Assessing H+ Movements in Sugar Uptake

The detection of H⁺ movements associated with initial sugar uptake was assessed by adding sugar pulses to unbuffered cell suspensions^{62 (link)}. Bacteria (OD₆₀₀ of 1.0) were harvested by centrifugation (5000 g, 5 min, 4 °C), washed with ice-cold PBS, resuspended in 0.9% NaCl to a final concentration of 10⁶/ml and kept in 37 °C for 3 h for starvation. After starvation, 500 µl of WT, mutants, complement strains, and 0.9% NaCl (negative control) were seeded into a 24-well plate (Seahorse XF24 Cell Culture Microplates, Agilent). After acquiring baseline measurements, the following drug was used for an extracellular pH test: 30 mM fructose (port A). Cells were assessed for extracellular alkalinization rate using the Seahorse XF analyzer (Agilent)^{63 (link)}.