Molecular cloning and characterization of two serine proteinase genes fromthe crayfish plague fungus, Aphanomyces astaci

Two novel genes encoding the serine proteinases, subtilisin (AaSP1) and try psin (AaSP2), from Aphanomyces astaci were identified. Based on the amino a cid consensus sequences around the catalytic triad of these serine proteina ses, degenerated oligonucleotides were designed for isolation of serine pro teinase genes from a genomic DNA library. The AaSP1 gene encodes a full-len gth protein of 515 amino acids as a large precursor of 56 kDa. After cleava ge of a predicted leader sequence of 18 residues and a prepeptide of 133 am ino acids, the mature enzyme of 364 amino acids is generated with a calcula ted molecular mass of 39 kDa and a pI of 6.0. The primary sequence of AaSP1 showed similarity to both bacterial subtilisin and fungal subtilisin-like serine proteinases. Southern blot analysis of AaSP1 revealed the presence o f at least two subtilisin genes in the A. astaci genome. Northern blot anal ysis indicated that the size of AaSP1 transcript was 1.6 kb, The AaSP2 gene encodes a prepropeptide of 276 amino acids with a molecular mass of 29 kDa , A mature protein of 237 amino acids is probably generated after cleavage of a 17-residue signal peptide and a 21-amino-acid prepeptide with a predic ted molecular mass of 25 kDa and a pI of 6.0. The primary sequence of AaSP2 showed similarity to trypsin enzymes from various organisms. Southern blot analysis revealed the presence of multiple trypsin genes in the A. astaci genome. Northern blot analysis indicated that the size of AaSP2 transcript was 1.0 kb, The regulation of AaSP2 transcription was not controlled by nit rogen catabolic repression. However, the expression of AaSP2 was found to b e specifically induced by crayfish plasma, implying a role in pathogenesis toward the crayfish host. (C) 2001 Academic Press.