FIG 5.

DpaA is active in cells. (A) The wild type (BW25113) or mutants with single or multiple gene deletions (BW25113 ΔdpaA, BW25113 Δlpp, and BW25113 ΔldtA ΔldtB ΔldtC) expressing DpaA or DpaA(C143A) from a plasmid, or carrying the empty plasmid, were grown in LB medium and harvested, and the muropeptide composition was determined. Only expression of active DpaA, and not the inactive DpaA(C143A), reduced the Tri-LysArg peak when present. (B) Muropeptide profiles of BW25113 ΔldtD ΔldtE ΔdpaA (lacking LDTs involved with 3-3 cross-link formation and DpaA) expressing DpaA, DpaA(C143A), or no DpaA version. Chromatographs were cropped above 250 mAU; the uncropped HPLC chromatographs of panels A and B are shown in Fig. S3. (C) Quantification of the total tripeptides and Tri-LysArg in the chromatograms shown in panels A and B. The values are means ± variations from two independent biological replicates, except BW25113 ΔldtA ΔldtB ΔldtC/pDpaA, which was prepared once. Significance was measured by a two-tailed, homoscedastic t test. ns (not significant), P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.