Rachel Jones

Abstract:

Bacteria from the phylum Bacteroidota move by gliding across surfaces. Movement of extracellular adhesins attached to a helically-arranged periplasmic track causes propulsion of cells in a corkscrewing motion. The gliding track is propelled by a rotary motor that also powers the Type 9 Secretion System (T9SS) that exports the gliding adhesins to the cell surface. Flavobacterium johnsoniae is the model organism used to study Bacteroidota gliding. In this organism, sustained gliding requires the major adhesin SprB as well as the proteins RemF, RemG, SprC, SprD, and SprF that are encoded by the sprB operon.

In this thesis, I demonstrate that SprC is exported to the cell surface by the T9SS and subsequently anchored to the gliding track by the outer membrane protein SprD. Movement of SprC and SprD along the gliding track is independent of RemF and RemG but requires SprC export. SprC accumulates at the leading pole of individual cells actively gliding on glass and in cells at the leading edge of colonies spreading on agar. Based on these observations and structure predictions, I propose a model where SprD acts as a hub, binding SprC at its extracellular face whilst its periplasmic domain binds RemF and RemG. Accumulation of the SprCDRemFG complex at the leading pole then allows selective activation of the adhesins moving in one direction along the cell body. 

During this project, attempts were made to develop a microscope and corresponding data analysis pipeline suitable for 3D single particle tracking across a large field of view in order to analyse the behaviour of gliding components in actively gliding cells. Preliminary data from this microscope show that a single SprB molecule can take overlapping paths over the cell pole on successive circuits. However, modification of the setup to image over a reduced area may be necessary to achieve accurate tracking of single molecules in moving cells.

The architecture of the gliding track and the proteins that comprise it has heretofore been enigmatic. Here, single particle tracking of SprB was used to expand upon biochemical data to show that the protein GldK forms part of the gliding track and that the outer membrane protein SprF connects SprB to the track via a disulfide bond to GldK. Imaging experiments show that the mechanism of adhesin movement does not require the disulfide linkage between GldK and SprF to be reversible. These observations, together with my evidence for overlapping SprB trajectories, leads me to propose a new model for the organisation of the gliding track in which the `track’ is in reality short antiparallel filaments of GldJ and GldK.

Previous genetic studies have identified GldD, GldH, and GldI as three lipoproteins required for the stability of the GldJ and GldK components of the gliding track. Here I demonstrate that these proteins form a complex. 

The mechanism by which the gliding adhesins bind to surfaces is unknown. Because most characterized bacterial surface adhesins bind to polysaccharides, I investigated the potential role of secreted polysaccharides in mediating surface adhesion by F. johnsoniae. I characterise the phenotype of strains with deletions of two predicted polysaccharide exporters. Neither exporter is required for gliding on glass. One appears to export capsular polysaccharides whilst the other exports polysaccharides that are important for initial adhesin-dependent adhesion of cells to glass.