Fig. 2.

Environmental pH controls speB expression via SIP signaling pathway. a The SIP* mutant strain was grown to early stationary phase (STAT, A₆₀₀ ~1.7) and harvested by centrifugation. Bacteria were suspended in THY broth adjusted to indicated pH, supplemented with synthetic SIP and incubated for 1 h. The fold-changes in speB transcript levels relative to the unsupplemented SIP* mutant strain are shown. P values (***P < 0.001) of the indicated samples were compared to unsupplemented GAS growth. P values were determined by t test. The amino acid sequence of the synthetic peptide SIP is shown in the inset. b Confocal microscopy images of isogenic SIP* mutant strain either unsupplemented or supplemented with indicated synthetic peptides in medium adjusted to indicated pH. For each sample, bright field, fluorescence field, merged images, and magnified views are shown. c The SIP* mutant strain was grown to STAT phase (A₆₀₀ ~1.7). Cells were transferred to THY broth adjusted to indicated pH and supplemented with either the indicated synthetic peptide or the carrier for the synthetic peptides (DMSO). After 1 h incubation, fluorescence measurements were obtained from clarified cell lysates using excitation and emission wavelengths of 480 and 520 nm, respectively. The changes in relative fluorescence units relative to the unsupplemented isogenic SIP* mutant strain are shown. The amino acid sequence of the synthetic peptide SIP with fluorescein modification at its amino-terminus (FITC-SIP) is shown in the inset. d RopB–SIP-binding constants assessed in binding buffer adjusted to indicated pH. e The relationship between pH and the ratio of cFSE intensities at wavelength 490 to 438 nm. The calibration curve with observed fluorescence ratio between fluorescence intensities at wavelength 490 to 435 nm in buffers adjusted to indicated pH (black triangles) is shown. The ratio of fluorescence intensities at wavelength 490 to 435 nm for cFSE-loaded GAS incubated in buffers adjusted to indicated pH are marked on the calibration curve (red circles). f The calculated GAS intracellular pH values in each tested extracellular pH values as determined by the equation derived from the calibration curve