Studies of Self-assembly Properties of Bacillus Spore Coat Proteins

Daniela Krajcikova¹, Wan Qiang², Haiyan Qiao³, Jilin Tang³, Per A. Bullough² and Imrich Barak¹

¹Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia

²Krebs Institute for Bimolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, United Kingdom

³State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, China

When challenged by starvation, Bacillus subtilis produce a dormant cell type, called a spore. Spores are distinctive by a unique capability to withstand extreme environmental conditions. Fundamental importance to spore resistance but also for its germination is the coat. The coat is proteinaceous multilayered shell that provides mechanical integrity and excludes large toxic molecules while allowing small nutrient molecules to access germination receptors beneath the coat. Spore coat is formed by over 70 proteins, ranging in size from about 6 to 70 kDa. The process of the coat assembly which is rather poorly understood, represents a central objective of this research. We have focused on a small group of proteins, called morphogenetic which act in controlling the deposition of the various coat components around the forming spore, namely SpoIVA, SpoVID, SafA and CotE. We are also interested in proteins localized directly on the spore surface - in the outermost part of the coat called crust CotY, CotZ, CotV and CotW. In our previous work we examined the protein-protein interactions between these proteins using genetic, biochemical and biophysical methods (Krajcikova et al., 2009, Mullerova et al. 2009, Qiao et al., 2012, Qiao et al., 2013). Besides confirming the previously revealed interaction between SpoVID and SafA (Costa et al. 2006) we showed that there are also direct contacts between the other key morphogenetic proteins SpoVID-SpoIVA, SpoVID-CotE and SpoVID-SpoIVA. Our data imply that due to the physical association the crucial morphogenetic proteins can form a basic skeleton where other coat proteins would be attached. We have also revealed that the crust proteins CotY, CotZ and CotW, CotV form a protein complex and most of the proteins have a high tendency to make the homo-oligomers.

Consequently we investigated the self-assembly properties of spore coat proteins by electron microscopy. Currently, there are no structural information about the individual coat components. In order to gain insights into the structure of the spore coat of B. subtilis we have prepared a set of recombinant coat proteins including CotY, CotE, CotZ, CotV, CotW, SafA and SpoVID and analyzed these proteins both as single species and in combination. A number of self-assembled structures including two-dimensional crystals and helical fibers were discovered thus clearly indicating that coat proteins have an intrinsic tendency for self-‐organization into higher order assemblies. We conclude that these assemblies could play a role in coat formation.

Costa, T., Isidro, A.L., Moran, Jr. C.P. Henriques A.O. Interaction between Coat Morphogenetic Proteins SafA and SpoVID. J. Bacteriol. 2006, 188 (22) 7731–7741

Krajcikova, D., Lukacova, M., Mullerova, D., Cutting, S.M., Barak, I., 2009. Searching for Protein-Protein Interactions within the Bacillus subtilis Spore Coat. J. Bacteriol. 191, 3212-3219.

Mullerova, D., Krajcikova, D., Barak, I., 2009. Interactions between Bacillus subtilis early spore coat morphogenetic proteins. FEMS Microbiol. Lett. 299, 74-85.

Qiao, H., Krajcikova, D., Liu, C., Li, Y., Wang, H., Barak, I., Tang, J., 2012. The interactions of spore-coat morphogenetic proteins studied by single-molecule recognition force spectroscopy. Chem. Asian J. 7, 725-731.

Qiao H, Krajcikova D, Xing C, Lu B, Hao J, Ke X, Wang H, Barak I, Tang J.

Study of the interactions between the key spore coat morphogenetic proteins CotE and SpoVID. J Struct Biol. 2013;181(2):128-35.

This work was supported by the grant Vega 2/0131/14 from the Slovak Academy of Sciences to DK and by the grant from the Slovak Research and Development Agency under the contract APVV-00335-10 to IB