| Summary: | <p>Surface layers, or S-layers, are a proteinaceous component of the prokaryotic cell envelope, found in almost all archaea and most bacteria. They constitute a two-dimensional, paracrystalline lattice that encompasses the entire cell, representing the outermost component of the cell envelope. Since their identification in the 1950s, S-layers have gained attention for their unique but diverse structural properties, symmetry, self-assembly, and range of biological functions. The projects described within this thesis seek to expand on our understanding of some of the most fundamental S-layer properties, including metal-binding, self-assembly, subcellular localisation, and integration of recently secreted surface layer proteins (SLPs) into the expanding S-layer. </p>
<p>Using a combination of light microscopy, structural, and cryoelectron microscopy techniques, this thesis provides insight into how the S-layer producing, Gram-negative bacterium Caulobacter crescentus utilises extracellular Ca<sup>2+</sup> to facilitate S-layer biogenesis. Using an engineered strain of C. crescentus expressing a SpyTag peptide on the constituent SLP, RsaA, exposed to the extracellular milieu, we observed a strong dependence of RsaA integration into the S-layer on Ca<sup>2+</sup> availability, characterised by loss or gain of fluorescence from a SpyCatcher-FP conjugate. Further to light microscopy, cryoelectron tomography of C. crescentus cells shows Ca<sup>2+</sup> depletion results in aggregation and detachment of the S-layer, showing Ca<sup>2+</sup> by RsaA binding stabilising S-layer proteins and architecture, facilitating oligomerisation, and enabling attachment to the LPS. This binding is further contextualised by mapping the exact locations of Ca<sup>2+</sup> ions within the structure of RsaACTD crystals using long wavelength X-ray diffraction. Ca<sup>2+</sup> ions can be accurately mapped at consistent locations across the RsaACTD structure, particularly at the central pore and between β sheets that make up the interlocking arms of the hexamer, the latter of which is consistent of toxin-related RTX domains. Interestingly, two canonical Ca<sup>2+</sup> binding positions in RsaACTD show an absence of any cations or a loosely bound K<sup>+</sup>, suggesting flexibility in some binding sites. </p>
<p>The fluorescent labelling of RsaA was further exploited to expand on the spatiotemporal localisation of S-layer biogenesis. A growing number of studies are revealing potential localisation of SLP integration to the midcell and cell poles, with similar spatiotemporal profiles to proteins typically associated with cell growth, development, and division. Using pulse-chase labelling, this study shows that flagellated swarmer cells, known to be divisionally redundant, exhibit polar labelling of new S-layer material. Due to the asymmetric nature of C. crescentus cell development, one pole will give rise to a stalk (also labelled as being covered in new S-layer) before new RsaA integration is localised to the midcell. This re-localisation is consistent with the redistribution of cell growth and the production of new cellular material to the pre-empted division site. This localisation pattern is unperturbed by induced filamentation of C. crescentus cells using sublethal concentrations of cephalexin but can be made highly irregular or completely displaced using A22, which inhibits cell elongation and the cytoskeletal protein MreB. Additionally, when co-labelled with the fluorescent D-amino acid HADA, a marker for highest levels of peptidoglycan maturation in the cell (in this case, the site of division or stalk), there is observable colocalisation of S-layer biogenesis and cell wall turnover. </p>
<p>Finally, this thesis explores the potential applications of cryogenic correlative light and electron microscopy (cryo-CLEM) to investigate S-layer biogenesis. Vitrified, dual-labelled C. crescentus cells were imaged under liquid nitrogen using widefield light microscopy, followed by data collection via cryoelectron tomography (cryo-ET) on regions of new S-layer insertion. Using cross-correlation techniques and the previously resolved RsaA hexamer structure, resolved by cryo-EM, to create a map along the S-layer to identify faults in lattice. In several locations, possible events of RsaA insertion were identified at regions of high curvature and membrane distortion, particularly at the base of the stalk, previously identified as a major point for S-layer expansion.</p>
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