Outer-membrane translocation of bulky small molecules by passive diffusion

The outer membrane (OM) of gram-negative bacteria forms a protective layer around the cell that serves as a permeability barrier to prevent unrestricted access of noxious substances. The permeability barrier of the OM results partly from the limited pore diameters of OM diffusion channels. As a consequence, there is an “OM size-exclusion limit,” and the uptake of bulky molecules with molecular masses of more than ∼600 Da is thought to be mediated by TonB-dependent, active transporters. Intriguingly, the OM protein CymA from Klebsiella oxytoca does not depend on TonB but nevertheless mediates efficient OM passage of cyclodextrins with diameters of up to ∼15 öÅÅ. Here we show, by using X-ray crystallography, molecular dynamics simulations, and single-channel electrophysiology, that CymA forms a monomeric 14-stranded β-barrel with a large pore that is occluded on the periplasmic side by the N-terminal 15 residues of the protein. Representing a previously unidentified paradigm in OM transport, CymA mediates the passive diffusion of bulky molecules via an elegant transport mechanism in which a mobile element formed by the N terminus acts as a ligand-expelled gate to preserve the permeability barrier of the OM. Significance The outer membrane (OM) of gram-negative bacteria forms a protective layer on the outside of the cell that prevents unrestricted access of harmful compounds. For the acquisition of ions and nutrients, the OM contains two types of transport proteins: passive diffusion channels and active transporters. Due to the limited diameters of passive diffusion channels, bulky molecules such as iron–siderophores and complex oligosaccharides are assumed to be taken up exclusively by active transporters. Here we assert that this assumption is incorrect. Using a combination of biophysical and computational approaches, we show that the OM protein CymA (cyclodextrin metabolism A) from Klebsiella oxytoca represents a previously unidentified paradigm in OM transport by mediating the passive diffusion of cyclic oligosaccharides (cyclodextrins) with diameters of ∼15 öÅ.