THE SERRATIA-MARCESCENS HEMOLYSIN IS SECRETED BUT NOT ACTIVATED BY STABLE PROTOPLAST-TYPE L-FORMS OF PROTEUS-MIRABILIS

The outer-membrane protein ShlB of Serratia marcescens activates and s ecretes hemolytic ShlA into the culture medium. Without ShlB, inactive ShlA (termed ShlA) remains in the periplasm. Since Proteus mirabilis L-form cells lack an outer membrane and a periplasm, it was of intere st to determine in which compartment recombinant ShlA and ShlB are lo calized and whether ShlB activates ShlA. The cloned shlB and shlA gen es were transcribed in P. mirabilis stable L-form cells by the tempera ture-inducible phage T7 RNA polymerase. Radiolabeling, Western blottin g, and complementation with C-terminally truncated ShlA (ShlA255) iden tified inactive ShlA in the culture supernatant. ShlB remained cell-b ound and did not activate ShlA without integration in an outer membran e. Although hemolytic ShlA added to L-form cells had access to the cyt oplasmic membrane, it did not affect L-form cells. Synthesis of the la rge ShlA protein (165 kDa) in P. mirabilis L-form cells under phage T7 promoter control demonstrates that L-form cells are suitable for the synthesis and secretion of large recombinant proteins. This property a nd the easy isolation of released proteins make L-form cells suitable for the biotechnological production of proteins.