Structural and functional characterization of the surface layer of Caulobacter crescentus

Proteinaceous surface layers (S-layers) have been identified in hundreds of different species belonging to all major phylogenetic groups of Bacteria and most Archaea and form a geometrically arranged paracrystalline lattice. Despite their wide abundance, few structural and functional studies have been performed on S-layers. Obtaining high-resolution structural models has been hampered by the aggregation properties of S-layers, whereas only a few functions have been identified. This thesis focuses on the structure and the function of the hexagonal S-layer of the environmental bacterium Caulobacter crescentus. In the first part of my thesis, an array of new methods was devised and tested to stabilize the S-layer protein (RsaA). Protein concentrations of an N-terminally truncated protein (RsaAΔ0-222) could be increased up to 7 mg/mL while maintaining the protein in the monomeric form. Stable protein samples were found to crystallize in the presence of Ca²⁺ and Sr²⁺. However, crystals were not thick enough (mainly formed in two dimensions), as x-ray diffraction experiments showed diffraction patterns with low resolutions. Hence, obtaining high-quality crystals remains a challenge. The second part of my thesis demonstrates that the S-layer serves as a resistance mechanism to cationic antimicrobial peptides, which are produced by virtually all living organisms, including bacteria, fungi, plants and animals. S-layer positive strains were shown to be less susceptible to antimicrobial peptides than S-layer negative strains in MIC and killing assays. This protective effect was also observed when S-layer positive and negative strains were grown in biofilms. S-layer mediated resistance to antimicrobial peptides was further confirmed using epifluorescence microscopy. Addition of exogenous S-layer protein significantly increased the resistance of an S-layer negative strain to antimicrobial peptides. Overall, these results identify a new, previously unrecognized role for the S-layer as a resistance mechanism against antimicrobial peptides.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat