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The Serratia marcescens hemolysin is secreted but not activated by stable protoplast-type L-forms of Proteus
S Sieben1, R Hertle, J Gumpert
1Mikrobiologie/Membranphysiologie, Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany.
Abstract:
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.
Insights
Proteus mirabilis L-form cells can synthesize and secrete large recombinant proteins like ShlA, even without an outer membrane. This finding highlights their potential for biotechnological protein production.
Area of Science:
- Microbiology
- Biotechnology
Background:
- The outer-membrane protein ShlB from Serratia marcescens is essential for activating and secreting the hemolytic protein ShlA.
- Inactive ShlA (ShlA*) accumulates in the periplasm without ShlB.
- Proteus mirabilis L-form cells lack an outer membrane and periplasm, presenting a unique system to study protein localization and activation.
Purpose of the Study:
- To investigate the localization and activation of recombinant ShlA* and ShlB in Proteus mirabilis L-form cells.
- To assess the suitability of L-form cells for the synthesis and secretion of large recombinant proteins.
Main Methods:
- Cloning and expression of shlB and shlA genes in P. mirabilis L-form cells using T7 RNA polymerase.
- Radiolabeling and Western blotting to detect and localize proteins.
- Complementation assays using C-terminally truncated ShlA (ShlA255).
Main Results:
- Inactive ShlA* was identified in the culture supernatant of L-form cells.
- ShlB remained cell-bound and did not activate ShlA* without an outer membrane.
- Hemolytic ShlA did not affect L-form cells even when accessing the cytoplasmic membrane.
Conclusions:
- Proteus mirabilis L-form cells are capable of synthesizing and secreting large recombinant proteins, such as the 165 kDa ShlA.
- The absence of an outer membrane in L-form cells does not impede secretion into the supernatant.
- L-form cells offer a promising platform for the biotechnological production of secreted recombinant proteins due to efficient synthesis and easy isolation.