Characterization of factors in pathogenicity of Erwinia carotovora subsp. carotovora.

Erwinia carotovora subsp.carotovora (Ecc) strain EC 14 was subjected to transposon (Tn5) mutagenesis and the resultant mutants were screened on enzyme detection medium for production of pectolytic enzymes, cellulase, protease, phospholipase and DNase.Out of 6,637 mutants tested 36 showed changes in one or more enzyme activities.Two mutants were completely deficient in either DNase or protease activity.When maceration produced by the protease deficient mutant was compared to maceration of the wild type on potato slices, Ecc strain EC14 prt produced 6% less macerated tissue, but this difference was not significant above P=0.50.One major extracellular protease was isolated from Ecc strain EC14 using DEAE (DE53) cellulose column chromatography and isoelectric focusing.The extracellular protease had a pi = 4.95, a molecular weight of 53,000 and its optimum pH for activity was 7. Using a cAMP deficient mutant Ecc strain EC1491 it was determined that protease was not under cAMP control.A 2.5 kb piece of DNA from Ecc strain EC14 was subcloned from the hybrid protease cosmid pCA7 into pBR325.The resultant plasmid (pFSD produced protease in E. coli strain HB101.Plasmid pFSl was subjected to Tn5 transpositional mutagenesis and a protease deficient mutant was selected.Attempts to transform Ecc strain EC14 with plasmid pFSl::Tn5 were unsuccessful.Two Erwinia herbicola (EM and two E. coli strains were transformed with hybrid plasmids pDRl or pDR30 w hich contained pectolytic genes of Ecc strain EC14.When intracellular and extracellular v 12. Isoelectric focusing profile of Ecc strain EC 14 extracellular protease activity peak from DEAE cellulose (DE53) column chromatography.5513.Protease activity substrate overlay of Ecc strain EC 14 and E. coli strain S-101 intracellular and extracellular protein fractions after electophoresis on an ultrathin polyacrylamide gel.56 14.The optimum pH for Ecc strain EC 14 extracellular protease activity.57 x 15.Relative extracellular protease (A) and pectate lyase (B) activity produced by fee strain EC 14 in culture supernatants .58 16.Growth pattern of Ecc strain EC 14 in LB broth and NaPPMS medium incubated at 30 C.59 17. Endonuclease restriction of plasmids pFSl, pCA7, and pBR325.60