J. Bacteriol., 07 1996, 3899-3907, Vol 178, No. 13
Copyright © 1996, American Society for Microbiology

Disruption of the serine proteinase gene (sep) in Aspergillus flavus leads to a compensatory increase in the expression of a metalloproteinase gene (mep20)

MV Ramesh and PE Kolattukudy
Neurobiotechnology Center, Ohio State University, Columbus, Ohio 43210, USA.

The serine proteinase gene (sep) in Aspergillus flavus was disrupted by homologous recombination with a hygromycin resistance gene as the marker. The gene-disrupted mutant GR-2 contained a single-copy insertion of the marker gene and did not express the sep gene. Serine proteinase activity, 36-kDa protein labeled by 3H- diisopropylfluorophosphate, and immunologically detectable proteinase were not detected in the culture 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) product than did the wild type. Furthermore, mep20 mRNA levels, measured by RNase protection assay, in the mutant were higher than those in the wild type. Inhibition of the serine proteinase by Streptomyces subtilisin inhibitor (SSI) in the culture medium of wild-type A.flavus also resulted in an elevation of mep20 gene products. Although no serine proteinase activity could be detected, the level of elastinolytic activity 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 that the expression of the genes encoding serine and metalloproteinases are controlled by a common regulatory system and the fungus has a mechanism to sense the status of extracellular proteolytic activities.

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