Figure 4. MurAA is stabilized in the absence of IreB or ClpC and by the N188K substitution.

Immunoblot analysis of whole cell lysates prepared from E. faecalis cultures pre- and post-chloramphenicol treatment to evaluate degradation of MurAA over time. Whole cell lysates were subjected to SDS-PAGE and immunoblotting analysis using a MurAA anti-serum. MurAA signal was normalized to total protein in each lane (total protein images shown in Supplemental Figure 8a) and compared to t₀. MurAA half-life (t_1/2) was calculated by fitting the normalized signal intensity over time to first order decay. The half-life for the ΔclpC and ΔireB strains was not determined (n.d.) due to nearly complete stabilization of MurAA over the course of the experiment. Quantification was done for three biological replicates and mean ± standard error is reported. Stars represent statistical differences relative to wild-type (*, p=0.01 to 0.05; **, p=0.001 to 0.01) based on Two-way ANOVA with Dunnett’s multiple comparisons. One representative immunoblot is shown for each strain. Strains are wild-type, OG1; ΔclpC, CM35; ΔireB, JL367; murAA N188K, CM3; murAA A115T, CM2).