Extended Data Fig. 1: S. aureus strains without dltX cannot survive on tunicamycin but still produce LTAs.
From: Mechanism of d-alanine transfer to teichoic acids shows how bacteria acylate cell envelope polymers
a, Chemical structures of three repeat units of Staphylococcus aureus LTA (left) and one repeat unit of Streptococcus pneumoniae LTA (right)2. d-Alanine esters are shown in blue. b, Full set of controls for the spot titer assay shown in Fig. 1c. S. aureus strains were grown on tryptic soy agar (TSA) plates containing the indicated compounds: dimethyl sulfoxide (DMSO; 1.25 µL per 1.00 mL of TSA), isopropyl-β-d-1-thiogalactopyranoside (IPTG; 1.00 mM), and/or tunicamycin (tunic.; 1.0 µg/mL). Tunic. inhibits WTA biosynthesis, IPTG was used to induce expression of the indicated plasmid-borne cassette, and DMSO was used as a vehicle control. The image of the plate with tunicamycin and IPTG has been reproduced here for clarity. The plasmid harboring dltXABCD is leaky thus resulting in growth of the sixth strain even in the absence of IPTG. c, An α-LTA western blot indicates that all of the strains tested in b and Fig. 1c produce LTAs at roughly equivalent levels to wild-type S. aureus. The strain in Lane 2 (4S5)75 is a mutant that lacks LtaS and contains a suppressor mutation that permits growth in the absence of LTAs. It does not produce any LTAs. The LTAs themselves run as a smear from ~15–37 kDa. d, Spot titer assay results for a partially different set of S. aureus strains. Here, the complementation constructs are under anhydrotetracycline (aTc)-inducible control, and the overall cassettes are genomically integrated. The TSA plates here contained DMSO, tunic., and aTc as indicated, with the concentrations of DMSO and tunic. being the same as for b and that for aTc being 0.4 µM. In these strains, the dltXABCD complementation construct does not appear leaky. e, SDS-PAGE autoradiography of LTAs from S. aureus cells grown in the presence of d-[14C]alanine again shows that dltX is required for LTA d-alanylation. Here, the same set of strains from d was used. f, An α-LTA western blot indicates that all strains tested in d and e produce LTAs at roughly equivalent levels to wild-type S. aureus.
