INCOMPLETE ACTIVATION OF ESCHERICHIA-COLI HEMOLYSIN (HLYA) DUE TO MUTATIONS IN THE 3'-REGION OF HLYC

Mutational analysis of the carboxy-terminal region of Escherichia coli HlyC was performed by site-directed mutagenesis. Replacement of resid ue Val-127 or Lys-129 reduced the activity of HlyC to about 30 or 60%, respectively, of that of the wild type, while replacement of Gly-128 reduced the activity to less than 1% of the wild-type level, Complete inactivation of HlyC was caused by a double mutation, replacement of G ly-128 with valine and of Lys-129 with isoleucine. Analysis of culture supernatants from mutants with reduced hemolytic activity by two-dime nsional gel electrophoresis revealed the production and simultaneous s ecretion of nonacylated, monoacylated, and fully acylated HlyA forms, demonstrating impairment of the acylation reaction, possibly due to a decreased affinity of HlyC for the individual HlyA acylation sites.