Metal trafficking via siderophores in Gram-negative bacteria: Specificities and characteristics of the pyoverdine pathway

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Abstract

Under iron-limiting conditions, fluorescent pseudomonads secrete fluorescent siderophores called pyoverdines (Pvd), which form complexes with iron that are then taken up by the bacteria. Pvds consist of a fluorescent chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and containing one of the bidentate groups involved in iron chelation, linked to a peptide moiety containing the other two bidentate groups required for binding to Fe3+. More than 100 different Pvds have been identified, with different peptide sequences, forming a wide family of siderophores. In the human opportunistic pathogen Pseudomonas aeruginosa, Pvd is necessary for infection and is considered to be a virulence factor. This review focuses on the mechanisms underlying iron uptake by the Pvd pathway in pseudomonads, taking into account recent biochemical and biophysical studies and recently solved 3D-structures of the Pvd outer membrane transporter FpvA in four different loading states. These data are discussed and compared with the mechanisms of siderophore–Fe uptake reported for other Gram-negative bacteria.

Introduction

Iron is an essential element for the growth of most micro-organisms. It acts as a cofactor for the redox-dependent enzymes involved in most cellular processes, including electron transfer, RNA synthesis and resistance to reactive oxygen intermediates [1]. Under aerobic conditions, free iron abundance is limited by the very low solubility of ferric hydroxide. Bacteria and fungi have therefore developed efficient ferric ion-chelating agents, called siderophores, to scavenge iron from the extracellular environment and import it, making it possible to maintain adequate intracellular levels of iron [2], [3], [4]. Pseudomonas, a widespread bacterial genus, may be classified into five genetic homology groups [5]. One of these groups – fluorescent pseudomonads – is characterised by the production of yellow-green, water-soluble compounds called pyoverdins (Pvds) in iron-deficient conditions [6], [7]. More than 100 different Pvds have been identified, forming a wide class of siderophores with a great diversity of structures. Not all of these molecules have been studied to the same extent. The Pvd produced by the human opportunistic pathogen Pseudomonas aeruginosa (PvdI, Fig. 1) is the archetype of this group of molecules, the pathogenicity of this bacterium ensuring its notoriety. P. aeruginosa infections are severe, and are frequently lethal in immunocompromised patients and patients with cystic fibrosis. During infections, this bacterium produces Pvd as a means of obtaining to iron, in conditions of strong competition with the host. This siderophore is therefore considered to be a virulence factor, essential for bacterial virulence [8].

The intrinsic fluorescence of Pvds has made it possible to obtain large amounts of data on this iron uptake system in P. aeruginosa. The spectral characteristics of iron-free Pvd or Pvd loaded with Ga and FpvA (outer membrane transporter of Pvd) are almost ideal for the observation of FRET (fluorescence resonance energy transfer) between Pvd and the Trp residues of its bacterial membrane transporter, when these two molecules are in close contact, such as during the binding of Pvd [9], [10]. FRET is one of the most powerful techniques available for monitoring protein ligand interactions over time. These functional data obtained and the structures of FpvA in four loading states (FpvA, FpvA–Pvd, FpvA–Pvd–Fe and FpvA–Pvd–Ga [11], [12], [13]) indicate that this iron uptake pathway has certain features in common with other Gram-negative siderophore uptake pathways and haemophore trafficking, but that it also differs from these pathways. We review here the specific features and characteristics of the Pvd uptake pathway. The FpvAI transporter is also involved in a signalling cascade controlling the expression of fpvA and genes related to Pvd biosynthesis. This cascade involves a transmembrane signalling system induced by the binding of the siderophore to the outer membrane transporter, and the extracytoplasmic function (ECF) sigma factor/anti-sigma factor pairs, FpvI/FpvR and PvdS/FpvR [8], [14], [15], [16], [17], [18], [19], [20]. This aspect of the PvdI uptake pathway will not be discussed here (for review see [18]).

Section snippets

Pyoverdine

All Pvds consist of a chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline, with a peptide moiety bound to the chromophore and a side chain bound to the nitrogen atom at position C-3 of the chromophore. In most cases, this side chain is a diacid of the Krebs cycle, such as succinic, malic or α-ketoglutaric acid or one of their amide derivatives. The composition and length of the peptide are unique to each strain. Determinations of about 50 primary structures of Pvd have shown that the

General structure of siderophore outer membrane transporters

Ferric-siderophore complexes form in the extracellular medium and are then captured at the cell surface by their cognate outer membrane transporters (MW between 75 and 90 kDa), with a kd of 0.3–50 nM. Each of the three structurally different Pvds identified (Pvd type I, II and III) in P. aeruginosa strains is recognized by a specific receptor, FpvAI-III at the outer membrane [22], [23], [24]. FpvB has also been identified as an alternative PvdI–Fe receptor (FpvAI) in P. aeruginosa[41]. FpvAI is

Biological relevance of apo Pvd Binding to FpvAI

Under iron limitation, large amounts of Pvds are produced by fluorescent pseudomonads. Based on the affinity of apo PvdI for FpvAI, fluorescence microscopy observations of P. aeruginosa and functional studies, all FpvAI transporters at the cell surface are loaded with apo PvdI in the absence of iron [9], [11], [46]. This ability to bind apo siderophore is common to other siderophore outer membrane transporters, as described above [43], [44], [45].

The biological function of this binding remains

Formation of an FpvAI–PvdI–Fe complex

Formation of a transporter–siderophore–iron complex is the first step for iron uptake across the outer membrane via siderophores. In the case of the PvdI/FpvAI system, there are two major obstacles to the formation of such a complex. First, the apo PvdI already bound to FpvAI must be released. This dissociation is regulated by the TonB machinery and the protonmotive force of the inner membrane [48]. The second problem is the production of large amounts of PvdI by the bacteria in conditions of

The cellular location of PvdI–Fe dissociation

Analyses of the P. aeruginosa genome have shown it to have far fewer ABC transporter genes than outer membrane transporter genes (32 putative outer membrane transporters, http://www.pseudomonas.com) [7], [39]. Eleven of these outer membrane transporters are expressed in the absence of iron and are potentially involved in the uptake of this metal [81]. The others may be involved in the transport of nutriments like vitamine B12, sucrose or maltodextrins [82], [83]. Concerning ABC transporters or

Conclusion

All the available data for the PvdI uptake pathway and other siderophore uptake pathways clearly show that, whatever the nature and size of the siderophore, a similar mechanism of translocation across the outer membrane must be used. All these uptake pathways involve a siderophore outer membrane transporter in which a channel is formed by a two-gate system. Subsequent events may differ according to the bacterium and the siderophore concerned. Iron may be released from the siderophore into the

Abbreviations

    OMT

    outer membrane transporter

    PvdI

    pyoverdine, siderophore produced by Pseudomonas aeruginosa

    FpvAI

    PvdI–Fe outer membrane transporter in P. aeruginosa

    FptA

    pyochelin outer membrane transporter in P. aeruginosa

    FhuA

    ferrichrome–Fe outer membrane transporter in E. coli

    FepA

    enterobactin–Fe outer membrane transporter E. coli

    FecA

    citrate–Fe outer membrane transporter E. coli

    BtuB

    vitamine B12 outer membrane transporter E. coli

    Cir

    catecholate outer membrane transporter E. coli

    HasR

    haemophore outer membrane

Acknowledgements

I.J.S. was supported by the Centre National de la Recherche Scientifique, the Association Vaincre la Mucoviscidose (French Association against Cystic Fibrosis) and a grant from the ANR (Agence Nationale de Recherche, ANR-05-JCJC-0181-01).

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