AMINO-ACID REPLACEMENTS IN THE SERRATIA-MARCESCENS HEMOLYSIN SHLA DEFINE SITES INVOLVED IN ACTIVATION AND SECRETION

The haemolysin of Serratia marcescens (ShlA) is translocated through t he cytoplasmic membrane by the signal peptide-dependent export apparat us. Translocation across the outer membrane (secretion) is mediated by the ShlB protein. Only the secreted form of ShlA is haemolytic. ShlB also converts in vitro inactive ShlA (ShlA), synthesized in the absen ce of ShlB, into the haemolytic form (a process termed activation). To define regions in ShlA involved in both processes, ShlA derivatives w ere isolated and tested for secretion and activation. Analysis of C-te rminally truncated proteins (ShlA') assigned the secretion signal to t he amino-terminal 238 residues of ShlA. Trypsin cleavage of a secreted ShlA' derivative yielded a 15 kDa N-terminal fragment, by which a hae molytically inactive ShlA protein could be activated in vitro. It is suggested that the haemolysin activation site is located in this N-ter minal fragment. Replacement of asparagine-69 and asparagine-109 by iso leucine yielded inactive haemolysin derivatives. Both asparagine resid ues are part of two short sequence motifs, reading Ala-Asn-Pro-Asn, wh ich are critical to both activation and secretion. These point mutants as well as N-terminal deletion derivatives which were not activated b y ShlB were activated by adding a non-haemolytic N-terminal fragment s ynthesized in an ShlB+ strain (complementation). Apparently the activa ted N-terminal fragment substituted for the missing activation of the ShlA derivatives and directed them into the erythrocyte membrane, wher e they formed pores. It is concluded that activation is only required for initiation of pore formation, and that in vivo activation and secr etion are tightly coupled processes. Complementation may also indicate that haemolysin oligomers form the pores.