Escherichia coli alpha-hemolysin (HlyA) is heterogeneously acylated in vivo with 14-, 15-, and 17-carbon fatty acids

alpha -Hemolysin (HlyA) is a secreted protein virulence factor observed in certain uropathogenic strains of Escherichia coil. The active, mature form of HlyA is produced by posttranslational modification of the protoxin that is mediated by acyl carrier protein and an acyltransferase, HlyC. We have n ow shown using mass spectrometry that these modifications, when observed in protein isolated in vivo, consist of acylation at the E-amino groups of tw o internal lysine residues, at positions 564 and 690, with saturated 14- (6 8%), 15- (26%), and 17- (6%) carbon amide-linked side chains. Thus, HlyA ac tivated in vivo consists of a heterogeneous family of up to nine different covalent structures, and the substrate specificity of the HlyC acyltransfer ase appears to differ from that of the closely related CyaC acyltransferase expressed by Bordetella pertussis.