Iron uptake by fungi: contrasted mechanisms with internal or external reduction.

Almost all iron uptake by fungi involves reduction from Fe(III) to Fe(II) in order to facilitate ligand exchange. This leads to two mechanisms: uptake before reduction, or reduction before uptake. Many fungi secrete specific hydroxamate siderophores when short of iron. The mechanism with uptake before reduction is described in the context of siderophore synthesis and usage, since it applies to many (but not all) siderophores. The hydroxamate functional group is synthesized from ornithine by N5 hydroxylation and acylation. In most fungal siderophores, two or three modified ornithines are joined together by a non-ribosomal peptide synthetase. The transcription of these genes is regulated by an iron activated repressor. There is evidence that the iron-free siderophore may be stored in intracellular vesicles until secretion is required. After loading with iron, re-entry is likely to be via a proton symport. In some fungi, siderophores are used for iron storage. The iron is liberated by an NADPH-linked reductase. The second mechanism starts with Fe(III) reduction. In yeast, this is catalysed by an NADPH-linked transmembrane reductase, which has homology with the NADPH oxidase of neutrophils. There are two closely similar reductases with overlapping roles in Fe(III) and Cu(II) reduction, while the substrates for reduction include Fe(III)-siderophores. External reductants, which may be important in certain fungi, include 3-hydroxyanthranilic acid, melanin, cellobiose dehydrogenase and 2,5-dimethylhydroquinone. In yeast, a high-affinity iron uptake pathway involves reoxidation of Fe(II) to Fe(III), probably to confer specificity for iron. This is catalysed by a copper protein which has homology with ceruloplasmin, and is closely coupled to Fe(III) transport. The transcription of these genes is regulated by an iron-inhibited activator. Because of its copper requirement, the high-affinity pathway is blocked by disruption of genes for copper metabolism. A low-affinity uptake transports Fe(II) directly and is important in anoxic growth. In many fungi, mechanisms with internal or external reduction are both important. The external reduction is applicable to almost any Fe(III) complex, while internal reduction is more efficient at low iron but requires a siderophore permease through which toxins might enter. Both mechanisms require close coupling of Fe(III) reduction and Fe(II) utilization in order to minimize production of active oxygen.

The purMN genes of Rhizobium leguminosarum and a superficial link with siderophore production.

We isolated a mutant of R. leguminosarum initially on the basis of reduced production of the siderophore vicibactin on chrome azurol sulfonate (CAS)/agar indicator plates. The mutation was in the purMN operon and the mutant was shown to be an adenine auxotroph and defective for nodulation of peas. The siderophore defect appears to be trivial, being due to diminished growth of the auxotroph on agar-based minimal medium, which contains unknown contaminant(s) that allow it grow poorly. Transcriptional fusions showed that purMN was transcribed at relatively high levels in media containing purines. Expression was enhanced, approximately twofold, if purines were omitted.

Is the fur gene of Rhizobium leguminosarum essential?

Using primers corresponding to conserved regions of the bacterial regulatory gene fur, a homologue of this gene from the genome of Rhizobium leguminosarum biovar viciae, the nitrogen-fixing symbiont of peas, was isolated and sequenced. The fur gene is normally expressed constitutively, independent of the presence of Fe in the medium, but in one Rhizobium strain it was transcribed at a low level. Attempts to isolate a fur knockout mutant failed, suggesting that the gene is essential for free-living growth. In other bacteria, certain fur mutations confer manganese resistance; however, none of the manganese-resistant mutants of R. leguminosarum which we isolated was corrected by the cloned fur gene. When the cloned R. leguminosarum fur gene was introduced into a fur mutant of Escherichia coli, it caused some Fe-dependent reduction in the amount of siderophore, indicating that it can function heterologously.

Expression of the exoY gene, required for exopolysaccharide synthesis in Agrobacterium, is activated by the regulatory ros gene.

Some mutants of Agrobacterium radiobacter, defective in exopolysaccharide synthesis, were phenotypically complemented by two different regions of cloned chromosomal DNA. One of these had been shown to contain a gene termed ros, a novel class of transcriptional regulator. The other contains a gene termed exoY which encodes a glycosyltransferase that is involved in one of the early steps in exopolysaccharide synthesis. Mutations in ros reduced the expression of exoY and a model to account for the complementation of certain exo alleles by both ros and exoY is presented. TnphoA insertions into exoY which expressed alkaline phosphatase activity were isolated and mapped, confirming the membrane location of the exoY gene product. Some of these mutations were dominant, causing merodiploids to be non-mucoid. exoY is linked to two genes, one encoding an omega-aminotransferase and the other encoding an aldehyde dehydrogenase.

The Rhizobium leguminosarum tonB gene is required for the uptake of siderophore and haem as sources of iron.

In the N2-fixing bacterium Rhizobium leguminosarum, mutations in a homologue of tonB (tonB(Rl)) block the import of vicibactin and haem as iron sources in free-living bacteria. TonB(Rl) mutants were normal for growth with ferric dicitrate and slightly reduced for growth with haemoglobin as sole iron sources. The deduced TonB(Rl) product is larger than that of (for example) Escherichia coli, on account of an extended N-terminal domain. Transcription of tonB(Rl) was enhanced in low-Fe growth conditions; this was not controlled by Fur, nor RpoI, an Fe-regulated extracytoplasmic sigma factor. Upstream of tonB(Rl) and transcribed divergently is an operon, hmuPSTUV, whose products are homologous to ABC transporters involved in haem uptake in pathogenic bacteria. Expression of hmuPSTUV was enhanced in low-Fe conditions, and hmu mutants show slightly diminished growth on haem as sole Fe source, suggesting that there is more than one system for the uptake of this molecule. hmuPSTUV expression appears to be from three closely linked promoters. Downstream of hmuPSTUV, a gene that may encode an extracytoplasmic sigma factor was identified, but this gene, rpoZ, did not affect the transcription of tonB(Rl) or hmuPSTUV. Mutations in tonB(Rl), hmu genes and rpoZ did not affect symbiotic N(2) fixation in peas.