Cloning and Characterization of a Gene for Extracellular Protease of Erwinia Carotovora Subsp. Carotovora

Abstract

Erwinia carotovora subsp. carotovora (Ecc) produces a number of extracellular degradative enzymes including pectate lyase, polygalacturonase, pectin lyase, phospholipase C, cellulase, and protease. The role and function of the enzymes in pathogenesis, individually or in concert, are not fully understood. In order to elucidate the importance of protease (PRT) and study its properties, we have isolated Ecc strain 71 DNA segments that specify protease. A gene bank was constructed in Escherichia coli (HB101) using chromosomal DNA of Ecc71, partially digested with PstI, cloned in the cosmid vector pHC79. Transductants were screened on nutrient gelatin agar for protease activity. After chloroform treatment of the assay plates, three Prt⁺ clones were identified by the presence of an opaque halo surrounding the colony. All were active on Azocasein or Azocoll, and detectable PRT activity was found to be entirely periplasmic. One clone (HB10l/pAKC216) was chosen for subsequent investigations. Subcloning of pAKC216 yielded pAKC217 which carried a 6 kb insert, with a single EcoRI site, that still encoded PRT. The protease has been partially purified from Ecc71 supernatant and HB10l/pAKC217 periplasmic fluid by (NH₄)₂SO₄ precipitation and column chromatography. We will present the findings of our investigations into the properties of the enzymes and their importance in pathogenesis.