The pho-controlled outer membrane porin PhoE does not contain specific binding sites for phosphate or polyphosphates

Area of Science:

• Molecular biology
• Biophysics
• Membrane protein function

Background:

• PhoE-porins are outer membrane proteins crucial for nutrient uptake and antibiotic resistance in bacteria.
• Understanding porin selectivity and function is vital for developing new antimicrobial strategies.

Purpose of the Study:

• To investigate the effect of polyphosphates on the selectivity and size of reconstituted PhoE-porins.
• To elucidate the mechanism underlying polyphosphate-mediated changes in porin function.

Main Methods:

• Reconstitution of purified PhoE-porins into black lipid bilayer membranes.
• Electrophysiological measurements to determine pore selectivity and single-channel conductance.
• In vitro and in vivo experiments assessing the impact of polyphosphates and Mg2+ on porin function and antibiotic permeability.

Main Results:

• Polyphosphates shifted PhoE-porin selectivity from anion to cation, without affecting pore size.
• Polyphosphate-induced inhibition of pore conductance required the presence of Mg2+, suggesting chelate formation.
• These effects were not specific to phosphate, as other polyvalent anions like citrate mimicked the results.
• In vivo experiments confirmed that Mg2+ is necessary for polyphosphate to affect outer membrane permeability to beta-lactam antibiotics.

Conclusions:

• PhoE-porins lack specific binding sites for phosphate or polyphosphates.
• Anion selectivity arises from an excess of positive charges within the pore.
• Polyphosphate-mediated functional changes are likely due to interactions with divalent cations like Mg2+.