Dual control of hydrogen cyanide biosynthesis by the global activator GacA in Pseudomonas aeruginosa PAO1

Abstract

The global response regulator GacA of Pseudomonas aeruginosa PAO1 positively controls the production of the quorum sensing signal molecule N-butanoyl-homoserine-lactone (C4-HSL) and hence the synthesis of several C4-HSL-dependent virulence factors, including hydrogen cyanide (HCN). This study presents evidence that GacA positively influences the transcription of the rhlI gene, specifying C4-HSL synthase, explaining the quorum sensing-dependent transcriptional control of the HCN biosynthetic genes (hcnABC). In addition, GacA was found to modulate hcn gene expression positively at a post-transcriptional level involving the hcnA ribosome-binding site. Thus, the activating effect of GacA on cyanogenesis results from both transcriptional and post-transcriptional mechanisms.

GacS/GacA two-component system, Secondary metabolism, Hydrogen cyanide, Quorum sensing, Pseudomonas aeruginosa

1 Introduction

Among bacteria that utilize quorum sensing regulation as part of their pathogenic lifestyle, the opportunistic pathogen Pseudomonas aeruginosa is perhaps the best understood example in terms of the mechanisms involved in the expression of pathogenicity [1,2]. Cell density-dependent regulation of P. aeruginosa is essentially mediated by the signal molecules N-(3-oxo-dodecanoyl)-homoserine lactone (3-oxo-C12-HSL; produced by the LasI enzyme) and N-butanoyl-homoserine lactone (C4-HSL; produced by the RhlI enzyme). These signals interact with the transcriptional regulators LasR and RhlR, respectively, to activate the expression of genes encoding virulence factors, including hydrogen cyanide (HCN) [3,4]. The LasR/LasI and RhlR/RhlI systems are organized in a regulatory cascade; a further effector, the Pseudomonas quinolone signal, modulates the expression of both systems [5].

C4-HSL production in P. aeruginosa is under positive control of the GacS/GacA system [6]. This two-component system is conserved in many Gram-negative bacteria. GacS is a transmembrane sensor protein, which responds to an unknown signal by autophosphorylation; phosphotransfer from GacS to the amino-terminal domain of the response regulator GacA occurs subsequently. This mechanism has been shown recently for the GacS/GacA homologs BarA and UvrY in Escherichia coli [7]. The GacS/GacA system also regulates N-acyl-HSL synthesis in a biocontrol strain of Pseudomonas aureofaciens [8] and in the plant pathogen Pseudomonas syringae [9]. In P. aeruginosa a gacA mutant exhibits strongly decreased virulence in the nematode Caenorhabditis elegans, in the plant Arabidopsis thaliana, and in burnt mice [10,,12]. The mechanisms by which GacA regulates C4-HSL synthesis and virulence are only partially understood; it has been shown that GacA has a positive effect on lasR and rhlR transcription [6]. Recently, Blumer et al. [13] have demonstrated that in P. fluorescens CHA0, a bacterium that does not produce N-acyl-HSL, GacA control manifests itself at the level of translation of target genes. This control involves a region close to the ribosome-binding site (RBS) of target genes such as the hcnABC structural genes for HCN synthase. The RBS region of the P. fluorescens hcnA gene might be recognized by a translational regulatory complex including the RNA-binding protein RsmA [13]. One function of GacA could be to alleviate RsmA-mediated translational repression of target genes, e.g. by inducing the synthesis of a regulatory RNA capable of sequestering RsmA [14,15]. However, the direct GacA targets are still elusive.

Here we report that in P. aeruginosa, GacA controls the expression of the hcnABC genes [16] at two levels: first, via a transcriptional activation of the rhlI gene and second, via a post-transcriptional mechanism involving the RBS region of the hcnA gene.

2 Materials and methods

2.1 Bacterial strains and growth conditions

P. aeruginosa strains used were the wild-type PAO1 and PAO6281 (gacA::ΩSp^R/Sm^R) [6]. Bacterial strains were routinely grown in nutrient yeast broth (NYB) or on nutrient agar plates at 37°C [6]. When required, tetracycline was added to the medium at a concentration of 25 μg ml⁻¹ (E. coli) or 125 μg ml¹ (P. aeruginosa) and spectinomycin/streptomycin were added at a concentration of 1000 μg ml¹.

2.2 DNA manipulation and cloning procedures

DNA cloning and plasmid preparations were performed according to standard methods [17]. Plasmids pME3826 (carrying a translational hcnA ′-′lacZ fusion) and pME3850.1 (carrying a transcriptional hcnA –lacZ fusion), have been described [16]. The construction of the translational rhlI ′-′lacZ fusion on vector pME6010 [18], resulting in plasmid pME3846 (Fig. 1) will be described elsewhere. To generate plasmid pME3855 (Fig. 1), a 1.24-kb BamHI/PstI fragment from pME3840 [16] containing the rhlI promoter and almost the entire coding region of rhlI (lacking the last nine codons) was fused in frame with the ′lacZ reporter from pNM481 [19] carried by the vector pME6010 [18]. Plasmid pME3861 containing the rhlI gene under the control of the tac promoter (Fig. 1) was obtained as follows: primer R3M (5′-GCGCGGTACCTGTGTGTGCTGGTAT-3′), which anneals to the region around the +1 transcription start site, and primer R2 (5′-AAAACTGCAGCGGAAAGCCCTTCCAGCG-3′), which is complementary to codons 9 to 14 of rhlI, were used to PCR amplify the rhlI leader region from pME3846. The resulting 141-bp fragment was cleaved with KpnI and PstI (artificial sites, underlined) and cloned into a pUK21 derivative containing the tac promoter [13]. A 1.14-kb BglII/PstI fragment of this construct (containing the tac promoter) was then used to replace the 0.7-kb BamHI/PstI fragment of plasmid pME3846 (containing the native promoter), thereby creating plasmid pME3861. The insertion of three additional bases into the RBS of the translational rhlI ′-′lacZ fusion (on pME3846) was generated by the overlap extension method [20] using primer MR1 (5′-GCCGGCACGACACGGACAGGACTTGGT-3′) to introduce the mutation (underlined). Using pMP21 [21] as the template, two PCR products of 742 bp and 200 bp were generated with the primers R2 plus R1 (5′-GCTGGAGCGATACCAGATGCA-3′), which anneals 702 nt upstream of the rhlI translational start site, and MR1 plus a primer (lacZ) annealing within the lacZ sequence (5′-TGCTGCAAGGCGATTAAGTGGG-3′), respectively. A mixture of these two PCR products was used as the template to amplify the rhlI region containing the mutation in the RBS by the use of primers R1 and lacZ; the resulting product was cut with BamHI and PstI and inserted into pME3846, creating plasmid pME3862 (Fig. 1). The promoter probe vector pME6522 [13] containing a promoterless lacZ gene was used to construct a transcriptional rhlI –lacZ fusion on plasmid pME3864 as follows. The rhlI promoter region was amplified by PCR using the EcoRI-tagged primer R4T (5′-GCTCGAATTCCGATCCTCAACGGCCT-3′), which anneals 560 bp upstream from the +1 transcriptional start site, and the PstI-tagged primer R5T (5′-GCTCCTGCAGATGAGGGGGAAGACTAAAG-3′), which anneals to the +1 transcriptional start site (K. Winzer, personal communication). The 580-bp amplified region was digested with EcoRI and PstI and cloned into pME6522 [13]. In plasmid pME3843 [13] the hcnA promoter of plasmid pME3826 [16] has been replaced by the constitutively expressed tac promoter. The 76-bp KpnI/PstI fragment of pME3843 containing the hcn 5′ untranslated region and the first nine codons of the hcnA coding region, was substituted by a synthetic KpnI/PstI linker carrying a mutated RBS (CACACAGG), resulting in plasmid pME3860.

1

Translational and transcriptional lacZ fusions used to study GacA control of rhlI expression. All constructs shown are derived from vector pME6010 [18]. The relevant rhlI sequence [24] is shown below. S/D, putative Shine Dalgarno sequence, indicated in bold; the rhlI transcriptional start site (K. Winzer, personal communication) is indicated by an arrow; a potential lux box (RhlR- and/or LasR-binding site) is underlined. rhlI sequences are shown by heavy black lines, ptac and lacZ by white boxes; artificially introduced restriction sites are marked with *. The 3-bp ACA insertion in pME3862 is boxed. β-Galactosidase activities were determined in P. aeruginosa PAO1 (wild-type=wt) and PAO6281 (gacA) grown in NYB to an OD₆₀₀ of 1.01.2. Data are the means±standard deviation of three measurements.

2.3 β-Galactosidase assay

P. aeruginosa cells were grown at 37°C with shaking at 180 rpm in 50-ml flasks containing 20 ml NYB supplemented with 0.05% Triton X-100. The media were inoculated with 10⁷ cells ml¹. Cultures were sampled at different time points and assayed for β-galactosidase-specific activities according to the method of Miller [17].

3 Results

3.1 Regulation of rhlI gene expression by GacA

To find out how the GacS/GacA system modulates C4-HSL production, we studied the expression of the rhlI gene using transcriptional and translational lacZ fusions (carried by pME3846 and pME3864, respectively; see Fig. 1), in the wild-type strain PAO1 and the gacA mutant PAO6281. Both lacZ fusions were expressed progressively to high levels with increasing cell densities and introduction of a gacA mutation resulted in an approximately 2–3-fold reduction of expression throughout growth in rich medium (Fig. 2A,B). These data indicate that GacA essentially influences rhlI expression at the transcriptional level.

2

Influence of GacA on rhlI gene expression. β-Galactosidase activities resulting from (A) a translational rhlI ′–′lacZ fusion on pME3846 (squares) and (B) a transcriptional rhlI –lacZ fusion on pME3864 (circles) were determined in the wild-type P. aeruginosa PAO1 (filled symbols) and in the gacA mutant PAO6281 (open symbols) growing in NYB. Each value is the mean±standard deviation from three experiments.

Several controls were done. (i) When the rhlI promoter was replaced by the tac promoter (ptac), as in pME3861, no GacA-dependent regulation was found, as expected (Fig. 1). (ii) A 3-bp (ACA) insertion in the RBS sequence of rhlI, in pME3862, had no effect on GacA control (Fig. 1). As will be shown below, a similar insertion into the RBS of hcnA abolishes post-transcriptional GacA control. (iii) A rhlI ′–′lacZ translational fusion constructed near the 3′ end of rhlI, in pME3855, showed the same GacA dependence as a translational fusion made near the 5′ end of rhlI (in pME3846), indicating that the rhlI coding sequence is irrelevant to GacA control.

The expression of the rhlI gene is activated by RhlR in the presence of C4-HSL [22], presumably via the recognition of the lux box in the rhlI promoter (Fig. 1). Since GacA positively controls rhlR transcription [6], the positive effect of GacA on rhlI expression is likely to be mediated by RhlR.

3.2 GacA modulates hcn gene transcription essentially via the RhlR/C4-HSL system

The hcn promoter is activated by both the LasR/oxo-C12-HSL and the RhlR/C4-HSL systems in P. aeruginosa [16]. A gacA mutation has little influence on the amount of oxo-C12-HSL produced [6]. Using a translational lasI ′–′lacZ fusion, we compared lasI expression in the wild-type PAO1 and in the gacA mutant PAO6281. Expression was the same in both strains, within the experimental error (data not shown). Thus, GacA-dependent activation of hcn transcription is mediated essentially by the RhlR/C4-HSL system. To monitor this effect, we used a transcriptional hcn –lacZ fusion carried by plasmid pME3850.1, whose expression is solely dependent on quorum sensing [16]. As shown in Fig. 3, this construct was indeed expressed at lower levels in a gacA mutant than in the wild-type. However, although the difference was significant, it was smaller than that expected from previous measurements of the product, HCN [6]. Therefore, we concluded that GacA exerted an additional control on hcn gene expression, i.e. downstream of transcription.

3

Influence of GacA on hcn gene transcription. β-Galactosidase activities resulting from a transcriptional hcn –lacZ fusion on pME3850.1 was determined in the P. aeruginosa wild-type PAO1 (squares) and the gacA mutant PAO6281 (triangles). Each value is the mean±standard deviation from three experiments.

3.3 GacA additionally regulates hcn gene expression at a post-transcriptional level

In P. fluorescens, a 3-bp (ACA) insertion in the RBS region of the hcnA gene from this organism abolishes GacA control [13]. The untranslated hcnA leader sequences of P. fluorescens and P. aeruginosa are similar, albeit not identical [13]. Therefore, we tested in P. aeruginosa (i) whether a translational hcnA ′–′lacZ fusion would still be regulated by the GacS/GacA system after replacement of the native hcn promoter by the tac promoter and (ii) whether an analogous 3-bp insertion in the RBS region would interfere with GacA control. As shown by the ptac constructs pME3843 and pME3860 (Fig. 4), both assumptions proved correct. We conclude that the GacS/GacA system also has an impact on translation of the hcnA target gene in P. aeruginosa, via an unidentified pathway.

4

Post-transcriptional, GacA-dependent regulation of P. aeruginosa translational hcnA ′–′lacZ fusions in P. aeruginosa. All constructs were made in the vector pME6010 as described in Section 2. The hcn region is indicated in boldface; the 3-bp ACA insertion in the hcn RBS of pME3860 is boxed; artificially introduced restriction sites are marked with *. β-Galactosidase activities were determined in P. aeruginosa PAO1 (wild-type=wt), and PAO6281 (gacA⁻) grown in NYB to an OD₆₀₀ of 1.0–1.2. Activities are the means±standard deviation from three measurements.

4 Discussion

In this study we have investigated an apparent dilemma. On the one hand, our previous observations have indicated that the global activator GacA positively influences the transcription of the lasR and rhlR genes as well as the production of C4-HSL and HCN [6] and that LasR and RhlR both activate the hcn promoter in P. aeruginosa [16]. On the other hand, we have reported that in P. fluorescens (an organism apparently devoid of N-acyl-HSLs), GacA control of hcn gene expression does not involve the hcn promoter, but only the RBS region [13,23]. We now show that in P. aeruginosa the GacS/GacA regulatory system has a dual role: this system upregulates the hcn promoter, essentially by activating the RhlR/C4-HSL part of the quorum sensing machinery, and also has a positive effect on hcn translation, via another, unknown mechanism. By analogy with the situation in P. fluorescens [13], this latter mechanism may involve the RNA-binding protein RsmA. Experiments to clarify this point are currently being done.

Chancey et al. [8] have observed a similar complex regulatory effect of the GacS/GacA system on the production of phenazine antibiotics in P. aureofaciens. In this bacterium, the GacS/GacA system controls transcription of the N-hexanoyl-homoserine lactone (C6-HSL) synthase gene phzI. However, the addition of exogenous C6-HSL does not complement a gacA or a gacS mutant of P. aureofaciens for phenazine production, indicating that the regulation of phenazine production by the GacS/GacA system also occurs downstream of PhzR/PhzI control, presumably at the level of the phz structural genes [8].

We note that the rhlI –lacZ transcriptional fusion on pME3864 displayed a much higher activity than did the translational fusion pME3846 (Fig. 1). Furthermore, the expression of the rhlI ′–′lacZ translational fusion driven from the strong tac promoter on pME3861 was also very low (Fig. 1). It appears that translational expression of the rhlI gene may be limited either by restricted translation initiation or by poor mRNA stability. Since a transcriptional lacZ fusion constructed within the rhlI structural gene is expressed at high levels in P. aeruginosa PAO1 [25], it is more likely that the low expression of the translational rhlI ′–′lacZ fusion results from poor translation initiation.

In conclusion, global control of secondary metabolism by the GacS/GacA system in P. aeruginosa does not operate via a simple, linear signal transduction pathway, but involves at least two layers of regulation. In the first, quorum sensing is modulated. In the second, which depends on the RBS of target gene, N-acyl-HSLs are probably not required.

Acknowledgements

We thank Cornelia Reimmann for discussion and Klaus Winzer for communication of unpublished results. This work was supported by the Swiss National Foundation for Scientific Research (31-56608.99) and the European Biotechnology project BIO4CT960119.