Extended Data Fig. 5: Length alterations at the C-terminus of DltX are not well-tolerated.

a, Full set of controls for the spot titer assay shown in Fig. 3d. S. aureus strains were grown on TSA plates containing the indicated compounds: DMSO at 1.25 µL per 1.00 mL of TSA, IPTG at 1.00 mM, anhydrotetracycline (aTc) at 0.4 µM, and tunicamycin at 1.0 µg/mL. Tunic. inhibits wall teichoic acid biosynthesis, IPTG was used to induce expression of the indicated plasmid-borne cassette (here, null refers to a strain with an empty IPTG-inducible cassette in the plasmid), aTc was used to induce expression of the indicated chromosomally integrated cassette, and DMSO was used as a vehicle control for the tunic. The image of the plate with tunic., IPTG, and aTc has been reproduced here for clarity. Here, the bracketed sequences in the superscripts of the aTc-inducible cassettes represent the C-terminal motif sequence of the given DltX variant. Red letters denote changes from the wild-type sequence, and a red hyphen denotes the deletion of a residue. b, A spot titer assay shows that, of all the DltX mutants we have tested that do not allow for growth of a dltX-null strain on tunicamycin when expressed at low levels (from a chromosomally integrated aTc-inducible cassette), only the DltX mutant with an alanine added onto the C-terminus can complement when expressed at high levels (from an IPTG-inducible cassette on a medium copy number plasmid). Here, dltX* indicates that the plasmid encodes for a variant of DltX with the C-terminal sequence indicated in the ‘DltX C-term.’ column. Again, red letters denote changes from the wild-type sequence, and a red hyphen denotes the deletion of a residue. Both dltX^wt (wild-type) and all dltX* here encode an N-terminal FLAG tag.