Biomedical subjects

M Fèvre

Publications and source records attributed to M Fèvre.

At least 19 recordsLinked to original sources

PubMed2003

Characterization of a SNF1 homologue from the phytopathogenic fungus Sclerotinia sclerotiorum.

In yeast, the SNF1 gene product is essential for the release of catabolic repression. We report the isolation and characterization of an SNF1 homologue from the necrotrophic pathogen Sclerotinia sclerotiorum. Ss snf1 encodes a 765-amino-acid protein in which the catalytic domain has an overall identity with the yeast proteins varying from 55 to 76% while the C-terminal half of Ss SNF1 has a weak homology of about 20% with the yeast sequences. Reverse transcription-polymerase chain reaction showed that its transcripts were weakly and constitutively expressed in planta and in vitro regardless of the nature of the carbon sources and of the presence or absence of glucose. Expression of Ss snf1 in yeast cells allowed the snf1 mutant cells to utilize sucrose, raffinose or glycerol for growth while expression of the Ss snf1 catalytic domain did not restore growth on raffinose or glycerol. Ss SNF1 is structurally homologous to Snf1p, suggesting that the interactions between the kinase and the accessory subunits to activate the enzymatic complex are conserved in fungi.

Amino Acid Sequence↗

PubMed2002

Purification and characterization of a glucoamylase secreted by the plant pathogen Scierotinia sclerotiorum.

Among the lytic enzymes secreted by the phytopathogen fungus Sclerotinia sclerotiorum, a starch-degrading enzyme has been isolated and characterized. This glycoprotein of 72 kDa is composed of several isoforms ranging from pI 4.8 to 5.4. The enzymatic parameters have been determined. Specificity studies together with the analysis of the reaction products show that it is an alpha-1,4-glucanohydrolase. This result is also corroborated by the analysis of the N-terminal and two inner amino acids sequences that are very similar to fungal glucoamylase genes or enzymes so far sequenced.

Amino Acid Sequence↗

PubMed2001

aspS encoding an unusual aspartyl protease from Sclerotinia sclerotiorum is expressed during phytopathogenesis.

The gene aspS encoding an aspartyl protease has been cloned from Sclerotinia sclerotiorum by screening a genomic library with a PCR-amplified fragment of the gene. The open reading frame of 1368 bp interrupted by one intron would encode a preproprotein of 435 amino acids. The catalytic aspartyl residues characteristic of aspartyl proteases are conserved; however, the active-site motif (DSG) in the N-terminal lobe is unusual in that Ser replaced Thr used in the active-site motif (DTG) of the C-terminal lobe and in all other fungal aspartyl proteases. RT-PCR revealed that aspS expression in axenic culture is not subjected to catabolite repression and demonstrated that aspS is expressed from the beginning of infection of sunflower cotyledons.

Amino Acid Sequence↗

PubMed2000

Mutation of a putative AMPK phosphorylation site abolishes the repressor activity but not the nuclear targeting of the fungal glucose regulator CRE1.

In filamentous ascomycetes, glucose repression is mediated by CRE1, a zinc-finger protein related to Miglp from yeast. Five putative AMPK phosphorylation motifs identified in the glucose repressor from the phytopathogenic fungus Sclerotinia sclerotiorum were mutated in a GFP::CRE1 translational fusion. Complementation experiments in Aspergillus nidulans and fluorescence microscopy analyses showed that mutation of one site (Ser266) abolishes the repressor activity of the fusion protein but not its nuclear targeting, suggesting that an AMPK protein kinase may be involved in the function of the fungal glucose repressor.

AMP-Activated Protein Kinases↗

PubMed1999

The glucose repressor CRE1 from Sclerotinia sclerotiorum is functionally related to CREA from Aspergillus nidulans but not to the Mig proteins from Saccharomyces cerevisiae.

We isolated the putative glucose repressor gene cre1 from the phytopathogenic fungus Sclerotinia sclerotiorum. cre1 encodes a 429 amino acid protein 59% similar to the carbon catabolite repressor CREA from Aspergillus nidulans. In addition to the overall amino acid sequence relatedness between CRE1 and CREA proteins, cre1 can functionally complement the A. nidulans creAd30 mutation as assessed by repression of the alcohol dehydrogenase I gene expression. The CREI region carrying the two zinc fingers is also very similar to the DNA binding domains of the Saccharomyces cerevisiae glucose repressors Mig1p and Mig2p. Despite the presence in the CRE1 protein of several motifs involved in the regulation of Miglp activity, cre1 cannot complement mig deficiencies in S. cerevisiae. These data suggest that glucose repression pathways may have evolved differently in yeasts and filamentous fungi.

Amino Acid Sequence↗

PubMed1999

Isolation and expression of a nitrogen regulatory gene, nmc, of Penicillium roqueforti.

The nmc gene, encoding a global nitrogen regulator, has been cloned and characterized from Penicillium roqueforti, a fungus used in the dairy industry. The deduced amino acid sequence predicts a protein of 860 amino acids in length whose zinc finger DNA binding domain is at least 94% identical to those of the homologous fungal proteins. Northern blot analysis showed that nmc expression is induced by nitrogen starvation and not repressed by variation of the external pH.

Amino Acid Sequence↗

PubMed1999

Expression of a catalytic domain of a Neocallimastix frontalis endoxylanase gene (xyn3) in Kluyveromyces lactis and Penicillium roqueforti.

A cDNA fragment encoding the A catalytic domain of the Neocallimastix frontalis endoxylanase XYN3 was amplified and cloned by the polymerase chain reaction technique. The xyn3A DNA fragment was inserted between the Saccharomyces cerevisiae phosphoglycerate kinase gene promoter and terminator sequences on a multicopy episomal plasmid for Kluyveromyces lactis. The XYN3A domain was successfully expressed in K. lactis and functional endoxylanase was secreted by the yeast cells with the K. lactis killer toxin secretion signal. The XYN3A domain was also expressed in a strain of Penicillium roqueforti as a fusion protein (ShBLE::XYN3A) of the phleomycin-resistance gene product and the endoxylanase. Active endoxylanase was efficiently secreted from the fungal cells with the Trichoderma viride cellobiohydrolase (CBH1) secretion signal and processed by a related KEX2 endoprotease of the secretion pathway. Several differently glycosylated forms of the recombinant enzymes were secreted by the yeast and the filamentous fungus.

Amino Acid Sequence↗

PubMed1999

Expression and compartmentation of the glucose repressor CRE1 from the phytopathogenic fungus Sclerotinia sclerotiorum.

The glucose repressor from the phytopathogenic fungus Sclerotinia sclerotiorum is encoded by the cre1 gene. Polyclonal antibodies were raised against a fusion protein (gluthathione S-transferase) GST-CRE1 in order to study cre1 expression. Western blot analyses revealed that CRE1 synthesis is regulated by the nature of the extracellular carbon source. High CRE1 levels are induced by glucose and remain stable after transfer into pectin medium, suggesting the existence of post-translational mechanisms which inactivate CRE1 to allow transcription of glucose-repressed genes. Subcellular fractionation demonstrated that CRE1 is localized in the nuclei of glucose grown hyphae and in the cytoplasm when glucose is removed from the culture medium. CRE1 fused to green fluorescent protein (GFP) was introduced into Aspergillus nidulans. Fluorescence microscopy showed the nuclear localization of the GFP-CRE1 fusion protein according to the presence of glucose in the culture medium, suggesting homologous post-translational regulations of glucose repressors in fungi. We propose that filamentous fungi regulate the activity of the glucose repressor by controlling its nuclear translocation.

Ascomycota↗

PubMed1998

Multiplicity and expression of xylanases in the rumen fungus Neocallimastix frontalis.

The time course production of xylanolytic enzymes by the rumen anaerobic fungus Neocallimastix frontalis was studied during growth on different carbon sources and revealed using isoelectric focusing and immunoblotting. A constant low level of endoxylanase expression was observed in glucose medium. A high level of xylanase activity was detected in methyl glucoside medium corresponding to the induction of new isoforms which were repressed by the presence of glucose. beta-Xylosidases were constitutively produced at a high level and remained mainly associated to the fungal cells. Polyclonal antibodies raised against the endoxylanases XYLI and XYLII revealed that XYLI was secreted to the different culture media showing a characteristic pattern of constitutive expression, while anti-XYLII recognized several polypeptides larger than XYLII indicating the production of multiple antigenically related enzymes during growth on the inducing substrate.

Animals↗

PubMed1998

Purification and characterization of two endopolygalacturonases secreted during the early stages of the saprophytic growth of Sclerotinia sclerotiorum.

Two endopolygalacturonases (endo-PGs) secreted at the early stage of cultures of Sclerotinia sclerotiorum grown on polygalacturonate medium, were purified to apparent homogeneity, using ion exchange chromatography. They are glycoproteins of an apparent weight of 42 and 41.5 kDa and a pI of 4.8. The two purified isoforms found in early cultures were not detected in late cultures. Purification of the isoforms secreted at different stages of growth revealed that the increase of polygalacturonase activity during the culture corresponds to a sequential production of enzymes and to the successive replacement of isoforms by new enzymes.

Ascomycota↗

PubMed1997

Transient expression of the beta-glucuronidase gene after biolistic transformation of the anaerobic fungus Neocallimastix frontalis.

The rumen anaerobic fungus Neocallimastix frontalis was biolistically transformed using plasmids containing the bacterial beta-glucuronidase gene (GUS) fused to the promoter sequences of the enolase gene from N. frontalis. Multiple copies of the plasmids were precipitated onto tungsten particles and delivered into zoosporangia and a mycelial mat by a helium-driven biolistic device. Transformants were detected by histochemical assay for beta-glucuronidase. It was found that the enolase promoter sequences tested were responsible for the transient expression of the beta-glucuronidase gene. This is the first study presenting results on the transformation of an anaerobic fungus.

Anaerobiosis↗

PubMed1997

Transformation of the oomycete Saprolegnia monoïca to hygromycin-B resistance.

Saprolegnia monoïca was stably transformed to hygromycin resistance using the plasmids pTH210 and pHAM34H containing a bacterial phosphotransferase gene fused to regulatory sequences from genes of another Oomycete. Vectors pBT6, pCM54, used to transform higher fungi, yielded no stable transformants. DNA hybridizations indicated that transformation resulted from a single-copy integration of the transforming vector. Development of non-resistant subcultures from transformed colonies revealed that the transgene could become silent and was not uniformly expressed in the transformants.

DNA, Fungal↗

PubMed1997

A mitochondrial-like targeting signal on the hydrogenosomal malic enzyme from the anaerobic fungus Neocallimastix frontalis: support for the hypothesis that hydrogenosomes are modified mitochondria.

The hydrogenosomal malic enzyme (ME) was purified from the anaerobic fungus Neocallimastix frontalis. Using reverse genetics, the corresponding cDNA was isolated and characterized. The deduced amino acid sequence of the ME showed high similarity to ME from metazoa, plants and protists. Putative functional domains for malate and NAD+/NADP+ binding were identified. Phylogenetic analysis of the deduced amino acid sequence of the new ME suggests that it is homologous to reference bacterial and eukaryotic ME. Most interestingly, the cDNA codes for a protein which contains a 27-amino-acid N-terminus which is not present on the purified mature protein. This presequence shares features with known mitochondrial targeting signals, including an enrichment in Ala, Leu, Ser, and Arg, and the presence of an Arg at position-2 relative to amino acid 1 of the mature protein. This is the first report of a mitochondrial-like targeting signal on a hydrogenosomal enzyme from an anaerobic fungus and provides support for the hypothesis that hydrogenosomes in Neocallimastix frontalis might be modified mitochondria.

Amino Acid Sequence↗

PubMed1996

scsB, a cDNA encoding the hydrogenosomal beta subunit of succinyl-CoA synthetase from the anaerobic fungus Neocallimastix frontalis.

A clone containing a Neocallimastix frontalis cDNA assumed to encode the beta subunit of succinyl-CoA synthetase (SCSB) was identified by sequence homology with prokaryotic and eukaryotic counter-parts. An open reading frame of 1311 bp was found. The deduced 437 amino acid sequence showed a high degree of identity to the beta-succinyl-CoA synthetase of Escherichia coli (46%), the mitochondrial beta-succinyl-CoA synthetase from pig (48%) and the hydrogenosomal beta-succinyl-CoA synthetase from Trichomonas vaginalis (49%). The G + C content of the succinyl-CoA synthetase coding sequence (43.8%) was considerably higher than that of the 5' (14.8%) and 3' (13.3%) non-translated flanking sequences, as has been observed for other genes from N. frontalis. The codon usage pattern was biased, with only 34 codons used and a strong preference for a pyrimidine (T) in the third positions of the codons. The coding sequence of the beta-succinyl-CoA synthetase cDNA was cloned in an E. coli expression vector encoding a 6(His) tag. The recombinant protein was purified by affinity binding and used to produce polyclonal antibodies. The anti-succinyl-CoA synthetase serum recognized a 45 kDa protein from a N. frontalis fraction enriched for hydrogenosomes and similar polypeptides in two related anaerobic fungi, Piromyces rhizinflata (45 kDa) and Caecomyces communis (47 kDa). Immunocytochemical experiments suggest that succinyl-CoA synthetase is located in the hydrogenosomal matrix. Staining for SCS activity in native electrophoretic gels revealed a band with an apparent molecular weight of approximately 330 kDa. The C-terminus of the succinyl-CoA synthetase sequence was devoid of the typical targeting signals identified so far in microbody proteins, indicating that N. frontalis uses a different signal for sorting SCSB into hydrogenosomes. Based on comparisons with other proteins we propose a putative N-terminal targeting signal for succinyl-CoA synthetase of N. frontalis that shows some of the features of mitochondrial targeting sequences.

Amino Acid Sequence↗

PubMed1996

A 34-kilodalton polypeptide is associated with 1,3-beta-glucan synthase activity from the fungus Saprolegnia monoica.

Three major polypeptides of 34, 48 and 50 kDa which appear to copurify with 1,3-beta-glucan synthase activity were isolated by glycerol gradient centrifugation of Chaps-solubilized proteins from the fungus Saprolegnia monoica. The antiserum produced against the 34-kDa polypeptide revealed by protein immunoblotting that this polypeptide copurified with 1,3-beta-glucan synthase during enzyme purification. This antiserum adsorbs the enzymatic activity as well as the 48- and 50-kDa polypeptides. These results indicate that the 34-kDa peptide is a component of the multisubunit protein complex involved in 1,3-beta-glucan synthase activity.

Animals↗

PubMed1996

Molecular karyotype of the phytopathogenic fungus Sclerotinia sclerotiorum.

Molecular techniques have been used to characterize different field isolates of Sclerotinia sclerotiorum, an ubiquitous phytopathogen. Chromosomal DNA resolved by pulsed-field gel electrophoresis (PFGE) revealed that S. sclerotiorum contains at least 16 chromosomes ranging from 1.5 Mb to 4.0 Mb. The size of the haploid genome was estimated to be 43.5 Mb. Six field isolates with different levels of virulence on sunflower germlings or green beans were differentiated by random amplification of polymorphic DNA (RAPD), and analysed by clamped homogeneous electric field electrophoresis. This analysis revealed few chromosome-length polymorphisms among these strains. Chromosomal DNA hybridization indicated that the endopolygalacturonase-encoding pg1 gene is localized on the smallest chromosome of all the strains, whereas the ribosomal DNA mapped to different-sized chromosomes. The less-aggressive strain was characterized by the presence of a supernumary small band, presumably consisting of dsRNA. In contrast to numerous other phytopathogenic fungi, this study reveals a strong karyotypic stability among the strains of S. sclerotiorum which may be preserved by the sexual mode of reproduction of this species

Ascomycota↗

PubMed1996

Expression of the Sclerotinia sclerotiorum polygalacturonase pg1 gene: possible involvement of CREA in glucose catabolite repression.

Northern-blot analysis of RNA isolated from Sclerotinia sclerotiorum grown on either glucose or polygalacturonate as the sole carbon source showed that pg1, encoding a neutral polygalacturonase, was not expressed during growth in both media. In contrast, transcripts of this gene were detected during infection of sunflower germlings. Analysis of the promoter sequence revealed a number of cis-acting sequences known to regulate the expression of many fungal promoters. Protein-DNA-binding experiments showed that proteins extracted from mycelia grown on polygalacturonate or glucose interacted with different regions of the promoter. The GST-CREA fusion protein, containing the two zinc fingers of the Aspergillus nidulans repressor CREA involved in carbon catabolite repression, forms several complexes with DNA fragments carrying the consensus 5'-SYGGRG-3'. These results suggest that a CREA homolog may be involved in the regulation of pg1.

Amino Acid Sequence↗

PubMed1996

Molecular characterization of xyn3, a member of the endoxylanase multigene family of the rumen anaerobic fungus Neocallimastix frontalis.

Different cDNAs designated xyn3 and xyn4 were isolated from an expression library of the anaerobic rumen fungus Neocallimastix frontalis. Xyn3 was further characterized and was shown to contain a single open reading frame of 1821 bp coding for a protein, XYN3, of 607 amino acids (Mr 66 000). The predicted primary structure of XYN3 consisted of two large reiterated regions of 223 amino acids with a high degree of identity (88.3%). Each domain of XYN3, XYN3A and XYN3B, showed significant homology with fungal and bacterial xylanases belonging to the endoxylanase family 11. XYN3 and XYN3A were cloned in a bacterial expression plasmid harbouring a 6 His-C terminal tag and the recombinant proteins XYN3 and XYN3A were purified from Escherichia coli. The recombinant proteins had Mr of 66 800 and 34 000 respectively and hydrolysed xylan to xylo-oligosaccharides. Analysis of truncated forms of XYN3 confirmed that the full-length protein contained two catalytic domains displaying similar substrate specificity. Western-blot analysis using antiserum raised against XYN3A showed that the N. frontalis xylanase was not submitted to post-translational maturation. XYN3A antiserum recognized similar polypeptides in the culture medium of two other rumen fungi, Piromyces rhizinflata and Caecomyces communis.

Amino Acid Sequence↗