Structural studies of prokaryotic surface layer proteins

Thesis

Many prokaryotic organisms are encapsulated by a flexible two-dimensional proteinaceous surface layer (S-layer) consisting of repeating units of surface layer proteins (SLPs). SLPs are often the highest copy-number macromolecules in these cells, playing key roles in essential cellular functions including cell-shape maintenance, protection against predators and interaction with the environment. Using recently developed electron cryomicroscopy (cryo-EM) and electron cryotomography (cryo-ET) techniques, I have determined the structures of several S-layers across domains of life, from archaeal and bacterial model organisms and investigated their molecular function.

In the Gram-negative bacteria model organism Caulobacter crescentus, the S-layer is anchored to the cell envelope via lipopolysaccharide (LPS). Using cellular cryo-ET coupled with subtomogram averaging (STA) I resolved a 3.5 Å resolution in-situ structure of the complete S-layer, showing how the native S-layer is anchored at the tip of the lipopolysaccharide.

Combining structural studies with deep-homology bioinformatics (in collaboration) for Haloferax volcanii archaeal and Deinococcus radiodurans bacterial SLPs, I showed that immunoglobulin containing S-layers are widespread in prokaryotes. Additionally, I structurally characterised an abundant outer membrane β-barrel protein from D. radiodurans, elucidating its multidomain architecture and how it tethers the outer membrane to the underlying peptidoglycan layer and thereby shapes and maintains the cell envelope.

Finally, I elucidated the in-situ structure of the S-layer of the ammonia-oxidising archaeon Nitrosopumilus maritimus. Using whole cell isothermal titration calorimetry and molecular dynamics simulations (in collaboration), I showed that this S-layer is required for ammonium enrichment from a dilute marine environment, facilitating the conversion of ammonium oxidation to nitrite.

Taken together, these findings revealed common principles of S-layer arrangement and assembly, advancing our understanding of this important and widespread cellular organelle.

Authors: Von Kügelgen, AIB

• DOI: 10.5287/ora-j18boxvqk
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: cryo-EM; Nitrosopumilus maritimus; Caulobacter crescentus; S-layer; Haloferax volcanii; cryo-ET; Deinococcus radiodurans; subtomogram averaging; prokaryotic cell surfaces
• Subjects: Structural biology; Microbiology; Halophilic microorganisms