Outer-membrane translocation of bulky small molecules by passive diffusion

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Gram-negative bacteria possess an outer membrane (OM) acting as a permeability barrier.
• This barrier limits the uptake of molecules larger than 600 Da, typically requiring TonB-dependent active transporters.
• The OM protein CymA from Klebsiella oxytoca mediates passage of larger molecules without TonB.

Purpose of the Study:

• To elucidate the transport mechanism of the OM protein CymA.
• To understand how CymA facilitates the passage of bulky molecules across the bacterial outer membrane.
• To identify a novel mechanism for outer membrane transport in bacteria.

Main Methods:

• X-ray crystallography to determine CymA structure.
• Molecular dynamics simulations to analyze protein dynamics.
• Single-channel electrophysiology to study pore function.

Main Results:

• CymA forms a monomeric 14-stranded beta-barrel with a large pore.
• The N-terminal residues of CymA act as a mobile gate, occluding the pore on the periplasmic side.
• CymA mediates passive diffusion of molecules up to 15 Å in diameter.
• This represents a new paradigm in OM transport, distinct from TonB-dependent systems.

Conclusions:

• CymA utilizes a unique ligand-expelled gate mechanism for passive transport of bulky molecules.
• This mechanism preserves the outer membrane's permeability barrier while allowing entry of larger substances.
• CymA reveals a previously unknown pathway for nutrient uptake in gram-negative bacteria.