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

Abstract

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.