Extended Data Fig. 8: Survival and LTA d-alanylation assays for wild-type and mutant DltB.

a, Lysozyme susceptibility survival assay. For DltB residues used in both LTA d-alanylation and survival assays, corresponding DltB residue numbers in two species are listed. The endogenous dlt operon was deleted in the B. subtilis strain and complemented with an ectopic copy of the wild-type dlt operon without tag on DltB. Representative images of serial dilutions of cells plated on LB agar (left) and LB agar supplemented with 30 µg ml⁻¹ of lysozyme (right). The genotype of the dltB gene is indicated above the corresponding column of serial dilutions. Dilutions of cells are indicated on the y axis. Mutation of the critical histidine (His328) and residues of DltB involved in binding with DltC(V297/F298) increase the susceptibility to lysozyme of B. subtilis. b, Per cent survival of B. bacillus variants towards lysozyme treatment. This was calculated by dividing the colony-forming units (CFUs) from lysozyme plates by the CFUs from LB-only plates. Data are mean ± s.d. of three biological replicates. The genotype of dltB is indicated at the bottom. B. subtilis strains containing untagged DltB show a similar lysozyme susceptibility pattern to those containing Flag-tagged DltB. c, LTA d-alanylation assay. In experiment 1, the assay time was 120 min after ¹⁴C-d-alanine was added, whereas for experiments 2 and 3, the assay time was 30 min. Experiments 2 and 3 are two parallel assays for LTA d-alanylation detection. AMSA represents m-AMSA, a DltB inhibitor.