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 pre-proenzyme 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.
BonantsP.J.M., FittersP. F. L., ThijsH., Den-BelderE., WaalwijkC., HenflingW. D. M. . 1995; A basic serine protease from Paecilomyces lilacinus with biological activity against Meloidogyne hapla eggs. Microbiology 141:775–784
ChiangTY., MarzlufG. A.1994; DNA recognition by the NIT2 nitrogen regulatory protein: importance of the number, spacing and orientation of GATA core elements and their flanking sequences upon NIT2 binding. Biochemistry 33:576–582
CohenB.L., DruckerH.1977; Regulation of exocellular protease in Neurospora crassa:, induction and repression under conditions of nitrogen starvation. Arch Biochem Biophys 182:601–613
DavidowL. S., O'DonnellM. M., KaczmarekF. S., PereiraD. A., DezeeuwJ. R., FrankeA. E.1987; Cloning and sequencing of the alkaline extracellular protease gene of Yarroma lipolytica. J Bacteriol 169:4621–4629
El-GogaryS., LeiteA., CrivellaroO., EveleighD. E., El-DorryH.1989; Mechanism by which cellulose triggers cellobiohydrolase I gene expression in Trichoderma reesei. Proc Natl Acad Sci USA 86:6138–6141
GargasA., TaylorJ. W.1995; Phylogeny of discomycetes and early radiations of the apothecial ascomycotina inferred from SSU rDNA sequence data. Exp Mycol 19:7–15
GeremiaR. A., GoldmanG. A., JacobsD., VilaS. B., ArdilesW., Van MontaguM.M., Herrera-EstrellaA.1993; Molecular characterization of the proteinase-encoding gene Prb1 related to mycoparasitism by Trichoderma harzianum. Mol Microbiol 8:603–613
GunkelF.A., GassenH. G.1989; Proteinase K from Triti- rachium album limber. Characterization of the chromosomal gene and expression of the cDNA in Escherichia coli. Eur J Biochem 179:184–194
GurrS. J., UnklesS. E., KinghornJ. R.1987; The structure and organization of nuclear genes of filamentous fungi. In Gene Structure in Eukaryotic Microbes. Special Publication of the Society for General Microbiology22 pp. 93–139 Edited by KinghornJ. R. Oxford: IRL Press;
IsogaiT., FukagawaT. I. M., KohsakaH. K. M., AokiH., ImanakaH.1991; Cloning and nucleotide sequences of the complementary and genomic DNAs for the alkaline protease from Cephalosporium acremonium. Agrie Biol Chem 55:471–477
Jaton-OgayK., SuterM., CrameriR., FalchetteR., FatihA., MonodM.1992; Nucleotide sequence of a genomic and a cDNA clone encoding an extracellular alkaline protease of Aspergillus fumigatus. FEMS Microbiol Lett 92:163–168
JoshiL., St LegerR. J. , BidochkaM. J.1995; Cloning of a cuticle-degrading protease from the entomopathogenic fungus Beauveria bassiana. FEMS Microbiol Lett 125:211–218
Lopez-LlorcaL.V.1990; Purification and properties of extracellular proteases produced by the nematophagous fungus Verti- cillium suchlasporium. Can J Microbiol 36:530–537
Nordbring-HertzB., BrinckC.1974; Qualitative characterization of some peptides inducing morphogenesis in the nematode trapping fungus Arthrobotrys oligospora. Physiol Plant 31:59–63
PatersonI. C., CharnleyA. K., CooperR. M., ClarksonJ. M.1993; Regulation of production of a trypsin-like protease by the insect pathogenic fungus Metarhivfum anisopliae. FEMS Microbiol Lett 109:323–328
RiviereL. R., FlemmingM., EliconeC., TempestP.1991; Study and applications of the effects of detergents and chaotropes on enzymatic protolysis. In Techniques in Protein Chemistry11 pp. 171–179 Edited by VillafrancaJ. J. London: Academic Press;
SegersR., ButtT. M., KerryB. R., PeberdyJ. F.1994; The nematophagous fungus Verticillium chlamydosporium Goddard produces a chymoelastase-like protease which hydrolyses host nematode proteins in situ.. Microbiology 140:2715–2723
SegersR., ButtT. M., KeenJ. N., KerryB. R., PeberdyJ. F.1995; The subtilisins of the invertebrate my copathogens Verti- cillium chlamydosporium and Metarhivfum anisopliae are serologically and functionally related. FEMS Microbiol Lett 126:227–231
St LegerR.J., CharnleyA. K., CooperR. M.1987; Characterization of cuticle-degrading proteases produced by the entomo- pathogen Metarhisfum anisopliae.. Arch Biochem Biophys 253:221–232
St LegerR.J., DurrandsP. K., CooperR. M., CharnleyA. K.1988; Regulation of production of proteolytic enzymes by the entomopathogenic fungus Metarhiyium anisopliae. Arch Microbiol 150:413–416
St LegerR.J., FrankD. C., RobertsD. W., StaplesR. C.1992; Molecular cloning and regulatory analysis of the cuticle-degrading protease structural gene from the entomopathogenic fungus Metarhivfum anisopliae. Eur J Biochem 204:991–1001
TatsumiH., OgawaY., MurakamiS., IshidaY., MurakamiK., MasakiA., KawabeH., ArimuraE., MotaiH.1989; A full length cDNA clone for the alkaline protease from Aspergillus oryzae : structural analysis and expression in Saccharomyces cerevisiae.. Mol Gen Genet 219:33–38
TaylorJ. W., SwannE. C., BerbeeM. L.1994; Molecular evolution of ascomycete fungi: phylogeny and conflict. In Asco- mycete Systematics: Problems and Perspectives in the Nineties pp. 201–212 Edited by HawksworthD. L. New York: Plenum Press;
Tigano-MilaniM..S., SamsonR. A., MartinsI., SobralB. W. S.1995; DNA markers for differentiating isolates of Paecilomyces lilacinus. Microbiology 141:239–245
TunlidA., JanssonS.1991; Proteases and their involvement in the infection and immobilization of nematodes by the nematophagous fungus Arthrobotrys oligospora. Appl Environ Microbiol 57:2868–2872
TunlidA., RosénS., EkB., RaskR.1994; Purification and characterization of an extracellular serine protease from the nematode-trapping fungus Arthrobotrys oligospora. Microbiology 140:1687–1695
YakuraK., KatoA., TanifujiS.1984; Length heterogeneity of the large spacer of Vicia faba rDNA is due to the differing number of a 325 bp repetitive sequence elements. Mol Gen Genet 193:400–405