On the role of penicillin-binding protein SpoVD in endospore cortex assembly

Abstract: Bacteria of the genera Bacillus and Clostridium can form endospores as a strategy to survive unfavourable environmental conditions. Endospore formation involves synthesis of cortex, a thick layer of modified peptidoglycan that surrounds the spore. This layer is required for heat resistance of the spore and mutant spores lacking the cortex layer can be identified by a simple heat shock assay. B. subtilis SpoVD is a class B, high molecular weight, penicillin-binding protein (PBP) essential for spore cortex peptidoglycan synthesis. The exact role of the protein in cortex assembly is nknown but it most likely catalyses the formation of cross-links between glycan strands in nascent peptidoglycan. SpoVD deficient strains produce heat sensitive spores without cortex layer. Two conserved cysteine residues (Cys332 and Cys351) in the transpeptidase domain of SpoVD seem important for activity of the enzyme. They can form an intramolecular disulfide bond and this is catalysed by the membrane-bound thiol-disulfide oxidoreductase BdbD. The disulfide bond in SpoVD is located close to the transpeptidase active site and blocks the function of the protein. The bond is broken by the action of StoA, a sporulation-specific membrane-bound thiol-disulfide oxidoreductase. Based on these findings a thiol-based redox switch regulation of SpoVD activity was proposed in 2010. The aim of this PhD project was to elucidate the function of SpoVD in cortex synthesis and to find out the physiological role of the proposed switch and the two cysteine residues in SpoVD. In depth investigation of the process of cortex assembly contribute to our understanding of peptidoglycan synthesis in general. This is of considerable medicinal interest since, e.g., bacterial cell wall synthesis is an effective target for many antibiotics in clinical use, such as penicillins and cephalosporins and eventual new drugs. I demonstrate, by the use of a constructed SpoVD active site mutant strain, that synthesis of cortex explicitly depends on the transpeptidase activity of the protein. I show that the C-terminal PASTA domain of SpoVD is not important for the function of the protein in cortex synthesis. My results from in vitro experiments with several isolated protein variants strengthen the view that SpoVD is a specific target for StoA. My findings, supported by data available in the literature, indicate that the two cysteine residues in SpoVD affect the dynamics of the transpeptidase domain. Finally, I propose a revised model for the function of BdbD and StoA in modulation of the redox state of SpoVD, where BdbD and StoA are suggested to act (mainly) during the folding of newly synthesised SpoVD.