Siderophore Activity Among Members of the <Emphasis Type="Italic">Legionella</Emphasis> Genus

Members of the Legionella genus are ubiquitous aquatic bacteria and the etiologic agents of Legionnaires disease, a potentially fatal form of pneumonia. Using the chrome azurol S (CAS) assay, we previously determined that Legionella pneumophila secretes a siderophore (legiobactin) when it is grown in a low-iron, chemically defined medium (CDM). In the present study, we examined 29 other species of Legionella for their ability to produce CAS-reactive material when grown in deferrated CDM. Although some of the species did not grow in CDM, the majority replicated and secreted CAS reactivity, suggesting that siderophores are conserved among the legionellae.     

lbtA and lbtB are required for production of the Legionella pneumophila siderophore legiobactin

Under iron stress, Legionella pneumophila secretes legiobactin, a nonclassical siderophore that is reactive in the chrome azurol S (CAS) assay. Here, we have optimized conditions for legiobactin expression, shown its biological activity, and identified two genes, lbtA and lbtB, which are involved in legiobactin production. lbtA appears to be iron repressed and encodes a protein that has significant homology with siderophore synthetases, and FrgA, a previously described iron-regulated protein of L. pneumophila. lbtB encodes a protein homologous with members of the major facilitator superfamily of multidrug efflux pumps. Mutants lacking lbtA or lbtB were defective for legiobactin, producing 40 to 70% less CAS reactivity in deferrated chemically defined medium (CDM). In bioassays, mutant CDM culture supernatants, unlike those of the wild type, did not support growth of iron-limited wild-type bacteria in 2',2'-dipyridyl-containing buffered charcoal yeast extract (BCYE) agar and a ferrous iron transport mutant on BCYE agar without added iron. The lbtA mutant was modestly defective for growth in deferrated CDM containing the iron chelator citrate, indicating that legiobactin is required in conditions of severe iron limitation. Complementation of the lbt mutants restored both siderophore expression, as measured by the CAS assay and bioassays, and bacterial growth in deferrated, citrate-containing media. The lbtA mutant replicated as the wild type did in macrophages, amoebae, and the lungs of mice. However, L. pneumophila expresses lbtA in the macrophage, suggesting that legiobactin, though not required, may play a dispensable role in intracellular growth. The discovery of lbtAB represents the first identification of genes required for L. pneumophila siderophore expression.     

Discovery of a nonclassical siderophore, legiobactin, produced by strains of Legionella pneumophila

The mechanisms by which Legionella pneumophila, a facultative intracellular parasite and the agent of Legionnaires' disease, acquires iron are largely unexplained. Several earlier studies indicated that L. pneumophila does not elaborate siderophores. However, we now present evidence that supernatants from L. pneumophila cultures can contain a nonproteinaceous, high-affinity iron chelator. More specifically, when aerobically grown in a low-iron, chemically defined medium (CDM), L. pneumophila secretes a substance that is reactive in the chrome azurol S (CAS) assay. Importantly, the siderophore-like activity was only observed when the CDM cultures were inoculated to relatively high density with bacteria that had been grown overnight to log or early stationary phase in CDM or buffered yeast extract. Inocula derived from late-stationary-phase cultures, despite ultimately growing, consistently failed to result in the elaboration of siderophore-like activity. The Legionella CAS reactivity was detected in the culture supernatants of the serogroup 1 strains 130b and Philadelphia-1, as well as those from representatives of other serogroups and other Legionella species. The CAS-reactive substance was resistant to boiling and protease treatment and was associated with the <1-kDa supernatant fraction. As would also be expected for a siderophore, the addition of 0.5 or 2.0 microM iron to the cultures repressed the expression of the CAS-reactive substance. Interestingly, the supernatants were negative in the Arnow, Csáky, and Rioux assays, indicating that the Legionella siderophore was not a classic catecholate or hydroxamate and, hence, might have a novel structure. We have designated the L. pneumophila siderophore legiobactin.     

Response of the cyanobacterium,Oscillatoria tenuis,to low iron environments: the effect on growth rate and evidence for siderophore production

Cyanobacteria vary in their ability to grow in media contaning low amounts of biologically available iron. Some strains, such as Oscillatoria tenuis, are well adapted to thrive in low-iron environments. We investigated the mechanism of iron scavenging in O. tenuis and found that this cyanobacterium has a siderophore-mediated iron transport system that differs significantly from the traditional hydroxamate-siderophore transport system reported from other cyanobacteria. Unlike other cyanobacteria, this strain produces two types of siderophores, a hydroxamate-type siderophore and a catechol-type siderophore. Production of these two siderophores is expressed at two different iron levels in the medium, suggesting two different iron regulated uptake systems. We compared the production of each siderophore with the growth rate of the culture and found that the production of the catechol siderophore enhances the growth rate of the cyanobacterium, whereas the cells maintain lower than maximal growth rates when only the hydroxamate-type siderophore is being produced.Abbreviation EDDA ethylene diamine di-(o-hydroxyphenylacetic acid)     

Effects of iron level on the anatagonistic action of siderophores from non-pathogenicStaphylococcus spp

The production, detection and the effects of iron concentration on the siderophore production ofStaphylococcus strains used as meat starter cultures were studied. Non-pathogenicStaphylococcus strains produce extracellular low molecular weight compounds which exhibited positive reactivity when measured by a universal detection method for siderophores. The production of siderophores was very closely associated with the iron concentration in the medium, and very low additions considerably reduced siderophore production. Although the production of siderophores was highly iron-dependent, the antimicrobial activity of spent medium fromStaphylococcus cultures against selected yeasts and moulds remained considerable under high iron concentrations.     

Alcaligin siderophore production by Bordetella bronchiseptica strain RB50 is not repressed by the BvgAS virulence control system

A previous study found that alcaligin siderophore production by Bordetella bronchiseptica strain RB50 is Bvg repressed. In contrast, we report that alcaligin production by RB50 does not require Bvg phenotypic phase modulation and that isogenic Bvg(Con) and Bvg(-) phase-locked mutants both produce alcaligin in response to iron starvation.     

Iron and pH‐responsive FtrABCD ferrous iron utilization system of Bordetella species

A putative operon encoding an uncharacterized ferrous iron transport (FtrABCD) system was previously identified in cDNA microarray studies. In growth studies using buffered medium at pH values ranging from pH 6.0 to 7.6, Bordetella pertussis and Bordetella bronchiseptica FtrABCD system mutants showed dramatic reductions in growth yields under iron‐restricted conditions at pH 6.0, but had no growth defects at pH 7.6. Supplementation of culture medium with 2 mM ascorbate reductant was inhibitory to alcaligin siderophore‐dependent growth at pH 7.6, but had a neglible effect on FtrABCD system‐dependent iron assimilation at pH 6.0 consistent with its predicted specificity for ferrous iron. Unlike Bordetella siderophore‐dependent and haem iron transport systems, and in agreement with its hypothesized role in transport of inorganic iron from periplasm to cytoplasm, FtrABCD system function did not require the TonB energy transduction complex. Gene fusion analysis revealed that ftrABCD promoter activity was maximal under iron‐restricted growth conditions at acidic pH. The pH of human airway surface fluids ranges from pH 5.5 to 7.9, and the FtrABCD system may supply ferrous iron necessary for Bordetella growth in acidic host microenvironments in which siderophores are ineffective for iron retrieval.     

Iron-dependent growth of and siderophore production by two heterotrophic bacteria isolated from brackish water of the southern Baltic Sea

Iron is indispensable to the growth and metabolism of all marine organisms, including bacteria. In this work, we investigated and compared the influence of iron(III) concentration on the growth of and siderophore production by two heterotrophic bacteria – Micrococcus luteus and Bacillus silvestris.Our results showed that the iron concentration strongly influences the growth of both species. The growth curves were different for each iron concentration and each strain. M. luteus grew more rapidly than B. silvestris, but produced a roughly four times smaller quantity of siderophores. Both M. luteus and B. silvestris secreted hydroxamate-type siderophores and α-keto/α-hydroxy acids, but did not produce catecholates.This paper is probably the first to report on siderophore production by B. silvestris and M. luteus isolated from seawater. Moreover, the influence of different iron concentrations on the growth of and siderophore production in these bacteria has been documented. This provides further evidence indicating iron bioavailability as the actual reason for siderophore release by biota.     

Characterization of ‘Schizokinen’; a dihydroxamate-type siderophore produced by Rhizobium leguminosarum IARI 917

The Rhizobia comprise one of the most important groups of beneficial bacteria, which form nodules on the roots (rarely on the stems) of leguminous plants. They live within the nodules and reduce atmospheric nitrogen to ammonia, which is further assimilated by plants into required nitrogenous compounds. The Rhizobia in return obtain nutrition from the plant. Rhizobia are free-living soil bacteria and have to compete with other microorganisms for the limited available iron in the rhizosphere. In order to acquire iron Rhizobia have been shown to express siderophore-mediated iron transport systems. Rhizobium leguminosarum IARI 917 was investigated for its ability to produce siderophore. It was found to produce a dihydroxamate type siderophore under iron restricted conditions. The siderophore was purified and chemically characterized. The ESMS, MS/MS and NMR analysis indicate the dihydroxamate siderophore to be ‘schizokinen’, a siderophore reported to be produced by Bacillus megaterium that shares a similar structure to ‘rhizobactin 1021’ produced by Sinorhizobium meliloti 1021. This is the first report of production of schizokinen by a strain of R. leguminosarum, therefore it was carefully investigated to confirm that it is indeed ‘schizokinen’ and not a degradation product of ‘rhizobactin 1021’. Since ferric–siderophore complexes are transported across the outer membrane (OM) into the periplasm via an OM receptor protein, R. leguminosarum IARI 917 was investigated for the presence of an OM receptor for ‘ferric–schizokinen’. SDS PAGE analysis of whole cell pellet and extracted OM fractions indicate the presence of a possible iron-repressible OM receptor protein with the molecular weight (MW) of approximately 74 kDa.     

Legionella pneumophila in the Environment: The Occurrence of a Fastidious Bacterium in Oligotrophic Conditions

Legionella pneumophila, a micro-organism encountered in aquatic environments, can cause serious intracellular infections among humans. Since the bacterium is ubiquitous in aquatic habitats, it appears to be impossible to prevent L. pneumophila from entering man-made water systems. However, many questions concerning the survival and/or growth in the environment, the partners and opponents of L. pneumophila remain unanswered. This review focuses on the factors governing the ecology of L. pneumophila, since there is considerable divergence and even contradiction in literature on its environmental requirements. A key question to be resolved is the discrepancy between the fastidious nature of L. pneumophila in axenic cultures (e.g. 400 mg l⁻¹ L-cysteine and 250 mg l^-1 ferric iron) and the nutritionally poor environments in which it is commonly detected. It is assumed that dense microbial communities, as occurring in sediments and biofilms – but not likely in surface and drinking water, – can provide the necessary growth requirements for L. pneumophila. However, most of the studies concerning L. pneumophila have led to the general opinion that the organism can only multiply in the aquatic environment as a parasite in certain protozoa. The discovery of the non-classical siderophore legiobactin also indicates that the iron requirement for survival and autonomous growth is not as high as has been assumed. It thus appears that in order to control Legionella in the environment, focus should be on the eradication of microbial hotspots in which L. pneumophila resides.     

Siderophore-mediated iron uptake in Alcaligenes eutrophus CH34 and identification of aleB encoding the ferric iron-alcaligin E receptor.

Siderophore production in response to iron limitation was observed in Alcaligenes eutrophus CH34, and the corresponding siderophore was named alcaligin E. Alcaligin E was characterized as a phenolate-type siderophore containing neither catecholate nor hydroxamate groups. Alcaligin E promoted the growth of siderophore-deficient A. eutrophus mutants under iron-restricted conditions and promoted 59Fe uptake by iron-limited cells. However, the growth of the Sid- mutant AE1152, which was obtained from CH34 by Tn5-Tc mutagenesis, was completely inhibited by the addition of alcaligin E. AE1152 also showed strongly reduced 59Fe uptake in the presence of alcaligin E. This indicates that a gene, designated aleB, which is involved in transport of ferric iron-alcaligin E across the membrane is inactivated. The aleB gene was cloned, and its putative amino acid sequence showed strong similarity to those of ferric iron-siderophore receptor proteins. Both wild-type strain CH34 and aleB mutant AE1152 were able to use the same heterologous siderophores, indicating that AleB is involved only in ferric iron-alcaligin E uptake. Interestingly, no utilization of pyochelin, which is also a phenolate-type siderophore, was observed for A. eutrophus CH34. Genetic studies of different Sid- mutants, obtained after transposon mutagenesis, showed that the genes involved in alcaligin E and ferric iron-alcaligin E receptor biosynthesis are clustered in a 20-kb region on the A. eutrophus CH34 chromosome in the proximity of the cys-232 locus.     

Purification, spectroscopic analysis and biological activity of the macrocyclic dihydroxamate siderophore alcaligin produced by Bordetella pertussis and Bordetella bronchiseptica

Hydroxamate siderophores of virulent Bordetella pertussis and Bordetella bronchiseptica strains were purified using a simple large-scale isolation procedure, and identified by various spectroscopic techniques as the macrocyclic dihydroxamate siderophore trivially known as alcaligin, 1,8(S),11,18(S)-tetrahydroxy-1,6,11,16-tetraazacycloeicosane-2,5,12,15-tetrone, which was previously isolated from the taxonomically-related bacterial species Alcaligenes denitrificans subsp. xylosoxydans. Alcaligin purified from iron-depleted cultures of B. pertussis and B. bronchiseptica exhibited specific growth-promoting activity under iron-restricted conditions for Bordetella indicator strains, and were active in [⁵⁵Fe]ferric alcaligin transport assays. Evidence suggests that several C ₂-symmetric conformations of alcaligin exist simultaneously in both methanolic and aqueous solution.     

Siderophore Cross-Utilization Amongst Rhizospheric Bacteria and the Role of Their Differential Affinities for Fe3+ on Growth Stimulation Under Iron-Limited Conditions

The majority of bacteria isolated from rhizospheres of Arachis hypogea (Groundnut) and Vigna radiata (Mung bean) predominantly produced catechol-type siderophores except for a few fluorescent pseudomonads that produced hydroxamates in addition to catecholates. The rhizospheric isolates differed in their ability to cross-utilize siderophores produced by other rhizospheric isolates (heterologous); some were highly proficient at utilizing heterologous siderophores, while others were poor cross-utilizers. Isolate G9, which utilized hydroxamate as well as catecholate siderophores, was found to be an efficient siderophore cross-utilizer, while isolates G2 and G6 were poor-utilizers of catecholate and non-utilizers of hydroxamate siderophores. Growth stimulation of two isolates G9 and G6 was seen when grown in the presence of externally supplied heterologous siderophores, which they cross-utilized. The iron-regulated outer membrane protein (IROMP) profiles differed for the most cross-utilizer and the least cross-utilizer strains, but in both the cases no new outer membrane proteins (OMP) were induced in response to the exogenous siderophores supplied. The growth of the organisms in the presence of heterologous siderophores that they failed to cross-utilize led to growth inhibition in the case of isolate G9. This appears to be due to a lower affinity of the siderophore of G9 as compared to the exogenously supplied G6 siderophore. A simple method was devised to measure relative affinities of respective siderophores for iron based on CAS solution decolorization by the siderophore preparations. The effect on the growth of the differential affinities of the siderophores for iron and the interactions of the organisms through cross-utilization is also discussed.     

Detection of siderophores in growing cultures ofPseudomonas spp.

Summary The siderophores produced byPseudomonas fluorescens andP. chlororaphis were detected from the culture supernatants in MM9 and modified King's medium by the universal CAS assay at wavelengths 620–690 nm. The CAS assay was applied to detectPseudomonas siderophores directly in situ, during their production phase, in modified King's medium. Optimum results were detected with a final CAS concentration of 0.025 mM and an iron concentration of 1.25 M. The problems of the method are discussed with respect to the absorbance spectrum, the toxicity of the HDTMA detergent, the influence of the iron concentration and the complexity of media for siderophore production.     

bvg Repression of alcaligin synthesis in Bordetella bronchiseptica is associated with phylogenetic lineage.

Recent studies have shown that Bordetella bronchiseptica utilizes a siderophore-mediated transport system for acquisition of iron from the host iron-binding proteins lactoferrin and transferrin. We recently identified the B. bronchiseptica siderophore as alcaligin, which is also produced by B. pertussis. Alcaligin production by B. bronchiseptica is repressed by exogenous iron, a phenotype of other microbes that produce siderophores. In this study, we report that alcaligin production by B. bronchiseptica RB50 and GP1SN was repressed by the Bordetella global virulence regulator, bvg, in addition to being Fe repressed. Modulation of bvg locus expression with 50 mM MgSO4 or inactivation of bvg by deletion allowed strain RB50 to produce alcaligin. In modulated organisms, siderophore production remained Fe repressed. These observations contrasted with our previous data indicating that alcaligin production by B. bronchiseptica MBORD846 and B. pertussis was repressed by Fe but bvg independent. Despite bvg repression of alcaligin production, strain RB50 was still able to acquire Fe from purified alcaligin, suggesting that expression of the bacterial alcaligin receptor was not repressed by bvg. We tested 114 B. bronchiseptica strains and found that bvg repression of alcaligin production was strongly associated with Bordetella phylogenetic lineage and with host species from which the organisms were isolated.     

High-performance liquid chromatography of siderophores from fungi

Summary A reversed-phase HPLC separation of iron(III) chelates of 16 representative fungal siderophores including ferrichromes, coprogens and triacetylfusarinine C was established in order to investigate siderophore production of fungi. For comparison purposes, the widely used bacterial siderophore ferrioxamine B was included. Culture filtrates of the fungiPenicillium resticulosum, Fusarium dimerum, Aspergillus fumigatus andNeurospora crassa were quantitatively analyzed for the presence of known and unknown siderophores after growth in low-iron culture media and adsorption on XAD-2 columns using this HPLC separation system. Photodiode array detection allowed the distinction between siderophores and non-siderophores. According to their ultraviolet/visible spectra, a further classification of the siderophores into four types due to the number of anhydromevalonic acid residues per molecule (0–3) was possible.     

Siderophore production and utilization byRhizobium trifolii

Summary Several strains ofRhizobium trifolii were tested for their ability to synthesize and utilize phenolate or hydroxamate types of siderophores. None of the nodulating strains ofR. trifolii was able to produce detectable amounts of siderophores. Only the non-nodulating strainR. trifolii AR6 formed a phenolate siderophore, which stimulated the growth of the siderophore-negative mutant AR65. Other strains ofR. trifolii could not utilize iron from exogenously supplied Desferal, pseudobactin or citrate. The siderophore fromR. trifolii AR6 and 2,3-dihydroxybenzoic acid slightly stimulated the growth of someR. trifolii strains.     

Growth and siderophore production inBradyrhizobium (lupin) strains under iron limitation

SixBradyrhizobium (lupin) strains were evaluated for their ability to produce siderophores using four chemical assays. Two strains gave positive reactions with chrome azurol S assay (CAS) and produced hydroxamate-type siderophores. The other four strains gave negative results for siderophore production using the four assays. Generation time, growth yield and hydroxamate production of one strain (WPBS 3201 D) were affected by the iron concentration of the culture medium and the previous culture history of the cells. Resuspension of washed cells grown previously in media supplemented with 0 and 20 μmol/L Fe into differing iron regimes (0, 0.5, 1, 2, 4, 8, 10, 15 and 20 μmol/L Fe) suggest that the extent of hydroxamate production depended on the growth history of the cells. Cells pregrown in 20 μmol/L Fe produced a high amount of hydroxamates compared with cells pregrown in iron-free medium when resuspended in medium containing up to 4 μmol/L Fe. Cells pregrown in 20 μmol/L Fe were more sensitive to iron repression than those pregrown in 0.5 μmol/L Fe. Mannitol was the best carbon source for siderophore production. Siderophore synthesis was inhibited by 4-chloromercuribenzenesulfonic acid, 2,4-dinitrophenol, sodium azide and MgCl₂ suggesting that an energized membrane and a mercapto group are essential and required for hydroxamate synthesis in strain WPB5 3201 D.     

Hydroxamate Production as a High Affinity Iron Acquisition Mechanism in Paracoccidioides Spp

Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity.