Untersuchung von spaeten Schritten der Proteinsekretion bei den Gram-positiven Bakterien Bacillus subtilis und Staphylococcus carnosus

The secretory expression of heterologous proteins in Gram-positive bacteria is often hampered due to several bottlenecks in the secretion pathway. Many of these bottlenecks regard the 'late steps' of protein secretion. These steps comprise (a) the processing of the signal peptide by type I signal peptidases, (b) the degradation of the cleaved signal peptide by signal peptide peptidases, (c) the folding of the mature export protein within the cell envelope and finally (d) the release of the native protein from the thick cell wall into the growth medium. Several aspects of these processes were analysed in this thesis, using Bacillus subtilis and Staphylococcus carnosus as Gram-positive model organisms. The following results were obtained: (1) S. carnosus possesses two genes that encode proteins with significant homology to type I signalpeptidases: sipA and sipB. Interestingly the SipA-protein lacks a conserved serine-lysine-dyad that is essential for endopeptidase activity. Accordingly it was found that SipA does not possess processing activity. Currently the physiological function of this protein is not clear. On the other hand the sipB-gene of S. carnosus encodes a functional type I signalpeptidase. Both, SipA and SipB, are anchored to the plasmamembrane via an N-terminal membran segment, with the main part of the protein residing in the periplasmic space. (2) Using the OmpA-protein from E. coli as a heterologous model protein the folding potential of a secretory protein was identified as an important determinant for its proteolytic stability within and its efficient release from the Gram-positive cell envelope. For the first time a direct correlation between folding and degradation of an export protein in this compartment could be demonstrated. (3) Results obtained with the OmpA-protein indicate that the cell envelope of B. subtilis contains a 'quality control system' that monitors protein folding in this compartment and - if necessary - degrades misfolded export proteins. This system consists of several proteases. CtpA, a homologue of the periplasmic protease Tsp from E. coli is obviously one of them. (4) In an approach to investigate signal peptide peptidase-like proteases from B. subtillis the TepA-Protein was identified as a new determinant of bacterial protein secretion. TepA (for 'translocation enhancing protein') is a previously unknown serine protease that is probably specific for Gram-positive bacteria. Since TepA shows significant homology not only to signal peptide peptidase but also to the ubiquitous heat shock protease ClpP, the protein could be involved in the degradation of cleaved signal peptides or might, in analogy to ClpP, play a regulatory role in protein secretion. (orig.)Available from TIB Hannover: RA 831(3745) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman