Isabelle J. Schalk

New roles for bacterial siderophores in metal transport and tolerance

Siderophores are chelators with extremely strong affinity for ferric iron and are best known for their capacity to feed microorganisms with this metal. Despite their preference for iron, they can also chelate numerous other metals with variable affinities. There is also increasing evidence that metals other than iron can activate the production of siderophores by bacteria, thereby implicating siderophores in the homeostasis of metals other than iron and especially heavy metal tolerance. This article considers this new concept that siderophores play a role in protecting bacteria against metal toxicity and discusses the possible contribution of these chelators to the transport of biological relevant metals in addition to iron.

New insights into the metal specificity of the Pseudomonas aeruginosa pyoverdine–iron uptake pathway

Pyoverdine (PvdI) is the major siderophore secreted by Pseudomonas aeruginosa PAOI in order to get access to iron. After being loaded with iron in the extracellular medium, PvdI is transported across the bacterial outer membrane by the transporter, FpvAI. We used the spectral properties of PvdI to show that in addition to Fe(3+), this siderophore also chelates, but with lower efficiencies, all the 16 metals used in our screening. Afterwards, FpvAI at the cell surface binds Ag(+), Al(3+), Cd(2+), Co(2+), Cu(2+), Fe(3+), Ga(3+), Hg(2+), Mn(2+), Ni(2+) or Zn(2+) in complex with PvdI. We used Inductively Coupled Plasma-Atomic Emission Spectrometry to monitor metal uptake in P. aeruginosa: TonB-dependent uptake, in the presence of PvdI, was only efficient for Fe(3+). Cu(2+), Ga(3+), Mn(2+) and Ni(2+) were also transported into the cell but with lower uptake rates. The presence of Al(3+), Cu(2+), Ga(3+), Mn(2+), Ni(2+) and Zn(2+) in the extracellular medium induced PvdI production in P. aeruginosa. All these data allow a better understanding of the behaviour of the PvdI uptake pathway in the presence of metals other than iron: FpvAI at the cell surface has broad metal specificity at the binding stage and it is highly selective for Fe(3+) only during the uptake process.

Iron-free pyoverdin binds to its outer membrane receptor FpvA in Pseudomonas aeruginosa: a new mechanism for membrane iron transport.

Under iron limitation, Pseudomonas aeruginosa secretes a fluorescent siderophore called pyoverdin, which, after complexing iron, is transported back into the cell via its outer membrane receptor FpvA. Previous studies demonstrated co‐purification of FpvA with iron‐free PaA and reported similar binding affinities of iron‐free pyoverdin and ferric‐pyoverdin to purified FpvA. The fluorescence resonance energy transfer between iron‐free PaA and the FpvA receptor here reveals the existence of an FpvA–pyoverdin complex in P. aeruginosa in vivo, suggesting that the pyoverdin‐loaded FpvA is the normal state of the receptor in the absence of iron. Using tritiated ferric‐pyoverdin, it is shown that iron‐free PaA binds to the outer membrane but is not taken up into the cell, and that in vitro and, presumably, in vivo ferric‐pyoverdin displaces the bound iron‐free pyoverdin on FpvA–PaA to form FpvA–PaA‐Fe complexes. In vivo, the kinetics of formation of this FpvA–PaA‐Fe complex are more than two orders of magnitude faster than in vitro and depend on the presence of TonB. In P. aeruginosa, two tonB genes have been identified (tonB1 and tonB2). TonB1 is directly involved in ferric‐pyoverdin uptake, and TonB2 seems to be able partially to replace TonB1 in its role in iron acquisition. However, no effect of TonB1 or TonB2 on the apparent affinity of free pyoverdin to FpvA was observed, and a 17‐fold difference was measured between the affinities of the two forms of pyoverdin (PaA and PaA‐Fe) to FpvA in the absence of TonB1 or TonB2. The mechanism of iron uptake in P. aeruginosa via the pyoverdin pathway is discussed in view of these new findings.

Presence of the siderophores pyoverdine and pyochelin in the extracellular medium reduces toxic metal accumulation in Pseudomonas aeruginosa and increases bacterial metal tolerance

In order to get access to iron, Pseudomonas aeruginosa strain PAO1 produces two major siderophores pyoverdine (PVD) and pyochelin (PCH). Both siderophores are able to chelate many other metals in addition to iron. However, despite this property, only iron is transported efficiently into the bacteria by the PVD and PCH uptake pathways. Growth studies with P. aeruginosa strains showed a lower sensitivity to toxic metals for the siderophore-producing strain than for the mutants unable to produce siderophores. Moreover, addition of PVD or PCH to the growth medium of a siderophore-deficient strain considerably reduced the toxicity of toxic metals present at concentrations of 100 µM in iron-limited and iron-supplemented growth conditions. Measurement by Inductively Coupled Plasma-Atomic Emission Spectrometry of the concentration of metals present in bacteria incubated with metals in the presence or absence of PVD or PCH indicated that both siderophores were able to sequester metals from the extracellular medium of the bacteria, decreasing metal diffusion into the bacteria. Pyoverdine was able to sequester Al(3+) , Co(2+) , Cu(2+) , Eu(3+) , Ni(2+) , Pb(2+) , Tb(3+) and Zn(2+) from the extracellular medium, and PCH, Al(3+) , Co(2+) , Cu(2+) , Ni(2+) , Pb(2+) and Zn(2+) . Moreover, the presence of 100 µM Cu(2+) and Ni(2+) increased PVD production by 290% and 380%, respectively, in a medium supplemented with iron. All these data suggest that PVD and PCH may contribute to P. aeruginosa resistance to heavy metals.

The Pseudomonas aeruginosa Pyochelin-Iron Uptake Pathway and Its Metal Specificity

Pyochelin (Pch) is one of the two major siderophores produced and secreted by Pseudomonas aeruginosa PAO1 to assimilate iron. It chelates iron in the extracellular medium and transports it into the cell via a specific outer membrane transporter, FptA. We used the fluorescent properties of Pch to show that this siderophore chelates, in addition to Fe(3+) albeit with substantially lower affinities, Ag(+), Al(3+), Cd(2+), Co(2+), Cr(2+), Cu(2+), Eu(3+), Ga(3+), Hg(2+), Mn(2+), Ni(2+), Pb(2+), Sn(2+), Tb(3+), Tl(+), and Zn(2+). Surprisingly, the Pch complexes with all these metals bound to FptA with affinities in the range of 10 nM to 4.8 microM (the affinity of Pch-Fe is 10 nM) and were able to inhibit, with various efficiencies, Pch-(55)Fe uptake in vivo. We used inductively coupled plasma atomic emission spectrometry to follow metal uptake by P. aeruginosa. Energy-dependent metal uptake, in the presence of Pch, was efficient only for Fe(3+). Co(2+), Ga(3+), and Ni(2+) were also transported, but the uptake rates were 23- to 35-fold lower than that for Fe(3+). No uptake was seen for all the other metals. Thus, cell surface FptA has broad metal specificity at the binding stage but is much more selective for the metal uptake process. This uptake pathway does not appear to efficiently assimilate any metal other than Fe(3+).

Pyoverdine biosynthesis and secretion in Pseudomonas aeruginosa: implications for metal homeostasis.

Pyoverdines are siderophores produced by fluorescent Pseudomonads to acquire iron. At least 60 different pyoverdines produced by diverse strains have been chemically characterized. They all consist of a dihydroquinoline-type chromophore linked to a peptide. These peptides are of various lengths and the sequences are strain specific. Pyoverdine biosynthesis in Pseudomonas aeruginosa and fluorescent Pseudomonads is a complex process involving at least 12 different proteins, starting in the cytoplasm and ending in the periplasm. The cellular localization of pyoverdine precursors was recently shown to be consistent with their biosynthetic enzymes. In the cytoplasm, pyoverdine appears to be assembled at the inner membrane and particularly at the old cell pole of the bacterium. Mature pyoverdine is uniformly distributed throughout the periplasm, like the periplasmic enzyme PvdQ. Secretion of pyoverdine involves a recently identified ATP-dependent efflux pump, PvdRT-OpmQ. This efflux system does not only secrete newly synthesized pyoverdine but also pyoverdine that already transported iron into the bacterial periplasm and any pyoverdine-metal complex other than ferri-pyoverdine present in the periplasm. This review considers how these new insights into pyoverdine biosynthesis and secretion contribute to our understanding of the role of pyoverdine in iron and metal homeostasis in fluorescent Pseudomonads.

Pseudomonas fluorescens CHA0 Produces Enantio-pyochelin, the Optical Antipode of the Pseudomonas aeruginosa Siderophore Pyochelin

The siderophore pyochelin is made by a thiotemplate mechanism from salicylate and two molecules of cysteine. In Pseudomonas aeruginosa, the first cysteine residue is converted to its D-isoform during thiazoline ring formation whereas the second cysteine remains in its L-configuration, thus determining the stereochemistry of the two interconvertible pyochelin diastereoisomers as 4 ′R, 2 ″R, 4 ″R (pyochelin I) and 4 ′R, 2 ″S, 4 ″R (pyochelin II). Pseudomonas fluorescens CHA0 was found to make a different stereoisomeric mixture, which promoted growth under iron limitation in strain CHA0 and induced the expression of its biosynthetic genes, but was not recognized as a siderophore and signaling molecule by P. aeruginosa. Reciprocally, pyochelin promoted growth and induced pyochelin gene expression in P. aeruginosa, but was not functional in P. fluorescens. The structure of the CHA0 siderophore was determined by mass spectrometry, thin-layer chromatography, NMR, polarimetry, and chiral HPLC as enantio-pyochelin, the optical antipode of the P. aeruginosa siderophore pyochelin. Enantio-pyochelin was chemically synthesized and confirmed to be active in CHA0. Its potential biosynthetic pathway in CHA0 is discussed.

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

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.

Recognition of iron-free siderophores by TonB-dependent iron transporters.

TonB‐dependent iron transporters reside in the outer membranes of Gram‐negative bacteria, transporting ferric‐complexes into the periplasm by a mechanism requiring proton motive force and an integral inner membrane complex, TonB–ExbB–ExbD. Certain TonB‐dependent transporters contain an additional domain at the N‐terminus, which interacts with an inner membrane regulatory protein and a cytoplasmic sigma factor to induce transcription of iron transport genes when a ferric‐ligand is bound at the extracellular surface of the transporter. Transport of the ferric‐ligand is apparently not necessary for transcription induction. Recent biophysical and crystallographic experiments have shown that this subclass of TonB‐dependent iron transporters can bind iron‐free ligands, whereas only the ferric‐ligands are transported into the periplasm. This review focuses on the ligand binding properties of these transporters and includes a discussion of the biological function of the additional domain, the mechanism of transcription induction and the mechanism of ferric‐ligand transport.

Real Time Fluorescent Resonance Energy Transfer Visualization of Ferric Pyoverdine Uptake in Pseudomonas aeruginosa A ROLE FOR FERROUS IRON

To acquire iron, Pseudomonas aeruginosa secretes a major fluorescent siderophore, pyoverdine (PvdI), that chelates iron and shuttles it into the cells via the specific outer membrane transporter, FpvAI. We took advantage of the fluorescence properties of PvdI and its metal chelates as well as the efficient FRET between donor tryptophans in FpvAI and PvdI to follow the fate of the siderophore during iron uptake. Our findings with PvdI-Ga and PvdI-Cr uptake indicate that iron reduction is required for the dissociation of PvdI-Fe, that a ligand exchange for iron occurs, and that this dissociation occurs in the periplasm. We also observed a delay between PvdI-Fe dissociation and the rebinding of PvdI to FpvAI, underlining the kinetic independence of metal release and siderophore recycling. Meanwhile, PvdI is not modified but recycled to the medium, still competent for iron chelation and transport. Finally, in vivo fluorescence microscopy revealed patches of PvdI, suggesting that uptake occurs via macromolecular assemblies on the cell surface.