(A) Lysates from wild-type M. smegmatis treated +/- benzyl alcohol (BA) were sedimented in a sucrose density gradient. Density of the cellular material is quantified in Figure 3—figure supplement 1. (B) Top, wild-type M. smegmatis was incubated or not with benzyl alcohol or dibucaine, then labeled with alkDADA; merged images correspond to fluorescent image with the corresponding phase contrast. Bottom, the distribution of peptidoglycan labeling from wild-type M. smegmatis that was incubated with BA or dibucaine (DB) for the indicated time was quantitated as in Figure 2A, except that signal intensity was not normalized. The changes in fluorescence are further quantified by flow cytometry in Figure 3—figure supplement 5. (C) Top left, DivIVA-eGFP-ID M. smegmatis was either treated with benzyl alcohol, depleted of DivIVA, or both, and the peptidoglycan precursors from whole cells were biotinylated as in Figure 2C. Bottom left, biotin-derived chemiluminescence was quantified by densitometry; signal is expressed as % of untreated DivIVA-eGFP-ID (first lane). Right, DivIVA-eGFP-ID M. smegmatis was treated as in the left panel but labeled with alkDADA, subjected to CuAAC, and analyzed by flow cytometry. MFI, median fluorescence intensity values for a representative experiment. Error bars denote standard deviation of technical triplicates. (D) Phylogenetic tree constructed with 16S rDNA sequences (rate of mutation not considered). Taxonomic groups matched with colors to their levels with only diverging points shown. Shapes and growth modes illustrated for select species. (E) Left, different bacteria treated +/- benzyl alcohol followed by alkDADA incubation. Arrowheads highlight irregular patches of peptidoglycan. Insets are magnified. Where applicable, E. coli was pre-incubated with A22. Right, bacteria were treated with benzyl alcohol, translation-inhibiting kanamycin, or peptidoglycan-acting phosphomycin or ampicillin and then labeled as in (B) and analyzed by flow cytometry (see Materials and methods). MFI values were normalized to untreated controls. Experiments were performed three to nine times in triplicate. Error bars denote standard deviation of biological replicates. *p<0.05; **p<0.005; ***p<0.0005; ****p<0.00005, ratio paired t-tests and one-way ANOVA with Dunnet’s test for non-normalized MFI of biological replicates. Scale bars, 5 µm.