Structural studies of the Type IX secretion system inner membrane complex

Thesis

The Type IX Secretion System (T9SS) is a protein transport system found exclusively in the Bacteroidetes phylum of Gram-negative bacteria. The T9SS transports folded substrates across the outer membrane, including virulence factors of human pathogens, polysaccharide utilisation enzymes of environmental bacteria, and cell-surface adhesin molecules involved in Bacteroidetes gliding motility

The inner membrane proteins GldL and GldM use the proton-motive force to energise both the T9SS and the gliding motility apparatus. I present structures from five different species of the transmembrane core of the GldLM complex, solved using electron cryo-microscopy. The GldLM complex has two copies of GldM with one transmembrane helix each, surrounded by five copies of GldL with two transmembrane helices each. Most of the cytoplasmic domain of GldL was unresolved. I also present a low-resolution structure of the full length orthologous PorLM complex from Porphyromonas gingivalis.

Several polar residues are present in the transmembrane helices of GldL and GldM, suggesting that they may have a role in conducting protons across the inner membrane. I generated mutant strains where these polar residues were substituted with non-polar alternatives and found that some of these strains were deficient in T9SS and gliding motility. A universally conserved glutamate residue in GldL was essential to T9SS and gliding motility activity. In the structures of GldLM, one copy of this glutamate residue appears to form a salt bridge with a conserved arginine residue in one copy of GldM. I propose that a proton flow-dependent alternation of which copy of GldM is involved in salt bridge formation drives rotation of the GldM dimer, which spans the periplasm to connect the inner membrane and outer membrane components of the T9SS.

These results reveal the GldLM complex to be a third class of ion-driven rotary motor after the ATP synthase and the MotAB flagellar stator family.

Authors: Hennell James, R

• DOI: 10.5287/ora-qq5na86ej
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: bacterial secretion; cryoEM; gliding motility; type IX secretion system
• Subjects: Electron microscopes; Bacteria; Bacterial proteins; structural biology