ShlB mutants of Serratia marcescens allow uncoupling of activation and secretion of the ShlA hemolysin

The ShlB protein in the outer membrane of Serratia marcescens secretes hemo lytic ShlA protein into the culture medium. In the absence of ShlB, nonhemo lytic ShlA remains in the periplasm. ShlB mutants were isolated in which se cretion was uncoupled from activation. Mutants with a tetrapeptide insertio n after residues 136 or 224 of mature ShlB and a mutant with an insertion a fter residue 154 and a deletion secreted inactive ShlA. In vitro, secreted nonhemolytic ShlA was converted into hemolytic ShlA by isolated wild-type S hlB and by complementation with an N-terminal ShlA fragment of 255 residues (ShlA-255). The isolation of secretion-competent, but activation-negative mutants indicates that secretion alone is not sufficient for activation of ShlA. Rather, ShlB is required for activation and secretion, and the mutant s define sites in ShlB which are involved in activation. According to a pre dicted transmembrane model of ShlB, the mutations that retain secretion com petence but abolish activation competence are located in the most prominent surface loop and the following transmembrane loop. In one tetrapeptide ins ertion mutant, ShlB-332, most of the ShlA remained cell-associated in an in active form and low amounts (6 %) were hemolytic. Secreted inactive ShlA de grees was completely degraded by trypsin, in contrast to hemolytic ShlA, wh ich was cleaved into two fragments of 60 and 100 kDa. This result indicates that the conformational change from a highly trypsin-sensitive to a highly trypsin-resistant protein with only a single cleavage site in a polypeptid e of 1578 residues occurs upon activation of ShlA and not during secretion.