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- Construction of plasmid-based expression and secretion vectors and study of the immobilization of proteins on the surface of Bacillus subtilis cells (2006)
- Plasmids are useful tools to study gene expression and their function. However, most of the available plasmids for Bacillus subtilis suffer from structural instability because of their rolling-circle replication mechanism. In this work, stable plasmids have been constructed allowing expression of recombinant proteins in the cytoplasm and their secretion into the culture supernatant. The author has also established an experimental system to immobilize proteins on the cell wall of B. subtilis. The sorting of surface proteins to the cell wall in B. subtilis has been investigated. A first generation of plasmids, the series of plasmid-based expression vectors pHCMCs has been constructed allowing stable intracellular expression of recombinant proteins in B. subtilis cells. These expression vectors are based on the recently described Escherichia coli - B. subtilis shuttle vector pMTLBs72 that uses the theta mode of replication. Three different controlable promoters have been inserted into the shuttle vector: PgsiB that can be induced by heat, acid shock, and by ethanol, and PxylA and Pspac that respond to the addition of xylose and IPTG, respectively. The versatility of these expression vectors was demonstrated by fusing their promoters to a reporter gene and by overexpression the gene of the HtpG (a heat shock protein) protein with three of them. All recombinant vectors exhibited full structural stability. A second generation of plasmids, two plasmid-based expression vectors have been constructed, where one plasmid allows intracellular production of recombinant proteins while the second directs the proteins into the culture medium. Both vectors use the strong promoter preceding the groESL operon (codes for the essential heat shock proteins GroES and GroEL) of B. subtilis fused to the lac operator allowing their induction by addition of IPTG. While the background level of expression of these expression cassettes was very low in the absence of the inducer, an induction factor of about 1300 was measured. When the genes htpG and pbpE (coding for a penicillin-binding protein) were fused to the groE promoter, the amount of recombinant protein produced after addition of IPTG represented 10 and 13%, respectively, of the total cellular protein. To obtain secretion of recombinant proteins, the coding region for the signal peptide of the amyQ gene encoding the amylase from Bacillus amyloliquefaciens was fused to the groE promoter. High-level secretion of amyQ amylase and cellulase A and B of Clostridium thermocellum was demonstrated. Gram-positive bacteria code for one or more enzymes termed sortases that catalyze the covalent anchoring of substrate proteins on their cell wall. They recognize an amino acid sequence designated sorting motif, present close to the C-terminal end of the substrate proteins, cleave within this motif, and catalyze anchoring of the polypeptide chain to the peptide crossbridge linking the peptidoglycan strands in a transpeptidation reaction. B. subtilis has been reported to code for two putative sortases, YhcS and YwpE, but the sorting sequences recognized by them are yet unknown. To be able to immobilize proteins on the surface of B. subtilis cells, the srtA gene coding for sortase A of Listeria monocytogenes that recognizes a known sorting motif was introduced into B. subtilis. L. monocytogenes and B. subtilis share the same peptide crossbridge. Next, the coding region of the amylase gene was fused to the C-terminal region of Staphylococcus aureus fibronectin binding protein B (FnBPB) containing the sorting sequence including its sorting motif (LPETG). Covalent linkage could be proven by treatment of the cells with lysozyme and by immunofluorescence microscopy. Up to 240,000 molecules of amylase could be immobilized per cell, 24 times more than previously reported for other bacterial species. To study the influence of the distance between the sorting motif and the C-terminus of the amylase (AmyQ) on the activity of the enzyme, the length of the spacer was varied. It turned out that the highest activity was measured with a spacer length of 123 aa residues. To elucidate whether the putative sortases YhcS and YwpE of B. subtilis can retain the two potential substrates of the sortase YfkN and YhcR, the yhcS and/or ywpE knockout strains were constructed and the translational fusions between AmyQ and N-terminal of YfkN or YhcR, both harbouring the 123-aa spacers, were generated resulting in AmyQ-YfkN123 and AmyQ-YhcR123, respectively. The results demonstrated that YhcS could retain the fusion AmyQ-YhcR123 on the cell wall and YwpE seems to assist YhcS to perform its functions. YhcS could recognize the sequence containing the sorting motif LPDTS from YhcR but not LPETG from FnBPB. YhcR could be a substrate of YhcS, while it is not clear whether YfkN is a cell wall protein. A model for the covalent anchoring of proteins on the cell wall by sortases is presented.