Fig. 2: Periplasmic secretion of DbfQ is essential for its regulation of DbfS activity.
a Left: Representative confocal microscopy images of DbfQ-mNG in V. cholerae expressed from an ectopic locus. Right: As in left panel, except the secretion signal of DbfQ has been removed (dbfQ∆sec-mNG). b PhoA-LacZα fusions expressed in E. coli were used to assess DbfQ periplasmic localization. LacZ activity, which is non-functional in the periplasm, was assessed colorimetrically via the addition of Red-Gal (6-chloro-3-indolyl-β-D-galactoside), a beta-galactosidase substrate that turns red upon cleavage. Simultaneously, PhoA activity, which is functional in the periplasm but not the cytoplasm, is observed via the addition of X-Pho (5-bromo-4-chloro-3-indolyl-phosphate), which turns blue in the presence of phosphatase activity. Thus, redness is a measure of cytoplasmic localization, and blue pigmentation is representative of periplasmic localization. Controls included untagged, cytoplasmically expressed phoA-lacZα as well as a fusion of the periplasmic secretion signal of E. coli maltose binding protein (MBP) to phoA-lacZα (secMBP-phoA-lacZα). Results are representative of N = 3 biological replicates. c Quantification of PdbfQRS-lux reporter activity for secretion and cleavage mutants. Data are presented as means ± SD of peak RLU normalized to the average peak value of WT. Points represent individual replicates of N = 2 biological replicates and 3 technical replicates. Statistical analysis was performed using one-way ANOVA (P < 10−5) with Dunnett’s multiple comparisons test to compare each mutant to WT. P < 10−5 in all cases. ****P < 0.0001. d Western blot analysis of the FLAG-tagged WT DbfQ (cleaved) and the cleavage site mutants DbfQA31W and DbfQA31Y. Data are representative of N = 3 biological replicates.
