Characterization of siderophore-mediated iron transport inGeotrichum candidum,a non-siderophore producer

Summary Geotrichum candidum is capable of utilizing iron from hydroxamate siderophores of different structural classes. The relative rates of iron transport for ferrichrome, ferrichrysin, ferrioxamine B, fusigen, ferrichrome A, rhodotorulic acid, coprogen B, dimerium acid and ferrirhodin were 100%, 98%, 74%, 59%, 49%, 35%, 24%, 12% and 11% respectively. Ferrichrome, ferrichrysine and ferrichrome A inhibited [⁵⁹Fe]ferrioxamine-B-mediated iron transport by 71%, 68% and 28% respectively when added at equimolar concentrations to the radioactive complex. The inhibitory mechanism of [⁵⁹Fe]ferrioxamine B uptake by ferrichrome was non-competitive (K _i 2.4 M), suggesting that the two siderophores do not share a common transport system. Uptake of [⁵⁹Fe]ferrichrome, [⁵⁹Fe]rhodotorulic acid and [⁵⁹Fe]fusigen was unaffected by competition with the other two siderophores or with ferrioxamine B. Thus,G. candidum may possess independent transport systems for siderophores of different structural classes. The uptake rates of [¹⁴C]ferrioxamine B and⁶⁷Ga-desferrioxamine B were 30% and 60% respectively, as compared to [⁵⁹Fe]ferrioxamine B. The specific ferrous chelates, dipyridyl and ferrozine at 6 mM, caused 65% and 35% inhibition of [⁵⁹Fe]ferrioxamine uptake. From these results we conclude that, although about 70% of the iron is apparently removed from the complex by reduction prior to being transported across the cellular membrane, a significant portion of the chelated ligand may enter the cell intact. The former and latter mechanisms seem not to be mutually exclusive.     

Ferrioxamine transport mutants and the identification of the ferrioxamine receptor protein (FoxA) inErwinia herbicola (Enterobacter agglomerans)

Summary Iron deprivation ofErwinia herbicola (Enterobacter agglomerans) induces the biosynthesis of six high-M _r outer-membrane proteins and large amounts of ferrioxamine E. Mutagenesis withN-methyl-N-nitro-N-nitrosoguanidine and selection with ferrimycin A yielded mutants ofE. herbicola K4 (wild type), defective in the expression of a 76-kDa outer-membrane protein, as determined by SDS/polyacrylamide gel electrophoresis. While in bioassays wild-type cells showed growth promotion in the presence of ferrioxamines (B, D₁, D₂, E, G), enterobactin, citrate, ferrichrome and coprogen, these mutants failed to respond to ferrioxamines. Moreover, experiments with⁵⁵Fe-labelled siderophores confirmed that iron transport mediated by ferrioxamine E and B in the mutants was completely inhibited, whereas iron transport by other hydroxamate siderophores, such as ferrichrome and coprogen was unaffected. The results are evidence that the 76-kDa protein in the outer membrane represents the receptor protein (FoxA) for ferrioxamines inE. herbicola.     

Desferrioxamine-dependent iron transport in Erwinia amylovora CFBP1430: cloning of the gene encoding the ferrioxamine receptor FoxR

Iron deprivation of Erwinia amylovora CFBP1430, a species causing fire blight on Pomoïdeae, was shown to induce the production of siderophores of the desferrioxamine (dfo) family and two outer membrane polypeptides with apparent molecular weight of about 70 and 80 kDa, respectively. Cyclic dfo E was characterized as the major metabolite. Phage MudII_pR13 insertional mutagenesis and screening on CAS-agar medium yielded three dfo non-producing and one overproducing clones. These clones failed to grow in the presence of the Fe(III) chelator EDDHA and were determined further as dfo and ferrioxamine transport negative mutants, respectively. The transport mutant which appeared to lack the 70 kDa polypeptide in the outer membrane allowed the purification of dfo E. Growth under iron limitation of dfo negative mutants was stimulated with ferrioxamine E and B but not with other ferrisiderophores tested. The host DNA sequence flanking the left terminal part of the MudII_pR13 prophage responsible for the transport mutation was cloned and used to probe a parental gene library by DNA-DNA hybridization. Two recombinant cosmids restoring the transport mutation to normal were identified. Both cosmids also conferred the ability to utilize ferrioxamine B and E as iron sources on a FhuE¹ mutant of Escherichia coli. This correlated with the production of an additional polypeptide of 70 kDa in the outer membrane of E. coli transconjugants, thus confirming that this protein serves the ferrioxamine receptor function (FoxR) in E. amylovora.R. Kachadourian and A. Dellagi have contributed equally to this work.     

FhuF, part of a siderophore-reductase system

FhuF is a cytoplasmic 2Fe-2S protein of Escherichia coli loosely associated with the cytoplasmic membrane. E. coli fhuF mutants showed reduced growth on plates with ferrioxamine B as the sole iron source, although siderophore uptake was not defective in transport experiments. Removal of iron from coprogen, ferrichrome, and ferrioxamine B was significantly lower in fhuF mutants compared to the corresponding parental strains, which suggested that FhuF is involved in iron removal from these hydroxamate-type siderophores. A redox potential E(1/2) of -310 +/- 25 mV relative to the normal hydrogen electrode was determined for FhuF by EPR redox titration; this redox potential is sufficient to reduce the siderophores coprogen and ferrichrome. M?ssbauer spectra revealed that FhuF in its [Fe(2+)-Fe(3+)] state is also capable of direct reduction of ferrioxamine B-bound ferric iron, thus proving its reductase function. This is the first report on a bacterial siderophore-iron reductase which in vivo seems to be specific for a certain group of hydroxamates.     

Iron(III) hydroxamate transport across the cytoplasmic membrane ofEscherichia coli

Summary Transport of iron(III) hydroxamates across the inner membrane into the cytoplasm ofEscherichia coli is mediated by the FhuC, FhuD and FhuB proteins and displays characteristics typical of a periplasmic-binding-protein-dependent transport mechanism. In contrast to the highly specific receptor proteins in the outer membrane, at least six different siderophores of the hydroxamate type and the antibiotic albomycin are accepted as substrates. AfhuB mutant (deficient in transport of substrates across the inner membrane) which overproduced the periplasmic FhuD 30-kDa protein, bound [⁵⁵Fe] iron(III) ferrichrome. Resistance of FhuD to proteinase K in the presence of ferrichrome, aerobactin, and coprogen indicated binding of these substrates to FhuD. FhuD displays significant similarity to the periplasmic FecB, FepB, and BtuE proteins. The extremely hydrophobic FhuB 70-kDa protein is located in the cytoplasmic membrane and consists of two apparently duplicated halves. The N-and C-terminal halves [FhuB(N) and FhuB(C)] were expressed separately infhuB mutants. Only combinations of FhuB(N) and FhuB(C) polypeptides restored sensitivity to albomycin and growth on iron hydroxamate as a sole iron source, indicating that both halves of FhuB were essential for substrate translocation and that they combined to form an active permease. In addition, a FhuB derivative with a large internal duplication of 271 amino acids was found to be transport-active, indicating that the extra portion did not disturb proper insertion of the active FhuB segments into the cytoplasmic membrane. A region of considerable similarity, present twice in FhuB, was identified near the C-terminus of 20 analyzed hydrophobic proteins of periplasmic-binding-protein-dependent systems. The FhuC 30 kDa protein, most likely involved in ATP binding, contains two domains representing consensus sequences among all peripheral cytoplasmic membrane proteins of these systems. Amino acid replacements in domain I (LysGlu and Gln) and domain II (AspAsn and Glu) resulted in a transport-deficient phenotype.     

Identification of siderophores and siderophore-mediated uptake of iron in Stemphylium botryosum

Under iron-deficient conditions Stemphylium botryosum f. sp. lycopersici produces three major siderophores; dimerum acid, coprogen B and an unidentified monohydroxamate siderophore designated as A. The system of siderophores mediating uptake of iron was characterized. It exhibits active transport, saturation kinetics and an optimum at pH 6 and 30°. The rate of iron uptake via dimerum acid and coprogen B was four times higher than siderophore A. S. botryosum was capable of taking up iron from hydroxamate siderophores produced by other fungi, e.g. ferrichrome, fusigen, rhodotorulic acid but not ferrioxamine B. Double labelling experiments suggest that ferric coprogen B accumulates in mycelial cells as an intact chelate.     

Fusarinines and dimerum acid, mono- and dihydroxamate siderophores from Penicillium chrysogenum, improve iron utilization by strategy I and strategy II plants

Cucumber, as a strategy I plant, and Maize as a strategy II plant, were cultivated in hydroponic culture in the presence of a ferrated siderophore mixture (1 M) from a culture of Penicillium chrysogenumisolated from soil. The siderophore mixture significantly improved the iron status of these plants as measured by chlorophyll concentration to the same degree as a 100-fold higher FeEDTA supply. Analysis of the siderophore mixture from P. chrysogenum by HPLC and electrospray mass spectrometry revealed that besides the trihydroxamates, coprogen and ferricrocin, large amounts of dimerum acid and fusarinines were present which represent precursor siderophores or breakdown products of coprogen. In order to prove the iron donor properties of dimerum acid and fusarinines for plants, purified coprogen was hydrolyzed with ammonia and the hydrolysis products consisting of dimerum acid and fusarinine were used for iron uptake by cucumber and maize. In short term experiments radioactive iron uptake and translocation rates were determined using ferrioxamine B, coprogen and hydrolysis products of coprogen. While the trihydroxamates revealed negligible or intermediate iron uptake rates by both plant species, the fungal siderophore mixture and the ammoniacal hydrolysis products of coprogen showed high iron uptake, suggesting that dimerum acid and fusarinines are very efficient iron sources for plants. Iron reduction assays using cucumber roots or ascorbic acid also showed that iron bound to hydrolysis products of coprogen was more easily reduced compared to iron bound to trihydroxamates. Ligand exchange studies with epi-hydroxymugineic acid and EDTA showed that iron was easily exchanged between coprogen hydrolysis products and phytosiderophores or EDTA. The results indicate that coprogen hydrolysis products are an excellent source for Fe nutrition of plants.     

Confirmation of Occurrence of Hydroxamate Siderophores in Soil by a Novel Escherichia coli Bioassay

The occurrence of ferrichrome-type hydroxamate siderophores in soil was confirmed. In the presence of the iron-scavenging chelator ethylenediamine[di(o-hydroxyphenylacetic)acid], soil extract stimulated the growth of an Escherichia coli strain possessing the ferrichrome transport protein (TonA) but did not stimulate growth of a strain lacking this protein (TonA⁻). The siderophore concentration in a 1:1 (soil-water) extract was estimated to be approximately 78 nM. Specificity of the assay was supported by the absence of significant differential strain responses to ferric citrate, ferric 2,3-dihydroxybenzoate, enterochelin, ferrioxamine B, coprogen, and triacetylfusigen.     

Characterization of ferrioxamine E as the principal siderophore ofErwinia herbicola (Enterobacter agglomerans)

Summary Several strains of the enterobacterial groupErwinia herbicola (Enterobacter agglomerans) were screened for siderophore production. After 3 days of growth in a low-iron medium, all strains studied produced hydroxamate siderophores. The retention values of the main siderophore during thin-layer chromatography on silica gel plates and on HPLC reversed-phase columns were identical with those of an authentic sample of ferrioxamine E (norcardamine). Gas-chromatographic analysis of the HI hydrolyzate yielded succinic acid and 1,5-diaminopentane in equimolar amounts; fast-atom-bombardment (FAB) mass spectroscopy showed a molecular mass of 653 Da. Iron from⁵⁵Fe-labelled ferrioxamine E was well taken up by iron-starved cells ofE. herbicola (K _m=0.1 M,V _max=8 pmol mg^–1 min^–1). However, besides ferrioxamine E (100%), several exogenous siderophores such as enterobactin (94.5%), ferric citrate (78.5%), coprogen (63.5%) and ferrichrome (17.5%) served as siderophores, suggesting the presence of multiple siderophore receptors in the outer membrane ofE. herbicola.     

Iron transport in Streptomyces pilosus mediated by ferrichrome siderophores, rhodotorulic acid, and enantio-rhodotorulic acid

Streptomyces pilosus is one of several microbes which produce ferrioxamine siderophores. In the accompanying paper (G. Müller and K. Raymond, J. Bacteriol. 160:304-312), the mechanism of iron uptake mediated by the endogenous ferrioxamines B, D1, D2, and E was examined. Here we report iron transport behavior in S. pilosus as mediated by the exogenous siderophores ferrichrome, ferrichrysin, rhodotorulic acid (RA), and synthetic enantio-RA. In each case iron acquisition depended on metabolic energy and had uptake rates comparable to that of [55Fe]ferrioxamine B. However, the synthetic ferric enantio-RA (which has the same preferred chirality at the metal center as ferrichrome) was twice as effective in supplying iron as was the natural ferric RA complex, suggesting that stereospecific recognition at the metal center is involved in the transport process. Iron uptake mediated by ferrichrome and ferric enantio-RA was strongly inhibited by kinetically inert chromic complexes of desferrioxamine B. These inhibition experiments indicate that iron from these exogenous siderophores is transported by the same uptake system as ferrioxamine B. Since the ligands have no structural similarity to ferrioxamine B except for the presence of three hydoxamate groups, we conclude that only the hydroxamate iron center and its direct surroundings are important for recognition and uptake. This hypothesis is supported by the fact that ferrichrome A and ferrirubin, which are both substituted at the hydroxamate carbonyl groups, were not (or were poorly) effective in supplying iron to S. pilosus.     

Enzymatic release of iron from sideramines in fungi. NADH: Sideramine oxidoreductase in Neurospora crassa

Young mycelia of the fungus Neurospora crassa contain a soluble NADH-linked sideramine reductase, which may be responsible for liberating iron in vivo from accumulated sideramines during iron-deficient cultivation. The enzymes can be assayed using a soluble supernatant fraction, EDTA, and an atmosphere of pure nitrogen. The enzyme is stable without loss of activity up to 45°C and has an optimum of activity at pH 7.0. Besides coprogen (K_m = 100 μM, V = 2.8 nmol/min. per mg protein), some other ferrichrome-type compounds are reduced. However, ferrichrome, ferrirubin, coprogen B and ferrioxamine are poor substrates. When the mucelia were grown in a medium containing 10^?5 M ferric iron, the activity of the reductase was found to be only 30% of that found under low iron conditions. The enzyme is inhibited by oxygen, SH-alkylating agents and partly by some detergents. Unlike the reductase of N. crassa, the corresponding enzyme from Aspergillus fumigatus revealed low reduction of coprogen and high reduction of ferrichrome, indicating genus-dependent specificities of sideramine reduction enzymes in fungi. The participation of acids of the citric acid cycle as natural iron acceptors during strong iron deficiency is studied and confirmed by iron uptake measurements on isolated mitochondria.     

Utilization of microbial siderophores in iron acquisition by oat

Iron uptake by oat (Avena sativa cv Victory) was examined under hydroponic chemical conditions that required direct utilization of microbial siderophores for iron transport. Measurements of iron uptake rates by excised roots from the hydroxamate siderophores, ferrichrome, ferrichrome A, coprogen, ferrioxamine B (FOB), and rhodotorulic acid (RA) showed all five of the siderophores supplied iron, but that FOB and RA were preferentially utilized. FOB-mediated iron uptake increased four-fold when roots were preconditioned to iron stress and involved an active, iron-stress induced transport system that was inhibited by 5 millimolar sodium azide or 0.5 millimolar dinitrophenol. Kinetic studies indicated partial saturation with an apparent K_m of 5 micromolar when FOB was supplied at 0.1 to 50 micromolar concentrations. Whole plant experiments confirmed that 5 micromolar FOB was sufficient for plant growth. Siderophore-mediated iron transport was inhibited by Cr-ferrichrome, an analog of ferrated siderophore. Our results confirm the existence of a microbial siderophore iron transport system in oat which functions within the physiological concentrations produced and used by soil microorganisms.     

Iron acquisition systems for ferric hydroxamates, haemin and haemoglobin in Listeria monocytogenes

Listeria monocytogenes is a Gram-positive bacterium that causes severe opportunistic infections in humans and animals. We biochemically characterized, for the first time, the iron uptake processes of this facultative intracellular pathogen, and identified the genetic loci encoding two of its membrane iron transporters. Strain EGD-e used iron complexes of hydroxamates (ferrichrome and ferrichrome A, ferrioxamine B), catecholates (ferric enterobactin, ferric corynebactin) and eukaryotic binding proteins (transferrin, lactoferrin, ferritin, haemoglobin). Quantitative determinations showed 10-100-fold lower affinity for ferric siderophores (Km approximately 1-10 nM) than Gram-negative bacteria, and generally lower uptake rates. Vmax for [59Fe]-enterobactin (0.15 pMol per 10(9) cells per minute) was 400-fold lower than that of Escherichia coli. For [59Fe]-corynebactin, Vmax was also low (1.2 pMol per 10(9) cells per minute), but EGD-e transported [59Fe]-apoferrichrome similarly to E. coli (Vmax=24 pMol per 10(9) cells per minute). L. monocytogenes encodes potential Fur-regulated iron transporters at 2.031 Mb (the fur-fhu region), 2.184 Mb (the feo region), 2.27 Mb (the srtB region) and 2.499 Mb (designated hupDGC region). Chromosomal deletions in the fur-fhu and hupDGC regions diminished iron uptake from ferric hydroxamates and haemin/haemoglobin respectively. In the former locus, deletion of fhuD (lmo1959) or fhuC (lmo1960) strongly reduced [59Fe]-apoferrichrome uptake. Deletion of hupC (lmo2429) eliminated the uptake of haemin and haemoglobin, and decreased the virulence of L. monocytogenes 50-fold in mice. Elimination of srtB region genes (Deltalmo2185, Deltalmo2186, Deltalmo2183), both sortase structural genes (DeltasrtB, DeltasrtA, DeltasrtAB), fur and feoB did not impair iron transport. However, deletion of bacterioferritin (Deltafri, lmo943; 0.97 Mb) decreased growth and altered iron uptake: Vmax of [59Fe]-corynebactin transport tripled in this strain, whereas that of [59Fe]-apoferrichrome decreased 20-fold.     

Conversion of the coprogen transport protein FhuE and the ferrioxamine B transport protein FoxA into ferrichrome transport proteins

The FhuA protein of Escherichia coli K-12 transports ferrichrome and the structurally related antibiotic albomycin across the outer membrane and serves as a receptor for the phages T1, T5, and φ80 and for colicin M. In this paper, we show that chimeric proteins consisting of the central part of FhuA and the N- and C-terminal parts of FhuE (coprogen receptor) or the N- and/or C-terminal parts of FoxA (ferrioxamine B receptor), function as ferrichrome transport proteins. Although the hybrid proteins contained the previously identified gating loop of FhuA, which is the principal binding site of the phages T5, T1, and φ80, only the hybrid protein consisting of the N-terminal third of FoxA and the C-terminal two thirds of FhuA conferred weak phage sensitivity to cells. Apparently, the gating loop is essential, but not sufficient for wild-type levels of ferrichrome transport and for phage sensitivity. The properties of FhuA-FoxA hybrids suggest different regions of the two receptors for ferric siderophore uptake.     

Functional expression of Escherichia coli fhuA gene in Rhizobium spp. of Cajanus cajan provides growth advantage in presence of Fe3+: ferrichrome as iron source

Cajanus cajan rhizobial isolates were found to be unable to utilize iron bound to ferrichrome, desferrioxamine B or rhodotorulic acid, all being hydroxamate type siderophores. A broad host range expression vector containing the Escherichia coli fhuA gene, encoding the outer membrane receptor for Fe-ferrichrome, was constructed. The plasmid construct (pGR1), designed to express fhuA under the lac promoter of E. coli, complemented E. coli MB97 ΔfhuA mutant for ferri-ferrichrome utilization and also allowed Rhizobium spp. ST1 and Rhizobium spp. IC3123 to grow using iron bound to ferrichrome. Sensitivity to the antibiotic albomycin, transported via the FhuA receptor, was found in case of MB97 as well as rhizobial transformants harboring pGR1. The rhizobial transformants expressing fhuA showed growth stimulation when co-inoculated with Ustilago maydis, a fungal species known to produce ferrichrome under iron starved conditions. Growth stimulation was also observed in the presence of externally supplied ferrichrome. The significance of these findings in terms of the potential for improving the survivability of rhizobial bioinoculant strains in natural soils is discussed.     

Soluble and membrane-bound ferrisiderophore reductases of Escherichia coli K-12

After uptake of microbial ferrisiderophores, iron is assumed to be released by reduction. Two ferrisiderophore-reductase activities were identified in Escherichia coli K-12. They differed in cellular location, susceptibility to amytal, and competition between oxygen and ferrichrome-iron(III) reduction. The ferrisiderophore reductase associated with the 40,000×g sediment (membrane-bound enzyme) was inhibited by 10 mM amytal in contrast to the ferrisiderophore reductase present in the 100,000×g supernatant (soluble enzyme). Reduction by the membrane-bound enzyme followed sigmoid kinetics, but was biphasic in the case of the soluble enzyme. The soluble reductase could be assigned to a protein consisting of a single polypeptide of M _r 26000. Reduction of iron(III) by the purified enzyme depended on the addition of NADH or NADPH which were equally active reductants. The cofactor FMN and to a lesser degree FAD stimulated the reaction. Substrate specificity of the soluble reductase was low. In addition to the hydroxamate siderophores arthrobactin, schizokinen, fusigen, aerobactin, ferrichrome, ferrioxamine B, coprogen, and ferrichrome A, the iron(III) complexes of synthetic catecholates, dihydroxy benzoic acid, and dicitrate, as well as carrier-free iron(III) were accepted as substrates. Both ferrisiderophore reductases were not controlled by the fur regulatory system and were not suppressed by anaerobic growth.Abbreviations DHB dihydroxybenzoic acid - MECAM 1,3,5-N,N,N-tris-(2,3-dihydroxybenzoyl)-triamino-methylbenzene - MECAMS 2,3-dihydroxy-5-sulfonyl-derivative of MECAM     

Role of Nitrosomonas europaea NitABC iron transporter in the uptake of Fe3+-siderophore complexes

Nitrosomonas europaea has a single three-gene operon (nitABC) encoding an iron ABC transporter system (NitABC). Phylogenetic analysis clustered the subunit NitB with Fe³⁺-ABC transporter permease components from other organisms. The N. europaea strain deficient in nitB (nitB::kan) grew well in either Fe-replete or Fe-limited media and in Fe-limited medium containing the catecholate-type siderophore, enterobactin or the citrate-based dihydroxamate-type siderophore, aerobactin. However, the nitB::kan mutant strain was unable to grow in Fe-limited media containing either the hydroxamate-type siderophores, ferrioxamine and ferrichrome or the mixed-chelating type siderophore, pyoverdine. Exposure of N. europaea cells to a ferrichrome analog coupled to the fluorescent moiety naphthalic diimide (Fhu-NI) led to increase in fluorescence in the wild type but not in nitB::kan mutant cells. Spheroplasts prepared from N. europaea wild type exposed to Fhu-NI analog retained the fluorescence, while spheroplasts of the nitB::kan mutant were not fluorescent. NitABC transports intact Fe³⁺-ferrichrome complex into the cytoplasm and is an atypical ABC type iron transporter for Fe³⁺ bound to ferrioxamine, ferrichrome or pyoverdine siderophores into the cytoplasm. The mechanisms to transport iron in either the Fe³⁺ or Fe²⁺ forms or Fe³⁺ associated with enterobactin or aerobactin siderophores into the cell across the cytoplasmic membrane are as yet undetermined.     

Transport across the outer membrane of Escherichia coli K12 via the FhuA receptor is regulated by the TonB protein of the cytoplasmic membrane

Summary Point mutations in the “TonB box” offhuA were suppressed by point mutations in thetonB gene, suggesting both a functional and physical interaction between the FhuA receptor protein in the outer membrane and the TonB protein in the cytoplasmic membrane ofEscherichia coli K12. Mutations influA were classified into four types according to the extent by which they impaired mutant cells in their growth on ferrichrome as sole iron source and in their sensitivity to the antibiotic albomycin and to colicin M. ThetonB mutation with a glutamine to leucine replacement at position 165 was less efficient in restoring the FhuA functions than the glutamine to lysine exchange at the same position. The better the coupling between FhuA and TonB the poorer was the inhibition of phage T1 binding to FhuA by ferrichrome. A working model is proposed in which the TonB protein assumes different conformations in response to the energized state of the cytoplasmic membrane and thereby allosterically regulates the activity of the FhuA receptor. This model implies an intermembrane coupling between two proteins in adjacent membranes.     

The Bradyrhizobium japonicum fsrB gene is essential for utilization of structurally diverse ferric siderophores to fulfill its nutritional iron requirement

In Bradyrhizobium japonicum, iron uptake from ferric siderophores involves selective outer membrane proteins and non-selective periplasmic and cytoplasmic membrane components that accommodate numerous structurally diverse siderophores. Free iron traverses the cytoplasmic membrane through the ferrous (Fe²⁺) transporter system FeoAB, but the other non-selective components have not been described. Here, we identify fsrB as an iron-regulated gene required for growth on iron chelates of catecholate- and hydroxymate-type siderophores, but not on inorganic iron. Utilization of the non-physiological iron chelator EDDHA as an iron source was also dependent on fsrB. Uptake activities of ⁵⁵Fe³⁺ bound to ferrioxamine B, ferrichrome or enterobactin were severely diminished in the fsrB mutant compared with the wild type. Growth of the fsrB or feoB strains on ferrichrome were rescued with plasmid-borne E. coli fhuCDB ferrichrome transport genes, suggesting that FsrB activity occurs in the periplasm rather than the cytoplasm. Whole cells of an fsrB mutant are defective in ferric reductase activity. Both whole cells and spheroplasts catalyzed the demetallation of ferric siderophores that were defective in an fsrB mutant. Collectively, the data support a model whereby FsrB is required for reduction of iron and its dissociation from the siderophore in the periplasm, followed by transport of the ferrous ion into the cytoplasm by FeoAB.     

Pyoverdine-mediated iron transport Fate of iron and ligand inPseudomonas aeruginosa

Summary Incubated in the presence of [⁵⁵Fe]ferri[¹⁴C]pyoverdine, iron-poorPseudomonas aeruginosa accumulated more⁵⁵Fe than¹⁴C over a 60-min period. Distribution studies showed (a) more¹⁴C than⁵⁵Fe in the soluble fraction during the first 20 min, (b) approximately 60% of the⁵⁵Fe associated with the membranes at 60 min, and (c) approximately 85% of the¹⁴C in the soluble fraction at 60 min. Cells osmotically shocked after incubating with [⁵⁵Fe]ferri[¹⁴C]pyoverdine for 60 min released⁵⁵Fe but not¹⁴C, suggesting separation of metal and ligand in the periplasmic space. Whereas the mechanism of dissociation of iron and ligand is not known, the decrease in transport observed in the presence of dipyridyl suggests involvement of reduction in this process. Transport of iron was energized by the proton motive force instead of by intracellular levels of ATP. The hydrogen ion gradient was the major driving force of transport. Cyanide-poisoned cells accumulated more¹⁴C than⁵⁵Fe over 60 min. Here, iron accumulated in the soluble fraction instead of on the membranes.