Extended Data Figure 3. Dipeptide probes differentially and specifically label PG of diverse gram-positive bacteria allowing live-cell experiments.

Phase contrast and epifluorescence microscopy of B. subtilis (a–c), Streptococcus pneumoniae (d), and Streptomyces venezuelae (e). a, Five minute and 60 minute aliquots were taken from wild-type B. subtilis grown with 0.5 mM alkyne containing EDA-DA, DA-EDA or as a positive control with EDA. These aliquots together with unlabeled controls were ‘clicked’ to Alexa Fluor 488 azide and imaged. When the alkyne is on the N-terminus (EDA-DA), labeling is comparable to EDA. On the other hand, the labeling with C-terminal tag (DA-EDA) is much fainter. b, B. subtilis grown with 0.5 mM alkyne containing EDA-DA or the L-enantiomer control ELA-LA for 45 min and clicked as above indicates that the labeling is D-enantiomer specific. The partial lysis of the cells visible in phase contrast is caused by 70% EtOH fixation. c–d, When live B. subtilis and S. pneumoniae labeled with azide containing ADA-DA and DA-ADA (0.4 mM and 1.6 mM for c and 0.5 mM for d) were clicked to Alexa Fluor 488 DIBO alkyne using a non-toxic procedure, the signals from N-terminally tagged dipeptide ADA-DA were much higher than the signal from DA-ADA labeled cells. (c) Interestingly, the signal from DA-ADA can be elevated to the ADA-DA level, if the labeling is performed in a ΔdacA, D,D-carboxpeptidase-null mutant of B. subtilis. Since copper-free click-chemistry is not toxic to cells, a pulse-chase experiment was done, which shows the trapping of old PG at the poles of the cells (lower panel). e, When polarly growing S. venezuelae cells are grown with the blue fluorescent D-amino acid HADA (2 h, 0.5 mM)¹⁹ for several generations and briefly pulsed with EDA-DA (10 min, 0.5 mM) and clicked, the signal from EDA-DA complements the signal from HADA. This result shows that dipeptide probes label the cell wall at sites of new PG synthesis. Fluorescent images (a–d) were taken and processed in the same manner for comparison. In ‘Adjusted’ images, signal intensities were lowered for comparison of labeling patterns. All experiments were conducted in biological duplicates, and images are representative of 2–5 fields viewed per condition/time point/replicate.