FIG 2.

pH drop assays. Strains SK36 (MMZ1612), manL, and manLComp were cultured to the late exponential phase (OD₆₀₀, 0.8) in 50 ml BHI medium, harvested by centrifugation, and used immediately for assay (A) or frozen at −80°C and then thawed and assayed at least 1 day later (B). Each sample was washed once with 50 ml cold water, resuspended in a solution containing 50 ml KCl and 1 mM MgCl₂, and normalized to an OD₆₀₀ of 4.5. The assay was initiated by the addition of 50 mM glucose, and pH was monitored and recorded at 30-s intervals for at least 40 min. Each curve includes the average and standard deviation (error bars) of three biological replicates. For statistical analysis, the pH values were first converted to proton concentrations, and the area under the curve value was calculated for each sample, followed by Student's t test for assessment of statistical significance (**, P < 0.01).