SEQUENCE-ANALYSIS AND REGULATION OF A GENE ENCODING A CUTICLE-DEGRADING SERINE-PROTEASE FROM THE NEMATOPHAGOUS FUNGUS ARTHROBOTRYS OLIGOSPORA

The nematode trapping fungus Arthrobotrys oligospora produces an extra cellular serine protease (designated PII) that immobilizes free-living nematodes in bioassays and hydrolyses proteins of the nematode cuticl e. Peptides were isolated from PII and partly sequenced. Three interna l peptide sequences were used to design synthetic oligonucleotides, wh ich allowed the subsequent isolation of the gene encoding PII from a g enomic library. The deduced amino acid sequence indicated that PII is synthesized as a preproenzyme containing the mature enzyme, a signal s equence and a propeptide that are removed before the enzyme is secrete d into the medium. The primary sequence of PII displayed a high degree of similarity with several other serine proteases of ascomycetes belo nging to the subtilisin family. Northern analysis demonstrated that PI I was expressed when the fungus was starved of nitrogen and carbon and that the expression was significantly stimulated by the addition to t he medium of various soluble and insoluble proteins, including fragmen ts of nematode cuticle. The levels of the mRNA as well as the proteoly tic activity of PII were repressed in the presence of more easily meta bolized forms of nitrogen (including ammonia, nitrate and amino acids) or glucose. The activity of the enzyme was almost completely inhibite d by the peptide Phe-Val, as well as by the amino acid Phe, without a corresponding decrease in mRNA level. Notably, peptides with similar s tructures are known to be secreted by the host (nematode) and to stimu late the production of infection structures (traps) of the fungus.