Amino acid replacements in the Serratia marcescens haemolysin ShIA define sites involved in activation and secretion.
The haemolysin of Serratia marcescens (ShIA) is translocated through the cytoplasmic membrane by the signal peptide-dependent export apparatus. Translocation across the outer membrane (secretion) is mediated by the ShIB protein. Only the secreted form of ShIA is haemolytic. ShIB also converts in vitro inactive ShIA (ShIA*), synthesized in the absence of ShIB, into the haemolytic form (a process termed activation). To define regions in ShIA involved in both processes, ShIA derivatives were isolated and tested for secretion and activation. Analysis of C-terminally truncated proteins (ShIA) assigned the secretion signal to the amino-terminal 238 residues of ShIA. Trypsin cleavage of a secreted ShIA' derivative yielded a 15 kDa N-terminal fragment, by which a haemolytically inactive ShIA* protein could be activated in vitro. It is suggested that the haemolysin activation site is located in this N-terminal fragment. Replacement of asparagine-69 and asparagine-109 by isoleucine yielded inactive haemolysin derivatives. Both asparagine residues are part of two short sequence motifs, reading Ala-Asn-Pro-Asn, which are critical to both activation and secretion. These point mutants as well as N-terminal deletion derivatives which were not activated by ShIB were activated by adding a non-haemolytic N-terminal fragment synthesized in an ShIB+ strain (complementation). Apparently the activated N-terminal fragment substituted for the missing activation of the ShIA derivatives and directed them into the erythrocyte membrane, where they formed pores. It is concluded that activation is only required for initiation of pore formation, and that in vivo activation and secretion are tightly coupled processes. Complementation may also indicate that haemolysin oligomers form the pores.
Schönherr
R
R
Mikrobiologie II, Tübingen, Germany.
Tsolis
R
R
Focareta
T
T
Braun
V
V
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D005816:Genetic Complementation Test; D005838:Genotype; D006460:Hemolysin Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D006461:Hemolysis; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010957:Plasmids; D017354:Point Mutation; D012706:Serratia marcescens / Q000235:genetics / Q000378:metabolism*; D014357:Trypsin
D001426:Bacterial Proteins; D006460:Hemolysin Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; C089911:ShlA protein, Serratia marcescens; D014357:Trypsin