Synthetic dyes decolourisation by white-rot fungi: Development of original microtitre plate method and screening.

A microtitre plate-based method was developed for a fast screening of numerous fungal strains for their ability to decolourise textile dyes. In 3 days, this method allowed to estimate significant fungal decolourisation capability by measuring the absorbance decrease on up to ten dyes. More than 325 white-rot fungi (WRF) strains belonging to 76 fungal genera were compared with regards to their capability to decolourise five azo and two anthraquinone dyes as well as the dyes mixture. The most recalcitrant dyes belonged to the azo group. Several new species unstudied in the bioremediation field were found to be able to efficiently decolourise all the dyes tested.

Structural and functional analysis of deficient mutants in subunit I of cytochrome c oxidase from Saccharomyces cerevisiae.

Four point mutations in subunit I of cytochrome c oxidase from Saccharomyces cerevisiae that had been selected for respiratory incompetence but still contained spectrally detectable haem aa3 were analysed. The isolated mutant enzymes exhibited minor band shifts in their optical spectra and contained all eleven subunits. However, steady state activities were only a few percent compared to wild type enzyme. Using a comprehensive experimental approach, we first checked the integrity of the enzyme preparations and then identified the specific functional defect. The results are discussed using information from the recently solved structures of cytochrome c oxidase at 2.8 A. Mutation 167N is positioned between haem a and a conserved glutamate residue (E243). It caused a distortion of the EPR signal of haem a and shifted its midpoint potential by 54 mV to the negative. The high-resolution structure suggests that the primary reason for the low activity of the mutant enzyme could be that asparagine in position 67 might form a stable hydrogen bond to E243, which is part of a proposed proton channel. Cytochrome c oxidase isolated from mutant T316K did not meet our criteria for homogeneity and was therefore omitted from further analysis. Mutants G352V and V380M exhibited an impairment of electron transfer from haem a to a3 and ligand binding to the binuclear centre was affected. In mutant V380M also the midpoint potential of CuB was shifted by 65 mV to the positive. The results indicated for these two mutants changes primarily associated with the binuclear centre, possibly associated with an interference in the routes and/or sites of protonation which are required for stable formation of the catalytic intermediates. This interpretation is discussed in the light of the high resolution structure.

Functional mapping reveals the importance of yeast cytochrome b C-terminal region in assembly and function of the bc1 complex.

Genetic and molecular analyses have been undertaken for four respiratory deficient mutants (mit-). The four mutations affect the C-terminal region of apocytochrome b. The frameshift (L263STOP) and non-sense (Q338STOP) mutations give rise to a truncated apocytochrome b. The mutant G337R conserves only 32% of its NADH oxidase activity which suggests that the presence of a positively charged amino acid in the transmembranous helix 7 of cytochrome b alters, either directly or indirectly, the bc1 function, without affecting its assembly. The mutation G352V has a 65% loss of cytochrome b spectral content and prevents all of the mitochondrial respiratory activity. This leads us to believe that the glycine, conserved in position 352, may play a crucial role in bc1 complex function.

Endophytic fungi from leaves of Centella asiatica: occurrence and potential interactions within leaves.

Fungal endophytes were isolated from leaves of Centella asiatica (Apiaceae) collected at Mangoro (middle eastern region of Madagascar, 200 km from Antananarivo). Forty- five different taxa were recovered. The overall foliar colonization rate was 78%. The most common endophytes were the non-sporulating species 1 (isolation frequency IF 19.2%) followed by Colletotrichum sp.1 (IF 13.2%), Guignardia sp. (IF 8.5%), Glomerella sp. (IF 7.7%), an unidentified ascomycete (IF 7.2%), the non-sporulating species 2 (IF 3.7%) and Phialophora sp. (IF 3.5%). Using sequences of the ribosomal DNA internal transcribed spacer (ITS) regions, major endophytes (IF > 7%) were identified as xylariaceous taxa or as Colletotrichum higginsianum, Guignardia mangiferae and Glomerella cingulata. Results from in vitro fungal disk experiments showed a strong inhibitory activity of the xylariaceous non-sporulating species 1 against G. mangiferae and C. higginsianum and of C. higginsianum against G. mangiferae. This can be explained by antagonism between dominant taxa.

Nuclearly inherited diuron-resistant mutations conferring a deficiency in the NADH--or succinate--ubiquinone oxidoreductase activity in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, diuron, antimycin and myxothiazol block the respiratory pathway at the bc1 complex level. Nuclearly inherited mutations located at the DIU3 and DIU4 loci confer in vitro resistance to diuron and cross-resistance to antimycin and myxothiazol at the NADH oxidase level. The mutant strains do not exhibit diuron resistance at the quinol-cytochrome-c oxidoreductase level. Thus, the apparent resistance does not seem to be the result of a modification of the inhibitory sites. Instead, the quinone reduction rate was found to be altered in the mutant. The diu3 mutations lead to a deficiency of the NADH--ubiquinone oxidoreductase activity, and the diu4 mutations to a deficiency of the succinate--ubiquinone oxidoreductase activity. On the basis of the model of Kröger and Klingenberg, a decrease of quinone reduction could explain the resistance to the bc1 complex inhibitors. Thus, the apparent resistance to the bc1 complex inhibitors was found to be due to a modification of the electron transfer kinetics.