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