Extended Data Fig. 7: DltB, DltD, and DltX levels are comparable between samples in the DltD d-alanylation in vitro reconstitution assay, and active forms of all three proteins plus DltA and DltC allow for rapid d-alanylation of DltD.
From: Mechanism of d-alanine transfer to teichoic acids shows how bacteria acylate cell envelope polymers
a, A Coomassie-stained SDS-PAGE gel from an in vitro reconstitution experiment run in parallel to the one shown in Fig. 4b but with cold d-alanine rather than d-[14C]alanine. Lane 4 contains all five active proteins, whereas Lanes 3, 5, and 6 are missing the indicated protein(s). Lanes 7–9 contain four active proteins and the indicated mutant. b, A Coomassie-stained SDS-PAGE gel (run using a Tris-glycine gel rather than a Bis-Tris gel as in a) of purified StDltBDX complex, specifically containing wild-type DltB, inactive DltX (Y56F), and wild-type DltD, provided as a reference for comparison to the samples in a. This suggests the impurity below DltB in a may be a degradation product. c, SDS-PAGE autoradiograph from a time course of the in vitro reconstitution with DltA, DltC, and DltBDX. For the ‘10 sec.’ time point, the DltBDX complex was added to the DltC charging reaction, the sample was mixed by pipetting up and down several times, and then immediately 4x XT sample buffer was added and mixed followed by flash freezing of the sample in liquid nitrogen. For all other time points, after the DltBDX complex was added to the DltC charging reaction, samples were incubated at 30 °C for the indicated amount of time before addition of SDS-PAGE loading buffer and immediate flash freezing in liquid nitrogen.
