Fatty acids are essential components of almost all biological membranes. Additionally, they are important in energy storage, as second messengers during signal transduction, and in post-translational protein modification. De novo synthesis of fatty acids is essential for almost all organisms, and entails the iterative elongation of the growing fatty acid chain through a set of reactions conserved in all kingdoms. During our work on the biosynthesis of secondary metabolites, a 450-kDa protein was detected by SDS-PAGE of enriched fractions from mycelial lysates from the basidiomycete Omphalotus olearius. Protein sequencing of this protein band revealed the presence of peptides with homology to both α and β subunits of the ascomycete fatty acid synthase (FAS) family. The FAS encoding gene of O. olearius was sequenced. The positions of its predicted 21 introns were verified. The gene encodes a 3931 amino acids single protein, with an equivalent of the ascomycetous β subunit at the N-terminus and the α subunit at the C-terminus. This is the first report on an FAS protein from a homobasidiomycete and also the first fungal FAS which is comprised of a single polypeptide.

The plant pathogenic fungus Magnaporthe grisea excretes siderophores of the coprogen-type for iron acquisition and uses ferricrocin for intracellular iron storage. In the present report we characterize mutants with defects in extracellular siderophore biosynthesis. Deletion of the M. grisea SSM2 gene, which encodes a non-ribosomal peptide synthetase, resulted in a loss of the production of all coprogens. The mutant strains had a reduced growth rate, produced fewer conidia and were more sensitive to oxidative stress. Ferricrocin production was not affected. Upon deletion of M. grisea OMO1, a gene predicted to encode an L-ornithine-N(5)-monooxygenase, no siderophores of any type were detected, the strain was aconidial, growth rate was reduced and sensitivity to oxidative stress was increased. Abundance of several proteins was affected in the mutants. The Deltassm2 and Deltaomo1 mutant phenotypes were complemented by supplementation of the medium with siderophores or reintroduction of the respective genes.

SUMMARY Iron is an essential element for the growth of nearly all organisms. In order to overcome the problem of its low bioavailability, microorganisms (including fungi) secrete siderophores, high-affinity iron chelators. As the acquisition of iron is also a key step in infection processes, siderophores have been considered as potential virulence factors in several host-pathogen interactions. Most fungi produce siderophores of the hydroxamate-type, which are synthesized by non-ribosomal peptide synthetases (NRPSs). Magnaporthe grisea, the causal agent of rice blast disease, produces ferricrocin as intracellular storage siderophore and excretes coprogens. In the M. grisea genome we identified SSM1, an NRPS gene, and a gene encoding an l-ornithine N5-monooxygenase (OMO1) that is clustered with SSM1 and responsible for catalysing the first step in siderophore biosynthesis, the N(5) hydroxylation of ornithine. Disruption of SSM1 confirmed that the gene encodes ferricrocin synthetase. Pathogenicity of these mutants towards rice was reduced, suggesting a role of this siderophore in pathogenicity of M. grisea.

An analysis of siderophores produced by Magnaporthe grisea revealed the presence of one intracellular storage siderophore, ferricrocin, and four coprogen derivatives secreted into the medium under iron depletion. Structural analysis showed that the compounds are coprogen, coprogen B, 2-N-methylcoprogen and 2-N-methylcoprogen B. Siderophore production under low and high iron conditions was quantified.

K28 is a viral A/B toxin that traverses eukaryotic cells by endocytosis and retrograde transport through the secretory pathway. Here we show that toxin retrotranslocation from the endoplasmic reticulum (ER) requires Kar2p/BiP, Pdi1p, Scj1p, Jem1p, and proper maintenance of Ca(2+) homeostasis. Neither cytosolic chaperones nor Cdc48p/Ufd1p/Npl4p complex components or proteasome activity are required for ER exit, indicating that K28 retrotranslocation is mechanistically different from classical ER-associated protein degradation (ERAD). We demonstrate that K28 exits the ER in a heterodimeric but unfolded conformation and dissociates into its subunits as it emerges into the cytosol where beta is ubiquitinated and degraded. ER export and in vivo toxicity were not affected in a lysine-free K28 variant nor under conditions when ubiquitination and proteasome activity was blocked. In contrast, toxin uptake from the plasma membrane required Ubc4p (E2) and Rsp5p (E3) and intoxicated ubc4 and rsp5 mutants accumulate K28 at the cell surface incapable of toxin internalization. We propose a model in which ubiquitination is involved in the endocytic pathway of the toxin, while ER-to-cytosol retrotranslocation is independent of ubiquitination, ERAD and proteasome activity.

Under iron deprivation Omphalotus olearius was found to produce the hydroxamate siderophore ferrichrome A. A gene cluster consisting of three genes: fso1, a nonribosomal peptide synthetase whose expression is enhanced in the absence of iron; omo1, a l-ornithine-N5-monooxygenase; and ato1, an acyltransferase probably involved in the transfer of the methylglutaconyl residue to N5-hydroxyorinithine was identified. The fso1 sequence is interrupted by 48 introns and its derived protein sequence has a similar structure to the homologous genes of Ustilago maydis and Aspergillus nidulans. This is the first report of a nonribosomal peptide synthetase gene and a biosynthetic gene cluster in homobasidiomycetes.

Angewandte Molekularbiologie, Universita$ t des Saarlandes, FR 8.3, Geba$ ude 2, Postfach 151150, D-66041 Saarbru$ cken, Germany K28 killer strains of Saccharomyces cerevisiae are permanently infected with a cytoplasmic persisting dsRNA virus encoding a secreted α/β heterodimeric protein toxin that kills sensitive cells by cell-cycle arrest and inhibition of DNA synthesis. In vivo processing of the 345 aa toxin precursor (preprotoxin; pptox) involves multiple internal and carboxy-terminal cleavage events by the prohormone convertases Kex2p and Kex1p. By site-directed mutagenesis of the preprotoxin gene and phenotypic analysis of its in vivo effects it is now demonstrated that secretion of a biological active virus toxin requires signal peptidase cleavage after Gly36 and Kex2p-mediated processing at the α subunit N terminus (after Glu-Arg49), the α subunit C terminus (after Ser-Arg149) and at the β subunit N terminus (after Lys-Arg245). The mature C terminus of the β subunit is trimmed by Kex1p, which removes the terminal Arg345 residue, thus uncovering the toxin’s endoplasmic reticulum targeting signal (HDEL) which – in a sensitive target cell – is essential for retrograde toxin transport. Interestingly, both toxin subunits are covalently linked by a single disulfide bond between α-Cys56 and β-Cys340, and expression of a mutant toxin in which β-Cys340 had been replaced by Ser340 resulted in the secretion of a non-toxic α/β heterodimer that is blocked in retrograde transport and incapable of entering the yeast cell cytosol, indicating that one important in vivo function of β-Cys340 might be to ensure accessibility of the toxin’s β subunit C terminus to the HDEL receptor of the target cell.