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 extracellular serine protease (designated PII) that immobilizes free-living nematodes in bioassays and hydrolyses proteins of the nematode cuticle. Peptides were isolated from PII and partly sequenced. Three internal peptide sequences were used to design synthetic oligonucleotides, which allowed the subsequent isolation of the gene encoding PII from a genomic library. The deduced amino acid sequence indicated that PII is synthesized as a preproenzyme containing the mature enzyme, a signal sequence and a propeptide that are removed before the enzyme is secreted into the medium. The primary sequence of PII displayed a high degree of similarity with several other serine proteases of ascomycetes belonging to the subtilisin family. Northern analysis demonstrated that PII was expressed when the fungus was starved of nitrogen and carbon and that the expression was significantly stimulated by the addition to the medium of various soluble and insoluble proteins, including fragments of nematode cuticle. The levels of the mRNA as well as the proteolytic activity of PII were repressed in the presence of more easily metabolized forms of nitrogen (including ammonia, nitrate and amino acids) or glucose. The activity of the enzyme was almost completely inhibited by the peptide Phe-Val, as well as by the amino acid Phe, without a corresponding decrease in mRNA level. Notably, peptides with similar structures are known to be secreted by the host (nematode) and to stimulate the production of infection structures (traps) of the fungus.
Ahman
J
J
Department of Microbial Ecology, Lund University, Sweden. Johan.Ahman@mbioekol.lu.se
Ek
B
B
Rask
L
L
Tunlid
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D003904:Mitosporic Fungi / Q000201:enzymology* / Q000235:genetics* / Q000472:pathogenicity; D008969:Molecular Sequence Data; D009348:Nematoda / Q000382:microbiology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics*
D017931:DNA Primers; D004271:DNA, Fungal; D012333:RNA, Messenger; D012697:Serine Endopeptidases