DISRUPTION OF THE SERINE PROTEINASE GENE (SEP) IN ASPERGILLUS-FLAVUS LEADS TO A COMPENSATORY INCREASE IN THE EXPRESSION OF A METALLOPROTEINASE GENE (MEP20)

The serine proteinase gene (sep) in Aspergillus flavus was disrupted b y homologous recombination with a hygromycin resistance gene as the ma rker. The gene-disrupted mutant GR-2 contained a single copy insertion of the marker gene and did not express the sep gene. Serine proteinas e activity, 36-kDa protein labeled by H-3-diisopropylfluorophosphate, and immunologically detectable proteinase were not detected in the cul ture fluid of GR-2, Despite the absence of the serine proteinase, the total elastinolytic activity levels in the mutant and the wild-type A. flavus were comparable. Immunoblots revealed that the mutant secreted greater amounts of an elastinolytic metalloproteinase gene (mep20) pr oduct than did the wild type. Furthermore, mep20 mRNA levels, measured by RNase protection assay, in the mutant were higher than those in th e wild type. Inhibition of the serine proteinase by Streptomyces subti lisin inhibitor (SSI) in the culture medium of wild-type A, flavus als o resulted in an elevation of mep 20 gene products, Although no serine proteinase activity could be detected, the level of elastinolytic act ivity of the SSI-treated culture was comparable to that of the control . Immunoblots revealed that the addition of SSI caused an elevation in the levels of metalloproteinase and its mRNA. These results suggest t hat the expression of the genes encoding serine and metalloproteinases are controlled by a common regulatory system and the fungus has a mec hanism to sense the status of extracellular proteolytic activities.