Journal: mBio / Year: 2022 Title: Structures of the Type IX Secretion/Gliding Motility Motor from across the Phylum . Authors: Rory Hennell James / Justin C Deme / Alicia Hunter / Ben C Berks / Susan M Lea / Abstract: Gliding motility using cell surface adhesins, and export of proteins by the type IX secretion system (T9SS) are two phylum-specific features of the Bacteroidetes. Both of these processes are ...Gliding motility using cell surface adhesins, and export of proteins by the type IX secretion system (T9SS) are two phylum-specific features of the Bacteroidetes. Both of these processes are energized by the GldLM motor complex, which transduces the proton motive force at the inner membrane into mechanical work at the outer membrane. We previously used cryo-electron microscopy to solve the structure of the GldLM motor core from Flavobacterium johnsoniae at 3.9-Å resolution (R. Hennell James, J. C. Deme, A. Kjaer, F. Alcock, et al., Nat Microbiol 6:221-233, 2021, https://dx.doi.org/10.1038/s41564-020-00823-6). Here, we present structures of homologous complexes from a range of pathogenic and environmental species at up to 3.0-Å resolution. These structures show that the architecture of the GldLM motor core is conserved across the phylum, although there are species-specific differences at the N terminus of GldL. The resolution improvements reveal a cage-like structure that ties together the membrane-proximal cytoplasmic region of GldL and influences gliding function. These findings add detail to our structural understanding of bacterial ion-driven motors that drive the T9SS and gliding motility. Many bacteria in the phylum use the type IX secretion system to secrete proteins across their outer membrane. Most of these bacteria can also glide across surfaces using adhesin proteins that are propelled across the cell surface. Both secretion and gliding motility are driven by the GldLM protein complex, which forms a nanoscale electrochemical motor. We used cryo-electron microscopy to study the structure of the GldLM protein complex from different species, including the human pathogens Porphyromonas gingivalis and Capnocytophaga canimorsus. The organization of the motor is conserved across species, but we find species-specific structural differences and resolve motor features at higher resolution. This work improves our understanding of the type IX secretion system, which is a virulence determinant in human and animal diseases.
UniProtKB: Gliding motility-associated protein GldL
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
1.0 mg/mL
Buffer
pH: 8 Component:
Concentration
Formula
Name
100.0 mM
NH2C(CH2OH)3.HCl
Tris-HCl
150.0 mM
NaCl
sodium chloride
0.01 %
C47H88O22
Lauryl Maltose Neopentyl Glycol
1.0 mM
C10H16N2O8
Ethylenediaminetetraacetic acid
Grid
Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Details: 15 mA
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Wait time 10 s Blot time 2 s.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 56.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 111727
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)
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Atomic model buiding 1
Details
A homology model based on the structure of FjoGldLM (PDB 6YS8) was used as a starting model and modified to fit the EM density using Coot. Refinement was carried out using Phenix.
Refinement
Space: REAL / Protocol: AB INITIO MODEL / Overall B value: 63.2
Output model
PDB-7sax: Structure of GldLM, the proton-powered motor that drives Type IX protein secretion and gliding motility in Sphingobacterium wenxiniae
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