The organisation and dynamics of Escherichia coli outer membrane proteins

Thesis

The outer membrane of E. coli is a component of the cell envelope with significant physiological importance. This multifunctional membrane allows the influx of nutrients and simultaneously excludes toxic compounds, such as antibiotics, which must cross this membrane to elicit their cytotoxic effect. The composition and general structure of this membrane are well known, an asymmetric lipid bilayer containing LPS in the outer leaflet, phospholipids in the inner leaflet, densely populated by integral β-barrel outer membrane proteins and extensively linked to the underlying peptidoglycan through certain lipoylated inner leaflet outer membrane proteins. Little is known, however, about the lateral organisation and dynamics of the proteins in this membrane. In this thesis I investigate the organisation and dynamics of two highly abundant outer membrane proteins: the general porin, OmpF, and the lipoprotein, Pal.

OmpF distribution—visualised with Colicin N fluorescent fusion proteins—could be sorted into distinct categories related to the cell cycle, and these distributions arose due to the biased insertion of OmpF at mid-cell. This biased mid-cell insertion also caused old OmpF to localise to the cell poles during growth. Superresolution investigations showed that at fine resolutions, OmpF displayed a significant degree of clustering in the outer membrane and was highly restricted in its diffusion (D_app: 0.0018 µm²s⁻¹).

Pal, investigated by PALM-SPT, was shown to accumulate at the septum of dividing cells, revealing a key mechanism by which this protein stabilises the outer membrane during division. Pal diffusion was highly restricted (0.0042 µm2s−1) as a result of peptidoglycan binding, demonstrated by an almost 5 fold increase in diffusion coefficient (0.0205 µm²s⁻¹) upon removal of the PG binding domain.

This work shows that integral β-barrel OMP distribution and dynamics are dictated by the subcellular biased assembly of these proteins into a membrane that does not permit lateral diffusion. In addition, analysis of Pal demonstrates the vastly different diffusive environment of the inner leaflet of the outer membrane and the contribution of underlying cell envelope components on protein diffusion.

Authors: Inns, P

• DOI: 10.5287/ora-gwynj54p4
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Bacteriocins; Outer Membrane Protein; OMPs; Superresolution Microscopy; Colicins; Membrane Biology; OmpF; Microscopy; Microbiology
• Subjects: Microbiology; Microscopy; Biochemistry