A new regulator linking quorum sensing and iron uptake in Pseudomonas aeruginosa

Pierre Cornelis and Séverine Aendekerk

Laboratory of Microbial Interactions, Department of Cellular and Molecular Interactions, Flanders Interuniversity Institute of Biotechnology, Vrije Universiteit Brussel, Institute of Molecular Biology, Pleinlaan 2, B-1050 Brussels, Belgium

Correspondence
Pierre Cornelis
(pcornel{at}vub.ac.be)

Pseudomonas aeruginosa is a ubiquitous Gram-negative {gamma}-proteobacterium capable of causing disease in plants, animals and humans (Cao et al., 2001a). Different extracellular virulence factors, including proteases, a toxin and secondary metabolites, are produced both in a cell-density-dependent manner, via cell-to-cell communication or ‘quorum sensing’ (QS) (Withers et al., 2001; Smith & Iglewski, 2003a), and in a growth-phase-dependent manner (Diggle et al., 2003). Also, iron limitation as encountered in the host induces the production of siderophores and virulence factors (Lamont et al., 2002). Understanding how these different regulatory networks interact is vital in order to be able to model and predict bacterial behaviour in novel conditions.

Typically, QS in Gram-negative bacteria involves the production of N-acyl-homoserine lactones (AHLs), which circulate in and out of the cell where they associate with a regulator protein of the so-called LuxR family (Fuqua et al., 1996). In P. aeruginosa, two different AHL synthases produce N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL), the product of the LasI synthase, and N-butanoyl-L-homoserine lactone (C4-HSL), the product of the RhlI synthase (de Kievit & Iglewski, 2000; Cámara et al., 2002). The cognate LuxR regulators are LasR for 3-oxo-C12-HSL and RhlR for C4-HSL. The LasI–LasR system exerts a control on the RhlI–RhlR system, imposing a hierarchy in the QS regulation (de Kievit & Iglewski, 2000; Cámara et al., 2002). The regulator in association with its cognate AHL binds to so-called ‘lux boxes' (called las boxes in the case of P. aeruginosa) upstream of the promoters of genes regulated by QS (Whiteley & Greenberg, 2001). A third, entirely different, signal molecule, 2-heptyl-3-hydroxy-4(1H)-quinolone, termed the Pseudomonas quinolone signal (PQS), and which closely resembles the 4-quinolone family of synthetic antimicrobials, has also been shown to regulate the expression of some virulence factors, such as the production of the redox active phenazine compound pyocyanin (Pesci et al., 1999; McKnight et al., 2000; Gallagher et al., 2002; D'Argenio et al., 2002; Diggle et al., 2003; Déziel et al., 2004). PQS functions as a regulatory link between the las and rhl AHL-dependent QS systems and is needed for the expression of some rhl-regulated genes (Diggle et al., 2003; Déziel et al., 2004).

Another level of complexity is added by the existence of other regulators influencing the QS regulatory circuit, such as the global Vfr and GacA regulators (Albus et al., 1997; Reimmann et al., 1997), the RsaL, QscR (a third LuxR-type regulator) and MvaT repressors (de Kievit et al., 1999; Chugani et al., 2001; Diggle et al., 2002) and the MfvR (PqsR) regulator (Diggle et al., 2003; Déziel et al., 2004). Moreover, the sigma factor RpoS has recently been shown to influence the transcription of a large proportion of QS-regulated genes (Schuster et al., 2004). Three different microarray-based transcriptome analyses of all P. aeruginosa open reading frames (n=5570) have shown that between 163 and 388 genes could be QS-regulated (Schuster et al., 2003; Wagner et al., 2003; Hentzer et al., 2003). In this issue, Juhas et al. (2004) describe a fourth LuxR regulator, which they term VqsR (virulence and quorum-sensing regulator). Although vqsR gene expression is low when the cells are grown in LB medium, its inactivation negates the production of AHLs. The same authors also demonstrate that vqsR expression is enhanced by the presence of H2O2 or serum in the medium.

VqsR and QS
A transcriptome analysis revealed that a high proportion of the genes regulated by QS in P. aeruginosa are repressed in a vqsR mutant. Table 1 lists 56 genes down-regulated in a vqsR mutant in the presence of serum or H2O2 and also found as being QS-regulated in at least two of the above-mentioned transcriptome analyses. Interestingly, the vast majority of these genes (n=44) belong to the category induced by human serum, four belong to the category of genes induced by H2O2, while eight are detected under both conditions.


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Table 1. QS-regulated genes in P. aeruginosa based on transcriptional fusions (Whiteley et al., 1999) or transcriptome analyses (Wagner et al., 2003; Schuster et al., 2003; Hentzer et al., 2003) compared to the genes down-regulated in the absence of VqsR (Juhas et al., 2004)

The shaded rows indicate the genes that are also regulated by iron (Table 2).

 
Ten of these genes had already been identified by Whiteley et al. (1999) as being QS-regulated using a lacZ transcription fusion approach (shown in Table 1).

The LasR-, RhlR- and VqsR-dependent genes include those encoding extracellular proteins, regulators, enzymes involved in the production of secondary metabolites, post-translational modification and chaperones, and lectins, with the remaining group representing genes encoding hypothetical proteins.

VqsR and iron
Perhaps the most interesting finding of Juhas et al. (2004) is the importance of VqsR for the full expression of some iron-regulated genes (Table 2). In P. aeruginosa, iron availability not only regulates the production of the siderophores pyochelin and pyoverdine, but also of some virulence factors, including the extracellular protease PrpL and the exotoxin A (Lamont et al., 2002; Ravel & Cornelis, 2003). In fluorescent pseudomonads, the expression of pyoverdine biosynthesis genes depends on the alternative sigma factor PvdS, the gene of which is controlled by the general iron-co-factored repressor Fur (Lamont et al., 2002; Ravel & Cornelis, 2003).


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Table 2. Genes known to be induced by iron limitation as detected by transcriptome analyses (Ochsner et al., 2002; Palma et al., 2003) which show a lower expression in a vqsR mutant (Juhas et al., 2004)

The shaded rows indicate the genes that are also regulated by QS (Table 1).

 
In the vqsR mutant, the pvdS gene is down-regulated together with the transcription of other genes belonging to the pyoverdine biosynthetic locus (reviewed by Ravel & Cornelis, 2003). Likewise, the expression of the genes for the biosynthesis and uptake of the second P. aeruginosa siderophore, pyochelin, are clearly reduced in the vqsR mutant (Table 2). In total, 25 genes found previously to be iron-regulated using a microarray analysis (Ochsner et al., 2002; Palma et al., 2003) are also repressed in a vqsR mutant. Out of these iron-regulated genes, a few are also regulated by QS (mvfR, aprF, rsaL, prpL). Interestingly, MvfR (PqsR) has been described as a regulator for the production of virulence factors (Cao et al., 2001b) and the signal molecule PQS (Diggle et al., 2003; Déziel et al., 2004), while RsaL is a repressor of the lasI gene (de Kievit et al., 1999).

Previously, links were suggested between the QS and iron regulons (Whiteley et al., 1999), but were not confirmed by transcriptome analyses (Schuster et al., 2003; Wagner et al., 2003; Hentzer et al., 2003). The most plausible explanation for these discrepancies is the fact that media used to grow the cells contained iron(III) above a concentration of 10 µM resulting in repression of most of the iron-limitation-induced genes (Vasil, 2003). It would be very interesting in the future to undertake transcriptome studies under conditions of iron limitation. In strong support of the data of Juhas et al. (2004), Arevalo-Ferro et al. (2003) demonstrated a similar decreased production of iron proteins involved in haem uptake and the FptA protein corresponding to the receptor for pyochelin in a recently published proteome analysis of QS-regulated genes.

Conclusions
The QS network in P. aeruginosa has been the subject of extensive studies during recent years and also has attracted a lot of attention because it could form an interesting target for the development of drugs that could antagonize the production of and/or the response to signal molecules (Hentzer et al., 2003; Hentzer & Givskov, 2003; Smith & Iglewski, 2003b). Likewise, it is known that siderophores play an important role in the virulence of P. aeruginosa because they can provide the bacteria with the necessary iron for their growth, but also because they can act as signal molecules for the production of virulence factors (Lamont et al., 2002). With the discovery of VqsR a clear link has been established between the QS and iron regulons. The challenge for the future will be to integrate different signals (AHLs, PQS, siderophores) and regulators (LasR, RhlR, QscR, VqsR, RsaL, Vfr, GacA, MvfR, Fur, PvdS) in order to get a better picture of the incredible adaptability of P. aeruginosa to changing environmental conditions.

Acknowledgements
S. A. is a recipient of a PhD fellowship from IWT-Flanders.

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