ATP-driven active transport in right-side-out bacterial membrane vesicles

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• Active transport is crucial for nutrient uptake in bacteria.
• Bacterial membrane vesicles can be engineered to study transport mechanisms.

Purpose of the Study:

• To investigate the mechanism of phosphoenolpyruvate-driven active transport in Salmonella typhimurium.
• To clone and characterize the phosphoglycerate transport system.

Main Methods:

• Preparation of Salmonella typhimurium membrane vesicles loaded with pyruvate kinase and ADP.
• Assaying active transport of proline and serine in the presence of phosphoenolpyruvate.
• Inhibition studies using carbonyl cyanide-m-chlorophenylhydrazone and N,N' dicyclohexylcarbodiimide.
• Cloning of the phosphoglycerate transport system gene into E. coli.

Main Results:

• Phosphoenolpyruvate-driven transport of proline and serine was observed in prepared vesicles.
• The transport was sensitive to protonophore and H+-ATPase inhibitors, indicating a proton motive force.
• D-lactate-driven transport showed different sensitivities to inhibitors and anoxia.
• Cloning confirmed the role of the phosphoglycerate transporter in this process.

Conclusions:

• A mechanism involving the phosphoglycerate transporter, pyruvate kinase, and H+-ATPase explains phosphoenolpyruvate-driven active transport.
• The generated proton electrochemical gradient drives H+/solute symport.
• The cloned phosphoglycerate transport system functions in E. coli vesicles.