The Serratia marcescens hemolysin is secreted but not activated by stable protoplast-type L-forms of Proteus...

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.