Spore Germination Requires Ferrichrome Biosynthesis and the Siderophore Transporter Str1 in Schizosaccharomyces pombe.

Spore germination is a process whereby spores exit dormancy to become competent for mitotic cell division. In Schizosaccharomyces pombe, one critical step of germination is the formation of a germ tube that hatches out the spore wall in a stage called outgrowth. Here, we show that iron deficiency blocks the outgrowth of germinating spores. The siderophore synthetase Sib1 and the ornithine N5-oxygenase Sib2 participate in ferrichrome biosynthesis, whereas Str1 functions as a ferrichrome transporter. Expression profiles of sib1+ , sib2+ , and str1+ transcripts reveal that they are induced shortly after induction of germination and their expression remains upregulated throughout the germination program under low-iron conditions. sib1Δ sib2Δ mutant spores are unable to form a germ tube under iron-poor conditions. Supplementation with exogenous ferrichrome suppresses this phenotype when str1+ is present. Str1 localizes at the contour of swollen spores 4 hr after induction of germination. At the onset of outgrowth, localization of Str1 changes and it moves away from the mother spore to primarily localize at the periphery of the new daughter cell. Two conserved Tyr residues (Tyr553 and Tyr567) are predicted to be located in the last extracellular loop region of Str1. Results show that these amino acid residues are critical to ensure timely completion of the outgrowth phase of spores in response to exogenous ferrichrome. Taken together, the results reveal the essential requirement of ferrichrome biosynthesis to promote outgrowth, as well as the necessity to take up ferrichrome from an external source via Str1 when ferrichrome biosynthesis is blocked.

A Branch Point of Streptomyces Sulfur Amino Acid Metabolism Controls the Production of Albomycin.

Albomycin (ABM), also known as grisein, is a sulfur-containing metabolite produced by Streptomyces griseus ATCC 700974. Genes predicted to be involved in the biosynthesis of ABM and ABM-like molecules are found in the genomes of other actinomycetes. ABM has potent antibacterial activity, and as a result, many attempts have been made to develop ABM into a drug since the last century. Although the productivity of S. griseus can be increased with random mutagenesis methods, understanding of Streptomyces sulfur amino acid (SAA) metabolism, which supplies a precursor for ABM biosynthesis, could lead to improved and stable production. We previously characterized the gene cluster (abm) in the genome-sequenced S. griseus strain and proposed that the sulfur atom of ABM is derived from either cysteine (Cys) or homocysteine (Hcy). The gene product, AbmD, appears to be an important link between primary and secondary sulfur metabolic pathways. Here, we show that propargylglycine or iron supplementation in growth media increased ABM production by significantly changing the relative concentrations of intracellular Cys and Hcy. An SAA metabolic network of S. griseus was constructed. Pathways toward increasing Hcy were shown to positively impact ABM production. The abmD gene and five genes that increased the Hcy/Cys ratio were assembled downstream of hrdBp promoter sequences and integrated into the chromosome for overexpression. The ABM titer of one engineered strain, SCAK3, in a chemically defined medium was consistently improved to levels ∼400% of the wild type. Finally, we analyzed the production and growth of SCAK3 in shake flasks for further process development.

Optimization of triacetylfusarinine C and ferricrocin productions in Aspergillus fumigatus.

Iron is an essential element for all microorganisms. Bacteria and fungi produce versatile siderophores for binding and storing this essential transition metal when its availability is limited in the environment. The aim of the study was to optimize the fermentation medium of Aspergillus fumigatus for siderophore production. Triacetyl-fusarinine C and ferricrocin yields were dependent on glucose and glycine supplementations as well as the initial pH of the culture media. The optimal fermentation medium for triacetylfusarinine C production contained 8% glucose, 0.4% glycine and the initial pH was set to 5.9. Meanwhile, maximal ferricrocin yields were recorded in the presence of 10% glucose, 0.5% glycine and at an initial pH of 7.4. Under optimized fermentation conditions, the yields for triacetylfusarinine C and ferricrocin increased up to 2.9 g/l culture medium and 18.9 mg/g mycelium, respectively.

Albomycin is an effective antibiotic, as exemplified with Yersinia enterocolitica and Streptococcus pneumoniae.

Albomycin belongs to the class of sideromycins, compounds composed of iron carriers linked to antibiotic moieties. Albomycin was found to be active against bacteria that have a functional ferric hydroxamate transport system meaning that bacteria will actively transport albomycin until they die. We examined the activity spectrum of albomycin for bacterial pathogens and found that Enterobacteriaceae except species of Proteus and Morganella were sensitive. Resistance in the two genera was due to the lack of the ferric hydroxamate transport system. Among Gram-positive bacteria, Staphylococcus aureus and Streptococcus pneumoniae were highly sensitive, whereas Streptococcus agalactiae, Streptococcus pyogenes, and Staphylococcus epidermidis were resistant. The in vivo efficacy of albomycin was examined in mice infected with S. pneumoniae or Yersinia enterocolitica. A single dose of 10mg albomycin/kg body weight reduced the colony-forming units of Y. enterocolitica by three to four orders of magnitude. A single dose of 1mg albomycin/kg body weight was sufficient to clear S. pneumoniae infections in mice. In direct competition experiments with wild-type S. pneumoniae and its albomycin-resistant mutant, the recovery rate of the mutant was lower than for the wild-type indicating that the mutant had reduced fitness in the mouse model. We conclude that albomycin is effective in clearing infections caused by both Gram-positive and Gram-negative bacteria in a mouse model. Albomycin treatment reduces the bacterial load allowing the immune system to remove residual albomycin-resistant bacteria, and as such would make albomycin-based antibiotics an adjunct to treatment. The ferrichrome transport system serves as a Trojan horse to get albomycin into bacteria.

The Aspergillus fumigatus siderophore biosynthetic gene sidA, encoding L-ornithine N5-oxygenase, is required for virulence.

Aspergillus fumigatus is the leading cause of invasive mold infection and is a serious problem in immunocompromised populations worldwide. We have previously shown that survival of A. fumigatus in serum may be related to secretion of siderophores. In this study, we identified and characterized the sidA gene of A. fumigatus, which encodes l-ornithine N(5)-oxygenase, the first committed step in hydroxamate siderophore biosynthesis. A. fumigatus sidA codes for a protein of 501 amino acids with significant homology to other fungal l-ornithine N(5)-oxygenases. A stable DeltasidA strain was created by deletion of A. fumigatus sidA. This strain was unable to synthesize the siderophores N',N",N'''-triacetylfusarinine C (TAF) and ferricrocin. Growth of the DeltasidA strain was the same as that of the wild type in rich media; however, the DeltasidA strain was unable to grow in low-iron defined media or media containing 10% human serum unless supplemented with TAF or ferricrocin. No significant differences in ferric reduction activities were observed between the parental strain and the DeltasidA strain, indicating that blocking siderophore secretion did not result in upregulation of this pathway. Unlike the parental strain, the DeltasidA strain was unable to remove iron from human transferrin. A rescued strain (DeltasidA + sidA) was constructed; it produced siderophores and had the same growth as the wild type on iron-limited media. Unlike the wild-type and rescued strains, the DeltasidA strain was avirulent in a mouse model of invasive aspergillosis, indicating that sidA is necessary for A. fumigatus virulence.

4'-phosphopantetheinyl transferase-encoding npgA is essential for siderophore biosynthesis in Aspergillus nidulans.

Aspergillus nidulans produces two major siderophores: it excretes triacetylfusarinine C to capture iron and contains ferricrocin as an intracellular iron-storage compound. Siderophore biosynthesis involves the enzymatic activity of nonribosomal peptide synthetases (NRPS). NRPS contain 4'-phosphopantetheine as an essential prosthetic group, which is attached by 4'-phosphopantetheinyl transferases. A. nidulans appears to possess at least one gene, npgA, encoding such an enzyme. Using a strain carrying a temperature-sensitive allele, cfwA2, we showed that NpgA is essential for biosynthesis of both the peptide bond-containing ferricrocin and the ester bond-containing triacetylfusarinene C. The cfwA2 strain was found to be iron-starved at the restrictive temperature during iron-replete conditions, consistent with the siderophore system being the major iron-uptake system-as we recently demonstrated. Northern analysis indicated that, in contrast to other genes which are involved in siderophore biosynthesis and uptake, expression of npgA is not controlled by the GATA-transcription factor SreA. It was shown previously that NpgA is required for biosynthesis of penicillin, pigment, and potentially lysine via the alpha-aminoadipate pathway. Supplementation with lysine plus triacetylfusarinine C restored normal growth of the cfwA2 strain at the restrictive temperature, suggesting that the growth defect of the mutant is mainly due to impaired biosynthesis of siderophores and lysine.

The siderophore system is essential for viability of Aspergillus nidulans: functional analysis of two genes encoding l-ornithine N 5-monooxygenase (sidA) and a non-ribosomal peptide synthetase (sidC).

The filamentous ascomycete A. nidulans produces two major siderophores: it excretes triacetylfusarinine C to capture iron and contains ferricrocin intracellularly. In this study we report the characterization of two siderophore biosynthetic genes, sidA encoding l-ornithine N(5)-monooxygenase and sidC encoding a non-ribosomal peptide synthetase respectively. Disruption of sidC eliminated synthesis of ferricrocin and deletion of sidA completely blocked siderophore biosynthesis. Siderophore-deficient strains were unable to grow, unless the growth medium was supplemented with siderophores, suggesting that the siderophore system is the major iron assimilatory system of A. nidulans during both iron depleted and iron-replete conditions. Partial restoration of the growth of siderophore-deficient mutants by high concentrations of Fe(2+) (but not Fe(3+)) indicates the presence of an additional ferrous transport system and the absence of an efficient reductive iron assmilatory system. Uptake studies demonstrated that TAFC-bound iron is transferred to cellular ferricrocin whereas ferricrocin is stored after uptake. The siderophore-deficient mutant was able to synthesize ferricrocin from triacetylfusarinine C. Ferricrocin-deficiency caused an increased intracellular labile iron pool, upregulation of antioxidative enzymes and elevated sensitivity to the redox cycler paraquat. This indicates that the lack of this cellular iron storage compound causes oxidative stress. Moreover, ferricrocin biosynthesis was found to be crucial for efficient conidiation.

Actinobacillus pleuropneumoniae serotype 7 siderophore receptor FhuA is not required for virulence.

A ferrichrome receptor, FhuA, was identified in Actinobacillus pleuropneumoniae serotype 7. An isogenic mutant with a deletion in the ferrichrome uptake receptor gene (fhuA) was constructed and examined in an aerosol infection model. The disease caused by the mutant was indistinguishable from disease induced by A. pleuropneumoniae serotype 7 wild-type; an isogenic mutant lacking expression of the exbB gene that is required for the uptake of transferrin-bound iron retained the ability to utilize ferrichrome, thereby indicating that an energy-coupling mechanism involved in ferrichrome transport remains to be identified.

Bioassay for siderophore utilization by Candida albicans.

A convenient plate assay which is sensitive to medium pH has been developed to evaluate potential siderophores of Candida albicans. Adding a siderophore to a filter paper disk on chemically defined Lee's agar (final pH 7.2) seeded with the test strain reversed the growth inhibitory effects of the supplemented (25-100 micrograms/ml) iron chelator ethylenediaminedi(o-hydroxyphenylacetic acid), to provide a zone of growth stimulation. This bioassay has been used to demonstrate the structure-activity relationships of ferrichrome and several water-soluble hydroxamate peptide building blocks of this natural siderophore. Of all compounds so evaluated, ferrichrome exhibited the best activity.