High-Resolution pH Imaging of Living Bacterial Cells To Detect Local pH Differences

Protons are utilized for various biological activities such as energy transduction and cell signaling. For construction of the bacterial flagellum, a type III export apparatus utilizes ATP and proton motive force to drive flagellar protein export, but the energy transduction mechanism remains unclear. Here, we have developed a high-resolution pH imaging system to measure local pH differences within living Salmonella enterica cells, especially in close proximity to the cytoplasmic membrane and the export apparatus. The local pH near the membrane was ca. 0.2 pH unit higher than the bulk cytoplasmic pH. However, the local pH near the export apparatus was ca. 0.1 pH unit lower than that near the membrane. This drop of local pH depended on the activities of both transmembrane export components and FliI ATPase. We propose that the export apparatus acts as an H⁺/protein antiporter to couple ATP hydrolysis with H⁺ flow to drive protein export.

The flagellar type III export apparatus is required for construction of the bacterial flagellum beyond the cellular membranes. The export apparatus consists of a transmembrane export gate and a cytoplasmic ATPase complex. The export apparatus utilizes ATP and proton motive force as the energy source for efficient and rapid protein export during flagellar assembly, but it remains unknown how. In this study, we have developed an in vivo pH imaging system with high spatial and pH resolutions with a pH indicator probe to measure local pH near the export apparatus. We provide direct evidence suggesting that ATP hydrolysis by the ATPase complex and the following rapid protein translocation by the export gate are both linked to efficient proton translocation through the gate.