in P. aeruginosa on the one hand (21, 25, 61) and to an overexpression of several quorum-sensing-regulated virulence genes (lasB, rhlAB, and hcnABC) on the other hand, it would have been difficult to predict the virulence properties of a P. aeruginosa rpoN mutant in an animal model. The work of Hendrickson et al. (21) showed that an rpoN mutant of strain PA14 can manifest pathogenicity differently depending on the host. For nematodes and burnt mice, the rpoN mutant was less virulent than the wild type, whereas both strains were equally able to kill wax moth larvae (21).

RpoN is required in P. aeruginosa strain CHA for type III secretion of exotoxin S and exotoxin T, and thus RpoN makes a contribution to cytotoxicity in this strain (10). Moreover, since many pathogenicity models use relatively high infectious doses, the adherence properties of P. aeruginosa in these models may be less important than in most clinical situations. To some extent, the decreased virulence of P. aeruginosa rpoN mutants in some models might then be a consequence of a decreased ability to utilize a large number of organic substrates, not just N sources (40), and to produce secreted toxins (10).

Acknowledgments

We thank Christoph Keel for discussion, Yoshifumi Itoh for supplying plasmid pRP1-rpoN and strain PAO4460, and Nazife Beqa for technical help. We also thank Barbara Iglewski for providing plasmids pTS400 and pECP60, Dan Hassett for informing us of his work on RpoN prior to publication, and Glaxo-SmithKline for generously supplying carbenicillin.

This study was supported by the Swiss National Foundation for Scientific Research (project 31-56608.99) and the program Génie Biomédical.

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