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 secretes 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 interest to determine in which compartment recombinant ShlA* and ShlB are localized and whether ShlB activates ShlA*. The cloned shlB and shlA genes were transcribed in P. mirabilis stable L-form cells by the temperature-inducible phage T7 RNA polymerase. Radiolabeling, Western blotting, and complementation with C-terminally truncated ShlA (ShlA255) identified inactive ShlA* in the culture supernatant. ShlB remained cell-bound and did not activate ShlA without integration in an outer membrane. Although hemolytic ShlA added to L-form cells had access to the cytoplasmic membrane, it did not affect L-form cells. Synthesis of the large 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 and the easy isolation of released proteins make L-form cells suitable for the biotechnological production of proteins.
Sieben
S
S
Mikrobiologie/Membranphysiologie, Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany.
Hertle
R
R
Gumpert
J
J
Braun
V
V
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D017123:Bacteriophage T7; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D006460:Hemolysin Proteins / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins; D011513:Proteus mirabilis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D011523:Protoplasts / Q000737:chemistry; D012705:Serratia / Q000378:metabolism*; D014158:Transcription, Genetic
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D006460:Hemolysin Proteins; D008565:Membrane Proteins; C089911:ShlA protein, Serratia marcescens; C107896:ShlB protein, Serratia marcescens