Three extracellular proteases from Cochliobolus carbonum: cloning and targeted disruption of ALP1

Three extracellular serine proteases (Alp1a, Alp1b, Alp2) from Cochliobolus carbonum were purified and characterized. Of eight carbon/protein substrates tested, total protease activity was highest when the fungus was grown on medium containing collagen. Alp1a and Alp1b are members of the trypsin family (EC 3.4.21.4), and Alp2 is a member of the subtilisin family (EC 3.4.21.62). Alp1a, Alp1b, and Alp2 have monomer molecular masses of 25, 30, and 38 kDa respectively. Alp1b is glycosylated, whereas Alp1a is not. The gene encoding Alp1a, ALP1, was isolated using PCR primers based on two amino acid sequences: One obtained directly from the N-terminus of Alp1a and another that is highly conserved in other trypsins. The transcriptional start site was determined using RACE and the intron structure and polyadenylation site were determined from a cDNA clone. An internal fragment of ALP1 was used to create Alp1a null mutants by transformation-mediated gene disruption. Total protease activity in the mutants was reduced by 35% to 45%. By chromatographic analysis, the mutants had lost two peaks of UV absorption and the two protease activities corresponding to Alp1a and Alp1b, which, together with the biochemical data, indicates that Alp1a and Alp1b are products of the same gene. The in vitro growth and diseases phenotypes of the ALP1 mutants were distinguishable from the wild-type strain; therefore, ALP1 is not by itself required for pathogenicity.