UCA 1064-A

The food- and airborne fungal genus Wallemia comprises seven xerophilic and halophilic species: W. sebi, W. mellicola, W. canadensis, W. tropicalis, W. muriae, W. hederae and W. ichthyophaga. All listed species are adapted to low water activity and can contaminate food preserved with high amounts of salt or sugar. In relation to food safety, the effect of high salt and sugar concentrations on the production of secondary metabolites by this toxigenic fungus was investigated. The secondary metabolite profiles of 30 strains of the listed species were examined using general growth media, known to support the production of secondary metabolites, supplemented with different concentrations of NaCl, glucose and MgCl2. In more than two hundred extracts approximately one hundred different compounds were detected using high-performance liquid chromatography-diode array detection (HPLC-DAD). Although the genome data analysis of W. mellicola (previously W. sebi sensu lato) and W. ichthyophaga revealed a low number of secondary metabolites clusters, a substantial number of secondary metabolites were detected at different conditions. Machine learning analysis of the obtained dataset showed that NaCl has higher influence on the production of secondary metabolites than other tested solutes. Mass spectrometric analysis of selected extracts revealed that NaCl in the medium affects the production of some compounds with substantial biological activities (wallimidione, walleminol, walleminone, UCA 1064-A and UCA 1064-B). In particular an increase in NaCl concentration from 5% to 15% in the growth media increased the production of the toxic metabolites wallimidione, walleminol and walleminone.

The sterol biosynthesis pathway of Arabidopsis produces a large set of structurally related phytosterols including sitosterol and campesterol, the latter being the precursor of the brassinosteroids (BRs). While BRs are implicated as phytohormones in post-embryonic growth, the functions of other types of steroid molecules are not clear. Characterization of the fackel (fk) mutants provided the first hint that sterols play a role in plant embryogenesis. FK encodes a sterol C-14 reductase that acts upstream of all known enzymatic steps corresponding to BR biosynthesis mutants. Here we report that genetic screens for fk-like seedling and embryonic phenotypes have identified two additional genes coding for sterol biosynthesis enzymes: CEPHALOPOD (CPH), a C-24 sterol methyl transferase, and HYDRA1 (HYD1), a sterol C-8,7 isomerase. We describe genetic interactions between cph, hyd1 and fk, and studies with 15-azasterol, an inhibitor of sterol C-14 reductase. Our experiments reveal that FK and HYD1 act sequentially, whereas CPH acts independently of these genes to produce essential sterols. Similar experiments indicate that the BR biosynthesis gene DWF1 acts independently of FK, whereas BR receptor gene BRI1 acts downstream of FK to promote post-embryonic growth. We found embryonic patterning defects in cph mutants and describe a GC-MS analysis of cph tissues which suggests that steroid molecules in addition to BRs play critical roles during plant embryogenesis. Taken together, our results imply that the sterol biosynthesis pathway is not a simple linear pathway but a complex network of enzymes that produce essential steroid molecules for plant growth and development.

Leishmania donovani promastigotes were cultured in the presence of an azasterol (20-piperidin-2-yl-5 alpha-pregnane-3 beta,20-diol) to determine the effects on sterol biosynthesis and cell proliferation. Inhibition of growth increased gradually with azasterol concentrations up to 5 micrograms/ml; concentrations of azasterol exceeding 5 micrograms/ml were lethal. Sterol biosynthesis was affected by the azasterol when administered at concentrations as low as 100 pg/ml. The primary site of action was the alkylation at C-24 of a delta 24-sterol precursor. The 24-alkylated sterols [ergosta-5,7,24(24(1))-trien-3 beta-ol and ergosta-5,7,22-trien-3 beta-ol] of the protozoan were replaced by delta 24-cholesta-type sterols which then accumulated in the cells. Administration of the azasterol together with a bis-triazole inhibitor of the 14 alpha-methylsterol 14-demethylase reaction, which operates in sterol biosynthesis, resulted in depletion of 24-alkylsterols and their replacement with predominantly 14 alpha-methylsterols lacking a 24-alkyl group. Continuous subculture of promastigotes in the presence of the azasterol resulted in gradual depletion of 24-alkylsterols and their complete replacement by delta 24-cholesta-type sterols. Transfer of the azasterol-treated cells to medium lacking azasterol resulted in a gradual restoration, after several subcultures, of the normal 24-alkylsterol pattern. The results indicate that, although 24-alkylsterols are normally produced by the protozoan, it can nevertheless survive with sterols possessing only the cholestane skeleton. Thus there is no absolute requirement for 24-alkylsterols to fulfil some essential 'sparking' role associated with cell growth in promastigotes.