RESULTS:

Production of basidiomycete atromentin-derived pigments like variegatic acid (pulvinic acid-type) and involutin (diarylcyclopentenone) from the brown-rotter Serpula lacrymans and the ectomycorrhiza-forming Paxillus involutus respectively is induced by complex nutrition and in the case of S. lacrymans bacteria. Pigmentation in S. lacrymans was stimulated by 13 different bacteria and cell-wall-damaging enzymes (lytic enzymes and proteases) but not by lysozyme or mechanical damage. The use of protease inhibitors with Bacillus subtilis or heat-killed bacteria during co-culturing with S. lacrymans significantly reduced pigmentation indicating that enzymatic hyphal damage and/or released peptides rather than mechanical injury was the major cause of systemic pigment induction. Conversely no significant pigmentation by bacteria was observed from P. involutus. We found additional putative transcriptional composite elements of atromentin synthetase genes in P. involutus and other ectomycorrhiza-forming species that were absent from S. lacrymans and other brown-rotters. Variegatic and its precursor xerocomic acid but not involutin in return inhibited swarming and colony biofilm spreading of Bacillus subtilis but did not kill B. subtilis. We suggest that dissimilar pigment regulation by fungal lifestyle was a consequence of pigment bioactivity and additional promoter motifs. The focus on basidiomycete natural product gene induction and regulation will assist in future studies to determine global regulators signalling pathways and associated transcription factors of basidiomycetes.

Summary: Proteolytic enzyme activities of the wood-decaying basidiomycetes Serpula lacrymans and Coriolus versicolor have been characterized using azocasein as substrate and by electrophoretic analysis with gelatin-containing polyacrylamide gels (gelatin-SDS-PAGE). In S. lacrymans intracellular and extracellular azocaseinase activity was optimal at pH 5-6 and was inhibited by pepstatin A. Gelatin-SDS-PAGE revealed two highly active proteinases S1 and S4 (apparent M r 65000 and 30000 respectively) and two less active enzymes S2 and S3 (apparent M r 47000 and 43000 respectively). S1 the predominant intracellular proteinase was present at all ages of the mycelium (tested up to 3 months). It is active over a broad pH range with highest activity around neutral pH. As S1 was partially inhibited by 110-phenanthroline the enzyme was considered to be a metalloproteinase although EDTA and phosphoramidon had no effect. A proteinase apparently identical to S1 was also detected in the medium of older cultures. S4 is a pepstatin-sensitive aspartic proteinase; its activity was highly pH-dependent and it was inactive in gelatin gels at pH 5-0 and above. S2 and S3 were identified as intracellular metalloproteinases present in relatively young and growing cultures. They were distinct from S1 as they were inhibited by EDTA and phosphoramidon. During starvation-induced autolysis of S. lacrymans proteinase S1 was the only enzyme present throughout (and the intracellular azocaseinase activity increased) which suggested a likely role of S1 in intra-hyphal protein mobilization. S4 is more likely to play a part in extracellular digestion of protein. The azocaseinase activities of cultures of C. versicolor were optimal at pH 7-0 (intracellular) and pH 5-6 (extracellular). Mycelial extracts gave one major band of proteinase activity in gelatin gels C1 (apparent M r 62-64000). Since the activity was sensitive to inhibitors of both serine and metalloproteinases there may have been overlapping bands due to enzymes of both types. Extracellular samples gave a more complex pattern (five bands C2-C6 M r 50000-100000). C2 and C4 are PMSF-sensitive proteinases C5 and C6 are probably metalloproteinases while C3 which was most active at pH 4-0 was unaffected by any of the inhibitors tested including pepstatin A. No aspartic proteinase equivalent to S. lacrymans S4 appeared to be produced by C. versicolor. From the information gained about the intracellular or extracellular location of these enzymes and the conditions under which they are active an in vivo role may be tentatively ascribed to some of them.

A novel basidiomycete yeast species represented by strain NYNU 2211328 was isolated from a leaf of Akebia trifoliata collected from the Baotianman Nature Reserve in Henan Province central China. Phylogenetic analysis of the D1/D2 domain of the large subunit rRNA gene and the internal transcribed spacer (ITS) region suggested that this strain is closely related to Naohidea sebacea CBS 8477 exhibiting the similarity values of 96.5% and 91.3% in the D1/D2 domain and the ITS region respectively. Physiologically the novel strain differs from N. sebacea in its ability to assimilate inulin trehalose and d-arabinose its inability to assimilate dl-lactate and its incapability of growth at 30 °C. Both phenotypic and phylogenetic analyses indicated that the isolated strain represents a novel species in the genus Naohidea and the name Naohidea akebiae fa. sp. nov. (holotype: CICC 33584; MycoBank number: MB 855585) is proposed.