Systemic anthrax, caused by inhalation or ingestion of Bacillus anthracis spores, is characterized by rapid microbial growth stages that require iron. Tightly bound and highly regulated in a mammalian host, iron is scarce during an infection. To scavenge iron from its environment, B. anthracis synthesizes by independent pathways two small molecules, the siderophores bacillibactin (BB) and petrobactin (PB). Despite the great efficiency of BB at chelating iron, PB may be the only siderophore necessary to ensure full virulence of the pathogen. In the present work, we show that BB is specifically bound by siderocalin, a recently discovered innate immune protein that is part of an antibacterial iron-depletion defense. In contrast, neither PB nor its ferric complex is bound by siderocalin. Although BB incorporates the common 2,3-dihydroxybenzoyl iron-chelating subunit, PB is novel in that it incorporates the very unusual 3,4-dihydroxybenzoyl chelating subunit. This structural variation results in a large change in the shape of both the iron complex and the free siderophore that precludes siderocalin binding, a stealthy evasion of the immune system. Our results indicate that the blockade of bacterial siderophore-mediated iron acquisition by siderocalin is not restricted to enteric pathogenic organisms and may be a general defense mechanism against several different bacterial species. Significantly, to evade this innate immune response, B. anthracis produces PB, which plays a key role in virulence of the organism. This analysis argues for antianthrax strategies targeting siderophore synthesis and uptake.bacillibactin ͉ Bacillus anthracis ͉ petrobactin ͉ siderocalin

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SummaryMany strains of mycobacteria produce two ferric chelating substances that are termed exochelin (an excreted product) and mycobactin (a cell-associated product). These agents may function as iron acquisition siderophores. To examine the genetics of the iron acquisition system in mycobacteria, ultraviolet (UV) and transposon (Tn611 ) mutagenesis techniques were used to generate exochelin-deficient mutants of Mycobacterium smegmatis strains ATCC 607 and LR222 respectively. Mutants were identified on CAS siderophore detection agar plates. Comparisons of the amounts of CAS-reactive material excreted by the possible mutant strains with that of the wild-type strains verified that seven UV mutant strains and two confirmed transposition mutant strains were deficient in exochelin production. Cell-associated mycobactin production in the mutants appeared to be normal. From the two transposon mutants, the mutated gene regions were cloned and identified by colony hybridization with an IS6100 probe, and the DNA regions flanking the transposon insertion sites were then used as probes to clone the wild-type loci from M. smegmatis LR222 genomic DNA. Complementation assays showed that an 8 kb Pst I fragment and a 4.8 kb Pst I/SacI subclone of this fragment complemented one transposon mutant (LUN2) and one UV mutant (R92). A 10.1 kb SacI fragment restored exochelin production to the other transposon mutant (LUN1). The nucleotide sequence of the 15.3 kb DNA region that spanned the two transposon insertion sites overlapped the 5Ј region of the previously reported exochelin biosynthetic gene fxbA and contained three open reading frames that were transcribed in the opposite orientation to fxbA. The corresponding genes were designated exiT, fxbB and fxbC. The deduced amino acid sequence of ExiT suggested that it was a member of the ABC transporter superfamily, while FxbB and FxbC displayed significant homology with many enzymes (including pristinamycin I synthetase) that catalyse non-ribosomal peptide synthesis. We propose that the peptide backbone of the siderophore exochelin is synthesized in part by enzymes resembling non-ribosomal peptide synthetases and that the ABC transporter ExiT is responsible for exochelin excretion.

Bacillus anthracis Sterne produced a catecholate siderophore named anthrachelin that was based on 3,4-dihydroxybenzoic acid (3,4-DHB, or protocatechuic acid), a catechol moiety previously unreported as a siderophore component. During iron restriction, both anthrachelin and free 3,4-DHB were excreted. Growth at 37 degrees C (as compared with 23 degrees C) decreased excretion of anthrachelin but not its precursor 3,4-DHB, suggesting that anthrachelin assembly is temperature regulated. A plasmidless strain also produced anthrachelin in an iron- and temperature-regulated fashion, indicating that anthrachelin genes are chromosomal. In addition to anthrachelin-mediated iron delivery, B. anthracis also used heme, hemoproteins, iron-transferrin, and certain heterologous siderophores (xenosiderophores) produced by other microorganisms as iron sources. Downregulation of anthrachelin production at the temperature of the mammalian host (which triggers toxin production in this pathogen) may focus the B. anthracis iron acquisition systems to exploit the iron sources prevailing in the infected host.

Aeromonas hydrophila 495A2 excreted two forms of amonabactin, a new phenolate siderophore composed of 2,3-dihydroxybenzoic acid, lysine, glycine, and either tryptophan (amonabactin T) or phenylalanine (amonabactin P). Supplementing cultures with L-tryptophan (0.3 mM) caused exclusive synthesis of amonabactin T, whereas supplements of L-phenylalanine (0.3 to 30 mM) gave predominant production of amonabactin P. The two forms of amonabactin were separately purified by a combination of production and polyamide column chromatographic methods. Both forms were biologically active, stimulating growth in iron-deficient medium of an amonabactin-negative mutant. Of 43 additional siderophore-producing isolates of the Aeromonas species that were tested, 76% (19 of 25) of the A. hydrophila isolates were amonabactin positive, whereas only 19% (3 of 16) of the A. sobria isolates and all (3 of 3) of the A. caviae isolates produced amonabactin, suggesting a predominant synthesis of amonabactin in certain Aeromonas species.Aeromonas hydrophila and related aeromonads are gramnegative, freshwater pathogens of fish and humans. In fish they cause a fatal hemorrhagic septicemia, and in humans they cause wound, soft tissue, and blood infections as well as acute gastroenteritis (2,(5)(6)(7)14 Purification of amonabactin. Amonabactin T and amonabactin P were separately purified from supernatants of A. hydrophila 495A2 after its growth in a low-iron minimal medium composed of the following (per liter): glucose, 5 g; (NH4)2HP04, 1 g; K2HPO4, 4 g; KH2PO4, 2.7 g. To lower metal contamination, the medium was treated with Chelex-100 (Bio-Rad Laboratories, Richmond, Calif.) by previously reported methods (1). After the Chelex-treated medium was filter sterilized, it was supplemented with filter sterilized solutions of high purity sulfate salts (Johnson-Matthey, Inc., Seabrook, N.H.) of magnesium (830 ,uM), manganese (40 ,uM), and iron (0.18 ,uM). were incubated at 30°C with aeration at 8 liters per min in a modified model 43-100 fermentor (The Virtis Co., Gardiner, N.Y.) in which exposed stainless steel components were coated with Teflon. For preparation of amonabactin T (the tryptophan-containing form of amonabactin), the medium was supplemented with 0.3 mM L-tryptophan, which caused exclusive production of amonabactin T. At maximum growth (usually after 12 to 18 h), the catecholate siderophore in the supernatant was readily detected by assay for dihydroxy phenolates (4) and by mixing 1 ml of supernatant with 0.005 ml of 1% ferric chloride, which resulted in a bluepurple color. The cells were removed by centrifuging the culture, and the phenolate(s) was adsorbed to polyamide (11) by passing the supernatant through a 5.5-by 13-cm column of polyamide (Woelm, Universal Adsorbents, Atlanta, Ga.) that had bren previously washed with methanol, acetone, and sufficient water to remove the organic solvents. The column then was washed with water (1.5 liters) and then washed with 500 ml of 100% acetone. The amonabactin T then was eluted with methanol (...

The mesophilic Aeromonas species are opportunistic pathogens that produce either of the siderophores amonabactin or enterobactin. Acquisition of iron for growth from Fe-transferrin in serum was dependent on the siderophore amonabactin; 50 of 54 amonabactin-producing isolates grew in heat-inactivated serum, whereas none of 30 enterobactin-producing strains were able to grow. Most isolates (regardless of siderophore produced) used haem as a sole source of iron for growth; all of 33 isolates grew with either haematin or haemoglobin and 30 of these used haemoglobin when complexed to human haptoglobin. Mutants unable to synthesize a siderophore used iron from haem, suggesting that this capacity was unrelated to siderophore production. Some members of the mesophilic Aeromonas species have evolved both siderophore-dependent and -independent mechanisms for acquisition of iron from a host.

Radioiron uptake from 59FeC13 by Streptococcus mutans OMZ176 was increased by anaerobiosis, sodium ascorbate, and phenazine methosulfate (PMS), although there was a 10-min lag before PMS stimulation was evident. The reductant ascorbate may have provided ferrous iron. The PMS was reduced by the cells, and the reduced PMS then may have generated ferrous iron for transport; reduced PMS also may have depleted dissolved oxygen. We conclude that S. mutans transports only ferrous iron, utilizing reductants furnished by glucose metabolism to reduce iron prior to its uptake.Streptococcus mutans is considered the major odontopathogen of human dental caries (11). However, the presence of S. mutans in the oral cavity does not imply disease in a simple cause-and-effect relationship; caries results from a complex interaction between host, microbial, and dietary factors (11). Some trace metals may shift this equilibrium to favor either the host or the microbe, and the presence and concentrations of these metals may be cariogenic or cariostatic (reviewed in reference 2). S. mutans has a superoxide dismutase that uses manganese as a cofactor but can substitute iron for manganese to produce active superoxide dismutase if manganese is absent (12, 13). Neither metal is required for anaerobic growth of S. mutans (13). Aerobic metabolic roles for iron in addition to its function as a cofactor for superoxide dismutase are suggested by experiments showing iron stimulation (nearly threefold) of S. mutans steady-state growth in manganese-containing medium (2). Iron-containing cytoplasmic fractions (other than superoxide dismutase) have been demonstrated in S. mutans (12). In other studies, iron was required for S. mutans growth (3), and an increase in colony size was obtained by adding iron to a solidified minimal medium (10). Lactoferrin (but not iron-saturated lactoferrin) increased the length of the S. mutans lag phase (5), suggesting that the organism is unable to obtain iron bound by lactoferrin.Transport of iron by S. mutans has not been extensively investigated. For efficient iron acquisition, many microorganisms produce siderophores that bind highly insoluble ferric iron, making it available for transport (14). Intracellular utilization of iron by S. mutans and the existence of specialized transport systems for iron in other organisms imply that S. mutans has membrane-associated iron uptake mechanisms. In the present studies, phenolate or hydroxamate siderophore production by S. mutans OMZ176 was not detected. The organism appeared to transport only reduced (ferrous) iron. Uptake of radioiron. For the radioiron uptake assays, the cells were grown aerobically without shaking in an atmosphere of 95% air-5% CO2 by the following procedure. A 0.1-ml amount of a 24-h culture of S. mutans OMZ176 in Todd-Hewitt broth (Difco Laboratories, Detroit, Mich.) was transferred to 10 ml of Chelex-100-treated FMC medium supplemented with magnesium, manganese, and iron. After 24 h of incubation, the entire 10-ml culture was transferred to 1 liter o...

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