Figure 2. ΔdacA1 mutant PG is less susceptible to cleavage by ShyA.

(A) Schematic representation of how DacA1 absence affects the pentapeptide content in the PG sacculus and as a result potential ShyA activity. (B) Schematic of experimental procedure. The PG sacculi from WT and ΔdacA1 cells were purified using the SDS precipitation method⁴⁷. Purified samples were then stained with Remazol Blue and used in sedimentation assays where PG fragmented by hydrolases remains soluble in the supernatant upon centrifugation, providing a quantitative readout of EP activity (C) RBB stained sacculi were treated as described in (B) with the addition of proteins indicated on the x-axis. Absorbance values were normalized against the buffer. Error bars represent standard deviation of two independent experiments. Significance was determined using a 2way ANOVA, **** corresponds to a P-value of <0.0001.