Structural studies of bacterial biofilm matrix fibres

Thesis

Many bacteria live in multicellular communities called biofilms. Within the biofilm, cells are embedded in a self-secreted extracellular biofilm matrix, which provides numerous benefits to bacteria inside. Biofilms also form during human infection, where the matrix provides protection against antibiotic treatment and the immune system. A major component of the biofilm matrix are protein fibres, which provide stability to the biofilm and scaffold its formation. Little is known about the structure of the biofilm matrix in general, and matrix fibres specifically. Here, structural and functional studies on biofilm matrix fibres are presented. An electron cryomicroscopy (cryo-EM) structure of the biofilm-promoting archaic Chaperone-Usher pilus CupE from Pseudomonas aeruginosa was solved, showing a zig-zag subunit architecture, where the majority of inter-subunit interactions are mediated by an N-terminal donor strand that extends into the following subunit and completes its Ig-like fold. Electron cryotomography (cryo-ET) imaging shows that the CupE pilus can adopt significant curvature in situ, which may aid in promoting cohesion between cells. Structural studies on the biofilm matrix fibre Fap from P. aeruginosa are presented, supporting previous work showing the fibres are amyloid. Moreover, a cryo-EM fibre structure of the major protein component of the Bacillus subtilis biofilm matrix, TasA, was solved. The structure shows that TasA fibres are not, like previously proposed, amyloid but instead polymerise through a donor strand, similar to Chaperone-Usher pili, but employing a different, previously undescribed assembly machinery. Finally, a compressed sensing algorithm is trialled for reconstruction of cryo-ET data of biological specimens, leading to improved direct visualisation and subtomogram averaging of macromolecules from small datasets, which may aid future studies in visualising the biofilm matrix in situ. Together, these findings will advance our structural understanding of biofilms.

Authors: Böhning, J

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: cryo-EM; cryo-ET; biofilm matrix fibres
• Subjects: biofilms; cryo-EM; structural biology