An Agrobacterium VirB10 mutation conferring a type IV secretion system gating defect

Area of Science:

• Microbiology
• Molecular Biology
• Structural Biology

Background:

• The Agrobacterium VirB/VirD4 type IV secretion system (T4SS) is crucial for DNA transfer.
• VirB10 is a key component of the T4SS core complex, spanning the cell envelope.
• VirB10 undergoes conformational changes linked to ATP hydrolysis, regulating substrate passage.

Purpose of the Study:

• To investigate the role of VirB10 in regulating substrate passage through the T4SS.
• To identify mutations in VirB10 that lead to unregulated substrate release.
• To elucidate the function of specific VirB10 domains in T4SS biogenesis.

Main Methods:

• Screening for Agrobacterium mutants with unregulated VirE2 substrate release.
• Characterizing the phenotype of VirB10 mutants, including ATP insensitivity and substrate transfer.
• Analyzing the effects of mutations on pilus production and cell envelope integrity.
• Comparing mutations in VirB10 with deletions in the antenna projection (AP) domain.

Main Results:

• A G272R mutation in VirB10 caused unregulated VirE2 release to the cell surface.
• The G272R mutation rendered VirB10 insensitive to ATP depletion but maintained DNA transfer.
• G272R blocked pilus production (Pil(-)) but not substrate transfer (Tra(+)).
• A partial deletion of the antenna projection (ΔAP) also resulted in a Tra(+) Pil(-) phenotype, releasing pilin monomers.

Conclusions:

• The G272 residue in VirB10 is critical for a gating mechanism controlling substrate passage across the outer membrane.
• VirB10's conformational state, influenced by ATP, regulates substrate translocation.
• Distinct mutations (G272R and ΔAP) disrupt pilus biogenesis at different stages.