Mutational analysis supports a role for multiple structural features in the <i>C</i>‐terminal secretion signal of <i>Escherichia coli haemolysin</i>

Abstract

Summary We have carried out an extensive mutational analysis of the C ‐terminal signal which targets the export of the 1024‐residue haemolysin protein (HlyA) of Escherichia coli across both bacterial membranes into the surrounding medium. Over 60 variants of the HlyA C ‐terminal 53‐amino‐acid sequence were created by oligonucleotide‐directed mutagenesis and fused to the HlyA N‐terminal 830 residues. Transport of the HlyA derivatives by the HlyB/HlyO system was compared with the wild‐type level and the data indicate that the HlyA C ‐terminal export signal lies within the last 48 amino acids and comprises three functional domains: an amphipathic, charged helix between residues 1,977 and R,996; a 13‐amino‐acid uncharged region from residue T,997 to S,1009; and an 8‐aminoacid hydroxylated tail at the extreme C ‐terminus. Analogous features were found in the C ‐terminal sequences of an extended family of haemolysins, leukotoxins and proteases which are secreted by HlyB/HlyD‐type translocators. In particular, all nine proteins which are secreted into the extracellular medium possess potential extended amphipathic helices. These results suggest a possible role for multiple regions of the HlyA C ‐terminal export signal in which the first two domains span the membranes and the third domain remains in the cytoplasm.