Iron Assimilation Research Articles

Characterization of siderophore‐mediated iron transport from Rhizobium leguminosarum bv. phaseoli.

A high‐affinity iron assimilation system mediated by siderophores may be required for efficient nitrogen fixation in the genus Rhizobium, in view of the role of iron enzymes in this process. Siderophores biosynthesized by three strains of Rhizobium leguminosarum bv phaseoli were purified, hydrolyzed and analyzed. Using paper chromatography, two well‐defined spots that, according to their relative mobility, were identified as the L‐amino acids: threonine and tyrosine. Using thin layer chromatography, 2,3‐dihydroxybenzoic acid (2,3‐DHBA) was identified in the hydrolyzed samples as well. The spectrophotometric profiles from 2,3‐DHBA itself and a purified siderophore preparation from one of the strains were similar. The FAB+ mass spectrum showed a maximum peak of 637 m/z that indicates the purified compound has a molecular mass of 636 Da. Analysis of our results indicate that the compound that matched that molecular mass may contain: 2,3‐DHBA, tyrosine and threonine in a proportion of 1:1:3, with two of the threonine bound by their methyl group. A 30 KDa cell membrane protein was identified in cells grown under iron‐starved conditions. The siderophore and membrane receptor protein, structural constituents of the high‐affinity iron transport system in this species were found different to the compounds previously described in this genus by other authors.

Iron supply and demand in the upper ocean

Iron is hypothesized to be a limiting micronutrient for ocean primary production. This paper presents an analysis of the iron budget in the upper ocean. The global distribution of annual iron assimilation by phytoplankton was estimated from distributions of satellite‐derived oceanic primary production and measured (Fe:C)cellular ratios. The distributions of iron supply by upwelling/mixing and aeolian deposition were obtained by applying (Fe:NO3)dissolved ratios to the nitrate supply and by assuming the soluble fraction of mineral aerosols. A lower bound on the rate of iron recycling in the photic zone was estimated as the difference between iron assimilation and supply. Global iron assimilation by phytoplankton for the open ocean was estimated to be 12 × 109 mol Fe yr−1. Atmospheric deposition of total Fe is estimated to be 96×109 mol Fe yr−1 in the open ocean, with the soluble Fe fraction ranging between 1 and 10% (or 1‐10 ×109 mol Fe yr−1). By comparison, the upwelling/entrainment supply of dissolved Fe to the upper ocean is small, ∼0.7×109 mol Fe yr−1. Uncertainties in the aeolian flux and assimilation may be as large as a factor of 5‐10 but remain difficult to quantify, as information is limited about the form and transformation of iron from the soil to phytoplankton incorporation. An iron stress index, relating the (Fe:N) demand to the (Fe :N) supply, confirms the production in the high‐nitrate low‐chlorophyll regions is indeed limited by iron availability.

Synergistic effect ofrib80 andhit Mutations on riboflavin biosynthesis and iron transport in the yeastPichia guilliermondii

Mutant strains of the yeast Pichia guilliermondii, carrying both rib80 and hit mutations in a haploid genome, were derived from previously obtained strains with defective rib80 or hit genes, exerting negative control of the riboflavin biosynthesis and iron transport in Pichia guilliermondii. The double mutant rib80hit strains exhibited an increased level of riboflavin biosynthesis and higher activities of GTP cyclohydrolase and riboflavin synthetase. Iron deficiency caused an additional increase in riboflavin overproduction. These results suggest the synergistic interaction of the rib80 and hit mutations. A combination of both mutations in a single genome did not affect iron assimilation by the cells: ferrireductase activity, the rate of 55Fe uptake, and the iron content in cells of the double mutants remained at the level characteristic of the parent strains.

The Legionella pneumophila iraAB locus is required for iron assimilation, intracellular infection, and virulence.

Legionella pneumophila, a facultative intracellular parasite of human alveolar macrophages and protozoa, causes Legionnaires' disease. Using mini-Tn10 mutagenesis, we previously isolated a L. pneumophila mutant that was hypersensitive to iron chelators. This mutant, NU216, and its allelic equivalent, NU216R, were also defective for intracellular infection, particularly in iron-deficient host cells. To determine whether NU216R was attenuated for virulence, we assessed its ability to cause disease in guinea pigs following intratracheal inoculation. NU216R-infected animals yielded 1,000-fold fewer bacteria from their lungs and spleen compared to wild-type-130b-infected animals that had received a 50-fold-lower dose. Moreover, NU216R-infected animals subsequently cleared the bacteria from these sites. While infection with 130b resulted in high fever, weight loss, and ruffled fur, inoculation with NU216R did not elicit any signs of disease. DNA sequence analysis revealed that the transposon insertion in NU216R lies in the first open reading frame of a two-gene operon. This open reading frame (iraA) encodes a 272-amino-acid protein that shows sequence similarity to methyltransferases. The second open reading frame (iraB) encodes a 501-amino-acid protein that is highly similar to di- and tripeptide transporters from both prokaryotes and eukaryotes. Southern hybridization analyses determined that the iraAB locus was largely limited to strains of L. pneumophila, the most pathogenic of the Legionella species. A newly derived mutant containing a targeted disruption of iraB showed reduced ability to grow under iron-depleted extracellular conditions, but it did not have an infectivity defect in the macrophage-like U937 cells. These data suggest that iraA is critical for virulence of L. pneumophila while iraB is involved in a novel method of iron acquisition which may utilize iron-loaded peptides.

Iron and Oxidative Stress in Bacteria

The appearance of oxygen on earth led to two major problems: the production of potentially deleterious reactive oxygen species and a drastic decrease in iron availability. In addition, iron, in its reduced form, potentiates oxygen toxicity by converting, via the Fenton reaction, the less reactive hydrogen peroxide to the more reactive oxygen species, hydroxyl radical and ferryl iron. Conversely superoxide, by releasing iron from iron-containing molecules, favors the Fenton reaction. It has been assumed that the strict regulation of iron assimilation prevents an excess of free intracellular iron that could lead to oxidative stress. Studies in bacteria supporting that view are reviewed. While genetic studies correlate oxidative stress with increase of intracellular free iron, there are only few and sometimes contradictory studies on direct measurements of free intracellular metal. Despite this weakness, the strict regulation of iron metabolism, and its coupling with regulation of defenses against oxidative stress, as well as the role played by iron in regulatory protein in sensing redox change, appear as essential factors for life in the presence of oxygen.

Metabolism and genetics of Helicobacter pylori: the genome era.

The publication of the complete sequence of Helicobacter pylori 26695 in 1997 and more recently that of strain J99 has provided new insight into the biology of this organism. In this review, we attempt to analyze and interpret the information provided by sequence annotations and to compare these data with those provided by experimental analyses. After a brief description of the general features of the genomes of the two sequenced strains, the principal metabolic pathways are analyzed. In particular, the enzymes encoded by H. pylori involved in fermentative and oxidative metabolism, lipopolysaccharide biosynthesis, nucleotide biosynthesis, aerobic and anaerobic respiration, and iron and nitrogen assimilation are described, and the areas of controversy between the experimental data and those provided by the sequence annotation are discussed. The role of urease, particularly in pH homeostasis, and other specialized mechanisms developed by the bacterium to maintain its internal pH are also considered. The replicational, transcriptional, and translational apparatuses are reviewed, as is the regulatory network. The numerous findings on the metabolism of the bacteria and the paucity of gene expression regulation systems are indicative of the high level of adaptation to the human gastric environment. Arguments in favor of the diversity of H. pylori and molecular data reflecting possible mechanisms involved in this diversity are presented. Finally, we compare the numerous experimental data on the colonization factors and those provided from the genome sequence annotation, in particular for genes involved in motility and adherence of the bacterium to the gastric tissue.

Acquisition, transport, and storage of iron by pathogenic fungi.

Iron is required by most living systems. A great variety of means of acquisition, avenues of uptake, and methods of storage are used by pathogenic fungi to ensure a supply of the essential metal. Solubilization of insoluble iron polymers is the first step in iron assimilation. The two methods most commonly used by microorganisms for solubilization of iron are reduction and chelation. Reduction of ferric iron to ferrous iron by enzymatic or nonenzymatic means is a common mechanism among pathogenic yeasts. Under conditions of iron starvation, many fungi synthesize iron chelators known as siderophores. Two classes of compounds that function in iron gathering are commonly observed: hydroxamates and polycarboxylates. Two major responses to iron stress in fungi are a high-affinity ferric iron reductase and siderophore synthesis. Regulation of these two mechanisms at the molecular level has received attention. Uptake of siderophores is a diverse process, which varies among the different classes of compounds. Since free iron is toxic, it must be stored for further metabolic use. Polyphosphates, ferritins, and siderophores themselves have been described as storage molecules. The iron-gathering mechanisms used by a pathogen in an infected host are largely unknown and can only be posited on the basis of in vitro studies at present.

Iron uptake by the yeast Pichia guilliermondii. Flavinogenesis and reductive iron assimilation are co-regulated processes.

Pichia guilliermondii cells overproduce riboflavin (vitamin B2) in responce to iron deprivation. The increase in ferrireductase activity in iron-starved P. guilliermondii cells correlated with the increase in flavin excretion. As in Saccharomyces cerevisiae, a typical b-type cytochrome spectrum was associated with the plasma membrane fraction of P. guilliermondii and the cell ferrireductase activity was strongly inhibited by diphenylene-iodonium, an inhibitor of flavoproteins, in both yeasts. Mutants of P. guilliermondii with increased ferrireductase activity were selected for further investigation of the relationship between iron reduction/uptake and flavin production. The obtained mutation has been called hit (high iron transport). A hit mutant with a single recessive mutation showed the following phenotype: high ferrireductase activity, increased rate of iron uptake and elevated flavinogenic activity. Cu(II) (50 microns) strongly inhibited the growth of the hit mutant compared to the wild-type. The mutant cells grown in copper-supplemented medium (5-25 microns) showed an increase of the ferrireductase activity (up to 2-3 fold). The copper content of the mutant cells grown under these conditions was also higher (1.5-2 fold) than that of the wild-type. The role of the HIT gene of P. guillermondii in the regulation of iron, copper and flavin metabolisms is discussed.

Iron assimilation in Chlamydomonas reinhardtii involves ferric reduction and is similar to Strategy I in higher plants

Iron assimilation in Chlamydomonas reinhardtii involves ferric reduction and is similar to Strategy I in higher plants

Iron assimilation in Chlamydomonas reinhardtii involves ferric reduction and is similar to Strategy I higher plants

The mechanism of adaptation to Fe-deficiency stress was investigated in the unicellular green alga, Chlamydomonas reinhardtii. Upon removal of nutri- Introduction tional Fe, the activity of a cell surface Fe(III)-chelate For higher plants Fe is an essential nutritional element reductase was increased by at least 15-fold within that is required in micromolar concentrations. In spite of 24 h. This increase was negatively correlated with the its widespread abundance in the Earth’s crust, Fe is Fe concentration in the growth media. Incubation poorly available in an oxidizing environment, which of cells in the presence of the Fe2+-specific chela- favours the formation of poorly soluble Fe(III )-oxides tor, bathophenanthrolinedisulphonic acid, led to an and -hydroxides at pH values above neutral. Nevertheless, increased Fe3+ reductase activity, even when suffi- excess Fe is toxic to the cells due to its ability to catalyse cient Fe was present. Growth of cells in Cu-free media the formation of reactive oxygen species such as superfor 48 h led to no statistically significant increase in oxide anions and hydroxyl radicals (Halliwell and Fe3+ reductase activity. The Fe(III)-chelate reductase Gutteridge, 1989). activity in Fe-starved cells was saturable with an In higher plants, two basic strategies for iron acquisiapparent K m of 31 mM and was inhibited by uncouplers tion have been observed (reviewed in Guerinot and Yi, of the transmembrane proton gradient but not by 1994). In grasses (Strategy II plants), Fe is taken up as SH-specific reagents.

Evidence for the importance of catechol-type siderophores in the iron-limited growth of a cyanobacterium

To compensate for low levels of available iron, cyanobacteria may produce siderophores to assist in the scavenging of iron from the environment. In this paper we examine the role of catechol -type siderophores produced by the halotolerant cyanobacterium Synechococcus sp. PCC 7002 in the acquisition of iron from a chelated source. To inhibit catecholtype siderophore mediated iron transport, bovine serum albumin (BSA) was added to iron -deficient and replete cultures. Batch culture growth rates and cellular photosynthetic pigments decreased markedly in iron -limited populations in the presence of BSA, with no apparent decreases in growth rate in the iron-replete cultures. These results are supported by experiments with continuous culture chemostats where the addition of BSA to steady -state cultures leads to the washout of cells from low -iron chemostats, indicating that the cellular growth rate was reduced. The addition of BSA to short-term iron assimilation experiments further demonstrates that the presence of BSA can induce uptake kinetics consistent with the activity of an “iron -shuttle,” while BSA itself has no affinity for iron. These results demonstrate that catechol -type siderophores associated with the surface of the cell play an important role as “iron custodians.” While the presence of these catechols introduces complexity in the iron -transport mechanism and decreases the maximum velocity of iron uptake during episodic pulses of iron, the presence of the catechol associated with the cell surface functions to increase the overall cellular affinity for iron in low-iron environments.

The early diagenesis of iron in pelagic sediments: a multidisciplinary approach

Biogeochemical reactions of iron within pelagic sediments from the eastern and western equatorial Atlantic are investigated by means of pore water chemistry, chemical leaching experiments and total elemental determinations, color reflectance spectroscopy, rock magnetic measurements and TEM observations of the magnetic fraction. Results indicate that in the presence of nitrate, ascorbate (a weak reducing agent) leachable iron decreased with depth from the sediment surface. Within this upper sediment region, iron assimilation by bacteria is indicated as magnetosomes were found by TEM observations throughout the whole core. Extractions with a strong reducing agent (dithionite), representing iron bound to iron oxides, correlated linearly with the concentration of highly coercive magnetic minerals and with the reflection intensity of red color within the same iron redox zone. The abrupt decrease of red color reflection intensity, relative to the amount of iron oxides below the iron redox boundary, is the result of a decrease in the specific surface area of iron oxyhydroxide/oxides. Magnetic parameters imply a smaller average grain size of the magnetic fraction below the iron redox boundary, arising from an increase in biogenic magnetite formed by magnetotactic (assimilatory) bacteria, dissolution of very fine-grained magnetite, and the gradual decrease of coarser grained terrigenous (titano-) magnetite with depth. The early diagenetically formed magnetic fraction withstands subsequent dissolution and gives pronounced peaks of magnetic parameters within sediments with high carbonate and low terrigenous matter content.

Influence of Copper Depletion on Iron Uptake Mediated by SFT, a Stimulator of Fe Transport

We recently identified a novel factor involved in cellular iron assimilation called SFT or Stimulator of Fe Transport (Gutierrez, J. A., Yu, J., Rivera, S., and Wessling-Resnick, M. (1997) J. Cell Biol. 149, 895-905). When stably expressed in HeLa cells, SFT was found to stimulate the uptake of both transferrin- and nontransferrin-bound Fe (iron). Assimilation of nontransferrin-bound Fe by HeLa cells stably expressing SFT was time- and temperature-dependent; both the rate and extent of uptake was enhanced relative to the activity of control nontransfected cells. Although the apparent Km for Fe uptake was unaffected by expression of SFT (5.6 versus 5.1 microM measured for control), the Vmax of transport was increased from 7.0 to 14.7 pmol/min/mg protein. Transport mediated by SFT was inhibitable by diethylenetriaminepentaacetic acid and ferrozine, Fe3+- and Fe2+-specific chelators. Because cellular copper status is known to influence Fe assimilation, we investigated the effects of Cu (copper) depletion on SFT function. After 4 days of culture in Cu-deficient media, HeLa cell Cu,Zn superoxide dismutase activity was reduced by more than 60%. Both control cells and cells stably expressing SFT displayed reduced Fe uptake as well; levels of transferrin-mediated import fell by approximately 80%, whereas levels of nontransferrin-bound Fe uptake were approximately 50% that of Cu-replete cells. The failure of SFT expression to stimulate Fe uptake above basal levels in Cu-depleted cells suggests a critical role for Cu in SFT function. A current model for both transferrin- and nontransferrin-bound Fe uptake involves the function of a ferrireductase that acts to reduce Fe3+ to Fe2+, with subsequent transport of the divalent cation across the membrane bilayer. SFT expression did not enhance levels of HeLa cell surface reductase activity; however, Cu depletion was found to reduce endogenous activity by 60%, suggesting impaired ferrireductase function may account for the influence of Cu depletion on SFT-mediated Fe uptake. To test this hypothesis, the ability of SFT to directly mediate Fe2+ import was examined. Although expression of SFT enhanced Fe2+ uptake by HeLa cells, Cu depletion did not significantly reduce this activity. Thus, we conclude that a ferrireductase activity is required for SFT function in Fe3+ transport and that Cu depletion reduces cellular iron assimilation by affecting this activity.

Purification and Characterization op a 31-Kilodalton Iron-Regulated Periplasmic Protein from Pasteurella Haenolytica A1

ABSTRACT A prominent iron-regulated periplasmic protein was purified from Pasteurella haemolytica grown in an iron-deficient chemically defined medium. The protein was purified by anion exchange chromatography and appeared as a single band by SDS-PAGE with a molecular weight of 32,000. A yield of five mg was obtained from 91 mg of protein extract. The iron-regulated protein existed as a monomer in the native state with an average molecular weight of 29,877 as determined by analytical ultracentrifugation. The protein had a molecular weight of 30,880 as determined by matrix-assisted laser desorption mass spectrometry, hence the protein is referred to as the 31 kDa protein. Isoelectric focusing showed four bands with pIs of 7.15, 6.8, 6.6, and 5.9, The secondary structure of the protein was determined by circular dichroism and contained 16% α-helical structure. The N-terminal sequence, EPFKVVTTFTVIQDIAQNVAGDKAT, showed a 95% identity with the 31 kDa iron-binding protein from Haemophilus influenzae. Isolation and characterization of iron-regulated proteins are of particular interest because of their potential roles in iron assimilation and microbial virulence.

Iron transport-mediated drug delivery using mixed-ligand siderophore-beta-lactam conjugates.

Assimilation of iron is essential for microbial growth. Most microbes synthesize and excrete low molecular weight iron chelators called siderophores to sequester and deliver iron by active transport processes. Specific outer membrane proteins recognize, bind and initiate transport of species-selective ferric siderophore complexes. Organisms most often have specific receptors for multiple types of siderophores, presumably to ensure adequate acquisition of the iron that is essential for their growth. Conjugation of drugs to synthetic hydroxamate or catechol siderophore components can facilitate active iron-transport-mediated drug delivery. While resistance to the siderophore-drug conjugates frequently occurs by selection of mutants deficient in the corresponding siderophore-selective outer membrane receptor, the mutants are less able to survive under iron-deficient conditions and in vivo. We anticipated that synthesis of mixed ligand siderophore-drug conjugates would allow active drug delivery by multiple iron receptor recognition and transport processes, further reducing the likelihood that resistant mutants would be viable. Mixed ligand siderophore-drug conjugates were synthesized by combining hydroxamate and catechol components in a single compound that could chelate iron, and that also contained a covalent linkage to carbacephalosporins, as representative drugs. The new conjugates appear to be assimilated by multiple active iron-transport processes both in wild type microbes and in selected mutants that are deficient in some outer membrane iron-transport receptors. The concept of active iron-transport-mediated drug delivery can now be extended to drug conjugates that can enter the cell through multiple outer membrane receptors. Mutants that are resistant to such conjugates should be severely impaired in iron uptake, and therefore particularly prone to iron starvation.

Trace Metal Uptake ByPichia Spartinae, an Endosymbiotic Yeast in the Salt Marsh Cord GrassSpartina Alterniflora

Abstract The ascosporogenous marine yeast Pichia spartinae is a dominant endosymbiont of the marsh grass Spartina alterniflora. Results of previous studies suggested that P. spartinae is involved in iron transport processes in the grass. of particular interest has been the mechanisms of metal uptake and metabolism by the yeast, and the ecological and plant biochemical significance of these processes. This investigation examined the uptake of iron and other metals (Zn, Cu, Cd, Ni, Pb, Cr) by P. spartinae, and provides data on possible mechanisms of this activity. the results suggest a) the yeast can assimilate divalent and trivalent forms of inorganic iron, as well as large organic-Fe(III) complexes, b) the uptake of inorganic trivalent iron under soluble iron-deficient conditions proceeded by a different mechanism than that of soluble Fe(II), with intracellular loadings of iron much increased under the former conditions; c) trivalent iron uptake is not mediated by hydroxamate siderophores at levels detectable by sensitive screening assays; d) the assimilation of some trace metals (Cu, Zn, Cd, Ni) is likely to be mediated by low molecular weight cysteine rich proteins, possibly metallothionein, and; e) siderophores from other fungi can provide iron for P. spartinae. the iron assimilation data suggested that multiple mechanisms are involved, and are influenced by the concentration and speciation of iron in the system. in general, iron assimilation mechanisms are comparable to those described for closely related yeasts, such as Saccharomyces cerevisiae. Among other things, these results indicated that future studies of trace metal mobilization and plant assimilation in salt marsh ecosystems must account for the activities of microbial symbionts associated with the plants.

Ferritin Uptake by Human Erythroid Precursors Is a Regulated Iron Uptake Pathway

Iron delivery to mammalian cells is traditionally ascribed to diferric transferrin (Tf). We recently reported that human erythroid precursor cells possess specific membranes receptors that bind and internalize acid isoferritin. Here we show that ferritin uptake by these cells is highly regulated and that the internalized ferritin-iron is used for home synthesis and thus, this process could constitute a physiological pathway for iron assimilation. Ferritin was internalized by a specific, saturable process, distinct from the uptake of iron associated with albumin. Ferritin uptake downregulated transferrin-receptor expression, indicating that internalized ferritin-iron was recognized as an integral part of the cellular iron content. Ferritin receptor expression was coordinated to cell development and was tightly regulated by cellular iron status. Receptor abundance was increased by iron-depletion and decreased by iron-loading, while the affinity of the ferritin receptor for acid isoferritin remained nearly constant (kd = 4.1 +/- 0.5 x 10(-6) mol/L). Under all experimental conditions, ferritin- and transferrin-receptor expression was closely coordinated, suggesting that these pathways possess a common regulatory element. It is concluded that ferritin uptake by erythroid cells constitutes an iron uptake pathway in addition to the classical transferrin uptake pathway.

Iron uptake and iron-repressible polypeptides in Yersinia pestis.

Pigmented (Pgm+) cells of Yersinia pestis are virulent, are sensitive to pesticin, adsorb exogenous hemin at 26 degrees C (Hms+), produce iron-repressible outer membrane proteins, and grow at 37 degrees C in iron-deficient media. These traits are lost upon spontaneous deletion of a chromosomal 102-kb pgm locus (Pgm-). Here we demonstrate that an Hms+ but pesticin-resistant (Pst(r)) mutant acquired a 5-bp deletion in the pesticin receptor gene (psn) encoding IrpB to IrpD. Growth and assimilation of iron by Pgm- and Hms+ Pst(r) mutants were markedly inhibited by ferrous chelators at 37 degrees C; inhibition by ferric and ferrous chelators was less effective at 26 degrees C. Iron-deficient growth at 26 degrees C induced iron-regulated outer membrane proteins of 34, 28.5, and 22.5 kDa and periplasmic polypeptides of 33.5 and 30 kDa. These findings provide a basis for understanding the psn-driven system of iron uptake, indicate the existence of at least one additional 26 degrees C-dependent iron assimilation system, and define over 30 iron-repressible proteins in Y. pestis.

Anaerobic biosynthesis of enterobactin Escherichia coli: regulation of entC gene expression and evidence against its involvement in menaquinone (vitamin K2) biosynthesis

In Escherichia coli, isochorismate is a common precursor for the biosynthesis of the siderophore enterobactin and menaquinone (vitamin K2). Isochorismate is formed by the shikimate pathway from chorismate by the enzyme isochorismate synthase encoded by the entC gene. Since enterobactin is involved in the aerobic assimilation of iron, and menaquinone is involved in anaerobic electron transport, we investigated the regulation of entC by iron and oxygen. An operon fusion between entC with its associated regulatory region and lacZ+ was constructed and introduced into the chromosome in a single copy. Expression of entC-lacZ was found to be regulated by the concentration of iron both aerobically and anaerobically. An established entC::kan mutant deficient in enterobactin biosynthesis was found to grow normally and synthesize wild-type levels of menaquinone under anaerobic conditions in iron-sufficient media. These results led to the demonstration of an alternate isochorismate synthase specifically involved in menaquinone synthesis encoded by the menF gene. Consistent with these findings, the entC+ strains were found to synthesize enterobactin anaerobically under iron-deficient conditions while the ent mutants failed to do so.

Legionella pneumophila mutants that are defective for iron acquisition and assimilation and intracellular infection.

Legionella pneumophila, a parasite of macrophages and protozoa, requires iron for optimal extracellular and intracellular growth. However, its mechanisms of iron acquisition remain uncharacterized. Using mini-Tn10 mutagenesis, we isolated 17 unique L. pneumophila strains which appeared to be defective for iron acquisition and assimilation. Eleven of these mutants were both sensitive to the iron chelator ethylenediamine di(o-hydroxyphenylacetic acid) and resistant to streptonigrin, an antibiotic whose lethal effect requires high levels of intracellular iron. Six mutants were also defective for the infection of macrophage-like U937 cells. Although none were altered in entry, mutants generally exhibited prolonged lag phases and in some cases replicated at slower rates. Overall, the reduced recoveries of mutants, relative to that of the wild type, ranged from 3- to 1,000-fold. Strain NU216, the mutant displaying the most severe lag phase and the slowest rate of replication, was studied further. Importantly, within U937 cells, NU216 was approximately 100-fold more sensitive than the wild type was to treatment with the Fe3+ chelator deferoxamine, indicating that it is defective for intracellular iron acquisition and assimilation. Furthermore, this strain was unable to mediate any cytopathic effect and was impaired for infectivity of an amoebal host. Taken together, the isolation of these mutants offers genetic proof that iron acquisition and assimilation are critical for intracellular infection by L. pneumophila.