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. 2023 Oct 20;12:RP84427. doi: 10.7554/eLife.84427

Figure 4. In vivo phenotypes of PrgB variants with point mutation(s) in the conserved site of the polymer adhesin domain.

PrgB variants are expressed from the pMSP3545S-MCS vector in the OG1RF pCF10ΔprgB or OG1RF pCF10 strain and analyzed in: (A) cellular aggregation, (B) biofilm formation, and (C) conjugation assays. OG1RF pCF10 carrying the empty vector serves as positive control and OG1RF pCF10ΔprgB with the empty vector as negative control. The height of each column represents the average of three independent experiments and the error bars indicate the standard error of the mean (SEM). Statistical significance between the PrgB variants were analyzed with one-way analysis of variance, with * indicating p < 0.05, ** indicating p < 0.01, and *** indicating p < 0.001.

Figure 4.

Figure 4—figure supplement 1. Comparison of the DNA-binding affinity of the wild-type polymer adhesion domain from PrgB (PrgB188–1234 WT) and its S442A and N444A variant (PrgB188–1234 S442A N444A).

Figure 4—figure supplement 1.

(A) For this mobility shift assay, protein was mixed and incubated with 50 nM DNA100 and applied to native gels. From left to right, DNA mixtures with 0, 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10, or 20 mM protein were loaded and the final lane is a negative control with only 20 mM protein (and no DNA). (B) Plot of the relative intensities of the unbound (not upshifted) DNA bands normalized against the protein-free condition (lane 1). The error is the standard deviation (N = 2).
Figure 4—figure supplement 1—source data 1. Raw tif files of the EMSAs in Figure 4—figure supplement 1A.